WO2025029677A2

WO2025029677A2 - Enzymes for fructose isomerization

Info

Publication number: WO2025029677A2
Application number: PCT/US2024/039877
Authority: WO
Inventors: Fernando Valle; Lu Yang; Ralph A. CACHO; Austin COLE
Original assignee: BP Corp North America Inc
Current assignee: BP Corp North America Inc
Priority date: 2023-07-28
Filing date: 2024-07-26
Publication date: 2025-02-06
Anticipated expiration: 2026-01-28
Also published as: WO2025029677A3

Abstract

The disclosure relates to polypeptides having the activity to isomerize fructose and glucose, such as at mesophilic temperatures. Recombinant microorganisms can be engineered to express the polypeptides. The recombinant microorganisms can be used in methods of isomerizing fructose and glucose, comprising culturing the recombinant microorganisms in a production medium comprising sucrose, fructose, and/or glucose. The culturing can be at mesophilic temperatures.

Description

ENZYMES FOR FRUCTOSE ISOMERIZATION

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional application no. 63/516,479, filed July 28, 2023, and United States provisional application no. 63/608,439, filed December 11 , 2023, the contents of which are incorporated herein in their entireties by reference thereto.

2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on June 17, 2024 is named BPC-013WO_SL.xml and is 31 ,232 bytes in size.

3. BACKGROUND

[0003] Sucrose is a plentiful and inexpensive material that can be isolated from various plant sources, such as sugarcane and sugar beet. Sucrose is a disaccharide comprising one glucose subunit and one fructose subunit.

[0004] Glucose isomerases are enzymes that catalyze the reversible isomerization of glucose to fructose. Such enzymes have been purified from microorganisms and used in the production of high fructose corn syrup (HFCS), which typically comprises 55% fructose instead of the 50% fructose found in hydrolyzed corn syrup. Production of HFCS from corn syrup is an in vitro process, and is typically conducted at temperatures of about 60°C or more.

[0005] There is a need in the art for efficient methods of isomerizing fructose and glucose in vivo at a temperature low enough for optimal growth and maintenance of most industriallyrelevant microorganisms.

4. SUMMARY

[0006] The present disclosure provides engineered enzymes with improved fructose isomerase activity. Engineered fructose isomerases of the present disclosure are described in Section 6.2 and Group 1 numbered embodiments 1 to 204 and 330 to 401. Exemplary engineered fructose isomerases of the present disclosure include those having an amino acid sequence comprising at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K compared to the amino acid sequence of SEQ ID NO:3.

[0007] Further examples of polypeptides, e.g., engineered fructose isomerases, of the present disclosure and nucleic acids encoding such polypeptides are also described in Group 2 numbered embodiments 1 to 17 and 45 to 107.

[0008] The fructose isomerases can have the activity of isomerizing fructose and glucose in vivo, e.g., at temperatures low enough for growth and maintenance of the microorganisms at temperatures that are cost-effective in industrial processes. In some embodiments, the temperatures are mesophilic temperatures, such as temperatures from 20°C to 40°C. The fructose isomerases can also have the activity of isomerizing fructose and glucose in vitro, e.g., at temperatures that require less energy expenditure than temperatures of about 60°C or more. Uses of engineered fructose isomerases of the present disclosure are described in Section 6.3 and Group 1 numbered embodiments 239 to 287.

[0009] Engineered fructose isomerases can be produced by engineering a microorganism (e.g., a microorganism lacking this capability) to express a fructose isomerase nucleotide sequence. The fructose isomerases can be used in vivo, e.g., by culturing recombinant microorganisms expressing fructose isomerase nucleotide sequences, or in vitro, e.g., after culturing recombinant microorganisms expressing fructose isomerase nucleotide sequences and purifying fructose isomerase polypeptides from recombinant microorganism biomass. Examples of microorganisms expressing fructose isomerases, are described in Section 6.5.1 and Group 1 numbered embodiments 213 to 234, and their use to isomerize fructose and glucose, optionally at mesophilic temperatures, is described in Section 6.8.3 and Group

1 numbered embodiments 240 to 270.

[0010] Further examples of recombinant microorganisms expressing polypeptides, e.g., engineered fructose isomerases, of the present disclosure are described in Group 2 numbered embodiments 18 to 20 and 110 to 112, and uses thereof are described in Group

2 numbered embodiments 22 to 28 and 113 to 119. [0011] Fructose and glucose isomerized by microorganisms of the disclosure can be provided by the hydrolysis of sucrose. In some embodiments, a microorganism of the disclosure can further be engineered to express a sucrose invertase nucleotide sequence. Examples of microorganisms configured to hydrolyze sucrose are described in Section 6.5.3 and Group 1 numbered embodiment 216.

[0012] Fructose, glucose, and/or sucrose can be non-phosphorylatively transported from the medium into a cell of the microorganism. In some embodiments, a microorganism of the disclosure can further be engineered to express a sucrose porin nucleotide sequence, a sucrose permease nucleotide sequence, a fructose porin nucleotide sequence, a fructose permease nucleotide sequence, a glucose porin nucleotide sequence, a glucose permease nucleotide sequence, or any two, three, four, five, or all six thereof. Examples of microorganisms configured to non-phosphorylatively transport sucrose, fructose, and/or glucose are described in Section 6.5.2 and Group 1 numbered embodiments 214 and 215.

[0013] Glucose produced by the microorganisms of the disclosure can subsequently enter into one or more cellular pathways. For example, glucose can be converted to 2-keto-3- deoxy-D-gluconate (KDG). In some embodiments, a microorganism of the disclosure can further be engineered to express a glucose dehydrogenase nucleotide sequence, a gluconate dehydratase nucleotide sequence, and optionally a gluconolactonase nucleotide sequence. Examples of microorganisms configured to convert glucose to KDG are described in Section 6.5.2 and Group 1 numbered embodiments 217 to 219, and their use is described in Section 6.8.4 and Group 1 numbered embodiments 271 to 287.

[0014] A microorganism of the disclosure can also further be engineered to have reduced phosphorylation of sucrose, fructose, and/or glucose. Doing so can reduce the mass of sugars shunted into other pathways, and is thereby expected to increasing the yield of glucose produced by isomerizing fructose. Examples of microorganisms configured to have reduced phosphorylation of sucrose, fructose, and/or glucose are described in Section 6.5.4 and Group 1 numbered embodiments 220 to 225.

[0015] Microorganisms of the disclosure can be engineered from parental microorganisms using known engineering techniques. Examples of parental microorganisms are described in Section 6.7 and Group 1 numbered embodiments 226 to 234. Examples of engineering techniques are described in Section 6.7.1. 5. BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 shows an exemplary pathway by which a recombinant microorganism according to some aspects of the present disclosure can hydrolyze sucrose to fructose and glucose (reaction [1]) and isomerize fructose and glucose (reaction [2]).

[0017] FIG. 2A schematically represents the effect of fructose on growth of E. coli strain SuA6. As described in Example 1 , because E. coli strain SuA6 lacks fructose isomerase activity, it cannot grow on fructose as its carbon source unless a fructose isomerase activity is engineered into it.

[0018] FIG. 2B schematically represents the effect of fructose on growth of E. coli strain SuA6 engineered to have a fructose isomerase activity.

[0019] FIG. 3 shows a schematic of plasmid pTrcHis2b-xylACT. XylA-CT: C. thermosulfurogenes xylose isomerase.

[0020] FIG. 4 shows an exemplary pathway by which a recombinant microorganism according to some aspects of the present disclosure can isomerize fructose and glucose and produce 2-keto-3-deoxygluconate (KDG) starting from glucose as precursor. Fructose can be isomerized to glucose by an engineered fructose isomerase of the disclosure (reaction 2). Glucose can be converted to gluconolactone by a glucose dehydrogenase (GDH) (reaction 3). Gluconolactone can spontaneously convert to gluconic acid, or this conversion can be catalyzed by a gluconolactonase (reaction 4). A gluconate dehydratase (GAD) can convert gluconic acid to KDG (reaction 5).

[0021] FIG. 5 shows an exemplary pathway by which a recombinant microorganism according to some aspects of the present disclosure can non-phosphorylatively transport extracellular sucrose into the cell and hydrolyze sucrose to glucose and fructose. Extracellular sucrose can be non-phosphorylatively transported into the periplasmic space by sucrose porin (activity A) and sucrose can be non-phosphorylatively transported from the periplasmic space into the cell by sucrose permease (activity B). Sucrose can be hydrolyzed (e.g., intracellularly) to fructose and glucose (reaction 1). Fructose and glucose can be isomerized (reaction 2) by an engineered fructose isomerase of the disclosure.

[0022] FIG. 6 shows an exemplary pathway by which a recombinant microorganism according to some aspects of the present disclosure can non-phosphorylatively transport extracel lular sucrose into the cell, hydrolyze intracellularly sucrose to fructose and glucose by a sucrose invertase (reaction 1), isomerize fructose and glucose by an engineered fructose isomerase of the disclosure, and produce 2-keto-3-deoxygluconate (KDG) starting from glucose as precursor. Activities A and B and reactions 1-5 are as shown in FIG. 4 and FIG. 5.

[0023] FIG. 7 graphically depicts the specific activity in vitro of twelve variants of the C. thermosulfurogenes XylA W139FA/186T double mutant using cell lysate, normalized relative to the activity of the double mutant.

6. DETAILED DESCRIPTION

6.1. Definitions

[0024] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. [0025] Various saccharides and compounds generated therefrom are chiral compounds, i.e., have D- and L- stereoisomers. Given that essentially all naturally-occurring saccharides have D- stereochemistry, saccharides may be referred to herein with or without the “D-“ prefix. In other words, unless the context dictates otherwise, the term “glucose” as used herein refers to D-glucose.

[0026] Fructose Isomerase. A fructose isomerase of the present disclosure is an enzyme that catalyzes the isomerization of fructose and glucose. An isomerase that can act on other monosaccharides in addition to fructose and glucose can be a fructose isomerase as described herein, provided it catalyzes the isomerization of fructose and glucose. A number of enzymes are known to catalyze isomerization of substrates other than fructose. Many of these enzymes can or potentially could also catalyze isomerization of fructose and are fructose isomerases as the term is used herein. Examples of such enzymes include xylose isomerase (which is known to catalyze isomerization between D-xylose and D-xylulose), also known as “XylA.” Exemplary XylAs include those from Actinoplanes missouriensis (UniProt identifier P12851), Escherichia coli (UniProt identifier P00944), Clostridium thermosulfurogenes (UniProt identifier P19148), Anoxybacillus kamchatkensis (UniProt identifier M4HQI7), Bacillus licheniformis (UniProt identifier P77832), B. coagulans (UniProt identifier G2TH70), Streptomyces rubiginosus (UniProt identifier P24300), S. olivochromogenes (UniProt identifier P15587), Thermotoga neapolitana (UniProt identifier P45687), Arthrobacter sp. (UniProt identifier P12070), Actinoplanes sp. (UniProt identifier p10654), and an uncultured bacterium (NCBI Protein Database ID AEL74969). Examples of such enzymes also include L-rhamnose isomerase (which is known to catalyze isomerization between L-rhamnose and L-rhamnulose), also known as “L-Rhi”. An exemplary L-Rhi is that from Pseudomonas stutzeri (UniProt identifier Q75WH8). Other examples of such enzymes include glucose-6-phosphate isomerase (GPI), also known as phosphoglucose isomerase/phosphoglucoisomerase (PGI) or phosphohexose isomerase (PHI), which is known to catalyze the isomerization of glucose-6-phosphate and fructose-6- phosphate. Exemplary PGIs or putative PGIs include those from Rhizobium meliloti (strain 1021) (UniProt identifier Q92UI1 and UniProt identifier Q92MQ8), E. coli MG1655 (UniProt identifier P0A6T1), Salmonella enterica serovar typhimurium (strain LT21 SGSC1412 / ATCC 700720) (UniProt identifier Q8ZMP7), Archaeoglobus fulgidus (UniProt identifier 028778), Methanosarcina mazei (UniProt identifier Q8PVJ5), and Pyrococcus furiosus (UniProt identifier P83194). Further examples of such enzymes include mannose-6- phosphate isomerase (ManA), also known as phosphomannose isomerase (PMI), which is known to catalyze the interconversion of fructose 6-phosphate and mannose-6-phosphate, an example of which is ManA from E. coli MG1655 (UniProt identifier P00946); D- glucoronate/D-galacturonate isomerase (UxaC), which is known to catalyze the interconversion of D-glucoronate and D-fructuronate, an example of which is UxaC from E. coli MG1655 (UniProt identifier P0A8G3); and 5-dehydro-4-deoxy-glucuronate isomerase (Kdul), which is understood to catalyze the interconversion of 5-dehydro-4-deoxy-D- glucuronate to 3-deoxy-D-glycero-2,5-hexodiulosonate, an example of which is Kdul from E. coli MG1655 (UniProt identifier Q46938).

[0027] Heterologous: The term “heterologous”, as used herein in relationship to a polypeptide (or amino acid sequence) or nucleic acid (or nucleotide sequence), refers to polypeptide (or amino acid sequence) or nucleic acid (or nucleotide sequence) that has been engineered into a microorganism. For example, in relation to a nucleic acid or nucleotide sequence, the nucleic acid or nucleotide sequence is deemed to be “heterologous” to a recombinant microorganism when the nucleic acid does not include a coding region having a nucleotide sequence not found in the parental microorganism of the recombinant microorganism, when a coding region encodes an amino acid sequence not found in the parental microorganism, and/or the nucleic acid includes a coding region operably linked to a regulatory region to which it is not operably linked in the parental microorganism. Similarly, in relation to a polypeptide or amino acid sequence, the polypeptide or amino acid sequence is deemed to be “heterologous” to a recombinant microorganism when the polypeptide is not found in the parental microorganism of the recombinant microorganism or the polypeptide has an amino acid sequence that is not found in the parental microorganism.

[0028] Nucleic Acid: The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.

[0029] Operably linked: In the context of transcriptional regulation, the term “operably linked” refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter, operator, or other regulatory region is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate recombinant cell or other expression system.

[0030] Parental cell, parental microorganism: The terms “parental cell” or “parental microorganism” are used interchangeably to refer to unicellular organisms which can be engineered to express one or more heterologous polypeptides or heterologous nucleic acids. A parental microorganism can be a bacterium, an archaeon, a fungus (e.g., a yeast), or any other unicellular organism. The adjective “parental” indicates that a recombinant cell or recombinant microorganism can be engineered by the introduction into a parental cell or parental microorganism of a heterologous nucleic acid or plurality of heterologous nucleic acids, such as nucleic acid(s) each comprising a coding region or plurality of coding regions each encoding a heterologous polypeptide. A parental microorganism can be a microorganism found in nature or a microorganism that is non-naturally occurring. In other words, a parental microorganism can comprise one or more genetic modifications (e.g., insertion, deletion, or modification of one or more coding regions and/or regulatory regions) relative to a strain thereof found in nature. In relationship to a recombinant microorganism of the disclosure generated through a series of engineering steps, the terms “parental cell” and “parental microorganism” can refer to an ancestral cell or organism incorporating any of the engineering steps, as well as a cell or microorganism without any of the engineering steps. Sometimes, for ease of reference and comparison, the terms “parental cell” and “parental microorganism” refer to a cell or microorganism which, if having genetic modifications, the genetic modification(s) do not relate to any of the pathway components specifically described herein.

[0031] Polypeptide, peptide, and protein: The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. A polypeptide herein may be identified by a name or by a percentage of sequence identity to a reference amino acid sequence. When a polypeptide is identified by a name indicative of an activity performed or enabled by the polypeptide, the name refers to any polypeptide capable of performing or enabling the activity.

[0032] Recombinant cell, recombinant microorganism: The terms “recombinant cell” and “recombinant microorganism” are used interchangeably to refer to a cell that has been genetically engineered. It should be understood that this term refers not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, a recombinant counterpart of a parental cell or parental microorganism includes progeny that are not identical to the initial recombinant cell or microorganism engineered from the parent cell or parental microorganism, but are still included within the scope of the terms “recombinant cell” or “recombinant microorganism” as used herein.

[0033] Sequence identity: “Sequence identity” in relation to nucleotide or amino acid sequence of a nucleic acid or polypeptide molecule, refers to the overall relatedness between two such sequence. Calculation of the percent sequence identity (nucleotide or amino acid sequence identity) of two sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid or amino acid sequence for optimal alignment). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Percent sequence identity can be determined manually once an alignment of nucleotide or amino acid sequences is generated. An alignment of query nucleotide or amino acid sequence and a reference nucleotide or amino acid sequence can be generated using the computer program ClustalW (version 1 .83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). ClustalW calculates the best match between a query and one or more reference sequences and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a reference sequence, or both, to maximize sequence alignments. For fast pair wise alignment of nucleotide sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For fast pairwise alignment of amino acid sequences, the following parameters are used: word size: 1 ; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. Unless indicated otherwise, the percent sequence identity between a reference nucleotide or amino acid sequence (e.g. a sequence with a defined SEQ ID NO: as disclosed herein) and a query nucleotide or amino acid sequence is calculated across the entire length of the reference sequence.

[0034] Transformation: The term “transformation” refers to the introduction of nucleic acid molecules into cells, e.g., into prokaryotic cells. In the context of the present disclosure, the term “transformation” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, e.g., into prokaryotic cells, such as into bacterial cells.

Such methods encompass, for example, electroporation, calcium phosphate precipitation, or nanoparticle-based transformation, among other techniques known to the person of ordinary skill in the art having the benefit of the present disclosure.

6.2. Engineered Fructose Isomerases

[0035] In one aspect, the present disclosure provides engineered fructose isomerases.

[0036] In some embodiments, engineered fructose isomerases of the present disclosure can isomerize fructose to glucose in vivo at mesophilic temperatures. Mesophilic temperatures are 20°C-45°C and includes subranges thereof (e.g., 20°C-25°C, 25°C-30°C, 30°C-35°C, 35°C-40°C, 40°C-45°C, or any combination of the foregoing) and particular temperatures therein, such as 37°C.

[0037] In some embodiments, a fructose isomerase of the present disclosure has an activity identified by EC numbers 5.3.1.5, 5.3.1.8, 5.3.1.9, 5.3.1.12, 5.3.1.14, and/or 5.3.1.17. In some embodiments, a fructose isomerase of the present disclosure has an activity identified by EC number 5.3.1.5.

[0038] In some embodiments, when fructose isomerases are heterologous to and are expressed by recombinant organisms that otherwise lack the ability to metabolize fructose, a recombinant organism expressing a fructose isomerase has greater growth than a nonexpressing control organism, as determined by the optical density at 600 nm (OD₆oo) after 3 days of growth in a medium containing fructose at the optimal temperature for growth of the organism.

[0039] In some embodiments, fructose isomerases of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to the mature sequence of at least one of Actinoplanes missouriensis xylose isomerase (XylA) having UniProt identifier P12851 (SEQ ID NO:1), Escherichia coli xylose isomerase (XylA) having UniProt identifier P00944 (SEQ ID NO:2), Clostridium thermosulf rogenes xylose isomerase (XylA) having UniProt identifier P19148 (SEQ ID NO:3), Anoxybacillus kamchatkensis xylose isomerase (XylA) having UniProt identifier M4HQI7 (SEQ ID NO:4), Bacillus licheniformis xylose isomerase (XylA) having UniProt identifier P77832 (SEQ ID NO:5), Bacillus coagulans xylose isomerase (XylA) having UniProt identifier G2TH70 (SEQ ID NO:6), Streptomyces rubiginosus xy\ose isomerase (XylA) having UniProt identifier P24300 (SEQ ID NO:7), Streptomyces olivochromogenes xylose isomerase (XylA) having UniProt identifier P15587 (SEQ ID NO:8), Thermotoga neapolitana xylose isomerase (XylA) having UniProt identifier P45687 (SEQ ID NO:9), uncultured bacteria xylose isomerase (XylA) having NCBI identifier AEL74969 (SEQ ID NQ:10), Pseudomonas stutzeri L- rhamnose isomerase (L-Rhi) having UniProt identifier Q75WH8 (SEQ ID NO:11), Arthrobacter sp. xylose isomerase (XylA) having UniProt identifier P12070 (SEQ ID NO:12), Rhizobium meliloti (strain 1021) putative glucose-6-phosphate isomerase (pgiA2) having UniProt identifier Q92UI1 (SEQ ID NO:13), E. coli MG1655 mannose-6-phosphate isomerase (ManA) having UniProt identifier P00946 (SEQ ID NO:14), Pseudomonas stutzeri L-rhamnose isomerase - E. coli optimized (IDT) (L-Rhi) having UniProt identifier Q75WH8 (SEQ ID NO:15), E. coli MG1655 glucose-6-phosphate isomerase (PGI) having UniProt identifier P0A6T1 (SEQ ID NO:16), E. coli MG1655 D-glucoronate/D-galacturonate isomerase (UxaC) having UniProt identifier P0A8G3 (SEQ ID NO:17), Rhizobium meliloti (strain 1021) putative glucose-6-phosphate isomerase (pgiA1) having UniProt identifier Q92MQ8 (SEQ ID NO: 18), Salmonella enterica serovar typhimurium (strain LT2 / SGSC1412 / ATCC 700720) glucose-6-phosphate isomerase (PGI) having UniProt identifier Q8ZMP7 (SEQ ID NO:19), E. coli MG1655 5-dehydro-4-deoxy-glucuronate isomerase (Kdul) having UniProt identifier Q46938 (SEQ ID NQ:20), Archaeoglobus fulgidus glucose-6- phosphate isomerase (PGI) having UniProt identifier 028778 (SEQ ID NO:21), Methanosarcina mazei glucose-6-phosphate isomerase (PGI) having UniProt identifier Q8PVJ5 (SEQ ID NO:22), Pyrococcus furiosus glucose-6-phosphate isomerase (PGI) having UniProt identifier P83194 (SEQ ID NO:23), or Actinoplanes sp. xylose isomerase (XylA) having UniProt identifier p10654 (SEQ ID NO:24). [0040] In some embodiments, fructose isomerases of the present disclosure differ from any one of SEQ ID NO: 1-24 by (a) at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions, deletions, or insertions and/or (b) up to 11 , up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 substitutions, deletions, or insertions. For example, the fructose isomerase can comprise an amino acid sequence having 3-10, 3-12, 3-15, 3-20, 4-10, 4-12, 4-15, or 4-20 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.ln particular embodiments, fructose isomerases can comprise an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3, with particular amino acid substitutions engineered in view of the information set forth below. Optionally, the amino acid sequences of the fructose isomerases can comprise at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:3. In some embodiments, the amino acid substitutions do not consist of W139F and V186T.

[0041] The active site of Clostridium thermosulfurogenes xylA comprises residues 101 and 104; the binding site for Co2+ (cofactor) comprises residues 232, 268, 271 , 296, 307, 309, and 339. Mutagenesis of residue 101 abolishes activity. The substitutions W139F and V186T enhance activity. Unexpectedly, and as described in Example 1, the inventors discovered that adding to or modifying the W139F/ V186T double mutant with one or more of the substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K further enhanced activity over the W139F/ V186T double mutant.

[0042] In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises both the substitutions W139F and/or V186T and optionally one or more substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0043] In some embodiments, polypeptides of the present disclosure comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3 and one or more of the amino acid substitutions: a) N19E or N19T; b) Y22F; c) A47S; d) Q59M; e) C99S or C99T; f) D128E; g) T141C or T141S; h) A142N, A142S, or A142T; i) L144C, L144K, or L144M; j) F150Y; k) S155A; l) V186C; m) F187L, F187Q, F187W, or F187Y; n) E203Q; o) N249H or N249S; p) F276Q; q) T299E; r) F363Y; s) K410E; t) R415K; u) Q417E or Q417K; and v) R424K; as compared to the amino acid sequence of SEQ ID NO:3.

[0044] In some embodiments, polypeptides of the present disclosure comprise at least two of the substitutions (a)-(v).

[0045] In some embodiments, polypeptides of the present disclosure comprise at least three of the substitutions (a)-(v).

[0046] In some embodiments, polypeptides of the present disclosure comprise at least four of the substitutions (a)-(v).

[0047] In some embodiments, polypeptides of the present disclosure comprise at least five of the substitutions (a)-(v).

[0048] In some embodiments, polypeptides of the present disclosure comprise up to six of the substitutions (a)-(v).

[0049] In some embodiments, polypeptides of the present disclosure comprise up to seven of the substitutions (a)-(v).

[0050] In some embodiments, polypeptides of the present disclosure comprise up to eight of the substitutions (a)-(v).

[0051] In some embodiments, polypeptides of the present disclosure comprise up to nine of the substitutions (a)-(v).

[0052] In some embodiments, polypeptides of the present disclosure comprise up to ten of the substitutions (a)-(v).

[0053] In some embodiments, polypeptides of the present disclosure further comprise one or both of the amino acid substitutions W139F and V186T. [0054] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, A142N, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0055] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, A142S, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0056] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions A47S, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0057] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99S, W139F, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0058] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99S, W139F, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0059] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, T141S, A142S, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

[0060] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, T141S, A142T, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

[0061] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0062] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, and V186C as compared to the amino acid sequence of SEQ ID NO:3. [0063] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0064] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions D128E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0065] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:3.

[0066] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, F150Y, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0067] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:3.

[0068] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:3.

[0069] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

[0070] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:3.

[0071] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3. [0072] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

[0073] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

[0074] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, L144C, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0075] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, L144K, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0076] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, L144M, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0077] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0078] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0079] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:3.

[0080] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:3. [0081] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:3.

[0082] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:3.

[0083] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0084] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

[0085] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0086] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, S155A, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0087] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141C, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0088] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141S, A142T, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0089] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141S, L144M, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3. [0090] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141S, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0091] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0092] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:3.

[0093] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Y22F, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

[0094] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0095] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0096] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions D128E, W139F, F150Y, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

[0097] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

[0098] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, D128E, W139F, V186T, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:3. [0099] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, A47S, D128E, W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

[0100] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0101] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions A47S, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0102] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0103] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions A47S, W139F, V186T, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0104] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions D128E, W139F, V186T, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

[0105] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, D128E, W139F, V186T, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0106] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and A142N. as compared to the amino acid sequence of SEQ ID NO:3.

[0107] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and A142S as compared to the amino acid sequence of SEQ ID NO:3.

[0108] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and A47S as compared to the amino acid sequence of SEQ ID NO:3.

[0109] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, C99S, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0110] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, C99S, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0111] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, C99T, T141S, A142S, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

[0112] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, C99T, T141S, A142T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

[0113] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, C99T, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0114] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, C99T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0115] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and C99T as compared to the amino acid sequence of SEQ ID NO:3.

[0116] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and D128E as compared to the amino acid sequence of SEQ ID NO:3.

[0117] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:3.

[0118] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F150Y as compared to the amino acid sequence of SEQ ID NO:3.

[0119] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:3.

[0120] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:3.

[0121] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

[0122] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187Y. [0123] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

[0124] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

[0125] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

[0126] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and L144C as compared to the amino acid sequence of SEQ ID NO:3.

[0127] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and L144K as compared to the amino acid sequence of SEQ ID NO:3. [0128] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and L144M as compared to the amino acid sequence of SEQ ID NO:3.

[0129] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N19E as compared to the amino acid sequence of SEQ ID NO:3.

[0130] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N19T as compared to the amino acid sequence of SEQ ID NO:3.

[0131] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:3.

[0132] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:3. [0133] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:3.

[0134] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:3.

[0135] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Q59M.

[0136] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

[0137] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0138] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and S155A as compared to the amino acid sequence of SEQ ID NO:3.

[0139] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and T141C as compared to the amino acid sequence of SEQ ID NO:3.

[0140] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, T141S, and A142T as compared to the amino acid sequence of SEQ ID NO:3.

[0141] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, T141S, L144M, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

[0142] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and T141S as compared to the amino acid sequence of SEQ ID NO:3.

[0143] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID N0:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:3.

[0144] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:3.

[0145] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Y22F as compared to the amino acid sequence of SEQ ID NO:3.

[0146] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0147] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0148] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, D128E, F150Y, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

[0149] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

[0150] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, N19E, D128E, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

[0151] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, N19E, A47S, D128E, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

[0152] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, N19E, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0153] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, A47S, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0154] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0155] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, A47S, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0156] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, D128E, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

[0157] In particular embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3; is identical to SEQ ID NO:3 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, D128E, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

[0158] The active site of Thermotoga neapolitana xylA comprises residues 101 and 104; the binding site for Co2+ (cofactor) comprises residues 232, 268, 271 , 296, 307, 309, and 339. In some embodiments, an engineered Thermotoga neapolitana xylA comprises one, two, or three amino acid substitutions V186T, L283P, and/or F187S as compared to the amino acid sequence of SEQ ID NO:9. In some embodiments, an engineered Thermotoga neapolitana xylA comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9. In some embodiments, an engineered Thermotoga neapolitana xylA is identical (i.e. , does not have a substitution relative) to SEQ ID NO:9 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339 of SEQ ID NO:9. In some particular embodiments, an engineered Thermotoga neapolitana xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:9; (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:9 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339, and/or (c) comprises the amino acid substitutions V186T, L283P, and F187S as compared to SEQ ID NO:9.

[0159] Alignment of the primary amino acid sequences or the tertiary structures of xylAs (e.g., SEQ ID NQ:1-10, 12, or 24) can identify residues of any xylA which are aligned with residues of SEQ ID NO:3 and/or SEQ ID NO:9 where the substitutions increased fructose isomerase activity.

[0160] In some embodiments, the aligned residues of any xylA can thus be substituted with the same amino acids of the substitutions of SEQ ID NO:3 and/or SEQ ID NO:9 set forth in particular embodiments above.

[0161] The active site of Actinoplanes missouriensis xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 292. In some embodiments, an engineered Actinoplanes missouriensis xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:1 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:1 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 292. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9. [0162] The active site of E. coli xylA comprises residues 101 and 104; and the binding site for Mg2+ (cofactor) comprises residues 232, 268, 271 , 296, 307, 309, and 339. In some embodiments, an engineered E. coli xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:2 and (b is identical (i.e. , does not have a substitution relative) to SEQ ID NO:2 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9.

[0163] The active site of Anoxybacillus kamchatkensis xylA comprises residues 99 and 102; and the binding site for Mg2+ (cofactor) comprises residues 230, 266, 269, 294, 305, 307, and 337. In some embodiments, an engineered Anoxybacillus kamchatkensis xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:4 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:4 at positions 99, 102, 230, 266, 269, 294, 305, 307, and 337. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9.

[0164] The active site of Bacillus licheniformis xylA comprises residues 99 and 102; and the binding site for Mg2+ (cofactor) comprises residues 230, 266, 269, 294, 305, 307, and 337. In some embodiments, an engineered Bacillus licheniformis xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:5 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:5 at positions 99, 102, 230, 266, 269, 294, 305, 307, and 337. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9.

[0165] The active site of Bacillus coagulans xylA comprises residues 100 and 103; and the binding site for Mg2+ (cofactor) comprises residues 231 , 267, 270, 295, 306, 308, and 338. In some embodiments, an engineered Bacillus coagulans xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:6 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:6 at positions 100, 103,

231 , 267, 270, 295, 306, 308, and 338. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9.

[0166] The active site of Streptomyces rubiginosus xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 287. In some embodiments, an engineered Streptomyces rubiginosus xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:7 and (b is identical (i.e., does not have a substitution relative) to SEQ ID NO:7 at positions 54, 57, 181, 217, 220, 245, 255, 257, and 287. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9.

[0167] The active site of Streptomyces olivochromogenes xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 287. In some embodiments, an engineered Streptomyces olivochromogenes xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:8 and (b) is identical (i.e. , does not have a substitution relative) to SEQ ID NO:8 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 287. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9.

[0168] The cofactor binding site of L-rhamnose isomerase of Pseudomonas stutzeri comprises residues 219, 254, 257, 281 , 289, 291, 298, and 327. In some embodiments, an engineered L-rhamnose isomerase of Pseudomonas stutzeri (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:11 or SEQ ID NO:15 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO: 11 or SEQ ID NO: 15 at positions 219, 254, 257, 281 , 289, 291 , 298, and 327.

[0169] The active site of Arthrobacter sp. (strain NRRL B3728) xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 293. In some embodiments, an engineered Arthrobacter sp. (strain NRRL B3728) xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:12 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:12 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 293. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:3 or V186T, L283P, or F187S of SEQ ID NO:9.

[0170] The binding site of Pyrococcus furiosus PGI for Fe cation (cofactor) comprises residues 88, 90, 97, and 146. The substitution T85Q may impart higher fructose isomerase activity. In some embodiments, an engineered Pyrococcus furiosus PGI (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:23; (b) is identical (i.e. , does not have a substitution relative) to SEQ ID NO:23 at positions 88, 90, 97, and 146; and/or comprises the substitution T85Q relative to SEQ ID NO:23.

[0171] Alignment of the primary amino acid sequences or the tertiary structures of PGIs (e.g. , SEQ I D NO: 13, 16, 18, 19, 21 , 22, or 23) can identify a residue of any PGI which is aligned with position 85 of SEQ ID NO:23.

[0172] In some embodiments, the residue of any PGI aligned with T85 of SEQ ID NO:23 can thus be substituted with Gin.

[0173] The binding sites of both Rhizobium meliloti pgiA1 and pgiA2 for Fe cation (cofactor) comprise residues 92, 94, 101 , and 140. In some embodiments, an engineered Rhizobium meliloti pgiA1 or pgiA2 (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:13 or SEQ ID NO:18 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO: 13 or SEQ ID NO: 18 at positions 92, 94, 101 , and 140.

[0174] The binding site of E. coli manA for Zn2+ (cofactor) comprises residues 97, 99, 134, and 255, and the active site is expected on the basis of similarity studies to comprise residue 274. In some embodiments, an engineered E. coli manA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:14 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:14 at positions 97, 99, 134, 255, and 274. [0175] The active site of E. coli PGI comprises residues 355, 386, and 514. In some embodiments, an engineered E. coli PGI (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 16 and (b) is identical (i.e. , does not have a substitution relative) to SEQ ID NO: 16 at positions 355, 386, and 514.

[0176] The binding site of E. coli Kdul for Zn2+ (cofactor) comprises residues 196, 198, 203, and 245. In some embodiments, an engineered E. coli Kdul (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NQ:20 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NQ:20 at positions 196, 198, 203, and 245.

[0177] The active site of Actinoplanes sp. (strain ATCC 31351 I 3876) xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 292. In some embodiments, an engineered Actinoplanes sp. (strain ATCC 31351 / 3876) xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:24 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:24 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 292..

[0178] Exemplary reference amino acid sequences for the engineered fructose isomerases of the present disclosure are provided in Table 1 :

6.3. Uses of Engineered Fructose Isomerases

[0179] The present disclosure provides engineered fructose isomerases. The engineered fructose isomerases can be produced by expression of nucleotide sequences encoding the engineered fructose isomerases in recombinant microorganisms.

[0180] In some embodiments, engineered fructose isomerases can be used in vivo, e.g., recombinant microorganisms expressing the engineered fructose isomerases can be configured to isomerize fructose and glucose. Accordingly, a recombinant microorganism of the disclosure may comprise means for isomerizing fructose and glucose. [0181] The isomerization of fructose and glucose can enhance a recombinant microorganism’s ability to survive or grow in media comprising fructose relative to a parental microorganism that lacks this ability. Alternatively, the isomerization of fructose and glucose can enhance a recombinant microorganism’s ability to survive or grow in media comprising glucose relative to a parental microorganism that lacks this ability.

[0182] The isomerization of fructose and glucose can enhance a cell’s ability to produce a product via a pathway to which fructose or glucose can be a feedstock. For example, in a recombinant microorganism in which glucose is converted to 2-keto-3-deoxygluconate (“KDG”), the isomerization of fructose and glucose in combination with the essentially irreversible conversion of glucose to KDG can drive the equilibrium of isomerization toward glucose, thereby increasing the yield of KDG from a given amount of starting material, e.g., sucrose, fructose, or glucose.

[0183] The isomerization of fructose and glucose can be useful in vitro. For example, a fructose isomerase can be purified from cells in which it is translated, and the purified fructose isomerase can be used to isomerize fructose and glucose in the production of high fructose corn syrup, other sweetening agents, or other products of processes to which fructose or glucose can be a feedstock.

6.4. Pathways Using Engineered Fructose Isomerases

[0184] The isomerization of fructose and glucose is independent of the cell’s source or sources of these monosaccharides, and the isomerized fructose or glucose can be used in any intracellular process making use of either monosaccharide. Accordingly, the fructose isomerases described in Section 6.2 can be included in a variety of intracellular biochemical pathways.

[0185] For example, fructose and/or glucose can be non-phosphorylatively transported into a cell from media comprising fructose and/or glucose, and subsequently isomerized. For another example, sucrose can be non-phosphorylatively transported into a cell from media comprising sucrose. Sucrose can be hydrolyzed to fructose and glucose, and the monosaccharides can subsequently be isomerized.

[0186] In some embodiments, such as is schematically depicted in FIG. 5, wherein the recombinant microorganism is a Gram-negative bacterium, non-phosphorylative transport of sucrose includes the activity of a sucrose porin (activity A) and a sucrose permease (activity B), and hydrolysis of sucrose to glucose and fructose includes the activity of a sucrose invertase enzyme (reaction 1). Isomerization of fructose and glucose by the activity of a fructose isomerase is shown as reaction 2.

[0187] The isomerization of fructose and glucose can provide fructose or glucose for biochemical processes of a recombinant microorganism. Those biochemical processes can include cellular survival and growth. Those biochemical processes can include production of KDG from glucose.

[0188] In some embodiments, such as is schematically depicted in FIG. 4, fructose can be isomerized to glucose by the activity of a fructose isomerase (reaction 2). Glucose can be converted to gluconolactone by the activity of a glucose dehydrogenase (GDH) (reaction 3). Gluconolactone can spontaneously convert to gluconic acid, or this conversion can be catalyzed by the activity of a gluconolactonase (reaction 4). The activity of a gluconate dehydratase (GAD) can convert gluconic acid to KDG (reaction 5).

[0189] In some embodiments, such as is schematically depicted in FIG. 6, the non- phosphorylative transport of sucrose, the hydrolysis of sucrose, the isomerization of fructose and glucose, and the production of KDG from glucose as precursor can be engineered into recombinant microorganisms.

[0190] Optionally, recombinant microorganisms of the disclosure are configured to have reduced phosphorylation of sucrose, glucose, and/or fructose, as described in Section 6.5.4.

[0191] Recombinant microorganisms configured to isomerize fructose and glucose include those described in Section 6.5. A recombinant microorganism can comprise nucleic acids comprising coding regions encoding one or more polypeptides described in Section 4.4 wherein the polypeptides are heterologous to the recombinant microorganism. A recombinant microorganism can be engineered starting from a parental microorganism described in Section 4.5, by techniques including those described in Section 4.6.1.

[0192] Recombinant microorganisms of the disclosure can be used in methods to isomerize fructose and glucose, as described in Section 4.7. The produced fructose or glucose can be used in other biochemical processes. For example, glucose can be used to produce KDG, as described in Section 6.8.4. [0193] Further details of recombinant microorganisms, polypeptides, pathways, methods, and uses of the disclosure are presented below.

6.5. Recombinant Microorganisms

[0194] In some aspects, the present disclosure relates to recombinant microorganisms configured to isomerize fructose and glucose. The recombinant microorganisms can optionally be further configured to non-phosphorylatively transport sucrose, fructose, and/ or glucose; to hydrolyze sucrose to fructose and glucose; and/or have reduced phosphorylation of sucrose, fructose, and/or glucose.

[0195] In some embodiments, recombinant microorganisms of the disclosure are configured to perform one or more of these activities at mesophilic temperatures (e.g., from 20°C to 40°C).

[0196] Recombinant microorganisms of the disclosure can be engineered from a parental microorganism known or hereafter discovered to be suitable for use in bioindustrial processes. Appropriate parental microorganisms include those described in Section 6.6, and suitable engineering techniques include those described in Section 6.7.1.

[0197] Generally, recombinant microorganisms of the disclosure will differ from parental microorganisms by the ability to isomerize fructose and glucose, optionally at mesophilic temperatures. This can be achieved by engineering parental microorganisms to express, by for example introducing one or more nucleotide sequences encoding one or more fructose isomerase variants, e.g., those described in Section 4.4, that impart the ability to isomerize fructose and glucose. Concurrently, parental cells can be engineered to increase uptake of sucrose, glucose, and/or fructose, and/or hydrolyze sucrose to glucose and fructose, by introducing one or more nucleotide sequence encoding one or more heterologous polypeptides that impart the desired ability.

[0198] The particular coding regions desirable for engineering into particular parental microorganisms to yield particular recombinant microorganisms can vary based on a number of factors, including but not necessarily limited to the activity or activities for which it is desired that the recombinant microorganisms be configured; any sub-activities the parental microorganisms are capable of performing, if any; other features of the parental microorganisms that may be relevant; or two or more thereof, among other factors that will be apparent to the person of ordinary skill in the art having the benefit of the present disclosure. Examples of such factors are discussed in Sections 6.5.1 to 6.5.4, both in general terms and with particular reference to exemplary parental microorganisms: E. coli K12 (American Type Culture Collection (ATCC) Accession Number 29425), E. coli W (ATCC 9637), Bacillus subtilis (e.g., B. subtilis 168, ATCC 23857), Pseudomonas putida (e.g., P. putida Migula, ATCC 12633), Klebsiella oxytoca (e.g., K. oxytoca (Flugge) Lautrop, ATCC 13182), Pantoea ananatis (e.g., P. ananatis (Serrano) Mergaert et al., ATCC TSD-232), Tatumella citrea (e.g., T. citrea (Kageyama et al.) Brady et al., ATCC 31623), Zymomonas mobilis e.g., Z. mobilis subsp. mobilis (Lindner) Kluyver and van Niel, ATCC 10988), and Corynebacterium glutamicum (e.g., C. glutamicum (Kinoshita et al.) Abe et al., ATCC 13032). A skilled artisan can readily apply the teachings provided with respect to the exemplary parental microorganisms to other species and strains of microorganisms.

6.5.1. Recombinant Microorganisms Configured to Isomerize Fructose and Glucose

[0199] The isomerization of fructose and glucose can be engineered into recombinant microorganisms by incorporating nucleic acids comprising coding regions encoding nucleotide sequences encoding fructose isomerases (“fructose isomerase nucleotide sequences”).

[0200] Features of parental microorganisms that can be considered when engineering recombinant microorganisms to gain or improve the function of a fructose isomerase include whether the parental microorganisms have fructose isomerase activity (at a desired level).

[0201] Turning to the exemplary parental microorganisms, all of them lack fructose isomerase activity. In some embodiments, a recombinant E. coli (e.g., E. coli K12 (or a strain derived therefrom, e.g., E. coli MG1655) or E. coli \N), B. subtilis, P. putida, K. oxytoca, P. ananatis, T. citrea, Z. mobilis, or C. glutamicum can be engineered to include a fructose isomerase activity.

6.5.2. Recombinant Microorganisms Configured to Non- Phosphorylatively Transport Sucrose, Fructose, and/or Glucose

[0202] The non-phosphorylative transport of sucrose, fructose, and/or glucose from the extracellular space into cells of recombinant microorganisms can be engineered into recombinant microorganisms by incorporating nucleic acids comprising coding regions encoding sucrose porins (“sucrose porin nucleotide sequences”), sucrose permeases (“sucrose permease nucleotide sequences”), fructose porins (“fructose porin nucleotide sequences”), fructose permeases (“fructose permease nucleotide sequences”), glucose porins (“glucose porin nucleotide sequences”), glucose permeases (“glucose permease nucleotide sequences”), any two, three, four, or five, or all six thereof.

[0203] A sucrose, fructose, or glucose porin is a beta barrel protein which, when present in the outer membrane of Gram-negative bacteria, permits passive diffusion of extracellular sucrose, fructose, and/or glucose into the periplasmic space. A porin that permits the passive diffusion of other molecules as well as sucrose, fructose, and/or glucose is a sucrose, fructose, or glucose porin as described herein, provided it permits the diffusion of sucrose, fructose, or glucose, respectively. Given the similarities in size, shape, composition, and hydrophobicity of sucrose, fructose, and glucose, a porin recognized as acting on one of these saccharides may also have activity on the others. For example, a sucrose porin may also permit the passive diffusion of fructose and/or glucose. For another example, a glucose porin may also permit the passive diffusion of sucrose and/or fructose. For yet another example, a fructose porin may also permit the passive diffusion of sucrose and/or glucose.

[0204] A porin that permits the passive diffusion of extracellular sucrose, fructose, and/or glucose through the single membrane of a Gram-positive bacterium is also a sucrose, fructose, or glucose porin as described herein.

[0205] A sucrose, fructose, or glucose permease is a member of major facilitator superfamily (MFS) and is a membrane protein which actively transports sucrose, fructose, and/or glucose across the membrane, such as from the periplasmic space through the inner membrane of Gram-negative bacteria or from the extracellular space through the cell membrane of Gram-positive bacteria and other microorganisms. A permease that actively transports other molecules as well as sucrose, fructose, and/or glucose is a sucrose, fructose, or glucose permease as described herein, provided it actively transports sucrose, fructose, or glucose, respectively. Given the similarities in size, shape, composition, and hydrophobicity of sucrose, fructose, and glucose, a permease recognized as acting on one of these saccharides may also have activity on the others. For example, a sucrose permease may also actively transport fructose and/or glucose. For another example, a glucose permease may also actively transport sucrose and/or fructose. For yet another example, a fructose permease may also actively transport sucrose and/or glucose.

[0206] Features of parental microorganisms that can be considered when engineering recombinant microorganisms to gain or improve the function of a porin or a permease include whether the parental microorganisms have a single membrane (e.g., the parental microorganisms are Gram-positive bacteria) or has an outer membrane and an inner membrane (e.g., the parental microorganisms are Gram-negative bacteria). In parental microorganisms, porins are generally found in the outer membranes of parental microorganisms having two membranes, and permeases are generally found in the inner membranes of parental microorganisms having two membranes or in the membranes of parental microorganisms having a single membrane.

[0207] In some embodiments, this general pattern can be mimicked, wherein recombinant microorganisms having outer and inner membranes can be engineered to localize a porin to the outer membrane, and recombinant microorganisms having one or two membranes can be engineered to localize a permease to the inner membrane or single membrane. In other embodiments, recombinant microorganisms can be engineered to localize a porin to an inner membrane or single membrane of the recombinant microorganisms, to localize a permease to an outer membrane when the recombinant microorganisms comprise outer and inner membranes, to localize both a porin and a permease to the same membrane or membranes, etc.

[0208] Of the exemplary parental microorganisms, parental E. coli K12 and parental P. putida lack both sucrose porin and sucrose permease activities. Parental Z. mobilis lacks sucrose permease activity and is not known to have sucrose porin activity. In some embodiments, a recombinant E. coli K12 or a strain derived therefrom such as or a strain E. coli MG1655, P. putida, or Z. mobilis can be engineered to include nucleic acid(s) encoding a sucrose, fructose, and/or glucose porin; nucleic acid(s) encoding a sucrose, fructose, and/or glucose permease, or nucleic acid(s) encoding both.

[0209] Parental E. coli \N lacks sucrose porin activity. In some embodiments, a recombinant E. coli W can be engineered to include nucleic acids encoding a sucrose, fructose, and/or glucose porin. [0210] Parental B. subtilis and C. glutamicum are Gram-positive, i.e., both lack an outer membrane, and both have a sucrose permease activity coupled to sucrose phosphorylation. In some embodiments, a recombinant B. subtilis or C. glutamicum can be engineered to localize a sucrose, fructose, and/or glucose porin to its single membrane, to supplement or replace its endogenous phosphorylation-coupled sucrose permease activity with a sucrose, fructose, and/or glucose permease activity that does not phosphorylate sucrose, or both.

[0211] Parental K. oxytoca has both sucrose porin and sucrose permease activity. In some embodiments, a recombinant K. oxytoca can be engineered to localize a sucrose, fructose, and/or glucose porin to its inner membrane, to localize a sucrose, fructose, and/or glucose permease to its outer membrane, to operably link either or both of a sucrose, fructose, and/or glucose porin coding region and a sucrose, fructose, and/or glucose permease coding region to a regulatory region with which it is not operably linked in parental K. oxytoca, or two or more thereof.

[0212] Parental P. ananatis and T. citrea are not known to have sucrose porin activity. Both have a sucrose permease activity coupled to sucrose phosphorylation. In some embodiments, a recombinant P. ananatis or T. citrea can be engineered to include nucleic acid(s) encoding a sucrose, fructose, and/or glucose porin, to supplement or replace its endogenous phosphorylation-coupled permease activity with a sucrose, fructose, and/or glucose permease activity that does not phosphorylate sucrose, or both.

6.5.3. Recombinant Microorganisms Configured to Hydrolyze Sucrose to Fructose and Glucose

[0213] The hydrolysis of sucrose to glucose and fructose can be engineered into recombinant microorganisms by incorporating nucleic acids comprising coding regions encoding sucrose invertases (“sucrose invertase nucleotide sequences”).

[0214] Features of parental microorganisms that can be considered when engineering recombinant microorganisms to gain or improve the function of a sucrose invertase include whether the parental microorganisms have sucrose invertase activity (at a desired level) and/or whether the sucrose invertase activity is intracellular or extracellular.

[0215] Recombinant microorganisms can have extracellular sucrose invertase activity. In further embodiments, recombinant microorganisms can further be engineered to non- phosphorylatively transport extracellular fructose, glucose, or both into the cell, by the engineering into the recombinant microorganism of a porin and/or a permease that transports fructose and/or glucose, as described in Section 6.5.2.

[0216] In some embodiments, the recombinant microorganisms have intracellular sucrose invertase activity.

[0217] Turning to the exemplary parental microorganisms, parental E. coli K12, B. subtilis, P. putida, P. ananatis, T. citrea, and C. glutamicum do not or are not known to have sucrose invertase activity. In some embodiments, a recombinant E. coli K12 or a strain derived therefrom such as or a strain derived therefrom, e.g., E. coli MG1655, B. subtilis, P. putida, P. ananatis, T. citrea, or C. glutamicum can be engineered to include a sucrose invertase activity.

[0218] Parental E. co// W and K. oxytoca are known to have sucrose invertase activity. In some embodiments, a recombinant E. coli W or K. oxytoca can be engineered to operably link a coding region encoding a sucrose invertase to a regulatory region to which the coding region is not operably linked in the parental microorganism.

[0219] Parental Z. mobilis is known to have extracellular sucrose invertase activity. In some embodiments, a recombinant Z. mobilis can be engineered to include an intracellular sucrose invertase activity, to operably link a coding region encoding a sucrose invertase to a regulatory region to which the coding region is not operably linked in the parental microorganism, or both.

6.5.4. Recombinant Microorganisms Configured to Have Reduced Phosphorylation of Sucrose, Glucose, and/or Fructose

[0220] Phosphorylation of sucrose, glucose, and/or fructose are common processes in many parental microorganisms. For example, parental microorganisms may retain intracellular glucose-6-phosphate better than intracellular glucose. For another example, phosphorylation of glucose to glucose-6-phosphate is the first step of the Embden- Meyerhof- Parnas pathway, culminating in the production of pyruvate for entry into the TCA cycle. While phosphorylation of sucrose, glucose, and/or fructose can be beneficial to parental microorganisms, phosphorylation of these sugars by recombinant microorganisms described herein under controlled conditions is undesirable, especially when the isomerization of non-phosphorylated fructose and glucose is intended. [0221] Hence, in some embodiments, recombinant microorganisms of the present disclosure can further comprise one or more genetic modifications which reduce phosphorylation of sucrose, glucose, and/or fructose. These modifications can include reduction of fructokinase activity, e.g., by deleting or disrupting a coding region encoding a fructokinase catalyzing the phosphorylation of fructose to fructose-1 -phosphate. For example, if a parental microorganism is an E. coli strain K12 or a strain derived therefrom, the mak gene can be deleted or disrupted.

[0222] Another modification can be reduction of glucose phosphotransferase (PTS) activity, e.g., by deleting or disrupting a coding region encoding one or more proteins involved in the uptake of extracellular glucose with concomitant transfer of a phosphate group to the glucose to yield a glucose-6-phosphate. For example, if a parental microorganism is an E. coli or a B. subtilis, one or more of the genes encoding Enzyme I, Enzyme IIA, Enzyme IIB, Enzyme IIC, or Histidine Protein, can be deleted or disrupted. Similar modifications include reduction of fructose PTS activity and mannose PTS activity. Still another modification can be reduction of gluconate kinase activity, e.g., by deleting or disrupting a coding region encoding a gluconokinase catalyzing the phosphorylation of gluconate to 6- phosphogluconate. For example, if a parental microorganism is an E. coli strain K12 or a strain derived therefrom, the gntK gene can be deleted or disrupted.

[0223] As should be apparent, the reduction of activity in a recombinant microorganism is determined relative to a parental microorganism.

6.6. Nucleic acids

[0224] In some embodiments, the present disclosure relates to nucleic acids comprising fructose isomerase nucleotide sequences.

[0225] Nucleic acids can further comprise regulatory regions operably linked to coding regions, generally with a 1 :1 correspondence of regulatory region to coding region.

[0226] Nucleic acids can further comprise one or more nucleic acid sequences that permit or enhance a construct’s ability to be introduced into a cell of a parental microorganism to yield a recombinant microorganism, to be selected for after introduction into the cell, to replicate independently of the genome of a recombinant microorganism, to be integrated into the genome of a recombinant microorganism, or two or more thereof. Hence, nucleic acids include but are not limited to plasmids.

[0227] In some embodiments, coding regions in nucleic acids can be modified (e.g., to add a peptide sequence to the N- or C-terminus of the transcribed product or to replace a peptide sequence in the transcribed product with a substitute peptide sequence, to localize the translated product of the coding region to a particular cellular location, to add or remove multimerization sites, to render the translated product more or less sensitive to interactions with molecules other than its intended substrate, or the like).

[0228] Any nucleotide sequences encoding polypeptides of interest can be optimized to increase the percentage of codons that are preferred by recombinant microorganisms, i.e., the codon from among each group of synonymous codons that is most prevalent in coding regions of a particular recombinant microorganism’s genome. Several methods for codon optimization are known in the art. In addition to increasing the percentage of preferred codons in optimized nucleotide sequences, in some embodiments, codon optimized sequences avoid nucleotide repeats and restriction sites.

[0229] In addition to nucleotide sequences encoding polypeptides described herein, coding regions in nucleic acids can further comprise a nucleotide sequence encoding one or more amino acid sequences each in a position that is N-terminal or C-terminal to the polypeptide amino acid sequence. Such other amino acid sequences can include His tags, polypeptide domains for inclusion in fusion proteins, and linkers, among others known to the person of ordinary skill in the art.

[0230] When nucleic acids comprise multiple coding regions, each coding region may have a unique regulatory region, or two or more coding regions may have identical or substantially identical regulatory regions. Each distinct regulatory region can comprise a constitutive promoter or an inducible promoter. In embodiments wherein one or more regulatory regions comprises an inducible promoter, nucleic acids can further comprise coding regions each encoding a repressor polypeptide, wherein the repressor polypeptide is involved in regulation of an inducible promoter. Additionally or alternatively, repressor polypeptides can be natively expressed by recombinant microorganisms. In embodiments comprising an inducible promoter, repressor polypeptides can be controlled by an inducing agent, such as a sugar (e.g., lactose) or an organic acid or a salt thereof (e.g., gluconate). [0231] In some embodiments, nucleic acids can comprise transposable regions, such that one or more coding regions (and optionally an operably-linked regulatory region of each) can be integrated into the chromosomes of the recombinant microorganisms.

[0232] Nucleic acids can comprise selectable markers or reporter genes, which can be used to identify cells which retain the selectable markers/reporter genes (and coding regions encoding polypeptides described herein) in non-integrated nucleic acids and/or integrated into the genome of recombinant microorganisms. Common selectable markers include genes encoding antibiotic resistance, fluorescence markers, enzymes catalyzing formation of a product that can be readily detected, and enzymes or cofactors required for cell survival or growth, among others.

[0233] In some embodiments, coding regions encoding fructose isomerases as described herein can also function as a selectable marker or reporter gene. In some embodiments, recombinant microorganisms may inherit from their parental microorganisms phenotypes such that the recombinant microorganisms can only survive or grow if they express a fructose isomerase. In some embodiments, recombinant microorganisms can be cultured under conditions such that, if they express fructose isomerase, a product can be detected.

[0234] Nucleotide sequences encoding amino acid sequences of any heterologous polypeptide can be codon optimized for the recombinant microorganism, /.e., the nucleotide sequences can comprise one or more codons which lead to more rapid translation and/or translation with fewer errors, which reduce the likelihood of a transcript forming secondary structures, or provide other advantages in expression of the polypeptides in recombinant microorganisms. In some embodiments, codon optimized nucleotide sequences can be generated using the Integrated DNA Technologies (IDT) algorithm (www.idtdna.com/pages/tools/codon-optimization-tool).

[0235] Each nucleic acid can be a vector, such as a plasmid (e.g., a pTrcHis2B plasmid). A pTrcHis2B plasmid comprises an origin of replication (ori), a lacl coding region operably linked to a laclq promoter, a site for incorporation of a coding region of interest (i) operably linked to a trc promoter (ii) in frame with a coding region encoding a 6xHis tag (SEQ ID NO: 25) and (iii) and the T1 and T2 transcription terminators from E. coli rrnB, and an AmpR coding region operably linked to an AmpR promoter. [0236] A particular plasmid, pTrcHis2b-XylACT, has the schematic structure shown in FIG.

3, wherein xylA-CT is a coding region encoding a protein differing from SEQ ID NO:3 (C. thermosulfurogenes xylA) by including the substitutions W139F and V186T as compared to the amino acid sequence of SEQ ID NO:3.

6.7. Parental Microorganisms

[0237] A parental microorganism that can be engineered into a recombinant microorganism of the disclosure can be any unicellular organism (e.g., a bacterium, an archaeon, or a fungus (e.g., a yeast), among others), in particular such an organism known or discovered to be suitable for use in isomerizing fructose and glucose.

[0238] In some embodiments, the microorganism is an E. coli strain. In particular embodiments, the E. coli strain is K12 or a strain derived therefrom, such as E. coli MG1655. In other particular embodiments, the microorganism is an E. coli \N.

[0239] In some embodiments, the microorganism is a Bacillus subtilis.

[0240] In some embodiments, the microorganism is a Pseudomonas putida.

[0241] In some embodiments, the microorganism is a Klebsiella oxytoca.

[0242] In some embodiments, the microorganism is a Pantoea ananatis.

[0243] In some embodiments, the microorganism is a Tatumella citrea.

[0244] In some embodiments, the microorganism is a Zymomonas mobilis.

[0245] In some embodiments, the microorganism is a Corynebacterium glutamicum.

[0246] In some embodiments, the microorganism is an E. coli SuA6 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS Hlcrr, Amak, AgntK, AidnK).

[0247] In some embodiments, the microorganism is an E. coli SuA7.1 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS Hlcrr, Amak, AidnK, Aglk).

[0248] In some embodiments, the microorganism is an E. coli SuA5_KmR (Apts, AgntK, AidnK, Aglk, AkdgK::KmR).

6.7.1. Engineering Methods

[0249] A parental microorganism can be engineered using techniques known in the art. For example, nucleic acid(s) comprising a coding region encoding a polypeptide specifically described herein can be introduced into a parental microorganism via techniques known in the art.

[0250] In some embodiments, a nucleic acid can be introduced into the microorganism by any appropriate transformation technique. The nucleic acid can be extrachromosomal, on a vector (typically a plasmid), such as a low copy number vector, an intermediate copy number vector, or a high copy number vector. The nucleic acid may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. Alternatively, the nucleic acid can be integrated in one or more copies into the genome of the cell. Integration into the cell’s genome can occur at random by non-homologous recombination, or at selected locations by homologous recombination, as is well known in the art.

[0251] Moreover, in some embodiments, a nucleic acid comprises a regulatory region and a coding region which are operably linked. Such a nucleic acid can be referred to as a “recombinant expression vector” or “expression vector.”

[0252] Various genome editing techniques, including but not limited to homologous recombination, CRISPR-Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), can be used to delete or disrupt genes in a parental microorganism or to operably link a coding region to a regulatory region to which it is not operably linked in a parental microorganism (which may change promoter strength, change whether a promoter is constitutive or inducible, or change which inducer molecule induces transcription of a coding region from an inducible promoter).

[0253] Additionally or alternatively, other techniques can be used in engineering a parental microorganism to yield a recombinant microorganism. Non-specific mutagens can be used to delete or disrupt genes in a parental microorganism, and cells can be screened for a phenotype indicative of deletion or disruption of a gene of interest. In some embodiments, the gene of interest is a gene involved in phosphorylation of sucrose, fructose, or glucose. Cells found to have the desired phenotype can then receive a heterologous nucleic acid encoding a polypeptide specifically described herein. 6.8. Methods of use of recombinant microorganisms

[0254] The present disclosure also relates to the use of a recombinant microorganism described herein in one or more methods. Specific methods include the isomerization of fructose and glucose.

6.8.1. Culture media

[0255] Generally, the methods comprise culturing a recombinant microorganism in a medium comprising a feedstock molecule of interest. Culturing can be in a batch mode or a continuous mode

[0256] Examples of known media that can be used in batch mode culturing include M9 medium and Hi-Def medium. The M9 medium can comprise the following: sodium phosphate dibasic heptahydrate, 1.28 w/v%; potassium phosphate monobasic, 0.3 w/v%; sodium chloride, 0.05 w/v%; ammonium chloride, 0.1 w/v%; glucose, 0.4 w/v%; MgSO4, 0.024 w/v%; and CaCI2, 0.001 w/v%. The Hi-Def medium can comprise ingredients known to the person of ordinary skill in the art, and it is commercially available (Teknova Inc. Hollister, CA.

[0257] In some embodiments, a culture medium comprises one or more precursor di- or polysaccharides that can be metabolized into fructose or glucose. Sucrose is an example of a precursor disaccharide that can be metabolized into fructose or glucose. Lactose and maltose are other examples of precursor disaccharides that can be metabolized into fructose or glucose, e.g., lactose can be metabolized into glucose (and galactose), and maltose can be metabolized into glucose.

[0258] In some embodiments, a culture medium comprises at least 0.1 w/v% precursor di- or polysaccharide(s), at least 0.2 w/v% precursor di- or polysaccharide(s), at least 0.3 w/v% precursor di- or polysaccharide(s), at least 0.4 w/v% precursor di- or polysaccharide(s), at least 0.5 w/v% precursor di- or polysaccharide(s), at least 0.6 w/v% precursor di- or polysaccharide(s), at least 0.7 w/v% precursor di- or polysaccharide(s), at least 0.8 w/v% precursor di- or polysaccharide(s), at least 0.9 w/v% precursor di- or polysaccharide(s), or at least 1 w/v% precursor di- or polysaccharide(s). A culture medium typically comprises less than 5 w/v% precursor di- or polysaccharide(s), more typically less than 2 w/v% precursor di- or polysaccharide(s) (e.g., a culture medium can comprise from 0.1 w/v% to 5 w/v% precursor di- or polysaccharide(s); from 0.1 w/v% to 2 w/v% precursor di- or polysaccharide(s); from 0.1 w/v% to 1 w/v% precursor di- or polysaccharide(s); or from 1 w/v% to 2 w/v% precursor di- or polysaccharide(s), among other possible ranges).

[0259] In some embodiments, a culture medium comprises sucrose, glucose, and/or fructose. A culture medium can comprise at least 0.1 w/v% sucrose, glucose, and/or fructose. In some embodiments, a culture medium comprises at least 0.1 w/v% sucrose, at least 0.2 w/v% sucrose, at least 0.3 w/v% sucrose, at least 0.4 w/v% sucrose, at least 0.5 w/v% sucrose, at least 0.6 w/v% sucrose, at least 0.7 w/v% sucrose, at least 0.8 w/v% sucrose, at least 0.9 w/v% sucrose, or at least 1 w/v% sucrose. A culture medium typically comprises less than 5 w/v% sucrose, more typically less than 2 w/v% sucrose (e.g., a culture medium can comprise from 0.1 w/v% to 5 w/v% sucrose; from 0.1 w/v% to 2 w/v% sucrose; from 0.1 w/v% to 1 w/v% sucrose; or from 1 w/v% to 2 w/v% sucrose, among other possible ranges).

[0260] In some embodiments, a culture medium comprises at least 0.1 w/v% glucose, at least 0.2 w/v% glucose, at least 0.3 w/v% glucose, at least 0.4 w/v% glucose, at least 0.5 w/v% glucose, at least 0.6 w/v% glucose, at least 0.7 w/v% glucose, at least 0.8 w/v% glucose, at least 0.9 w/v% glucose, or at least 1 w/v% glucose. A culture medium typically comprises less than 5 w/v% glucose, more typically less than 2 w/v% glucose (e.g., a culture medium can comprise from 0.1 w/v% to 5 w/v% glucose; from 0.1 w/v% to 2 w/v% glucose; from 0.1 w/v% to 1 w/v% glucose; or from 1 w/v% to 2 w/v% glucose, among other possible ranges).

[0261] In some embodiments, a culture medium comprises at least 0.1 w/v% fructose, at least 0.2 w/v% fructose, at least 0.3 w/v% fructose, at least 0.4 w/v% fructose, at least 0.5 w/v% fructose, at least 0.6 w/v% fructose, at least 0.7 w/v% fructose, at least 0.8 w/v% fructose, at least 0.9 w/v% fructose, or at least 1 w/v% fructose. A culture medium typically comprises less than 5 w/v% fructose, more typically less than 2 w/v% fructose (e.g., a culture medium can comprise from 0.1 w/v% to 5 w/v% fructose; from 0.1 w/v% to 2 w/v% fructose; from 0.1 w/v% to 1 w/v% fructose; or from 1 w/v% to 2 w/v% fructose, among other possible ranges).

[0262] Any culture medium can be M9 medium or Hi-Def medium supplemented with sucrose, glucose, and/or fructose. Such a medium may be referred to herein as a “production medium.” [0263] In some embodiments, a production medium comprises sucrose. In some embodiments, a production medium comprises fructose. In some embodiments, a production medium comprises glucose. In some embodiments, a production medium comprises any two or all three of sucrose, fructose, or glucose.

[0264] In some embodiments, a production medium comprises one or more precursor di- or polysaccharides that can be metabolized into fructose or glucose. In some embodiments, a production medium comprises fructose. In some embodiments, a production medium comprises glucose. In some embodiments, a production medium comprises any two or all three of precursor di- or polysaccharide(s), fructose, or glucose.

[0265] In some embodiments, wherein a culture medium comprises two or more carbon sources, the culture medium comprises at least 0.5 w/v% total carbon sources, at least 0.6 w/v% total carbon sources, at least 0.7 w/v% total carbon sources, at least 0.8 w/v% total carbon sources, at least 0.9 w/v% total carbon sources, or at least 1 w/v% total carbon sources. A culture medium typically comprises less than 5 w/v% total carbon sources, more typically less than 2 w/v% total carbon sources (e.g., a culture medium can comprise from 0.1 w/v% to 5 w/v% total carbon sources; from 0.1 w/v% to 2 w/v% total carbon sources; from 0.1 w/v% to 1 w/v% total carbon sources; or from 1 w/v% to 2 w/v% total carbon sources, among other possible ranges).

[0266] In some embodiments, a production medium comprises an inducer, i.e., a molecule which binds to a repressor and thereby induces translation of a coding region regulated by an inducible promoter.

[0267] In some embodiments of some methods described herein, it may be desirable to allow growth of a recombinant microorganism without expression of one or more polypeptides required to isomerize glucose and fructose until a desired biomass of the recombinant microorganism has been reached. This can be effected by use of a growth medium comprising a carbon source other than glucose, fructose, or sucrose. In some embodiments, a growth medium comprises glycerol, such as at least 0.1 w/v% glycerol, at least 0.2 w/v% glycerol, at least 0.3 w/v% glycerol, at least 0.4 w/v% glycerol, at least 0.5 w/v% glycerol, at least 0.6 w/v% glycerol, at least 0.7 w/v% glycerol, at least 0.8 w/v% glycerol, at least 0.9 w/v% glycerol, or at least 1 w/v% glycerol. A growth medium typically comprises less than 5 w/v% glycerol, more typically less than 2 w/v% glycerol (e.g., a growth medium can comprise from 0.1 w/v% to 5 w/v% glycerol; from 0.1 w/v% to 2 w/v% glycerol; from 0.1 w/v% to 1 w/v% glycerol; or from 1 w/v% to 2 w/v% glycerol, among other possible ranges).

[0268] Although glycerol can provide a carbon source for growth of a recombinant microorganism in a growth medium, glycerol can be included in a production medium. Typically, glycerol is included in a production medium at the same or lower concentration than in a growth medium.

[0269] In particular embodiments, a growth medium lacks added glucose, fructose, and/or sucrose, /.e., one or more of these sugars is not intentionally included in a growth medium. In particular embodiments, a growth medium comprises no more than 0.1 w/v% each of glucose, fructose, and/or sucrose.

[0270] The selection of particular concentrations of sucrose, glucose, fructose, and/or glycerol to include in a production medium and/or a growth medium can be made by the person of ordinary skill in the art having the benefit of the present disclosure as a routine matter.

[0271] For fed-batch and/or continuous mode culturing, the ranges of sucrose, glucose, fructose, glycerol, or combinations thereof given above can be initially provided to the medium. The consumption of the carbon source(s) during culturing can be repeatedly or continuously monitored and additional carbon source(s) can be provided as needed to sustain a desired respiratory coefficient, growth rate, or a rate of production of desired compound(s). The feed rate may be adjusted to avoid accumulation of carbon source(s), which may maximize output of desired compound(s) and minimize waste of carbon source(s). The person of ordinary skill in the art having the benefit of the present disclosure can select the medium composition and the amount of carbon source added thereto during the process to enable the production of desired product(s) to a desired concentration, such as at least 20 g/L, at least 50 g/L, or at least 100 g/L.

6.8.2. Culture conditions

[0272] Recombinant cells of the disclosure may be cultured under suitable conditions in a medium, such as a medium described in Section 6.8.1. In some embodiments, recombinant cells of the disclosure undergo fermentation. Fermentation conditions include batch, fed- batch and continuous fermentation. Classical batch fermentation is a closed system, wherein the composition of the medium is not subject to artificial alterations during fermentation. In fed-batch fermentation, the substrate is added in increments as fermentation progresses. In both classical batch fermentation and batch-fed fermentation, the product(s) remain in the bioreactor until the end of the process. Batch and fed-batch fermentation are common and well-known in the art. In continuous fermentation, a defined medium is added continuously to the bioreactor and an equal volume of product containing medium is removed simultaneously. Continuous fermentation aims to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology. The fermentation process is typically an aerobic fermentation process.

[0273] The fermentation process is typically run at a temperature that is optimal for growth of a recombinant microorganism. Fermentation for a mesophilic microorganism is typically carried out at a temperature within the range of from 20°C to 45°C, from 25°C to 40°C, from 35°C to 40°C, or from 30°C to 37°C. In embodiments wherein a recombinant microorganism is derived from one of the exemplary microorganisms described herein, culturing can comprise maintaining the recombinant microorganism at a mesophilic temperature. In some embodiments, the mesophilic temperature is selected from any of the foregoing ranges.

[0274] Fermentation is typically carried out at a pH in the range of 4 to 8, in the range of 5 to 7, or the range of 5.5 to 6.5. In certain embodiments, fermentation is carried out for a period of time within the range of from 8 to 240 hours, from 12 hours to 168 hours, from 16 hours to 144 hours, from 20 hours to 120 hours, from 24 hours to 72 hours, or from 46 to 48 hours.

6.8.3. Methods Of Isomerizing Fructose and Glucose

[0275] The present disclosure also relates to methods of producing glucose, comprising culturing, in production media comprising at least one of fructose or sucrose, at a mesophilic temperature, recombinant microorganisms configured to isomerize fructose and glucose at mesophilic temperatures as described herein. In some embodiments, the recombinant microorganisms comprise a nucleic acid comprising a nucleotide sequence encoding a fructose isomerase (e.g., a fructose isomerase as described in Section 6.2), wherein the fructose isomerase is heterologous to the microorganism. In some embodiments, the recombinant microorganisms further comprise a nucleic acid comprising a nucleotide sequence encoding a sucrose, glucose, and/or fructose porin; a sucrose, glucose, and/or fructose permease, and/or a sucrose invertase.

[0276] The present disclosure also relates to methods of isomerizing fructose and glucose, comprising culturing recombinant microorganisms expressing polypeptides as described herein in a production medium comprising fructose, glucose, and/or one or more precursor di- or polysaccharides that can be metabolized into fructose or glucose. In some embodiments, the culturing is at a mesophilic temperature.

[0277] The production media can be as described herein. In some embodiments, the production media comprise sucrose. Such production media may be desirable for use in embodiments wherein the recombinant microorganisms are configured to non- phosphorylatively transport sucrose from the production medium into a cell of the recombinant microorganism, and optionally hydrolyze sucrose to glucose and fructose.

[0278] In some embodiments, the production media comprise fructose. Such production media may be desirable for use in embodiments wherein the recombinant microorganisms are configured to non-phosphorylatively transport fructose from the production medium into a cell of the recombinant microorganism.

[0279] In some embodiments, the production media comprise glucose. Such production media may be desirable for use in embodiments wherein the recombinant microorganisms are configured to non-phosphorylatively transport glucose from the production medium into a cell of the recombinant microorganism.

[0280] The methods can further comprise growing recombinant microorganisms in growth media comprising a carbon source other than glucose, fructose, or sucrose, prior to culturing in production media. In some embodiments, the growth media comprise glycerol. In some embodiments, the growth media lack added glucose, fructose, and sucrose. In some embodiments, the growth media comprise no more than 0.1% each of glucose, fructose, and sucrose.

6.8.4. Methods Of Using Fructose and/or Glucose

[0281] Fructose or glucose produced by the methods described in Section 6.8.3 can be used for any desired purpose. In some embodiments, fructose and/or glucose can be purified from the recombinant microorganisms or from fructose isomerases used in vitro and used either individually or together in any proportion as a sweetening agent in foodstuffs. In some embodiments, fructose or glucose can be supplied to biochemical processes for the production of further products. The biochemical processes can be intracellular to the recombinant microorganisms or can be performed in other organisms. In some embodiments, the biochemical processes are intracellular to the recombinant microorganisms.

[0282] For example, recombinant microorganisms can be further configured to convert glucose to 2-keto-3-deoxygluconate (“KDG”), such as by being engineered to comprise nucleic acids comprising nucleotide sequences encoding glucose dehydrogenase, gluconate dehydratase, and optionally gluconolactonase. The action of these enzymes on glucose and products thereof increases the ratio of fructose to glucose, and thus the fructose isomerases of the recombinant microorganisms would be expected to convert fructose to glucose more than the reverse. Thus, the yield of KDG from fructose can be increased.

7. SPECIFIC EMBODIMENTS

[0283] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). The present disclosure is exemplified by the numbered embodiments set forth below.

[0284] Various aspects of the present disclosure are described in the embodiments set forth in the following numbered paragraphs of Group 1.

[0285] Group 1 :

1. A nucleic acid comprising a nucleotide sequence encoding an engineered fructose isomerase having at least one amino acid substitution as compared to a “reference” fructose isomerase of any one of SEQ ID NOS:1-24, optionally wherein

(a) the engineered fructose isomerase has at least 90% sequence identity to the reference fructose isomerase of any one of SEQ ID NOS: 1-24; and/or (b) the fructose isomerase has improved fructose isomerase activity as compared to the reference fructose isomerase having the amino acid sequence of any one of SEQ ID NOS: 1-24.

2. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3, optionally wherein the amino acid sequence comprises at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

3. The nucleic acid of embodiment 1 or embodiment 2, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.

4. The nucleic acid of any one of embodiments 1 to 3, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:3.

5. The nucleic acid of any one of embodiments 1 to 4, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:3.

6. The nucleic acid of any one of embodiments 1 to 5, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:3.

7. The nucleic acid of any one of embodiments 1 to 6, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3.

8. The nucleic acid of any one of embodiments 1 to 7, wherein the fructose isomerase comprises an amino acid sequence having amino acid substitutions W139F and/or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

9. The nucleic acid of any of embodiments 1 to 8, wherein the fructose isomerase comprises an amino acid sequence identical to (i.e. , does not have a substitution) at positions 101 , 104, 232, 268, 271, 296, 307, 309, and 339 as compared to the amino acid sequence of SEQ ID NO:3.

10. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A142N as compared to the amino acid sequence of SEQ ID NO:3.

11 . The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A142S as compared to the amino acid sequence of SEQ ID NO:3.

12. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A47S as compared to the amino acid sequence of SEQ ID NO:3.

13. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99S, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

14. The nucleic acid of any of embodimentsi to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99S, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

15. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, C99T, T141S, A142S, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

16. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, C99T, T141S, A142T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

17. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99T, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:3. 18. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

19. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and C99T as compared to the amino acid sequence of SEQ ID NO:3.

20. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and D128E as compared to the amino acid sequence of SEQ ID NO:3.

21 . The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:3.

22. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F150Y as compared to the amino acid sequence of SEQ ID NO:3.

23. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:3.

24. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:3.

25. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

26. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:3.

27. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3. 28. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

29. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

30. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144C as compared to the amino acid sequence of SEQ ID NO:3.

31 . The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144K as compared to the amino acid sequence of SEQ ID NO:3.

32. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144M as compared to the amino acid sequence of SEQ ID NO:3.

33. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N19E as compared to the amino acid sequence of SEQ ID NO:3.

34. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N19T as compared to the amino acid sequence of SEQ ID NO:3.

35. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:3.

36. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:3.

37. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:3. 38. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:3.

39. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q59M as compared to the amino acid sequence of SEQ ID NO:3.

40. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

41 . The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

42. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and S155A as compared to the amino acid sequence of SEQ ID NO:3.

43. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T141C as compared to the amino acid sequence of SEQ ID NO:3.

44. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, T141S, and A142T as compared to the amino acid sequence of SEQ ID NO:3.

45. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, T141S, L144M, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

46. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T141S as compared to the amino acid sequence of SEQ ID NO:3.

47. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:3. 48. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:3.

49. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Y22F as compared to the amino acid sequence of SEQ ID NO:3.

50. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

51 . The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

52. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, D128E, F150Y, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

53. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

54. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, D128E, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

55. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, A47S, D128E, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

56. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

57. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, A47S, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

58. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

59. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, A47S, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

60. The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, D128E, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

61 . The nucleic acid of any of embodiments 1 to 9, wherein the fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, D128E, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

62. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1.

63. The nucleic acid of embodiment 1 or embodiment 62, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1.

64. The nucleic acid of any one of embodiments 1 , 62, or 63, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ I D NO: 1.

65. The nucleic acid of any one of embodiments 1 , 62, 63, or 64, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ I D NO: 1.

66. The nucleic acid of any one of embodiments 1 , 62, 63, 64, or 65, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ I D NO: 1. 67. The nucleic acid of any one of embodiments 1 , 62, 63, 64, 65, or 66, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ I D NO: 1.

68. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.

69. The nucleic acid of embodiment 1 or embodiment 68, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2.

70. The nucleic acid of any one of embodiments 1 , 68, or 69, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:2.

71 . The nucleic acid of any one of embodiments 1 , 68, 69, or 70, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:2.

72. The nucleic acid of any one of embodiments 1 , 68, 69, 70, or 71 , wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:2.

73. The nucleic acid of any one of embodiments 1 , 68, 69, 70, 71, or 72, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2.

74. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4.

75. The nucleic acid of embodiment 1 or embodiment 74, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:4.

76. The nucleic acid of any one of embodiments 1 , 74, or 75, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:4.

77. The nucleic acid of any one of embodiments 1 and 74 to 76, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:4. 78. The nucleic acid of any one of embodiments 1 and 74 to 77, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:4.

79. The nucleic acid of any one of embodiments 1 and 74 to 78, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:4.

80. The nucleic acid of embodiment embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5.

81 . The nucleic acid of embodiment 1 or embodiment 80, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5.

82. The nucleic acid of any one of embodiments 1 , 68, and 81 , wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:5.

83. The nucleic acid of any one of embodiments 1 and 68 to 82, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:5.

84. The nucleic acid of any one of embodiments 1 and 68 to 83, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5.

85. The nucleic acid of any one of embodiments 1 and 68 to 84, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:5.

86. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6.

87. The nucleic acid of embodiment 1 or embodiment 86, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:6.

88. The nucleic acid of any one of embodiments 1 , 74, and 87, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:6. 89. The nucleic acid of any one of embodiments 1 and 74 to 88, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:6.

90. The nucleic acid of any one of embodiments 1 and 74 to 89, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:6.

91 . The nucleic acid of any one of embodiments 1 and 74 to 90, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:6.

92. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7.

93. The nucleic acid of embodiment 1 or embodiment 92, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:7.

94. The nucleic acid of any one of embodiments 1 , 92, and 93, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:7.

95. The nucleic acid of any one of embodiments 1 and 92 to 94, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:7.

96. The nucleic acid of any one of embodiments 1 and 92 to 95, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:7.

97. The nucleic acid of any one of embodiments 1 and 92 to 96, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:7.

98. The nucleic acid of embodiment 1 wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8.

99. The nucleic acid of embodiment 1 or embodiment 98, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:8. 100. The nucleic acid of any one of embodiments 1 , 98, and 99, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:8.

101. The nucleic acid of any one of embodiments 1 and 98 to 100, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:8.

102. The nucleic acid of any one of embodiments 1 and 98 to 101 , wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:8.

103. The nucleic acid of any one of embodiments 1 and 98 to 102, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:8.

104. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9.

105. The nucleic acid of embodiment 1 or embodiment 104, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9.

106. The nucleic acid of any one of embodiments 1 , 104, and 105, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:9.

107. The nucleic acid of any one of embodiments 1 and 104 to 106, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:9.

108. The nucleic acid of any one of embodiments 1 and 104 to 107, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:9.

109. The nucleic acid of any one of embodiments 1 and 104 to 108, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:9.

110. The nucleic acid of any one of embodiments 1 and 104 to 109, comprising at least one amino acid substitution V186T, L283P, or F187S. 111. The nucleic acid of embodiment 110, comprising at least two amino acid substitutions V186T, L283P, or F187S.

112. The nucleic acid of embodiment 110 or embodiment 111 , comprising amino acid substitutions V186T, L283P, and F187S.

113. The nucleic acid of any one of embodiments 1 and 104 to 112, comprising at least one amino acid substitution corresponding to an amino acid substitution W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K in the amino acid sequence of SEQ ID NO:3.

114. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10.

115. The nucleic acid embodiment 1 or embodiment 114, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:10.

116. The nucleic acid of any one of embodiments 1 , 114, and 115, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 10.

117. The nucleic acid of any one of embodiments 1 and 114 to 116, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10.

118. The nucleic acid of any one of embodiments 1 and 114 to 117, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10.

119. The nucleic acid of any one of embodiments 1 and 114 to 118, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10.

120. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11.

121. The nucleic acid of embodiments 1 or embodiment 120, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:11. 122. The nucleic acid of any one of embodiments 1 , 120, and 121 , wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ I D NO: 11.

123. The nucleic acid of any one of embodiments 1 and 120 to 122, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ I D NO: 11.

124. The nucleic acid of any one of embodiments 1 and 120 to 123, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ I D NO: 11.

125. The nucleic acid of any one of embodiments 1 and 120 to 124, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ I D NO: 11.

126. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12.

127. The nucleic acid of embodiment 1 or embodiment 126, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:12.

128. The nucleic acid of any one of embodiments 1 , 126, and 127, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 12.

129. The nucleic acid of any one of embodiments 1 and 126 to 128, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 12.

130. The nucleic acid of any one of embodiments 1 and 126 to 129, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12.

131. The nucleic acid of any one of embodiments 1 and 126 to 130, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12.

132. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. 133. The nucleic acid of embodiment 1 or embodiment 132, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:13.

134. The nucleic acid of any one of embodiments 1 , 132, and 133, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13.

135. The nucleic acid of any one of embodiments 1 and 132 to 134, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13.

136. The nucleic acid of any one of embodiments 1 and 132 to 135, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13.

137. The nucleic acid of any one of embodiments 1 and 132 to 136, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13.

138. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14.

139. The nucleic acid of embodiment 1 or embodiment 138, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:14.

140. The nucleic acid of any one of embodiments 1 , 138, and 139, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 14.

141. The nucleic acid of any one of embodiments 1 and 138 to 140, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 14.

142. The nucleic acid of any one of embodiments 1 and 138 to 141 , wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 14.

143. The nucleic acid of any one of embodiments 1 and 138 to 142, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 14. 144. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15.

145. The nucleic acid of embodiment 1 or embodiment 144, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:15.

146. The nucleic acid of any one of embodiments 1 , 144, and 145, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 15.

147. The nucleic acid of any one of embodiments 1 and 144 to 146, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 15.

148. The nucleic acid of any one of embodiments 1 and 144 to 147, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 15.

149. The nucleic acid of any one of embodiments 1 and 144 to 148, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 15.

150. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16.

151. The nucleic acid of embodiment 1 or embodiment 150, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:16.

152. The nucleic acid of any one of embodiments 1 , 150, and 151 , wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 16.

153. The nucleic acid of any one of embodiments 1 and 150 to 152, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 16.

154. The nucleic acid of any one of embodiments 1 and 150 to 153, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 16. 155. The nucleic acid of any one of embodiments 1 and 150 to 154, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 16.

156. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17.

157. The nucleic acid of embodiment 1 or embodiment 156, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:17.

158. The nucleic acid of any one of embodiments 1 , 156, and 157, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 17.

159. The nucleic acid of any one of embodiments 1 and 156 to 158, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 17.

160. The nucleic acid of any one of embodiments 1 and 156 to 159, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 17.

161. The nucleic acid of any one of embodiments 1 and 156 to 160, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 17.

162. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18.

163. The nucleic acid of embodiment 1 or embodiment 162, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18.

164. The nucleic acid of any one of embodiments 1 , 162, and 163, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 18.

165. The nucleic acid of any one of embodiments 1 and 162 to 164, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 18. 166. The nucleic acid of any one of embodiments 1 and 162 to 165, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 18.

167. The nucleic acid of any one of embodiments 1 and 162 to 166, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 18.

168. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19.

169. The nucleic acid of embodiment 1 or embodiment 168, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19.

170. The nucleic acid of any one of embodiments 1 , 168, and 169, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 19.

171. The nucleic acid of any one of embodiments 1 and 168 to 170, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 19.

172. The nucleic acid of any one of embodiments 1 and 168 to 171 , wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 19.

173. The nucleic acid of any one of embodiments 1 and 168 to 172, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 19.

174. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:20.

175. The nucleic acid of embodiment 1 or embodiment 174, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:20.

176. The nucleic acid of any one of embodiments 1 , 174, and 175, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:20. 177. The nucleic acid of any one of embodiments 1 and 174 to 176, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:20.

178. The nucleic acid of any one of embodiments 1 and 174 to 177, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:20.

179. The nucleic acid of any one of embodiments 1 and 174 to 178, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:20.

180. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:21.

181. The nucleic acid of embodiment 1 or embodiment 180, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:21.

182. The nucleic acid of any one of embodiments 1 , 180, and 181 , wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:21.

183. The nucleic acid of any one of embodiments 1 and 180 to 182, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:21.

184. The nucleic acid of any one of embodiments 1 and 180 to 183, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:21.

185. The nucleic acid of any one of embodiments 1 and 180 to 184, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:21.

186. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:22.

187. The nucleic acid of embodiment 1 or embodiment 186, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:22. 188. The nucleic acid of any one of embodiments 1 , 186, and 187, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:22.

189. The nucleic acid of any one of embodiments 1 and 186 to 188, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:22.

190. The nucleic acid of any one of embodiments 1 and 186 to 189, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:22.

191. The nucleic acid of any one of embodiments 1 and 186 to 190, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:22.

192. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23.

193. The nucleic acid of embodiment 1 or embodiment 192, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:23.

194. The nucleic acid of any one of embodiments 1 , 192, and 193, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:23.

195. The nucleic acid of any one of embodiments 1 and 192 to 194, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:23.

196. The nucleic acid of any one of embodiments 1 and 192 to 195, wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:23.

197. The nucleic acid of any one of embodiments 1 and 192 to 196, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:23.

198. The nucleic acid of embodiment 1, wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23. 199. The nucleic acid of embodiment 1 or embodiment 198, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:24.

200. The nucleic acid of any one of embodiments 1 , 198, and 199, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:24.

201. The nucleic acid of any one of embodiments 1 and 198 to 200, wherein the fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:24.

202. The nucleic acid of any one of embodiments 1 and 198 to 201 , wherein the fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:24.

203. The nucleic acid of any one of embodiments 1 and 198 to 202, wherein the fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:24.

204. The nucleic acid of any one of embodiments 1 and 50 to 203, comprising at least one amino acid substitution corresponding to an amino acid substitution W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K in the amino acid sequence of SEQ ID NO:3.

205. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 3-10 amino acid substitutions as compared to the reference amino acid sequence.

206. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 3-12 amino acid substitutions as compared to the reference amino acid sequence.

207. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 3-15 amino acid substitutions as compared to the reference amino acid sequence. 208. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 3-20 amino acid substitutions as compared to the reference amino acid sequence.

209. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 4-10 amino acid substitutions as compared to the reference amino acid sequence.

210. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 4-12 amino acid substitutions as compared to the reference amino acid sequence.

211. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 4-15 amino acid substitutions as compared to the reference amino acid sequence.

212. The nucleic acid of any one of embodiments 1 to 204, wherein the amino acid sequence comprises 4-20 amino acid substitutions as compared to the reference amino acid sequence.

213. A recombinant microorganism comprising the nucleic acid of any one of embodiments 1 to 212.

214. The recombinant microorganism of embodiment 213, further comprising at least one additional nucleic acid comprising a nucleotide sequence encoding a sucrose porin, a glucose porin, and/or a fructose porin.

215. The recombinant microorganism of embodiment 213 or embodiment 214, further comprising at least one additional nucleic acid comprising a nucleotide sequence encoding a sucrose permease, a glucose permease, and/or a fructose permease.

216. The recombinant microorganism of any one of embodiments 213 to 215, further comprising at least one additional nucleic acid comprising a nucleotide sequence encoding a sucrose invertase.

217. The recombinant microorganism of any one of embodiments 213 to 216, further comprising at least one additional nucleic acid comprising a nucleotide sequence encoding a glucose dehydrogenase.

218. The recombinant microorganism of any one of embodiments 213 to 217, further comprising at least one additional nucleic acid comprising a nucleotide sequence encoding a gluconolactonase. 219. The recombinant microorganism of any one of embodiments 213 to 218, further comprising at least one additional nucleic acid comprising a nucleotide sequence encoding a gluconate dehydratase.

220. The recombinant microorganism of any one of embodiments 213 to 219, which further has reduced glucokinase activity relative to a parental microorganism.

221. The recombinant microorganism of any one of embodiments 213 to 220, which further has reduced fructokinase activity relative to a parental microorganism.

222. The recombinant microorganism of any one of embodiments 213 to 221 , which further has reduced glucose phosphotransferase (PTS) activity relative to a parental microorganism.

223. The recombinant microorganism of any one of embodiments 213 to 222, which further has reduced fructose PTS activity relative to a parental microorganism.

224. The recombinant microorganism of any one of embodiments 213 to 223, which further has reduced mannose PTS activity relative to a parental microorganism.

225. The recombinant microorganism of any one of embodiments 213 to 224, which further has reduced gluconate kinase activity relative to a parental microorganism.

226. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is a Bacillus subtilis.

227. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is a Pseudomonas putida.

228. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is a Klebsiella oxytoca.

229. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is a Pantoea ananatis.

230. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is a Tatumella citrea.

231. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is a Zymomonas mobilis.

232. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is a Corynebacterium glutamicum.

233. The recombinant microorganism of any one of embodiments 213 to 225, wherein the recombinant microorganism is E. coli. 234. The recombinant microorganism of embodiment 233, wherein the E. coli is E. coli K12 or a strain derived therefrom (e.g., E. coli MG 1655) or E. coli W.

235. A composition comprising the recombinant microorganism of any one of embodiments 213 to 234 and a medium comprising sucrose, glucose, or fructose.

236. The composition of embodiment 235, wherein the medium comprises sucrose.

237. The composition of embodiment 235 or embodiment 236, wherein the medium comprises glucose.

238. The composition of any one of embodiments 235 to 237, wherein the medium comprises fructose.

239. A method of isomerizing fructose and glucose, comprising contacting, optionally at a mesophilic temperature, glucose and/or fructose with a fructose isomerase comprising an amino acid sequence encoded by a nucleic acid of any one of embodiments 1 to 204.

240. A method of isomerizing fructose and glucose, comprising culturing, in a production medium comprising sucrose, fructose, and/or glucose, optionally at a mesophilic temperature, a recombinant microorganism of any one of embodiments 213 to 234.

241. The method of embodiment 240, wherein the production medium comprises sucrose.

242. The method of embodiment 240 or embodiment 241 , wherein the production medium comprises from 0.1 w/v% to 5 w/v% sucrose, optionally 1 w/v% to 2 w/v% sucrose.

243. The method of any one of embodiments 240 to 242, wherein the production medium comprises fructose.

244. The method of any one of embodiments 240 to 243, wherein the production medium comprises from 0.1 w/v% to 5 w/v% fructose, optionally 1 w/v% to 2 w/v% fructose.

245. The method of any one of embodiments 240 to 244, wherein the production medium comprises glucose.

246. The method of any one of embodiments 240 to 245, wherein the production medium comprises from 0.1 w/v% to 5 w/v% glucose, optionally 1 w/v% to 2 w/v% glucose.

247. The method of any one of embodiments 240 to 246, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism. 248. The method of any one of embodiments 240 to 247, wherein the recombinant microorganism is capable of non-phosphorylatively transporting fructose from the production medium into a cell of the recombinant microorganism.

249. The method of any one of embodiments 240 to 248, wherein the recombinant microorganism is capable of non-phosphorylatively transporting glucose from the production medium into a cell of the recombinant microorganism.

250. The method of any one of embodiments 240 to 249, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, fructose, or sucrose, prior to culturing in the production medium.

251. The method of embodiment 250, wherein the growth medium comprises glycerol.

252. The method of embodiment 250 or embodiment 251 , wherein the growth medium comprises from 0.1 w/v% to 5 w/v% glycerol, optionally 1 w/v% to 2 w/v% glycerol.

253. The method of any one of embodiments 250 to 252, wherein the growth medium lacks added glucose, fructose, and sucrose.

254. The method of any one of embodiments 250 to 253, wherein the growth medium comprises no more than 0.1% glucose.

255. The method of any one of embodiments 250 to 254, wherein the growth medium comprises no more than 0.1% fructose.

256. The method of any one of embodiments 250 to 255, wherein the growth medium comprises no more than 0.1% sucrose.

257. A method of producing glucose, comprising culturing, in a production medium comprising at least one of fructose or sucrose, at a mesophilic temperature, a recombinant microorganism of any one of embodiments 213 to 234.

258. The method of embodiment 239, wherein the production medium comprises sucrose.

259. The method of embodiment 239 or embodiment 258, wherein the production medium comprises from 0.1 w/v% to 5 w/v% sucrose, optionally 1 w/v% to 2 w/v% sucrose.

260. The method of any one of embodiments 257 to 259, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism. 261. The method of any one of embodiments 239 to 260, wherein the production medium comprises fructose.

262. The method of any one of embodiments 239 to 261 , wherein the production medium comprises from 0.1 w/v% to 5 w/v% fructose, optionally 1 w/v% to 2 w/v% fructose.

263. The method of any one of embodiments 239 to 262, wherein the recombinant microorganism is capable of non-phosphorylatively transporting fructose from the production medium into a cell of the recombinant microorganism.

264. The method of any one of embodiments 239 to 263, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, fructose, or sucrose, prior to culturing in the production medium.

265. The method of embodiment 264, wherein the growth medium comprises glycerol.

266. The method of embodiment 264 or embodiment 265, wherein the growth medium comprises from 0.1 w/v% to 5 w/v% glycerol, optionally 1 w/v% to 2 w/v% glycerol.

267. The method of any one of embodiments 264 to 266, wherein the growth medium lacks added glucose, fructose, and sucrose.

268. The method of any one of embodiments 264 to 266, wherein the growth medium comprises no more than 0.1% glucose.

269. The method of any one of embodiments 264 to 268, wherein the growth medium comprises no more than 0.1% fructose.

270. The method of any one of embodiments 264 to 269, wherein the growth medium comprises no more than 0.1% sucrose.

271. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of embodiments 217to 219 in a production medium comprising sucrose, fructose, or glucose.

272. The method of embodiment 271 , wherein the production medium comprises sucrose.

273. The method of embodiment 271 or embodiment 272, wherein the production medium comprises from 0.1 w/v% to 5 w/v% sucrose, optionally 1 w/v% to 2 w/v% sucrose. 274. The method of any one of embodiments 271 to 273, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.

275. The method of any one of embodiments 271 to 274, wherein the production medium comprises fructose.

276. The method of any one of embodiments 271 to 275, wherein the production medium comprises from 0.1 w/v% to 5 w/v% fructose, optionally 1 w/v% to 2 w/v% fructose.

277. The method of any one of embodiments 271 to 276, wherein the recombinant microorganism is capable of non-phosphorylatively transporting fructose from the production medium into a cell of the recombinant microorganism.

278. The method of any one of embodiments 271 to 277, wherein the production medium comprises glucose.

279. The method of any one of embodiments 271 to 278, wherein the production medium comprises from 0.1 w/v% to 5 w/v% glucose, optionally 1 w/v% to 2 w/v% glucose.

280. The method of any one of embodiments 271 to 279, wherein the recombinant microorganism is capable of non-phosphorylatively transporting glucose from the production medium into a cell of the recombinant microorganism.

281. The method of any one of embodiments 271 to 280, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, fructose, or sucrose, prior to culturing in the production medium.

282. The method of embodiment 281 , wherein the growth medium comprises glycerol.

283. The method of embodiment 281 or embodiment 282, wherein the growth medium comprises from 0.1 w/v% to 5 w/v% glycerol, optionally 1 w/v% to 2 w/v% glycerol.

284. The method of any one of embodiments 281 to 283, wherein the growth medium lacks added glucose, fructose, and sucrose.

285. The method of any one of embodiments 281 to 283, wherein the growth medium comprises no more than 0.1% glucose.

286. The method of any one of embodiments 281 to 283 and 285, wherein the growth medium comprises no more than 0.1% fructose. 287. The method of any one of embodiments 281 to 283 and 286, wherein the growth medium comprises no more than 0.1% sucrose.

288. A recombinant microorganism comprising a nucleic acid or a plurality of nucleic acids encoding means for isomerizing fructose and glucose, optionally at mesophilic temperatures.

289. The recombinant microorganism of embodiment 288, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.

290. The recombinant microorganism of embodiment 288 or embodiment 289, further comprising a nucleic acid or a plurality of nucleic acids encoding means for non- phosphorylatively transporting fructose.

291. The recombinant microorganism of any one of embodiments 288 to 290, further comprising a nucleic acid or a plurality of nucleic acids encoding means for non- phosphorylatively transporting sucrose.

292. The recombinant microorganism of any one of embodiments 288 to 291 , further comprising a nucleic acid or a plurality of nucleic acids encoding means for non- phosphorylatively transporting glucose.

293. The recombinant microorganism of any one of embodiments 288 to 292, further comprising a nucleic acid or a plurality of nucleic acids encoding means for hydrolyzing sucrose to fructose and glucose.

294. A method of isomerizing fructose and glucose, comprising: culturing the recombinant microorganism of any one of embodiments 288 to 293 in a production medium comprising sucrose, fructose, or glucose.

295. The method of embodiment 294, wherein the production medium comprises sucrose.

296. The method of embodiment 294 or embodiment 295, wherein the production medium comprises from 0.1 w/v% to 5 w/v% sucrose, optionally 1 w/v% to 2 w/v% sucrose.

297. The method of any one of embodiments 294 to 296, wherein the production medium comprises fructose.

298. The method of any one of embodiments 294 to 297, wherein the production medium comprises from 0.1 w/v% to 5 w/v% fructose, optionally 1 w/v% to 2 w/v% fructose. 299. The method of any one of embodiments 294 to 298, wherein the production medium comprises glucose.

300. The method of any one of embodiments 294 to 299, wherein the production medium comprises from 0.1 w/v% to 5 w/v% glucose, optionally 1 w/v% to 2 w/v% glucose.

301. The method of any one of embodiments 294 to 300, wherein the culturing is at a temperature from 20°C to 40°C.

302. The method of any one of embodiments 294 to 301 , wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, fructose, or sucrose, prior to culturing in the production medium.

303. The method of embodiment 302, wherein the growth medium comprises glycerol.

304. The method of embodiment 302 or embodiment 303, wherein the growth medium comprises from 0.1 w/v% to 5 w/v% glycerol, optionally 1 w/v% to 2 w/v% glycerol.

305. The method of any one of embodiments 302 to 304, wherein the growth medium lacks added glucose, fructose, and sucrose.

306. The method of any one of embodiments 302 to 304, wherein the growth medium comprises no more than 0.1% glucose.

307. The method of any one of embodiments 302 to 304 and 306, wherein the growth medium comprises no more than 0.1% fructose.

308. The method of any one of embodiments 302 to 304 and 306 to 307, wherein the growth medium comprises no more than 0.1 % sucrose.

309. A recombinant microorganism comprising means for isomerizing fructose and glucose, optionally at mesophilic temperatures.

310. The recombinant microorganism of embodiment 309, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.

311. The recombinant microorganism of embodiment 309 or embodiment 310, further comprising means for non-phosphorylatively transporting fructose.

312. The recombinant microorganism of any one of embodiments 309 to 311 , further comprising means for non-phosphorylatively transporting sucrose. 313. The recombinant microorganism of any one of embodiments 309 to 312, further comprising means for non-phosphorylatively transporting glucose.

314. The recombinant microorganism of any one of embodiments 309 to 313, further comprising means for hydrolyzing sucrose to fructose and glucose.

315. A method of isomerizing fructose and glucose, comprising: culturing the recombinant microorganism of any one of embodiments 309 to 314 in a production medium comprising sucrose, fructose, or glucose.

316. The method of embodiment 315, wherein the production medium comprises sucrose.

317. The method of embodiment 315 or embodiment 316, wherein the production medium comprises from 0.1 w/v% to 5 w/v% sucrose, optionally 1 w/v% to 2 w/v% sucrose.

318. The method of any one of embodiments 315 to 317, wherein the production medium comprises fructose.

319. The method of any one of embodiments 315 to 318, wherein the production medium comprises from 0.1 w/v% to 5 w/v% fructose, optionally 1 w/v% to 2 w/v% fructose.

320. The method of any one of embodiments 315 to 319, wherein the production medium comprises glucose.

321. The method of any one of embodiments 315 to 320, wherein the production medium comprises from 0.1 w/v% to 5 w/v% glucose, optionally 1 w/v% to 2 w/v% glucose.

322. The method of any one of embodiments 315 to 321 , wherein the culturing is at a temperature from 20°C to 40°C.

323. The method of any one of embodiments 315 to 322, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, fructose, or sucrose, prior to culturing in the production medium.

324. The method of embodiment 323, wherein the growth medium comprises glycerol.

325. The method of embodiment 323 or embodiment 324, wherein the growth medium comprises from 0.1 w/v% to 5 w/v% glycerol, optionally 1 w/v% to 2 w/v% glycerol.

326. The method of any one of embodiments 323 to 325, wherein the growth medium lacks added glucose, fructose, and sucrose. 327. The method of any one of embodiments 323 to 325, wherein the growth medium comprises no more than 0.1% glucose.

328. The method of any one of embodiments 323 to 325 and 327, wherein the growth medium comprises no more than 0.1% fructose.

329. The method of any one of embodiments 323 to 325 and 327 to 328, wherein the growth medium comprises no more than 0.1 % sucrose.

330. A polypeptide comprising at least one amino acid substitution as compared to a "reference" polypeptide of any one of SEQ ID NOS: 1-24, optionally wherein

(a) the polypeptide has at least 90% sequence identity to the reference polypeptide of any one of SEQ ID NOS: 1-24; and/or

(b) the polypeptide has improved fructose isomerase activity as compared to the reference polypeptide having the amino acid sequence of any one of SEQ ID NOS: 1-24.

331. The polypeptide of embodiment 330, which comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3, optionally wherein the amino acid sequence comprises at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K compared to the amino acid sequence of SEQ ID NO:3.

332. The polypeptide of embodiment 330 or embodiment 331 , which comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3.

333. The polypeptide of any one of embodiments 330 to 332, which comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:3.

334. The polypeptide of any one of embodiments 330 to 333, which comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:3.

335. The polypeptide of any one of embodiments 330 to 334, which comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:3.

336. The polypeptide of any one of embodiments 330 to 335, which comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:3.

337. The polypeptide of any one of embodiments 330 to 336, which comprises an amino acid sequence having amino acid substitutions W139F and/or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

338. The polypeptide of any of embodiments 331 to 337, wherein the amino acid sequence is identical to (i.e., does not have a substitution) at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339 as compared to the amino acid sequence of SEQ ID NO:3.

339. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and A142N as compared to the amino acid sequence of SEQ ID NO:3.

340. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and A142S as compared to the amino acid sequence of SEQ ID NO:3.

341. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and A47S as compared to the amino acid sequence of SEQ ID NO:3.

342. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, C99S, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

343. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, C99S, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

344. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, C99T, T141S, A142S, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

345. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, C99T, T141S, A142T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

346. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, C99T, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:3. 347. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, C99T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

348. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and C99T as compared to the amino acid sequence of SEQ ID NO:3.

349. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and D128E as compared to the amino acid sequence of SEQ ID NO:3.

350. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:3.

351. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and F150Y as compared to the amino acid sequence of SEQ ID NO:3.

352. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:3.

353. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:3.

354. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

355. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:3.

356. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3. 357. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

358. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

359. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and L144C as compared to the amino acid sequence of SEQ ID NO:3.

360. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and L144K as compared to the amino acid sequence of SEQ ID NO:3.

361. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and L144M as compared to the amino acid sequence of SEQ ID NO:3.

362. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and N19E as compared to the amino acid sequence of SEQ ID NO:3.

363. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and N19T as compared to the amino acid sequence of SEQ ID NO:3.

364. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:3.

365. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:3.

366. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:3. 367. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:3.

368. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and Q59M as compared to the amino acid sequence of SEQ ID NO:3.

369. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

370. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

371. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and S155A as compared to the amino acid sequence of SEQ ID NO:3.

372. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and T141C as compared to the amino acid sequence of SEQ ID NO:3.

373. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, T141S, and A142T as compared to the amino acid sequence of SEQ ID NO:3.

374. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, T141S, L144M, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

375. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and T141S as compared to the amino acid sequence of SEQ ID NO:3.

376. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:3. 377. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:3.

378. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, and Y22F as compared to the amino acid sequence of SEQ ID NO:3.

379. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

380. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, Q59M, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

381. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, D128E, F150Y, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

382. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, Q59M, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

383. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, N19E, D128E, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

384. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, N19E, A47S, D128E, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

385. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, N19E, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

386. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, A47S, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3. 387. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, Q59M, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

388. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, A47S, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

389. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, D128E, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

390. The polypeptide of any of embodiments 331 to 338, wherein the amino acid sequence comprises the substitutions W139F, V186T, Q59M, D128E, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:3. The polypeptide of any one of embodiments 331 to 337, which comprises an amino acid sequence identical to (i.e., does not have a substitution) at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339 as compared to the amino acid sequence of SEQ ID NO:3.

391. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 3-10 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide.

392. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 3-12 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide.

393. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 3-15 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide.

394. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 3-20 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide.

395. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 4-10 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide. 396. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 4-12 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide.

397. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 4-15 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide.

398. The polypeptide of any one of embodiments 330 to 390, wherein the amino acid sequence comprises 4-20 amino acid substitutions as compared to the amino acid sequence of the reference polypeptide.

399. The polypeptide of any one of embodiments 330 to 398, which is intracellular, e.g., in a microorganism.

400. The polypeptide of any one of embodiments 330 to 398, which is purified.

401. The polypeptide of any one of embodiments 330 to 398, which is in a cell culture supernatant.

402. A method of isomerizing glucose and sucrose, comprising contacting glucose and/or sucrose with the polypeptide of any one of embodiments 330 to 401 under conditions under which isomerization occurs.

[0286] Various aspects of the present disclosure are described in the embodiments set forth in the following numbered paragraphs of Group 2.

[0287] Group 2:

1. A nucleic acid comprising a nucleotide sequence encoding a fructose isomerase comprising an amino acid sequence having at least 90% sequence identity and less than 100% sequence identity to one or more of SEQ ID NO: 1-20, wherein the fructose isomerase has at least three non-identical amino acids compared to SEQ ID NO:3.

2. The nucleic acid of embodiment 1 , wherein the fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.

3. The nucleic acid of embodiment 1 or embodiment 2, wherein the fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3. 4. The nucleic acid of any one of embodiments 1 to 3, wherein the fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:3.

8. The nucleic acid of any one of embodiments 1 to 7, wherein the fructose isomerase comprises an amino acid sequence comprising at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

9. The nucleic acid of any one of embodiments 1 to 8, comprising from 3-10 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.

10. The nucleic acid of any one of embodiments 1 to 8, comprising from 3-12 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.

11 . The nucleic acid of any one of embodiments 1 to 8, comprising from 3-15 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.

12. The nucleic acid of any one of embodiments 1 to 8, comprising from 3-20 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24. 13. The nucleic acid of any one of embodiments 1 to 8, comprising from 4-10 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.

14. The nucleic acid of any one of embodiments 1 to 8, comprising from 4-12 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.

15. The nucleic acid of any one of embodiments 1 to 8, comprising from 4-15 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.

16. The nucleic acid of any one of embodiments 1 to 8, comprising from 4-20 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:1-24.

17. A polypeptide encoded by the nucleic acid of any one of embodiments 1 to 16.

18. A recombinant microorganism comprising the nucleic acid of any one of embodiments 1 to 16 or the polypeptide of embodiment 17.

19. The recombinant microorganism of embodiment 18, wherein the recombinant microorganism is E. coli.

20. The recombinant microorganism of embodiment 19, wherein the E. coli is E. coli K12 or a strain derived therefrom (e.g., E. coli MG1655) or E. coli W.

21 . A composition, comprising the recombinant microorganism of any one of embodiments 18 to 20 and a medium comprising sucrose, glucose, or fructose.

22. A method of isomerizing fructose and glucose, comprising culturing, in a production medium comprising at least one of sucrose, fructose, or glucose, at a mesophilic temperature, a recombinant microorganism of any one of embodiments 18 to 20.

23. The method of embodiment 22, wherein the production medium comprises sucrose.

24. The method of embodiment 23, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism. 25. The method of any one of embodiments 22 to 24, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, fructose, or sucrose, prior to culturing in the production medium.

26. The method of embodiment 25, wherein the growth medium comprises glycerol.

27. The method of embodiment 25 or embodiment 26, wherein the growth medium lacks added glucose, fructose, and sucrose.

28. The method of embodiment 25 or embodiment 26, wherein the growth medium comprises no more than 0.1% of glucose, fructose, and/or sucrose.

29. A recombinant microorganism comprising a nucleic acid or a plurality of nucleic acids encoding means for isomerizing fructose and glucose, optionally at mesophilic temperatures.

30. The recombinant microorganism of embodiment 29, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.

31 . The recombinant microorganism of embodiment 29 or embodiment 30, further comprising a nucleic acid or a plurality of nucleic acids encoding means for non- phosphorylatively transporting fructose, sucrose, or glucose.

32. A method of isomerizing fructose and glucose, comprising: culturing the recombinant microorganism of any one of embodiments 29 to 31 in a production medium comprising sucrose, fructose, or glucose.

33. The method of embodiment 32, wherein the production medium comprises sucrose.

34. The method of embodiment 32 or embodiment 33, wherein the production medium comprises fructose.

35. The method of any one of embodiments 32 to 34, wherein the production medium comprises glucose.

36. The method of any one of embodiments 32 to 35, wherein the culturing is at a temperature from 20°C to 40°C.

37. A recombinant microorganism comprising means for isomerizing fructose and glucose, optionally at mesophilic temperatures. 38. The recombinant microorganism of embodiment 37, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.

39. The recombinant microorganism of embodiment 37 or embodiment 38, further comprising means for non-phosphorylatively transporting fructose, sucrose, or glucose.

40. A method of isomerizing fructose and glucose, comprising: culturing the recombinant microorganism of any one of embodiments 37 to 39 in a production medium comprising sucrose, fructose, or glucose.

41 . The method of embodiment 40, wherein the production medium comprises sucrose.

42. The method of embodiment 40 or embodiment 41 , wherein the production medium comprises fructose.

43. The method of any one of embodiments 40 to 42, wherein the production medium comprises glucose.

44. The method of any one of embodiments 40 to 43, wherein the culturing is at a temperature from 20°C to 40°C.

45. A polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3 and one or more of the amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:3:

(a) N19E or N19T;

(b) Y22F;

(c) A47S;

(d) Q59M;

(e) C99S or C99T;

(f) D128E;

(g) T141C or T141S;

(h) A142N, A142S, or A T;

(i) L144C, L144K, or L144M;

(j) F150Y;

(k) S155A;

(l) V186C; (m) F187L, F187Q, F187W, or F187Y;

(n) E203Q;

(o) N249H or N249S;

(p) F276Q;

(q) T299E;

(r) F363Y;

(s) K410E;

(t) R415K;

(u) Q417E or Q417K; and

(v) R424K.

46. The polypeptide of embodiment 45, which has at least two of the substitutions (a)-(v).

47. The polypeptide of embodiment 45 or embodiment 2, which has at least three of the amino acid substitutions (a)-(v).

48. The polypeptide of any one of embodiments 45 to 3, which has at least four of the amino acid substitutions (a)-(v).

49. The polypeptide of any one of embodiments 45 to 4, which has at least five of the amino acid substitutions (a)-(v).

50. The polypeptide of any one of embodiments 45 to 5, which has up to ten of the amino acid substitutions (a)-(v).

51. The polypeptide of any one of embodiments 45 to 6, which has up to nine of the amino acid substitutions (a)-(v).

52. The polypeptide of any one of embodiments 45 to 7, which has up to eight of the amino acid substitutions (a)-(v).

53. The polypeptide of any one of embodiments 45 to 8, which has up to seven of the amino acid substitutions (a)-(v).

54. The polypeptide of any one of embodiments 45 to 9, which has up to six of the amino acid substitutions (a)-(v).

55. The polypeptide of any one of embodiments 45 to 10, which further comprises one or both of the amino acid substitutions W139F and V186T as compared to the amino acid sequence of SEQ ID NO:3. 56. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, A142N, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

57. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, A142S, and V186T and as compared to the amino acid sequence of SEQ ID NO:3.

58. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions A47S, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

59. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99S, W139F, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

60. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99S, W139F, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

61 . The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, T141S, A142S, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

62. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, T141S, A142T, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

63. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

64. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

65. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3. 66. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions D128E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

67. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:3.

68. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, F150Y, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

69. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:3.

70. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:3.

71 . The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

72. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:3.

73. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

74. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

75. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:3. 76. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, L144C, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

77. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, L144K, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

78. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, L144M, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

79. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

80. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

81 . The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:3.

82. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:3.

83. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:3.

84. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:3.

85. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3. 86. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

87. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

88. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, S155A, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

89. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141C, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

90. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141S, A142T, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

91 . The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141S, L144M, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

92. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141S, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

93. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:3.

94. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:3.

95. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Y22F, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3. 96. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

97. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

98. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions D128E, W139F, F150Y, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

99. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

100. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, D128E, W139F, V186T, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

101. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, A47S, D128E, W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

102. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

103. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions A47S, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

104. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

105. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions A47S, W139F, V186T, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3. 106. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions D128E, W139F, V186T, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

107. The polypeptide of any of embodiments 45 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, D128E, W139F, V186T, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

108. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of embodiments 45 to 63.

109. The nucleic acid of embodiment 64, which is a plasmid.

110. A recombinant microorganism engineered to express the polypeptide of any one of embodiments 45 to 63.

111. A recombinant microorganism comprising the nucleic acid of embodiment 64 or embodiment 65.

112. The recombinant microorganism of embodiment 66 or embodiment 67, which is an E. coli.

113. A method of isomerizing fructose and glucose, comprising culturing the recombinant microorganism of any one of embodiments 66 to 68 in a production medium comprising fructose, glucose, and/or one or more precursor di- or polysaccharides that can be metabolized into fructose or glucose (e.g., sucrose).

114. The method of embodiment 69, wherein the culturing is at a mesophilic temperature.

115. The method of embodiment 69 or embodiment 70, in which the production medium comprises fructose.

116. The method of any one of embodiments 69 to 71, in which the production medium comprises glucose.

117. The method of any one of embodiments 69 to 72, in which the production medium comprises sucrose.

118. The method of any one of embodiments 69 to 117, in which the production medium comprises lactose.

119. The method of any one of embodiments 69 to 118, in which the production medium comprises maltose. 8. EXAMPLES

8.1. Example 1 : Improved Fructose Isomerization by Engineering of Clostridium Xylose Isomerase Double Mutant

[0288] The C. thermosulfurogenes xylose isomerase double with amino acid substitutions at W139F and V186T relative to SEQ ID NO:3 enabled higher growth of E. coli SuA6 cells relative to negative controls. Whether further engineering could improve the specific activity of the double mutant was investigated. Triple and higher order mutants were constructed by adding the following substitutions to the double mutant or replacing the Thr at position 186 of the double mutant with Cys:

[0289] All of the triple and higher order mutants listed in Table 2 demonstrated higher specific activity for fructose isomerization than the C. thermosulfurogenes xylose isomerase double mutant

[0290] The normalized specific activity of fructose isomerization of all variants listed in Table 2 was assayed in vitro using cell lysate at 25 mM fructose and 37°C. Normalized specific activity of a variant is defined as specific activity ([glucose]/[protein titer]) for the variant divided by specific activity of the W139F/V186T double mutant.

[0291] The initial turnover rates (1/s) for wild type Clostridium xylose isomerase, the V186T single mutant, the double mutant, and various higher order mutants were assayed using purified enzyme. The initial turnover rates are given in Table 3:

[0292] Table 3 shows multiple triple or higher order mutants had initial turnover rates about 20-60% higher than that of the W139F + V186T double mutant.

8.2. Example 2: Improved Fructose Isomerization by Additional Engineering of Clostridium Xylose Isomerase Double Mutant

[0293] The catalytic efficiency of the enzymes shown in Example 1, Table 3 can be estimated as k_cat/K_m, where k_cat is the initial turnover rate and K_m is the substrate concentration at which half of the enzyme active sites are occupied. The K_m of the Clostridium xylose isomerase mutants shown in Table 3 is on the order of 10 mM, which yields k_cat/K_m values on the order of 10² 1/s/M. This catalytic efficiency is much less than the median k_cat/K_m value of 4.1x10⁵ 1/s/M observed for bacterial enzymes involved in carbohydrate and energy metabolism. Thus, although the Clostridium xylose isomerase mutants listed in Table 3 may provide fructose isomerization activity at mesophilic temperatures in vivo that is sufficiently fast for E. coli SuA6 cells to grow on fructose, the catalytic efficiency may still be lower than is desired when glucose is used as a carbon and energy source in bioindustrial processes.

[0294] Using structural modeling, triple and higher order mutants of the Clostridium xylose isomerase incorporating the W139F + V186T substitutions are identified with a predicted K_cat of at least 2.00 1/s. A panel of about 20 triple and higher order mutants is expressed in a recombinant microorganism, purified, and the initial turnover rate of each member of the panel is assayed according to the methods described in Example 1.

8.3. Example 3: Improved Fructose Isomerization by Engineering of non- Clostridium Xylose Isomerases

[0295] The primary sequences and/or tertiary structures of xylose isomerases from organisms other than C. thermosulfurogenes (e.g., SEQ ID NO:1-2, 4-10, 12, or 24) are aligned with sequence and/or structure of the Clostridium enzyme to identify aligned residues as targets for substitutions corresponding to the Clostridium XylA substitutions W139F, V186T, and each of the those set forth in Table 2.

[0296] Using structural modeling, mutants of nor-Clostridium xylose isomerases incorporating one or more of the substitutions are identified with a predicted K_cat of at least 2.00 1/s. A panel of about 20 mutants is expressed in a recombinant microorganism, purified, and the initial turnover rate of each member of the panel is assayed according to the methods described in Example 1.

8.4. Example 4: Improved Fructose Isomerization Activity of Engineered Xylose Isomerases at Mesophilic Temperature in vivo

8.4.1. Materials and Methods

[0297] The C. thermosulfurogenes xylose isomerase comprising W139F and V186T substitutions relative to SEQ ID NO:3 was further mutated in silico by incorporating additional substitutions. Structural modeling was used to predict combinations of mutations that improved fructose isomerase activity.

[0298] Twelve fructose isomerases with predicted higher enzymatic activity (which had sitespecific mutations as indicated in Table 4) were selected for further work. Nucleic acid samples encoding the selected fructose isomerases were constructed and E. coli SuA6 cells were engineered therewith. The expression constructs incorporated a fructose isomerase coding region into a pTrcHis2b plasmid. Cells were grown in medium containing fructose for 3 days at 37°C. A vector-only construct was used as a negative control. An expression construct comprising a coding region for C. thermosulfurogenes W139FA/186T double mutant XylA was used as a positive control and for normalization of fructose isomerization activity.

[0299] The twelve fructose isomerases underwent in vitro assessments of normalized specific activity of cell lysates at 25mM fructose and 37°C, following the procedure of Example 1.

8.4.2. Results

[0300] Each tested sample had a normalized specific activity greater than that of the W139FA/186T double mutant, indicating that each sample had fructose isomerization activity and suggesting that the additional site-specific mutations increased the fructose isomerization activity of these samples over C. thermosulfurogenes W139FA/186T double mutant XylA (FIG. 7).

9. INCORPORATION BY REFERENCE

[0301] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0302] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed anywhere before the priority date of this application.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3 and one or more of the amino acid substitutions as compared to the amino acid sequence of SEQ ID NO:3:

(a) N19E or N19T;

(b) Y22F;

(c) A47S;

(d) Q59M;

(e) C99S or C99T;

(f) D128E;

(g) T141C or T141S;

(h) A142N, A142S, or A142T;

(i) L144C, L144K, or l_144M;

G) F150Y;

(k) S155A;

(l) V186C;

(m) F187L, F187Q, F187W, or F187Y;

(n) E203Q;

(o) N249H or N249S;

(p) F276Q;

(q) T299E;

(r) F363Y;

(s) K410E;

(t) R415K;

(u) Q417E or Q417K; and

(v) R424K.

2. The polypeptide of claim 1 , which has at least two of the substitutions (a)-(v).

3. The polypeptide of claim 1 or claim 2, which has at least three of the amino acid substitutions (a)-(v).

4. The polypeptide of any one of claims 1 to 3, which has at least four of the amino acid substitutions (a)-(v).

5. The polypeptide of any one of claims 1 to 4, which has at least five of the amino acid substitutions (a)-(v).

6. The polypeptide of any one of claims 1 to 5, which has up to ten of the amino acid substitutions (a)-(v).

7. The polypeptide of any one of claims 1 to 6, which has up to nine of the amino acid substitutions (a)-(v).

8. The polypeptide of any one of claims 1 to 7, which has up to eight of the amino acid substitutions (a)-(v).

9. The polypeptide of any one of claims 1 to 8, which has up to seven of the amino acid substitutions (a)-(v).

10. The polypeptide of any one of claims 1 to 9, which has up to six of the amino acid substitutions (a)-(v).

11 . The polypeptide of any one of claims 1 to 10, which further comprises one or both of the amino acid substitutions W139F and V186T as compared to the amino acid sequence of SEQ ID NO:3.

12. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, A142N, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

13. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, A142S, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

14. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions A47S, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

15. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99S, W139F, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

16. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99S, W139F, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

17. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, T141S, A142S, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

18. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, T141S, A142T, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

19. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

20. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, and V186C as compared to the amino acid sequence of SEQ ID NO:3.

21 . The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions C99T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

22. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions D128E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

23. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:3.

24. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, F150Y, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

25. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:3.

26. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:3.

27. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

28. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:3.

29. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

30. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

31 . The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

32. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, L144C, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

33. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, L144K, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

34. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, L144M, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

35. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

36. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

37. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:3.

38. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:3.

39. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:3.

40. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:3.

41 . The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

42. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

43. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

44. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, S155A, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

45. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141C, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

46. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141S, A142T, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

47. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141S, L144M, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:3.

48. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, T141S, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

49. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:3.

50. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:3.

51 . The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Y22F, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:3.

52. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

53. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

54. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions D128E, W139F, F150Y, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

55. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:3.

56. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, D128E, W139F, V186T, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:3.

57. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, A47S, D128E, W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:3.

58. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions N19E, Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

59. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions A47S, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

60. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

61 . The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions A47S, W139F, V186T, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

62. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions D128E, W139F, V186T, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:3.

63. The polypeptide of any of claims 1 to 11 , wherein the amino acid sequence comprises the amino acid substitutions Q59M, D128E, W139F, V186T, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:3.

64. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of claims 1 to 63.

65. The nucleic acid of claim 64, which is a plasmid.

66. A recombinant microorganism engineered to express the polypeptide of any one of claims 1 to 63.

67. A recombinant microorganism comprising the nucleic acid of claim 64 or claim 65.

68. The recombinant microorganism of claim 66 or claim 67, which is an E. coli.

69. A method of isomerizing fructose and glucose, comprising culturing the recombinant microorganism of any one of claims 66 to 68 in a production medium comprising fructose, glucose, and/or sucrose.

70. The method of claim 69, wherein the culturing is at a mesophilic temperature.

71 . The method of claim 69 or claim 70, in which the production medium comprises fructose.

72. The method of any one of claims 69 to 71, in which the production medium comprises glucose.

73. The method of any one of claims 69 to 72, in which the production medium comprises sucrose.