CN121127603A

CN121127603A - Strains and methods for producing mogrosides

Info

Publication number: CN121127603A
Application number: CN202480026622.3A
Authority: CN
Inventors: 赵正; 芮妮·马赛尔·德·钟; 约瑟夫·彼得鲁斯·约翰尼斯·施米茨; 妮可莱特·雅斯敏·布罗尔斯; 比安卡·伊丽莎白·玛丽亚·吉勒森
Original assignee: DSM IP Assets BV; Cargill Inc
Current assignee: DSM IP Assets BV; Cargill Inc
Priority date: 2023-04-20
Filing date: 2024-04-18
Publication date: 2025-12-12
Also published as: WO2024218244A1

Abstract

The present disclosure relates to a recombinant cell capable of producing one or more mogroside precursors and/or one or more mogrosides in a culture medium, wherein the cell has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product. Methods for producing one or more mogroside precursors and/or one or more mogrosides in a culture medium using such recombinant cells result in improved mogroside yield.

Description

Strains and methods for producing mogrosides

Technical Field

The present disclosure relates to recombinant cells capable of producing mogrosides or a mogroside precursor and to methods of producing such products.

Background

Mogrosides are a family of secondary metabolite compounds isolated from the plant momordica grosvenori (Siraitia grosvenorii) belonging to cucurbitaceae (Curcubitaceae) and grown almost exclusively in guangxi province, china. Such plants are commonly referred to as Momordica grosvenori (monk fruit/Luo Han Guo).

Mogrosides are glycosylated triterpene compounds sharing the same mogrol (mogrol) triterpene backbone, but the number of glucose moieties and the type of glycosidic linkages present are different. The chemical structures of several mogrosides are shown in figure 1.

Fructus Siraitiae Grosvenorii extract has long been used as natural sweetener in China. Mogroside V, mogroside IV and siamenoside I are the main components of the extract and are responsible for the intense sweetness of the extract, which may be 250 times higher than sucrose. Unfortunately, these compounds can only be present in very small amounts in fruits. In view of the increasing interest in natural sweeteners in the food industry and the difficulty in obtaining mogrosides from plants, new methods are needed that allow for the production of mogrosides in a sustainable and commercially viable manner.

Biosynthesis of some mogroside compounds has been described.

WO2013/076577 describes a method of producing a mogroside compound comprising contacting mogroside with a cell lysate prepared from a recombinant host expressing a UGT polypeptide to produce a mogroside compound.

WO2014/086842, WO2016/038617 and WO2016/050890 describe methods for producing mogrosides by means of enzymes. Various biosynthetic pathways useful for mogroside production and enzymes useful for mogroside production are provided, including recombinant cells capable of producing mogrols and/or mogrosides in a culture medium.

Several documents such as WO2014/086842, WO2016/038617 and WO2022/212917 describe that said recombinant hosts capable of producing mogrosides are preferably modified to reduce β -glucanase activity, which may lead to deglycosylation of the mogrosides. The recombinant host may be modified to reduce or even eliminate exo-1, 3-beta-glucanase activity. These documents describe yeast cells in which the EXG1 gene and/or EXG2 gene (both encoding an exo-1, 3- β -glucanase) is knocked out.

There remains a need for recombinant production systems that can accumulate high yields of desired highly glycosylated mogroside components, such as mogroside IV, mogroside V, mogroside VI, and siamenoside I. There is still a need to improve the production of mogroside compounds in recombinant hosts for commercial use.

Drawings

Figure 1 depicts the chemical structures of several mogroside compounds.

FIG. 2 depicts the biosynthetic pathway from acetyl-CoA to squalene.

Fig. 3 depicts the biosynthetic pathway from squalene to mogrol.

FIG. 4 depicts the biosynthetic pathway from mogrols to mogrosides.

Fig. 5 depicts the chemical structures of several mogroside precursors and intermediates in the biosynthetic pathway for the production of mogroside starting from squalene.

Fig. 6A and 6B depict normalized concentrations of mogroside I A ₁ and mogroside I E ₁, respectively, as determined in the same fermentation experiments for strains MOG001, MOG004, MOG003, MOG002, MOG006, and MOG 005.

Fig. 7A and 7B depict normalized concentrations of mogroside II a ₂ and mogroside III a ₁, respectively, as determined in the same fermentation experiments for strains MOG001, MOG004, MOG003, MOG002, MOG006 and MOG 005.

DESCRIPTION OF THE SEQUENCES

An illustration of the sequence is set forth in table 1.

Disclosure of Invention

Provided herein is a recombinant cell capable of producing one or more mogrosides and/or a mogroside precursor, e.g., capable of producing one or more mogrosides in a culture medium, wherein the cell has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product. The polypeptide capable of deglycosylating the mogroside product may be capable of hydrolyzing at least one of:

(a) A glycosidic bond between the carbon (C24 atom) at position 24 of the mogrol skeleton of mogroside and beta-1-glucose bound at said position, and/or

(B) A glycosidic bond between the carbon (C3 atom) at the 3-position of the mogrol skeleton of mogroside and β -1-glucose bound at said position.

Specifically, the polypeptide capable of deglycosylating the mogroside product may be selected from the group consisting of a polypeptide having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO. 1, and a polypeptide having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO. 2.

Also provided herein is a method of producing one or more mogrosides and/or mogroside precursors, the method comprising culturing a recombinant cell in a medium that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure under conditions suitable for producing the mogroside, mogroside precursor, and/or mogrol, and optionally isolating the one or more mogroside, mogroside precursor, and/or mogrol.

The present disclosure also provides a method of producing one or more mogrosides, the method comprising contacting mogrol, one or more mogroside precursors, or one or more mogroside substrates with a recombinant cell that has been modified to result in a lack of a polypeptide or a lysate or extract thereof capable of deglycosylating a mogroside product according to the present disclosure under conditions suitable for producing the one or more mogrosides, and optionally isolating the one or more mogrosides.

Also disclosed is:

-a fermentation broth comprising recombinant cells that have been modified to result in a lack of a polypeptide or a lysate or extract thereof capable of deglycosylating a mogroside product according to the present disclosure;

-a mogroside composition or sweetener composition obtainable by the process of the present disclosure;

-a food, beverage, pet food, feed, oral, pharmaceutical composition comprising a mogroside composition or sweetener composition according to the present disclosure.

General definition

In order that the present disclosure may be more readily understood, certain terms and methodologies are first defined. As used in the present application, each of the following terms shall have the following meanings unless explicitly stated otherwise herein. Additional definitions are set forth throughout the application. In case of conflict, the present application, including definitions, will control. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description and from the claims.

As used in this disclosure and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an element" may mean one element or more than one element, i.e., "at least one element.

The term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 standard deviation or within greater than 1 standard deviation according to practice in the art. Or "about" may mean a range of up to 20%. Furthermore, in particular with respect to biological systems or processes, the term may mean at most about a magnitude of a value or at most 5 times. When a particular value or composition is provided in the application and claims, unless otherwise indicated, the meaning of "about" or "consisting essentially of.

A "cell" as defined herein is an organism suitable for genetic manipulation and which may be cultivated at a cell density useful for industrial production of the product of interest. Suitable organisms may be microorganisms, for example microorganisms which may be maintained in a fermentation device. With respect to the present disclosure, it should be understood that cells (such as microorganisms, fungi, algae, or plants) also include synonyms or base names (basonyms) of such species having the same physiological properties, as defined by international prokaryote nomenclature (International Code of Nomenclature of Prokaryotes) or international nomenclature for algae, fungi, and plants (International Code of Nomenclature) (Melbourne method). The cells may be cells that are present in nature, or cells that have been genetically manipulated or subjected to classical mutagenesis derived from the parent cell.

The cells may be prokaryotic, archaebacteria or eukaryotic cells.

The prokaryotic cell may be, but is not limited to, a bacterial cell. The bacterial cell may be a gram negative bacterium or a gram positive bacterium. Examples of bacteria include, but are not limited to, bacteria belonging to the following genera: bacillus (e.g., bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus megaterium, bacillus halodurans, bacillus pumilus, acinetobacter, nocardia, bacillus flavescens, bacillus (Xanthobacter), escherichia (e.g., escherichia), coli (e.coli)), streptomyces (Streptomyces), erwinia (Erwinia), klebsiella (Klebsiella), serratia (Serratia) (e.g., serratia marcescens (S. marcessans)), pseudomonas (e.g., pseudomonas aeruginosa (P. Aeromonas), pseudomonas fluorescens (P. Fluoroscens)), salmonella (e.g., salmonella typhimurium (S. Tyrum), salmonella typhi (S. Tyrphi)), anabaena (Anabaena), acetobacter (Caulobactert), gluconobacter (Gluconobacter), rhodobacillus (Rhodobater), paracoccus (Paracoccus), brevibacterium (Brevibacterium), corynebacterium (Corynebacterium), rhizobium (Rhizobium) (Sinorhizobium)) Flavobacterium (Flavobacterium), klebsiella (Klebsiella), enterobacter (Enterobacter), lactobacillus (Lactobacillus), lactococcus (Lactobacillus), methylobacillus (Methylobacterium), and Staphylococcus (Staphylococcus). Bacteria also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria, green sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria).

Eukaryotic cells may be, but are not limited to, fungi (e.g., yeast or filamentous fungi), algae, plant cells, cell lines.

Eukaryotic cells may be fungi, such as filamentous fungi or yeasts. Filamentous fungal strains include, but are not limited to, the following strains: acremonium (Acremonium), aspergillus (Aspergillus) (e.g., aspergillus niger (A. Niger), aspergillus oryzae (A oryza), aspergillus nidulans (A. Nidulans)), agaricus (Agaricus), aureobasidium (Aureobasidium), coprinus (Coprinus), cryptococcus (Cryptococcus), leptosphaera (Corynaascus), chrysosporium (Chrysosporium), aureobasidium (Filibasidium), fusarium (Fusarium), humicola (Humicola), strychotum (Magnaporthe), monascus (Monascus), mucor (Mucor), myceliophthora (Mycelothora), mortierella (Mortierella), neocimum, pevularia (Neurospora), paecilomyces (Paecilomyces) (e.g., paecilomyces), penicillium chrysogenum (P. Chrysogenum), penicillium sarcinae (P. Camemberti)), rumex (Piromyces), phanerochaete (Phanerochaete), pleurotus (Pleurotus), pachybotrytis (Podospora), mirabilis (Pycnoporus), rhizopus (Rhizopus), schizophyllum (Schizophyllum), chaetomium (Sordaria), basket (Talaromyces), talaromyces (Rasamsonia) (e.g., emerson blue (Rasamsonia emersonii)), thermoascus (Thermoascus), thielavia, tolypocladium), schizophyllum (Tolypodium), trametes (Trametes) and Trichoderma (Trichoderma).

The yeast cells may be selected from the group consisting of Saccharomyces (e.g., saccharomyces cerevisiae), saccharomyces (S. Bayanus), pasteurella (S. Pastorianus), saccharomyces (S. Carlsbergensis), kluyveromyces, candida (e.g., candida rugosa (C. Rugosa), candida (C. Revkaufi), rhodotorula (C. Pulcherrima), candida tropicalis (C. Tropicalis), candida utilis (C. Utilis)), pichia (e.g., pichia pastoris (P. Pastoris)), schizosaccharomyces (Schizopus), issatchenkia (ISSATCHENKIA), zygomyces (Zgonomyces), hansenula (Hansena), klebsiella (Schvalia), schvalia (Kloeckera), yeast (Y. Lipolytica), and Yarrowia (Yeast) (e.g., yarrowia lipolytica).

The cells may be algae, microalgae or marine eukaryotes. The cell may be a cell of the class of the Rhizomucosae, preferably of the order thraustochytriales, more preferably of the family thraustochytriales, more preferably of a member of a genus selected from the group consisting of Citrus aurantiacus (Aurantucrytium), oblongichytrium, schizochytrium (Schizochytrium), humicola (Thraustochytrium) and Ulkenia, even more preferably of the genus Schizochytrium (Schizochytrium sp.) ATCC No. 20888.

The recombinant cells as disclosed herein may belong to one of the genera Saccharomyces, aspergillus, pichia, kluyveromyces, candida, hansenula (Hansenula), humicola (Humicola), issatchenkia, trichosporon (Trichosporon), brettanomyces (Brettanomyces), pachysolen, yarrowia, yamadazyma, or Escherichia, e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae) cells, yarrowia lipolytica cells, candida krusei (Candida krusei) cells, issatchenkia orientalis cells, or E.coli cells.

Thus, in one embodiment, the recombinant cell capable of producing mogrols or mogrosides (which has been modified to result in the absence of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure) may be a prokaryote, eukaryote or archaebacterium cell, in particular a plant cell or a cell selected from the group consisting of a saccharomyces cerevisiae cell, a yarrowia lipolytica cell, a candida krusei cell, an eastern issa cell, a pichia pastoris cell or an escherichia coli cell.

The term "control sequence" as used herein refers to a component that is involved in regulating expression of a coding sequence in a particular organism or in vitro. Examples of control sequences are transcription initiation sequences, termination sequences, promoters, leader sequences, signal peptides, prepeptides, prepropeptides or enhancer sequences, the sequence of the summer-darcino (Shine-Delgarno sequence), repressors or activators, efficient RNA processing signals, such as splicing and polyadenylation signals, sequences which stabilize cytoplasmic mRNA, sequences which enhance translation efficiency (e.g., ribosome binding sites), sequences which enhance protein stability, and sequences which enhance protein secretion when desired.

As used herein, the terms "culture fluid," "medium," and "growth medium" may be used interchangeably to refer to a liquid or solid that supports cell growth. Typically, the medium may comprise a carbon source (e.g., one or more of glucose, fructose, sucrose, xylose, glycerol, plant biomass, cellulose, hemicellulose, pectin, rhamnose, galactose, fucose, maltose, maltodextrin, ribose, ribulose, or starch, starch derivatives, lactose, fatty acids, triglycerides). Typically, the medium may also comprise a nitrogen source (e.g. urea) or an ammonium salt (e.g. ammonium sulphate, chloride, nitrate or phosphate). The culture broth may further comprise trace metals, vitamins, salts, amino acids, and the like. The trace metals may be divalent cations including, but not limited to, mn2+, mg2+, fe2+, cu2+, and the like.

When comparing the production of a particular mogroside from a recombinant cell lacking a polypeptide capable of deglycosylating a mogroside product with the production of the same mogroside from an otherwise identical cell not lacking the polypeptide, the phrase "culturing under the same conditions" refers to culturing both cells under the same conditions, wherein the amount and/or concentration of different mogrosides in both cells is measured in both cells using the same conditions. Preferably, the measurements are performed using the same assay and methodology, preferably in the same experiment.

As used herein, "cytochrome b5" or "CB5" refers to a protein comprising a lipid binding domain or a cytochrome b 5-like heme binding domain. In some embodiments, the lipid binding domain is a steroid binding domain. CB5 protein is a heme-binding protein or a lipid-binding protein. For example, CB5 may be a steroid binding protein. CB5 protein can be used as an electron transfer component of redox reactions. For example, CB5 may act as an obligatory electron donor in oxidation reactions. In some embodiments, CB5 may serve as an electron transport partner for cytochrome P450 (e.g., C11-hydroxylase). In some embodiments, CB5 may catalyze or promote electron transfer from NADPH to cytochrome P450 enzymes (e.g., C11-hydroxylase). In some other embodiments, CB5 may spatially interact with the P450 enzyme to support a conformation of the enzyme that promotes higher activity without the direct enzymatic action of CB5 itself.

The term "derived from" also includes the terms "derived from", "obtained from", "obtainable from", "isolated from" and "formed from," and generally means that one specified material is derived from or has characteristics that can be described with reference to another specified material. As used herein, a "cell-derived" substance (e.g., a nucleic acid molecule or polypeptide) preferably means that the substance is native to the microorganism.

The term "expression" when used in reference to a polynucleotide or polypeptide refers to any step involved in the production of the polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. The term "constitutive expression" when used in reference to a gene refers to the situation in which gene expression is under the control of a constitutive promoter that allows for continuous gene transcription. The term "induction of expression" when used in reference to a gene refers to a manner of regulating the expression of the gene, wherein a molecule, referred to as an inducer, regulates the expression of the gene by either a) binding to a gene promoter repressor protein, or b) binding to a gene promoter activator molecule, thereby allowing the RNA polymerase to perform gene transcription.

As used herein, the terms "fed-batch culture" or "semi-batch culture" are used interchangeably to refer to an operating technique in a biotechnology process wherein one or more nutrients (substrates) are fed (supplied) into a bioreactor during the culture and wherein the product remains in the bioreactor until the end of the run. In some embodiments, all nutrients are fed into the bioreactor.

In the context of the present disclosure, the terms "functional homolog", "functional equivalent" or "functional variant" may be used interchangeably. A functional homolog of a polypeptide is a polypeptide that has at least one biological function and/or at least one activity in common with the polypeptide. Typically, a functional homolog has a level of sequence similarity or identity to the amino acid sequence of the polypeptide, typically at least 50% sequence identity to the amino acid sequence of the polypeptide, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the polypeptide. The amino acid sequence of a functional homolog of a polypeptide may comprise one or more amino acid substitutions, deletions or additions if compared to the amino acid sequence of the polypeptide.

Functional homologs of a polypeptide (also referred to as reference polypeptides) may be naturally occurring polypeptides, such as homologs, orthologs or paralogs of the polypeptide. Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of a reference polypeptide. Sequence analysis may involve basic local alignment search tools such as protein BLAST, nucleotide BLAST, smartBLAST analysis of non-redundant databases using the amino acid sequence of a reference polypeptide. In some cases, the amino acid sequence is deduced from the nucleotide sequence, and in this case BLASTX may be used. Those polypeptides in the database that have greater than 40% sequence identity to the reference polypeptide are candidates for further evaluation of suitability as functional homologs of the polypeptides. Amino acid sequence similarity allows conservative amino acid substitutions, for example, substitution of one hydrophobic residue for another, or substitution of one polar residue for another. If desired, a manual inspection of these candidates may be performed to reduce the number of candidates to be further evaluated. Manual inspection may be performed by selecting those candidates that appear to have a domain (e.g., a conserved functional domain) present in the reference polypeptide. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels, rather than by using BLAST analysis. Conserved regions can be identified by locating the primary amino acid sequence of a reference polypeptide as a repeat, forming some secondary structure (e.g., helices and beta sheets), creating positively or negatively charged domains, or regions representing protein motifs or domains. For example, the InterPro database (see Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, Bileschi ML, Bork P, Bridge A, Colwell L, Gough J, Haft DH, Letunić I, Marchler-Bauer A, Mi H, Natale DA, Orengo CA, Pandurangan AP, Rivoire C, Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto SCE, Wu CH, Bateman A., 2023, "InterPro in 2022". Nucleic Acids Research,51: D418-D427） provides functional analysis of proteins by classifying them into families and predicting domains and important sites, and can be used to determine conserved regions in reference polypeptides. Conserved regions can also be determined by aligning sequences of identical or related polypeptides from closely related species.

Alternatively, the functional homologs may be synthetically produced using a variety of techniques known to those of skill in the art. For example, protein engineering tools known to those of skill in the art, such as directed evolution （Arnold, F. H. 2001. "Combinatorial and computational challenges for biocatalyst design." Nature 409:253-257;Powell, K. A., S. W. Ramer, S. B. del Cardayre, W. P. C. Stemmer, M. B. Tobin, P. F. Longchamp, and G. W. Huisman. 2001. "Directed evolution and biocatalysis." Angewandte Chemie-International Edition 40:3948-3959;Rohlin, L.、M. K. Oh and J. C. Liao. 2001. "Microbial pathway engineering for industrial processes: evolution, combinatorial biosynthesis and rational design." Current Opinion in Microbiology 4:330-335.） and/or rational design (Li, Q.S., U. Schwaneberg, M, fischer, J, schmitt, J, pleiss, S, lutz-Wahl and R. D. Schmid. 2001. "Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant." Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1545:114-121;Looger, L. L., M. A. Dwyer, J. J. Smith and H. W. Hellinga. 2003. "Computational design of receptor and sensor proteins with novel functions." Nature 423:185-190;Voigt, C. A., S. L. Mayo, F. H. Arnold and Z. G. Wang. 2001. "Computational method to reduce the search space for directed protein evolution." Proceedings of the National Academy of Sciences of the United States of America 98:3778-3783.） and/or designed divergent evolution (Yoshikuni, Y., T.E., ferrin and J.D., keasing.2006, "DESIGNED DIVERGENT evolution of enzyme function" Nature 440:1078-1082) may be used to generate amino acid sequences of potential functional homologs of polypeptides.

In this context, a "gene" is defined as a polynucleotide comprising an Open Reading Frame (ORF), which is the region of the gene that is to be transcribed and translated into a polypeptide, and its transcriptional control elements (promoter and terminator).

The term "heterologous" when used in reference to a polynucleotide (e.g., DNA or RNA), polypeptide, or protein, refers to a polynucleotide, polypeptide, or protein that does not naturally occur as part of a recombinant cell, genome, or DNA or RNA in which the polynucleotide, polypeptide, or protein naturally occurs, or is found to exist in different copy numbers, or is under the control of different control sequences, or is present in one or more locations in a different cell or genome or DNA or RNA than the polynucleotide, polypeptide, or protein is found in nature. The heterologous polynucleotide, polypeptide or protein is not endogenous to the cell into which the heterologous polynucleotide, polypeptide or protein is introduced, but is obtained from another cell, or is synthetically or recombinantly produced.

The term "homologous" when used in reference to a relationship between a given (recombinant) polynucleotide or polypeptide and a given host organism or host cell (e.g., a recombinant cell as disclosed herein) is understood to mean that the polynucleotide or polypeptide molecule is produced in nature by a recombinant cell, host cell or organism of the same species (e.g., the same variety or strain).

The term "isolated" or "recovered" as used herein means a product that is removed or purified from at least one component (e.g., a component present in the cells producing the product and/or fermentation broth or medium or crude extract or cell extract).

As used herein, the term "marker" refers to a gene encoding a trait or phenotype that allows selection or screening of recombinant microorganisms that contain the marker. The marker gene may be an antibiotic resistance gene whereby transformed cells may be selected from untransformed cells using an appropriate antibiotic. Alternatively, non-antibiotic resistance markers, such as auxotrophic markers (URA 3, TRP1, LEU 2) may be used. Recombinant cells transformed with the polynucleotide construct may be free of the marker gene. Methods for constructing recombinant cells free of recombinant marker genes are disclosed in EP-A-0 635 574 and are based on the use of bi-directional markers. Alternatively, a selectable marker (e.g., green fluorescent protein, lacZ, luciferase, chloramphenicol acetyl transferase, β -glucuronidase) can be incorporated into a polynucleotide construct as disclosed herein to allow selection of transformed cells. An exemplary label-free method for introducing heterologous polynucleotides is described in WO 0540186.

The phrase "measured under the same conditions" or "analyzed under the same conditions" means that the modified cells and the cells that have not been modified are cultured under the same conditions and the same conditions, preferably the same assay and/or methodology, are used in the modified cells and the unmodified cells, respectively, more preferably the amount and/or activity of the polypeptide that is absent or overexpressed in the modified cells compared to the unmodified cells is measured in the same experiment.

A "modification of the genome" of a recombinant cell is defined herein as any event that results in a change in a polynucleotide in the genome of the recombinant cell. A modification is interpreted as one or more modifications. Modifications can be introduced by, for example, classical strain improvement (e.g., random mutagenesis followed by selection). Modification may be achieved by introducing (inserting), substituting or removing (deleting) one or more nucleotides in the polynucleotide. The modification may be, for example, in a coding sequence or regulatory element required for transcription or translation of the polynucleotide. For example, nucleotides may be inserted or removed to result in the introduction of a stop codon, the removal of a start codon, or a change in the open reading frame of the coding sequence or a frame shift. Modification of the coding sequence or its regulatory elements may be accomplished by site-directed or random mutagenesis, DNA shuffling methods, DNA reassembly methods, gene synthesis (see, e.g., young and Dong, (2004), nucleic ACIDS RESEARCH, (7), electronic access to http:// nar.oujourn.org/cgi/reprint/32/7/e 59 or Gupta et al, (1968), proc. Natl. Acad. Sci USA, 60:1338-1344; scarpulla et al, (1982), anal. Biochem. 121:356-365; stemmer et al, (1995), gene 164:49-53) or PCR-generated mutagenesis according to methods known in the art. Examples of random mutagenesis procedures are well known in the art, such as chemical (e.g., NTG) mutagenesis or physical (e.g., UV) mutagenesis. Examples of directed mutagenesis procedures are the QuickChange @ site-directed mutagenesis kit (STRATAGENE CLONING SYSTEMS, la Jolla, calif.), the ALTERED SITES @ II in vitro mutagenesis system (Promega Corporation), or overlap extension by using PCR as described in Gene, 1989, month 15, day ;77(1):51-9.（Ho SN、Hunt HD、Horton RM、Pullen JK、Pease LR "Site-directed mutagenesis by overlap extension using the polymerase chain reaction（, by overlap extension using the polymerase chain reaction) or using PCR as described in Molecular Biology: current Innovations and Future trends (A.M. Griffin and H.G. Griffin editions, ISBN 1-898486-01-8;1995 Horizon Scientific Press, PO Box 1, wymondham, norfolk, U.K.).

The modification in the genome can be determined by comparing the polynucleotide sequence of the modified recombinant cell with the polynucleotide sequence of the unmodified recombinant cell. Polynucleotide sequencing and genomic sequencing can be performed using standard methods known to those skilled in the art, for example using Sanger sequencing techniques and/or next generation sequencing techniques, such as Illumina GA2, roche 454 et al, as described in Elaine R. Mardis (2008), Next-Generation DNA Sequencing Methods, Annual Review of Genomics and Human Genetics, 9: 387-402. （doi:10.1146/annurev.genom.9.081307.164359）.

Exemplary modification methods are based on techniques of gene replacement, gene deletion or gene disruption.

For example, in the case of a replacement polynucleotide, polynucleotide construct or expression cassette, the appropriate polynucleotide may be introduced at the target locus to be replaced. Suitable polynucleotides may be present on the cloning vector. An exemplary integrative cloning vector comprises a DNA fragment homologous to a polynucleotide and/or homologous to a polynucleotide flanking the locus to be replaced in order to target the integration of the cloning vector to the predetermined locus. To facilitate targeted integration, the cloning vector may be linearized prior to transformation of the microorganism. In some embodiments, linearization is performed such that at least one or either end of the cloning vector is flanked by polynucleotide sequences that are homologous to the polynucleotide (or flanking sequences) to be replaced. This process is called homologous recombination and this technique can also be used to achieve (partial) gene deletions or gene disruptions.

For example, for gene disruption, a polynucleotide corresponding to an endogenous polynucleotide may be replaced with a defective polynucleotide, which is a polynucleotide that is incapable of producing a (fully functional) protein. The defective polynucleotide replaces the endogenous polynucleotide by homologous recombination. It may be desirable that the defective polynucleotide also encodes a marker that can be used to select transformants in which the polynucleotide has been modified.

Alternatively, the modification of the recombinant microorganism to lack a polypeptide as disclosed herein may be performed by established antisense technology using a polynucleotide complementary to the polynucleotide encoding the polypeptide. More specifically, expression of a polynucleotide encoding a polypeptide as disclosed herein by a recombinant cell can be reduced or eliminated by introducing a polynucleotide having a sequence complementary to the sequence of the polynucleotide encoding the polypeptide, which polynucleotide can be transcribed in the recombinant cell and is capable of hybridizing to the mRNA encoding the polypeptide produced in the recombinant cell. The amount of protein translated is thus reduced or eliminated under conditions that allow hybridization of the complementary antisense polynucleotide to the polypeptide. Examples of antisense RNA expression are shown in Appl. environ. Microbiol. 2 nd month 2000; 66 (2): 775-82. (Characterization of a foldase, protein disulfide isomerase A in the protein secretory pathway of Aspergillus niger. Ngiam C, Jeenes DJ, Punt PJ, Van Den Hondel CA, Archer DB) or (Zrenner R、Willmitzer L、Sonnewald U. Analysis of the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA. Planta. (1993); 190(2):247-52.).

In addition, modifications, downregulation or inactivation of polypeptides capable of deglycosylating mogroside products may be obtained via RNA interference (RNAi) techniques (FEMS Microb. Lett. 237 (2004): 317-324). In this method, identical sense and antisense portions of a polynucleotide encoding a polypeptide to be affected (e.g., beta glucanase) are cloned into each other with a nucleotide spacer in between, and inserted into an expression vector. Upon transcription of such molecules, the formation of small nucleotide fragments will lead to targeted degradation of the mRNA to be affected. The elimination of a particular polypeptide capable of deglycosylating the mogroside product mRNA can be to varying degrees. RNA interference techniques described in WO2008/053019, WO2005/05672A1, WO2005/026356A1, oliveira et al ,"Efficient cloning system for construction of gene silencing vectors in Aspergillus niger" (2008) Appl. Microbiol. and Biotechnol. 80 (5): 917-924 and/or Barnes et al ,"siRNA as a molecular tool for use in Aspergillus niger" (2008) Biotechnology Letters 30 (5): 885-890 may be used for down-regulation, modification or inactivation of polynucleotides.

To increase the likelihood that the introduced enzyme is expressed in active form in a recombinant microorganism as disclosed herein, the corresponding encoding polynucleotide may be adapted to optimize codon usage of the polynucleotide of the selected recombinant microorganism. The adaptation of the polynucleotide encoding the enzyme to the codon usage of the selected recombinant microorganism may be expressed as a Codon Adaptation Index (CAI). Codon usage index is defined herein as a measure of the relative fitness of the codon usage of a gene towards that of a highly expressed gene. The relative fitness (w) of each codon is the ratio of the usage of each codon to the most abundant codon usage of the same amino acid. CAI index is defined as the geometric mean of these relative fitness values. Non-synonymous codons and stop codons (depending on the genetic codon) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li,1987, nucleic ACIDS RESEARCH 15:1281-1295; see also Jansen et al, 2003, nucleic Acids Res.31 (8): 2242-51). The adapted polynucleotide may have a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.

Recombinant cells as disclosed herein can be genetically modified with polynucleotides adapted for codon usage of recombinant microorganisms using codon pair optimization techniques well known to those skilled in the art. Codon pair optimisation is a method for producing a polypeptide in a recombinant cell, wherein a polynucleotide encoding the polypeptide has been modified with respect to its codon usage (in particular the codon pair used) to obtain improved expression of the polynucleotide encoding the polypeptide and/or improved production of the polypeptide. A codon pair is defined as a set of two immediately adjacent triplets (codons) in a coding sequence.

Further enhancement of the in vivo activity of enzymes in recombinant cells as disclosed herein can be obtained by well known methods such as error-prone PCR or directed evolution. Exemplary methods of directed evolution are described in WO03010183 and WO 03010311.

The term "mogroside precursor" as used herein refers to an intermediate compound in the mogroside biosynthetic pathway from squalene to mogrol, wherein the mogroside precursor comprises a cucurbitadienol or a mogrol backbone. The mogroside precursor may comprise one or more of cucurbitadienol, 11-hydroxy-cucurbitadienol, 24, 25-epoxy-cucurbitadienol, 11-hydroxy-24, 25-epoxy-cucurbitadienol, 24, 25-dihydroxy-cucurbitadienol and/or mogroside.

The term "naturally occurring" as used herein refers to a process, event or product that occurs in nature in its associated form. In contrast, "non-naturally occurring" refers to a process, event, or product whose presence or form involves man-made. The term "non-naturally occurring" is synonymous herein with "artificial". In general, the term "naturally occurring" with respect to a polypeptide or nucleic acid may be used interchangeably with the terms "wild-type" or "native". It refers to a polypeptide having the same amino acid sequence or polynucleotide sequence as in nature or a nucleic acid encoding the polypeptide, respectively. Naturally occurring polypeptides include native polypeptides, such as those naturally expressed or found in a particular cell. Naturally occurring polynucleotides include natural polynucleotides, such as those found naturally in the genome of a particular cell. In addition, a wild-type or naturally occurring sequence may refer to a sequence from which a variant or synthetic sequence is derived.

"Nucleic acid molecule" or "polynucleotide" (these terms are used interchangeably herein) is represented by a nucleotide sequence.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (including, for example, a coding sequence or another polynucleotide sequence) in a functional relationship. A polynucleotide is "operably linked" when it is in a functional relationship with another polynucleotide. For example, a promoter sequence or enhancer sequence is operably linked to a coding sequence if the promoter sequence or enhancer sequence affects the transcription of the coding sequence.

As used herein, the term "or" is understood to be inclusive unless specifically stated or apparent from the context. The term "and/or" as used in the phrase herein, e.g., "a and/or B," is intended to include both "a and B," a or B, "" a, "and" B. Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments A, B and C, A, B or C, A or B, B or C, A and B, B and C, A (alone), B (alone), and C (alone).

"Polypeptides" are represented by amino acid sequences.

As defined herein, a "polypeptide capable of deglycosylating a mogroside product" according to the present disclosure may be a glycoside hydrolase (EC: 3.2.1. -), e.g., an exo-acting glycoside hydrolase. Glycoside hydrolases are enzymes that catalyze the hydrolysis of glycosidic bonds of glycosides, leading to the formation of sugar hemiacetals or hemiketals and the corresponding free aglycones. Glycoside hydrolases are also known as glycosidases and sometimes also as glycosyl hydrolases. In general, glycoside hydrolases according to the present disclosure are capable of cleaving glucose moieties in an exo-type manner, i.e. are capable of cleaving glycosidic bonds and releasing glucose molecules. In general, glycoside hydrolases according to the present disclosure may be beta-glucosidase (EC 3.2.1.21), glucan 1, 3-beta-glucosidase (EC 3.2.1.58), glucan 1, 4-beta-glucosidase (EC 3.2.1.74). For the purposes of this disclosure, the terms "polypeptide capable of deglycosylating a mogroside product", "beta-glucanase", "Bei Dapu-glucanase", "beta-glucanase" may be used interchangeably (abbreviated BG).

As used herein, the term "promoter" refers to a polynucleotide fragment that functions to control transcription of one or more genes, is located upstream relative to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription initiation site, and any other polynucleotide fragment (including, but not limited to, transcription factor binding sites, repressors, and activator binding sites, and any other nucleotide sequence known to those of skill in the art for directly or indirectly regulating the amount of transcription from a promoter). A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under specific environmental or developmental conditions that may be regulated.

When used in reference to a nucleic acid or protein, the term "recombinant" means that the nucleic acid or protein has been sequence modified by manual intervention if compared to its native form. When referring to a cell, the term "recombinant" means that the genome of the cell has been sequence modified by human intervention if compared to its native form. The term "recombinant" is synonymous with "genetically modified".

As used herein, a "recombinant cell" is defined as a cell that is preferably genetically modified or transformed/transfected with one or more of the polynucleotides as defined elsewhere herein. The presence of one or more such polynucleotides alters the ability of a microorganism to produce one or more products. The untransformed/transfected or genetically modified cells are not recombinant cells and typically do not comprise one or more of the polynucleotides that enable the cell to produce a product (e.g., mogrosides). Thus, an untransformed/untransfected cell is typically a cell that does not naturally produce a product such as mogroside, but a cell that naturally produces a product (e.g., mogroside) and has been modified as disclosed herein (and thus has an altered ability to produce mogroside) is considered a recombinant cell as disclosed herein.

Within the context of the present disclosure, the term "recombinant cell lacking (producing) a polypeptide, e.g. a beta glucanase as described herein, means that the cell comprises a modification, preferably in its genome, which modification results in reduced or no production of the polypeptide, if compared to a parent cell that has not been modified, when analyzed under the same conditions. Alternatively or additionally thereto, the cell comprises a modification which results in a reduced or no activity (which may be enzymatic or other biological activity) of a (modified) polypeptide derived from a polypeptide as described herein, if compared to a parent cell which has not been modified, when assayed under the same conditions. Thus, a recombinant cell lacks (production of) a polypeptide as described herein when:

a) If the recombinant cell produces less polypeptide as defined herein or the recombinant cell does not produce polypeptide as defined herein compared to a parent cell that has not been modified and measured under the same conditions, and/or

B) Has a reduced level of expression or has a reduced level of translation of mRNA transcribed from a gene encoding the polypeptide;

b) When assayed under the same conditions, the recombinant cells produce polypeptides with reduced or no activity compared to cells that have not been modified.

The lack of production of a polypeptide as defined herein in a recombinant cell may be measured by determining the amount and/or (specific) activity of a related polypeptide produced by a genome-modified recombinant microorganism, and/or the lack of production of a polypeptide as defined herein in a recombinant cell may be measured by determining the amount of (episomal) mRNA transcribed from a gene encoding the polypeptide, and/or the lack of production of a polypeptide as defined herein in a recombinant cell may be measured by determining the amount of a product produced by a polypeptide in a genome-modified recombinant microorganism as defined above, and/or may be measured by gene or genome sequencing if compared to a genome-unmodified parent (recombinant) microorganism. The lack of production of the polypeptide may be measured using any assay available to those skilled in the art, such as transcriptional profiling, northern blotting, RT-PCR, Q-PCR, and Western blotting.

"Sequence identity" is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Typically, sequence identity or similarity is compared over the full length of the sequences compared, i.e., sequence identity or similarity is compared over the full length of the sequences for which sequence identity is determined. For the purposes of this disclosure, to determine the percent sequence homology or sequence identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps can be introduced in either of the two sequences being compared. This analogy can be performed over the full length of the sequences being compared. Or may be aligned over a shorter length, such as about 20, about 50, about 100 or more nucleotides/bases or amino acids. Sequence identity is the percentage of identical matches between two sequences over the reported alignment region.

A mathematical algorithm may be used to complete sequence comparison and percent sequence identity determination between two sequences. Those skilled in the art will appreciate that in fact several different computer programs can be used to align two sequences and determine identity between the two sequences (Kruskal, j.b. (1983), in d. Sankoff and j.b. Kruskal (edit), TIME WARPS, STRING EDITS AND macromolecules: the theory AND PRACTICE of sequence comparison, pages 1-44, an overview of sequence comparison in Addison Wesley). The percent sequence identity between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for aligning the two sequences. (Needleman, S.B. and Wunsch, C.D. (1970) J.mol. Biol. 48, 443-453). The amino acid sequence and the nucleotide sequence can be aligned by an algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For purposes of this disclosure, NEEDLE programs from EMBOSS package (version 2.8.0 or higher, EMBOSS: the European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. And Bleasby, A. TRENDS IN GENETICS 16, (6) pages 276-277, http:// EMBOSS. Bioinformation. Nl /) are used. For protein sequences, EBLOSUM62 was used for substitution matrix. For the nucleotide sequence, EDNAFULL was used. The optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that when using different algorithms, all these different parameters will produce slightly different results, but the overall percentage identity of the two sequences will not change significantly.

After alignment by the program NEEDLE as described above, the percent sequence identity between the query sequence and the sequences of the present disclosure is calculated as the number of corresponding positions in the alignment (which show the same amino acids or the same nucleotides in both sequences) divided by the total length of the alignment minus the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE by using NOBRIEF options and is labeled as "longest identity" in the output of the program.

The nucleic acid sequences and protein sequences as disclosed herein may further be used as "query sequences" to perform searches on public databases, for example, to identify other family members or related sequences. Such searches may be performed using the BLASTN and BLASTX programs of Altschul et al, (1990) J.mol. Biol. 215:403-10 (version 2.0). BLAST nucleotide searches can be performed using the BLASTN program with score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed using the BLASTX program with score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. To obtain a gap alignment for comparison purposes, gap BLAST may be used, as described in Altschul et al, (1997) Nucleic Acids Res.25 (17): 3389-3402. When using BLAST and Gapped BLAST programs, default parameters for the respective programs (e.g., BLASTX and BLASTN) can be used. See homepage http:// www.ncbi.nlm.nih.gov/, of the national center for biotechnology information (National Center for Biotechnology Information).

Detailed Description

The present disclosure provides a recombinant cell capable of producing one or more mogrosides and/or a mogroside precursor, e.g., capable of producing one or more mogrosides in a culture medium, wherein the cell has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product. The polypeptide capable of deglycosylating the mogroside product may be capable of hydrolyzing at least the following:

According to one embodiment, the polypeptide capable of deglycosylating a mogroside product according to the present disclosure may be a glycoside hydrolase (EC: 3.2.1. -), e.g., an exo-acting glycoside hydrolase. The polypeptide capable of deglycosylating the mogroside product (i.e., bei Dapu glycanase) may be selected from the group consisting of polypeptides having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO. 1, and polypeptides having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO. 2.

Mogrosides are glycosylated forms of the triterpene mogrols. The mogrol skeleton comprises hydroxyl groups which may form glycosyl bonds at carbons at position 3 (C3), position 11 (C11), position 24 (C24) and/or position 25 (C25). Typically, most related mogrosides comprise primary and/or secondary glycosylation at the C3 and/or C24 of the mogrol skeleton. For example, mogroside IE ₁ comprises a β -glycosidic bond (i.e., a bond) between the hydroxyl group at C3 of the mogroside backbone and the hydroxyl group at C1 of the glucose moiety. Mogroside IA ₁ comprises a β -glycosidic bond between a hydroxyl group at C24 of the mogrol backbone and a hydroxyl group at C1 of the glucose moiety. This glycosylation of a glucose moiety at position C3 and/or C24 of the mogrol skeleton is referred to as primary glycosylation. Mogroside IIE contains β -glycosidic linkages at both the C3 and C24 hydroxyl groups of the mogrol backbone. The glucose moiety at position C3 and/or C24 of the primary glycosylated mogroside may be further glycosylated by a beta-1, 6-glycosyl bond between the hydroxyl group at C6 of the primary glucose and the hydroxyl group at C1 of the second glucose moiety and/or by a beta-1, 2-glycosyl bond between the hydroxyl group at C2 of the primary glucose and the hydroxyl group at C1 of the second glucose moiety. Some mogrosides may contain beta-1, 4 glycosylation between the hydroxyl group at position C4 of the primary glucose moiety linked at position C3 of the mogrol skeleton and the hydroxyl group at position 1 of the second glucose moiety. The isosorbide IVE comprises such beta-1, 4 glycosylation. The beta 1,2, beta 1,4 and beta 1,6 glucosylation in mogroside molecules are referred to as secondary glycosylation. Figure 1 shows the structure of several known mogroside molecules.

Recombinant cells according to the present disclosure have been modified to result in the absence of a β -glucanase polypeptide.

In one aspect of the methods and recombinant cells according to the present disclosure, the beta glucanase disclosed herein is a polypeptide capable of hydrolyzing one or more bonds selected from the group consisting of:

(a) A glycosidic bond between the carbon (C24 atom) at position 24 of the mogrol skeleton of mogroside and the β -1-glucose moiety bound at said position;

(b) A glycosidic bond between the carbon (C3 atom) at position 3 of the mogrol skeleton of mogroside and the β -1-glucose moiety bound at said position;

(c) A beta 1,2 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C24 atom of the mogrol skeleton of mogroside;

(d) A beta 1,2 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C3 atom of the mogrol skeleton of mogroside;

(e) A beta 1,6 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C24 atom of the mogrol skeleton of mogroside;

(f) A beta 1,6 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C3 atom of the mogrol skeleton of mogroside;

(g) Beta 1,4 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C3 atom of the mogrol skeleton of mogroside.

Typically, the beta glucanase is capable of hydrolyzing at least the following

(A) A glycosidic bond between the carbon (C3 atom) at the 3-position of the mogrol skeleton of mogroside and beta-1-glucose bound at said position, and/or

(B) A glycosidic bond between the carbon (C24 atom) at position 24 of the mogrol skeleton of mogroside and the β -1-glucose bound at said position.

In another embodiment, the beta glucanase is capable of hydrolyzing at least the following:

(a) Beta 1,6 glycosidic bond between the second glucose molecule and beta-1-glucose bound at C3 atom of the mogrol skeleton of mogroside, and/or

(B) Beta 1,6 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C24 atom of the mogrol skeleton of mogroside.

In yet another embodiment, the beta glucanase is capable of hydrolyzing at least the following:

(a) Beta 1,2 glycosidic bond between the second glucose molecule and beta-1-glucose bound at C3 atom of the mogrol skeleton of mogroside, and/or

(B) Beta 1,2 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C24 atom of the mogrol skeleton of mogroside.

According to the present disclosure, the beta glucanase may be selected from a polypeptide having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO. 1, and a polypeptide having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO. 2. Thus, recombinant cells according to the present disclosure may be modified to lack polypeptides having the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2. Additionally or alternatively, recombinant cells according to the present disclosure may be modified to lack a functional homolog (also referred to as a functional equivalent or functional variant) of a polypeptide capable of deglycosylating a mogroside product according to SEQ ID No. 1 or according to SEQ ID No. 2. Additionally or alternatively, recombinant cells according to the present disclosure may be modified to lack more than one β -glucanase. For example, a recombinant cell may be modified to lack both a polypeptide according to SEQ ID NO. 1 and a polypeptide according to SEQ ID NO. 2. Alternatively, the recombinant cell may be modified to lack both a functional homolog of the polypeptide according to SEQ ID NO. 1 and a functional homolog of the polypeptide according to SEQ ID NO. 2. Alternatively, the recombinant cell may be modified to lack both the polypeptide according to SEQ ID NO. 1 and the functional homolog of the polypeptide according to SEQ ID NO. 2. Alternatively, the recombinant cell may be modified to lack both a functional homolog of the polypeptide according to SEQ ID NO. 1 and a polypeptide according to SEQ ID NO. 2.

Typically, a functional homolog of a polypeptide having an amino acid sequence according to SEQ ID No. 1 or SEQ ID No. 2 is a polypeptide capable of deglycosylating a mogroside product, e.g., by hydrolyzing one or more β -glycosidic bonds between two glucose moieties and/or between a glucose moiety and a mogrol backbone in a mogroside product, typically a polypeptide having at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of a polypeptide having an amino acid sequence according to SEQ ID No. 1 or SEQ ID No. 2.

The present inventors have found that in strains capable of producing one or more mogrosides and lacking EXG1 glucanase production, the production of certain mogrosides comprising a β -glycosidic bond at C3 and/or C24 of the mogrol backbone (e.g. mogroside IE ₁, mogroside IA ₁, mogroside IIIE, siamenoside I) remains limited. Surprisingly, when both cells are cultured under the same conditions, in a recombinant cell capable of producing one or more mogrosides and lacking the polypeptide according to SEQ ID No. 1, the production of mogrosides comprising a β -glycosidic linkage at C3 and/or C24 of the mogrol backbone is improved if compared to an otherwise identical recombinant cell that has not been modified to lack the polypeptide according to SEQ ID No. 1. In one embodiment, when both cells are cultured under the same conditions, in a recombinant cell capable of producing one or more mogrosides and lacking the polypeptide according to SEQ ID No. 1, the production of mogrosides comprising a β -glycosidic linkage at C3 of the mogrol backbone is improved if compared to an otherwise identical recombinant cell that has not been modified to lack the polypeptide according to SEQ ID No. 1. In another embodiment, the production of mogrosides comprising a β -glycosidic linkage at C24 of the mogrol backbone is improved in a recombinant cell capable of producing one or more mogrosides and lacking the polypeptide according to SEQ ID No. 1 when both cells are cultured under the same conditions, if compared to an otherwise identical recombinant cell that has not been modified to lack the polypeptide according to SEQ ID No. 1. In yet another embodiment, when both cells are cultured under the same conditions, in a recombinant cell capable of producing one or more mogrosides and lacking the polypeptide according to SEQ ID NO. 1, the production of mogrosides comprising a β -glycosidic linkage at C3 and C24 of the mogrol backbone is increased if compared to an otherwise identical recombinant cell that has not been modified to lack the polypeptide according to SEQ ID NO. 1. In the context of the present disclosure, increased mogroside production in a recombinant cell lacking a polypeptide (e.g., a polypeptide according to SEQ ID NO: 1 or SEQ ID NO: 2) if compared to an otherwise identical recombinant cell lacking the polypeptide (e.g., a polypeptide according to SEQ ID NO: 1 or SEQ ID NO: 2) refers to an increase in the yield or amount or concentration of mogroside produced by a recombinant cell lacking the polypeptide (e.g., a polypeptide according to SEQ ID NO: 1 or SEQ ID NO: 2) when both cells are cultured under the same conditions as compared to an otherwise identical recombinant cell that has not been modified. The lack of the polypeptide according to SEQ ID NO. 1 results in an increased production of mogrosides containing a beta-glycosidic bond at the C3 and/or C24 position of the mogrol backbone. Essentially, the modified recombinant cells are capable of producing more desired mogrosides under the same conditions as the unmodified cells.

Thus, a recombinant cell capable of producing one or more mogrosides that has been modified to result in the absence of one or more β -glucanases as disclosed herein may be modified to result in the absence of a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID No. 1, typically a polypeptide having an amino acid sequence that is at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of a polypeptide having an amino acid sequence according to SEQ ID No. 1, and a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID No. 2, typically a polypeptide having an amino acid sequence that is at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of a polypeptide having the amino acid sequence shown in SEQ ID No. 2.

The inventors have also found that for a recombinant strain capable of producing one or more mogrosides and lacking both the polypeptide according to SEQ ID No. 1 and the polypeptide according to SEQ ID No. 3 (encoding the EXG1 polypeptide), the production of e.g. mogroside II a ₂ is synergistically increased if compared to an otherwise identical recombinant cell which has not been modified to lack the polypeptides according to SEQ ID No. 1 and SEQ ID No. 3, for example, a mogroside having a higher degree of glycosylation (i.e. comprising a secondary glycosylated mogroside at the C3 and/or C24 position) when both cells are cultured under identical conditions.

In one embodiment of the method and recombinant cell according to the present disclosure, a recombinant cell according to the present disclosure capable of producing a mogroside precursor or one or more mogrosides is a cell, wherein the absence of a polypeptide capable of deglycosylating a mogroside product results in an increased production of one or more mogrosides when both cells are cultured under the same conditions, if compared to an otherwise identical recombinant cell without the polypeptide. In other words, when both cells are cultured under the same conditions, a recombinant cell lacking the polypeptide is able to produce more desired mogrosides under the same conditions as an otherwise identical recombinant cell not lacking the polypeptide.

Thus, recombinant cells that have been modified to result in the absence of a beta glucanase as disclosed herein capable of producing one or more mogrosides may be further modified to result in the absence of an EXG1 and/or EXG2 polypeptide, in particular a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID No. 3. In one embodiment, the recombinant cell may be modified to result in a lack of polypeptides having an amino acid sequence at least 50% identical to the amino acid sequence set forth in SEQ ID NO. 1, typically polypeptides having an amino acid sequence at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of a polypeptide having an amino acid sequence set forth in SEQ ID NO. 1, and polypeptides having an amino acid sequence at least 50% identical to the amino acid sequence set forth in SEQ ID NO. 3, typically polypeptides having an amino acid sequence at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of a polypeptide having an amino acid sequence set forth in SEQ ID NO. 3.

The inventors have also surprisingly found that in recombinant cells capable of producing one or more mogrosides and lacking the polypeptide according to SEQ ID No. 2, the production of mogrosides, e.g. mogroside IIIA ₁, comprising β -1,2 and/or β -1,6 glycosidic linkages can be increased if compared to otherwise identical recombinant cells lacking the EXG1 polypeptide (e.g. a polypeptide having an amino acid sequence according to SEQ ID No. 3). The increase in production of mogroside IIIA ₁ may be synergistically increased in a recombinant cell capable of producing one or more mogrosides and lacking a polypeptide according to SEQ ID NO: 2, which further lacks the production of a polypeptide according to SEQ ID NO: 3.

Thus, recombinant cells capable of producing a mogroside precursor or one or more mogrosides that have been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product may be modified to result in a lack of a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO. 2, typically a polypeptide having an amino acid sequence that is at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of a polypeptide having an amino acid sequence according to SEQ ID NO. 2, and a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO. 3, typically a polypeptide having an amino acid sequence that is at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 98% or at least 99% identical to the amino acid sequence of a polypeptide having the amino acid sequence shown in SEQ ID NO. 3.

In yet another embodiment, a recombinant cell capable of producing a mogroside precursor or one or more mogrosides that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product may be modified to result in a lack of a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 1, typically a polypeptide having an amino acid sequence that is at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of a polypeptide according to the amino acid sequence of SEQ ID NO: 1; and polypeptides having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO. 2, typically polypeptides having an amino acid sequence at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of a polypeptide according to the amino acid sequence of SEQ ID NO. 2, and polypeptides having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO. 3, typically polypeptides having an amino acid sequence at least 50%, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to the amino acid sequence of a polypeptide according to the amino acid sequence of SEQ ID NO. 3 Or at least 99% sequence identity.

Thus, in one embodiment of the method and recombinant cell according to the present disclosure, a recombinant cell is provided that is capable of producing one or more mogrosides and lacks the beta glucanase as disclosed above, wherein the production of the mogroside product of the cell is higher if compared to the production of a mogroside product of an otherwise identical recombinant cell that has not been modified to lack the beta glucanase when both cells are cultured under the same conditions. In one aspect, when both cells are cultured under the same conditions, the production of a mogroside product comprising a β -glycosidic linkage at C3 and/or C24 of the mogroside backbone is higher for a cell modified to lack the β -glucanase than for an otherwise identical recombinant cell that has not been modified to lack the β -glucanase. For example, in one aspect, when both cells are cultured under the same conditions, the production of mogroside IE ₁, mogroside IIE, and/or mogroside IA ₁ (i.e., yield, concentration, or amount) produced by a recombinant cell modified to lack beta glucanase, if compared to the production (i.e., yield, concentration, or amount) of the same mogroside produced by an otherwise identical recombinant cell that has not been modified to lack beta glucanase, Concentration or amount) is higher. In particular, mogroside IE ₁ and/or mogroside IA ₁ are produced (i.e., produced, concentrated or in amounts) higher. In another aspect, when both cells are cultured under the same conditions, the production of a mogroside product comprising a β -1, 2-glycosidic linkage between the second glucose molecule and β -1-glucose bound at the C3 and/or C24 atoms of the mogroside backbone is higher if produced by a cell modified to lack β -glucanase as compared to the production of an otherwise identical recombinant cell that has not been modified to lack a polypeptide capable of deglycosylating the mogroside product. For example, in another aspect, when two cells are cultured under the same conditions, if compared to the production (i.e., yield, concentration, or amount) of the same mogroside produced by an otherwise identical recombinant cell that has not been modified to lack beta glucanase, production of mogroside IIA, mogroside IIIA ₁, mogroside IIIE, mogroside V, mogroside VI, mogroside IVE and/or siamenoside I (i.e., yield), Concentration or amount) is higher. In particular, the production of mogroside IIIA ₁ can be increased. In yet another aspect, when both cells are cultured under the same conditions, the production of a mogroside product comprising a β -1, 6-glycosidic linkage between the second glucose molecule and β -1-glucose bound at the C3 atom and/or the C24 atom of the mogroside backbone is higher if produced by a cell modified to lack β -glucanase as compared to the production of an otherwise identical recombinant cell that has not been modified to lack a polypeptide capable of deglycosylating the mogroside product. For example, in yet another aspect, when two cells are cultured under the same conditions, if compared to the production (i.e., yield, concentration, or amount) of the same mogroside produced by an otherwise identical recombinant cell that has not been modified to lack beta glucanase, mogroside IIA ₁ produced by recombinant cells modified to lack the beta-glucanase mogroside IIA ₂, Mogroside IIIA ₂, mogroside IVE, mogroside IVA, mogroside III, mogroside IIIA ₁, mogroside V, mogroside VI and/or siamenoside I are produced (i.e. produced, concentrated or quantified) more. In particular, the production (i.e., yield, concentration or amount) of mogroside IIA ₂ may be increased.

The present disclosure also provides a recombinant cell capable of producing one or more mogrosides and lacking the beta glucanase as disclosed above, wherein the production of the mogroside product of the cell is higher when both cells are cultured under the same conditions, if compared to the production of an otherwise identical recombinant cell that has been modified to lack a polypeptide having the amino acid sequence shown in SEQ ID No. 3. In one aspect, when both cells are cultured under the same conditions, production of a mogroside product comprising a β -glycosidic linkage at C3 and/or C24 of the mogroside backbone is higher in cells modified to lack β -glucanase as compared to production of otherwise identical recombinant cells modified to lack a polypeptide having the amino acid sequence set forth in SEQ ID No. 3. In another aspect, when both cells are cultured under the same conditions, the production of a mogroside product comprising a β -1, 2-glycosidic linkage between the second glucose molecule and β -1-glucose bound at the C3 and/or C24 atom of the mogroside backbone is higher in a cell modified to lack β -glucanase as compared to the production of an otherwise identical recombinant cell modified to lack a polypeptide having the amino acid sequence shown in SEQ ID No. 3. In yet another aspect, when both cells are cultured under the same conditions, the production of a mogroside product comprising a beta-1, 6-glycosidic linkage between the second glucose molecule and beta-1-glucose bound at the C3 and/or C24 atom of the mogroside backbone is higher in a cell modified to lack beta glucanase as compared to the production of an otherwise identical recombinant cell modified to lack a polypeptide having the amino acid sequence shown in SEQ ID NO 3.

A polypeptide capable of deglycosylating a mogroside product as described herein, e.g., a polypeptide having an amino acid sequence as set forth in SEQ ID No. 1 or SEQ ID No. 2, or a polypeptide having an amino acid sequence at least 50% identical to any of SEQ ID No. 1 or SEQ ID No. 2, may be a polypeptide capable of deglycosylating a mogroside compound (which is a disaccharideated, trisaccharified, tetrasaccharified, penta-glycosylated or hexa-glycosylated mogroside compound) or an isomer thereof. The polypeptide may be capable of deglycosylating a mogroside product comprising mogroside IA ₁ (MogIA 1), mogroside IE ₁ (MogIE 1), mogroside IIA (MogIIA), mogroside IIA ₁ (MogIIA 1), mogroside IIA ₂ (MogIIA), mogroside IIE (MogIIE), mogroside IIIA ₁ (MogIIIA 1), mogroside IIIA ₂ (MogIIIA 2), mogroside IIIE (MogIIIE), mogroside III (MogIII), mogroside IVA (MogIVA), mogroside IVA (mogivie), mogroside V (MogV), mogroside VI (MogVI), mogroside VIA (MogVIA), mogroside VIA1 (MogVIa), mogroside VIB (MogVIB), siamenoside I (Sia), isosluo, isosorbide V (IsoV), or alpha-siamenoside I (isosia).

Thus, in one embodiment of the methods and recombinant cells according to the present disclosure, the recombinant cells capable of producing mogroside precursors or one or more mogrosides according to the present disclosure lack a polypeptide capable of deglycosylating a mogroside product, wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating a mogroside compound (which is a di-, tri-, tetra-, penta-, or hexa-glycosylated mogroside compound) or an isomer thereof, typically wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating at least one or more of the following: mogroside IA ₁, mogroside IE ₁, mogroside IIA ₁, mogroside IIA ₂, mogroside IIE, mogroside IIIA ₁, mogroside IIIA ₂, mogroside IIIE, mogroside III, mogroside IVA, mogroside IVE, mogroside V, mogroside VI, mogroside VIA ₁, mogroside VIB, siamenoside I, iso-mogroside IVE, iso-mogroside V, or alpha-siamenoside I.

Mogroside and/or a mogroside precursor may be synthesized starting from mogrol using one or more uridine 5' -diphosphate dependent (i.e., UDP-dependent) glycosyltransferase (UGT) polypeptides (EC: 2.4.1) (also referred to herein as UDP-glycosyltransferases). Thus, a recombinant cell according to the present disclosure capable of producing one or more mogrosides and/or mogroside precursors comprises a polynucleotide capable of (over) expressing at least one UGT polypeptide. A "UGT" polypeptide is a polypeptide capable of catalyzing the transfer of a glycosyl group from a UDP-sugar (glycosyl donor) to an acceptor molecule (e.g., mogrol or mogroside). As shown in fig. 4, mogroside synthesis begins with primary glycosylation of the mogrol molecule at the C3 carbon and/or the C24 carbon, producing mogroside IA ₁, mogroside IE ₁ and/or mogroside IIE by the action of one or more UGT polypeptides capable of catalyzing β -1 glycosylation of mogrol at positions C24, C3 and/or both C3 and C24, respectively. Or primary glycosylation may occur at position C11 or C25 of the mogrol skeleton. UGT polypeptides capable of primary glycosylation are herein indicated as primary UGT polypeptides. Thus, the primary UGT polypeptide may be capable of glycosylating a mogrol or mogroside compound at its C3 hydroxyl group, C24 hydroxyl group, C11 hydroxyl group, and/or C25 hydroxyl group. In other words, the primary UGT polypeptide may catalyze the glycosylation of hydroxyl groups in positions C3, C24, C11, and/or C25 of the mogrol backbone in mogrol or mogroside. as shown in FIG. 4, mogroside IA ₁, Mogroside IIE and/or mogroside IE ₁ may be converted to higher glycosylated mogroside compounds by one or more of beta-1, 2 and/or beta-1, 6 glycosylation of a hydroxyl group in the C2 'position of a glucose moiety (C24-O-glucose) attached to position C24 of the mogrol skeleton, and/or beta-1, 2 glycosylation of a hydroxyl group in the C2' position of a glucose moiety (C3-O-glucose) attached to position C3 of the mogrol skeleton, via a beta-1 glycosidic bond. UGT polypeptides capable of catalyzing beta-1, 2 and/or beta-1, 6 glycosylation of mogrosides are referred to as secondary UGT polypeptides. Starting from mogroside IA ₁, mogroside IIE and/or mogroside IE ₁, mogroside IIA1, mogroside IIA2, mogroside IIIA1, mogroside IIIA2, mogroside IIIE, mogroside III, mogroside IVA, One or more of mogroside IVE, mogroside V, mogroside VI, mogroside VIA1, mogroside VIB, siamenoside I, isosorbide IVE, isosorbide V, or alpha-siamenoside I.

Thus, in one aspect of the methods and recombinant cells according to the present disclosure, a recombinant cell capable of producing one or more mogroside and/or mogroside precursors and that has been modified to result in the lack of production of a beta glucanase according to the present disclosure is a cell comprising, capable of (over) expressing (e.g., under specific conditions) or (over) expressing one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of glycosylating a mogrol or mogroside compound at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group and/or C25 hydroxyl group, typically at its C3 hydroxyl group and/or C24 hydroxyl group;

(b) A polynucleotide encoding a polypeptide capable of β -1, 2-glycosylation of the C2 'hydroxyl group of the glucose moiety (C24-O-glucose) at the C24 position of a mogroside compound and/or capable of β -1, 2-glycosylation of the C2' hydroxyl group of the glucose moiety (C3-O-glucose) at the C3 position of a mogroside compound;

(c) A polynucleotide encoding a polypeptide capable of β -1, 6-glycosylation of the C6 'hydroxyl group of the glucose moiety at the C3 position of the mogroside compound and/or capable of β -1, 6-glycosylation of the C6' hydroxyl group of the glucose moiety at the C24 position of the mogroside compound;

In particular wherein the polypeptide according to (a), (b) or (c) is a uridine 5' -diphosphate dependent glycosyltransferase polypeptide (UGT polypeptide). In general, one or more of the polypeptides and/or polynucleotides according to (a), (b) or (c) may be a heterologous polypeptide and/or a heterologous polynucleotide. In general, one or more of the polynucleotides according to (a), (b) or (c) may be constitutively expressed. In another aspect, expression of one or more of the polynucleotides according to (a), (b) or (c) may be induced.

The polypeptide according to (a), (b) or (c), in particular the UGT polypeptide, may be any UGT polypeptide known in the art, e.g. any of the polypeptides having the amino acid sequences as depicted in table 2a to table 2 e.

In one aspect of the disclosure, a UGT polypeptide according to the disclosure can be any polypeptide having an amino acid sequence according to SEQ ID No. 6, or SEQ ID No. 14 to SEQ ID No. 18, or any of the amino acid sequences shown in tables 2a to 2e, or alternatively it can be a UGT polypeptide, typically a functional homolog thereof, having an amino acid sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of any polypeptide according to SEQ ID No. 6, or SEQ ID No. 14 to SEQ ID No. 18, or any amino acid sequence as shown in tables 2a to 2 e.

In one aspect of the disclosure, the polypeptide, particularly a UGT polypeptide capable of glycosylating a mogrol or mogroside compound at its C3 hydroxyl group, may be any polypeptide disclosed in the art, e.g., a UGT polypeptide having an amino acid sequence as shown in table 2a, or alternatively it may be a UGT polypeptide, e.g., a functional homolog thereof, having an amino acid sequence that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of any polypeptide having an amino acid sequence shown in table 2 a.

In another aspect of the disclosure, the polypeptide, particularly a UGT polypeptide capable of glycosylating a mogrol or mogroside compound at its C24 hydroxyl group, may be any polypeptide disclosed in the art, e.g., a UGT polypeptide having an amino acid sequence as shown in table 2b, or alternatively it may be a UGT, e.g., a functional homolog thereof, having an amino acid sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of any polypeptide having an amino acid sequence shown in table 2 b.

In yet another aspect of the disclosure, the polypeptide, particularly a UGT polypeptide capable of β -1, 2-glycosylation of the C2 'hydroxyl group of the glucose moiety (C24-O-glucose) at position C24 of mogroside and/or capable of β -1, 2-glycosylation of the C2' hydroxyl group of the glucose moiety (C3-O-glucose) at position C3 of a mogroside compound, may be any polypeptide disclosed in the art, e.g., a UGT polypeptide having an amino acid sequence as shown in table 2C, e.g., a functional homolog thereof, or alternatively it may be a UGT polypeptide having an amino acid sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any polypeptide having an amino acid sequence as shown in table 2C.

In yet another aspect of the disclosure, the polypeptide, particularly a UGT polypeptide capable of β -1, 6-glycosylation of the C6 'hydroxyl group of the glucose moiety at position C3 of the mogroside compound and/or capable of β -1, 6-glycosylation of the C6' hydroxyl group of the glucose moiety at position C24 of the mogroside compound, may be any polypeptide disclosed in the art, e.g., a UGT polypeptide having an amino acid sequence as shown in table 2d, or the amino acid sequence of the UGT polypeptide may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of any polypeptide having an amino acid sequence as shown in table 2 d.

Tables 2a through 2e provide non-limiting examples of the amino acid sequences of UGT polypeptides disclosed in the patent literature and their ability to perform specific glycosylation according to the patent literature.

Table 2a reports the amino acid sequence of UGT polypeptides having the ability to catalyze beta-1 glycosylation at the C3 carbon of the mogrol backbone in mogrol or mogroside, according to the patent literature to which they pertain. Table 2b reports the amino acid sequence of UGT polypeptides having the ability to catalyze beta-1 glycosylation at the C24 carbon of the mogrol backbone in mogrol or mogroside, according to the patent literature to which they pertain. Similarly, table 2C reports the amino acid sequence of UGT polypeptides having β -1,2 glycosylation activity at glucose located at C3 and/or C24 of the mogrol or mogroside backbone, and table 2d reports the amino acid sequence of UGT polypeptides having β -1,6 glycosylation activity at glucose located at C3 and/or C24 of the mogrol or mogroside backbone. Finally, table 2e reports the amino acid sequences of those UGT polypeptides for which specific UGT activity types are not indicated in the corresponding patent literature. Specific substrates are given in the table when known from the corresponding patent literature. For example, UGT94C9 can use MogIVE as a substrate to produce Mog V, indicated as "UGT94C9 (MogIVE)" in table 2 d. When known from the patent literature, the product of the UGT catalyzed reaction is also indicated. For example, UGT98 can convert MogIIE to MogIIIA2, indicated in table 2d as UGT98 (MogIIE →iiia2). The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 2a

TABLE 2b

TABLE 2c

TABLE 2d

TABLE 2e

The recombinant cells have been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure, and wherein the recombinant cells further comprise, in particular, wherein the recombinant cells are capable of (over) expressing (e.g., under specific conditions) or (over) expressing one or more nucleotides encoding one or more UGT polypeptides as disclosed herein, can be used in a method of producing one or more mogrosides, the method comprising contacting a mogrol or one or more mogroside substrates with the cells or a lysate or extract thereof under conditions suitable for producing the one or more mogrosides, optionally isolating the one or more mogrosides.

Accordingly, in one aspect, the present disclosure provides a method of producing one or more mogroside and/or mogroside precursors, the method comprising culturing a recombinant cell that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure and comprising the ability to express or express at least one polynucleotide encoding a UGT polypeptide as disclosed above under conditions suitable for the production of the mogroside and/or mogroside precursors, optionally isolating the one or more mogroside and/or mogroside precursors.

Depending on the type of mogroside desired to be produced and depending on the mogroside precursor used in the method, the cell will comprise, will be able to (over) express or will (over) express one or more polynucleotides encoding UGT polypeptides.

For example, a recombinant cell that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure and capable of producing MogIA a starting from mogrol may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its C24 hydroxyl group (e.g., one or more of the suitable polypeptides disclosed in table 2 b).

Recombinant cells that have been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure and capable of producing MogIE a starting from mogrol may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its C3 hydroxyl group (e.g., one or more of the suitable polypeptides disclosed in table 2 a).

Recombinant cells that have been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure and capable of producing MogIIE from mogrol may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its C3 hydroxyl group and at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its C24 hydroxyl group (e.g., one or more of the suitable polypeptides disclosed in table 2a and table 2b, respectively).

Recombinant cells that have been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure and capable of producing MogIIIE from mogrol may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol or mogroside at its C3 hydroxyl group and/or its C24 hydroxyl group (e.g., one or more of the suitable polypeptides disclosed in table 2a and table 2 b), and at least one polynucleotide encoding a polypeptide capable of β -1, 2-glycosylation of the C2' hydroxyl group of the glucose moiety at position C24 of a mogroside compound (e.g., one or more of the suitable polypeptides disclosed in table 2C).

A recombinant cell that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure and capable of producing MogIIIA a starting from mogrol may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol or mogroside at its C24 hydroxyl group (e.g., one or more of the suitable polypeptides disclosed in table 2 b), at least one polynucleotide encoding a polypeptide capable of β -1, 2-glycosylating the C2 'hydroxyl group of the glucose moiety at position C24 of a mogroside compound (e.g., one or more of the suitable polypeptides disclosed in table 2C), and at least one polynucleotide encoding a polypeptide capable of β -1, 6-glycosylating the C2' hydroxyl group of the glucose moiety at position C24 of a mogroside compound (e.g., one or more of the suitable polypeptides disclosed in table 2 d).

A recombinant cell that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure and capable of producing Sia from mogrol may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol or mogroside at its C3 hydroxyl group and its C24 hydroxyl group (e.g., one or more of the suitable polypeptides disclosed in table 2a and table 2b, respectively), at least one polynucleotide encoding a polypeptide capable of β -1, 2-glycosylating the C2 'hydroxyl group of the glucose moiety at position C24 of a mogroside compound (e.g., one or more of the suitable polypeptides disclosed in table 2C), and at least one polynucleotide encoding a polypeptide capable of β -1, 6-glycosylating the C2' hydroxyl group of the glucose moiety at position C24 of a mogroside compound (e.g., one or more of the suitable polypeptides disclosed in table 2 d).

FIG. 4 depicts putative mogroside biosynthesis pathways from mogrols to mogrosides, including the type of UGT polypeptide required for the production of each mogroside.

Research （Tang Q、Ma X、Mo C、Wilson IW、Song C、Zhao H、Yang Y、Fu W、Qiu D. "An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis." BMC Genomics (2011) 12:343）. has been conducted on putative mogroside biosynthesis in momordica grosvenori and some of the enzymes involved therein subsequently, methods of using recombinant cells to produce mogroside precursors and mogrosides have been described (see, e.g., WO2014/086842 A1, WO2016/038617 A1, WO2016/050890 A2).

Mogrosides have a triterpene skeleton. Triterpenes, such as mogrosides, are synthesized via the isoprenoid pathway by cyclization of 2, 3-epoxysqualene to produce the cucurbitane skeleton. The triterpene scaffold is then subjected to various transformations, such as oxidation mediated by cytochrome P450-dependent monooxygenases and other enzymes, substitution, and glycosylation mediated by UGT polypeptides as described above.

FIG. 3 depicts putative biosynthesis from squalene to mogrol. Squalene may in turn be formed by the mevalonate pathway (MVA pathway) starting from acetyl CoA (acetyl-CoA). The biosynthesis of squalene from acetyl-CoA is well known and is depicted in FIG. 2. Alternatively, the synthesis may be carried out starting from 1-deoxy-D-xylulose 5-phosphate via the (MEP/DOXP) pathway, which is an alternative metabolic pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (W. EISENREICH, A. Bacher, D. Arigoni and F. Rohdich. "Biosynthesis of isoprenoids via the non-mevalonate pathway." Cell. Mol. Life Sci. (2004) 61:1401-1426）.

Squalene is a biochemical precursor of steroids, an essential component in all cells. Therefore, squalene is synthesized in all cells. In order to enable the recombinant cells to produce momordica alcohol, the cells may be transformed with one or more polypeptides capable of catalyzing several steps (e.g., the steps depicted in fig. 3), which steps result in the synthesis of momordica alcohol.

Synthesis of mogrols from squalene may include one or more of the steps described below.

2, 3-Epoxysqualene can be obtained from squalene by the action of a polypeptide having squalene epoxidase activity. Squalene epoxidase can also catalyze the conversion of 2, 3-epoxysqualene to 2,3,22,23-dioxisqualene.

Cucurbitadienol may be obtained from 2, 3-epoxysqualene by the action of cucurbitadienol synthase. Cucurbitadienol synthase also catalyzes the cyclization of 2,3,22,23-dioxisqualene to 24, 25-epoxy-cucurbitadienol.

Cucurbitadienol, 24, 25-epoxy-cucurbitadienol, and 24, 25-dihydroxy-cucurbitadienol, respectively, may be converted to 11-hydroxy cucurbitadienol, 11-hydroxy-24, 25-epoxy-cucurbitadienol, and mogrol by a combination of one or more cytochrome P450 enzymes (CYPs) and at least one CYP activator, such as a Cytochrome P450 Reductase (CPR).

Cucurbitadienol and 11-hydroxy cucurbitadienol may be converted to 24, 25-epoxy-cucurbitadienol and 11-hydroxy-24, 25-epoxy-cucurbitadienol, respectively, by a combination of one or more cytochrome P450 enzymes and at least one CYP activator.

11-Hydroxy-24, 25-epoxy-cucurbitadienol and 24, 25-epoxy-cucurbitadienol, respectively, may be converted to mogrol and 24, 25-dihydroxy-cucurbitadienol, respectively, by one or more epoxide hydrolases (EPH).

WO2022/192688 A1 describes recombinant cells capable of producing mogrol precursors, mogrols and/or mogrosides, wherein the recombinant cells comprise a heterologous polynucleotide encoding a cytochrome b5 (CB 5) polypeptide. The recombinant cells are capable of producing more mogrol than control cells that do not comprise the heterologous polynucleotide.

Thus, synthesis of mogrol starting from squalene may comprise one or more steps, wherein a cytochrome b5 (CB 5) polypeptide, such as a CB5 polypeptide, may be used, such as those disclosed in WO2022/192688 A1.

Thus, in one embodiment of the method and recombinant cell according to the present disclosure, a recombinant cell is provided, which is capable of producing one or more mogroside and/or mogroside precursors, which recombinant cell has been modified to result in the absence of a beta glucanase according to the present disclosure, wherein the recombinant cell is further capable of producing mogrol, and further comprises, is capable of (over) expressing or (over) expressing one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of synthesizing 2, 3-epoxysqualene from squalene, or 2,3,22,23-dioxisqualene from 2, 3-epoxysqualene, in particular wherein said polypeptide is squalene epoxidase (SQE);

(b) A polynucleotide encoding a polypeptide capable of synthesizing cucurbitadienol from 2, 3-epoxysqualene, or synthesizing 24, 25-epoxycucurbitadienol from 2,3,22,23-epoxysqualene, in particular wherein said polypeptide is cucurbitadienol synthase (CDS);

(c) One or more polynucleotides encoding a polypeptide capable of synthesizing 11-hydroxy cucurbitadienol from cucurbitadienol, 11-hydroxy-24, 25-epoxy-cucurbitadienol from 24, 25-epoxy-cucurbitadienol or from 11-hydroxy cucurbitadienol, mogrol from 24, 25-dihydroxy-cucurbitadienol, or 24, 25-epoxy-cucurbitadienol from cucurbitadienol, in particular wherein the polypeptide is a cytochrome P450 enzyme (CYP 450);

(d) Polynucleotides encoding polypeptides capable of reducing a cytochrome P450 complex, in particular Cytochrome P450 Reductase (CPR);

(e) A polynucleotide encoding a polypeptide capable of synthesizing mogrol from 11-hydroxy-24, 25-epoxy-cucurbitadienol, or synthesizing 24, 25-dihydroxy-cucurbitadienol from 24, 25-epoxy-cucurbitadienol, in particular wherein the polypeptide is epoxide hydrolase (EPH);

(f) A polynucleotide encoding a cytochrome b5 polypeptide (CB 5).

In general, one or more of the polypeptides and/or polynucleotides according to (a), (b), (c), (d), (e) or (f) may be a heterologous polypeptide and/or a heterologous polynucleotide. In general, one or more of the polynucleotides according to (a), (b), (c), (d), (e) or (f) may be constitutively expressed. In another aspect, expression of one or more of the polynucleotides according to (a), (b), (c), (d), (e), or (f) may be induced.

Typically, a recombinant cell that has been modified to result in the lack of production of a beta-glucanase according to the present disclosure will comprise at least one polynucleotide selected from (a), (b), (c), (d), (e) or (f), or it will comprise at least two or more polynucleotides, preferably each polynucleotide from a different enzyme class selected from (a), (b), (c), (d), (e) or (f), e.g. at least three polynucleotides selected from (a), (b), (c), (d), (e) or (f), e.g. at least four polynucleotides selected from (a), (b), (c), (d), (e) or (f), preferably each polynucleotide from a different enzyme class, e.g. at least five polynucleotides selected from (a), (b), (c), (d), (e) or (f), preferably each polynucleotide from a different enzyme class, e.g. at least six polynucleotides selected from (a), (b), (c), (d), (e) or (f), preferably each polynucleotide from a different enzyme class), typically all polynucleotides (a), (b), (d), (e) or (f) (c), (d), (e) and (f).

In the methods and recombinant cells according to the present disclosure, polypeptides capable of synthesizing 2, 3-epoxysqualene from squalene, or 2,3,22,23-dioxisqualene from 2, 3-epoxysqualene, i.e., squalene epoxidases (SQEs), may be used.

Squalene epoxidase (EC 1.4.99.7) can catalyze the production of 2, 3-epoxysqualene (also known as oxidosqualene) from squalene, or 2,3,22,23-dioxisqualene (also known as dioxisqualene) from oxidosqualene, generally in the presence of NADPH. Thus, a recombinant cell capable of producing a mogroside precursor and/or one or more mogrosides according to the present disclosure may comprise, be capable of expressing or capable of expressing a polynucleotide encoding a polypeptide capable of synthesizing 2, 3-epoxysqualene from squalene or 2,3,22,23-dioxisqualene from 2, 3-epoxysqualene, in particular wherein the polypeptide is squalene epoxidase. Some recombinant cells may contain endogenous squalene epoxidase, in which case endogenous enzymes may be sufficient. Eukaryotic metabolism, such as yeast metabolism, presents endogenous oxidosqualene and squalene production pathways, and thus, if the recombinant host is eukaryotic, the steps may be endogenous to the recombinant host. However, it may be advantageous to enhance the expression of endogenous squalene epoxidase by any method known to the person skilled in the art.

Thus, in one embodiment, a recombinant cell capable of producing mogroside precursors and/or mogrosides that has been modified to result in the absence of a beta glucanase according to the present disclosure may comprise, be capable of (over) expressing or may be capable of (over) expressing at least one polynucleotide encoding a polypeptide capable of synthesizing 2, 3-epoxysqualene from squalene or 2,3,22,23-dioxisqualene from 2, 3-epoxysqualene, in particular wherein the polypeptide is squalene epoxidase (SQE). Table 3 provides a non-limiting example of SQE polypeptides that can be used in recombinant cells and methods according to the present disclosure. Thus, the SQE polypeptide may have an amino acid sequence according to SEQ ID NO. 23 herein, or according to any of the sequences disclosed in column 2 of Table 3 (under the heading "squalene epoxidase amino acid sequence"), or the SQE polypeptide may have an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with an amino acid sequence as shown in Table 3, or with any of the polypeptides according to SEQ ID NO. 23 herein.

Table 3 provides a non-limiting example of the amino acid sequence of the SQE polypeptides disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 3 Table 3

In the methods and recombinant cells according to the present disclosure, polypeptides, such as cucurbitadienol synthase (CDS), capable of synthesizing cucurbitadienol from 2, 3-epoxysqualene or synthesizing 24, 25-epoxy-cucurbitadienol from 2,3,22,23-epoxysqualene may be used. Cucurbitadienol synthase (EC: 5.4.99.8) is a triterpene cyclase, also known as oxidosqualene cyclase or triterpene cyclase, which has been isolated from Cucurbita (Cucurbina) plants (the Cucurbita M., adachi S and Ebizuka Y. "Cucurbitadienol synthase, the ﬁrst committed enzyme for cucurbitacin biosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterol biosynthesis" Tetrahedron (2004) 60:6995-7003）., as disclosed herein, may catalyze the conversion of an oxidosqualene (e.g., 2, 3-epoxysqualene or 2,3,22,23-dicarboxyl squalene) to a cucurbitadienol compound (e.g., cucurbitadienol and 24, 25-epoxy-cucurbitadienol). The activity of cucurbitadienol synthase may be measured by any method known in the art. For example, the activity of cucurbitadienol synthase may be measured in terms of the amount of cucurbitadienol compound produced per unit of enzyme. The skilled artisan may identify CDSs based on their ability to convert an oxidase to a cucurbitadienol compound. Alternatively, the novel CDS enzymes may be identified based on the presence of one or more domains in the protein that are associated with the activity of CDS based on a comparison of amino acid sequence to known CDS. The novel CDS may additionally be based on the three-dimensional structure of the novel CDS.

Thus, in one embodiment, a recombinant cell capable of producing mogroside precursors and/or mogrosides that has been modified to result in the absence of a beta glucanase according to the present disclosure may comprise, may be capable of (over) expressing or may be capable of (over) expressing at least one polynucleotide encoding a polypeptide capable of synthesizing cucurbitadienol from 2, 3-epoxysqualene or 24, 25-epoxysqualene from 2,3,22,23-epoxysqualene, in particular wherein the polypeptide is cucurbitadienol synthase (CDS). Table 4 provides a non-limiting example of CDS polypeptides that may be used in recombinant cells and methods according to the present disclosure. Thus, a CDS polypeptide may have an amino acid sequence according to SEQ ID No. 9 herein or according to any of the sequences disclosed in table 4, or a CDS polypeptide may have an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identical to the amino acid sequence of SEQ ID No. 9 or to any of the polypeptides having the amino acid sequences shown in table 4.

Table 4 provides a non-limiting list of amino acid sequences of CDS polypeptides disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 4 Table 4

In the methods and recombinant cells according to the present disclosure, cytochrome P450 polypeptides (abbreviated as CYP or CYP 450) may be used. CYP is a heme-b containing monooxygenase.

As disclosed herein, a cytochrome P450 polypeptide is defined as a heme-b containing enzyme that acts as a monooxygenase and catalyzes the use of the reducing capacity of NAD (P) (H), inserting one oxygen atom into the organic substrate RH while reducing another oxygen atom to water, as shown below:

RH + O2 + H⁺ + NAD(P)(H) → ROH + H2O + NAD(P)⁺

In cytochrome P450 polypeptides, the mechanism involves two sequential single electron transfer steps, in which electrons derived from NAD (P) (H) are transferred to the CYP heme center via one or more redox partners. Different systems have been classified and described in the literature, for example, hannem et al Biochimica et Biophysica Acta (2007) 1770:330-344; urlacer and Girhard, trends in Biotechnology (2012) 30 (1): 26-36; sadeghi and Gilardi, biotechnology AND APPLIED Biochemistry (2013) 60 (1): 102-110; roberts et al (2002) Journal of Bacteriology 184 (14), 3898-3908.

Depending on the number and organization of redox partners, the cytochrome P450 system can be organized into three topological types:

a) A 3-component system, i.e., a system in which electrons derived from NAD (P) (H) are transferred to P450 protein via 2 separate redox partners (e.g., FAD or FMN-containing flavodoxin reductase and ferredoxin bound to a thioiron cluster);

b) A 2-component system, for example, in which electrons derived from NAD (P) (H) are transferred via a separate redox partner (i.e., FMN/FAD-containing bisflavin reductase), also known as cytochrome P450 reductase (abbreviated CPR), to the P450 protein;

c) 1-component systems in which electrons derived from NAD (P) (H) are transferred to the P450 domain via a reductase domain fused to the P450 domain, such as a flavin reductase domain containing FMN and a ferredoxin domain (abbreviated Fdx) bound to the ferrosulfur cluster or as an alternative to a reductase containing FMN and Fdx, such as a reductase domain containing a FMN/FAD-containing bisflavin reductase (e.g., in Sadeghi et al, supra, fig. 1C);

CYP polypeptides according to the present disclosure may belong to a 2-component cytochrome P450 system (generally indicated as a class II P450 system, hannesman et al, sadeghi et al, supra) that requires a cytochrome P450 protein on the one hand, and a faD/FMN-containing di-flavin P450 reductase (also known as cytochrome P450 reductase or CPR) on the other hand, may be required to support the monooxygenase activity of the cytochrome P450 protein.

Or a CYP polypeptide according to the present disclosure may belong to a cytochrome P450 system that may require electron transfer protein flavodoxin reductase (FPR) and/or ferredoxin reductase (FDXR).

In methods and recombinant cells according to the present disclosure, a CYP polypeptide may be involved in hydroxylation of the C11 carbon of a mogroside precursor. These CYP enzymes can also be referred to as C11-hydroxylases. As used herein, a C11-hydroxylase polypeptide may be a CYP polypeptide capable of introducing a hydroxyl group at position 11 (i.e., at C11) of a mogroside precursor. In some embodiments, the C11-hydroxylase is capable of catalyzing the conversion of cucurbitadienol to 11-hydroxycucurbitadienol. In some other embodiments, the C11-hydroxylase is capable of catalyzing the conversion of 24, 25-epoxy-cucurbitadienol to 11-hydroxy-24, 25-epoxy-cucurbitadienol. In still other embodiments, the C11-hydroxylase is capable of catalyzing the conversion of 24, 25-dihydroxy-cucurbitadienol to mogrol. The CYP polypeptides according to the present disclosure may catalyze the oxidation of a mogroside precursor, such as the oxidation of cucurbitadienol to 24, 25-epoxy-cucurbitadienol, or the oxidation of 11-hydroxy cucurbitadienol to 11-hydroxy-24, 25-epoxy-cucurbitadienol.

Thus, in one embodiment, a recombinant cell capable of producing a mogroside precursor or mogroside that has been modified to result in the absence of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure may comprise, may (over) express or may (over) express at least one polynucleotide encoding a polypeptide capable of synthesizing 11-hydroxy cucurbitadienol from cucurbitadienol, 11-hydroxy-24, 25-epoxy-cucurbitadienol from 24, 25-dihydroxy-cucurbitadienol, mogrol from 24, 25-epoxy-cucurbitadienol, a polynucleotide encoding a polypeptide capable of synthesizing 24, 25-epoxy-cucurbitadienol from cucurbitadienol or 11-hydroxy-24, 25-epoxy-cucurbitadienol from 11-hydroxy-cucurbitadienol, in particular wherein the polypeptide is a cytochrome P450 enzyme (CYP 450). Table 5 provides a non-limiting example of CYP polypeptides that can be used in recombinant cells and methods according to the present disclosure. Thus, a CYP polypeptide can have an amino acid sequence according to any of the sequences disclosed in Table 5 or according to the amino acid sequences set forth in SEQ ID NO. 10 through SEQ ID NO. 12 herein, or the amino acid sequence of a CYP polypeptide can have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence according to the amino acid sequences set forth in SEQ ID NO. 10 through SEQ ID NO. 12 herein or with any of the polypeptides having the amino acid sequences set forth in Table 5.

Table 5 provides a non-limiting example of the amino acid sequence of the CYP polypeptides disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 5

In methods and recombinant cells according to the present disclosure, a cytochrome P450 polypeptide (CYP) may be used in combination with a Cytochrome P450 Reductase (CPR) to support monooxygenase activity.

Thus, in one embodiment, a recombinant cell capable of producing mogroside precursors and/or mogrosides that has been modified to result in the absence of a beta glucanase according to the disclosure may comprise, may be capable of (over) expressing or may be capable of (over) expressing at least one polynucleotide encoding a polypeptide capable of reducing a cytochrome P450 complex, in particular a Cytochrome P450 Reductase (CPR). Table 6 provides a non-limiting example of CPR polypeptides that can be used in recombinant cells and methods according to the present disclosure. Thus, a CPR polypeptide may have an amino acid sequence according to any of the sequences disclosed in Table 6 or as set forth in SEQ ID NO. 8 herein, or the amino acid sequence of a CPR polypeptide may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8 herein or to any of the polypeptides having an amino acid sequence set forth in Table 6.

Table 6 provides a non-limiting example of the amino acid sequence of CPR polypeptides disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 6

In methods and recombinant cells according to the present disclosure, epoxide hydrolase (EPH) may be used.

Epoxide hydrolase enzymes are enzymes of the EC 3.3.Xx family that catalyze the hydrolysis of epoxides to produce diols.

In the context of the present disclosure, epoxide hydrolase polypeptides as disclosed herein may catalyze the conversion of 11-hydroxy-24, 25-epoxy-cucurbitadienol to mogrol. Alternatively or additionally, epoxide hydrolase as disclosed herein can catalyze the conversion of 24, 25-epoxy-cucurbitadienol to 24, 25-dihydroxy-cucurbitadienol.

Thus, in one embodiment, a recombinant cell capable of producing a mogroside precursor and/or mogroside that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure may comprise, may be capable of (over) expressing or may be capable of (over) expressing at least one polynucleotide encoding a polypeptide capable of synthesizing mogrol from 11-hydroxy-24, 25-epoxy-cucurbitadienol or 24, 25-dihydroxy-cucurbitadienol from 24, 25-epoxy-cucurbitadienol, in particular wherein the polypeptide is an epoxide hydrolase (EPH). Table 7 provides a non-limiting example of EPH polypeptides that can be used in recombinant cells and methods according to the present disclosure. Thus, an EPH polypeptide may have an amino acid sequence according to SEQ ID NO. 13 herein or according to any of the sequences disclosed in Table 7, or the amino acid sequence of said polypeptide may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence according to SEQ ID NO. 13 herein or to any of the polypeptides having an amino acid sequence as shown in Table 7.

Table 7 provides a non-limiting example of the amino acid sequence of an EPH polypeptide disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 7

WO2022/192688 A1 discloses host cells useful for the production of mogroside precursors and/or mogrosides, wherein the host cells comprise a heterologous polynucleotide encoding cytochrome b5, wherein the host cells are capable of producing more mogrol than control cells that do not comprise the heterologous polynucleotide. Thus, in methods and recombinant cells according to the present disclosure, cytochrome b5 polypeptides (CB 5) may be used.

Thus, in one embodiment of the method or recombinant cell according to the present disclosure, a recombinant cell capable of producing a mogroside precursor and/or mogroside that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure may comprise, may be capable of (over) expressing or may (over) express at least one polynucleotide encoding a cytochrome b5 polypeptide (CB 5). Table 8 provides a non-limiting example of CB5 polypeptides that can be used in recombinant cells or methods according to the present disclosure. Thus, a CB5 polypeptide may have an amino acid sequence according to any of the sequences disclosed in Table 8 or according to the amino acid sequence set forth in SEQ ID NO. 7 herein, or the amino acid sequence of said polypeptide may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of any of the polypeptides having the amino acid sequence set forth in Table 8 or according to SEQ ID NO. 7 herein.

Table 8 provides a non-limiting example of the amino acid sequence of a CB5 polypeptide as disclosed in WO2022/192688 A1. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 8

In methods and recombinant cells according to the present disclosure, recombinant cells that have been modified to result in the absence of beta glucanases according to the present disclosure capable of producing mogroside precursors and/or mogrosides may comprise, may be capable of (over) expressing or may be (over) expressing one or more polynucleotides encoding squalene epoxidase, cucurbitadienol synthase, cytochrome P450 polypeptide, C11-hydroxylase, cytochrome P450 reductase, epoxide hydrolase, cytochrome b5 polypeptide and/or UGT polypeptide.

Thus, in one aspect, disclosed herein is a recombinant cell capable of producing mogroside precursors and/or mogrosides that has been modified to result in the absence of a beta glucanase according to the disclosure, wherein the recombinant cell comprises, is capable of (over) expressing or (over) expressing one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a squalene epoxidase polypeptide (SQE);

(b) A polynucleotide encoding a cucurbitadienol synthase polypeptide (CDS);

(c) A polynucleotide encoding a cytochrome P450 polypeptide (CYP);

(d) A polynucleotide encoding a cytochrome P450 reductase polypeptide (CPR);

(e) A polynucleotide encoding epoxide hydrolase (EPH);

(f) A polynucleotide encoding a cytochrome b5 polypeptide (CB 5);

(g) A polynucleotide encoding a UGT polypeptide.

Typically, one or more of the polypeptides and/or polynucleotides according to (a), (b), (c), (d), (e), (f) or (g) is a heterologous polypeptide and/or a heterologous polynucleotide. In general, one or more of the polynucleotides according to (a), (b), (c), (d), (e), (f) or (g) may be constitutively expressed. In another aspect, expression of one or more of the polynucleotides according to (a), (b), (c), (d), (e), or (f) may be induced.

Typically, a recombinant cell that has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure will comprise at least one polynucleotide selected from (a), (b), (c), (d), (e), (f) or (g), or it will comprise at least two or more polynucleotides, preferably each polynucleotide from a different enzyme class selected from (a), (b), (c), (d), (e), (f) or (g), e.g. at least three polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g), e.g. at least four polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g), preferably each polynucleotide from a different enzyme class, e.g. at least five polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g), preferably each polynucleotide from a different enzyme class, e.g. at least six polynucleotides selected from (a), (b), (c), (d), (e), a) or (g), (f) Or (g) (preferably each polynucleotide is from a different enzyme class), typically all polynucleotides (a), (b), (c), (d), (e), (f) and (g).

In general, recombinant cells capable of producing mogroside precursors and/or mogrosides that have been modified to result in the absence of a polypeptide capable of deglycosylating a mogroside product as described herein are described herein as potentially capable of producing one or more of 2, 3-epoxysqualene, 2,3,22,23-diepoxyiene, cucurbitadienol, 11-hydroxycucurbitadienol, 24, 25-epoxy-cucurbitadienol, 11-hydroxy-24, 25-epoxy-cucurbitadienol, and/or 24, 25-dihydroxy-cucurbitadienol. The chemical structures of some of the precursors and intermediates of mogroside in the mogroside biosynthetic pathway are shown in fig. 5.

As mentioned above, squalene is a biochemical precursor of mogrols. Squalene is synthesized from farnesyl diphosphate (FPP) in a reaction catalyzed by squalene synthase (SQS), which is believed to be carried out in the presence of NADPH as co-substrate. The recombinant cell may comprise an endogenous squalene synthase, in which case said endogenous enzyme may be sufficient. Eukaryotic metabolism, such as yeast metabolism, presents an endogenous squalene production pathway, and thus, if the recombinant host is eukaryotic, the steps may be endogenous to the recombinant host. However, it may be advantageous to enhance the expression of endogenous squalene synthase by any method known to the person skilled in the art.

Thus, in one embodiment of the method and recombinant cell according to the present disclosure, the recombinant cell capable of producing a mogroside precursor and/or mogroside that has been modified to result in the absence of a beta glucanase according to the present disclosure comprises, is capable of (over) expressing or is capable of (over) expressing a polynucleotide encoding a polypeptide capable of synthesizing squalene from farnesyl diphosphate (FPP), in particular wherein the polypeptide is squalene synthase (EC: 2.5.1.21) (SQS). Table 9 provides a non-limiting example of SQS polypeptides that can be used in recombinant cells and methods according to the present disclosure. Thus, the SQS polypeptide can have an amino acid sequence according to any of the sequences disclosed in Table 9 or according to the amino acid sequence shown in SEQ ID NO: 24 herein, or the amino acid sequence of the SQS polypeptide can have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 24 herein or to any of the polypeptides having the amino acid sequences shown in Table 9.

Table 9 provides a non-limiting example of the amino acid sequence of the SQS polypeptides disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 9

Farnesyl diphosphate (FPP) and geranyl diphosphate (GPP) may be substrates in the production of squalene from dimethylallyl Diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Thus, in one embodiment, the recombinant cell may be modified to increase production of farnesyl diphosphate and/or geranyl diphosphate.

Farnesyl diphosphate (FPP) may be synthesized from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP) by farnesyl diphosphate Synthase (FPPs). In turn, geranyl diphosphate (GPP) can be synthesized from dimethylallyl Diphosphate (DMAPP) and isopentenyl diphosphate (IPP) by the enzymes geranyl diphosphate synthase (GPPS) and/or enzymatically farnesyl diphosphate synthase (FPPS).

Thus, in one embodiment of the method or recombinant cell according to the present disclosure, the recombinant cell that has been modified to result in the absence of beta glucanase according to the present disclosure capable of producing mogroside precursors and/or mogrosides comprises, is capable of (over) expressing or is capable of (over) expressing a polynucleotide encoding a polypeptide capable of synthesizing Ninyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP), in particular wherein the polypeptide is farnesyl diphosphate synthase (EC: 2.5.1.10) (FPPS). Table 10 provides a non-limiting example of FPPS polypeptides that can be used in recombinant cells and methods according to the present disclosure. Thus, the FPPS polypeptide may have an amino acid sequence according to the amino acid sequence as set forth in SEQ ID NO. 20 herein or according to any of the sequences disclosed in Table 10, or the amino acid sequence of the FPPS polypeptide may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 20 herein or to the amino acid sequence of any of the polypeptides having the amino acid sequence as set forth in Table 10.

Table 10 provides a non-limiting example of the amino acid sequences of FPPS polypeptides disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

Table 10

In one embodiment, a recombinant cell capable of producing mogroside precursors and/or mogrosides, which has been modified to result in the absence of a beta glucanase according to the disclosure, comprises, is capable of (over) expressing or is capable of (over) expressing a polynucleotide encoding a polypeptide capable of synthesizing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP), in particular wherein the polypeptide is geranyl pyrophosphate synthase (EC: 2.5.1.1) (GPPs) and/or farnesyl pyrophosphate synthase (EC: 2.5.1.10) (FPPS). Non-limiting examples of FPPS polypeptides that can be used in recombinant cells and methods according to the present disclosure have been provided previously. Non-limiting examples of GGPS polypeptides may be any suitable GGPS known to those of skill in the art, and may be, for example, from a prokaryotic or eukaryotic source. Table 11 provides a non-limiting example of GPPS polypeptides that can be used in recombinant cells and methods according to the present disclosure. Thus, a GPPS polypeptide may have an amino acid sequence according to any of the sequences disclosed in table 11, or the amino acid sequence of a GPPS polypeptide may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of any of the polypeptides of the amino acid sequences as shown in table 11.

Table 11 provides a non-limiting example of the amino acid sequence of GPPS polypeptides disclosed in the patent literature. The SEQ ID NOs in the tables are those in the corresponding patent documents to which they are disclosed.

TABLE 11

Thus, in one aspect of the methods and recombinant cells according to the present disclosure, a recombinant cell capable of producing a mogroside precursor and/or mogroside, comprising, being capable of (over) expressing or (over) expressing one or more of the following recombinant polynucleotides, has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product according to the present disclosure:

(a) Polynucleotides encoding polypeptides capable of synthesizing geranyl diphosphate (GPP) from dimethylallyl Diphosphate (DMAPP) and isopentenyl diphosphate (IPP), in particular wherein the polypeptides are geranyl diphosphate synthase (EC: 2.5.1.1) (GPPS) and/or farnesyl diphosphate synthase (EC: 2.5.1.10) (FPPS);

(b) Polynucleotides encoding polypeptides capable of synthesizing Ninyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP), in particular wherein said polypeptides are farnesyl diphosphate synthases (EC: 2.5.1.10) (FPPS);

(c) A polynucleotide encoding a polypeptide capable of synthesizing squalene from farnesyl diphosphate (FPP), in particular wherein said polypeptide is squalene synthase (EC: 2.5.1.21) (SQS).

Typically, one or more of the polypeptides and/or polynucleotides according to (a), (b) or (c) is a heterologous polypeptide and/or a heterologous polynucleotide. In general, one or more of the polynucleotides according to (a), (b) or (c) may be constitutively expressed. In another aspect, expression of one or more of the polynucleotides according to (a), (b) or (c) may be induced.

In general, a cell may comprise, may be capable of (over) expressing or may (over) express all polynucleotides according to (a), (b) and (c), or according to (a) and (b), or according to (a) and (c), or according to (b) and (c).

In some embodiments according to the present disclosure, recombinant cells capable of producing mogroside precursors and/or mogrosides that have been modified to result in the absence of beta glucanase may comprise or (over) express one or more polynucleotides encoding polypeptides belonging to the mevalonate pathway.

The mevalonate pathway refers to a well-known biosynthetic pathway depicted in FIG. 2 that converts acetyl-CoA to isopentenyl diphosphate. This biosynthetic pathway is typically found in eukaryotic cells, while prokaryotic cells produce isopentenyl diphosphate via the MEP pathway, a non-mevalonate-dependent pathway that converts glyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP.

The mevalonate pathway comprises the steps of:

(a) Synthesis of acetoacetyl-CoA (AACoA) from acetyl-CoA (AcCoA) catalyzed by acetyl-CoA acetyltransferase (EC: 2.3.1.9) (AACT);

(b) Synthesis of hydroxymethylglutaryl coenzyme A (HMGCoA) from acetoacetyl coenzyme A (AACoA) catalyzed by hydroxymethylglutaryl coenzyme A synthase (EC: 2.3.3.10) (HMGS);

(c) Synthesis of Mevalonate (MVA) from hydroxymethylglutaryl-CoA (HMGCoA) catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase (EC: 1.1.1.34) (HMGR);

(d) Synthesis of mevalonate-5-phosphate (MVA-P) from Mevalonate (MVA) catalyzed by mevalonate kinase (EC: 2.7.1.36) (MK);

(e) Synthesis of mevalonate-5-diphosphate (MVA-PP) from mevalonate-5-phosphate (MVA-P) catalyzed by phosphomevalonate kinase (EC: 2.7.4.2) (PMK);

(f) Synthesis of isopentenyl diphosphate (IPP) from mevalonate-5-diphosphate (MVA-PP) catalyzed by mevalonate decarboxylase diphosphate (EC: 4.1.1.33) (MDD) and/or isopentenyl/dimethylallyl diphosphate synthase (EC: 1.17.1.2) (IPPS);

(g) Synthesis of dimethylallyl Diphosphate (DMAPP) from isopentenyl diphosphate (IPP) catalyzed by isopentenyl-diphosphate delta-isomerase (EC: 5.3.3.2) (IPI).

In one embodiment of the method and recombinant cell according to the present disclosure, a recombinant cell capable of producing mogroside precursors and/or mogrosides that has been modified to result in the absence of beta glucanase may comprise, may be capable of (over) expressing or may (over) express one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of synthesizing acetoacetyl-coa (AACoA) from acetyl-coa (AcCoA), in particular wherein the polypeptide is an acetyl-coa acetyltransferase (also known as acetoacetyl-coa thiolase) (EC: 2.3.1.9) (AACT);

(b) A polynucleotide encoding a polypeptide capable of synthesizing hydroxymethylglutaryl-coenzyme a (HMGCoA) from acetoacetyl-coenzyme a (AACoA), in particular wherein the polypeptide is hydroxymethylglutaryl-coenzyme a synthase (EC: 2.3.3.10) (HMGS);

(c) A polynucleotide encoding a polypeptide capable of synthesizing Mevalonate (MVA) from hydroxymethylglutaryl-coa (HMGCoA), in particular wherein the polypeptide is 3-hydroxy-3-methylglutaryl-coa reductase (EC: 1.1.1.34) (HMGR);

(d) A polynucleotide encoding a polypeptide capable of synthesizing mevalonate-5-phosphate (MVA-P) from Mevalonate (MVA), in particular wherein the polypeptide is mevalonate kinase (EC: 2.7.1.36) (MK);

(e) A polynucleotide encoding a polypeptide capable of synthesizing mevalonate-5-diphosphate (MVA-PP) from mevalonate-5-phosphate (MVA-P), in particular wherein the polypeptide is phosphomevalonate kinase (EC: 2.7.4.2) (PMK);

(f) Polynucleotides encoding polypeptides capable of synthesizing isopentenyl diphosphate (IPP) from mevalonate-5-diphosphate (MVA-PP), in particular wherein the polypeptides are mevalonate diphosphate decarboxylase (EC: 4.1.1.33) (MDD) and/or isopentenyl/dimethylallyl diphosphate synthase (EC: 1.17.1.2) (IPPS);

(g) A polynucleotide encoding a polypeptide capable of synthesizing dimethylallyl Diphosphate (DMAPP) from isopentenyl diphosphate (IPP), in particular wherein said polypeptide is an isopentenyl-diphosphate delta-isomerase (EC: 5.3.3.2) (IPI).

Typically, one or more of the polypeptides and/or polynucleotides according to (a), (b), (c), (d), (e), (f) or (g) is a heterologous polypeptide and/or a heterologous polynucleotide. In general, one or more of the polynucleotides according to (a), (b), (c), (d), (e), (f) or (g) may be constitutively expressed. In another aspect, expression of one or more of the polynucleotides according to (a), (b), (c), (d), (e), (f) or (g) may be induced. Typically, a recombinant cell that has been modified to result in the absence of a beta-glucanase according to the present disclosure will comprise at least one polynucleotide selected from (a), (b), (c), (d), (e), (f) or (g), or it will comprise at least two or more polynucleotides, preferably each polynucleotide from a different enzyme class selected from (a), (b), (c), (d), (e), (f) or (g), e.g. at least three polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g), e.g. at least four polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g), preferably each polynucleotide from a different enzyme class, e.g. at least five polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g), preferably each polynucleotide from a different enzyme class, e.g. at least six polynucleotides selected from (a), (b), (c), (d), (e), a), (f) Or (g) (preferably each polynucleotide is from a different enzyme class), typically all polynucleotides (a), (b), (c), (d), (e), (f) and (g).

Any acetyl-coa acetyltransferase known in the art may be used in the context of the present disclosure. Non-limiting examples of suitable acetoacetyl-CoA thiolase enzymes that may be used are the ERG10 enzyme of UniProtKB accession number P41338, or the enzyme of UniProtKB accession number P10551, or the enzyme of UniProtKB/Swiss-prot: Q6L8K7.1, or any acetyl-CoA acetyltransferase with which the amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

Hydroxymethylglutaryl-coa synthase known in the art can be used to convert acetoacetyl-coa to hydroxymethylglutaryl-coa. Suitable non-limiting examples include protein ERG13 UniProtKB accession number P54839 or YALI0F 30841P NCBI number xp_506052.1 or SEQ ID No. 21, or any HMG-CoA synthase polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

3-Hydroxy-3-methylglutaryl-CoA reductase polypeptides suitable for use in strains and methods according to the present disclosure are known to those of skill in the art. Suitable non-limiting examples are SEQ ID NO 25, the HMG1 polypeptide of UniProtKB accession number P12683 or the HMG2 gene of UniProtKB accession number P12684 providing non-limiting examples of HMG-CoA reductase or HMG-CoA reductase polypeptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

Any mevalonate kinase known in the art may be used in the context of the present disclosure. Examples of suitable, non-limiting mevalonate kinases are the ERG12 polypeptide of UniProtKB accession No. P07277 or the mevalonate kinase of GenBank accession No. QNP96798.1, or any MK polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

Non-limiting examples of phosphomevalonate kinases that can be used in the methods and strains of the present disclosure are any PMK polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a polypeptide having GenBank accession number GFP66625 or a polypeptide having GenBank accession number VBB79048.1, uniProtKB/Swiss-Prot number D4GXZ3.1, or an amino acid sequence.

Non-limiting examples of mevalonate decarboxylase that can be used in methods and recombinant cells according to the present disclosure are the polypeptide of GenBank accession number CAA66158.1 or the polypeptide of GenBank accession number QNQ00565.1 or KAG5364843.1 or AAT93171.1, non-limiting examples of isopentenyl-diphosphate delta-isomerase are the polypeptides of UniProtKB/Swiss-Prot number P15496.2 or GenBank number QNQ01368.1, AOW06629.1, or any mevalonate decarboxylase or isopentenyl diphosphate delta-isomerase having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

As shown in fig. 3, 2, 3-epoxysqualene (oxidosqualene) can be converted to lanosterol by the enzyme lanosterol synthase (ERG 7). Lanosterol is a precursor in sterol production in fungal cells. Sterols are tetracyclic triterpene lipids required for critical cellular functions of all eukaryotes, including maintenance of membrane fluidity, phagocytosis, stress tolerance, and cell signaling.

In one embodiment of the method and recombinant cell according to the present disclosure, when the recombinant cell produces an endogenous lanosterol synthase, the level of 2, 3-epoxysqualene may be increased by, for example, reducing the activity of the endogenous lanosterol synthase or reducing the expression of the endogenous lanosterol synthase. In recombinant hosts expressing endogenous lanosterol synthase, this can be achieved by making the cells deficient or partially deficient in lanosterol synthase production. Thus, in one embodiment according to the present disclosure, recombinant cells that have been modified to result in the absence of beta glucanase capable of producing mogroside precursors and/or mogrosides may include the absence of production of lanosterol synthase polypeptides.

Since lanosterol is an essential metabolite in eukaryotes, it is preferred that cells be generally deficient in lanosterol synthase production by any method known in the art that does not completely eliminate lanosterol synthase activity. For example, the recombinant cell may be modified such that it produces less lanosterol synthase, e.g., by reducing the level of lanosterol synthase expression or by reducing the level of translation of mRNA transcribed from the gene encoding lanosterol synthase. In one embodiment, the expression level of the gene encoding lanosterol synthase may be reduced by replacing the lanosterol synthase endogenous promoter with a weaker promoter than the endogenous promoter, or by other suitable means. Alternatively, the gene encoding the natural lanosterol synthase may be replaced with a gene that still encodes the natural lanosterol synthase, but is less easily translated into protein by recombinant cells due to different codon usage. Or the recombinant cell may be modified to produce a polypeptide having reduced lanosterol synthase activity. For example, a recombinant cell may be modified by replacing a gene encoding a natural lanosterol synthase with a gene encoding a modified lanosterol synthase having reduced activity compared to a natural or homologous lanosterol synthase. Non-limiting examples of suitable lanosterol synthases with reduced activity are described in WO2022/212917 and WO 2022/212924.

In one embodiment, a recombinant cell capable of producing a mogroside precursor and/or mogroside that has been modified to result in the lack of a beta glucanase as disclosed herein may be capable of producing one or more mogrosides selected from mogroside IA, mogroside IE ₁, mogroside IIA ₁, mogroside IIA ₂, mogroside IIE, mogroside IIIA ₁, mogroside IIIA ₂, mogroside IIIE, mogroside IV _A, mogroside IV _E, mogroside V, mogroside VI, mogroside VIA1, mogroside VIB, siamenoside I, iso-mogroside IVE, iso-mogroside V, or alpha-siamenoside I. In general, the recombinant cell may comprise, may be capable of (over) expressing or may (over) express one or more of the following:

(a) A polynucleotide encoding a squalene epoxidase polypeptide (SQE);

(b) A polynucleotide encoding a cucurbitadienol synthase polypeptide (CDS);

(c) Polynucleotides encoding cytochrome P450 polypeptides (CYPs), such as C11-hydroxylase polypeptides and/or cyclooxygenase;

(d) A polynucleotide encoding a cytochrome P450 reductase polypeptide (CPR);

(e) Polynucleotides encoding epoxide hydrolase (EPH)

(F) A polynucleotide encoding a cytochrome b5 polypeptide (CB 5).

(G) A polynucleotide encoding a UGT polypeptide, in particular one or more of the following UGT polypeptides:

i. UGT capable of glycosylating mogrol or mogroside compounds at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group and/or C25 hydroxyl group,

A UGT capable of beta-1, 2-glycosylation of the C2 'hydroxyl group of the glucose moiety (C24-O-glucose) at position C24 of the mogroside compound and/or a UGT capable of beta-1, 2-glycosylation of the C2' hydroxyl group of the glucose moiety (C3-O-glucose) at position C3 of the mogroside compound,

Beta-1, 6-glycosylation of the C6 'hydroxyl group of the glucose moiety at position C3 of the mogroside compound and/or beta-1, 6-glycosylation of the C6' hydroxyl group of the glucose moiety at position C24 of the mogroside compound,

And optionally one or more of the following

A. Geranyl pyrophosphate synthase (GPPS) and/or farnesyl pyrophosphate synthase (FPPS);

B. Farnesyl diphosphate synthase (FPPS);

C. Squalene synthase (SQS);

D. acetyl-CoA acetyltransferase (AACT);

E. Hydroxymethylglutaryl-coenzyme a synthase (HMGS);

F. 3-hydroxy-3-methylglutaryl-coa reductase (HMGR);

G. mevalonate Kinase (MK);

H. Phosphomevalonate kinase (PMK);

I. mevalonate Diphosphate Decarboxylase (MDD) and/or isopentenyl/dimethylallyl diphosphate synthase (IPPS);

J. Isopentenyl-diphosphate delta-isomerase (IPI).

Recombinant cells that have been modified to result in the lack of beta glucanase enzymes as disclosed herein capable of producing mogroside and/or mogroside precursors may be capable of producing one or more mogroside and/or mogroside precursors when cultured under suitable conditions, such as in the presence of a suitable carbon source and other nutrients.

Thus, in one aspect, the present disclosure relates to a method of producing one or more mogroside and/or mogroside precursors, the method comprising culturing in a medium a recombinant cell capable of producing a mogroside and/or a mogroside precursor as disclosed herein, under conditions suitable for producing the mogroside and/or a mogroside precursor, optionally isolating the one or more mogroside and/or mogroside precursors.

Typically, the recombinant cells are grown in a fermenter at a defined temperature, pH for a desired period of time, typically under conditions that are involved in gene expression in the biosynthetic pathways that produce mogrosides and/or mogroside precursors as disclosed herein. Recombinant cells can be grown in different modes of operation (e.g., batch, fed-batch, or continuous processes) in a bioreactor or fermenter.

Media for various recombinant cells are well known in the art. The medium used to culture the recombinant bacterial cells will depend on the nature of the recombinant cells. The culture medium typically comprises inorganic salts and compounds, amino acids, carbohydrates, vitamins, and other compounds necessary for the growth of the recombinant cells or that improve the health or growth of the recombinant cells or both. Optimization of bacterial and yeast cell growth can also be achieved by adding nutrients and supplements to the medium. Or the culture may be grown in a fermenter designed to control temperature, pH and aeration rate. The culture conditions are generally selected from the group consisting of aerobic, microaerophilic and anaerobic. Thus, oxygen and nitrogen can be flowed into the medium as needed.

Any of the recombinant cells disclosed in the present application can be cultured in any type of medium. As will be appreciated by one of ordinary skill in the art, the culture conditions or culture process may be optimized by routine experimentation.

In another aspect, the present disclosure relates to a method of producing one or more mogrosides, the method comprising contacting one or more mogroside precursors or one or more mogroside substrates with a recombinant cell capable of producing one or more mogroside and/or mogroside precursors, which has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product disclosed herein or a lysate or extract thereof, under conditions suitable for producing the one or more mogroside and/or mogroside precursors, and optionally isolating the one or more mogrosides.

Cell lysates can be prepared from the recombinant cells disclosed herein and used to contact a substrate so that a mogroside compound can be produced. For example, cell lysates may be prepared from recombinant cells disclosed herein that express one or more UGTs and used to contact mogrols such that mogroside compounds may be produced. In some embodiments, the mogroside compound may be produced using whole cells fed with a raw material comprising a precursor molecule (e.g., mogrol or mogroside), such as a mogroside extract comprising the mogrol or mogroside. The raw materials may be fed during or after cell growth. The whole cells may be in a medium as described above and further comprise a suspension or immobilized mogrol, a mogroside substrate or a mogroside. Whole cells may be in fermentation broth or reaction buffer. In some embodiments, permeabilizing agents may be required to efficiently transfer the substrate into the cell. The method starting from whole cells and mogrol or mogroside precursors is called "whole cell bioconversion". Methods for producing mogrosides by whole cell bioconversion are described for example in WO 2013/076577, WO 2014/086842 and WO 2016/050890. The mogroside substrate is typically a mogroside compound having a lower level of glycosylation if compared to the target mogroside product to be produced according to the method.

Methods according to the present disclosure may result in the production of one or more mogrosides, e.g., mogroside IA, mogroside IE1, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIIA1, mogroside IIIA2, mogroside IIIE, mogroside III, mogroside IVA, mogroside IVE, mogroside V, mogroside VI, mogroside VIA1, mogroside VIB, siamenoside I, siamenoside IVE, siamenoside V, or a-siamenoside I, in particular one or more of mogroside IIIE, mogroside IIIA, or siamenoside I.

In the methods according to the present disclosure, mogroside compounds and/or mogroside precursors may optionally be isolated or recovered from the reaction medium and optionally further purified using various techniques known to those of skill in the art. For example, after fermentation, the culture broth may be treated to kill the recombinant cells and remove the cells either before or after cell wall disruption. For example, cell-free lysates may be obtained by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris. Mechanical disruption of the dried broth material may also be performed, for example by sonication. The dissolved or suspended culture broth material may be filtered. The fermentation medium or cell-free lysate may optionally be treated to remove low molecular weight compounds, such as salts, and may optionally be dried and redissolved in a mixture of water and solvent prior to purification.

The supernatant or cell-free lysate may be purified, for example, by means of adsorption chromatography using different types of resins and elution solvents. The level of mogroside precursors and/or mogroside compounds in each fraction (including the flow-through) can then be analyzed by LC-MS. The fractions may then be combined and reduced in volume using a vacuum evaporator. Additional purification steps, such as additional chromatographic steps and crystallization, may be utilized if desired. For example, the mogroside compound may be isolated by methods not limited to ion exchange chromatography, reverse phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon column and/or decolorization steps.

The present disclosure further provides a fermentation broth comprising recombinant cells as disclosed herein or comprising a lysate or extract thereof.

The present disclosure also provides a mogroside composition or sweetener composition obtainable by a process as disclosed herein. The mogroside or sweetener composition may comprise mogroside IA, mogroside IE1, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIIA1, mogroside IIIA2, mogroside IIIE, mogroside IVA, mogroside IVE, mogroside V, mogroside VI, mogroside VIA1, mogroside VIB, siamenoside I, isosluo mogroside IVE, isosluo mogroside V, or alpha-siamenoside I, in particular one or more of mogroside IIIE, mogroside IIIA, or siamenoside I.

Further provided is mogrosides obtainable or obtained by a method as disclosed herein. The mogrosides can be used in any application known for such compounds. In particular, mogrosides or their compositions can be used, for example, as sweeteners, for example in foods or beverages. For example, mogrosides can be formulated in soft drinks as table sweeteners, chewing gums, dairy products such as yogurt (e.g., raw yogurt), cakes, cereal or grain-based foods, nutraceuticals, pharmaceuticals, edible gels, confectionery products, cosmetics, toothpastes or other oral compositions, and the like. In addition, mogrosides are useful not only in beverages, foods and other products dedicated for human consumption, but also in animal feeds and forage with improved characteristics. Thus further provided is a food, feed or beverage comprising said mogroside. In the manufacture of food, beverages, pharmaceutical, cosmetic, table products, chewing gum, conventional methods such as mixing, kneading, dissolving, pickling, infiltration, diafiltration, spraying, atomizing, infusion and other methods may be used.

The mogrosides obtained as disclosed herein can be used in dry or liquid form. Which may be added before or after the heat treatment of the food product. The amount of sweetener depends on the purpose of use. It may be added alone or in combination with other compounds.

Mogrosides produced according to the methods as disclosed herein can be blended with one or more additional non-caloric or caloric sweeteners. Such blending may be used to improve flavor or time characteristics or stability. A variety of non-caloric and caloric sweeteners are suitable for blending with mogrosides. For example, non-caloric sweeteners such as steviol glycosides, monatin, aspartame, acesulfame k, cyclamate, sucralose, sugar salts or erythritol. Caloric sweeteners suitable for blending with mogrosides include sugar alcohols and carbohydrates such as sucrose, glucose, fructose, and HFCS. Sweet amino acids such as glycine, alanine or serine may also be used.

Mogrosides can be used in combination with a sweetener inhibitor (e.g., a natural sweetener inhibitor). It may be combined with an umami taste enhancer such as an amino acid or a salt thereof.

Mogrosides can be combined with polyols or sugar alcohols, carbohydrates, physiologically active substances or functional ingredients (e.g. carotenoids, dietary fibers, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, phytosterols or stanols (phyto-and phytostanols), polyols, prebiotics, probiotics, metazoans, phytoestrogens, soy proteins, sulfides/thiols, amino acids, proteins, vitamins, minerals and/or substances classified based on health benefits such as cardiovascular, cholesterol lowering or anti-inflammatory.

Compositions comprising mogrosides may include flavoring agents, aromatic components, nucleotides, organic acids, organic acid salts, inorganic acids, bitter compounds, proteins or protein hydrolysates, surfactants, flavonoids, astringent compounds, vitamins, dietary fibers, antioxidants, fatty acids and/or salts.

Mogrosides as disclosed herein can be applied as high intensity sweeteners to produce zero-calorie, low-calorie or diabetic beverages and food products with improved taste characteristics. It can also be used in beverages, foods, medicines and other products where sugar cannot be used.

In addition, mogrosides as disclosed herein are useful not only in beverages, foods and other products dedicated for human consumption, but also in animal feeds and forage with improved characteristics.

Examples of products in which mogrosides as disclosed herein may be used as a sweet compound include alcoholic beverages such as vodka, wine, beer, white spirit, sake, etc., natural juices, refreshing beverages, soft drinks including carbonated soft drinks, diet beverages, zero calorie beverages, low calorie beverages and foods, yogurt drinks, instant juices, instant coffee, powdered instant beverages, canned products, syrups, fermented soybean pastes, soy sauce, vinegar, condiments, mayonnaise, tomato paste, curry, soups, instant broths, soy sauce powder, vinegar powder, various types of biscuits, scented rice cakes, salted biscuits, bread, chocolate, caramel, candies, chewing gum, jellies, puddings, candies and pickles, fresh cream, jams, orange pastes, sugar creams, milk powder, ice cream, popsicle, vegetables and fruits packaged in bottles, canned and cooked beans, cooked meats and foods in sweet pastes, agricultural foods, seafood, ham, frozen fish, canned foods, fish, cured fish, sausages, meat, dried foods, cured products, cured meat, cured foods, dried kelp, medicinal products, and the like. In principle it can have unlimited applications.

Non-limiting examples of sweet compositions include beverages, including non-carbonated and carbonated beverages such as cola, ginger juice soda, root beer, apple juice, fruit flavored soft drinks (e.g., citrus flavored soft drinks such as lemon lime or orange juice), soft drink powders, and the like, juices from fruits or vegetables, including juice from juice presses and the like, fruit juices containing fruit particles, fruit beverages, fruit juice containing beverages, fruit flavored beverages, vegetable juices, vegetable juice containing, and fruit and vegetable mixed juices, sports drinks, energy beverages, near water beverages, and the like (e.g., water with natural or synthetic flavors), tea or like beverages such as coffee, cocoa, black tea, green tea, oolong tea, and the like, milk component containing beverages such as milk beverages, milk component containing coffee, milk tea, fruit milk beverages, drinkable yogurt, lactobacillus beverages, and the like, and dairy products.

Generally, the amount of sweetener present in a sweetener composition will vary widely depending on the particular type of sweetener composition and the desired sweetness level thereof. One of ordinary skill in the art can readily determine the appropriate amount of sweetener to add to the sweetened composition, which can be used in dry or liquid form. Which may be added before or after the heat treatment of the food product. The amount of sweetener depends on the purpose of use. It may be added alone or in combination with other compounds.

In the manufacture of food, beverages, pharmaceutical, cosmetic, table products, chewing gum, conventional methods such as mixing, kneading, dissolving, pickling, infiltration, diafiltration, spraying, atomizing, infusion and other methods may be used.

Thus, the compositions as disclosed herein may be made by any method known to those of skill in the art. These methods include dry blending, spray drying, agglomeration, wet granulation, compaction, co-crystallization, and the like.

In solid form, mogrosides produced as disclosed herein may be provided to the consumer in any form suitable for delivery into the food to be sweetened, including sachets, packets, loose bags or boxes, cubes, tablets, sprays or dissolvable strips. The composition may be delivered in unit dosage or bulk form.

In liquid form, mogrosides produced as disclosed herein may be provided in any form suitable for consumer use, including fluid, semi-fluid, pasty and cream forms, and in any shape or form convenient to carry, dispense, store and/or transport using any suitable packaging material.

The composition may include various fillers, functional ingredients, colorants, and/or flavoring agents.

The reference to a patent document or other matter which is given as background herein is not to be taken as an admission that the document or matter was known or that the information contained in the document or matter was part of the common general knowledge at the priority date of any of the claims.

A non-limiting list according to embodiments of the present disclosure is shown below.

According to embodiments of the present disclosure

1. A recombinant cell capable of producing one or more mogrosides and/or a mogroside precursor, wherein the cell has been modified to result in a lack of a polypeptide capable of deglycosylating a mogroside product.

2. The recombinant cell of embodiment 1, wherein the polypeptide capable of deglycosylating a mogroside product is capable of hydrolyzing at least one of

3. The recombinant cell according to embodiment 1 or 2, wherein the polypeptide capable of deglycosylating the mogroside product may be selected from the group consisting of a polypeptide having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO.1 and a polypeptide having an amino acid sequence at least 50% identical to the amino acid sequence shown in SEQ ID NO. 2.

4. The recombinant cell of any one of the preceding embodiments, wherein the polypeptide capable of deglycosylating a mogroside product is a glycoside hydrolase (EC: 3.2.1. -), e.g., an exo-acting glycoside hydrolase.

5. The recombinant cell of any one of the preceding embodiments, wherein the polypeptide capable of deglycosylating a mogroside product is a polypeptide capable of hydrolyzing one or more bonds selected from the group consisting of:

6. The recombinant cell of embodiment 5, wherein the polypeptide capable of deglycosylating a mogroside product is capable of hydrolyzing at least the following

7. The recombinant cell of embodiment 5 or 6, wherein the polypeptide capable of deglycosylating a mogroside product is capable of hydrolyzing at least one of

(A) A beta 1,2 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C24 atom of the mogrol skeleton of mogroside;

(b) Beta 1,2 glycosidic bond between the second glucose molecule and beta-1-glucose bound at the C3 atom of the mogrol skeleton of mogroside.

8. The recombinant cell according to any one of the preceding embodiments, wherein the cell has been further modified to result in a lack of EXG1 and/or EXG2 polypeptides, in particular polypeptides wherein the amino acid sequence has an amino acid sequence which is at least 50% identical to the amino acid sequence shown in SEQ ID No. 3.

9. The recombinant cell of any one of the preceding embodiments, wherein the cell has a higher yield of mogroside product when both cells are cultured under the same conditions, if compared to the yield of mogroside product of an otherwise identical recombinant cell that has not been modified to lack the polypeptide capable of deglycosylating a mogroside product.

10. The recombinant cell of any one of the preceding embodiments, wherein the production of a mogroside product comprising a β -glycosidic linkage at C3 and/or C24 of the mogroside backbone is higher for the cell when both cells are cultured under the same conditions, if compared to the production of a mogroside product of an otherwise identical recombinant cell that has not been modified to lack the polypeptide capable of deglycosylating the mogroside product.

11. The recombinant cell of any one of the preceding embodiments, wherein the production of a mogroside product comprising a β -1, 2-glycosidic bond between a second glucose molecule and β -1-glucose bound at the C3 atom and/or the C24 atom of the mogroside backbone of the cell is higher if compared to the production of a mogroside product of an otherwise identical recombinant cell that is not modified to lack the polypeptide capable of deglycosylating the mogroside product when both cells are cultured under the same conditions.

12. The recombinant cell of any one of the preceding embodiments, wherein the production of a mogroside product comprising a β -1, 6-glycosidic bond between a second glucose molecule and β -1-glucose bound at the C3 atom and/or the C24 atom of the mogroside backbone of the cell is higher if compared to the production of a mogroside product of an otherwise identical recombinant cell that has not been modified to lack the polypeptide capable of deglycosylating the mogroside product when both cells are cultured under the same conditions.

13. The recombinant cell of any one of the preceding embodiments, wherein the production of mogroside product by the cell is higher when both cells are cultured under the same conditions than the production of a mogroside product by an otherwise identical recombinant cell that has not been modified to lack the polypeptide capable of deglycosylating a mogroside product as shown in SEQ ID No. 3.

14. The recombinant cell of any one of the preceding embodiments, wherein the production of a mogroside product comprising a beta-1, 2-glycosidic bond between a second glucose molecule and beta-1-glucose bound at the C3 atom and/or the C24 atom of the mogroside backbone of the cell is higher if compared to the production of a mogroside product of an otherwise identical recombinant cell modified to lack the polypeptide capable of deglycosylating the mogroside product as shown in SEQ ID No. 3 when both cells are cultured under identical conditions.

15. The recombinant cell of any one of the preceding embodiments, wherein the production of a mogroside product comprising a beta-1, 6-glycosidic bond between a second glucose molecule and beta-1-glucose bound at the C3 atom and/or the C24 atom of the mogroside backbone of the cell is higher if compared to the production of a mogroside product of an otherwise identical recombinant cell modified to lack the polypeptide capable of deglycosylating the mogroside product as shown in SEQ ID No. 3 when both cells are cultured under identical conditions.

16. The recombinant cell of any one of the preceding embodiments, wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating a mogroside compound that is a di-, tri-, tetra-, penta-, or hexa-glycosylated mogroside compound or an isomer thereof.

17. The recombinant cell of any one of the preceding embodiments, wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating at least one or more of mogroside IA, mogroside IE ₁, mogroside IIA ₁, mogroside IIA ₂, mogroside IIE, mogroside IIIA ₁, mogroside IIIA ₂, mogroside IIIE, mogroside III, mogroside IVA, mogroside IVE, mogroside V, mogroside VI, mogroside VIA ₁, mogroside VIB, siamenoside I, isosluo-mogroside IVE, isosluo-mogroside V, or alpha-siamenoside I.

18. The recombinant cell of any one of the preceding embodiments, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of glycosylating a mogrol or mogroside compound at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group and/or C25 hydroxyl group;

in particular wherein the polypeptide according to (a), (b) or (c) is a uridine diphosphate dependent glycosyltransferase polypeptide (UGT polypeptide).

19. The recombinant cell of any one of the preceding embodiments, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(c) A polynucleotide encoding a polypeptide capable of synthesizing 11-hydroxy cucurbitadienol from cucurbitadienol, 11-hydroxy-24, 25-epoxy-cucurbitadienol from 24, 25-epoxy-cucurbitadienol or from 11-hydroxy cucurbitadienol, mogrol from 24, 25-dihydroxy-cucurbitadienol, or 24, 25-epoxy-cucurbitadienol from cucurbitadienol, in particular wherein said polypeptide is a cytochrome P450 enzyme (CYP 450);

(f) A polynucleotide encoding a cytochrome b5 polypeptide (CB 5).

20. The recombinant cell of any one of embodiments 1-19, further comprising one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a squalene epoxidase polypeptide (SQE);

(b) A polynucleotide encoding a cucurbitadienol synthase polypeptide (CDS);

(c) A polynucleotide encoding a cytochrome P450 polypeptide (CYP);

(d) A polynucleotide encoding a cytochrome P450 reductase polypeptide (CPR);

(e) A polynucleotide encoding epoxide hydrolase (EPH);

(f) A polynucleotide encoding a cytochrome b5 polypeptide (CB 5);

(g) A polynucleotide encoding a UGT polypeptide.

21. The recombinant cell of any one of the preceding embodiments, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) Polynucleotides encoding polypeptides capable of synthesizing geranyl diphosphate (GPP) from dimethylallyl Diphosphate (DMAPP), in particular wherein the polypeptides are geranyl diphosphate synthase (EC: 2.5.1.1) (GPPS) and/or farnesyl diphosphate synthase (EC: 2.5.1.10) (FPPS);

(b) A polynucleotide encoding a polypeptide capable of synthesizing Ninyl diphosphate (FPP) from geranyl diphosphate (GPP), in particular wherein said polypeptide is farnesyl diphosphate synthase (EC: 2.5.1.10) (FPPS);

22. The recombinant cell of any one of the preceding embodiments, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of synthesizing acetoacetyl-coa (AACoA) from acetyl-coa (AcCoA), particularly wherein the polypeptide is an acetyl-coa acetyltransferase (EC: 2.3.1.9) (AACT);

23. The recombinant cell of any one of embodiments 1-22, wherein at least one of the one or more polypeptides is a heterologous polypeptide.

24. The recombinant cell of any one of the preceding embodiments, wherein the recombinant cell further comprises a lack of production of a lanosterol synthase polypeptide.

25. The recombinant cell of any one of the preceding embodiments, wherein the mogroside precursor is one or more of mogrol, cucurbitadienol, 11-hydroxy cucurbitadienol, 24, 25-epoxy-cucurbitadienol, 11-hydroxy-24, 25-epoxy-cucurbitadienol, and/or 24, 25-dihydroxy-cucurbitadienol.

26. The recombinant cell of any one of the preceding embodiments, wherein the mogroside is one or more of mogroside IA, mogroside IE ₁, mogroside IIA ₁, mogroside IIA ₂, mogroside IIE, mogroside IIIA ₁, mogroside IIIA ₂, mogroside IIIE, mogroside III, mogroside IV _A, mogroside IV _E, mogroside V, mogroside VI, mogroside VIA1, mogroside VIB, siamenoside I, iso-mogroside IVE, iso-mogroside V, or alpha-siamenoside I.

27. The recombinant cell of any one of the preceding embodiments, wherein the cell is a prokaryotic, eukaryotic, or archaeal cell.

28. The recombinant cell of embodiment 27, wherein the recombinant cell is a eukaryotic cell selected from a fungus (e.g., a yeast or filamentous fungus), an algae or a plant cell, or a cell selected from a saccharomyces cerevisiae cell, a yarrowia lipolytica cell, a candida krusei cell, an isaria orientalis cell, or a pichia pastoris cell.

29. The recombinant cell of embodiment 27, which is a prokaryotic cell, such as a bacterial cell or an E.coli cell.

30. A method of producing one or more mogrosides and/or mogroside precursors, the method comprising culturing the recombinant cell according to any of the preceding embodiments in a medium under conditions suitable for producing the mogroside and/or mogroside precursors, optionally isolating the one or more mogroside and/or mogroside precursors.

31. A method of producing one or more mogrosides, the method comprising contacting one or more mogroside precursors or one or more mogroside substrates with a recombinant cell according to any one of embodiments 1 to 30 or a lysate or extract thereof under conditions suitable for the production of the one or more mogrosides, optionally isolating the one or more mogrosides.

32. The method of embodiment 30 or 31, wherein the one or more mogrosides is selected from mogroside IA, mogroside IE ₁, mogroside IIA ₁, mogroside IIA ₂, mogroside IIE, mogroside IIIA ₁, mogroside IIIA ₂, mogroside IIIE, mogroside III, mogroside IV _A, mogroside IV _E, mogroside V, mogroside VI, mogroside VIA1, mogroside VIB, siamenoside I, iso-mogroside IVE, iso-mogroside V, or alpha-siamenoside I, in particular mogroside IIIE, mogroside IIIA, or siamenoside I.

33. A fermentation broth comprising the recombinant cell of any one of embodiments 1-29 or comprising a lysate or extract thereof.

34. A mogroside composition or sweetener composition obtainable by the method according to any one of embodiments 28 to 32.

35. A food product, beverage, pet food, feed, oral composition, pharmaceutical composition, the food product, beverage, pet food, feed, oral composition, pharmaceutical composition comprises a mogroside or sweetener composition according to embodiment 34.

The invention is further illustrated by the following non-limiting examples.

Examples

Genetic modification techniques

Standard genetic techniques, such as overexpression of enzymes in recombinant microorganisms, and additional genetic modification of recombinant microorganisms, are methods known in the art, such as described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or f. Ausubel et al, "Current protocols in molecular biology", john Wiley & Sons, inc. (2003). Methods for transformation and genetic modification of fungal host cells are known, for example, from EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671.

Methods for genetic manipulation and transformation of yarrowia lipolytica are described, for example, in:

Davidow L.S.、Apostolakos D.、O'Donnell M.M.、Proctor A.R.、Ogrydziak D.M.、Wing R.A.、Stasko I.、DeZeeuw J.R. "Integrative transformation of the yeast Yarrowia lipolytica" in Current Genetics (1985) 10: 39-48;

Fickersa P.、Le Dall M.T.、Gaillardin C.、Thonart P.、Nicaud J.M., "New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica" in J. Microbiol. Methods (2003) 55: 727-737;

Gao S.、Han L.、Zhu L.、Ge M.、Yang S.、Jiang Y.、Chen D.,"One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica" in Biotechnol. Lett. (2014) 36: 2523-2528;

Larroude M.、Rossignol T.、Nicaud J.-M.、Ledesma-Amaro R.,"Synthetic biology tools for engineering Yarrowia lipolytica" in Biotech. Advances (2018) 36: 2150-2164;

Gao S.、Tong Y.、Zhu L.、Ge M.、Zhang Y、Chen D.、Jiang Y.、Yang S. "Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production" in Metabolic Engineering (2017) 41: 192-201 Is a kind of medium.

Methods for genetic manipulation and transformation of the yeast Saccharomyces cerevisiae are described, for example, in:

gietz R.D. sum Schiestl R.H. "High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method" in Nature Protocols (2007) 2: 31-34;

Zhang Z.-X.、Wang L.-R.、Xu Y.-S.、Jiang W.-T.、Shi T.-Q.、Sun X.-M.、Huang H. "Recent advances in the application of multiplex genome editing in Saccharomyces cerevisiae" in Applied Microbiology and Biotechnology (2021) 105: 3873-3882;

Dangi A.K.、Dubey K.K.、Shukla P. "Strategies to Improve Saccharomyces cerevisiae: Technological Advancements and Evolutionary Engineering" in Indian J Microbiol (2017) 57(4): 378-386.

Assays for measuring Mogrosides (MOGs)

Liquid chromatography and mass spectrometry (LC-MS) assay

The mogrols and mogrosides were analyzed on a Vanquish DUO UHPLC system (Thermo Fisher) coupled with a Q Exactive Orbitrap mass spectrometer (Thermo Fisher) equipped with an electrospray ionization source operating in negative ion mode for all mogrols and mogrosides studied (table 12).

TABLE 12 mogrols and mogrosides measured in the assay

The chromatographic separation was achieved with a 2.1X105 mm 1.8 μm particle size Acquity UPLC HSS T3 column followed by a 2.1X105 mm 1.8 μm particle size Acquity UPLC HSS T3 column using a gradient elution with (A) 50mM ammonium acetate in LC-MS grade water and B) LC-MS grade acetonitrile as mobile phase. The 5 minute gradient starts at 20% B and increases linearly to 30% B over 0.4 minutes and then to 35% over 0.8 minutes, after which it increases linearly to 95% B over 1.3 minutes and remains at 95% B for 1.5 minutes, then re-equilibrates with 20% B for one minute. The flow rate was maintained at 0.63 ml/min, a5 μl sample injection was used, and the column temperature was set at 50 ℃. The desired components were quantified using an external calibration line for components in the range of 10-2000 ng/ml. The corresponding analytical samples are diluted accordingly so that the concentration of the desired component falls within the linear range of the calibration line. The final concentration of acetonitrile in the sample was 33%. The concentration of mogrol in the sample was calculated using a linear calibration line (nine points), with the origin forcing through the zero point. The remaining components (mogrosides) were determined by a one-point calibration.

Example 1 construction of Strain MOG001

Yarrowia lipolytica strain MOG001 was constructed starting from a yarrowia lipolytica strain in which the ku70 gene had been deleted (Δku70) to increase the efficiency of homologous recombination and integration of DNA fragments. MOG001 was constructed according to well-known genetic manipulation and transformation methods such as those disclosed herein in the "genetic modification techniques" section, using expression cassettes such as those disclosed in table 15. Yarrowia lipolytica strain MOG001 comprises one or more copies of a gene encoding a polypeptide required for the production of mogrosides and depicted in table 13.

Table 13. Polypeptide sequences of enzymes involved in the mogroside biosynthetic pathway and the polypeptide sequences of markers present in MOG 001.

The genes encoding the polypeptides of table 13 were expressed using the promoters and terminators listed in table 14.

TABLE 14 Polynucleotide sequences of promoters and terminators

EXAMPLE 2 construction of strains MOG002 to MOG006

Table 15 discloses yarrowia lipolytica strains lacking one or more beta glucanase polypeptides as used in the following experiments. The polypeptide sequences of the β -glucanase polypeptides are disclosed in table 16 a.

TABLE 15

Table 16a

The open reading frame encoding the β -glucanase in yarrowia lipolytica as set forth in table 16a was knocked out according to the method described in the "genetic modification techniques" section. Table 16b lists the specific regions where each dextranase was knocked out.

Table 16b

The correct knockdown of the desired glucanase was confirmed by colony PCR with primers listed in table 16c using KAPA2G Robust Hotstart PCR kit with dntps from KAPA Biosystems.

Table 16c

Example 4 production and quantification of mogroside in strains MOG001 to MOG006, preparation and quantification of mogroside samples

To determine the effect of deleting YALI0F01672g, YALI0B14289g and/or YALI0F05390g polypeptides, strains MOG002, MOG003, MOG004, MOG005 and MOG006 were grown with the parent strain MOG001 in shake flasks (0.5L, containing 60 ml medium) at 30 ℃ and 280 rpm for 2 days. The medium was modified for carbon and nitrogen sources based on Verduyn et al (Verduyn C, postma E, SCHEFFERS WA, van Dijken JP. Yeast, 7 months 1992; 8 (7): 501-517) as described in tables 17, 17a and 17 b.

TABLE 17a ^a microelement solution

TABLE 17b ^b vitamin solution

Subsequently, the pre-culture of 40 ml was transferred to a fermenter (starting volume of 0.4L) containing the medium listed in table 18, table 18a and table 18 b. During the incubation, the pH was controlled at 7 by adding ammonia (10 w/w%), the temperature was controlled at 30℃and the pO2 was controlled at 20% (relative to air saturation) by adjusting the stirrer speed. The glucose concentration was limited by controlling the 55 wt% glucose feed in the fermentor. After 144 hours of incubation, the culture broth was collected for sample preparation and quantitation of mogrosides.

TABLE 18 fermentation Medium composition

TABLE 18a ^a microelement solution

TABLE 18b ^b vitamin solution

After fermentation, samples for LC-MS quantification of mogrosides were prepared as follows. Several samples consisting of 1 ml whole fermentation broth were collected in 96 deep well plates. Each sample was homogenized after adding 500 μl of 100% acetonitrile and then centrifuged at 3700 rpm for 10 minutes. Subsequently, 150 μl of supernatant from each sample was transferred to a new 96-deep well plate and diluted with 300 μl of 33% acetonitrile. Finally, the samples were homogenized using a multichannel pipette and held in a deep-well plate, which was sealed before analysis. Quantitative determination of mogrosides was performed using the LC-MS method described above.

Example 5 Effect of beta-glucanase polypeptide deficiency in strains MOG002 to MOG006

To determine the effect of the deficiency of the β -glucanase polypeptides YALI0F01672g and YALI0B14289g, strains MOG001 to MOG006 were cultivated under the same conditions compared to the case of the deficiency of the prior art polypeptide EXG1 (YALI 0F 05390) and compared to the parent strain MOG001 without any β -glucanase, and the culture broth samples were collected for sample preparation and quantitative analysis of mogrosides as described in example 4.

For each fermentation experiment, the concentrations (in mg/L) of mogroside I A ₁ (Mog 1A 1), mogroside I E ₁ (Mog 1E 1), mogroside II A ₂ (Mog 2A 2) and mogroside III A ₁ (Mog 3A 1) produced by each strain were determined. For each of these mogrosides, the results for strains MOG001 to MOG006 were depicted using a bar graph, where the results were normalized to the strain with the highest yield (assigned a value of 1.0). The experimental results of mogroside I A ₁ (Mog 1 A1), mogroside I E ₁ (Mog 1E 1), mogroside II a ₂ (Mog 2 A2), and mogroside III a ₁ (Mog 3 A1) are plotted in fig. 6A, 6B, 7A, and 7B, respectively.

Primary glycosylation at C3 and C24 of the mogrol backbone by the action of UGT enzymes is the first step in mogroside biosynthesis. The inventors have surprisingly found that YALI0F01672g of the polypeptide is involved in the hydrolysis of the primary glycosidic bond at position C3 and possibly to a lesser extent at position C24 of the mogrol backbone.

Fig. 6B shows that in the strain that has not been modified to lack beta glucanase production (i.e., strain MOG 001), the amount of MOG1E1 is very low. In contrast, cells that have been made to lack production of the EXG1 polypeptide (strain MOG 004), production of MOG1E1 was still negligible, and the same was true for the strain made to lack YALI0B14289g polypeptide (strain MOG 003). Surprisingly, in the preparation of the strain (strain MOG 002) as lacking the production of YALI0F01672g polypeptide, the production of MOG1E1 was increased about 20-fold, indicating that this polypeptide is involved in the hydrolysis of the glycosidic bond between the C3 atom of the mogrol backbone of mogroside and the β -1-glucose moiety bound at said position. In the production of the strain (strain MOG 005) as lacking both the YALI0F01672g polypeptide and the EXG1 polypeptide, the production of MOG1E1 was also increased about 20-fold, confirming that the YALI0F01672g polypeptide (rather than the EXG1 polypeptide) was involved in the hydrolysis of the glycosidic bond between the C3 atom of the mogrol backbone of mogrosides and the β -1-glucose moiety bound at that position.

Fig. 6A shows that the amount of MOG1A1 is not very high in the strain that has not been modified to lack beta glucanase production as described herein (strain MOG 001). In contrast, cells that have been made to lack the EXG1 polypeptide (MOG 004), production of MOG1A1 was even lower than MOG001, and this was the case for strains made to lack the YALI0B14289g polypeptide (MOG 003). Surprisingly, in the preparation of the strain as lacking the production of YALI0F01672g polypeptide, if the production of MOG1A1 was increased about 2.5-5 fold compared to strain MOG001, it was shown that YALI0F01672g polypeptide (rather than the EXG1 polypeptide) was involved in the hydrolysis of the glycosidic bond between the C24 atom of the mogrol backbone of mogrosides and the β -1-glucose moiety bound at that position. Surprisingly, in cells comprising production of the EXG1 polypeptide and YALI0F01672g polypeptide (MOG 005 in fig. 6A), there was no production of MOG1 A1. The EXG1 polypeptide is known to be likely involved in the hydrolysis of beta-1, 2 and beta-1, 6 glycosidic linkages in mogrosides. Without being bound by theory, it was observed that cells lacking both EXG1 and YALI0F01672g polypeptide do not produce Mog1A1, probably due to the fact that in the absence of YALI0F01672g hydrolytic activity Mog1A1 becomes available as a substrate for other primary and secondary UGTs, resulting in the production of higher glycosylated mogrosides which are then no longer hydrolyzed to lower glycosylated mogrosides due to the absence of EXG1 and YALI0F01672g hydrolytic activity.

Fig. 7A shows normalized concentrations of MOG2A2 in cells MOG001 to MOG 006. This figure shows that mogroside II a ₂ production is limited (strains MOG004, MOG 003) or absent (MOG 002) in cells not lacking β -glucanase (MOG 001), or in cells lacking only one β -glucanase. However, if the strain MOG005 (fig. 7A) which lacks both EXG1 and YALI0F01672g polypeptides produced 100% more MOG2A2 than the cells which lacked only YALI0F01672g polypeptides (strain MOG 002), but produced much more MOG2A2 than the strain which lacked either EXG1 or YALI0B14289g polypeptides. These results support the hypothesis that Mog1E1, which became available due to the inhibition of YALI0F01672g hydrolytic activity, became the substrate for the secondary UGT, resulting in Mog2A2, which Mog2A2 was no longer hydrolyzed to lower degrees of glycosylation of mogrosides or mogrols in the absence of EXG1 activity in the cells.

Experiments have also shown that in cells capable of producing mogrosides (MOG 006 in fig. 7B), the lack of production of both polypeptides EXG1 and YALI0B14289g synergistically increases the production of higher glycosylated mogrosides (e.g. MOG3 A1). It is presumed that the polypeptide YALI0B14289g is active mainly in hydrolysis of beta-1, 2-glycosidic bond and beta-1, 6-glycosidic bond existing in mogroside, like EXG 1. This result suggests that downregulating multiple β -glucanases in cells may be beneficial to enhance the production of higher branched mogrosides.

In summary, it is evident that recombinant cells lacking one or more beta glucanases as disclosed herein are well suited for controlling the production of mogrosides with different glycosylation levels and exhibit several surprising advantages compared to the recombinant cells lacking EXG1 beta glucanase described in the background.

Claims

1. A recombinant cell capable of producing one or more mogrosides and/or mogroside precursors, said cell being modified to result in the absence of a polypeptide capable of deglycosylating mogroside products, said polypeptide capable of deglycosylating mogroside products being capable of hydrolyzing at least one of the following:

(a) The glycosidic bond between the carbon at position 24 (C24 atom) of the mogroside skeleton and the β-1-glucose bonded at that position; and/or

(b) The glycosidic bond between the carbon (C3 atom) at position 3 of the mogroside skeleton and the β-1-glucose bonded at that position.

2. The recombinant cell according to claim 1, wherein the polypeptide capable of deglycosylation of mogroside product is selected from: a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 1; and a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 2, or a combination of a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 1 and a polypeptide having an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO: 2.

3. The recombinant cell according to any one of the preceding claims, wherein the polypeptide capable of deglycosylation of mogroside products is a glycoside hydrolase (EC: 3.2.1-), such as an exonuclease.

4. The recombinant cell according to any one of the preceding claims, wherein the polypeptide capable of deglycosylation of mogroside product is a polypeptide capable of hydrolyzing one or more bonds selected from the following:

(a) The glycosidic bond between the carbon at position 24 (C24 atom) of the mogroside skeleton and the β-1-glucose moiety bonded at that position;

(b) The glycosidic bond between the carbon (C3 atom) at position 3 of the mogroside skeleton and the β-1-glucose moiety bonded at said position;

(c) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton of mogroside;

(d) The β-1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton of mogroside;

(e) The β-1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside backbone;

(f) The β-1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton;

(g) The β1,4 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton.

5. The recombinant cell according to claim 4, wherein the polypeptide capable of deglycosylation of mogroside products can be hydrolyzed to at least the following:

(a) The β-1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bound at the C3 atom of the mogroside backbone; and/or

(b) The β1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton.

6. The recombinant cell according to claim 4 or 5, wherein the polypeptide capable of deglycosylation of mogroside products is capable of hydrolyzing at least one of the following.

(a) The β-1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton of mogroside;

(b) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton.

7. The recombinant cell according to any one of the preceding claims, wherein the cell has been further modified to result in the absence of EXG1 and/or EXG2 polypeptides, particularly wherein the polypeptide has an amino acid sequence that is at least 50% identical to the amino acid sequence shown in SEQ ID NO:3.

8. The recombinant cells according to any one of the preceding claims, wherein when the two types of cells are cultured under the same conditions, the mogroside production of the cells is higher than that of recombinant cells that are otherwise identical but not modified to lack the polypeptide capable of deglycosylation of the mogroside product.

9. The recombinant cell according to any one of the preceding claims, wherein the polypeptide capable of deglycosylation of the mogroside product is capable of deglycosylation of the mogroside compound, wherein the mogroside compound is a disaccharidated, trisaccharidated, tetrasaccharidated, pentasaccharidated, or hexasaccharidated mogroside compound or its isomers, particularly wherein the polypeptide capable of deglycosylation of the mogroside product is capable of deglycosylation at least one or more of the following: mogroside IA, mogroside IE ₁ , mogroside IIA, mogroside IIA 1, mogroside IIA ₂ , mogroside IIE, mogroside IIIA ₁ , mogroside IIIA ₂ , mogroside IIIE, mogroside _III , mogroside IVA, mogroside IV, mogroside V, mogroside VI, mogroside VIA, mogroside VIa ₁ Monk fruit glycoside VIB, symbioside I, isomogroside IVE, isomogroside V or α-symbioside I.

10. The recombinant cell according to any one of the preceding claims, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of glycosylation of mogroside or mogroside compounds at their C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group and/or C25 hydroxyl group;

(b) Polynucleotides encoding polypeptides capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety (C24-O-glucose) at the C24 position of mogroside compounds and/or capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety (C3-O-glucose) at the C3 position of mogroside compounds.

(c) Polynucleotides encoding polypeptides capable of β-1,6-glycosylation of the C6' hydroxyl group of the glucose moiety at the C3 position of mogroside and/or capable of β-1,6-glycosylation of the C6' hydroxyl group of the glucose moiety at the C24 position of mogroside.

Specifically, the polypeptide described in (a), (b) or (c) is a uridine diphosphate-dependent glycosyltransferase polypeptide (UGT polypeptide).

11. The recombinant cell according to any one of the preceding claims, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of synthesizing 2,3-epoxysqualene from squalene, or 2,3-epoxysqualene from 2,3-epoxysqualene, wherein the polypeptide is, in particular, squalene epoxyenase (SQE).

(b) A polynucleotide encoding a polypeptide capable of synthesizing cucurbitacinol from 2,3-epoxysqualene or 2,4,25-epoxycucurbitacinol from 2,3,22,23-diepoxysqualene, wherein the polypeptide is, in particular, a cucurbitacinol synthase (CDS).

(c) A polynucleotide encoding a polypeptide capable of synthesizing 11-hydroxycucurbita alcohol from cucurbita alcohol, 11-hydroxy-24,25-epoxy-cucurbita alcohol from 24,25-epoxy-cucurbita alcohol or from 11-hydroxycucurbita alcohol, mogroside from 24,25-dihydroxy-cucurbita alcohol, or 24,25-epoxy-cucurbita alcohol from cucurbita alcohol, particularly wherein said polypeptide is a cytochrome P450 enzyme (CYP450).

(d) Polynucleotides encoding peptides that can reduce cytochrome P450 complexes, particularly cytochrome P450 reductase (CPR);

(e) Encoding a polynucleotide capable of synthesizing mogroside from 11-hydroxy-24,25-epoxy-cucurbitadienol, or 24,25-dihydroxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol, wherein the polypeptide is, in particular, an epoxide hydrolase (EPH).

(f) Polynucleotides encoding cytochrome b5 polypeptide (CB5).

12. The recombinant cell according to any one of claims 1 to 11, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding squalene epoxygenase polypeptide (SQE);

(b) Polynucleotides encoding cucurbitacin synthase polypeptide (CDS);

(c) Polynucleotides encoding cytochrome P450 polypeptide (CYP);

(d) Polynucleotides encoding cytochrome P450 reductase polypeptide (CPR);

(e) Polynucleotides encoding epoxide hydrolase (EPH);

(f) Polynucleotides encoding cytochrome b5 polypeptide (CB5);

(g) Polynucleotides encoding UGT polypeptide.

13. The recombinant cell according to any one of the preceding claims, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of synthesizing gerany diphosphate (GPP) from dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is gerany diphosphate synthase (EC:2.5.1.1) (GPPS) and/or farnesyl diphosphate synthase (EC:2.5.1.10) (FPPS).

(b) A polynucleotide encoding a polypeptide capable of synthesizing farnesyl diphosphate (FPP) from gerany diphosphate (GPP), particularly wherein said polypeptide is farnesyl diphosphate synthase (EC:2.5.1.10) (FPPS).

(c) A polynucleotide encoding a polypeptide capable of synthesizing squalene from farnesyl diphosphate (FPP), wherein said polypeptide is squalene synthase (EC:2.5.1.21) (SQS).

14. The recombinant cell according to any one of the preceding claims, wherein the recombinant cell further comprises one or more of the following recombinant polynucleotides:

(a) A polynucleotide encoding a polypeptide capable of synthesizing acetyl-CoA (AACoA) from acetyl-CoA, particularly wherein said polypeptide is acetyl-CoA acetyltransferase (EC: 2.3.1.9) (AACT).

(b) A polynucleotide encoding a polypeptide capable of synthesizing hydroxymethylglutaryl-CoA (HMGCoA) from acetyl-CoA, particularly wherein said polypeptide is hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) (HMGS).

(c) A polynucleotide encoding a polypeptide capable of synthesizing mevalonic acid (MVA) from hydroxymethylglutaryl-CoA (HMGCoA), particularly wherein said polypeptide is 3-hydroxy-3-methylglutaryl-CoA reductase (EC: 1.1.1.34) (HMGR).

(d) A polynucleotide encoding a polypeptide capable of synthesizing mevalonate-5-phosphate (MVA-P) from mevalonate (MVA), particularly wherein said polypeptide is mevalonate kinase (EC:2.7.1.36) (MK).

(e) A polynucleotide encoding a polypeptide capable of synthesizing mevalonate-5-bisphosphate (MVA-PP) from mevalonate-5-phosphate (MVA-P), particularly wherein said polypeptide is phosphate mevalonate kinase (EC: 2.7.4.2) (PMK).

(f) A polynucleotide encoding a polypeptide capable of synthesizing isopentenyl diphosphate (IPP) from mevalonate-5-bisphosphate (MVA-PP), particularly wherein said polypeptide is mevalonate diphosphate decarboxylase (EC: 4.1.1.33) (MDD) and/or isopentenyl/dimethylallyl diphosphate synthase (EC: 1.17.1.2) (IPPS).

(g) A polynucleotide encoding a polypeptide capable of synthesizing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP), particularly wherein said polypeptide is isopentenyl diphosphate δ-isomerase (EC: 5.3.3.2) (IPI).

15. The recombinant cell according to any one of the preceding claims, wherein the recombinant cell further comprises the production of a lanosterol synthase polypeptide lacking the presence of lanosterol.

16. The recombinant cells according to any one of the preceding claims, wherein the mogroside precursor is one or more of cucurbitacin, mogroside, 11-hydroxycucurbitacin, 24,25-epoxy-cucurbitacin, 11-hydroxy-24,25-epoxy-cucurbitacin and/or 24,25-dihydroxy-cucurbitacin.

17. The recombinant cells according to any one of the preceding claims, wherein the mogroside is one or more of mogroside IA, mogroside IE ₁ , mogroside IIA, mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIE, mogroside IIIA ₁ , mogroside IIIA ₂ , mogroside _IIIE , mogroside III, mogroside IVA, mogroside IVE, mogroside V, mogroside _VI , mogroside VIA, mogroside VIa1, mogroside VIB, sarcoside I, isomogroside IV, isomogroside V, or α-sarcoside I.

18. The recombinant cell according to any one of the preceding claims, wherein the cell is a prokaryotic, eukaryotic (e.g., yeast, filamentous fungus or plant cell) or archaea cell, particularly selected from Saccharomyces cerevisiae cells, Yerovia lipolytica cells, Candida krusei cells, Isaac's orientalis cells, Pichia pastoris cells or Escherichia coli cells.

19. A method for producing one or more mogrosides and/or mogroside precursors, the method comprising culturing recombinant cells according to any one of claims 1 to 18 in a culture medium under conditions suitable for producing the mogrosides and/or mogroside precursors; optionally isolating the one or more mogrosides and/or mogroside precursors.

20. A method for producing one or more mogrosides, the method comprising contacting one or more mogroside precursors or one or more mogroside substrates with recombinant cells or their lysates or extracts according to any one of claims 1 to 19 under conditions suitable for producing the one or more mogrosides; optionally separating the one or more mogrosides.

21. The method according to claim 19 or 20, wherein the one or more mogrosides are selected from mogroside IA, mogroside IE ₁ , mogroside IIA, mogroside IIA ₁ , mogroside IIA ₂ , mogroside IIE, mogroside IIIA ₁ , mogroside IIIA ₂ , mogroside IIIE, mogroside _III , mogroside IVA, mogroside IVE, mogroside V, mogroside VI, mogroside _VIA , mogroside VIa1, mogroside VIB, sarcoside I, isomogroside IV, isomogroside V or α-sarcoside I, particularly mogroside IIIE, mogroside IIIA or sarcoside I.