EP4698673A1

EP4698673A1 - Strains and methods for the production of mogrosides

Info

Publication number: EP4698673A1
Application number: EP24718546.5A
Authority: EP
Inventors: Zheng Zhao; René Marcel de Jong; Jozef Petrus Johannes Schmitz; Nicolette Jasmijn Broers; Bianca Elisabeth Maria Gielesen
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: 2026-02-25
Also published as: WO2024218244A1; CN121127603A

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 deficiency 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 cell led to improved production of mogrosides.

Description

STRAINS AND METHODS FOR THE PRODUCTION OF MOGROSIDES

Field

The present disclosure relates to recombinant cells capable to produce mogrosides, or mogroside precursors and to methods to produce such products.

Background

Mogrosides are a family of secondary metabolite compounds isolated from the plant Siraitia grosvenorii, belonging to the Curcubitaceae family and almost exclusively growing in the Guangxi province of China. The plant is commonly known as monk fruit or Luo Han Guo.

Mogrosides are glycosylated triterpene compounds sharing the same mogrol triterpene backbone and differing in the number of glucose moieties and the type of glycosidic bonds present. The chemical structure of several mogrosides is shown in Fig. 1.

Monk fruit extracts have been used since a long time as natural sweeteners in China. Mogroside V, Mogroside IV and Siamenoside I are between the major components in said extracts and responsible for their intense sweetness, which can be 250 times higher than sucrose. Unfortunately, these compounds can only be found in tiny amounts in the fruit. Given the growing interest for natural sweeteners in the food industry and the difficulties of obtaining mogrosides from the plant, there is a need for new methods which allow the production of mogrosides in a sustainable and commercially viable way.

The biosynthesis of some mogrosides compounds has been described.

WO2013/076577 describes a method of producing a mogroside compound, said method comprising contacting mogrol 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 with the aid of enzymes. Various biosynthetic pathways useful for mogroside production and enzymes useful for mogroside production are provided, including recombinant cells capable of producing mogrol 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 beta-glucanase activity, which may result in de-glycosylation of mogrosides. The recombinant host may be modified to reduce or even abolish exo-1 ,3-beta-glucanase activity. These documents describe yeast cells wherein the EXG1 gene and/or of the EXG2 gene, both of which are encoding an exo-1 ,3-beta-glucanase, are knocked out.

There is still a need for a recombinant production system that can accumulate high yields of the desired highly glycosylated mogroside components, such as Mogroside IV, Mogroside V, Mogroside VI and Siamenoside I. There also remains a need for improved production of mogroside compounds in recombinant hosts for commercial uses.

Description of the Figures

Figure 1 depicts the chemical structure of several mogroside compounds.

Figure 2 depicts the biosynthetic pathway from Acetyl Coenzyme A to Squalene.

Figure 3 depicts the biosynthetic pathway from Squalene to Mogrol.

Figure 4 depicts the biosynthetic pathway from Mogrol to Mogrosides.

Figure 5 depicts the chemical structure of several mogroside precursors and intermediates in the biosynthetic pathway to the production of mogrosides starting from squalene.

Figures 6A and 6B depict the normalized concentration, respectively of Mogroside I Ai and Mogroside I Ei as determined in identical fermentation experiments for strain MOG001 , MOG004, MGG003, MGG002, MGG006, and MGG005.

Figures 7A and 7B depict the normalized concentration, respectively of Mogroside II A2, and Mogroside III A1 as determined in identical fermentation experiments for strain MGG001 , MGG004, MGG003, MGG002, MGG006, and MGG005.

Description of Sequences

A description of the sequences is set out in Table 1 .

Summary

Provided herein is a recombinant cell capable of producing one or more mogrosides and/or mogroside precursors, such as capable of producing one or more mogrosides in a culture medium, wherein the cell has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product. Said polypeptide capable of deglycosylating a mogroside product may be capable of hydrolyzing at least one of

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

(b) the glycosidic bond between the carbon at position 3 (C3 atom) of the mogrol backbone of a mogroside and a beta-1 -glucose bound at said position.

Particularly said polypeptide capable of deglycosylating a mogroside product may be selected from a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 ; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2.

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

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

Also disclosed are: a fermentation broth comprising a recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure or a lysate or an extract thereof; a mogroside composition or sweetener composition obtainable by a method according to the disclosure; a food product, a beverage, a pet-food, a feed, an oral, a pharmaceutical composition comprising the mogroside composition or sweetener composition according to the disclosure.

General definitions

In order that the present disclosure can be more readily understood, certain terms and methodologies are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Unless defined otherwise, all 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 is related.

Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are 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 the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise. As an 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" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of should be assumed to be within an acceptable error range for that particular value or composition.

A “cell” as defined herein is an organism suitable for genetic manipulation and which may be cultured at cell densities useful for industrial production of a target product. A suitable organism may be a microorganism, for example one which may be maintained in a fermentation device. With regard to the present disclosure, it is understood that cells, such as e.g., microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). A cell may be a cell found in nature or a cell derived from a parent cell after genetic manipulation or classical mutagenesis. A cell may be a prokaryotic, archaebacterial or eukaryotic cell.

A prokaryotic cell may, but is not limited to, a bacterial cell. Bacterial cell may be Gram-negative or Gram-positive bacteria. Examples of bacteria include, but are not limited to, bacteria belonging to the genus Bacillus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus), Acinetobacter, Nocardia, Xanthobacter, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa, P. fiuorescens), Salmonella (e.g., S. typhimurium, S. typhi), Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Paracoccus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, 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 nonsulfur bacteria.

A eukaryotic cell may be, but is not limited to, fungus (e.g. a yeast or a filamentous fungus), an algae, a plant cell, a cell line.

A eukaryotic cell may be a fungus, such as a filamentous fungus or yeast. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus (e.g. A. niger, A oryzae, A. nidulans), Agaricus, Aureobasidium, Coprinus, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium (e.g. P. chrysogenum, P. camemberti), Piromyces, Phanerochaete Pleurotus, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia (e.g. Rasamsonia emersonii), Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma.

Yeast cells may be selected from the genera: Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), Kluyveromyces, Candida (e.g., C. rugosa, C. revkaufi, C. pulcherrima, C. tropical is, C. util is), Pichia (e.g., P. pastoris), Schizosaccharomyces, Issatchenkia, Zygosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia (e.g., Y. lipolytica, formerly classified as Candida lipolytica).

The cell may be an algae, a microalgae or a marine eukaryote. The cell may be a Labyrinthulomycetes cell, preferably of the order Thraustochytriales, more preferably of the family Thraustochytriaceae, more preferably a member of a genus selected from the group consisting of Aurantiochytrium, Oblongichytrium, Schizochytrium, Thraustochytrium, and Ulkenia, even more preferably Schizochytrium sp. ATCC# 20888.

The recombinant cell as disclosed herein may belong to one of the genera Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadazyma or Escherichia, for example a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an Issatchenkia orientalis cell or an Escherichia coll cell.

Therefore, in one embodiment a recombinant cell capable of producing mogrol or mogrosides which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure may be a procaryote, eukaryote or archaeal cell, particularly a plant cell or a cell selected from a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an Issatchenkia orientalis cell, Pichia pastoris or an Escherichia coll cell.

The term “control sequence” as used herein refers to components involved in the regulation of the expression of a coding sequence in a specific organism or in vitro. Examples of control sequences are transcription initiation sequences, termination sequences, promoters, leaders, signal peptides, propeptides, prepropeptides, or enhancer sequences; Shine-Delgarno sequences, repressor or activator sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.

As used herein, the terms "culture broth," "culture medium," and "growth medium" can be used interchangeably to refer to a liquid or solid that supports growth of a cell. Typically, the culture medium may comprise a carbon source (e.g. one or more of glucose, fructose, sucrose, xylose, glycerol, plant biomass, celluloses, hemicelluloses, pectin, rhamnose, galactose, fucose, maltose, maltodextrin, ribose, ribulose, or starch, starch derivatives, lactose, fatty acids, triglycerides). Typically, the culture medium may also comprise a nitrogen source such as urea, or an ammonium salt such as ammonium sulphate, ammonium chloride, ammonium nitrate or ammonium phosphate. A culture broth may further comprise trace metals, vitamins, salts, amino acids, etc.. The trace metals may be divalent cations, including, but not limited to Mn2+, Mg2+, Fe2+, Cu2+ etcetera.

When comparing the production of a specific mogroside by a recombinant cell that is deficient in a polypeptide capable of deglycosylating a mogroside product to the production of the same mogroside by an otherwise identical cell that is not deficient in said polypeptide, the wording “cultured under the same conditions” refers to the cultivation of both cells under identical conditions, wherein the amount and/or concentration of the mogroside in which the two cells differ is measured in both cells using the same conditions. Preferably, the measurement is carried out using the same assay and methodology, preferably within the same experiment.

As used herein, a "cytochrome b5" or"CB5" refers to a protein that comprises a lipid binding domain or cytochrome b5-like heme binding domain. In some embodiments, a lipid binding domain is a steroid binding domain. CB5 proteins are heme- or lipid- binding proteins. For example, a CB5 may be a steroid binding protein. CB5 proteins may serve as an electron transfer component of a redox reaction. For example, a CB5 may function as an obligate electron donor in an oxidative reaction. In some embodiments, a CB5 may serve as an electron-delivery partner for a cytochrome P450 (e.g., a C11 -hydroxylase). In some embodiments, a CB5 may catalyze or promote electron transfer from NADPH to a cytochrome P450 enzyme (e.g., a C11 -hydroxylase). In some other embodiments, a CB5 may sterically interact with a P450 enzyme to support an enzyme conformation that promotes higher activity, without a direct enzymatic role of the CB5 itself.

The term "derived from" also includes the terms "originates from," "obtained from," "obtainable from," "isolated from," and "created from," and typically indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material. As used herein, a substance (e.g., a nucleic acid molecule or polypeptide) "derived from" a cell preferably means that the substance is native to that microorganism.

The terms “expression”, “express”, “expressing”, when used in reference to a polynucleotide or polypeptide refers to any step involved in the production of (a) polypeptide(s) 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 a situation wherein gene expression is under control of a constitutive promoter which allows for continuous gene transcription. The term “induced expression” when used in reference to a gene refers to a way of regulating gene expression wherein a molecule called inducer regulates the expression of a gene by either a) binding to a gene promoter repressor protein or b) by binding to a gene promoter activators molecule, therefore allowing RNA polymerase to perform gene transcription.

As used herein, the term "fed-batch culture" or "semi-batch culture" are used interchangeably to refer to as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. In some embodiments, all the nutrients are fed into the bioreactor.

In the context of the present disclosure the terms “functional homologue”, “functional equivalent” or “functional variant” can be used interchangeably. Functional homologue of a polypeptide is a polypeptide having at least one biological function and/or one activity in common with the polypeptide. Typically, the functional homologue has a certain level of sequence similarity or identity with the amino acid sequence of the polypeptide, typically at least 50% sequence identity with 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 with the amino acid sequence of the polypeptide. The amino acid sequence of a functional homologue of a polypeptide can comprise one or more amino acid substitutions, deletions or additions if compared with the amino acid sequence of the polypeptide.

A functional homolog of a polypeptide (also indicated as reference polypeptide) can be a natural occurring polypeptide, such as a homologue, orthologue, or paralogue of the polypeptide. Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of the reference polypeptide. Sequence analysis can involve basic local alignments search tools such as Protein BLAST, Nucleotide BLAST, SmartBLAST analysis of non- redundant databases using the amino acid sequence of the reference polypeptide. Amino acid sequence is, in some instances, deduced from the nucleotide sequence and in such case BLASTX can be used. Those polypeptides in the database that have greater than 40 percent sequence identity with the reference polypeptide are candidates for further evaluation for suitability as functional homologues of the polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in the reference polypeptide, e.g., conserved functional domains. 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 a region within the primary amino acid sequence of the reference polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. 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, Letunic 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 the reference polypeptide. Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.

Alternatively, a functional homologue can be produced synthetically using multiple techniques known to those skilled in the art. For example, the amino acid sequence of a potential functional homolog of a polypeptide can be generated using protein engineering tools known to those skilled 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-lnternational 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. Keasling. 2006. “Designed divergent evolution of enzyme function.” Nature 440:1078-1082). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptidepolypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.

Herein, a “gene” is defined as a polynucleotide containing an open reading frame (ORF) together with its transcriptional control elements (promoter and terminator), the ORF being the region on the gene that will be transcribed and translated into the polypeptide.

The term "heterologous" when used with respect to a polynucleotide (such as DNA or RNA), polypeptide or protein refers to a polynucleotide, polypeptide or protein that does not occur naturally as part of the recombinant cell, genome or DNA or RNA in which it is present, or that is found in a different number of copies, or under the control of a different control sequence, or in a cell or location or locations in the genome or DNA or RNA that differ from that in which it is found in nature. Heterologous polynucleotides, polypeptides or proteins are not endogenous to the cell into which they are introduced but have been obtained from another cell or synthetically or recombinantly produced.

The term "homologous" when used to indicate the relation between a given (recombinant) polynucleotide or polypeptide and a given host organism or host cell such as the recombinant cell as disclosed herein, is understood to mean that in nature the polynucleotide or polypeptide molecule is produced by a recombinant cell, host cell or organism of the same species, such as of the same variety or strain. The term "isolated" or “recovered” referring to a product as used herein means a product that is removed or purified from at least one component, e.g. components present in the cell where the product is produced and or the fermentation broth or medium or crude or cell extract.

As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a recombinant microorganism containing the marker. The marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Alternatively, or also, non-antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1 , LEU2). The recombinant cells transformed with the polynucleotide constructs may be marker gene free. Methods for constructing recombinant marker gene free recombinant cells are disclosed in EP-A-0 635 574 and are based on the use of bidirectional markers. Alternatively, a screenable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into the polynucleotide constructs as disclosed herein allowing to screen for transformed cells. An exemplary marker-free method for the introduction of heterologous polynucleotides is described in W00540186.

The wording “measured under the same conditions” or “analyzed under the same conditions” means that the modified cell and the cell that has not been modified are cultivated under the same conditions and that the amount and/or activity of the polypeptide in which the modified cell is deficient or overexpressed, compared to the not modified cell, is measured in the modified cell and in the not modified cell, respectively, using the same conditions, preferably by using the same assay and/or methodology, more preferably within the same experiment.

“Modification of a genome” of a recombinant cell is herein defined as any event resulting in a change in a polynucleotide in the genome of the recombinant cell. A modification is construed as one or more modifications. Modification can be introduced by e.g. classical strain improvement such as random mutagenesis followed by selection. Modification may be accomplished by the introduction (insertion), substitution or removal (deletion) of one or more nucleotides in a polynucleotide. This modification may for example be in a coding sequence or a regulatory element required for the 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 or a frameshift of the open reading frame of a coding sequence. The modification of a coding sequence or a regulatory element thereof may be accomplished by site-directed or random mutagenesis, DNA shuffling methods, DNA reassembly methods, gene synthesis (see for example Young and Dong, (2004), Nucleic Acids Research 32, (7) electronic access http://nar.oupjournals.Org/cgi/reprint/32/7/e59 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 in accordance with methods known in the art. Examples of random mutagenesis procedures are well known in the art, such as for example chemical (NTG for example) mutagenesis or physical (UV for example) mutagenesis. Examples of directed mutagenesis procedures are the QuickChange® site-directed mutagenesis kit (Stratagene Cloning Systems, La Jolla, CA), the ‘The Altered Sites® II in vitro Mutagenesis Systems’ (Promega Corporation) or by overlap extension using PCR as described in Gene. 1989 Apr 15;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”) or using PCR as described in “Molecular Biology: Current Innovations and Future Trends.” (Eds. A.M. Griffin and H.G. Griffin. ISBN 1-898486-01- 8;1995 Horizon Scientific Press, PO Box 1 , Wymondham, Norfolk, U.K.).

A modification in the genome can be determined by comparing the polynucleotide sequence of the modified recombinant cell to the polynucleotide sequence of the non-modified recombinant cell. Sequencing of a polynucleotide and genome sequencing can be done using standard methods known to the person skilled in the art, for example using Sanger sequencing technology and/or next generation sequencing technologies such as Illumina GA2, Roche 454, etc. as reviewed 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 methods of modification are based on techniques of gene replacement, gene deletion, or gene disruption.

For example, in case of replacement of a polynucleotide, polynucleotide construct or expression cassette, an appropriate polynucleotide may be introduced at the target locus to be replaced. The appropriate polynucleotide may be present on a cloning vector. Exemplary integrative cloning vectors comprise a DNA fragment, which is homologous to the polynucleotide and I or has homology to the polynucleotides flanking the locus to be replaced for targeting the integration of the cloning vector to this pre-determined locus. To promote 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 homologous to the polynucleotide (or flanking sequences) to be replaced. This process is called homologous recombination and this technique may also be used to achieve (partial) gene deletion or gene disruption.

For example, for gene disruption, a polynucleotide corresponding to the endogenous polynucleotide may be replaced by a defective polynucleotide, that is a polynucleotide that fails to produce a (fully functional) protein. By homologous recombination, the defective polynucleotide replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker, which may be used for selection of transformants in which the polynucleotide has been modified.

Alternatively, modification due to which the recombinant microorganism has a deficiency in a polypeptide as disclosed herewith may be performed by established anti-sense techniques using a polynucleotide complementary to the polynucleotide encoding said polypeptide. More specifically, expression of the polynucleotide encoding a polypeptide as disclosed herewith by a recombinant cell may be reduced or eliminated by introducing a polynucleotide with a sequence complementary to the sequence of the polynucleotide encoding said polypeptide which may be transcribed in the recombinant cell and is capable of hybridizing to the mRNA coding for said polypeptide produced in the recombinant cell. Under conditions allowing the complementary anti-sense polynucleotide to hybridize to the said polypeptide, the amount of protein translated is thus reduced or eliminated. An example of expressing an antisense-RNA is shown in Appl. Environ. Microbiol. 2000 Feb; 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 uridinediphosphateglucose pyrophosphorylase and its inhibition by antisense RNA. Planta. (1993); 190(2):247-52.). Furthermore, modification, downregulation or inactivation of a polypeptide capable of deglycosylating a mogroside product may be obtained via the RNA interference (RNAi) technique (FEMS Microb. Lett. 237 (2004): 317-324). In this method, identical sense and antisense parts of the polypeptide (e.g. beta glucanase) encoding polynucleotide which expression is to be affected, are cloned behind each other with a nucleotide spacer in between, and inserted into an expression vector. After such a molecule is transcribed, formation of small nucleotide fragments will lead to a targeted degradation of the mRNA, which is to be affected. The elimination of the specific polypeptide capable of deglycosylating a mogroside product mRNA can be to various extents. The RNA interference techniques described in W02008/053019, W02005/05672A1 ,

W02005/026356A1 , 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 downregulation, modification or inactivation of a polynucleotide.

To increase the likelihood that the introduced enzymes are expressed in active form in a recombinant microorganism as disclosed herein, the corresponding encoding polynucleotide may be adapted to optimize codon usage to that of the chosen recombinant microorganism. The adaptiveness of the polynucleotides encoding the enzymes to the codon usage of the chosen recombinant microorganism may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) 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; also see: Jansen et al., 2003, Nucleic Acids Res. 31 (8):2242-51). An adapted polynucleotide may have a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.

The recombinant cell as disclosed herein may be genetically modified with (a) polynucleotide(s) which is (are) adapted to the codon usage of the recombinant microorganism using codon pair optimization technology which is well known to those skilled in the art. Codon-pair optimization is a method for producing a polypeptide in a recombinant cell, wherein the polynucleotides encoding the polypeptide have been modified with respect to their codon-usage, in particular the codon-pairs that are used, to obtain improved expression of the polynucleotide encoding the polypeptide and/or improved production of the polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.

Further improvement of the activity of the enzymes in vivo in a recombinant cell as disclosed herein, can be obtained by well-known methods like error prone PCR or directed evolution. An exemplary method of directed evolution is described in W003010183 and W003010311.

The term “mogroside precursor” as used herewith refers to an intermediate compound in the mogroside biosynthetic pathway from squalene to mogrol, wherein the mogroside precursor comprises a cucurbitadienol or mogrol backbone. A 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 mogrol.

The term "naturally-occurring" as used herein refers to processes, events, or products that occur in their relevant form in nature. By contrast, "not naturally-occurring" refers to processes, events, or products whose existence or form involves the hand of man. The term "non-naturally occurring is herein synonymous with "man-made". Generally, the term “naturally-occurring” with regard to polypeptides or nucleic acids can be used interchangeably with the term "wild-type" or “native”. It refers to polypeptide or nucleic acids encoding a polypeptide, having an amino acid sequence or polynucleotide sequence, respectively, identical to that found in nature. Naturally occurring polypeptides include native polypeptides, such as those polypeptides naturally expressed or found in a particular cell. Naturally occurring polynucleotides include native polynucleotides such as those polynucleotides naturally found in the genome of a particular cell. Additionally, a sequence that is wild-type or naturally occurring may refer to a sequence from which a variant or a synthetic sequence is derived.

A “nucleic acid molecule” or “polynucleotide” (the 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 (comprising e.g. a coding sequence or another polynucleotide sequence) in a functional relationship. A polynucleotide is "operably linked" when it is placed into a functional relationship with another polynucleotide. For instance, a promoter sequence or enhancer sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase 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 C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

A “polypeptide” is represented by an amino acid sequence.

As defined herewith, a “polypeptide capable of deglycosylating a mogroside product” according to the disclosure may be a glycoside hydrolase (EC:3.2.1 .-), such as an exo-acting glycoside hydrolase. Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Typically, the glycoside hydrolase according to the disclosure may be capable of cleaving the glucose moiety in an exo fashion, i.e. may be capable of cleaving a glycosidic bond and releasing a glucose molecule. Typically, the glycoside hydrolase according to the disclosure may be a p-glucosidase (EC 3.2.1 .21), a glucan 1 ,3-p-glucosidase (EC 3.2.1 .58), a glucan 1 ,4-p-glucosidase (EC 3.2.1.74). For the purpose of this disclosure the terms “polypeptide capable of deglycosylating a mogroside product”, “beta-glucanase”, “p-glucanase”, “beta glucanase”, “p glucanase”, may be used interchangeably (abbreviated as BG).

As used herein, the term "promoter" refers to a polynucleotide fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other polynucleotide fragments, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skilled in the art to act directly or indirectly to regulate the amount of transcription from the 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 which can be regulated.

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

As used herein, a “recombinant cell” is defined as a cell which is preferably genetically modified or transformed/transfected with one or more of the polynucleotides as defined elsewhere herein. The presence of the one or more such polynucleotides alters the ability of the microorganism to produce one or more products. A cell that is not transformed/transfected or genetically modified, is not a recombinant cell and does typically not comprise one or more of the polynucleotides enabling the cell to produce a product, e.g. a mogroside. Hence, a non-transformed/non-transfected cell is typically a cell that does not naturally produce a product such as mogroside, although a cell which naturally produces a product, e.g. a mogroside, and which has been modified as disclosed herein (and which thus has an altered ability to produce a mogroside) is considered a recombinant cell as disclosed herein.

Within the context of the present disclosure the term “recombinant cell deficient in (the production of) a polypeptide”, such as a beta glucanase as described herein, means the cell comprises a modification, preferably in its genome, which results in a reduced or no production of the polypeptide if compared to the parent cell that has not been modified, when analyzed under the same conditions. Alternatively, or in addition thereto, the cell comprises a modification which results in a (modified) polypeptide derived from the polypeptide as described herein with decreased or no activity (which activity may be enzymatic or other biological activity), if compared to the parent cell that has not been modified, when analyzed under the same conditions. Therefore, a recombinant cell is deficient in (the production of) a polypeptide as described herein when a) it produces less of the polypeptide as defined herein or it produces no polypeptide as defined herein if compared with the parent cell which has not been modified and measured under the same conditions; and/or b) has a reduced expression level or has a reduced translation level of the mRNA transcribed from a gene encoding the polypeptide; b) it produces a polypeptide with decreased or no activity if compared to the cell that has not been modified, when analyzed under the same conditions.

Deficiency in production of a polypeptide as defined herein in a recombinant cell may be measured by determining the amount and/or (specific) activity of the relevant polypeptide produced by the recombinant microorganism modified in its genome and/or it may be measured by determining the amount of (free) mRNA transcribed from a gene encoding the polypeptide and/or it may be measured by determining the amount of a product produced by the polypeptide in a recombinant microorganism modified in its genome as defined above and/or it may be measured by gene or genome sequencing if compared to the parent (recombinant) microorganism which has not been modified in its genome. Deficiency in the production of said polypeptide can be measured using any assay available to the skilled person, such as transcriptional profiling, Northern blotting, RT- PCR, Q-PCR and Western blotting.

“Sequence identity” is herein defined as a 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. Usually, sequence identities or similarities are compared over the whole length of the sequences compared, i.e. sequence identities or similarities are compared over the whole length of the sequence in respect of which the sequence identity is determined. For the purpose of this disclosure, to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this disclosure the NEEDLE program from the EMBOSS package was used (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) pp276 — 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the disclosure is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as “longest-identity”.

The nucleic acid and protein sequences as disclosed herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTX programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the disclosure. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

Detailed description

The disclosure provides a recombinant cell capable of producing one or more mogrosides and/or mogroside precursors, such as capable of producing one or more mogrosides in a culture medium, wherein the cell has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product. Said polypeptide capable of deglycosylating a mogroside product may be capable of hydrolyzing at least

According to one embodiment the polypeptide capable of deglycosylating a mogroside product according to the disclosure may be a glycoside hydrolase (EC:3.2.1 .-), such as an exoacting glycoside hydrolase. The polypeptide capable of deglycosylating a mogroside product (namely, beta glucanase) may be selected from a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 ; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2.

Mogrosides are glycosylated forms of the triterpene Mogrol. The mogrol backbone comprises hydroxylic groups which may undergo a glycosylic bond at the carbon at position 3 (C3), at position 11 (C11), at position 24 (C24) and/or at position 25 (C25). Typically, most relevant mogrosides comprise primary and/or secondary glycosylations at the C3 and/or the C24 of the mogrol backbone. For example, Mogroside IEi comprises a beta glycosidic linkage (i.e., bond) between the hydroxyl group at C3 of the mogrol backbone and the hydroxyl group at C1 of a glucose moiety. Mogroside IAi comprises a beta glycosidic linkage between the hydroxyl group at C24 of the mogrol backbone and the hydroxyl group at C1 of a glucose moiety. This glycosylation of one glucose moiety at position C3 and/or C24 of the mogrol backbone is called primary glycosylation. Mogroside HE comprises a beta glycosidic linkage both at the hydroxyl group of C3 and of C24 of the mogrol backbone. A glucose moiety at position C3 and/or C24 of a primary glycosylated mogroside can be further glycosylated through a beta-1 ,6-glycosydic bond between the hydroxyl at C6 of the primary glucose and the hydroxyl at C1 of a second glucose moiety and/or through a beta-1 ,2-glycosydic bond between the hydroxyl at C2 of the primary glucose and the hydroxyl at C1 of a second glucose moiety. Some mogrosides may comprise a beta-1 ,4 glycosylation between the hydroxyl at position C4 of a primary glucose moiety linked at position C3 of the mogrol backbone and the hydroxyl at position 1 of a second glucose moiety. Isomogroside IVE comprises such a beta-1 ,4 glycosylation. Said beta 1 ,2, beta 1 ,4 and beta 1 ,6 glucosylation in a mogroside molecule are known as secondary glycosylation. Figure 1 shows the structure of several known mogroside molecules.

The recombinant cell according to the disclosure has been modified to result in a deficiency of a beta glucanase polypeptide.

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

(a) the glycosidic bond between the carbon at position 24 (C24 atom) of the mogrol backbone of a mogroside and a beta-1 -glucose moiety bound at said position; (b) the glycosidic bond between the carbon at position 3 (C3 atom) of the mogrol backbone of a mogroside and a beta-1 -glucose moiety bound at said position;

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

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

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

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

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

Typically, said beta glucanase is capable of hydrolyzing at least

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

(b) the glycosidic bond between the carbon at position 24 (C24 atom) of the mogrol backbone of a mogroside and a beta-1 -glucose bound at said position.

In another embodiment said beta glucanase is capable of hydrolyzing at least:

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

(b) the beta 1 ,6 glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C24 atom of the mogrol backbone of a mogroside.

In yet another embodiment said beta glucanase is capable of hydrolyzing at least:

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

(b) the beta 1 ,2 glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C24 atom of the mogrol backbone of a mogroside.

According to the disclosure the beta glucanase may be selected from a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 ; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2. Therefore the recombinant cell according to the disclosure may be modified to be deficient in a polypeptide with an amino acid sequence set out in SEQ ID NO: 1 or set out in SEQ ID NO: 2. Alternatively or additionally, the recombinant cell according to the disclosure may be modified to be deficient in a functional homologue (also known as functional equivalent or functional variant) of the polypeptide capable of deglycosylating a mogroside product according to SEQ ID NO:1 or according to SEQ ID NO: 2. Alternatively or additionally, the recombinant cell according to the disclosure may be modified to be deficient in more than one beta- glucanase. For example, the recombinant cell may be modified to be deficient in both the polypeptide according to SEQ ID NO: 1 and the polypeptide according to SEQ ID NO: 2. Alternatively the recombinant cell may be modified to be deficient both in a functional homologue of the polypeptide according to SEQ ID NO: 1 and a functional homologue of the polypeptide according to SEQ ID NO: 2. Alternatively the recombinant cell may be modified to be deficient both in a polypeptide according to SEQ ID NO: 1 and a functional homologue of the polypeptide according to SEQ ID NO: 2. Alternatively the recombinant cell may be modified to be deficient both in a functional homologue of the polypeptide according to SEQ ID NO: 1 and in a polypeptide according to SEQ ID NO: 2.

Typically, the functional homologue of a polypeptide with amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:2 is a polypeptide capable of deglycosylating a mogroside product, such as by hydrolyzing one or more of the beta glycosidic bonds between two glucose moieties in the mogroside and/or between a glucose moiety and the mogrol backbone, typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:2.

The inventors have found that in a strain capable of producing one or more mogrosides and deficient in the production of the EXG1 glucanase, the production of certain mogrosides comprising a beta-glycosidic linkage at the C3 and/or at the C24 of the mogrol backbone (such as Mogroside IEi, Mogroside IAi, Mogroside HIE, Siamenoside I) is still limited. Surprisingly, in a recombinant cell capable of producing one or more mogrosides and deficient in a polypeptide according to SEQ ID NO: 1 , the production of mogrosides comprising one beta-glycosidic linkage at the C3 and/or at the C24 of the mogrol backbone is improved if compared with an otherwise identical recombinant cell which has not been modified to be deficient in a polypeptide according to SEQ ID NO: 1 , when both cell are cultured under the same conditions. In one embodiment in a recombinant cell capable of producing one or more mogrosides and deficient in a polypeptide according to SEQ ID NO: 1 , the production of mogrosides comprising a beta-glycosidic linkage at the C3 of the mogrol backbone is improved if compared with an otherwise identical recombinant cell which has not been modified to be deficient in a polypeptide according to SEQ ID NO: 1 , when both cells are cultured under the same conditions. In another embodiment, in a recombinant cell capable of producing one or more mogrosides and deficient in a polypeptide according to SEQ ID NO: 1 , the production of mogrosides comprising a beta-glycosidic linkage at the C24 of the mogrol backbone is improved if compared with an otherwise identical recombinant cell which has not been modified to be deficient in a polypeptide according to SEQ ID NO: 1 , when both cell are cultured under the same conditions. In yet another embodiment, in a recombinant cell capable of producing one or more mogrosides and deficient in a polypeptide according to SEQ ID NO: 1 , the production of mogrosides comprising a beta-glycosidic linkage at the C3 and at the C24 of the mogrol backbone is improved if compared with an otherwise identical recombinant cell which has not been modified to be deficient in a polypeptide according to SEQ ID NO: 1 , when both cell are cultured under the same conditions. In the context of the present disclosure, the improved production of mogroside in a recombinant cell deficient in a polypeptide (such as the polypeptide according to SEQ ID NO: 1 or 2) if compared to an otherwise identical recombinant cell not deficient in said polypeptide refers to the increase in the yield or amount of mogroside or concentration that is produced by the recombinant cell which is deficient in said polypeptide (such as the polypeptide according to SEQ ID NO: 1 or 2) when compared to an otherwise identical recombinant cell that has not been modified, when both cells are cultured under the same conditions. The deficiency of the polypeptide according to SEQ ID NO: 1 leads to an increase in the production of mogrosides that contain a beta-glycosidic linkage at the C3 and/or C24 positions of the mogrol backbone. Essentially, the modified recombinant cell is able to produce more of the desired mogrosides under the same conditions as the unmodified cell.

Therefore, a recombinant cell capable of producing one or more mogrosides which has been modified to result in a deficiency of one or more beta glucanase as herewith disclosed, may be modified to result in a deficiency of both a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 , typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 1 ; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2, typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 2.

The inventors have also found that a recombinant strain capable of producing one or more mogrosides and which is deficient in both a polypeptide according to SEQ ID NO: 1 and a polypeptide according to SEQ ID NO: 3 (encoding an EXG1 polypeptide), the production of more highly glycosylated mogrosides (i.e. mogrosides comprising secondary glycosylation at the C3 and/or C24 position), such as Mogroside II A2 is synergistically improved if compared with an otherwise identical recombinant cell which has not been modified to be deficient in a polypeptide according to SEQ ID NO: 1 and SEQ ID NO: 3, when both cells are cultured under the same conditions.

In one embodiment of the methods and recombinant cells according to the disclosure, the recombinant cell capable of producing mogroside precursor(s) or one or more mogrosides according to the disclosure is a cell wherein the deficiency in a polypeptide capable of deglycosylating a mogroside product results in increased production of one or more mogrosides if compared with an otherwise identical recombinant cell which is not deficient in said polypeptide, when both cells are cultured under the same conditions. In other words, the recombinant cell deficient in the polypeptide is able to produce more of the desired mogrosides under the same conditions as an otherwise identical recombinant cell which is not deficient in the polypeptide, when both cells are cultured under the same conditions.

Therefore, a recombinant cell capable of producing one or more mogrosides which has been modified to result in a deficiency of a beta glucanase as herewith disclosed, may be further modified to result in a deficiency of an EXG1 and/or EXG2 polypeptide, particularly a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 3. In one embodiment, the recombinant cell may be modified to result in a deficiency of both a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 , typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 1 ; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 3, typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 3.

The inventors have also surprisingly found that in a recombinant cell capable of producing one or more mogrosides and deficient in a polypeptide according to SEQ ID NO: 2, the production of mogrosides comprising beta-1 ,2 and/or beta-1 ,6 glycosidic linkage, such Mogroside 11 IAi can be improved if compared to the production of an otherwise identical a recombinant cell which is deficient in an EXG1 polypeptide (e.g. the polypeptide with amino acid sequence according to SEQ ID NO: 3). The improvement in the production of Mogroside lllAi can be synergistically increased in a recombinant cell capable of producing one or more mogrosides and deficient in a polypeptide according to SEQ ID NO: 2 which is further deficient in the production of the polypeptide according to SEQ ID NO: 3.

Therefore, the recombinant cell capable of producing a mogroside precursor or one or more mogrosides which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product, may be modified to result in a deficiency of both a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2, typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 2; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 3, typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 3.

In yet another embodiment, the recombinant cell capable of producing a mogroside precursor or one or more mogrosides which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product, may be modified to result in a deficiency of a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 , typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 1 , and of a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2, typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 2, and of a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 3, typically a polypeptide with an amino acid sequence with 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 with the amino acid sequence of the polypeptide with amino acid sequence according to SEQ ID NO: 3.

Accordingly, in one embodiment of the methods and recombinant cells according to the disclosure, a recombinant cell is provided which is capable of producing one or more mogrosides and which deficient in a beta glucanase as disclosed herein above, wherein the production of mogroside product by said cell is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the beta glucanase, when both cells are cultured under the same conditions. In one aspect, the production of mogroside product comprising a beta-glycosidic linkage at the C3 and/or at the C24 of the mogrol backbone by the cell modified to be deficient in a beta glucanase is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the beta glucanase, when both cells are cultured under the same conditions. For example, in one aspect the production i.e. yield, concentration or amount of Mogroside IEi , Mogroside HE and/or Mogroside IAi, produced by the recombinant cell modified to be deficient in a beta glucanase is higher if compared with the production i.e. yield, concentration or amount of the same mogroside(s) produced by an otherwise identical recombinant cell which has not been modified to be deficient in the beta glucanase, when both cells are cultured under the same conditions. Particularly the production i.e. yield, concentration or amount of Mogroside IEi and/or Mogroside IAi is higher. In another aspect, the production of mogroside product comprising a beta-1 ,2-glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom and/or at the C24 atom of the mogroside backbone by the cell modified to be deficient in a beta glucanase is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the polypeptide capable of deglycosylating a mogroside product, when both cells are cultured under the same conditions. For example, in another aspect the production i.e. yield, concentration or amount of Mogroside HA, Mogroside lllAi, Mogroside HIE, Mogroside V, Mogroside VI, Mogroside IVE and/or Siamenoside I produced by the recombinant cell modified to be deficient in a beta glucanase is higher if compared with the production i.e. yield, concentration or amount of the same mogroside(s) produced by an otherwise identical recombinant cell which has not been modified to be deficient in the beta glucanase, when both cells are cultured under the same conditions. Particularly the production of Mogroside lllAi may be improved. In yet another aspect, the production of mogroside product comprising a beta-1 ,6-glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom and/or at the C24 atom of the mogroside backbone by the cell modified to be deficient in a beta glucanase is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the polypeptide capable of deglycosylating a mogroside product, when both cells are cultured under the same conditions. For example, in yet another aspect the production i.e. yield, concentration or amount of Mogroside IIAi , Mogroside IIA2, Mogroside IIIA2, Mogroside IVE, Mogroside IVA, Mogroside III, Mogroside IIIA1, Mogroside V, Mogroside VI and/or Siamenoside I produced by the recombinant cell modified to be deficient in a beta glucanase is higher if compared with the production i.e. yield, concentration or amount of the same mogroside(s) produced by an otherwise identical recombinant cell which has not been modified to be deficient in the beta glucanase, when both cells are cultured under the same conditions. Particularly, the production i.e. yield, concentration or amount of Mogroside I IA2 may be improved.

The disclosure also provides a recombinant cell capable of producing one or more mogrosides and deficient in a beta glucanase as disclosed herein above wherein the production of mogroside product by said cell is higher if compared with the production by an otherwise identical recombinant cell which has been modified to be deficient in a polypeptide with the amino acid sequence set out in SEQ ID NO: 3, when both cells are cultured under the same conditions. In one aspect, the production of mogroside product comprising a beta-glycosidic bond at the C3 and/or at the C24 of the mogrol backbone by the cell modified to be deficient in a beta glucanase is higher if compared with the production by an otherwise identical recombinant cell which has been modified to be deficient in a polypeptide with the amino acid sequence set out in SEQ ID NO: 3, when both cells are cultured under the same conditions. In another aspect, the production of mogroside product comprising a beta-1 ,2-glycosidic bond between a second glucose molecule and a beta-1- glucose bound at the C3 atom and/or at the C24 atom of the mogroside backbone by the cell modified to be deficient in a beta glucanase is higher if compared with the production by an otherwise identical recombinant cell which has been modified to be deficient in a polypeptide with the amino acid sequence set out in SEQ ID NO: 3, when both cells are cultured under the same conditions. In yet another aspect, the production of mogroside product comprising a beta-1 ,6- glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom and/or at the C24 atom of the mogroside backbone by the cell modified to be deficient in a beta glucanase is higher if compared with the production by an otherwise identical recombinant cell which has been modified to be deficient to be deficient in a polypeptide with the amino acid sequence set out in SEQ ID NO: 3, when both cells are cultured under the same conditions. The polypeptide capable of deglycosylating a mogroside product as described herewith, for example a polypeptide with the amino acid sequences set out in SEQ ID NO: 1 or 2; or a polypeptide with an amino acid sequence at least 50% identical to either one of SEQ ID NO:1 or 2, may be a polypeptide capable of deglycosylating a mogroside compound which is a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, a penta-glycosylated or exa-glycosylated mogroside compound, or an isomer thereof. The polypeptide may be capable of deglycosylating a mogroside product comprising one or more of Mogroside IAi (MoglA1>, Mogroside IEi (MoglEI), Mogroside IIA (MogllA), Mogroside IIAi (MogllAI), Mogroside IIA2 (MogllA2), Mogroside HE (MogllE), Mogroside IIIA1 (MoglllAI), Mogroside IIIA2 (MoglllA2), Mogroside HIE (MoglllE), Mogroside III (Moglll), Mogroside IVA (MogIVA), Mogroside IVE (MogIVE), Mogroside V (MogV), Mogroside VI (MogVI), Mogroside VIA (MogVIA), Mogroside Vla1 (MogVIal), Mogroside VIB (MogVIB), Siamenoside I (Sia), Isomogroside IVE (IsolVE), Isomogroside V (IsoV) or a-Siamenoside I (aSia).

Therefore, in one embodiment of the methods and recombinant cells according to the disclosure, the recombinant cell capable of producing mogroside precursor(s) or one or more mogrosides according to the disclosure which is deficient in a polypeptide capable of deglycosylating a mogroside product is a cell wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating a mogroside compound which is a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, a penta-glycosylated or exa-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: Mogroside IA1, Mogroside IE1, Mogroside IIA, Mogroside IIA1, Mogroside IIA2, Mogroside HE, Mogroside IIIA1 , Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vlai, Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I.

Mogrosides and/or mogroside precursors 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 indicated herewith as UDP-Glycosyltransferases). Therefore, the recombinant cell capable of producing one or more mogrosides and/or mogroside precursor(s) according to the disclosure comprises, is capable of (over)expressing or (over)expresses, at least one polynucleotide encoding a 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, such as mogrol or mogroside. As shown in Figure 4, the mogroside synthesis starts with the primary glycosylation of the mogrol molecule at the carbon at C3 and/or at the carbon at C24, leading to Mogroside IA1 , Mogroside IE1 , and/or Mogroside HE, by the action of one or more UGT polypeptides capable of catalyzing the p-1 glycosylation of the mogrol at position C24, C3, and/or both C3 and C24, respectively. Alternatively, primary glycosylation may occur at position C11 or C25 of the mogrol backbone. A UGT polypeptide capable of primary glycosylation is herewith indicated as a primary UGT polypeptide. A primary UGT polypeptide may therefore be capable of glycosylating mogrol or a mogroside compound at its 03 hydroxyl group, C24 hydroxyl group, C11 hydroxyl group, and/or C25 hydroxyl group. In other words, a primary UGT polypeptide can catalyze the glycosylation of a hydroxyl group in position C3, C24, C11 and/or C25 of mogrol or of a mogrol backbone in a mogroside. As shown in Figure 4, Mogroside IAi , Mogroside HE and/or Mogroside I Ei can be converted to higher glycosylated mogroside compounds by the activity of one or more UGT polypeptides capable of catalyzing the p-1 ,2 glycosylation and/or the p-1 ,6 glycosylation of the hydroxyl group in C2' position of a glucose moiety linked to position C24 of the mogrol backbone (a 024-O-glucose) (via a p-1 glycosidic bond) and/or or capable of catalyzing the p-1 ,2 glycosylation and/or the p-1 ,6 glycosylation of the hydroxyl group in C2' position of a glucose moiety linked at position 03 of the mogrol backbone (a 03-O-glucose) (via a p-1 glycosidic bond). UGT polypeptides capable of catalyzing the p-1 ,2 glycosylation and/or p-1 ,6 glycosylation of a mogroside are indicated as secondary UGT polypeptides. Starting from Mogroside IAi , Mogroside HE and/or Mogroside I Ei , by the action of one or more secondary UGT polypeptides one or more of Mogroside IIA, Mogroside IIA1 , Mogroside IIA2, Mogroside IIIA1 , Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I may be obtained.

Hence, in one aspect of the methods and recombinant cells according to the disclosure, the recombinant cell capable of producing one or more mogrosides, and/or mogroside precursors, and which has been modified to result in a deficiency in the production of a beta glucanase according to the disclosure is a cell comprising, capable of (over)expressing (such as under specific conditions), or (over)expressing, one or more of the following recombinant polynucleotides:

(a) a polynucleotide encoding a polypeptide capable of glycosylating mogrol or a mogroside compound at its 03 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group, and/or C25 hydroxyl group, typically at it C3 hydroxyl group and/or C24 hydroxyl group;

(b) a polynucleotide encoding a polypeptide capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 (a 024-O-glucose) of a mogroside compound and/or or capable of beta-1 ,2-glycosylation of the 02' hydroxyl group of a glucose moiety at position C3 (a 03-O-glucose) of a mogroside compound;

(c) a polynucleotide encoding a polypeptide capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glucose moiety at position C3 of a mogroside compound and/or capable of beta-1 ,6-glycosylation of the 06' hydroxyl group of a glycose moiety at position C24 of a mogroside compound; particularly wherein the polypeptide according to (a), (b) or (c) is a uridine 5’-diphosphate- dependent glycosyl transferase polypeptide (UGT polypeptide). Typically, one or more of the polypeptides and/or polynucleotides according to (a), (b) or (c) may be heterologous polypeptides and/or heterologous polynucleotides. Typically, one or more of the polynucleotides according to (a), (b) or (c) may be constitutively expressed. In another aspect the expression of one or more of the polynucleotides according to (a), (b) or (c) may be induced. A polypeptide, particularly a UGT polypeptide, according to (a), (b) or (c) can be any UGT polypeptide known in the art, such as any of the polypeptides with amino acid sequence as depicted in Table 2a to 2e.

In one aspect of the disclosure, a UGT polypeptide according to the disclosure may be any polypeptide with the amino acid sequence according to SEQ ID NO: 6, or 14 to 18 herein, or any of the amino acid sequences indicated in Table 2a to 2e or, alternatively it may be a UGT polypeptide, typically a functional homolog thereof, with an amino acid sequence which 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 identity with the amino acid sequence of any polypeptide with the amino acid sequence according to SEQ ID NO: 6, or 14 to 18 herein, or with any amino acid sequence as indicated in Table 2a to 2e.

In one aspect of the disclosure, a polypeptide, particularly a UGT polypeptide capable of glycosylating mogrol or a mogroside compound at its C3 hydroxyl group may be any polypeptide disclosed in the art, such as a UGT polypeptide with amino acid sequence as indicated in Table 2a or, alternatively it may be a UGT polypeptide, such as a functional homolog thereof, with an amino acid sequence which 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 identity with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 2a.

In another aspect of the disclosure, a polypeptide, particularly a UGT polypeptide capable of glycosylating mogrol or a mogroside compound at its C24 hydroxyl group may be any polypeptide disclosed in the art, such as a UGT polypeptide with amino acid sequence as indicated in Table 2b or alternatively it may be a UGT, such as a functional homolog thereof, polypeptide with an amino acid sequence which 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 identity with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 2b.

In yet another aspect of the disclosure, a polypeptide, particularly a UGT polypeptide capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 (a 024-O-glucose) of a mogroside and/or or capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C3 (a 03-O-glucose) of a mogroside compound, may be any polypeptide disclosed in the art, such as a UGT polypeptide, such as a functional homolog thereof, with amino acid sequence as indicated in Table 2c or alternatively it may be a UGT polypeptide with an amino acid sequence which 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 identity with the amino acid sequence of any polypeptide with amino acid sequence as indicated Table 2c. In yet another aspect of the disclosure, a polypeptide, particularly a UGT polypeptide capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glucose moiety at position C3 of a mogroside compound and/or capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glycose moiety at position C24 of a mogroside compound, may be any polypeptide disclosed in the art, such as a UGT polypeptide with amino acid sequence as indicated in Table 2d or the UGT polypeptide may have an amino acid sequence which 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 identity with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 2d.

Table 2a to 2e provide non-limiting examples of amino acid sequences of UGT polypeptides disclosed in patent documents and their ability to perform specific glycosylations according to the patent documents.

Table 2a reports amino acid sequences of UGT polypeptides which according to the patent document to which they belong have the ability to catalyze p-1 glycosylation at C3 carbon of the mogrol backbone in a mogrol or mogroside. Table 2b reports amino acid sequences of UGT polypeptides which according to the patent document to which they belong have the ability to catalyze p-1 glycosylation at C24 carbon of the mogrol backbone in a mogrol or mogroside. Similarly, Table 2c reports amino acid sequences for UGT polypeptides with p-1 ,2 glycosylation activity at the glucose located at C3 and/or C24 of mogrol or mogroside backbone, and table 2d reports amino acid sequences for UGT polypeptides with p-1 ,6 glycosylation activity at the glucose located at C3 and/or C24 of mogrol or mogroside backbone. Finally, Table 2e reports the amino acid sequences of those UGT polypeptides for which the specific type of UGT activity is not indicated in the corresponding patent document. When known from the corresponding patent document, specific substrates are given in the tables. For example, UGT94C9 being able to use MogIVE as a substrate to produce Mog V is indicated as “UGT94C9 (MogIVE)” in table 2d. When, known from the patent document, the product of the reaction catalysed by the UGT is also indicated. For example, UGT98 being able to convert MogllE into MoglllA2 is indicated as UGT98 (MogllE-> IIIA2) in table 2d. The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed.

Table 2a

Table 2b

Table 2c

Table 2d Table 2e

A recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and wherein the recombinant cell further comprises, particularly wherein the recombinant cell is capable of (over)expressing (such as under specific conditions) or (over)expresses, one or more nucleotides encoding one or more UGT polypeptides as disclosed herewith, may be used in a method of producing one or more mogrosides comprising contacting mogrol, or one or more mogroside substrates with said cell, or a lysate or extract thereof under conditions suitable to produce said one or more mogrosides, optionally isolating said one or more mogrosides.

Therefore, in one aspect the disclosure provides a method of producing one or more mogrosides and/or mogroside precursors comprising culturing a recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and comprising, being capable of expressing or expressing at least one polynucleotide encoding for a UGT polypeptide as disclosed herein above under conditions suitable to the production of said mogrosides and/or mogroside precursors, optionally isolating said one or more mogrosides and/or mogroside precursors.

Depending on the type of mogrosides that one wishes to produce and depending on the mogroside precursor used in the method, the cell will comprise, will be capable of (over)expressing or will (over)express one or more polynucleotides encoding a UGT polypeptide.

For example, a recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and capable of producing MoglAI starting from mogrol, may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its C24 hydroxyl group, such as one or more of the suitable polypeptides disclosed in Table 2b.

A recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and capable of producing MoglEI starting from mogrol, may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its C3 hydroxyl group, such as one or more of the suitable polypeptides disclosed in Table 2a.

A recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and capable of producing MogllE starting from mogrol, may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its 03 hydroxyl group and at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol at its C24 hydroxyl group, such as one or more of the suitable polypeptides disclosed in Table 2a and 2b, respectively.

A recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and capable of producing MoglllE starting from mogrol, may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol or mogroside at its 03 hydroxyl group and/or at its C24 hydroxyl group, such as one or more of the suitable polypeptides disclosed in Table 2a and 2b, and at least a polynucleotide encoding a polypeptide capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 of a mogroside compound, such as one or more of the suitable polypeptides disclosed in Table 2c.

A recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and capable of producing MoglllAI starting from mogrol, may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol or mogroside at its C24 hydroxyl group, such as one or more of the suitable polypeptides disclosed in Table 2b, at least a polynucleotide encoding a polypeptide capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 of a mogroside compound, such as one or more of the suitable polypeptides disclosed in Table 2c, and at least a polynucleotide encoding a polypeptide capable of beta-1 ,6-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 of a mogroside compound, such as one or more of the suitable polypeptides disclosed in Table 2d.

A recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure and capable of producing Sia starting from mogrol, may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol or mogroside at its C3 hydroxyl group and at its C24 hydroxyl group, such as one or more of the suitable polypeptides disclosed in Table 2a and 2b, respectively, at least a polynucleotide encoding a polypeptide capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 of a mogroside compound, such as one or more of the suitable polypeptides disclosed in Table 2c, and at least a polynucleotide encoding a polypeptide capable of beta-1 ,6-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 of a mogroside compound, such as one or more of the suitable polypeptides disclosed in Table 2d.

Figure 4 depicts a putative mogroside biosynthetic pathway from Mogrol to mogrosides, including the types of UGT polypeptides needed to produce each mogroside.

The putative mogroside biosynthesis in Siraitia grosvenorii and some of the enzymes involved therein has been studied (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). Subsequently, methods to use recombinant cells to produce mogroside precursors and mogrosides have been described (see for example WO2014/086842 A1 , WO2016/038617 A1 , WO2016/050890 A2).

Mogrosides have a triterpenoid backbone. Triterpenes, such as mogrosides, are synthesized via the isoprenoid pathway by cyclization of 2,3-epoxysqualene leading to a cucurbitane backbone. The triterpenoid backbone subsequently undergoes various transformations such as oxidations, substitutions, mediated by cytochrome P450-dependent monooxygenases and other enzymes, and glycosylations mediated by UGT polypeptides as described herein above.

The putative biosynthesis of Mogrol from squalene is depicted in Figure 3. Squalene may in turn be formed through the mevalonate pathway (MVA pathway) starting from acetyl-Coenzyme A (Acetyl-CoA). The biosynthesis of squalene from Acetyl-CoA is well known and is depicted in Figure 2. Alternatively, the synthesis may start from 1-deoxy-D-xylulose 5-phosphate through 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 nonmevalonate pathway.” Cell. Mol. Life Sci. (2004) 61 :1401-1426).

Squalene is the biochemical precursor of steroids which are essential components in all cells. Squalene is therefore synthesized in all cells. For a recombinant cell to be able to produce mogrol, a cell may be transformed with one or more polypeptides capable to catalyze the several steps, such as those depicted in Figure 3, which lead to the synthesis of Mogrol.

The synthesis of Mogrol starting from squalene may comprise one or more steps described hereafter.

2,3-Epoxysqualene may be obtained from squalene by the action of a polypeptide with squalene epoxidase activity. Squalene epoxidase may also catalyze the conversion of 2,3- epoxysqualene into 2,3,22,23-diepoxysqualene.

Cucurbitadienol may be obtained from 2,3-epoxysqualene by the action of cucurbitadienol synthase. Cucurbitadienol synthase may also catalyze the cyclization of 2,3,22,23- diepoxysqualene into 24,25-epoxy-curcubitadienol.

Cucurbitadienol, 24,25-epoxy-curcubitadienol and 24,25-dihydroxy-cucurbitadienol respectively, may be converted to 11-hydroxy cucurbitadienol, 11 -hydroxy-24, 25-epoxy- curcubitadienol, and to Mogrol, respectively, by one or more cytochrome P450 enzyme (CYP) in combination with at least one CYP activator, such as cytochrome P450 reductase (CPR).

Cucurbitadienol and 11-hydroxy cucurbitadienol, respectively, may be converted to 24,25- epoxy cucurbitadienol and 11 -hydroxy-24, 25-expoxy cucurbitadienol, respectively by one or more cytochrome P450 enzymes in combination with 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 described recombinant cells capable to produce mogrol precursors, mogrol and/or mogrosides wherein the recombinant cell comprises a heterologous polynucleotide encoding a cytochrome b5 (CB5) polypeptide. Said recombinant cell is capable of producing more mogrol than a control cell that does not comprise said heterologous polynucleotide.

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

Thus, in one embodiment of the methods and recombinant cells according to the disclosure, a recombinant cell is provided which is capable of producing one or more mogrosides and/or mogroside precursors which has been modified to result in a deficiency of a beta glucanase according to the disclosure, wherein the recombinant cell is further capable of producing Mogrol and further comprises, is capable of (over)expressing or (over)expresses one or more of the following recombinant polynucleotides: (a) a polynucleotide encoding a polypeptide capable of synthesizing 2,3-epoxy squalene from squalene, or 2,3,22,23-diepoxy squalene from 2,3-epoxy squalene, particularly wherein said polypeptide is a squalene epoxidase (SQE);

(b) a polynucleotide encoding a polypeptide capable of synthesizing cucurbitadienol from 2,3-epoxy squalene, or 24,25-epoxy-cucurbitadienol from 2,3,22,23-diepoxy squalene, particularly wherein said polypeptide is a 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, particularly wherein said polypeptide is a cytochrome P450 enzyme (CYP450);

(d) a polynucleotide encoding a polypeptide capable of reducing a cytochrome P450 complex, particularly a cytochrome P450 reductase (CPR);

(e) a polynucleotide encoding a polypeptide capable of synthesizing mogrol from 1 1- hydroxy-24, 25-epoxy-cucurbitadienol, or 24,25-dihydroxy-cucurbitadienol from 24,25-epoxy- cucurbitadienol, particularly wherein said polypeptide is an epoxide hydrolase (EPH);

(f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5).

Typically, one or more of the polypeptides and/or polynucleotides according to (a), (b), (c),

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

Typically a recombinant cell which has been modified to result in a deficiency in the production of a beta glucanase according to the 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 category of enzymes, selected from (a), (b), (c), (d),

(e), or (f), such as at least three polynucleotides selected from (a), (b), (c), (d), (e), or (f), such as at least four polynucleotides selected from (a), (b), (c), (d), (e), or (f) (preferably each polynucleotide from a different category of enzymes), such as at least five polynucleotides selected from (a), (b), (c), (d), (e), or (f) (preferably each polynucleotide from a different category of enzymes), such as at least six polynucleotides selected from (a), (b), (c), (d), (e), or (f) (preferably each polynucleotide from a different category of enzymes), typically all the polynucleotides (a), (b), (c), (d), (e), and (f).

In the methods and recombinant cells according to the disclosure, a polypeptide capable of synthesizing 2,3-epoxy squalene from squalene, or 2,3,22,23-diepoxy squalene from 2,3-epoxy squalene, a squalene epoxidase (SQE) may be used.

Squalene epoxidase (EC 1 .4.99.7) may catalyze production of 2,3-epoxy squalene (also known as oxido-squalene) from squalene, or production of 2,3,22,23-diepoxy squalene (also known as dioxido-squalene) from oxido squalene, typically in the presence of NADPH. Therefore, a recombinant cell capable to produce mogroside precursor(s) and/or one or more mogrosides according to the disclosure may comprise, may be able to express or may express a polynucleotide encoding a polypeptide capable of synthesizing 2,3-epoxy squalene from squalene, or 2,3,22,23- diepoxy squalene from 2,3-epoxy squalene, particularly wherein said polypeptide is a squalene epoxidase. Some recombinant cells may comprise an endogenous squalene epoxidase, in which case the endogenous enzyme may suffice. Endogenous oxido-squalene and dioxido-squalene production pathways exist in eukaryotes metabolism, such as yeast metabolism, and accordingly, if the recombinant host is a eukaryote, then said step may be endogenous to the recombinant host. However, it may be advantageous to enhance expression of the endogenous squalene epoxidase by any method known to those skilled in the art.

Therefore, in one embodiment the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase according to the disclosure may comprise, may be capable of (over)expressing or may (over)express at least one polynucleotide encoding a polypeptide capable of synthesizing 2,3- epoxy squalene from squalene, or 2,3,22,23-diepoxy squalene from 2,3-epoxy squalene, particularly wherein said polypeptide is a squalene epoxidase (SQE). Non limiting examples of SQE polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 3. Therefore, the SQE polypeptide may have an amino acid sequence according to SEQ ID NO: 23 herein, or an amino acid sequence according to any sequence 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 which is 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 with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 3 or with the amino acid sequence according to SEQ ID NO: 23 herein.

Table 3 provides non-limiting examples of amino acid sequences of SQE polypeptides disclosed in patent documents. The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed.

Table 3

In the methods and recombinant cells according to the disclosure, a polypeptide capable of synthesizing cucurbitadienol from 2,3-epoxy squalene, or 24,25-epoxy-cucurbitadienol from 2,3,22,23-diepoxy squalene may be used, such as a cucurbitadienol synthase (CDS). Cucurbitadienol synthase (EC:5.4.99.8), a triterpene cyclase also known as oxidosqualene cyclase or triterpene cyclase, has been isolated from Cucurbita plants (Shibuya 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). A cucurbitadienol synthase as disclosed herewith may catalyze the conversion of oxidosqualene (such as 2,3-epoxysqualene or 2,3,22,23-diepoxysqualene) into cucurbitadienol compounds (such as cucurbitadienol and 24,25-epoxy-cucurbitadienol). The activity of cucurbitadienol synthase may be measured with any methods known in the art. For example, the activity of a CDS may be measured from the amount of cucurbitadienol compound produced per unit of enzyme. CDS enzymes may be identified by those skilled in the art by their ability to convert oxidosqualene into cucurbitadienol compounds. Alternatively new CDS enzymes may also be identified based on the comparison of the amino acid sequence with known CDS, e.g., based on the presence of one or more domains in the protein which are associated with CDS activity. In addition, new CDS enzymes may as well be identified based on the comparison of the three- dimensional structure with those of known CDS.

Accordingly, in one embodiment, the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase according to the disclosure may comprise, may be capable of (over)expressing or may (over)express at least one polynucleotide encoding a polypeptide capable of synthesizing cucurbitadienol from 2,3-epoxy squalene, or 24,25-epoxy-cucurbitadienol from 2,3,22,23-diepoxy squalene, particularly wherein said polypeptide is a cucurbitadienol synthase (CDS). Non limiting examples of CDS polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 4. Therefore, the CDS polypeptide may have an amino acid sequence according to SEQ ID NO: 9 herein or according to any sequence disclosed in Table 4, or the CDS polypeptide may have an amino acid sequence which is has 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 of SEQ ID NO: 9 or of any polypeptide with amino acid sequence as indicated in Table 4.

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

Table 4

In the methods and recombinant cells according to the disclosure cytochrome P450 polypeptides (abbreviated CYP or CYP450) may be used. CYPs are heme-b-containing monooxygenases.

As disclosed herein a cytochrome P450 polypeptide is defined as a heme-b-containing enzymes acting as monooxygenases and catalyzing the insertion of one atom of oxygen into an organic substrate RH with the concomitant reduction of the other oxygen atom into water, using the reducing power of NAD(P)(H) as shown below:

RH + 02 + H⁺ + NAD(P)(H) ->• ROH + H2O + NAD(P)⁺

In cytochrome P450 polypeptides, the mechanism involves two successive one-electron transfer steps where electrons originating from NAD(P)(H) are transferred to the CYP heme center via one or more redox partners. The different systems have been classified and described in literature, for example in Hanneman et al. in Biochimica et Biophysica Acta (2007) 1770: 330-344; Urlacher and Girhard in Trends in Biotechnology (2012) 30(1): 26-36; Sadeghi and Gilardi in Biotechnology and Applied Biochemistry (2013) 60(1): 102-110; Roberts et al. (2002) Journal of Bacteriology 184(14), 3898-3908.

Based on the number and organization of the redox partners, the cytochrome P450 systems can be organized in three types of topologies: a) 3-component systems, i.e. systems wherein electrons originating from NAD(P)(H) are transferred to the P450 protein via 2 separate redox partners, such as e.g. a FAD or FMN- containing flavodoxin reductase and a Ferredoxin protein bound to a Sulphur-iron cluster; b) 2-component systems such as that wherein electrons originating from NAD(P)(H) are transferred to the P450 protein via a separate redox partner, i.e. a FMN/FAD-containing diflavin reductase, wherein the FMN/FAD-containing di-flavin reductase is also indicated as cytochrome P450 reductase (abbreviated CPR); c) 1 -component systems wherein electrons originating from NAD(P)(H) are transferred to the P450 domain via reductase domains which are fused to the P450 domain, such as e.g. FMN-containing flavin reductase domain and a Ferredoxin domain (abbreviated Fdx) bound to a Sulphur-iron cluster or, in alternative to the FMN-containing reductase and Fdx e.g., a reductase domain containing an FMN/FAD-containing di-flavin reductase, (such as in Figure 1 C of Sadeghi et al., vide supra);

CYP polypeptides according to the disclosure may belong to 2-component cytochrome P450 systems (often indicated as Class II P450 systems, Hanneman et al., Sadeghi et al., vide supra), which require the cytochrome P450 protein on the one hand and may require a FAD/FMN- containing di-flavin P450 reductase (also indicated as cytochrome P450 reductase or CPR) to support the monooxygenase activity of the cytochrome P450 protein on the other hand.

Alternatively, CYP polypeptides according to the disclosure may belong to cytochrome P450 systems which may require electron transfer proteins flavodoxin reductase (FPR) and/or ferredoxin reductase (FDXR).

In the methods and recombinant cells according to the disclosure CYP polypeptides may be involved in the hydroxylation of the C11 carbon of a mogroside precursor. These CYP enzymes may be also known as C11 -hydroxylases. As used herein a C11 -hydroxylase polypeptide may be a CYP polypeptide capable of introducing a hydroxyl group at the carbon at position 11 (i.e., at the C11) of mogroside precursor. In some embodiments, a C11 -hydroxylase is capable of catalyzing the conversion of cucurbitadienol into 11-hydroxy cucurbitadienol. In some other embodiments, a C11 -hydroxylase is capable of catalyzing the conversion of 24,25-epoxy-cucurbitadienol into 11- hydroxy-24,25-epoxy-cucurbitadienol. In yet other embodiments, a C1 1 -hydroxylase is capable of catalyzing the conversion of 24,25-dihydroxy-cucurbitadienol into Mogrol. CYP polypeptides according to the disclosure may catalyze the oxidation of mogroside precursors such as the (ep)oxidation of cucurbitadienol to 24,25-epoxy-cucurbitadienol or the (ep)oxidation of 11- hydroxycucurbitadienol to 11 -hydroxy-24, 25-epoxy-cucurbitadienol.

Accordingly, in one embodiment the recombinant cell capable of producing mogroside precursor(s) or mogroside(s) which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure may comprise, may be capable of (over)expressing or may (over)express at least a polynucleotide encoding a polypeptide capable of synthesizing 11-hydroxy cucurbitadienol from cucurbitadienol, 11 -hydroxy-24, 25-epoxy- cucurbitadienol from 24,25-epoxy-cucurbitadienol, Mogrol from 24,25-dihydroxy-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, particularly wherein said polypeptide is a cytochrome P450 enzyme (CYP450). Non limiting examples of CYP polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 5. Therefore, the CYP polypeptide may have an amino acid sequence according to any sequence disclosed in Table 5 or according to the amino acid sequences set out in SEQ ID NO: 10 to 12 herewith, or the CYP polypeptide may have an amino acid sequence which is has 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 SEQ ID NO: 10 to 12 herewith or with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 5.

Table 5 provides non-limiting examples of amino acid sequences of CYP polypeptides disclosed in patent documents. The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed. Table s

In the methods and recombinant cells according to the disclosure, Cytochrome P450 polypeptides (CYP) may be used in conjunction with cytochrome P450 reductase (CPR) to support the monooxygenase activity.

Accordingly, in one embodiment the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase according to the disclosure may comprise, may be capable of (over)expressing or may (over)express at least a polynucleotide encoding a polypeptide capable of reducing a cytochrome P450 complex, particularly a cytochrome P450 reductase (CPR). Non limiting examples of CPR polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 6. Therefore, the CPR polypeptide may have an amino acid sequence according to any sequence disclosed in Table 6 or according to the amino acid sequence set out in SEQ ID NO: 8 herewith or the CPR polypeptide may have an amino acid sequence which is has 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 set out in SEQ ID NO: 8 herewith or the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 6.

Table 6 provides non-limiting examples of amino acid sequences of CPR polypeptides disclosed in patent documents. The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed.

Table 6

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

Epoxide hydrolases are enzymes of the family EC 3.3.x.x. which catalyze the hydrolysis of an epoxide yielding a glycol.

In the context of the present disclosure, an epoxide hydrolases polypeptide as disclosed herewith may catalyze the conversion of 11 -hydroxy-24, 25-epoxy-cucurbitadienol to mogrol. In alterative or in addition an epoxide hydrolase as disclosed herewith may catalyze the conversion of 24, 25-epoxy-cucurbitadienol to 24,25-dihydroxy-cucurbitadienol.

Accordingly, in one embodiment the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure may comprise, may be capable of (over)expressing or may (over)express at least a 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, particularly wherein said polypeptide is an epoxide hydrolase (EPH). Non limiting examples of EPH polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 7. Therefore, the EPH polypeptide may have an amino acid sequence according to SEQ ID NO: 13 herein or according to any sequence disclosed in Table 7, or the polypeptide may have an amino acid sequence which has 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 SEQ ID NO: 13 herewith or with an amino acid sequence according to any polypeptide with amino acid sequence as indicated in Table 7.

Table 7 provides non-limiting examples of amino acid sequences of EPH polypeptides disclosed in patent documents. The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed. Table 7

WO2022/192688 A1 discloses host cells useful for producing mogroside precursor(s) and/or mogrosides wherein said host cell comprises a heterologous polynucleotide encoding a cytochrome b5, wherein the host cell is capable of producing more mogrol than a control cell that does not comprise the heterologous polynucleotide. Therefore, in the methods and recombinant cells according to the disclosure, cytochrome b5 polypeptides (CB5) may be used.

Accordingly, in one embodiment according to the methods or recombinant cells of the disclosure, the recombinant cell capable of producing mogroside precursor(s) and/or mogrosides which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure may comprise, may be capable of (over)expressing or may (over)express at least a polynucleotide encoding for a cytochrome b5 polypeptide (CB5). Non limiting examples of CB5 polypeptides which can be used in the recombinant cells or methods according to the disclosure are provided in Table 8. Therefore, the CB5 polypeptide may have an amino acid sequence according to any sequence disclosed in Table 8 or according to the amino acid sequence set out in SEQ ID NO: 7 herein, or the polypeptide may have an amino acid sequence which is has 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 of any polypeptide with amino acid sequence as indicated in Table 8 or according to SEQ ID NO: 7 herein. Table 8 provides non-limiting examples of amino acid sequences of CB5 polypeptides as disclosed in WO2022/192688 A1. The SEQ ID NOs in the table are those in the corresponding patent document in which they are disclosed.

Table 8

In the methods and recombinant cells according to the disclosure, a recombinant cell capable of producing mogroside precursors) and/or mogrosides which has been modified to result in a deficiency of a beta glucanase according to the disclosure may comprise, may be capable of (over)expressing or may (over)express one or more polynucleotides coding for a squalene epoxidase, a cucurbitadienol synthase a cytochrome P450 polypeptide, a C11 -hydroxylase a cytochrome P450 reductase, an epoxide hydrolase a cytochrome b5 polypeptide and/or a UGT polypeptide.

Therefore, in one aspect, it is herewith disclosed a recombinant cell capable of producing mogroside precursors and/or mogrosides which has been modified to result in a deficiency of a beta glucanase according to the disclosure, wherein the recombinant cell comprises, is capable of (over)expressing or is (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 an epoxide hydrolase (EPH);

(f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5);

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

(a), (b), (c), (d), (e) or (f) may be induced.

Typically a recombinant cell which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the 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 category of enzymes, selected from (a), (b), (c), (d), (e), (f) or (g), such as at least three polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g), such as at least four polynucleotides selected from (a),

(b), (c), (d), (e), (f) or (g) (preferably each polynucleotide from a different category of enzymes), such as at least five polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g) (preferably each polynucleotide from a different category of enzymes), such as at least six polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g) (preferably each polynucleotide from a different category of enzymes), typically all the polynucleotides (a), (b), (c), (d), (e), (f) and (g).

Typically, a recombinant cell capable of producing mogroside precursors and/or mogrosides which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product as described herewith may be capable of producing one or more of the following: 2,3-epoxysqualene, 2,3,22,23-diepoxysqualene, cucurbitadienol, 11-hydroxy cucurbitadienol, 24,25-epoxy-cucurbitadienol, 11 -hydroxy-24, 25-epoxy-cucurbitadienol, and/or 24,25-dihydroxy-cucurbitadienol. The chemical structure of some mogroside precursors and intermediates in the biosynthetic pathway to mogrosides is shown in Figure 5.

As described herein above, squalene is the biochemical precursors of Mogrol. Squalene is synthesized from Farnesyl diphosphate (FPP) in a reaction catalyzed by a Squalene Synthase (SQS), which is thought to proceed in the presence of NADPH as a co-substrate. Recombinant cells may comprise an endogenous squalene synthase, in which case the endogenous enzyme may suffice. Endogenous squalene production pathways exist in eukaryotes metabolism, such as yeast metabolism, and accordingly, if the recombinant host is a eukaryote, then said step may be endogenous to the recombinant host. However, it may be advantageous to enhance expression of the endogenous squalene synthase by any method known to those skilled in the art.

Therefore, in one embodiment of the methods and recombinant cells according to the disclosure, the recombinant cell capable of producing mogroside precursors) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase according to the disclosure comprises, is capable of (over)expressing or (over)expresses a polynucleotide encoding a polypeptide capable of synthesizing Squalene from Farnesyl diphosphate (FPP), particularly wherein said polypeptide is a squalene synthase (EC:2.5.1 .21) (SQS). Non limiting examples of SQS polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 9. Therefore the SQS polypeptide may have an amino acid sequence according to any sequence disclosed in Table 9 or according to SEQ ID NO: 24 herewith, or the SQS polypeptide may have an amino acid sequence which is has 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 set out in SEQ ID NO: 24 herewith or with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 9.

Table 9 provides non-limiting examples of amino acid sequences of SQS polypeptides disclosed in patent documents. The SEQ ID NOs in the table are those in the corresponding patent documents in 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). Therefore, in one embodiment the recombinant cell may be modified to increase production of farnesyl diphosphate and/or geranyl diphosphate.

Farnesyl diphosphate (FPP) can be synthesized from Geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP) by the enzyme Farnesyl diphosphate synthase (FPPS). In turn, Geranyl diphosphate (GPP) may be synthesized from Dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) by the enzyme Geranyl diphosphate synthase (GPPS) and/or the enzyme Farnesyl diphosphate synthase (FPPS).

Therefore, in one embodiment according to the methods or recombinant cells according to the disclosure, the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase according to the disclosure comprises, is capable of (over)expressing or (over)expresses a polynucleotide encoding a polypeptide capable of synthesizing Farnesyl diphosphate (FPP) from Geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP), particularly wherein said polypeptide is a farnesyl diphosphate synthase (EC:2.5.1 .10) (FPPS). Non limiting examples of FPPS polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 10. Therefore, the FPPS polypeptide may have an amino acid sequence according to the amino acid sequence set out in SEQ ID NO: 20 herewith or according to any sequence disclosed in Table 10, or the FPPS polypeptide may have an amino acid sequence which is has 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 set out in SEQ ID NO: 20 herein or with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 10.

Table 10 provides non-limiting examples of amino acid sequences of FPPS polypeptides disclosed in patent documents. The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed.

Table 10

In one embodiment, the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase according to the disclosure comprises, is capable of (over)expressing or (over)expresses a polynucleotide encoding a polypeptide capable of synthesizing Geranyl diphosphate (GPP) from Dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is a geranyl diphosphate synthase (EC:2.5.1.1) (GPPS) and/or a farnesyl diphosphate synthase (EC:2.5.1 .10) (FPPS). Non limiting examples of FPPS polypeptides which can be used in the recombinant cells and methods according to the disclosure have been provided herein before. Non limiting examples of GGPS polypeptides may be any suitable GGPS known to the person skilled in the art and may e.g., be from prokaryotic or eukaryotic origin. Non limiting examples of GPPS polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 11. Therefore, the GPPS polypeptide may have an amino acid sequence according to any sequence disclosed in Table 11 , or the GPPS polypeptide may have an amino acid sequence which is has 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 of any polypeptide with amino acid sequence as indicated in Table 11 .

Table 11 provides non-limiting examples of amino acid sequences of GPPS polypeptides disclosed in patent documents. The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed.

Table 11

Therefore, in one aspect of the methods and recombinant cells according to the disclosure, the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product according to the disclosure comprises, is capable of (over)expressing or (over)expresses one or more of the following recombinant polynucleotides:

(a) a polynucleotide encoding a polypeptide capable of synthesizing Geranyl diphosphate (GPP) from Dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), particularly wherein said polypeptide is a geranyl diphosphate synthase (EC:2.5.1.1) (GPPS) and/or a farnesyl diphosphate synthase (EC:2.5.1 .10) (FPPS);

(b) a polynucleotide encoding a polypeptide capable of synthesizing Farnesyl diphosphate (FPP) from Geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP), particularly wherein said polypeptide is a farnesyl diphosphate synthase (EC:2.5.1.10) (FPPS);

(c) a polynucleotide encoding a polypeptide capable of synthesizing Squalene from Farnesyl diphosphate (FPP), particularly wherein said polypeptide is a squalene synthase (EC:2.5.1 .21) (SQS).

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

Typically, the cell may comprise, may be capable of (over)expressing or may (over)express all the 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 disclosure, the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase may comprise or (over)express one or more polynucleotides coding for polypeptides belonging to the mevalonate pathway.

The mevalonate pathway refers to the well-known biosynthetic pathway depicted in Figure 2 which converts acetyl Coenzyme A to Isopentenyl diphosphate. Such biosynthetic pathway is normally present in eukaryotic cells while procaryotic cells produce Isopentenyl diphosphate via the MEP pathway, a mevalonic acid-independent pathway that converts glyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP.

The mevalonate pathway comprises the following steps:

(a) the synthesis of Acetoacetyl-Coenzyme A (AACoA) from Acetyl Coenzyme A (AcCoA) catalyzed by an acetyl-CoA acetyltransferase (EC:2.3.1.9) (AACT); (b) the synthesis of Hydroxymethylglutaryl-Coenzyme A (HMGCoA) from Acetoacetyl- Coenzyme A (AACoA) catalyzed by an Hydroxymethylglutaryl-Coenzyme A synthase (EC:2.3.3.10) (HMGS);

(c) the synthesis of mevalonic acid (MVA) from Hydroxymethylglutaryl-Coenzyme A (HMGCoA) catalysed by a 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC:1 .1 .1 .34) (HMGR);

(d) the synthesis of Mevalonate-5-phosphate (MVA-P) from Mevalonic acid (MVA) catalyzed by a mevalonate kinase (EC:2.7.1 .36) (MK);

(e) the synthesis of Mevalonate-5-diphosphate (MVA-PP) from Mevalonate-5- phosphate (MVA-P) catalyzed by a phosphomevalonate kinase (EC:2.7.4.2) (PMK);

(f) the synthesis of Isopentenyl diphosphate (IPP) from Mevalonate-5-diphosphate (MVA-PP), catalyzed by a diphosphomevalonate decarboxylase (EC:4.1 .1 .33) (MDD) and/or an isopentenyl/dimethylallyl diphosphate synthase (EC:1 .17.1 .2) (IPPS);

(g) the synthesis of Dimethylallyl diphosphate (DMAPP) from Isopentenyl diphosphate (IPP), catalyzed by an isopentenyl-diphosphate delta-isomerase (EC:5.3.3.2) (IPI).

In one embodiment of the methods and recombinant cells according to the disclosure, the recombinant cell capable of producing mogroside precursors and/or mogrosides which has been modified to result in a deficiency of a 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-Coenzyme A (AACoA) from Acetyl Coenzyme A (AcCoA), particularly wherein said 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), particularly wherein said polypeptide is an Hydroxymethylglutaryl-Coenzyme A synthase (EC:2.3.3.10) (HMGS);

(c) a polynucleotide encoding a polypeptide capable of synthesizing mevalonic acid (MVA) from Hydroxymethylglutaryl-Coenzyme A (HMGCoA), particularly wherein said polypeptide is a 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC:1 .1 .1 .34) (HMGR);

(d) a polynucleotide encoding a polypeptide capable of synthesizing Mevalonate-5- phosphate (MVA-P) from Mevalonic acid (MVA), particularly wherein said polypeptide is a 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), particularly wherein said polypeptide is a phosphomevalonate kinase (EC:2.7.4.2) (PMK);

(f) a polynucleotide encoding a polypeptide capable of synthesizing Isopentenyl diphosphate (IPP) from Mevalonate-5-diphosphate) (MVA-PP), particularly wherein said polypeptide is a diphosphomevalonate decarboxylase (EC:4.1 .1 .33) (MDD) and/ or an 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 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) are heterologous polypeptides and/or heterologous polynucleotides. Typically, one or more of the polynucleotides according to (a), (b), (c) (d), (e), (f) or (g) may be constitutively expressed. In another aspect the 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 which has been modified to result in a deficiency of a beta glucanase according to the 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 category of enzymes, selected from (a), (b), (c), (d), (e), (f) or (g), such as at least three polynucleotides selected from (a), (b), (c),

(d), (e), (f) or (g), such as at least four polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g) (preferably each polynucleotide from a different category of enzymes), such as at least five polynucleotides selected from (a), (b), (c), (d), (e), (f) or (g) (preferably each polynucleotide from a different category of enzymes), such as at least six polynucleotides selected from (a), (b), (c), (d),

(e), (f) or (g) (preferably each polynucleotide from a different category of enzymes), typically all the polynucleotides (a), (b), (c), (d), (e), (f) and (g).

Any acetyl-CoA acetyltransferase known in the art may be used in the context of this disclosure. Suitable, non-limiting examples of Acetoacetyl-CoA thiolase which can be used are the ERG10 enzyme UniProtKB Accession Nos. P41338, or the enzyme with UniProtKB Accession no. P10551 or the enzyme with UniProtKB/Swiss-Prot: Q6L8K7.1 or any acetyl-CoA acetyltransferase with an amino acid sequence which is has 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.

Hydroxymethylglutaryl-Coenzyme A synthases known in the art can be used to convert acetoacetyl Coenzyme A into Hydroxymethylglutaryl-Coenzyme A. Suitable, non-limiting examples include the protein ERG13 UniProtKB Accession Nos. P54839 or the YALI0F30481 p NCBI No. XP_506052.1 or SEQ ID NO: 21 or any HMG-CoA synthase polypeptide with an amino acid sequence which is has 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-coenzyme A reductase polypeptides suitable to be applied in the strains and method according to the disclosure are known to those skilled in the art. Suitable e, non-limiting examples are SEQ ID NO: 25 herewith, the HMG1 polypeptide with UniProtKB Accession No. P12683 or the HMG2 gene UniProtKB Accession No. P12684 provide non-limiting example of an HMG-CoA reductase or HMG-CoA reductase polypeptides with an amino acid sequence which is has 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 this disclosure. Suitable, non-limiting examples of mevalonate kinase are ERG12 polypeptide with UniProtKB Accession Nos. P07277 or the mevalonate kinase with GenBank accession no. QNP96798.1 or any MK polypeptide with an amino acid sequence which is has 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 kinase which can be used in the methods and strains according to the disclosure are the polypeptide with GenBank accession no. GFP66625 or polypeptide with GenBank accession no. VBB79048.1 , polypeptide with UniProtKB/Swiss-Prot No D4GXZ3.1 or any PMK polypeptide with an amino acid sequence which is has 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 diphosphomevalonate decarboxylase which can be used in the methods and recombinant cells according to the disclosure are the polypeptide with GenBank accession no. CAA66158.1 or the polypeptide with GenBank accession no. QNQ00565.1 or KAG5364843.1 , or AAT93171.1 , non-limiting examples of isopentenyl-diphosphate delta- isomerase are the polypeptide with UniProtKB/Swiss-Prot No. P15496.2 or GenBank No. QNQ01368.1 , AOW06629.1 or any diphosphomevalonate decarboxylase or isopentenyl- diphosphate delta-isomerase with an amino acid sequence which is has 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 Figure 3, 2,3-Epoxysqualene (oxido-squalene) may be converted into lanosterol by the enzyme lanosterol synthase (ERG7). In fungal cells lanosterol is a precursor in the production of sterols. Sterols are tetracyclic triterpenoid lipids that are required by all eukaryotes for critical cellular functions including maintaining membrane fluidity, phagocytosis, stress tolerance, and cell signaling.

In one embodiment of the methods and recombinant cells according to the disclosure, when the recombinant cell produces an endogenous lanosterol synthase, the level of 2,3-Epoxysqualene may be increased by e.g. reducing the activity or by lowering the expression of an endogenous lanosterol synthase. In recombinant hosts expressing an endogenous lanosterol synthase, this may be achieved by rendering the cell deficient or partially deficient in the production of lanosterol synthase. Therefore, in one embodiment according to the disclosure a recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase may comprise a deficiency in the production of a lanosterol synthase polypeptide.

Since lanosterol is an essential metabolite in eukaryotes preferably the cell may be typically made deficient in the production of lanosterol synthase by any method known in the art which does not fully eliminate lanosterol synthase activity. For example, the recombinant cell may be modified so that it produces less lanosterol synthase, e.g. by reducing the expression level or by reducing the translation level of the mRNA transcribed from a gene encoding the lanosterol synthase. In one embodiment the expression level of the gene encoding for the lanosterol synthase may be reduced by replacing the lanosterol synthase endogenous promoter with a promoter which is weaker than the endogenous one or other suitable methods. Alternatively, one can replace the gene coding for the native lanosterol synthase with a gene which, while still coding for the native lanosterol synthase, due to the different codon usage, is less easily translated into protein by the recombinant cell. Alternatively, the recombinant cell may be modified to produce a polypeptide with decreased lanosterol synthase activity. For example, the recombinant cell may be modified by replacing the gene coding for a native lanosterol synthase with a gene coding for a modified lanosterol synthase with reduced lanosterol synthase activity if compared with the native or homologous lanosterol synthase. Non-limiting examples of suitable lanosterol synthase with reduced activities are described in WO2022/212917 and in WO2022/212924.

In one embodiment, the recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase as disclosed herewith may be capable of producing one or more mogrosides selected from Mogroside IA, Mogroside IEi, Mogroside IIA, Mogroside IIAi, Mogroside IIA2, Mogroside HE, Mogroside IIIA1, Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I. Typically, the recombinant cell may comprise, may be capable of (over)expressing or may (over)express one or more of:

(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), such as a C11- hydroxylase polypeptide and/or an epoxidase;

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

(e) a polynucleotide encoding an epoxide hydrolase (EPH)

(f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5).

(g) a polynucleotide encoding a UGT polypeptide, particularly one or more of the following UGT polypeptides: i. a UGT capable of glycosylating mogrol or a mogroside compound at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group, and/or C25 hydroxyl group, ii. a UGT capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 (a 024-O-glucose) of a mogroside compound and/or or capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C3 (a 03-O-glucose) of a mogroside compound,

Hi. a UGT capable of beta-1 ,6-glycosylation of the 06' hydroxyl group of a glucose moiety at position C3 of a mogroside compound and/or capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glycose moiety at position C24 of a mogroside compound, and optionally one or more of the following

A. a geranyl diphosphate synthase (GPPS) and/or a farnesyl diphosphate synthase (FPPS);

B. a farnesyl diphosphate synthase (FPPS);

C. a squalene synthase (SQS);

D. an acetyl-CoA acetyltransferase (AACT);

E. an Hydroxymethylglutaryl-Coenzyme A synthase (HMGS);

F. a 3-hydroxy-3- methylglutaryl-coenzyme A reductase (HMGR);

G. a mevalonate kinase (MK);

H. a phosphomevalonate kinase (PMK);

I. a diphosphomevalonate decarboxylase (MDD) and/ or an isopentenyl/dimethylallyl diphosphate synthase (IPPS);

J. an isopentenyl-diphosphate delta-isomerase (IPI).

A recombinant cell capable of producing mogroside precursor(s) and/or mogroside(s) which has been modified to result in a deficiency of a beta glucanase as disclosed herewith may be capable of producing one or more mogrosides and/or mogroside precursors when cultured under suitable conditions such as in the presence of a suitable carbon source and other nutrients.

Therefore, in one aspect, the disclosure relates to a method of producing one or more mogrosides and/or mogroside precursors comprising culturing a recombinant cell capable of producing mogrosides and/or mogroside precursors as disclosed herewith in a culture medium under conditions suitable to the production of said mogrosides and/or mogroside precursors, optionally isolating said one or more mogrosides and/or mogroside precursors.

Typically, the recombinant cell is grown in a fermenter at defined temperature(s), pH and for a desired period of time, typically under conditions in which the genes involved in the biosynthetic pathway to produce mogrosides and/or mogroside precursors as disclosed herewith are expressed. The recombinant cell may be grown in a bioreactor or in a fermenter under different operation modes such batch, fed batch, or continuous process.

Culture media used for various recombinant cells are well known in the art. Culture media used to culture recombinant bacterial cells will depend on the identity of the recombinant cell. Culture media generally comprise inorganic salts and compounds, amino acids, carbohydrates, vitamins and other compounds that are either necessary for the growth of the recombinant cell or improve health or growth or both of the recombinant cell. Growth optimization of bacterial and yeast cells can also be achieved by the addition of nutrients and supplements into a culture media. Alternatively, the cultures can be grown in a fermenter designed for temperature, pH control and controlled aeration rates. Culture conditions are generally selected from aerobic, microaerobic, and anaerobic. Therefore, oxygen and nitrogen can be flown into the media as necessary.

Any of the recombinant cells disclosed in this application can be cultured in media of any type. The conditions of the culture or culturing process can be optimized through routine experimentation as would be understood by one of ordinary skill in the art.

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

A cell lysate may be prepared from the recombinant cell disclosed herewith and be used to contact a substrate, such that mogroside compounds can be produced. For example, a cell lysate can be prepared from the recombinant cell disclosed herewith and expressing one or more UGTs and used to contact Mogrol, such that mogroside compounds can be produced. In some embodiments, mogroside compounds may be produced using whole cells that are fed with raw materials that contain precursor molecules, e.g., Mogrol or mogrosides, such as monk fruit extracts comprising the same. The raw materials may be fed during cell growth or after cell growth. The whole cells may be in culture medium as described herein above and further comprising Mogrol, a mogroside substrate or a mogroside, in a suspension or immobilized. The whole cells may be in fermentation broth or in a reaction buffer. In some embodiments a permeabilizing agent may be required for efficient transfer of substrate into the cells. A method starting from whole cells and Mogrol or mogroside precursors is known as “whole cell bioconversion”. Methods to produce mogrosides via whole cell bioconversion are described in e.g. WO 2013/076577, WO 2014/086842 and WO 2016/050890. The mogroside substrate is typically a mogroside compound with a lower level of glycosylation if compared to the target mogroside product to be produced according to the method.

The methods according to the disclosure may lead to the production of one or more mogrosides such as one or more of Mogroside IA, Mogroside IE1 , Mogroside HA, Mogroside I IA1 , Mogroside IIA2, Mogroside HE, Mogroside IIIA1 , Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I, particularly Mogroside HIE, Mogroside 11 IA or Siamenoside I.

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

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 levels of mogroside precursors and/or mogroside compounds in each fraction, including the flow-through, can then be analyzed by LC -MS. Fractions may then be combined and reduced in volume using a vacuum evaporator. Additional purification steps may be utilized, if desired, such as additional chromatography steps and crystallization. For example, mogroside compounds may be isolated by methods not limited to ion exchange chromatography, reversed-phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon columns and/or decoloring steps.

The disclosure further provides a fermentation broth comprising a recombinant cell as disclosed herewith or comprising a lysate or an extract thereof.

The disclosure also provides a mogroside composition or sweetener composition obtainable from a method as disclosed herewith. The mogroside or sweetener composition may comprise one or more of Mogroside IA, Mogroside IE1 , Mogroside IIA, Mogroside IIA1 , Mogroside IIA2, Mogroside HE, Mogroside IIIA1 , Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I, particularly Mogroside HIE, Mogroside IIIA or Siamenoside I. Furthermore, food product, beverage, feed, oral composition, pharmaceutical composition, feed composition, pet food composition comprising a mogroside or sweetener compositions as disclosed herewith are also provided.

Further provided is a mogroside obtainable or obtained by a method as disclosed herein. The mogroside, may be used in any application known for such compounds. In particular, mogrosides or compositions thereof may for instance be used as a sweetener, such as in a food or a beverage. For example, mogrosides may be formulated in soft drinks, as a table-top sweetener, chewing gum, dairy product such as yoghurt (e.g. plain yoghurt), cake, cereal or cereal-based food, nutraceutical, pharmaceutical, edible gel, confectionery product, cosmetic, toothpastes or other oral cavity composition, etc. In addition, a mogroside can be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics. Further provided is thus such foodstuff, feed or beverage which comprises said mogroside. During the manufacturing of foodstuffs, drinks, pharmaceuticals, cosmetics, table-top products, chewing gum the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods can be used.

The mogroside obtained as disclosed herein may be used in dry or liquid forms. It can be added before or after heat treatment of food products. The amount of the sweetener depends on the purpose of usage. It can be added alone or in combination with other compounds.

Mogrosides produced according to the method as disclosed herein may be blended with one or more further non-calorific or calorific sweeteners. Such blending may be used to improve flavor or temporal profile or stability. A wide range of both non-calorific and calorific sweeteners may be suitable for blending with mogrosides. For example, non-calorific sweeteners such as steviol glycosides, monatin, aspartame, acesulfame salts, cyclamate, sucralose, saccharin salts or erythritol. Calorific sweeteners suitable for blending with mogrosides include sugar alcohols and carbohydrates such as sucrose, glucose, fructose and HFCS. Sweet tasting amino acids such as glycine, alanine or serine may also be used.

The mogroside can be used in combination with a sweetener suppressor, such as a natural sweetener suppressor. It may be combined with an umami taste enhancer, such as an amino acid or a salt thereof.

The mogroside can be combined with a polyol or sugar alcohol, a carbohydrate, a physiologically active substance or functional ingredient (such as a carotenoid, dietary fiber, fatty acid, saponin, antioxidant, nutraceutical, flavonoid, isothiocyanate, phenol, plant sterol or stand (phytosterols and phytostanols), a polyol, a prebiotic, a probiotic, a postbiotic, a phytoestrogen, soy protein, sulfides/thiols, amino acids, a protein, a vitamin, a mineral, and/or a substance classified based on a health benefits, such as cardiovascular, cholesterol-reducing or anti-inflammatory substance.

A composition comprising a mogroside may include a flavoring agent, an aroma component, a nucleotide, an organic acid, an organic acid salt, an inorganic acid, a bitter compound, a protein or protein hydrolysate, a surfactant, a flavonoid, an astringent compound, a vitamin, a dietary fiber, an antioxidant, a fatty acid and/or a salt.

A mogroside as disclosed herein may be applied as a high intensity sweetener to produce zero calorie, reduced calorie or diabetic beverages and food products with improved taste characteristics. Also, it can be used in drinks, foodstuffs, pharmaceuticals, and other products in which sugar cannot be used.

In addition, a mogroside as disclosed herein may be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics. Examples of products where a mogroside as disclosed herein can be used as a sweetening compound include alcoholic beverages such as vodka, wine, beer, liquor, sake, etc.; natural juices, refreshing drinks, soft drinks including carbonated soft drinks, diet drinks, zero calorie drinks, reduced calorie drinks and foods, yogurt drinks, instant juices, instant coffee, powdered types of instant beverages, canned products, syrups, fermented soybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchups, curry, soup, instant bouillon, powdered soy sauce, powdered vinegar, types of biscuits, rice biscuit, crackers, bread, chocolates, caramel, candy, chewing gum, jelly, pudding, preserved fruits and vegetables, fresh cream, jam, marmalade, flower paste, powdered milk, ice cream, sorbet, vegetables and fruits packed in bottles, canned and boiled beans, meat and foods boiled in sweetened sauce, agricultural vegetable food products, seafood, ham, sausage, fish ham, fish sausage, fish paste, deep fried fish products, dried seafood products, frozen food products, preserved seaweed, preserved meat, tobacco, medicinal products, and many others. In principle, it can have unlimited applications.

The sweetened composition comprises a beverage, non-limiting examples of which include non-carbonated and carbonated beverages such as colas, ginger ales, root beers, ciders, fruit- flavored soft drinks (e.g., citrus-flavored soft drinks such as lemon-lime or orange), powdered soft drinks, and the like; fruit juices originating in fruits or vegetables, fruit juices including squeezed juices or the like, fruit juices containing fruit particles, fruit beverages, fruit juice beverages, beverages containing fruit juices, beverages with fruit flavorings, vegetable juices, juices containing vegetables, and mixed juices containing fruits and vegetables; sport drinks, energy drinks, near water and the like drinks (e.g., water with natural or synthetic flavorants); tea type or favorite type beverages such as coffee, cocoa, black tea, green tea, oolong tea and the like; beverages containing milk components such as milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages, drinkable yogurt, lactic acid bacteria beverages or the like; and dairy products.

Generally, the amount of sweetener present in a sweetened composition varies widely depending on the particular type of sweetened composition and its desired sweetness. Those of ordinary skill in the art can readily discern the appropriate amount of sweetener to put in the sweetened composition can be used in dry or liquid forms. It can be added before or after heat treatment of food products. The amount of the sweetener depends on the purpose of usage. It can be added alone or in combination with other compounds.

During the manufacturing of foodstuffs, drinks, pharmaceuticals, cosmetics, table-top products, chewing gum the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods can be used.

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

In solid form, a mogroside produced as disclosed herein can be provided to consumers in any form suitable for delivery into the comestible to be sweetened, including sachets, packets, bulk bags or boxes, cubes, tablets, mists, or dissolvable strips. The composition can be delivered as a unit dose or in bulk form.

In liquid form, a mogroside produced as disclosed herein can be provided in any form suitable for use by the consumer, including in fluid, semi-fluid, paste and cream forms, and in any appropriate packing using any appropriate packing material in any shape or form which is convenient to carry, dispense, store, and/or transport.

The composition may include various bulking agents, functional ingredients, colorants, and/or flavors.

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

A non-limiting list of embodiments according to the disclosure is displayed hereafter.

Embodiments according to the disclosure

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

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

3. A recombinant cell according to embodiment 1 or 2 wherein said polypeptide capable of deglycosylating a mogroside product is selected from a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 ; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2.

4. A recombinant cell according to any one of the preceding embodiments wherein the polypeptide capable of deglycosylating a mogroside product is a glycoside hydrolase (EC:3.2.1 .-), such as an exo-acting glycoside hydrolase.

5. A recombinant cell according to any one of the preceding embodiments wherein the polypeptide capable of deglycosylating a mogroside product is a polypeptide capable of hydrolyzing one or more of the bonds selected from: (a) the glycosidic bond between the carbon at position 24 (C24 atom) of the mogrol backbone of a mogroside and a beta-1 -glucose moiety bound at said position;

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

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

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

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

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

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

6. A recombinant cell according to embodiment 5 wherein the polypeptide is capable of deglycosylating a mogroside product is capable of hydrolyzing at least

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

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

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

8. A recombinant cell according to any one of the preceding embodiments wherein the cell has been further modified to result in a deficiency of an EXG1 and/or EXG2 polypeptide, particularly wherein a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 3.

9. A recombinant cell according to any one of the preceding embodiments wherein the production of mogroside product by said cell is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the polypeptide capable of deglycosylating a mogroside product, when both cells are cultured under the same conditions. 10. A recombinant cell according to any one of the preceding embodiments wherein the production of mogroside product comprising a beta-glycosidic bond at the C3 and/or at the C24 of the mogrol backbone by said cell is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the polypeptide capable of deglycosylating a mogroside product, when both cells are cultured under the same conditions.

11. A recombinant cell according to any one of the preceding embodiments wherein the production of mogroside product comprising a beta-1 ,2-glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom and/or at the C24 atom of the mogroside backbone by said cell is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the polypeptide capable of deglycosylating a mogroside product, when both cells are cultured under the same conditions.

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

13. A recombinant cell according to any one of the preceding embodiments wherein the production of mogroside product by said cell is higher if compared with the production by an otherwise identical recombinant cell which has been modified to be deficient in a polypeptide capable of deglycosylating a mogroside product set out in SEQ ID NO: 3, when both cells are cultured under the same conditions.

14. A recombinant cell according to any one of the preceding embodiments wherein the production of mogroside product comprising a beta-1 ,2-glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom and/or at the C24 atom of the mogroside backbone by said cell is higher if compared with the production by an otherwise identical recombinant cell which has been modified to be deficient in a polypeptide capable of deglycosylating a mogroside product set out in SEQ ID NO: 3, when both cells are cultured under the same conditions.

15. A recombinant cell according to any one of the preceding embodiments wherein the production of mogroside product comprising a beta-1 ,6-glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom and/or at the C24 atom of the mogroside backbone by said cell is higher if compared with the production by an otherwise identical recombinant cell which has been modified to be deficient in a polypeptide capable of deglycosylating a mogroside product set out in SEQ ID NO: 3, when both cells are cultured under the same conditions.

16. A recombinant cell according to any one of the preceding embodiments wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating mogroside compounds which is a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, a pentaglycosylated or exa-glycosylated mogroside compound, or an isomer thereof.

17. A recombinant cell according to 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 IEi, Mogroside IIA, Mogroside IIAi, Mogroside I IA2, Mogroside HE, Mogroside IIIA1, Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vlai, Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I.

18. A recombinant cell according to 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 Mogrol or a mogroside compound at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group, and/or C25 hydroxyl group;

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

(c) a polynucleotide encoding a polypeptide capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glucose moiety at position C3 of a mogroside compound and/or capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glycose moiety at position C24 of a mogroside compound; particularly wherein the polypeptide according to (a), (b) or (c) is a uridine diphosphate dependent glycosyl transferase polypeptide (UGT polypeptide).

19. A recombinant cell according to 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 2,3- epoxysqualene from squalene, or 2,3,22,23-diepoxysqualene from 2,3- epoxysqualene, particularly wherein said polypeptide is a squalene epoxidase (SQE);

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

(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, particularly wherein said polypeptide is a cytochrome P450 enzyme (CYP450);

(e) a polynucleotide encoding a polypeptide capable of synthesizing mogrol from 1 1- hydroxy-24, 25-epoxy-cucurbitadienol, or 24,25-dihydroxy-cucurbitadienol from

24.25-epoxy-cucurbitadienol, particularly wherein said polypeptide is an epoxide hydrolase (EPH)

(f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5).

20. A recombinant cell according to any one of embodiments 1 to 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 an epoxide hydrolase (EPH);

(f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5);

(g) a polynucleotide encoding a UGT polypeptide.

21 . A recombinant cell according to 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 Geranyl diphosphate (GPP) from Dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is a geranyl diphosphate synthase (EC:2.5.1 .1) (GPPS) and/or a farnesyl diphosphate synthase (EC:2.5.1.10) (FPPS);

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

(c) a polynucleotide encoding a polypeptide capable of synthesizing Squalene from Farnesyl diphosphate (FPP), particularly wherein said polypeptide is a squalene synthase (EC:2.5.1 .21) (SQS). 22. A recombinant cell according to 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- Coenzyme A (AACoA) from Acetyl Coenzyme A (AcCoA), particularly wherein said polypeptide is an acetyl-CoA acetyltransferase (EC:2.3.1.9) (AACT);

(b) a polynucleotide encoding a polypeptide capable of synthesizing Hydroxymethylglutaryl-Coenzyme A (HMGCoA) from Acetoacetyl-Coenzyme A (AACoA), particularly wherein said polypeptide is an Hydroxymethylglutaryl-Coenzyme A synthase (EC:2.3.3.10) (HMGS);

(c) a polynucleotide encoding a polypeptide capable of synthesizing mevalonic acid (MVA) from Hydroxymethylglutaryl-Coenzyme A (HMGCoA), particularly wherein said polypeptide is a 3-hydroxy-3- methylglutaryl-coenzyme A reductase (EC:1 .1 .1 .34) (HMGR);

23. A recombinant cell according to any one of embodiments 1 to 22 wherein at least one of the one or more polypeptides is a heterologous polypeptide.

24. A recombinant cell according to any one of the preceding embodiments wherein the recombinant cell further comprising a deficiency in the production of a lanosterol synthase polypeptide.

25. A recombinant cell according to 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. A recombinant cell according to any one of the preceding embodiments wherein the mogroside is one or more of Mogroside IA, Mogroside IEi, Mogroside IIA, Mogroside IIAi, Mogroside IIA2, Mogroside HE, Mogroside IIIA1, Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I.

27. A recombinant cell according to any one of the preceding embodiments wherein the cell is procaryote, eukaryote or archaeal cell.

28. A recombinant cell according to embodiment 27 wherein the recombinant cell is an eukaryotic cell selected from a fungus (e.g. yeast or a filamentous fungus), an algae or aa plant cell, or a cell selected from a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an Issatchenkia orientalis cell, or Pichia pastoris

29. A recombinant cell according to embodiment 27 which is a prokaryotic cell, such as a bacterial cell, or an Escherichia coli cell.

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

31 . A method of producing one or more mogrosides comprising contacting one or more mogrosides 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 to produce said one or more mogrosides, optionally isolating said one or more mogrosides.

32. A method according to embodiment 30 or 31 wherein the one or more mogrosides is selected from Mogroside IA, Mogroside IE1, Mogroside IIA, Mogroside IIA1, Mogroside IIA2, Mogroside HE, Mogroside IIIA1, Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I, particularly Mogroside HIE, Mogroside 11 IA or Siamenoside I.

33. A fermentation broth comprising a recombinant cell according to any one of embodiments 1 to 29 or comprising a lysate or an extract thereof.

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

35. A food product, beverage, a pet food, feed, oral composition, pharmaceutical composition comprising 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 a recombinant microorganism as well as for additional genetic modification of recombinant microorganism, are known methods in the art, such as described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", John Wiley & Sons, Inc. (2003). Methods for transformation and genetic modification of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671 .

Methods for genetic manipulation and transformation of the yeast Yarrowia lipolytica are for example described 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 p-carotene production” in Metabolic Engineering (2017) 41 : 192-201 .

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

Gietz R.D. and 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. Assay for measuring Mogrosides (MOGs)

Liquid Chromatography with Mass Spectrometer (LC-MS) determination

Mogrol and mogrosides were analysed on a Vanquish DUO UHPLC system (Thermo Fisher) coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher) equipped with an electrospray ionization source operated in the negative ion mode for all mogrol and mogrosides (Tab.12) studied.

Table 12: Mogrol and mogrosides measured in the assay

Name CAS Number Standard supplier

Mogrol 88930-15-8 MedChemExpress

Mogroside I A1 88901-46-6 MedChemExpress

Mogroside I E1 88901-39-7 MedChemExpress

Mogroside II A 1613527-65-3 MedChemExpress

Mogroside II A1 88901-44-4 MedChemExpress

Mogroside II A2 88901-45-5 MedChemExpress

Mogroside II E 88901-38-6 MedChemExpress

Mogroside III 130567-83-8 MedChemExpress

Mogroside III A1 88901-42-2 MedChemExpress

Mogroside III E 88901-37-5 MedChemExpress

Mogroside IV 89590-95-4 MedChemExpress

Mogroside IV A 88901-41-1 MedChemExpress

Mogroside IV E 88915-64-4 MedChemExpress

Mogroside V 88901-36-4 MedChemExpress

11-oxo-mogroside V 126105-11-1 MedChemExpress

Siamenoside I _ 126105-12-2 _ Sigma Aldrich _

The chromatographic separation was achieved with a 2.1 x 100 mm 1.8pm particle size, Acquity UPLC® HSS T3 column, after a 2.1 x 5 mm 1.8pm particle size, Acquity UPLC® HSS T3 guard column, using a gradient elution with (A) 50 mM ammonium acetate in LC-MS grade water, and B) LC-MS grade acetonitrile as mobile phases. The 5-minute gradient started from 20% B linearly increasing to 30% B in 0.4 minutes and then 35% in 0.8 minutes, afterwards linearly increased to 95% B in 1 .3 minutes and kept there for 1 .5 minutes, then re-equilibrating with 20% B for one minute. The flow rate was kept at 0.63 ml/min, using an injection volume of 5 pl and the column temperature was set to 50°C. The desired components were quantified using an external calibration line of the components in the range of 10-2000 ng/ml. The corresponding samples analysed were diluted accordingly so the concentration of the desired components fell in the linear range of calibration line. The end concentration of acetonitrile in the sample was 33%. The concentration of the mogrol in the samples was calculated using a linear calibration line (nine points) whereby the origin was forced through zero. The other components (mogrosides) were determined by one point calibration.

Example 1. Construction of strains MOG001

Yarrowia lipolytica strain MOG001 was constructed starting from a Yarrowia lipolytica strain wherein the ku70 gene had been deleted (Aku70) to improve homologous recombination and integration efficiency of DNA fragments. MOG001 was built according to well-known genetic manipulation and transformation methods as those disclosed herein in the “Genetic modification techniques” section, using expression cassettes as those disclosed in Table 15. Yarrowia lipolytica strain MOG001 comprised one or multiple copies of the genes encoding for the polypeptides required to produce mogrosides and depicted in Table 13.

Table 13. Polypeptide sequences of the enzymes involved in the biosynthetic pathway of mogrosides and of markers present in MGG001 .

The genes coding for the polypeptides of Table 13 are expressed using promoters and terminators listed in Table 14. Table 14. Polynucleotide sequences of promoters and terminators

Example 2. Construction of strains MOG002 to MOG006

The Yarrowia lipolytica strains deficient in one or more beta glucanase polypeptide as used in the following experiments are disclosed in Table 15. The polypeptide sequences of the beta- glucanase polypeptides are disclosed in Table 16a.

Table 15 Table 16a

The open reading frames encoding the beta-glucanases in Yarrowia lipolytica as listed in Table 16a, were knocked out according to methods described in the “Genetic modification techniques” section. The specific regions that were knocked out for each glucanase are listed in Table 16b.

Table 16b

Correct knock out of the desired glucanases were confirmed by colony PCR with primers listed in Table 16c using KAPA2G Robust Hotstart PCR kit with dNTP’s from KAPA Biosystems.

Table 16c Example 4. Production of mogrosides in strains MOG001 to MOG006, mogroside sample preparation and quantification

To establish the effect of the deletion of the YALI0F01672g, YALI0B14289g and/or YALI0F05390g polypeptide, strains MOG002, MOG003, MGG004, MGG005 and MGG006 were cultivated, together with parent strain MGG001 , in shake-flasks (0.5 L with 60 ml medium) for 2 days at 30°C and 280 rpm. The medium was based on Verduyn et al. (Verduyn C, Postma E, Scheffers WA, Van Dijken JP. Yeast, 1992 Jul;8(7):501 -517) with modifications in the carbon and nitrogen sources as described in Table 17, 17a and 17b.

Table 17. Preculture medium composition

Table 17a. ^aTrace elements solution

Table 17b. ^bVitamins solution

Subsequently, 40 ml of the pre-cultures were transferred into fermenters (starting volume 0.4 L) containing the medium as set out in Tables 18, 18a and 18b. During cultivation, the pH was controlled at 7 by addition of ammonia (10 w/w%), the temperature was controlled at 30°C, and the pO2 was controlled at 20% (relative to air saturation) by adjusting the stirrer speed. The glucose concentration was kept limited by controlled 55 wt% glucose feed to the fermenter. After 144 hours of cultivation, the broths were collected for sample preparation and quantification of mogrosides.

Table 18. Fermentation medium composition

Table 18a. ^aTrace elements solution

Table 18b. ^bVitamin solution

After fermentation, samples for LC-MS quantification of mogrosides were prepared as follows. Several samples consisting of 1 ml of whole broth were collected in a 96 deep-well plate. Each sample was homogenized after adding 500 pL 100% acetonitrile, followed by 10 minutes centrifugation at 3700 rpm. Afterwards, 150 pL of supernatant from each sample were transferred to a new 96 deep-well plate and diluted with 300 pL 33% acetonitrile. At last, the samples were homogenized using a multichannel pipette and kept in deep well plate which was sealed before analysis. The quantitative determination of mogrosides was performed using the LC-MS method described herein above.

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

To establish the effect of the deficiency of the beta glucanase polypeptides YALI0F01672g and YALI0B14289g, in comparison with deficiency of the prior art polypeptide EXG1 (YALI0F05390) and in comparison, with parent strain MOG001 which is not deficient in any beta glucanases, strains MGG001 to MGG006 were cultured under identical conditions and broths samples were collected for sample preparation and quantification of the mogrosides as described in Example 4.

For each fermentation experiment the concentrations (in mg/L) of Mogroside I Ai (Mog1A1), Mogroside I Ei (Mog1 E1), Mogroside II A2 (Mog2A2) and Mogroside III A1 (Mog3A1) produced by each strain were determined. For each of these mogrosides the results for the strains MGG001 to MGG006 were depicted using a bar plot where the results were normalized in respect to the highest producing strain to which a value of 1 .0 was assigned. The results of the experiments for Mogroside I A1 (Mog1A1), Mogroside I E1 (Mog1 E1), Mogroside II A2 (Mog2A2) and Mogroside III A1 (Mog3A1) are plotted in Figure 6A, 6B, 7A and 7B, respectively.

The primary glycosylation at C3 and C24 of the mogrol backbone by the action of a UGT enzyme is the first step in the biosynthesis of mogrosides. The present inventors have surprisingly found that YALI0F01672g polypeptide is involved in the hydrolysis of the primary glycosidic bond at position C3 and, probably in lesser extent, at position C24 of the mogrol backbone.

Figure 6B shows that in a strain which has not been modified to be deficient in the production of a beta glucanase (i.e., strain MGG001) the amount of Mog1 E1 is very low. By comparison, a cell which has been made deficient in the production of EXG1 polypeptide (strain MGG004) the production of Mog1 E1 remains negligible and the same can be said for a strain made deficient in YALI0B14289g polypeptide (strain MGG003). Surprisingly, in a strain which is made deficient in the production of YALI0F01672g polypeptide (strain MGG002) the production of Mog1 E1 is improved by ~20 times, indicating the involvement of this polypeptide in the hydrolysis of the glycosidic bond between the C3 atom of the mogrol backbone of a mogroside and a p-1- glucose moiety bound at said position. In a strain which is made deficient in the production of both YALI0F01672g polypeptide and the EXG1 polypeptide (strain MOG005) the production of Mog1 E1 is also improved by ~20 times, confirming that the YALI0F01672g polypeptide (but not the EXG1 polypeptide) is involved in the hydrolysis of the of the glycosidic bond between the C3 atom of the mogrol backbone of a mogroside and a p-1 -glucose moiety bound at said position.

Figure 6A shows that in a strain which has not been modified to be deficient in the production of a beta glucanase as described herewith (strain MGG001) the amount of Mog1 A1 is not very high. By comparison, a cell which has been made deficient in the EXG1 polypeptide (MGG004) the production of Mog1A1 is even lower than in MGG001 and the same can be said for a strain made deficient in YALI0B14289g polypeptide (MGG003). Surprisingly, in a strain which is made deficient in the production of YALI0F01672g polypeptide the production of Mog1A1 is improved by ~2.5-5 times if compared with strain MGG001 , indicating involvement of the YALI0F01672g polypeptide (but not of the EXG1 polypeptide) in the hydrolysis of the glycosidic bond between the C24 atom of the mogrol backbone of a mogroside and a p-1 -glucose moiety bound at said position. Surprisingly a cell comprising a deficiency in the production of both EXG1 polypeptide and of YALI0F01672g polypeptide (MGG005 in Figure 6A) the production of Mog1A1 is absent. It is known that EXG1 polypeptide is possibly involved in the hydrolysis of p-1 ,2 and p- 1 ,6 glycoside bonds in mogrosides. Without being bound by a theory, the observation that a cell deficient in both EXG1 and YALI0F01672g polypeptides does not produce Mog1A1 is probably due to the fact that, in the absence of YALI0F01672g hydrolytic activity, Mog1A1 becomes available as substrate for other primary and secondary UGT’s leading to the production of more highly glycosylated mogrosides which, in turn, due to the absence of the EXG1 and YALI0F01672g hydrolytic activity, are not anymore hydrolyzed to less glycosylated mogrosides.

Figure 7A shows the normalized concentration of Mog2A2 in cells MGG001 to MGG006. This figure shows that in a cell which is not deficient in beta glucanases (MGG001), or in a cell which is deficient in only one beta glucanase the production of Mogroside II A2 is limited (strains MGG004, MGG003) or absent (MGG002). However, strain MGG005 (Figure 7A), which is deficient in both the EXG1 and YALI0F01672g polypeptides, produces 100% more Mog2A2 if compared to the cell which is only deficient YALI0F01672g (strain MGG002) and much more Mog2A2 if compared to the strains deficient in either EXG1 or YALI0B14289g polypeptide. These results support the hypothesis that the Mog1 E1 which becomes available due to the suppression of YALI0F01672g hydrolytic activity becomes substrate for secondary UGT’s leading to the production of Mog2A2 which, in the absence of EXG1 activity in the cell, is not anymore hydrolyzed to less glycosylated mogrosides or mogrol.

The experiments have also shown that the deficiency in the production of both the polypeptide EXG1 and YALI0B14289g in a cell capable of producing mogrosides (MGG006 in figure 7B) synergistically improve the production of more highly glycosylated mogrosides such as Mog3A1 . It is hypothesized that polypeptide YALI0B14289g, likewise EXG1 , is mainly active in the hydrolysis of beta-1 ,2- and beta-1 ,6-glycosidic bonds present in mogrosides. This result shows that it may be beneficial to downregulate multiple beta glucanases in the cell in order to improve the production of more highly branched mogrosides. In conclusion, it is clear that the recombinant cell which are deficient in one or more beta glucanase as disclosed herein are very suitable to steer the production of mogrosides with different level of glycosylation and display several surprising advantages if compared to the recombinant cells which are deficient in the EXG1 beta glucanase described in the background art.

Claims

1. A recombinant cell capable of producing one or more mogrosides and/or mogroside precursors wherein the cell has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product, wherein the polypeptide capable of deglycosylating a mogroside product is capable of hydrolyzing at least one of

2. A recombinant cell according to claim 1 wherein said polypeptide capable of deglycosylating a mogroside product is selected from a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 ; and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2, or from a combination of a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 1 and a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 2.

3. A recombinant cell according to any one of the preceding claims wherein the polypeptide capable of deglycosylating a mogroside product is a glycoside hydrolase (EC:3.2.1 .-), such as an exo-acting glycoside hydrolase.

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

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

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

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

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

(f) the beta 1 ,6 glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom of the mogrol backbone of a mogroside; (g) the beta 1 ,4 glycosidic bond between a second glucose molecule and a beta-1 -glucose bound at the C3 atom of the mogrol backbone of a mogroside.

5. A recombinant cell according to claim 4 wherein the polypeptide which is capable of deglycosylating a mogroside product is capable of hydrolyzing at least

6. A recombinant cell according to claim 4 or 5 wherein the polypeptide which is capable of deglycosylating a mogroside product is capable of hydrolyzing at least one of

7. A recombinant cell according to any one of the preceding claims wherein the cell has been further modified to result in a deficiency of an EXG1 and/or EXG2 polypeptide, particularly wherein a polypeptide with an amino acid sequence at least 50% identical to the amino acid sequence set out in SEQ ID NO: 3.

8. A recombinant cell according to any one of the preceding claims wherein the production of mogroside product by said cell is higher if compared with the production by an otherwise identical recombinant cell which has not been modified to be deficient in the polypeptide capable of deglycosylating a mogroside product, when both cells are cultured under the same conditions.

9. A recombinant cell according to any one of the preceding claims wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating mogroside compounds which is a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, a pentaglycosylated or exa-glycosylated mogroside compound, or an isomer thereof, particularly wherein the polypeptide capable of deglycosylating a mogroside product is capable of deglycosylating at least one or more of: Mogroside IA, Mogroside IEi, Mogroside IIA, Mogroside IIAi , Mogroside I IA2, Mogroside HE, Mogroside IIIA1, Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vlai, Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I.

10. A 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 glycosylating mogrol or a mogroside compound at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group, and/or C25 hydroxyl group;

(b) a polynucleotide encoding a polypeptide capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 (a C24-0-glucose) of a mogroside compound and/or or capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C3 (a C3-0-glucose) of a mogroside compound;

11. A 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 epoxy squalene from squalene, or 2,3,22,23 diepoxy squalene from 2,3 epoxy squalene, particularly wherein said polypeptide is a squalene epoxidase (SQE);

(b) a polynucleotide encoding a polypeptide capable of synthesizing cucurbitadienol from 2,3 epoxy squalene, or 24,25 epoxy-cucurbitadienol from 2,3,22,23 di-epoxy squalene, particularly wherein said polypeptide is a cucurbitadienol synthase (CDS);

(f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5).

12. A recombinant cell according to any one of claims 1 to 11 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 an epoxide hydrolase (EPH);

(f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5);

(g) a polynucleotide encoding a UGT polypeptide.

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

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

(d) a polynucleotide encoding a polypeptide capable of synthesizing Mevalonate-5- phosphate (MVA-P) from Mevalonic acid (MVA), particularly wherein said polypeptide is a 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), particularly wherein said polypeptide is a phosphomevalonate kinase (EC:2.7.4.2) (PMK);

15. A recombinant cell according to any one of the preceding claims wherein the recombinant cell further comprises a deficiency in the production of a lanosterol synthase polypeptide.

16. A recombinant cell according to any one of the preceding claims wherein the mogroside precursor is one or more of cucurbitadienol, mogrol, 11 -hydroxy cucurbitadienol, 24,25- epoxy-cucurbitadienol, 11 -hydroxy-24, 25-epoxy-cucurbitadienol, and/or 24,25-dihydroxy- cucurbitadienol.

17. A recombinant cell according to any one of the preceding claims wherein the mogroside is one or more of Mogroside IA, Mogroside IEi, Mogroside IIA, Mogroside IIAi, Mogroside IIA2, Mogroside HE, Mogroside IIIA1, Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I.

18. A recombinant cell according to any one of the preceding claims wherein the cell is procaryote, eukaryote (such as a yeast, a filamentous fungus or a plant cell) or archaeal cell, particularly a cell selected from a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an Issatchenkia orientalis cell, Pichia pastoris or an Escherichia coli cell.

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

20. A method of producing one or more mogrosides comprising contacting one or more mogrosides precursors or one or more mogroside substrates with a recombinant cell according to any one of claims 1 to 19, or a lysate or extract thereof under conditions suitable to produce said one or more mogrosides, optionally isolating said one or more mogrosides.

21. A method according to claim 19 or 20 wherein the one or more mogrosides is selected from Mogroside IA, Mogroside IEi, Mogroside IIA, Mogroside IIAi, Mogroside IIA2, Mogroside HE, Mogroside IIIA1, Mogroside IIIA2, Mogroside HIE, Mogroside III, Mogroside IVA, Mogroside IVE, Mogroside V, Mogroside VI, Mogroside VIA, Mogroside Vla1 , Mogroside VIB, Siamenoside I, Isomogroside IVE, Isomogroside V or a-Siamenoside I, particularly Mogroside HIE, Mogroside IIIA or Siamenoside I.