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WO2017190056A1 - Conversion de composés de 1-carbone en produits - Google Patents

Conversion de composés de 1-carbone en produits Download PDF

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WO2017190056A1
WO2017190056A1 PCT/US2017/030203 US2017030203W WO2017190056A1 WO 2017190056 A1 WO2017190056 A1 WO 2017190056A1 US 2017030203 W US2017030203 W US 2017030203W WO 2017190056 A1 WO2017190056 A1 WO 2017190056A1
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coa
coli
aldehyde
microorganism
conversion
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Ramon Gonzalez
James M. CLOMBURG
Alexander CHOU
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William Marsh Rice University
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William Marsh Rice University
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Definitions

  • the invention relates to biotechnological methods for the production of industrial ly relevant chemicals.
  • methods for the biological production of products containing at least one terminal alcohol, carboxyl group, amine or alkyl group directly by the assimilation of single carbon units is described.
  • Microbes have been designed and engineered to synthesize products of interest using feedstocks as diverse as sugars, glycerol, carbon dioxide, carbon monoxide, formate, methanol, and methane.
  • feedstocks as diverse as sugars, glycerol, carbon dioxide, carbon monoxide, formate, methanol, and methane.
  • I -carbon feedstocks such conversions are made possible by a general network of metabolic pathways that are organized as shown in FIG. 1 and include specialized pathways for carbon fixation, central metabolism, and product synthesis. This type of metabolic architecture has been exploited in all metabolic engineering efforts conducted to date to develop microbes for industrial applications.
  • This architecture is also commonly limited to elongation of a carbon backbone by a minimum of two carbons per step, which is a result of the aforementioned use of common metabolic intermediates.
  • This disclosure describes an alternative platform for the byconversion of 1 -carbon substrates to products of interest, which consists of a single engineered metabolic pathway that allows for the direct assimilation of one-carbon compounds into chemical products containing at least one terminal alcohol, carboxyl group, amine or alkyl .
  • Chemicals containing terminal alcohol, carboxyl , amine or alkyl groups have broad uses in the chemical industry including, but not limited to, applications such as fuels, cosmetics, and polymers.
  • the engineered pathway designed from the "bottom-up " , defines a new metabolic architecture that consolidates carbon fixation, central metaboli sm, and product synthesis into single pathway ( FIG. 2-7).
  • the pathway uses single carbon extension units, which bypasses the need for the production of common metabolic intermediates and allows for elongation of a carbon backbone iteratively in single carbon increments.
  • This synthetic pathway is inspired by the catabolic a- oxidation pathway (one carbon shortening of fatty acids), and based on the key recent finding in our laboratory that the enzyme 2-bydroxyl-acyl-CoA lyase (HACL, part of the a-oxidation pathway) is reversible and able to catalyze C-C bond formation between formyl-CoA and an aldehyde.
  • HACL 2-bydroxyl-acyl-CoA lyase
  • An enzyme from the alpha oxidation pathway ( hy droxy 1 -acy 1 -Co A lyases or HACLs) is driven in the reverse direction and thus used to catalyze the key condensation (C-C bond formation) reaction.
  • a series of dehydration and reduction reactions then converts the molecule to a form suitable for successiv e rounds of elongation (FIG. 3-4).
  • products containing at least one terminal alcohol, carboxyl, amine or alkyl are derived solely from single carbon molecules.
  • products can be derived from a combination of single carbon molecules and multi -carbon molecules.
  • These additions can also aid the throughput of the designed pathway and all ow for the production of more v aried products that contain at least one terminal alcohol, carboxyl, amine or alkyl .
  • bacteria , "strain” and the like may be used interchangeably and all such designations include their progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it ill be clear from the context.
  • a "cell, " "microbe,” etc. is generally understood to include a culture of such cell s, as the work described herein is done in cultures having 10 9"15 cells.
  • “homolog” means an enzyme ith at least 40% identity to one of the listed sequences and also hav ing the same general catalytic activ ity, although of course Km, Kcat, and the like can vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it i s typical for bacterial sequences to diverge significantly (40- 60%), yet still be identifiable as homologs, while mammalian species tend to diverge less (80-90%).
  • proteins herein can be understood to include reference to the gene encoding such protein.
  • a claimed "permease" protein can include the related gene encoding that permease.
  • Another way of finding suitable enzymes/proteins for use in the invention i s to consider other enzymes with the same EC number, since these numbers are assigned based on the reactions performed by a given enzyme.
  • An enzyme that thus be obtained e.g., from AddGene or from the author of the work describing that enzyme, and tested for functionality as described herein.
  • NCBITM provides codon usage databases for optimizing DNA sequences for protein expression in various species. Using such databases, a gene or cDNA may be "optimized" for expression in / ⁇ note ' . coli, yeast, algal or other species using the codon bias for the species in which the gene will be expressed.
  • % identity number of aligned residues in the query sequence/length of reference sequence. Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-250, and available through the NCBI website. The default parameters were used, except the filters were turned OFF.
  • operably associated or “operably linked” , as used herein, refer to functionally coupled nucleic acid or amino acid sequences.
  • Recombinant is relating to, derived from, or containing genetically engineered material .
  • the genome was intentionally manipulated by the hand- of-man in some way.
  • Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, by knock-out, by adding stop codon s, by frame shift mutation, and the like.
  • null or “knockout” what is meant is that the mutation produces undetectable active protein.
  • a gene can be completely (100%) reduced by knockout or removal of part of all of the gene sequence.
  • Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can al so completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein. All null mutants herein are signified by ⁇ .
  • “Overexpression " or "overex pressed” is defined herein to be at least 150%) of protein activity as compared with an appropriate control species, or any expression in a species that lacks the activ ity altogether. Preferably, the activity is increased 100-500%. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resi stant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. All overexpressed genes or proteins are signified herein by "+”.
  • endogenous or “native” means that a gene originated from the species in question, without regard to subspecies or strain, although that gene may be naturally or intentionally mutated, or placed under the control of a promoter that results in overexpression or controlled expression of said gene.
  • genes from Clostridia would not be endogenous to Escherichia, but a plasmid expressing a gene from E. coli or would be considered to be endogenous to any genus of Escherichia, even though it may now be ov erexpressed.
  • Expression vectors are used in accordance with the art accepted definition of a plasmid, virus or other propagatable sequence designed for protein expression in cells. There are thousands of such vectors commercially available, and typically each has an origin of replication (ori); a multiple cloning site; a selectable marker; ribosome binding sites; a promoter and often enhancers; and the needed termination sequences. Most expression vectors are inducible, although constitutive expression vectors also exist.
  • inducible means that gene expression can be controlled by the hand-of-man, by adding e.g., a ligand to induce expression from an inducible promoter.
  • exemplary inducible promoters include the lac operon inducible by IPTG, the yeast AOXl promoter inducible w ith methanol, the strong LAC4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesi s may still occur even in expression vectors with tightly controlled promoters.
  • an "integrated sequence” means the sequence has been integrated into the host genome, as opposed to being maintained on an expression vector. It will still be expressible, and preferably is inducible as well .
  • FIG. 1 Current 'top-down' metabolic engineering approach based on editing existing architecture of natural metabolism .
  • FIG. 2 Single carbon manipulation reactions for the generation of formyl-CoA. Representative enzymes for each reaction are given in the legend.
  • FIG. 3 A pathway for the assimilation of single carbon molecules into products containing at least one terminal alcohol . Referred to as Scheme 3 A . Representative enzymes for each reaction are given in the legend.
  • FIG. 4 A pathway for assimilation of single carbon molecules into products containing at least one terminal alcohol . Referred to as Scheme 4B. Representative enzymes for each reaction are giv en in the legend.
  • FIG. 5 A pathway for the assimilation of single carbon molecules into products containing at least one carboxyl group. Referred to as Scheme 5 A. Representative enzymes for each reaction are given in the legend.
  • FIG. 6 A pathway for assimilation of single carbon molecules into products containing at least one carboxyl group. Referred to as Scheme 6B. Representative enzymes for each reaction are giv en in the legend.
  • FIG. 7 A pathway for the assimilation of single carbon molecules into products containing at least one terminal amine. Referred to as Scheme 7 A. Representative enzymes for each reaction are given in the legend.
  • FIG. 8 A pathway for assimilation of single carbon molecules into products containing at least one terminal amine.
  • Scheme 8B Representativ e enzymes for each reaction are giv en in the legend.
  • 100411 FIG. 9 A pathway for the assimilation of single carbon molecules into products containing at least one terminal alkyl group.
  • Scheme A Representative enzymes for each reaction are given in the legend.
  • FIG. 10 A pathway for assimilation of single carbon molecules into products containing at least one terminal alkyl group. Referred to as Scheme 10B. Representative enzymes for each reaction are given in the legend.
  • FIG. 1 A method for the production of products from solely single carbon molecules via Scheme 1 1 A ( FIG. 3).
  • FIG. 12 A method for the production of products from single carbon molecules and a supplied priming molecule (Scheme 12AB).
  • FIG. 13 A method for the production of products from single carbon molecules and an additionally supplied, unrelated multi-carbon substrate ( Scheme 3AB).
  • FIG. 14 Examples of functional groups ( R- groups) for the described products and primers. Groups can also be used in combinations. Not a comprehensive list.
  • FIG. 15. Vector construct containing the gene encoding N-terminal
  • FIG. 6 SDS-PAGE showing expression and purification of Lmo l 1 79 from / ⁇ , " . coli.
  • FIG. 17. Time course of absorbance at 340 nm corresponding to the production of NADU in the assay of Lmol 179.
  • FIG. 19A S. cerevisiae and FIG. 19B E. coli expression vector constructs containing the gene encoding an amino-terminal 6X HIS-tagged hydroxyl-acyl- CoA lyase HACL 1 from Homo sapiens.
  • FIG. 20 SDS-PAGE showing the purification of hydroxy 1-acyl-Co A lyase HACL 1 from S. cerevisiae. Lane 1 corresponds to the protein ladder. Lane 2 is the S. cerevisiae cell extract. Lane 3 corresponds to purified HACL 1 .
  • FIG. 21 GC-FID chromatograms of pentadecanal content in HACLl degradative reaction mixtures after extraction with hexane. Top: pentadecanal standard; Middle: HACLl assay sampled; Bottom: no enzyme control. In samples containing HACLl, a pentadecanal peak is seen, while there is no peak in the sample in which enzyme was omitted.
  • FIG. 22 GC-FID chromatograms of HACLl synthetic reaction mixtures after hydrolysis of acyl-CoAs and denvatization. From top to bottom : no enzyme control; HACLl sample; 2-hydroxyhexadecanoyl-CoA standard; 2-hydroxyhexanoic acid standard. HACLl was incubated with pentadecanal and formyl-CoA and was capable of li gating the molecules to 2-hydroxyhexadecanoyl-CoA as indicated by the peaks corresponding to the standards.
  • FIG. 18 and FIG. 19 prove that the enzyme can run in reverse, as required for the synthetic pathway.
  • FIG. 23 HPLC chromatogram of HACL 1 synthetic reaction samples incubated with acetal delu de and formyl-CoA. Solid line: HACLl sample; Dashed line: no enzyme control .
  • the expected product of the ligation of acetal dehyde and formyl-CoA is lactoyl-CoA, which would be expected to be hydrolyzed to its acid form, lactate.
  • a peak for lactate (26 min) was observed in samples containing purified HACLl and was not observed in samples that did not contain enzyme.
  • FIG. 24 HPLC chromatogram of HACLl synthetic reaction samples incubated with formaldehyde and formyl-CoA. Solid line: no enzyme control; Dashed line: HACL 1 sample; Dotted line: glycol ate standard. The expected product of the ligation of formaldehyde and formyl-CoA i s glycolyl-CoA, which would be expected to be hydrolyzed to its acid form, glycolate. A peak matching a glycolate standard (25.2 min) was observed in samples containing purified HACL 1 and was not observed in samples that did not contain enzyme.
  • FIG. 25 NMR spectra of HACL 1 assay samples incubated with acetal dehyde and formyl-CoA. (A) HACL 1 sample. (B) No enzyme control . A peak corresponding to lactate was identified in the sample containing HACL 1 .
  • FIG. 26A-B V ector constructs encoding oxaly -CoA decarboxylases from FIG. 20 A Oxalohacter formigenes and FIG. 20 B E. coli.
  • FIG. 27 Vector construct encoding benzaldehyde lyase from
  • FIG. 28 Vector construct containing the gene encoding LcdABC from
  • LcdABC is an example of a 2- hydroxyacyl-CoA dehydratase.
  • FIG. 29 Eadie-Hofstee plot for the determination of Euglena gracilis
  • EgTER enzyme kinetics.
  • EgTER is an example of transenoyl-CoA reductase.
  • FIG. 30 Time course of absorbance at 340 nm corresponding to the consumption of N ADU in the assay of MhpF.
  • FIG. 31 Time course of absorbance at 340 nm corresponding to the consumption of NADH in the assay of FucO.
  • FIG. 32 HPLC chromatogram of the in vitro assembly of the transfer) oyl -Co A reductase and acyl-CoA reductase steps of Scheme B.
  • a peak corresponding to butyraldehyde was present in samples containing Treponema denticola TER (TdTER) and ./ ⁇ , " . coli MhpF (solid, thin line), but not the control containing TdTER only (dashed line).
  • FIG. 33 Production of the 1 ,2-diol ethylene glycol in vivo using an engineered strain of E. coli.
  • FIG. 34 Production of 2-hydroxy acids and 1 ,2-diol s in the forms of gly colic acid and ethylene glycol using an assembled reaction mixture in vitro.
  • FIG. 35 Production of the 2-hydroxy acid gly colic acid in vivo using an engineered strain of E. coli.
  • FIG. 36 Production of 2-hydroxyacids glycolic acid and lactic acid using an assembled reaction mixture in vitro, demonstrating products of different carbon chain length.
  • FIG. 37 Assay of purified diol dehydratase KoPddABC coupled with aldehyde dehydrogenase AldB.
  • the sample containing KoPddABC blue
  • the sample without KoPddABC had no noticeable change in absorbance.
  • FIG. 38. (A) SDS/'PAGE gel showing expression and purification of histidine tagged CV2025.
  • FIG. 39 Alkyl compound synthesis from aldehyde intermediates through the use of aldehyde decarboxylase ORF 1 593 from Synechococcus elongatus PCC7942.
  • A Purification of n-terminal His-tagged ORF 1 593 from / ⁇ dress ' . coli. Lane 1 : Protein ladder; Lane 2: Purified protein after dialysis; Lane 3 : Purified protein; Lane 4: Crude extract of MG I 655 expressing ORF 1 593 from pUCBB-pTrc-ntH6_orf 1 593.
  • B GC-FID chromatogram demonstrating undecane production from dodecanal with purified ORF 1 593. Black: full reaction; Green: No enzyme control; Red: No substrate control; Blue: No PMS/NADH control .
  • Table 2 Summary of enzymes that have been characterized in vitro.
  • the first function of the pathway is to produce formyl-CoA. and is illustrated in FIG. 2.
  • Single carbon molecules of various reduction lev els are intercon verted by the illustrated reactions to produce formyl-CoA, the single carbon unit used to extend a carbon backbone.
  • Detail s regarding the reactions and exemplary enzymes that accompli sh the first function can be found in TABLE 1.
  • Methane can be oxidized to methanol ( FIG. 2, reaction 1 ) by a suitable methane monooxygenase.
  • Methanol can be oxidized to formaldehyde ( FIG. 2, reaction 2) by a suitable methanol dehydrogenase.
  • Formaldehyde can be oxidized to form ⁇ -Co A ( FIG.
  • Formate can be converted to formyl-CoA either directly ( FIG. 2, reaction 7) by a suitable acetyl -Co A synthetase or through the intermediate formyl- phosphate ( FIG. 2, reaction 5-6) by a suitable formate kinase and phosphate acetyl- transferase.
  • the provision of formyl-CoA can be accomplished from either formaldehyde, by the expression of an acylating aldehyde dehydrogenase, or from formate, by a suitable acetyl -Co A synthetase or combined formate kinsane and phosphate acetyl-transferase.
  • Combinations of the above reactions can be used to generate formyl-CoA from other single carbon molecules.
  • an implementation that makes use of methane would include the expression of a methane monooxygenase, a methanol dehydrogenase, and an acylating aldehyde dehydrogenase.
  • Even more combinations of the described reactions and accompanying enzymes can be used to allow for implementations that use a mixture of single carbon units, for example a combination of methane and carbon dioxide through all of the described reactions.
  • the second function of the pathway is the iterative elongation of a carbon backbone by the single carbon unit formyl-CoA, known as an "extender unit” herein. This is illustrated in FIG. 3- 10. Details regarding the reactions and exemplary enzymes that accomplish the second function can be found in TABLE 1.
  • formyl-CoA i s condensed with an aldehyde to give a 2 - h y d rox y a cy 1 -C o A that is one carbon longer than the initial aldehyde (e.g., FIG. 3, reaction 1) by a suitable 2-hydroxyacy -CoA lyase.
  • the 2- hy droxy acyl -Co A is then reduced to a 2 -hydroxy aldehyde (e.g., FIG. 3, reaction 2) by a suitable acyl-CoA reductase.
  • the 2-hy droxy al dehy de is further reduced to a 1 ,2-diol (e.g., FIG. 3, reaction 3) by a suitable 1 ,2-diol oxidoreductase.
  • the diol can be dehydrated to give an aldehyde (e.g., FIG. 3, reaction 4) by a suitable diol dehydratase, which is capable of further elongation .
  • formyl-CoA is condensed with an aldehyde to give a 2-hydroxyacyl-CoA that is one carbon longer than the initial aldehyde (e.g., FIG.
  • reaction 1 by a suitable 2-hydroxyacyl-CoA lyase.
  • the 2- hydroxyacyl-CoA is then dehydrated to give a tran s-2-enoy 1 -C o A (e.g., FIG. 4, reaction 2) by a suitable 2-hydroxyacyl-CoA dehydratase.
  • the trans-2-enoyl-CoA is then reduced to an acyl- CoA (e.g., FIG. 4, reaction 3) by a suitable trans-2-enoyl-CoA reductase.
  • the acyl- CoA can be reduced to give an aldehyde (e.g., FIG. 4, reaction 4) by an acyl-CoA reductase, which is capable of further elongation.
  • the final function of the pathway is the formation of the products that contain at least one terminal alcohol, carboxyl, amine or alkyl .
  • Detail s regarding the reactions and exemplary enzymes that accomplish the third function can be found in TABLE 1.
  • terminal alcohols can be derived from the aldehyde intermediate by reduction of the aldehyde group to an alcohol group ( FIG. 3-4, reaction 5) by a suitable alcohol dehydrogenase.
  • the 1 ,2-diol also already contains a terminal alcohol group, and is thus a representative product.
  • terminal alcohol s can be derived from the aldehyde intermediate by reduction of the aldehyde group to an alcohol group ( FIG. 3-4, reaction 5) by a suitable alcohol dehydrogenase.
  • the acyl-CoA. intermediates can also be reduced to a terminal alcohol ( FIG. 4, reaction 6) by a suitable alcohol-forming coenzyme- A.
  • a carboxyl group can be derived from the aldehyde intermediates by oxidation of the aldehyde group to a carboxyl group (FIG. 5/6, reaction 5) by a suitable aldehyde dehydrogenase.
  • a 2-hydroxycarboxylic acid can be obtained by oxidation of the 2-hydroxy aldehyde.
  • a carboxyl group can be derived from the aldehyde intermediate by oxidation of the aldehyde group ( FIG. 5/6, reaction 5) by a suitable aldehyde dehydrogenase.
  • the acyl-CoA. intermediates can al so be converted to a carboxylic acid (FIG.
  • terminal amines can be derived from the aldehyde intermediates by the transfer of an amine group ( FIG. 7/8, reaction 5) by a suitable transaminase.
  • terminal amines can be derived from the aldehyde intermediate by the transfer of an amine group ( FIG. 7/8, reaction 5) by a suitable transaminase.
  • the acyl- Co A intermediates can also be reduced to aldehydes ( FIG. 8, reaction 4) by a suitable acyl- CoA reductase, where, in combination with a suitable transaminase, results in 2- hydroxyamines from 2-hydroxyacyl-CoA or unsaturated amines from tran s-2-enoy 1 -Co A .
  • terminal alkyl groups can be derived from the aldehyde intermediates decarbonylation ( FIG. 9/10, reaction 5) by a suitable aldehyde decarbonylase.
  • terminal alkyl s can be derived from the aldehyde intermediate by decarbonylation ( FIG. 9/10, reaction 5) by a suitable aldehyde decarbonylase.
  • the acy -CoA intermediates can also be reduced to aldehydes ( FIG. 10, reaction 4) by a suitable acyl-CoA reductase, followed by decarbonylation resulting in a 2-hydroxy alkyl from a 2-hydroxyacyl-CoA or an alkene from trans-2-enoy -CoA.
  • the described pathway is provided within the context of a microbial host.
  • the pathway in a living system is generally created by transforming the microbe with one or more expression vector(s) containing a gene encoding one or more of the needed enzymes, but the genes can also be added to the chromosome by recombineering, homologous recombination, and similar techniques.
  • the needed protein is endogenous, as is the case in some instances, it may suffice as is, but i s usual ly overexpressed for better functionality and control over the level of active enzyme.
  • one or more, or all, such genes are under the control of an inducible promoter.
  • yeasts are a common species used for microbial manufacturing, and many species can be successfully transformed.
  • the alpha oxidation pathway is present in yeast and the alpha-oxidation enzyme hy drox yl-acyl -C o A lyases (HACL), which is a key part of this inv ention was successfully expressed in yeast Saccharomyces.
  • Other species include but are not limited to Candida, Aspergillus, Arxida adeninivorans, Candida boidinii, Hansenida polymorpha (Pichia angnsta), Kluyveromyces lactis, Pichia pastoris, and Yarrow ia lipolytica, to name a few.
  • microalga Pavlova lutheri is already being used as a source of economically valuable docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), and Crypthecodinium cohnii i s the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.
  • DHA docosahexaenoic
  • EPA eicosapentaenoic acids
  • Crypthecodinium cohnii i the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.
  • a number of databases include vector information and/or a repository of vectors and can be used to choose expression v ectors suitable for the chosen host species. See e.g., AddGene.org, which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues.
  • PlasmID PI asm id Information Database
  • DNASU having over 191,000 plasmids.
  • a collection of cloning vectors of E. coli is also kept at the National Institute of Genetics as a resource for the biological research community. Furthermore, vectors (including particular ORFS therein) are usually available from colleagues.
  • the enzymes can be added to the genome or via expression vectors, as desired. Preferably, multiple enzymes are expressed in one vector or multiple enzymes can be combined into one operon by adding the needed signals between coding regions. Further improvements can be had by overexpressing one or more, or even all of the enzymes, e.g., by adding extra copies to the cell via pi asm id or other vector.
  • Initial experiments may employ expression plasmids hosting 3 or more ORFs for convenience, but it may be preferred to insert operons or individual genes into the genome for stability reasons.
  • culturing of the developed strains can be performed to evaluate the effectiv eness of the pathway at its intended goal—the production of products from single carbon compounds.
  • the organism can be cultured in a suitable grow th medium, and can be evaluated for product formation on single carbon substrates, from methane to O:, either alone or in combination with multi-carbon molecules.
  • the products produced by the organism can be measured by HP I or GC, and indicators of performance such as growth rate, productivity, titer, yield, or carbon efficiency can be determined.
  • a cell free in vitro version of the pathway can be constructed.
  • the overall pathway can be assembled by combining the necessary enzymes in a reaction mixture.
  • the pathway can be assessed for its performance independently of a host.
  • single carbon molecules such as carbon dioxide, formate, formaldehyde, methanol, methane, and carbon monoxide are solely used in the production of products containing at least one terminal alcohol, carboxyl, amine, alkyl or derivatives thereof.
  • formyl-CoA is produced from single carbon molecules as usual, but formaldehyde, a one carbon aldehyde, serves as the initial aldehyde that is elongated. This initial aldehyde will be referred to as the "primer " or "priming aldehyde.
  • formaldehyde is already provided when molecules at the same reduction level or more reduced (formaldehyde, methanol, methane) are used. From molecules that are more oxidized, such as formate and carbon dioxide, some of the produced formyl-CoA. can be reduced to give formaldehyde (reverse of the reaction shown). When using formaldehyde as the primer. Scheme A must be used for elongation, because Scheme B requires a third carbon to proceed with further rounds of elongation.
  • the priming aldehyde can be provided along with single carbon molecules for elongation. This is illustrated in FIG. 12.
  • the product containing at least one terminal alcohol can be made to also contain other interesting functional groups.
  • a ⁇ -phenyl aldehyde can serve as the priming aldehyde, which i s converted to an elongated ⁇ -phenyl alcohol through the described invention.
  • Some exemplary functional groups are given in FIG. 14. In this embodiment, it is not necessary that the priming aldehyde is exogenously added.
  • the 2-hy droxy al dehy de intermediate in Scheme A can serve as a priming aldehyde for further rounds of elongation to giv e polyols.
  • the trans-2-enoyl-CoA. can be converted to a trans-2-enaldehyde and used for further rounds of elongation to give polyunsaturated products.
  • the priming aldehyde can be derived from provided unrelated multi-carbon molecules or substrates, which allow for product formation along with provided single carbon molecules. This is illustrated in FIG. 13. These unrelated multi-carbon molecules, i .e. multi-carbon molecules that do not contain at least one aldehyde group, must first be converted to a suitable priming aldehyde.
  • the pathway will be implemented in an engineered microbial host that i s engineered to be able to convert the multi-carbon molecules into a priming aldehyde.
  • Some exemplary substrates include glucose and other sugars or glycerol and other sugar alcohols, which may be converted to priming aldehydes via a pathway such as glycolysis, resulting in the production of acetaldehyde or succinic semialdehyde. This embodiment can further increase the diversity of products produced by the pathway.
  • the unrelated multi- carbon substrates may provide additional carbon and energy for microbial survival .
  • Enzymes of interest are expressed from vectors such as pCDFDuet- 1
  • the genes can be amplified by PCR using primers designed with 15-25 base pairs of homology for the appropriate vector cut site.
  • pCDFDuet- 1 can be linearized with Ncol and EcoRI. Enzymes that wi ll be purified by Ni-NTA column w il l make use of the 6X-HIS tag in pCDFDuet- 1 .
  • the vector can be linearized using only EcoRI in this case.
  • the PCR product can be inserted into the vector using e.g., the In-
  • Transformants can be selected on solid media containing the appropriate antibiotic.
  • Plasmid DNA can be isolated using any suitable method, including QIAprep Spin Miniprep Kit (QIAGEN, Limburg), and the construct confirmed by PCR and sequencing.
  • Confirmed constructs can be transformed by e.g., electroporation into a host strain such as E. coli for expression, but other host species can be used with suitable expression vectors and possible codon optimization for that host species.
  • Expression of the desired enzymes from the constructed strain can be conducted in liquid culture, e.g., shaking flasks, bioreactors, chemostats, fermentation tanks and the like.
  • Gene expression is typically induced by the addition of a suitable inducer, when the culture reaches an OD550 of approximately 0.5-0.8. Induced cells can be grown for about 4-8 hours, at which point the cells can be pelleted and saved to -20°C. Expression of the desired protein can be con finned by running cell pellet samples on SDS-PAGE.
  • the expressed enzyme can be directly assayed in crude cell lysates, simply by breaking the cells by chemical, enzymatic, heat or mechanical means. Depending on the expression level and activity of the enzyme, however, purification may be required to be able to measure enzyme activity over background levels. Purified enzymes can also allow for the in vitro assembly of the pathway, allowing for its controlled characterization . N- terminal or C-terminal HIS-tagged proteins can be purified using e.g., a Ni-NTA Spin Kit (Qiagen, Venlo, Limburg) following the manufacturer's protocol, or other methods could be used. The H I S-tag system was chosen for convenience only, and other tags are available for purification uses. Further, the proteins in the final assembled pathway need not be tagged if they are for in vivo use. Tagging was conv enient, however, for the enzyme characterization work performed herein.
  • reaction conditions for enzyme assays can vary greatly with the type of enzyme to be tested. In general, howev er, enzyme assays follow a similar general protocol . Purified enzyme or crude lysate is added to suitable reaction buffer. Reaction buffers typically contain salts, necessary enzyme cofactors, and are at the proper pH. Buffer compositions often change depending on the enzyme or reaction type. The reaction is initiated by the addition of substrate, and some aspect of the reaction related either to the consumption of a substrate or the production of a product is monitored.
  • Spectrophotometri c assays are convenient because they allow for the real time determination of enzyme activ ity by measuring the concentration dependent absorbance of a compound at a certain wavelength.
  • GC Gas chromatography
  • Internal standards typically one or more molecules of similar type not involved in the reaction, is added to the reaction mixture, and the reaction mixture i s extracted with an organic solvent, such as hexane.
  • Fatty acid samples for example, can be dried under a stream of nitrogen and converted to their tri methyl si lyl derivatives using BSTFA and pyridine in a 1 : 1 ratio. After 30 minutes incubation, the samples are once again dried and re-suspended in hexane to be applied to the GC. Aldehyde samples do not need to be derivatized. Samples can be run e.g., on a Varian CP-3800 gas chromatograph (VARIAN ASSOCIATES, INC., Palo Alto, CA) equipped with a flame ionization detector and 1IP-5 capillary column (AGILENT TECHNOLOGIES, CA).
  • VARIAN ASSOCIATES INC., Palo Alto, CA
  • the pathway can be constructed in vivo with greater confidence.
  • the strain construction for the in vivo pathway operation should allow for the ell-defined, controlled expression of the enzymes of the pathway.
  • E. coli, B. subtilus or yeast will be a host of choice for the in vivo pathway, but other hosts could be used.
  • the Duet system (MERCK KGaA, Germany ), allows for the simultaneous expression of up to eight proteins by induction with IPTG in E. coli, and initial experiments will use this host.
  • Pathway enzymes can al so be inserted into the host chromosome, allowing for the maintenance of the pathway without requiring antibiotics to ensure the continued upkeep of pi asm ids.
  • genes that can be placed on the chromosome as chromosomal expression does not require separate origins of replication as is the case with pi asm id expression.
  • DNA constructs for chromosomal integration usual ly include an antibiotic resistance marker with flanking FRT sites for removal, as described by Datsenko and Wanner, a well characterized promoter, a ribosome binding site, the gene of interest, and a transcriptional terminator.
  • the overal 1 product i s a linear DNA fragment with 50 base pairs of homology for the target site on the chromosome flanking each side of the construct.
  • the Flp-FRT recombination method i s only one system for adding genes to a chromosome, and other systems are available, such as the RecBCD pathway, the RecF pathway, RecA recombinase, non-homologous end joining (NHEJ), Cre- Lox recombination, TYR recombinases and integrases, SER resolvases/invertases, SER integrases, P C31 Integrase, and the like.
  • Chromosomal modifications in E. coli can also achieved by the method of recombineering, as originally described by Datsenko and Wanner, or using new gene editing tools, such as CRISPR/CAS.
  • the cells are prepared for electroporation following standard techniques, and the cells transformed with linear DNA that contains flanking 50 base pair targeting homology for the desired modification site.
  • a two-step approach can be taken using a cassette that contains both positive and negative selection markers, such as the combination of cat and sacB.
  • the cat-sacB cassette with targeting homology for the desired modification site is introduced to the cells.
  • the cat gene provides resistance to chloramphenicol, which allows for positive recombinants to be selected for on solid media containing chloramphenicol.
  • a positive isolate can be subjected to a second round of recombineering introducing the desired DNA construct with targeting homology for sites that correspond to the removal of the cat-sac B cassette.
  • the sacB gene encodes for an enzyme that provides sensitivity to sucrose.
  • P 1 phage ly sates can be made from isolates confirmed by PGR and sequencing. The lysates can be used to transduce the modification into desired strains, as described prev iously.
  • Engineered strains expressing the designed pathway can be cultured under the following or similar conditions.
  • Overnight cultures started from a single colony can be used to inoculate flasks containing appropriate media. Cultures are grown for a set period of time, and the culture media analyzed. The conditions will be highly dependent on the specifications of the actual pathway and what exactly is to be tested. For example, the ability for the pathway to be used for autotrophic grow th can be tested by the use of formate or formaldehyde as a substrate in MOPS minimal media, as described by Neidhardt, supplemented with appropriate antibiotics, and inducers. Mixotrophic growth can be characterized by the addition of both single carbon compounds and glucose or glycerol .
  • Genome scale modeling allows for the identification of additional modifications to the host strain that might lead to improved performance. Deletion of competing pathways, for example, might increase carbon flux through the engineered pathway for product production.
  • Plasmids also referred to as vectors in each case contain at least one promoter, a ribosome binding site for each gene, the gene(s) of interest, at least one terminator, an origin of replication, and an antibiotic resistance marker. Exemplary plasmids are shown in FIG. 15, 19, and 16-28.
  • hysteria monocytogenes Lmo l 1 79 was cloned (FIG. 15), expressed, and purified (FIG. 16) in E. coli as described above.
  • the purified enzyme was evaluated for its ability to convert formaldehyde into the extender unit formyl-CoA. Enzyme assays were performed in 23 niM potassium phosphate buffer pH 7.0, 1 inM CoASH, 0.5 mM N AD " , 20 mM 2-mercaptoethanol, and 50 mM formaldehyde. The reaction was monitored by measuring absorbance at 340 nm, corresponding to the production of NADU.
  • Co A compounds were extracted from the reaction mixture by solid phase extraction (SPE) using a CI 8 column, and the mass of the extracted Co As were determined by ESI -TOP MS.
  • a pi asm id containing the codon optimized gene encoding human HIS- tagged HACL 1 was constructed as described.
  • the resulting construct, FIG. 19, was transformed into S. cerevisiae InvSC 1 (Life Tech. ).
  • the resulting strain was cultured in 50 mL of SC-URA media containing 2% glucose at 30°C for 24 hours.
  • the cells were pelleted and the required amount of cell s were used to inoculate a 250 mL culture volume of SC-URA media containing 0.2% galactose, 1 mM MgCl 2 , and 0.1 mM thiamine to 0.4 OD600. After 20 hours incubation with shaking at 30°C, the cells were pelleted and saved.
  • a pi asm id containing the codon optimized gene encoding 6X HIS-tagged HACL 1 from Homo sapiens was constructed as described above.
  • the resulting construct was transformed into E. coli BL21(DE3) for expression.
  • the resulting strain was cultured in LB media containing 50 ug/mL spectinomycin. When the culture reached an OD550 of approximately 0.6, expression was induced by addition of 0.4 mM IPTG. Cells were harvested by centrifugation after overnight incubation at room temperature. [001221 When needed, the cell pellets from S.
  • OD600 OD600 of approximately 100 in a buffer containing 50 mM potassium phosphate pH 7.4, 0.1 mM thiamine pyrophosphate, 1 mM MgC , 0.5 mM AEBSF, 10 inM imidazole, and 250 units of Benzonase nuclease.
  • To the cell suspension approximately equal volumes of 425-600 ⁇ glass beads were added. Cells were broken in four cycles of 30 seconds of vortexing at 3000 rpm followed by 30 seconds on ice. The glass beads and cell debris were pelleted by centrifugation and supernatant containing the cell extract was collected.
  • human HACL 1 was purified and tested for its native catabolic activity by assessing its ability to cleave 2-hydroxyhexadecanoyl-CoA to pentadecanal and formyl-CoA.
  • Enzyme assays were performed in 50 mM tris-HCl pH 7.5, 0.8 mM MgCL, 0.02 mM TPP, 6.6 ⁇ BSA, and 0.3 mM 2-hydroxyhexadecanoyl-CoA .
  • the assay mixtures were incubated for one hour at 37°C, after which the presence of pentadecanal was assessed by extraction with hexane and analysis by GC-FID.
  • pentadecanal was produced in the sample containing HACL 1 , but not in the control sample, which did not contain HACL 1 , indicating that the protein was expressed and purified in an active form.
  • HACL 1 The ability of purified HACL 1 to run in the anabolic direction (reverse from the physiological direction ) was also determined.
  • An aldehyde and formyl-CoA were tested for ligation in a buffer comprised of 60 mM potassium phosphate pH 5.4, 2.5 mM MgCl 2 , 0.1 mM TPP, 6.6 ⁇ BSA, 5 mM aldehyde, 20% DMSO, approximately 1 mM freshly prepared formyl-CoA, and approximately 0.5 nig/mL purified HACL 1 .
  • the reaction was allowed to take place at room temperature for 16 hours, after which acy -CoAs were hydrolyzed to their corresponding acids by adjusting to pH > 12.0.
  • samples were analyzed by I I PLC.
  • longer products for example the production of 2-hydroxyhexadecanoic acid from pentadecanal
  • samples were acidified with HC1 and extracted with diethyl ether.
  • the extracted diethyl ether was evaporated to dryness under a stream of nitrogen and derivatized by the addition of 1 : 1 BSTFA:pyridine. After incubation at 70°C for 30 min, these samples were analyzed by GC-FID.
  • HACL 1 was shown to catalyze the ligation of these molecules to 2-hydroxyhexadecanoyl-CoA as hypothesized.
  • the chromatogram of the sample containing enzyme shows similar peaks to the 2- hydroxyhexadecanoyl-CoA spiked standard, which are absent from the no-enzyme control .
  • HACL 1 is capable of catalyzing the ligation of aldehydes with chain lengths ranging at least from C 1 - C 1 5 with formyl-CoA, making it suitable for the engineered iterative pathway.
  • 2-hydroxyhexadecanoyl-CoA was prepared by the n- hydroxy succi ni m i de method (Blecher, 1981).
  • the n-hydroxysuccinimide ester of 2-hydroxyhexadecanoic acid is prepared by reacting n-hydroxysuccinimide with the acid in the presence of dicyclohexylcarbodiimide.
  • the product was filtered and puri ied by recrystallization from methanol to give pure n-hydroxysuccinimide ester of 2- hydroxyhexadecanoic acid.
  • the ester was reacted with CoA-SH in presence of thioglycolic acid to give 2-hydroxyhexadecanoyl-CoA .
  • the 2-hydroxyhexadecanoyl-CoA was purified by precipitation using perchloric acid, filtration, and washing the filtrate with perchloric acid, diethyl ether, and acetone.
  • Formyl-CoA was prepared by first forming formic ethylcarbonic anhydride as previously described (Parasaran & Tarbell, 1964). Briefly, formic acid (0.4 mmol ) and ethyl chloroformate (0.4 mmol ) were combined in 4 ml., anhydrous diethyl ether and cooled to -20°C. 0.4 mmol tri ethyl amine was added to the mixture and the reaction was allowed to proceed at -2Q°C for 30 minutes. The reaction mixture was filtered over glass wool to give a solution containing formic ethyl carbonic anhydride in diethyl ether.
  • Lmo l 1 79 an acyl- CoA reductase from hysteria monocytogenes, HACL1 , a 2-hydroxyacyl-CoA lyase from Homo sapiens, and FucO, a 1 ,2-diol oxidoreductase from Escherichia coli, were cloned, expressed, and purified as described above.
  • a reaction mixture was assembled comprised of 60 mM potassium phosphate pH 7.4, 2.5 mM MgCl 2 , 0.1 mM TPP, 2.5 mM CoASH, 5 mM NAD " , 50 mM formaldehyde, 0.5 mM DTT, 0.1 g/L HACLl, 0.5 g/L Lmol 179, and 0.5 g/L FucO. After overnight incubation at room temperature, the reaction was terminated by addition of 1% sulfuric acid. Precipitant was pelleted by centrifugation and the supernatant was analyzed by HPLC.
  • the pathway is assembled in vivo using E. coli as the host organism.
  • Lmo l 1 79, HACL 1 , and FucO were cloned as described above to give the plasmids pCDFDuet- 1 -P 1 -ntH6-HACL 1 and pETDuet- 1 -P 1 -Lmo 1 1 79-FucO.
  • coli MG1655(DE3) was engineered with knockouts of the genes glcD, frmA , fdhF, fdnG, fdoG using standard methods described above.
  • This MG1655(DE3) AglcD AfrmA AfdhF fdnG AfdoG is hereby referred to as AC440.
  • AC440 was transformed with the plasmids pCDFDuet- 1 -P 1 -ntH6-FIACL 1 and pETDuet- 1 -P 1 -Lmo 1 1 79-FucO using standard methods described above to give the strain AC440 pCDFDuet- 1 -P I -ntH6-H ACL 1 -P2-AMA pETDuet- 1-P l-Lmol 179, hereby referred to as AC529.
  • MOPS medium Nidhardt et al ., 1974 with 125 mM MOPS
  • MOPS-LB-glycerol 1 5 uM thiamine
  • MOPS medium Na 2 HP0 4 in place of K 2 HP0 4 (2.8 mM), 5 mM ( H 4 ) 2 S0 4 , 30 mM NH 4 C1 and 15 uM thiamine, hereby referred to as MOPS medium with no carbon source.
  • the cells were resuspended to 25 OD in the MOPS medium with no carbon source, to which 50 mM formaldehyde was added. After 24 hours incubation at room temperature, cells were pelleted and the supernatant was analyzed by HPLC.
  • KoPddABC a diol dehydratase from Klebsiella oxytoca, and YqhD, an alcohol dehydrogenase from E. coli, were cloned, expressed, and purified.
  • Cell extracts of E. coli expressing KoPddABC were prepared by resuspending a pellet of said E. coli to an OD 550 of 40 in 60 mM potassium phosphate buffer pFI 7.4 with 200 mM 1 ,2-ethanediol . 1 ml., of the cell suspension was added to 0.75 g of glass beads and the cells were disrupted for 3 minutes using a cell disruptor (Scientific Industries, Bohemia, NY, USA).
  • the cell debris and glass beads were pelleted by centrifugation and the supernatant comprising the cell extract was used for assays.
  • the cell extract was incubated at 30°C for 3 hours in the presence of 10 ⁇ coenzyme B 12.
  • the reaction w as terminated by the addition of 1% sulfuric acid, and the precipitant was pelleted by centrifugation.
  • the supernatant was analyzed by HPLC.
  • Acetaldehyde was detected in extracts of cells expressing KoPddABC, indicating that the dehydratase can convert 1 ,2-diols to their corresponding aldehydes.
  • / ⁇ , ' . coli YqhD and FucO were expressed from A SKA collection strains and the cell extracts were assayed for activity for acetaldehyde reduction to ethanol by monitoring NAD(P)H oxidation at 340 nni. Both cell extracts of YqhD and FucO were capable of reducing acetaldehyde with specific activities of 0.1 10 ⁇ 0.001 ⁇ /mg/min and 0.381 ⁇ 0.000 ⁇ /mg/min, respectively.
  • FucO was also tested for its ability to reduce longer chain aldehydes propionaldehyde and butyraldehyde to 1 -propanol and 1-butanol, resulting in specific activities of 3.35 ⁇ 0.07 ⁇ /rng/min and 3.864 ⁇ 0.008 ⁇ /rng/min, respectively. These results demonstrate that normal alcohols can be produced via this reaction pathway.
  • the purpose of this example is to demonstrate the synthesis of products containing a carboxylic acid group from one carbon molecules using the pathway referred to as Scheme 5 A .
  • 2-hydroxyacids are obtained by prov iding a suitable aldehyde dehydrogenase.
  • the pathway is assembled in vitro. Lmol l79, HACLl, and AldA, an aldehyde dehydrogenase from E. coli, were cloned, expressed, and purified as described above.
  • a reaction mixture was assembled comprised of 60 mM potassium phosphate pH 7.4, 2.5 mM MgCl 2 , 0.1 mM TPP, 2.5 mM CoASH, 15 mM NAD ⁇ , 50 mM formaldehyde, 0.5 mM DTT, 0.2 g/L HACL l , 0.5 g/L Lmo l 1 79, 0.5 g/L AldA . After overnight incubation, the reaction was terminated by addition of 1% sulfuric acid. Precipitant was pelleted by centrifugation and the supernatant was analyzed by HPLC. Glycolic acid (2- hydroxyacetic acid) was detected at concentration of 4.6 mM (0.35 g/L ), demonstrating 2- hydroxy acid production from the pathway ( FIG. 34).
  • a reaction mixture was assembled comprised of 60 mM potassium phosphate pH 7.4, 2.5 mM MgCL, 0.1 mM TPP, 2.5 mM CoASH, 12.5 mM AT P, 5 mM NADH, 5 mM acetaldehyde, 50 mM formic acid, 0.5 mM DT T , 0.5 g/L ACS, 0.2 g/L HACL l , 0.5 g/L Lmo l 1 79, 0.5 g/L AldA. After overnight incubation at room temperature, the reaction was terminated by addition of 1% sulfuric acid. Precipitant was pel leted by centrifugation and the supernatant was analyzed by HPLC.
  • Lactic acid 2-h droxy propi oni c acid
  • the pathway is assembled in vivo using E. coli as the host organism.
  • Non-functionalized carboxylic acids such as acetic acid
  • Scheme 5 A can also be produced from Scheme 5 A through the reaction step catalyzed by diol dehydratase by supplying a suitable aldehyde dehydrogenase.
  • Lmo l 1 79, HACL 1 , fucO, KoPddABC, and AldB an aldehyde dehydrogenase from E. coli, were cloned, expressed, and purified.
  • a reaction mixture containing 60 mM potassium phosphate pH 7.4, 0.5 niM NADP " 200 niM 1 ,2-ethanediol, 1 5 uM B 1 2, 0.1 g/L AldB, and 40 mg L KoPddABC was prepared and acetaldehyde oxidation to acetate was monitored by corresponding NADP " reduction to NADPH at 340 nm. Reduction of NADP was obser ed in a sample containing KoPddABC but was not observed in a sample where KoPddABC was omitted, indicating that the diol could be converted to the aldehyde and further oxidized to the acid form (FIG. 37).
  • 2-hydroxy amines can be produced from the 2-hydroxy aldehyde resulting from the reaction step catalyzed by a suitable acyl-CoA reductase acting on 2- hydroxy acyl -Co A .
  • Non-functionalized terminal amines can be produced from n-aldehydes resulting from the reaction step catalyzed by diol dehydratase. It has been demonstrated in above examples that KoPddABC can act on 1 ,2-diols to give said aldehydes.
  • Transaminase activity was determined by incubating the purified enzyme with methylbenzylamine (MBA) (amino donor) with and without the addition of pyruvate as the amino group acceptor in a standard assay mixture (Enzyme and Microbial Technology 4 1 , 628-637, 2007).
  • Alkyl compounds can be produced from aldehyde intermediates such as those resulting from the reaction step catalyzed by diol dehydratase. It has been demonstrated in above examples that KoPddABC can act on 1 ,2-diol s to give said aldehydes.
  • the in vitro aldehyde decarbonylase assay contained 50 mM HEPES (pH7.2), 100 mM KG, 10% glycerol, 1 mM THP, 80 ⁇ ammonium iron sulfate, Phenazine methosulfate 160 ⁇ , ⁇ , NADH 1 .6 mM, BSA 1 mg/mL, 5 mM aldehyde (Dodecanal ) and purified protein in a final volume of 400 ⁇ ]_. Reactions were carried out at 37°C, 200 rpm, 2 hours and were stopped by adding 400 ⁇ , of ethyl acetate containing 50 mg/L tridecane as internal standard.
  • Samples were analyzed by GC using the following parameters: run time: 27 min; column: Innowax (length 30 m, I D. 0.25 mm ID, film 0.25 ⁇ ); inlet: 250°C splitless; carrier gas: Helium, 1.0 niL/min flow; oven temp: 45°C hold 5 min, 250°C at 25°C/min, 250°C hold 10 min.
  • LcdABC has been characterized by Hofmeister and Buckel for the dehydration of 2-hydroxybutyryl-CoA to crotonyl-CoA, with specific activity corresponding to 1.21 ⁇ 0.08 ⁇ /min/mg protein (Hofmeister & Buckel, 1992).
  • the genes encoding LcdABC were cloned as described above (FIG. 28).
  • trans-2-enoyl-CoA is then reduced to the saturated acyl -Co A by a trans-2-enoyl-CoA reductase.
  • EgTER E. gracilis
  • in vitro assays were performed by monitoring the loss of NADH absorbance in the presence of 100 mM Tris HCL pH 7.5 and 0.2 mM NADH in a final volume of 200 ⁇ at 25°C. This revealed that the enzyme i s capable of catalyzing the conversion of crotonyl-CoA.
  • the assay mixture consisted of 100 mM MOPS pH 7.5, 6 mM DTT, 5 mM MgSO.,, 0.3 mM Fe(NH 4 ) 2 (S0 4 ) 2 , 0.3 mM NADH, and 0.2 mM butyryl-CoA.
  • the reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of N ADH, as shown in FIG. 30.
  • MhpF was capable of catalyzing the conv ersion with a specific activ ity of 0.009 ⁇ 0.003 ⁇ /min/mg protein.
  • Treponema denticola Ter 0.1 g/L
  • MhpF 7.5 mM NADH
  • 1 .7 mM crotony -CoA was added to the media for use as a primer in the new reaction pathway.
  • Reaction with Lmo l 1 79 contained 0. 1 5 g/L TdTer, 0.9 g/L Lmo l 1 79, 7.5 mM NADH and 1 .7 mM crotonyi-CoA.
  • the assay was monitored by measuring production of butyraldehyde with HPLC.
  • Alcohols can be produced in this scheme from aldehyde intermediates using a suitable alcohol dehydrogenase.
  • FucO was expressed from an A SKA collection strain and purified as described abov e. Purified FucO was assayed in a buffer containing 100 mM Tris-HCl pH 7.5, 0.3 niM NADH, and 10 mM butyraldehyde. The reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of N ADH, as shown in FIG. 31. FucO was capable of catalyzing the reduction of butyraldehyde to butanol with a specific activity of 5.08 ⁇ 0.08 ⁇ /min/mg protein. Amines and alky Is can also be produced in this scheme from aldehyde intermediates. These have been demonstrated in previous examples.
  • Binstock, IF. & Schulz, H. ( 198 1 ) Fatty acid oxidation complex from

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Abstract

L'invention concerne des procédés biotechnologiques de production de produits chimiques industriellement pertinents. En particulier, l'invention concerne des procédés de production biologique de produits contenant au moins un alcool terminal, un groupe carboxyle, une amine ou un groupe alkyle directement par assimilation de motifs à un seul carbone à l'aide de micro-organismes.
PCT/US2017/030203 2016-04-28 2017-04-28 Conversion de composés de 1-carbone en produits Ceased WO2017190056A1 (fr)

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CN108795956A (zh) * 2018-05-04 2018-11-13 华南农业大学 GmMDH12基因在促进大豆结瘤固氮能力方面的应用
CN111363713A (zh) * 2020-03-24 2020-07-03 华东理工大学 一种提高聚羟基丁酸乳酸酯中乳酸组分含量的基因工程大肠杆菌的构建方法及应用
WO2021038095A1 (fr) * 2019-08-31 2021-03-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Peptides et procédés pour la formation d'une liaison carbone-carbone
EP3980534A4 (fr) * 2019-06-04 2023-08-30 Ramon Gonzalez Production de molécules 2-hydroxyacyl-coa et leurs dérivés
US12391937B2 (en) 2014-10-29 2025-08-19 Mojia Biotech Pte. Ltd. Biosynthesis of products from 1-carbon compounds

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US20130288320A1 (en) * 2012-04-27 2013-10-31 Bioamber Inc. Methods and microorganisms for increasing the biological synthesis of difunctional alkanes
US20140322774A1 (en) * 2011-11-03 2014-10-30 Easel Biotechnologies, Llc Microbial production of n-butyraldehyde
WO2016069929A1 (fr) * 2014-10-29 2016-05-06 William Marsh Rice University Biosynthèse de produits à partir de composés à un seul atome de carbone
US20160257932A1 (en) * 2013-11-18 2016-09-08 Rubius Therapeutics, Inc. Genetically engineered enucleated erythroid cells comprising a phenylalanine ammonia lyase receiver polypeptide

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US20140322774A1 (en) * 2011-11-03 2014-10-30 Easel Biotechnologies, Llc Microbial production of n-butyraldehyde
US20130288320A1 (en) * 2012-04-27 2013-10-31 Bioamber Inc. Methods and microorganisms for increasing the biological synthesis of difunctional alkanes
US20160257932A1 (en) * 2013-11-18 2016-09-08 Rubius Therapeutics, Inc. Genetically engineered enucleated erythroid cells comprising a phenylalanine ammonia lyase receiver polypeptide
WO2016069929A1 (fr) * 2014-10-29 2016-05-06 William Marsh Rice University Biosynthèse de produits à partir de composés à un seul atome de carbone

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12391937B2 (en) 2014-10-29 2025-08-19 Mojia Biotech Pte. Ltd. Biosynthesis of products from 1-carbon compounds
CN108795956A (zh) * 2018-05-04 2018-11-13 华南农业大学 GmMDH12基因在促进大豆结瘤固氮能力方面的应用
CN108795956B (zh) * 2018-05-04 2021-04-23 华南农业大学 GmMDH12基因在促进大豆结瘤固氮能力方面的应用
EP3980534A4 (fr) * 2019-06-04 2023-08-30 Ramon Gonzalez Production de molécules 2-hydroxyacyl-coa et leurs dérivés
US12359236B2 (en) 2019-06-04 2025-07-15 Mojia Biotech Pte. Ltd. Production of 2-hydroxyacyl-CoAs and derivatives thereof
WO2021038095A1 (fr) * 2019-08-31 2021-03-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Peptides et procédés pour la formation d'une liaison carbone-carbone
CN111363713A (zh) * 2020-03-24 2020-07-03 华东理工大学 一种提高聚羟基丁酸乳酸酯中乳酸组分含量的基因工程大肠杆菌的构建方法及应用

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