Cells and methods for the production of glycosylated compounds

Abstract

The present disclosure relates generally to the production of glycosylated compounds. Provided is a recombinant cell and a method for enzymatically modifying a glycosyl acceptor. The method comprises contacting a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising a heterologous enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor; thereby producing a modified glycosyl acceptor. Also provided is a composition obtainable by a method of the disclosure, and the use thereof.

Description

[0001] CELLS AND METHODS FOR THE PRODUCTION OF GLYCOSYLATED COMPOUNDS

[0002] Field

[0003] The present disclosure relates generally to the production of glycosylated compounds. Provided is a method and a recombinant cell for enzymatically glycosylating compounds such as a terpenoid.

[0004] Background

[0005] Secondary metabolic pathways in plants and microbes generate a plethora of small molecules such as terpenoids, flavonoids, and polyketides. Most of these molecules exist in nature as glycosides, in which sugar residues act as a decisive factor in their architectural complexity and bioactivity.

[0006] For example, terpenoids (also known as isoprenoids) are one of the largest groups of secondary metabolites and exist ubiquitously in plants. They exist mainly as glycosides in which sugar moieties are attached to their core backbones. They are recognized for their multiple industrially desirable characteristics such as sweeteners, flavors, aromas, colorants, pharmaceuticals and nutraceuticals.

[0007] Terpenoids are derived from two isomeric five-carbon compounds, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In plants, DMAPP and IPP are synthesized through two distinct and independent biochemical pathways, the mevalonic acid (MVA) pathway localized in cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway localized in plastids. In the downstream terpenoid pathway, DMAPP and IPP are condensed to produce geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP) and geranylfarnesyl diphosphate (GFPP). These prenyl diphosphate intermediates are converted by specific enzymes, including terpene synthases, to produce the various terpenoids. Based on the number of isoprene unit, terpenoids are classified into C5 hemiterpenoids, C10 monoterpenoids, C15 sesquiterpenoids, C20 diterpenoids, C25 sesterterpenoids, C30 triterpenoids, C40 tetraterpenoids, and C>40 polyterpenoids.

[0008] Examples of terpenoids include mogrosides which are C30 triterpenoids and have recently gained commercial significance as alternative high-intensity sweeteners. Said compounds are found in the fruit of Siraitia grosvenorii (also known as monk fruit, monkfruit or luohan guo) which is a plant belonging to the Curcubitaceae family and almost exclusively growing in the Guangxi province of China. Mogrosides all share the same mogrol backbone, but mainly differ in their glycosylation pattern (including the number of glucose moieties). In plants, the glycosylation of mogrosides is mainly mediated by uridine-5'-diphosphate (UDP)-glycosyltransferases (UGTs) which use nucleotide-activated sugars as donors (e.g. UDP-glucose) and typically catalyze the formation p-glycosidic bonds. Depending on the glycosylation pattern, the sweetness level and taste profile can vary among the different mogroside compounds. Mogroside V is the most abundant found in monk fruit. It is estimated to be several hundred times sweeter than sucrose while providing negligible calories. Other mogrosides, such as Mogroside IV, Mogroside III, and Mogroside II, also contribute to the overall sweetness of monk fruit, but these are generally not as sweet as Mogroside V and their sweetness may be accompanied by off-flavors (e.g. aftertaste, bitterness). More recently, Siamenoside I (SI) was isolated from monk fruit and was identified to have even superior sweetness to Mogroside V. Manufacturers often use a combination of mogrosides to achieve a balanced sweetness profile while minimizing any potential off-flavors, taking advantage of the strengths of each compound.

[0009] With the increasing need of developing new glycosylated compounds with tailored properties, engineering the glycosylation pattern of said compounds has received considerable interest. For example, in the context of terpenoids for use as sweeteners, engineered glycosylation of said compounds has been explored to modify their taste profile (e.g. making them more palatable and closer to the taste of table sugar), to improve their sweetness and solubility and / or to contribute to their stability and shelf-life. One recent example is a modified mogroside as described in WO2018204483 A2, W02020096905 A1 , W02020096907 A1 and Xu et al. (Xu et al. (2021), Food Chemistry 359:129938). Said compound, was referred as a-Siamenoside I or a-SI was obtained by in vitro enzymatic a-glycosylation of Mogroside HIE, wherein Mogroside HIE was contacted with soluble starch (as glucose donor) and a cell-free lysate of E. coli comprising a recombinant cyclodextrine glycosyltransferase (CGTase). Compared to Mogroside HIE, a-SI showed an increased sweetness intensity and an improved taste quality, thereby promoting a-SI as another potential low-calorie sweetener. Xu et al. also concluded that a-glycosylation, like p-glycosylation which typically occur in the formation of mogrosides in plants, can also enhance the sweet taste of mogrosides.

[0010] Both in vitro and in vivo biocatalytic glycosylation tools have emerged for engineering the glycosylation of compounds such as terpenoids. Purified biocatalysts are the core ingredients in the in vitro methods. However, obtaining them in pure form is laborious and expensive, and at times, immobilization is required to improve their activity and stability. Furthermore, in some cases such as in the case of UGTs for the glycosylation of mogrosides, expensive activated donor molecules (e.g. UDP-glucose) are required. Alternatively, in vivo biocatalytic glycosylation tools have also emerged for the production and glycosylation of terpenoids. For example, WO2014 / 086842, WO2016 / 038617 and WO2016 / 050890 describe recombinant cells and methods capable of producing mogrol and / or mogrosides. However, while these recombinant microbial production systems appear to be adapted for the production of various p-glycosylated terpenoids, they are not suitable for the production of a-glycosylated terpenoids. From an industrial perspective, given the growing interest in the global industrial market, there is still therefore a need for more robust microbial production systems that enable the production of various glycosylated compounds (incl. a-glycosylated and / or p-glycosylated terpenoids) in a sustainable, controlled and commercially viable way.

[0011] Description of the Figures

[0012] Figure 1 depicts the chemical structure of several mogroside compounds.

[0013] Figure 2 depicts the biosynthetic pathway from Acetyl Coenzyme A to Squalene.

[0014] Figure 3 depicts the biosynthetic pathway from Squalene to Mogrol.

[0015] Figure 4 depicts the biosynthetic pathway from Mogrol to Mogrosides.

[0016] Figure 5 depicts the chemical structure of several mogroside precursors in the biosynthetic pathway to the production of mogrosides starting from squalene.

[0017] Figure 6 depicts a schematic of the one or more reactions which may occur in the recombinant cell according to the disclosure.

[0018] Figure 7 depicts in vitro Compound (I) formation by various DexT using M3E as substrate. A part of the supplemented M3E appeared to be converted to other components that were not further analysed, leading to a mass gap.

[0019] Figure 8 depicts in vitro Compound (I) formation by various truncated DexT using M3E as substrate. Compound (I) production levels of the reference DexT, i.e. least truncated DexT variant per species, were set to 100%.

[0020] Figure 9 depicts in vitro Compound (I) formation by various DexT in absence and presence of an alpha-glycosidase. Samples were treated without (-) and with (+) an alpha-glycosidase enzyme (dextranase) to remove any alpha-glycosylated Compound (I). Non-specific M3E glycosylation and poly a-glycosylated Compound (I) were not measured explaining the mass gap in the analysis.

[0021] Figure 10 depicts in vitro Compound (I) formation by engineered DexT. Non-specific a- glycosylation and poly a-glycosylated Compound (I) were not analysed, explaining the mass gap in the analysis.

[0022] Figure 11 depicts M3E and Siamenoside production by Yarrowia lipolytica SIR001 (wherein no Yl- BGL2 is overexpressed) and SIR002 (with overexpressed YI-BGL2).

[0023] Figure 12 depicts in vivo Compound (I) production using intracellular (A), extracellular membrane anchored (B) and extracellular (C) localized DexT mutants. The strains were cultivated in absence (-) or presence (+) of sucrose. Compound(l) production levels are relative to the M3E levels produced in the cultures without sucrose supplementation which were set to 100%.

[0024] Figure 13 depicts the in vivo Compound (I) production using extracellular localized DexT variants in combination with (+) or without (-) an overexpressed alpha-glycosidase enzyme (AGL). Compound (I) production levels of strains comprising the AGL gene are relative to the Compound (I) formation levels produced in the strains without the overexpressed AGL which were set to 100%. Figure 14 depicts in-situ sucrose production by MOG production strains without and with overexpression of heterologous sucrose synthesis genes. Figure 15 depicts Mog3E and Compound (I) production of DexT overexpressing strains lacking (-) or comprising (+) heterologous sucrose synthesis genes. Values are relative to the M3E production levels of the strain without the in-situ sucrose synthesis genes. Non-specific M3E glycosylation and poly a-glycosylated Compound (I) were not measured explaining the mass gap in the analysis.

[0025] Description of Sequences

[0026] A description of the sequences is set out in Table 1 .

[0027] Summary

[0028] The disclosure provides herein a method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor comprising contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with a recombinant cell comprising, capable of (over)expressing or (over)expressing a polynucleotide encoding at least one (heterologous) enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions allows the at least one (heterologous) enzyme to catalyze the formation of a glycosidic bond, such as an a-glycosidic bond, between the transferred glycosyl group and the glycosyl acceptor; thereby, producing a modified glycosyl acceptor. Typically said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme.

[0029] Another aspect of the disclosure provides herein a method for producing a modified glycosyl acceptor comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising, capable of (over)expressing or (over)expressing a polynucleotide encoding for at least one (heterologous) enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor and / or a precursor thereof, wherein said contacting under suitable conditions comprises culturing the recombinant cell in a suitable culture medium in the presence of the glycosyl donor and the glycosyl acceptor and / or a precursor thereof, optionally wherein said contacting under suitable conditions allows the recombinant cell to express the at least one (heterologous enzyme) , wherein said contacting under suitable conditions allows the at least one (heterologous) enzyme to catalyze the formation of a glycosidic bond, preferably an a-glycosidic bond, between the transferred glycosyl group and the glycosyl acceptor; thereby producing the modified glycosyl acceptor, and optionally, isolating the modified glycosyl acceptor produced therefrom.

[0030] Another aspect of the disclosure provides herein a method for producing a modified glycosyl acceptor comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising, capable of (over)expressing or (over)expressing a polynucleotide encoding for at least one (heterologous) enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions comprises contacting the recombinant cell, the glycosyl donor and the glycosyl acceptor and / or a precursor thereof in a reaction mixture, wherein said contacting under suitable conditions allows the at least one (heterologous) enzyme to catalyze the formation of a glycosidic bond, preferably of an a-glycosidic bond, between the transferred glycosyl group and the glycosyl acceptor; thereby producing a modified glycosyl acceptor, and optionally, isolating the modified glycosyl acceptor produced therefrom. Optionally said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme.

[0031] The glycosidic bond is typically an a-glycosidic bond. The modified glycosyl acceptor (e.g. modified terpenoid) is typically an a-glycosylated glycosyl acceptor (e.g. a-glycosylated terpenoid).

[0032] A method of the disclosure for producing a modified glycosyl acceptor may be a fermentation, a biotransformation or a combination thereof.

[0033] In said method, the at least one heterologous enzyme may be an intracellular and / or an extracellular enzyme.

[0034] In said method, the recombinant cell is a typically living cell or a whole cell.

[0035] Typically, in the methods according to the disclosure “contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with the recombinant cell” may occur as follows: a) the glycosyl donor and the glycosyl acceptor come into contact inside (intracellularly to) the recombinant cell; and / or b) the glycosyl donor and the glycosyl acceptor come into contact outside (extracellularly to) the recombinant cell.

[0036] Also disclosed are:

[0037] A recombinant cell for use in the methods according to the disclosure, such as a recombinant cell comprising at least one (heterologous) polynucleotide encoding at least one enzyme capable of catalyzing a transglycosylation (such as transglucosylation) reaction, such as the transfer of a glycosyl group from a glycosyl donor (e.g. sucrose) to a glycosyl acceptor (e.g. a terpenoid such as a mogroside), wherein the recombinant cell is deficient in an enzyme capable of hydrolyzing the glycosyl donor (such as an invertase) and, optionally wherein the cell is deficient in a p-glucanase, optionally wherein the cell comprises at least one polynucleotide sequence encoding an a-glycosidase and / or at least one polynucleotide encoding a p- glucosidase. Typically, the at least one enzyme capable of catalyzing a transglycosylation reaction is an enzyme capable of catalyzing an a-glycosidic bond between a glycosyl group from a glycosyl donor (e.g. sucrose) and a glycosyl acceptor (e.g. a terpenoid such as a mogroside).

[0038] A reaction mixture comprising the recombinant cell according to the disclosure.

[0039] A culture medium comprising the recombinant cell according to the disclosure.

[0040] A fermentation broth comprising the recombinant cell according to the disclosure.

[0041] A composition obtained or obtainable by the method according to the disclosure.

[0042] A food product, a beverage, a pet-food, a feed, an oral or a pharmaceutical composition comprising the composition according to the disclosure.

[0043] Use of the recombinant cell according to the disclosure to produce a modified glycosyl acceptor (e.g. a modified terpenoid such as a modified mogroside), e.g. an a- glycosylated glycosyl acceptor (e.g. a-glycosylated terpenoid such as a-glycosylated mogroside).

[0044] In one embodiment of the disclosure, the a-glycosylated glycosyl acceptor is an a- glycosylated mogroside, more in particular an a-1 ,6-glycosylated Mogroside HIE.

[0045] Preferably, the a-glycosylated mogroside is Compound (I) (CAS 2419125-32-7) having the structure of:

[0046] Compound (I)

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Throughout the present disclosure, the words “comprise”, “include” and “having” and variations such as “comprises”, “comprising”, “includes” and “including” are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0051] 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”.

[0052] 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. 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).

[0053] 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.

[0054] According to the various aspects and embodiments disclosed herein in the context of a cell, the present invention preferably does not include processes for cloning human beings, processes for modifying the germ line genetic identity of human beings and / or uses of human embryos for industrial or commercial purposes.

[0055] A “nucleic acid molecule” or “polynucleotide” (the terms are used interchangeably herein) is represented by a nucleotide sequence.

[0056] A “polypeptide” is represented by an amino acid sequence.

[0057] The term “expression”, “expressed”, “ ex press (es)”, or “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.

[0058] “Expression of a gene” encompasses “heterologous expression” and “over-expression” and involves transcription of the gene and translation of the mRNA into a polypeptide (e.g. an enzyme). Overexpression refers to the production of the gene product as measured by levels of mRNA, polypeptide and / or enzyme activity in transgenic cells or organisms that exceeds levels of production in non-transformed cells or organisms of a similar genetic background.

[0059] Herein, a “gene” is defined as a polynucleotide containing an open reading frame (ORF) together with its transcriptional control sequences. The ORF is the polynucleotide region on the gene that encodes a polypeptide (e.g. an enzyme) and that will be transcribed and translated into said polypeptide.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] The term "homologous" or “endogenous” when used to indicate the relation between a given (recombinant) polynucleotide (such as DNA or RNA) or polypeptide and a given host organism or host cell such as the cell as disclosed herein, is understood to mean that in nature the polynucleotide or polypeptide molecule is produced by a host cell or organism of the same species, such as of the same variety or strain.

[0064] The term "heterologous" when used with respect to a polynucleotide (such as DNA or RNA) or a polypeptide refers to a polynucleotide or polypeptide that does not occur naturally as part of the host cell such as the cell as disclosed herein. In other words, heterologous polynucleotides or polypeptides are not endogenous to the cell into which they are introduced but have been obtained from another cell or synthetically or recombinantly produced.

[0065] 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.

[0066] 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.

[0067] The term “recombinant” is synonymous with “genetically modified”. The term “recombinant” when used in reference to a polynucleotide or polypeptide indicates that the polynucleotide or polypeptide 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 cell has been genetically modified in its sequence if compared to its native form or transformed / transfected with one or more polynucleotides by human intervention. The presence of the one or more polynucleotides alters the ability of the microorganism to produce one or more products.

[0068] 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. Hence, a non-transformed / non-transfected cell is typically a cell that does not naturally produce a product, although a cell which naturally produces a product and which has been modified as disclosed herein is considered a recombinant cell as disclosed herein.

[0069] The term “deficient in (the production of) a polypeptide (e.g. an enzyme)” when used in reference to a cell, typically means that the cell comprises a modification, preferably in its genome, which results in a reduced or no production of the polypeptide (e.g. an enzyme) 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 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. In this context, a 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; and / or b) it has a reduced expression level or has a reduced translation level of the mRNA transcribed from a gene encoding the polypeptide; and / or c) 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.

[0070] Deficiency in production of a polypeptide as defined herein in a cell may be measured by determining the amount and / or (specific) activity of the relevant polypeptide produced by the cell 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 cell modified in its genome as defined above and / or it may be measured by gene or genome sequencing if compared to the parent cell 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.

[0071] Within the context of the present disclosure the term “deficient in (the production of) a polypeptide (e.g. an enzyme)” when used in reference to a cell, may also mean that the cell naturally lacks said polypeptide (e.g. an enzyme). For example, Yarrowia lipolytica is naturally unable to metabolize sucrose because it lacks (or is deficient in) an invertase enzyme required for the cleavage of the disaccharide.

[0072] 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.

[0073] As defined herewith, an “enzyme capable of catalyzing the transfer of a glycosyl group from a glycosyl donor (e.g. sucrose) to a glycosyl acceptor (e.g. a terpenoid)” refers to an enzyme capable of catalyzing a transglycosylation reaction. Said enzyme preferentially belongs to glycoside hydrolase family 70 (GH70), glycoside hydrolase family 13 (GH13) and / or glycoside hydrolase family 77 (GH77). GH70 enzymes are involved in the synthesis of a-glucans, which are polysaccharides composed of a-linked glucose units. Examples of GH70 enzymes include glucansucrases (E.C. 2.4.1 .5) which are enzymes that synthesize various glucans from sucrose. GH13 enzymes comprises transglycosidases. Examples of GH13 enzymes include cyclomaltodextrin glucanotransferases or CGTases (E.C. 2.4.1 .19) which typically cleaves a-1 ,4- glucosidic linkages in the substrate (e.g. starch) to create a new a-1 ,4-glucosidic bond in an acceptor molecule (e.g. a terpenoid). The GH77 family is related to the GH13 family.

[0074] In the context of the disclosure, the term “glycosylation” or “glycosylation reaction” refers to an enzyme-catalyzed reaction in which a glycosyl group is covalently attached, typically through the anomeric carbon in such glycosyl group, to a hydroxyl or other functional groups of another molecule (i.e. a glycosyl acceptor); thereby, resulting in the formation of a glycosidic bond. Depending on the functional group, there are different types of glycosylation, including N- glycosylation, S-glycosylation and O-glycosylation. In the context of the present disclosure the glycosylation is typically an O-glycosylation and the glycosidic bond is typically a O-glycosidic bond.

[0075] The term “transglycosylation” or “transglycosylation reaction” refers to a specific type of glycosylation reaction that involves the transfer of a glycosyl group from one molecule (i.e. a glycosyl donor such as sucrose) to another (i.e. a glycosyl acceptor such as a terpenoid) that contains hydroxyl or other functional groups; thereby, resulting in the formation of a glycosidic bond. Enzymatic transglycosylation can be catalyzed by glycosyltransferases and glycoside hydrolases also known as glycosidases.

[0076] The term “glycosyl group” is used herein to refer to a sugar molecule (e.g. a glucose molecule) or a sugar moiety (e.g. a glucose moiety).

[0077] In the context of the disclosure, the term “glycosyl donor”, “glycosyl donor molecule”, “donor molecule”, “sugar donor”, “glucose donor”, “sugar donor molecule”, “glucose donor molecule” can be used interchangeably. “Glycosyl donor” is used herein to refer to a molecule that donates a glycosyl group in a glycosylation reaction (e.g. transglycosylation reaction). The glycosyl donor can be an activated donor (i.e. a sugar nucleotide e.g. UDP-glucose or GDP-mannose) or a nonactivated sugar (i.e. a sugar or glycan with one or more glycosidic linkages, e.g. sucrose, lactose, starch).

[0078] The term “glycosyl acceptor” or “glycosyl acceptor molecule” or “acceptor molecule” (e.g. a terpenoid or glycosylated terpenoid) is used herein to refer to a molecule that receives a glycosyl group during the process of glycosylation reaction (e.g. transglycosylation reaction). In glycosylation reactions, the glycosyl acceptor typically contains a functional group (e.g. hydroxyl group) that can react with the glycosyl group. The resulting reaction forms a glycosidic bond between the glycosyl donor and the glycosyl acceptor.

[0079] The term “glycosidic bond”, in particular “O-glycosidic bond”, is used herein to refer to a type of covalent bond that forms between the anomeric carbon atom of a glycosyl donor (e.g. C1 of glucose moiety) and an oxygen atom of a glycosyl acceptor molecule, often another sugar (e.g. a glucose) or a non-sugar moiety (e.g. a mogrol backbone). Said glycosidic bond is typically formed through a condensation reaction when the hydroxyl group on the anomeric carbon of a sugar molecule reacts with a hydroxyl group or a carboxylic group of an acceptor molecule, resulting in the elimination of a water molecule.

[0080] The specific type of glycosidic bond formed depends on which hydroxyl (-OH) group is involved in the linkage and the orientation of the anomeric carbon. The two common types of glycosidic bonds are a- and p-glycosidic bonds. In an “a-glycosidic bond”, the -OH group on the anomeric carbon of the sugar molecule is oriented below the plane of the ring. This results in a downward projection of the linked substituents. In a “p-glycosidic bond”, the -OH group on the anomeric carbon of the sugar molecule is oriented above the plane of the ring. This results in an upward projection of the linked substituents.

[0081] In an a-x,y-glycosidic bond, the numbers x and y referto the carbon atoms involved in the glycosidic bond. For example, in an a-1 ,6-glycosidic bond, the bond may be formed between the anomeric carbon at position 1 (C1 ; x = 1) of one sugar molecule and the hydroxyl group attached to the carbon at position 6 (C6; y = 6) of another sugar molecule wherein the bond is in an a configuration.

[0082] In the context of the disclosure, the terms “terpenoid”, “terpenoid compound”, “isoprenoid”, “isoprenoid compound”, “terpene”, “terpene compound” are used interchangeably herein. A “terpenoid” refers to a compound derived from two isomeric five-carbon compounds, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In plants, DMAPP and IPP are synthesized through two distinct and independent biochemical pathways, the mevalonic acid (MVA) pathway localized in cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway localized in plastids. In the downstream terpenoid pathway, DMAPP and IPP are condensed to produce prenyl diphosphate substrates, such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP) and geranylfarnesyl diphosphate (GFPP). These prenyl diphosphate intermediates are utilized by terpene synthases to produce the various terpenoids. Based on the number of isoprene unit, terpenoids are classified into Cs hemiterpenoids, C10 monoterpenoids, C15 sesquiterpenoids, C20 diterpenoids, C25 sesterterpenoids, C30 triterpenoids, C40 tetraterpenoids, and C>40 polyterpenoids. Examples of terpenoids include mogrosides which are C30 triterpenoids and have recently gained commercial significance as alternative high-intensity sweetener.

[0083] The terms “mogroside” and “mogroside compound” are used interchangeably herein and refer to a family of C30 triterpene glycosides. Non-limiting exemplary examples of mogrosides include such as Mogroside V, Siamenoside I, Mogroside IVE, Iso-mogroside V, Mogroside HIE, 11- Deoxy-mogroside V, 11-Oxo-mogroside V, Mogroside VI, Mogroside IVA, Mogroside HA, Mogroside IIA1 , Mogroside IIA2, Mogroside IA, 11-oxo-Mogroside VI, 11-oxo-Mogroside HIE, 11- oxo-Mogroside IVE, Mogroside IE, Mogrol, 11-oxo-mogrol, Mogroside HE, Mogroside IIIA2, and Mogroside III, which have been identified from the fruits of Siraitia grosvenorii (Swingle) that are responsible for the sweetness of the fruits.

[0084] In the context of the disclosure, the term “modified glycosyl acceptor” (e.g. “modified terpenoid” or “modified mogroside”) refers to a glycosyl acceptor (e.g. a terpenoid or a mogroside) that is modified with at least one additional glycosyl group. The glycosyl acceptor may be modified at multiple positions. The modification may involve different type of glycosidic bond (e.g. a- and / or p-glycosidic bond).

[0085] As used herein, the term “prenyl diphosphate” is used interchangeably with “prenyl pyrophosphate”.

[0086] The “mevalonate pathway” or the “mevalonic acid pathway” (also known as the “isoprenoid pathway” or “HMG-CoA reductase pathway”) is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five- carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Key enzymes are acetoacetyl-CoA thiolase (indicated AACT in Fig. 2), HMG-CoA synthase (indicated as HMGS in Fig. 2), HMG-CoA reductase (indicated as HMGR in Fig. 2), mevalonate kinase (indicated as MVK in Fig. 2), phosphomevalonate kinase (indicated as PMVK in Fig. 2), a mevalonate diphosphate decarboxylase (indicated as MDD in Fig. 2), and an isopentenyl diphosphate isomerase (indicated as IPI in Fig. 2).

[0087] The term “terpene synthase” refers to an enzyme that catalyzes the cyclization of prenyl diphosphate substrates, such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and geranylfarnesyl diphosphate (GFPP), leading to the formation of various (multi)cyclic terpene structures. Terpene synthases are classified into different types based on the types of products they generate. For example, monoterpene synthases produce monoterpenes, sesquiterpene synthases produce sesquiterpenes, and diterpene synthases produce diterpenes. Terpene synthases can also be classified into two classes, i.e. Type I and Type II, based on structure and catalytic nature. Type I class of enzymes possess a highly conserved DDXXD motif (a domain) and a less conserved NSE / DTE motif metal (Mg2+or Mn2+) binding domain required for the catalysis. Type II class of enzyme possess the DXDD motif (p domain) close to N-terminus, which is vital for protonation, and cyclization of GGPP to convert into copalyl diphosphate / cyclic diterpene diphosphate. Another conserved motif R(R)X8W, was found to be located downstream to the N-terminal of many TPS gene. Terpene synthases are also classified based on their gene structures, where Class I contains 12-14 introns, Class II contains nine introns, and Class III contains six introns. According to a recent classification based on sequence similarities and functional features, TPS gene families can be further classified into eight subfamilies, namely TPS-a, TPS-b, TPS-c, TPS-d, TPS-e, TPS-f, TPS-g, and TPS-h, where TPS-c belongs to Type II TPS and others belongs to Type I.

[0088] The term “precursor” when referring to a compound, is a compound that precedes another in a metabolic pathway or a synthetic process. In other words, a precursor molecule may be transformed into another molecule through one or more chemical reaction steps and / or one or more biological (e.g. enzymatic) process steps. For example, Mogroside HE is a precursor of mogroside HIE.

[0089] In the context of the disclosure, the term “contacting” refers to the interaction or association between a biocatalyst (e.g. an enzyme, a cell), a glycosyl donor and a glycosyl acceptor. The process of contacting involves the physical association of the biocatalyst with the glycosyl donor and glycosyl acceptor in a way that allows the catalytic reaction to occur.

[0090] In the context of the disclosure, the term “living cell” (such as disclosed herein) refers to a unit of life, bounded by a selectively permeable lipid bilayer known as the cell membrane, which separates its internal (“intracellular”) milieu from the external (“extracellular”) environment. The cell contains genetic material, typically DNA, which encodes the instructions for cellular structure and function. The genetic material is organized into chromosomes within a membrane-bound nucleus in eukaryotic cells or exists in a nucleoid region in prokaryotic cells. The ability to carry out essential cellular functions, respond to stimuli, and maintain a degree of metabolic activity are considered indicative of a living state. For the sake of clarity, a “living cell” is not a lysed cell, not a dead cell, not a crude cell extract, not a cell free extract. A “living cell” may be a “proliferating cell” or a “resting cell”. A “living cell” may be an “intact cell” or a “permeabilized cell”.

[0091] The term “proliferating cell” refers herein to a cell that is undergoing cell division, a process in which a single parent cell divides to produce two or more daughter cells. The term “resting cell” refers herein to a cell that is in a non-dividing or quiescent state. Resting cells are viable cells, meaning they are alive and capable of resuming growth (i.e. proliferating state) when conditions become more favorable.

[0092] The term “intact cell” refers herein to a cell having an undisturbed and fully functional cell membrane, maintaining its natural barrier properties. In other words, intact cells maintain their natural membrane integrity.

[0093] In the context of the disclosure, the term “permeabilized”, “permeabilization”, “alteration of the cell membrane” or the like when referring to a cell, refers herein to a cell whose cell membrane has been altered intentionally or inadvertently to allow the passage of certain substances that would not normally cross the membrane. In the context of the disclosure, the permeabilizing of cells may be to facilitate the entry or exit of specific molecules, such a glycosyl donor (e.g. sucrose) and / or a terpenoid, inside or outside the cell. In the context of the disclosure, a “permeabilized cell” is a living cell. Methods to permeabilize cells are well known in the art.

[0094] In the context of the present disclosure, the term “extracellular” or “outside the cell” can be used interchangeably. The term “extracellular” when referring to a polypeptide (e.g. an enzyme) and / or any other molecules (e.g. a glycosyl donor such as sucrose, a glycosyl acceptor such as a terpenoid) indicates that said polypeptide and / or molecules are located outside the cell, the cell being a whole cell. This space is usually taken to be outside the plasma membrane and / or cell wall of the cell and occupied by fluid (e.g. culture medium, fermentation broth, reaction buffer). Said extracellular polypeptide and / or molecules may be provided (i.e. fed) to the cell extracellularly. For example, said extracellular polypeptide and / or molecules may be added in the fluid (or liquid) where the cell is present, e.g. in the culture medium, the fermentation broth, the reaction buffer. Alternatively, or additionally, said extracellular polypeptide and / or molecules may be produced by the cell and subsequently secreted or transported outside the cell. For example, when “extracellular” refers to a polypeptide being expressed by a cell, said polypeptide may be “secreted free (i.e., fully released) outside the cell” or “displayed at the surface of the cell. The term “extracellular” is used in contrast to “intracellular” or “inside the cell”.

[0095] The term “secretion”, “secreted”, “secrete(s)” or “secreting”, when used in reference to a polypeptide (e.g. an enzyme), refers to the process by which a cell releases said polypeptide into the extracellular environment or transport them to specific cellular compartments. Proteins can be secreted outside the cell through various mechanisms. In the context of the present disclosure, the polypeptide may be secreted “free outside the cell” or “displayed at the surface of the cell”. The term “displayed at the surface of the cell” or “surface displayed” when referring to a polypeptide (e.g. an enzyme) indicates that said polypeptide is presented and anchored on the extracellular surface of the cell. For this, the gene encoding the polypeptide of interest is fused with a gene that encodes a surface-display anchor protein. This anchor protein is typically a component of the cell envelope that facilitates the attachment of the protein to the cell surface. The engineered fusion gene is introduced into the cells (such as bacteria, yeast, or mammalian cells) using transformation (for bacteria and yeast) or transfection (for mammalian cells). The cells express the fusion protein, resulting in the simultaneous expression of the anchor protein and polypeptide of interest. The anchor protein guides the display of the polypeptide of interest on the cell surface.

[0096] The term “free outside the cell” when referring to a polypeptide (e.g. enzyme) is used in contrast to “displayed at the surface of the cell”. Said polypeptide does not contain a surface-display anchor protein. Said polypeptide may interact with the extracellular surface of the cell but is not anchored to said surface as per definition above.

[0097] In the context of the present disclosure, the terms “intracellular” and “inside the cell” can be used interchangeably. The term “intracellular” when referring to a polypeptide (e.g. an enzyme) and / or any other molecules (e.g. a glycosyl donor such as sucrose, a glycosyl acceptor such as a terpenoid) indicates that said polypeptide and / or molecules are located inside the cell, the cell being a whole cell. The term “intracellular” is used in contrast to “extracellular”.

[0098] In the context of the disclosure, the term “whole cell” when referring to a cell expressing an enzyme (such as the cell disclosed herein) and “whole cell biocatalyst” may be used interchangeably and refer to living cells, typically microorganisms like bacteria or yeast, which are used as catalysts to perform specific biochemical reactions. Typically, the whole cell biocatalyst comprises single or multiple enzymes for the conversion of substrates into desired products. Said whole cell biocatalyst can be genetically modified or selected to carry out specific reactions, such as the production of desired products or precursors. A whole cell may be used in fermentation process and / or biotransformation process.

[0099] The term “biotransformation”, “biotransformation process and the like as used herein refer to a process wherein a compound (e.g., a terpenoid) is modified by a living organism. This can involve the introduction, modification, or removal of specific groups (e.g., a glycosyl group) in the compound, thereby resulting in a modified compound (e.g., a modified terpenoid).

[0100] The terms “fermentation process”, “fermentation”, “fermenting” and the like as used herein refers to a process wherein a microorganism, such as bacteria, yeast or fungi, is used to convert a carbon source (e.g., a carbohydrate) into a desired product, e.g., a modified terpenoid. The carbon source typically serves as the substrate that microorganisms use for growth and energy production. The choice of carbon source depends on the microorganism and the desired end product. Glucose is one of the most commonly used carbon sources in fermentation as it is a simple sugar that can be readily metabolized by many microorganisms.

[0101] A “batch fermentation” is defined herein as a fermentation wherein all nutrients are added at the start of a fermentation and the fermentation process continues until the desired product(s) is obtained or the fermentation is stopped.

[0102] A “fed-batch fermentation” or “semi-batch fermentation” is a fermentation wherein the initial growth medium is added to a bioreactor at the beginning (similar to a batch process), but additional nutrients, substrates, or feedstocks are intermittently or continuously introduced to the bioreactor during cultivation and in which the desired product(s) remains in the bioreactor until the end of the run.

[0103] A “continuous fermentation” is a fermentation wherein nutrients are continuously added to the fermentation and wherein the desired product(s) or the fermentation broth is continuously removed from the bioreactor.

[0104] As used herein, the terms "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 medium may further comprise trace metals, vitamins, salts, amino acids, etc. The trace metals may be divalent cations, including, but not limited Mn2+, Mg2+, Fe2+, Cu2+ etc.

[0105] The term “fermentation broth” is a liquid medium containing nutrients and microorganisms used in the process of fermentation.

[0106] As used herein, the term “reaction mixture” in the context of an enzymatic reaction (using e.g., enzyme, whole cell, cell lysate, cell extract) refers to the combination of all components involved in the reaction. Among others, the reaction typically includes the enzyme(s), substrate(s), buffer solution, salt and ions, products.

[0107] The term “isolated” or “recovered” when referring to a product as used herein means that the product that is removed or separated from at least one component, e.g., components present in the cell where the product is produced, the fermentation broth, the culture medium, the crude, cell-free extract and / or the reaction mixture. As used herein, “purified” when referring to a product means that the product is present at a higher amount relative to other compounds typically found with the indicated compound (e.g., in its natural environment).

[0108] “Purification” as described herein, can refer to methods for extracting a product from a cell lysate and / or a supernatant, wherein the cell is excreting the product. “Lysate” as described herein, comprises the cellular content of a cell after disruption of the cell wall and cell membranes and can include proteins, sugars, and mogrosides, for example. Purification can involve ammonium sulfate precipitation to remove proteins, salting to remove proteins, hydrophobic separation (HPLC), and use of an affinity column. In view of the products produced by the methods herein, affinity media is contemplated for the removal of specific mogrosides with an adsorbent resin.

[0109] “HPLC” as described herein is a form of liquid chromatography that can be used to separate compounds that are dissolved in solution. Without being limiting the HPLC instruments can comprise of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds can then be separated by injecting a sample mixture onto the column. The different components in the mixture pass can pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. There are several columns that can be used. Without being limiting the columns can be normal phase columns, reverse phase columns, size exclusion type of columns, and ion exchange columns.

[0110] A “sweetener”, “sweet flavoring agent”, “sweet flavor entity”, “sweet compound,” or “sweet tasting compound,” as used herein refers to a compound or physiologically acceptable salt thereof that elicits a detectable sweet flavor in a subject. A “sweet modifier,” as used herein refers to a compound or physiologically acceptable salt thereof that enhances, modifies, or improves the perception of sweetness.

[0111] “Modification of a genome” of a cell is herein defined as any event resulting in a change in a polynucleotide in the genome of the 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 ortranslation 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.oupjoumals.org / cgi / reprint / 32Z7 / e59 or Gupta et al. (1968), Proc. Natl. Acad. Sci USA, 60: 1338-1344; Scarpulla et a / . (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.).

[0112] A modification in the genome can be determined by comparing the polynucleotide sequence of the modified cell to the polynucleotide sequence of the non-modified 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:10.1 146 / annurev.genom.9.081307.164359).

[0113] Exemplary methods of modification are based on techniques of gene replacement, gene deletion, or gene disruption.

[0114] 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 / 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 priorto 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.

[0115] 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 modified cell 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.).

[0116] Furthermore, modification, downregulation or inactivation of a polypeptide 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. invertase) 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 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.

[0117] To increase the likelihood that the introduced enzymes are expressed in active form in a cell as disclosed herein, the corresponding encoding polynucleotide may be adapted to optimize codon usage to that of the chosen modified cell. The adaptiveness of the polynucleotides encoding the enzymes to the codon usage of the chosen cell 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 cell as disclosed herein may be genetically modified with (a) polynucleotide(s) which is (are) adapted to the codon usage of said cell 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 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.

[0118] Further improvement of the activity of the enzymes in vivo in a 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.

[0119] 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.

[0120] “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. 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.

[0121] 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.

[0122] 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”.

[0123] 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 etal. (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 / .

[0124] In the context of the present disclosure the terms “functional homologue”, “functional equivalent” and “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.

[0125] 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.

[0126] 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.

[0127] Detailed description

[0128] In the context of the present disclosure, it has been surprisingly found that a heterologous enzyme capable of transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor could be functionally expressed in a recombinant cell, both intracellularly and / or extracellularly (free outside the cell or displayed at the outside surface of the cell), thereby enabling a robust cell production platform and method for the intracellular ( / n vivo) production and / or extracellular production of modified glycosyl acceptors, in particular a-glycosylated glycosyl acceptors (e.g. a- glycosylated terpenoids such as a-glycosylated mogrosides).

[0129] It is therefore an object of the present disclosure to provide a method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor (and / or a precursor thereof) comprising contacting under suitable conditions the glycosyl donor and the glycosyl acceptor (and / or a precursor thereof) with a recombinant cell comprising, capable of (over)expressing or (over)expressing a polynucleotide encoding at least one (heterologous) enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor (and / or a precursor thereof), wherein said contacting under suitable conditions allows the at least one (heterologous) enzyme to catalyze the formation of a glycosidic bond, such as an a -glycosidic bond, between the transferred glycosyl group and the glycosyl acceptor (and / or a precursor thereof); thereby, producing a modified glycosyl acceptor. Typically said contacting under suitable conditions allows the recombinant cell to express the at least one (heterologous) enzyme.

[0130] It is further an object of the present disclosure to provide a method for producing a modified glycosyl acceptor comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor (and / or a precursor thereof) with a recombinant cell comprising, capable of (over)expressing or (over)expressing a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor (and / or a precursor thereof), wherein said contacting under suitable conditions comprises culturing said recombinant cell in a suitable culture medium in the presence of the glycosyl donor and the glycosyl acceptor and / or a precursor thereof , wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze formation of a glycosidic bond between the transferred glycosyl group and the glycosyl acceptor (and / or a precursor thereof), thereby, producing the modified glycosyl acceptor and optionally, isolating the modified glycosyl acceptor produced therefrom. Typically said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme.

[0131] Another aspect of the disclosure provides herein a method for producing a modified glycosyl acceptor comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor (and / or a precursor thereof) with a recombinant cell comprising, capable of (over)expressing or (over)expressing at least one heterologous enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor (and / or a precursor thereof), wherein said contacting under suitable conditions comprises contacting the recombinant cell, the glycosyl donor and the glycosyl acceptor (and / or a precursor thereof) in a reaction mixture, and wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyzes formation of a glycosidic bond between the transferred glycosyl group and the glycosyl acceptor (and / or a precursor thereof), thereby producing a modified glycosyl acceptor, and optionally, isolating the modified glycosyl acceptor produced therefrom. Typically said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme.

[0132] Typically, in the methods according to the disclosure “contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with the recombinant cell” may imply that: a) the glycosyl donor and the glycosyl acceptor come into contact inside (intracellularly to) the recombinant cell; typically, the at least one heterologous enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor is also present intracellularly (i.e., said heterologous enzyme is an intracellular enzyme) and / or that b) the glycosyl donor and the glycosyl acceptor come into contact outside (extracellularly to) the recombinant cell; typically, the at least one heterologous enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor is also present extracellularly (i.e., said heterologous enzyme is an extracellular enzyme).

[0133] The glycosidic bond may be an a-glycosidic bond. The glycosyl acceptor may be a terpenoid or glycosylated terpenoid, and the modified glycosyl acceptor (e.g. modified terpenoid or modified glycosylated terpenoid) may be an a-glycosylated glycosyl acceptor (e.g. a-glycosylated terpenoid, such as a-glycosylated mogroside).

[0134] A method of the disclosure for producing a modified glycosyl acceptor may be a fermentation, a biotransformation or a combination thereof.

[0135] In said method, the at least one heterologous enzyme may be an intracellular and / or an extracellular enzyme.

[0136] In said method, the recombinant cell may be a living cell or a whole cell.

[0137] Figure 6 depicts the reactions occurring in the methods and cells according to the present disclosure.

[0138] Enzyme for the transfer of a glycosyl group from a glycosyl donor to a glycosyl acceptor

[0139] In the methods according to the present disclosure, a recombinant cell comprises, is capable of (over)expressing or (over)expresses a polynucleotide encoding at least one heterologous enzyme capable of catalyzing or catalyzing the transfer of a glycosyl group from a glycosyl donor to a glycosyl acceptor.

[0140] In one embodiment of the disclosure, the heterologous enzyme capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor is an enzyme capable of catalyzing a glycosylation reaction, more specifically a transglycosylation reaction. Said enzyme may be a glycosyltransferase and / or a glycoside hydrolase (i.e. a glycosidase). Preferably, said enzyme belongs to glycoside hydrolase family 70 (GH70), glycoside hydrolase family 13 (GH13) and / or glycoside hydrolase family 77 (GH77).

[0141] In a further embodiment, the heterologous enzyme capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor is preferably a glycosyltransferase, more preferably a non- Leloir glycosyltransferase. One of the advantages of non-Leloir glycosyltransferases it that they use commercially available donors, such as non-activated sugars as donors, when catalyzing the transglycosylation reaction. Examples of non-Leloir glycosyltransferases include, but are not limited to, glucansucrases belonging to family GH70 and cyclomaltodextrin glucanotransferases (CGTases) belonging to family GH13. Together with enzymes from the family GH77, said enzymes form clan GH-H, sharing mechanistic, structural, and evolutionary characteristics.

[0142] Glucansucrases typically use sucrose as a glycosyl donor and catalyze reactions via an a- retaining double-displacement mechanism. First, the a-glycosidic linkage of the donor sucrose is cleaved, resulting in the formation of a p-glycosyl-enzyme intermediate. Second, a glycosyl acceptor (e.g. a terpenoid) attacks the p-glycosyl-enzyme intermediate, after which the glycosyl moiety is transferred to the acceptor with retention of the a-anomeric configuration; thereby, resulting in the formation of an a-glycosidic bond.

[0143] Examples of glucansucrases include, but are not limited to, dextransucrases (E.C. 2.4.1 .5), alternansucrases (E.C. 2.4.1.140), mutansucrases (E.C. 2.4.1.5) and reuteransucrases (E.C. 2.4.1.5). Accordingly, in another embodiment of the disclosure, the heterologous enzyme capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor is an enzyme selected from the group of dextransucrases, alternansucrases, mutansucrases and reuteransucrases.

[0144] CGTases (E.C. 2.4.1.19) use typically use starch as a glycosyl donor and, similarly to glucansucrases, catalyze an a-glycosidic bond during the glycosylation reaction.

[0145] Accordingly, in another embodiment, the heterologous enzyme capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor is an enzyme capable of catalyzing an a- glycosidic bond between the glycosyl group and the glycosyl acceptor.

[0146] In an acceptor reaction as described herein above, the glycosyl group may be transferred to a hydroxyl group present in the backbone of the glycosyl acceptor (e.g. mogrol backbone) and / or to a hydroxyl group of a sugar moiety linked to the backbone of said glycosyl acceptor; thereby, producing a modified glycosyl acceptor comprising the glycosyl group. When the reaction takes place between the glycosyl group and a sugar moiety present in the backbone of the glycosyl acceptor, the heterologous enzyme of the present disclosure may be capable of catalyzing a-1 ,3- glycosidic bonds, a-1 ,6-glycosidic bonds and / or a-1 ,4-glycosidic bonds. In one example, said enzyme is a dextransucrase and is capable of catalyzing a-1 ,6-glycosidic bonds and / or a-1 ,3- glycosidic bonds. In another example, said enzyme is a CGTase and is capable of catalyzing a- 1 ,4-glycosidic bonds.

[0147] Enzyme for the transfer of a glycosyl group from a glycosyl donor to a glycosyl acceptor: dextransucrases (DexT)

[0148] In one preferred embodiment of the disclosure, the heterologous enzyme capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor is a glucansucrase, preferably a dextransucrase (E.C. 2.4.1 .5). Any dextransucrase can be used in the methods and recombinant cells according to the disclosure. Suitable, non-limiting examples of dextransucrases which can be used in the methods and recombinant cells according to the disclosure are those set out in SEQ ID NO: 1 , 2, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46 or any of the Dextransucrase variants disclosed in Table 2 hereafter.

[0149] According to all aspects and embodiments herein, at least one DexT enzyme, truncation or engineered variant thereof as disclosed herein can be used alone or in combination in a (trans)glycosylation reaction conducted in vivo (i.e., at least partially via fermentation) or in vitro (biocatalytic outside of a living cell, i.e., after enzyme synthesis in vivo or synthetic), or in a combination of at least one in vivo process and at least one in vitro process (see e.g., Example 5), to obtain inter alia Compound (I) molecules with higher yields and / or quality in comparison to prior art methods.

[0150] Certain dextransucrases, alone or in combination, show progressive activity, leading to either Compound (I) molecules with one or more additional a1-6 glycosides attached to the first a1-6 glucose already present in Compound (I) (poly a-glycosylated Compound (I)), or show non-specific activity leading to mogrosides containing distinct alpha glycosidic linkages leading to compounds other than Compound (I), for example, enzymes as shown in Table 16 and detailed in Example 1.

[0151] The inventors surprisingly found that DexT size reduction by means of truncations can improve functional expression and production of Compound(l). Favorable DexT truncation variants are shown, but not limited, to those as detailed in Table 17 (cf. Example 2) that can be used alone or in combination for an even improved production of Compound(l).

[0152] In certain embodiments, a DexT or mutant or variant thereof according to the various aspects of the present disclosure, for example, as shown in Table 18, but not limited thereto, can be favorably combined with an a / pha-glycosidase enzyme (AGL) / dextranase to achieve even higher levels of Compound(l) are observed for the samples with AGL addition versus without AGL in a variety of biocatalytic processes. The inventors have surprisingly found that alpha-glycosidase enzyme is able to catalyze the hydrolysis of poly alpha-glycosylated Compound (I) formed by the progressive activity of DexTto improve Compound (I) yield.

[0153] In yet another embodiment, an engineered DexT mutant / variant as described hereafter can be used, for example, but not limited thereto, as shown in Table 19, Table 20, and Table 21 alone or in combination, to achieve an improved production of Compound(l).

[0154] In yet a further embodiment, at least one DexT enzyme, mutant or variant as disclosed herein can be expressed in a cell capable of producing mogrosides and which is deficient or lacks certain beta- glucanase activity to achieve an even higher amount of Mog3E substrate, and subsequently, of Compound (I). In certain preferred embodiments, a strain according to the disclosure can be engineered to produce sucrose in s / tu that can be favorably used with at least one DexT enzyme, mutant or variant as disclosed herein, for example, but not limited thereto, as shown in Table 20-21 , alone or in combination, optionally together with an AGL enzyme activity present.

[0155] In yet another preferred embodiment, at least one DexT enzyme, mutant or variant as disclosed herein, preferably at least one of mutant ID 18, 226, 232, 251 and 291 can be used in combination with in situ production of sucrose during in-vivo, fermentative production of Compound (I) formation using DexT to obtain remarkable amounts of Compound (I) in a convenient way in such a suitable production strain in a streamlined and reliable, and thus cost-efficient, process.

[0156] For example, said dextransucrases may be derived from Lactobacillus reuteri, such Lreul 80 gtf180 (SEQ ID NO: 1). Truncated versions at the N- and / or C-terminus of this Lreul 80 gtf180 can also be successfully used, such as Lreu-DexT.orf (SEQ ID NO: 2), Lreu_DexT.orf_0028 (SEQ ID NO: 24), Lreu_DexT.orf_0001 (SEQ ID NO: 38), Lreu_DexT.orf_0002 (SEQ ID NO: 39), Lreu_DexT.orf_0003 (SEQ ID NO: 40), Lreu_DexT.orf_0004 (SEQ ID NO: 41), and Lreu_DexT.orf_0005 (SEQ ID NO: 42). In alternative, variants comprising one or more amino acid modifications in respect to a DexT derived from Lactobacillus reuteri can be used, such as any of DexT variants of Lreu-DexT.orf (SEQ ID NO: 2), i.e. mutant_0014 to mutant_0197 in Table 2.

[0157] Dextransucrases derived from Oenococcus species are other suitable examples, such as Oesp_DexT.orf (SEQ ID NO: 28), truncated versions thereof at the N- and / or C-terminus, such as Oesp_DexT.orf_0001 (SEQ ID NO: 29), Oesp_DexT.orf_0002 (SEQ ID NO: 30), Oesp_DexT.orf_0003 (SEQ ID NO: 31), Oesp_DexT.orf_0004 (SEQ ID NO: 32), Oesp_DexT.orf_0005 (SEQ ID NO: 33), Oesp_DexT.orf_0006 (SEQ ID NO: 34), and variants of the latter, such as mutant _0198 to mutant_0223 of Table 2.

[0158] Other dextransucrases are those derived from Streptococcus downei, such as the full-length wild type enzyme GTFS_STRDO Dextransucrase (SEQ ID NO: 25), Sdow_DexT.orf (SEQ ID NO: 26), Sdow_DexT.orf_001 (SEQ ID NO: 27), Sdow_DexT.orf_002 (SEQ ID NO: 43),

[0159] Sdow_DexT.orf_003 (SEQ ID NO: 44), Sdow_DexT.orf_004 (SEQ ID NO: 45),

[0160] Sdow_DexT.orf_005 (SEQ ID NO: 46), and mutants of Sdow_DexT.orf_001 , i.e. mutant_0224 to mutant_0291 of Table 2.

[0161] Yet other dextransucrases are derived from Leuconostoc citreum, such as Lcit_DexT.orf (SEQ ID NO: 18), Lcit_DexT.orf_0001 (SEQ ID NO: 19), Lcit_DexT.orf_0002 (SEQ ID NO: 20), Lcit_DexT.orf_0003 (SEQ ID NO: 21), Lcit_DexT.orf_0004 (SEQ ID NO: 22), Lcit_DexT.orf_0005 (SEQ ID NO: 23) and mutants of Lcit_DexT.orf_0003, i.e. mutant_0001 to mutant_0013 of Table 2, or dextransucrases derived from Streptococcus mutans, such as GTFD_STRMU dextran sucrase (SEQ ID NO: 35), Smut_DexT.orf (SEQ ID NO: 36), and Smut_DexT.orf_0001 (SEQ ID NO: 37).

[0162] Functional homologues of the DexT enzymes disclosed herein above may also be used in the methods and recombinant cells according to the disclosure. For example, said DexT enzyme may comprise an amino acid sequence having at least 40%, at least 45%, 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, or at least 100% sequence identity with the amino acid sequence according to any of the following: SEQ ID NO: 1 , 2, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, or 46 or to any of mutants described in Table 2 hereafter, i.e. mutant_0001 to mutant_0291.

[0163] Enzyme for the transfer of a glycosyl group from a glycosyl donor to a glycosyl acceptor: Dextransucrase Variants

[0164] According to one aspect of the disclosure, the dextransucrase to be used in the methods and recombinant cells according to the disclosure is a dextransucrase variant of a parent polypeptide. As used herein, the terms “variant, or “mutant” can be used interchangeably. They can refer to either polypeptides or nucleic acids. Variants include substitutions, insertions, deletions, truncations, transversions, and / or inversions, at one or more locations relative to a parent sequence. Variants can be made for example by site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombination or synthetic approaches. Variant polypeptides may differ from a parent polypeptide by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology / identity with a reference polypeptide, for example the parent polypeptide. Preferably, variant polypeptides have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 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 even at least 99% amino acid sequence identity with a reference polypeptide, e.g. a parent polypeptide. Methods for determining percent identity are known in the art and described herein. Generally, the variants retain the characteristic nature of the parent polypeptide, but have altered properties in some specific aspects.

[0165] According to an embodiment the variant comprises an amino acid sequence which, when aligned with the amino acid sequence of the parent polypeptide (or of any reference polypeptide, e.g. the polypeptide according to the amino acid sequence of SEQ ID NO: 21 , SEQ ID NO: 2, SEQ ID NO: 34 or SEQ ID NO: 26), comprises at least one modification of an amino acid residue corresponding to any of the amino acids in the amino acid sequence of the parent polypeptide.

[0166] The wording “Amino acid sequence aligned with the amino acid sequence set out in the sequence of the parent polypeptide” (or aligned with the amino acid sequence set out in the sequence of the reference polypeptide, e.g. the polypeptide according to the amino acid sequence of SEQ ID NO: 21 , SEQ ID NO: 2, SEQ ID NO: 34 or SEQ ID NO: 26) means that the variant amino acid sequence and the amino acid sequence of the parent polypeptide (or e.g. set out in SEQ ID NO: 21 , SEQ ID NO: 2, SEQ ID NO: 34 or SEQ ID NO: 26) are aligned by a suitable method which allows a) comparison of the sequences with each other and b) identification of the positions in the amino acid sequence of the variant wherein either the same amino acid is present (identical position), or another amino acid is present (substitution), or one or more extra amino acids are present (insertion or extension) or no amino acid is present (deletion or truncation) if compared with the amino acid sequence of the parent polypeptide or reference polypeptide (e.g. the one set out in SEQ ID NO: 21 , SEQ ID NO: 2, SEQ ID NO: 34 or SEQ ID NO: 26). Therefore, a modification in the context of the disclosure is either a substitution, an addition or a deletion, of an amino acid residue corresponding to any of amino acids present in the parent polypeptide or a reference polypeptide when aligned with a suitable method. A suitable method allowing comparison of two amino acid sequences may be any suitable Pairwise Sequence Alignment method known to those skilled in the art, preferably a Global Pairwise Sequence Alignment method. A preferred Global Pairwise Sequence Alignment method is the EMBOSS Needle method based on the Needleman- Wunsch alignment algorithm (aiming at finding the optimum alignment (including gaps) of the two sequences along their entire length) (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453) as described herein. In one embodiment the amino acid sequence is aligned with the amino acid sequence of the parent polypeptide or of a reference polypeptide (as e.g. set out in SEQ ID NO: 21 , SEQ ID NO: 2, SEQ ID NO: 34 or SEQ ID NO: 26) using the NEEDLE program from the EMBOSS package, using EBLOSUM62 as a substitution matrix, with a gap-open penalty of 10 and a gap extension penalty of 0.5.

[0167] In one aspect there is disclosed a variant dextransucrase (of a parent polypeptide), wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 21 , comprises at least one modification of an amino acid residue corresponding to any of amino acids: 274, 275, 276, 319, 322, 406, 478, 481 , said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 21 . In one embodiment said modification is a substitution, an addition or a deletion, of an amino acid residue corresponding to any of amino acids: 274, 275, 276, 319, 322, 406, 478, 481 , said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 21 . In one embodiment, said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 21 , comprises one or more of: F or L at position 274, Y, F, K, or S at position 275, W at position 276, P at position 319, A at position 322, Q at position 406, Y at position 478, A at position 481 , said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 21 . In one embodiment said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 21 , comprises the following amino acid substitutions or combinations of amino acid substitutions at positions: -481 , preferably the substitution present in mutant_0001 of Table 2; -319 and 322, preferably the substitution indicated in mutant_0002 of Table 2;

[0168] -276 and 319, preferably the substitution present in mutant_0003 of Table 2;

[0169] -274 and 319, preferably the substitution present in mutant_0004 or mutant-0005 of Table 2;

[0170] -322 and 478, preferably the substitution present in mutant_0006 of Table 2;

[0171] -406 and 478, preferably the substitution present in mutant_0007 of Table 2;

[0172] -319, 406 and 478, preferably the substitution present in mutant_0008 of Table 2;

[0173] -275 and 276, preferably the substitution present in mutant_0009, mutant_0010 or mutant_001 1 of Table 2;

[0174] -275, 276 and 319, preferably the substitution present in mutant_0012 or mutant_0013 of Table 2. In one embodiment the parent polypeptide of said dextransucrase variant comprises or is the polypeptide with the amino acid sequence set out in SEQ ID NO: 21 or a polypeptide which 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 set out in SEQ ID NO: 21 .

[0175] In one embodiment the dextransucrase variant 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 set out in SEQ ID NO: 21.

[0176] According to another aspect of the disclosure, the dextransucrase to be used in the methods and the recombinant cells according to the disclosure is a dextransucrase variant of a parent polypeptide, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one modification of an amino acid residue corresponding to any of amino acids:

[0177] 196, 197, 199, 201 , 215, 227, 228, 239, 242, 282, 289, 290, 326, 328, 334, 346, 349, 398, 399, 400, 401 , 402, 417, 715, 721 , 726, 763, 788, 867, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 2. In one embodiment said modification is a substitution, an addition or a deletion, of an amino acid residue corresponding to any of amino acids:

[0178] 196, 197, 199, 201 , 215, 227, 228, 239, 242, 282, 289, 290, 326, 328, 334, 346, 349, 398, 399, 400, 401 , 402, 417, 715, 721 , 726, 763, 788, 867, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 2. In one embodiment, said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises one or more of

[0179] F at position 196, S at position 197, F at position 199, W or F at position 201 , P at position 215, M or Q at position 227, P at position 228, P at position 239, A at position 242, S at position 282, Y or W at position 289, M at position 290, L, F, Y or Q at position 326, F, L, S, M or H at position 328, F at position 334, F at position 346, H at position 349, Y at position 398, E or D at position 399, V, S or L at position 400, E, A, D, F, G, H, L, M, N, S, T, V, W or Y at position 401 , T or S at position 402, H at position 417, R at position 715, T or Q at position 721 , W at position 726, M at position 763, R at position 788, S at position 867, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 2.

[0180] In one embodiment said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises the following amino acid substitutions or combinations of amino acid substitutions at positions:

[0181] -239, preferably the substitution present in mutant_0014 of Table 2;

[0182] -401 , preferably the substitution present in mutant_0015 of Table 2;

[0183] -398, preferably the substitution present in mutant_0016 of Table 2;

[0184] -239 and 401 , preferably the substitutions present in mutant_0017 of Table 2;

[0185] -239 and 398, preferably the substitutions present in mutant_0018 of Table 2;

[0186] -398 and 401 , preferably the substitutions present in mutant_0019 of Table 2;

[0187] -242, preferably the substitution present in mutant_0020 of Table 2;

[0188] -326, preferably the substitution present in mutant_0021 or mutant_0022 of Table 2;

[0189] -242 and 401 , preferably the substitutions present in mutant_0023 of Table 2;

[0190] -326 and 401 , preferably the substitutions present in mutant_0024 or mutant_0025 of Table 2;

[0191] -289, preferably the substitution present in mutant_0026 or mutant_0027 of Table 2;

[0192] -201 , preferably the substitution present in mutant_0028 of Table 2;

[0193] -201 and 401 , preferably the substitutions present in mutant_0029 of Table 2;

[0194] -201 , 242 and 401 , preferably the substitutions present in mutant_0030 of Table 2;

[0195] -201 , 326 and 401 , preferably the substitutions present in mutant_0031 of Table 2;

[0196] -239 and 242, preferably the substitutions present in mutant_0032 of Table 2;

[0197] -239 and 326, preferably the substitutions present in mutant_0033 or mutant_0034 of Table 2;

[0198] -239, 242 and 326, preferably the substitutions present in mutant_0035 or mutant_0036 of Table 2;

[0199] -239 and 201 , preferably the substitutions present in mutant_0037 or mutant_01 12 of Table 2;

[0200] -239, 201 and 242, preferably the substitutions present in mutant_0038 of Table 2;

[0201] -239, 201 and 326, preferably the substitutions present in mutant_0039 or mutant_0040 of Table 2;

[0202] -239, 201 , 242 and 326, preferably the substitutions present in mutant_0041 of Table 2;

[0203] -239, 398 and 242, preferably the substitutions present in mutant_0042 of Table 2;

[0204] -239, 398 and 326, preferably the substitutions present in mutant_0043 or mutant_0044 of Table 2;

[0205] -239, 398, 242 and 326, preferably the substitutions present in mutant_0045 or mutant_0046 of Table 2;

[0206] -239, 398 and 201 , preferably the substitutions present in mutant_0047 or mutant_0115 of Table 2;

[0207] -239, 398, 201 and 242, preferably the substitutions present in mutant_0048 of Table 2; -239, 398, 201 and 326, preferably the substitutions present in mutant_0049 or mutant_0050 of Table 2;

[0208] -239, 398, 201 , 242 and 326, preferably the substitutions present in mutant_0051 of Table 2;

[0209] -239, 401 , 398 and 242, preferably the substitutions present in mutant_0052 of Table 2;

[0210] -239, 401 , 398 and 326, preferably the substitutions present in mutant_0053 or mutant_0054 of Table 2;

[0211] -239, 401 , 398, 242 and 326, preferably the substitutions present in mutant_0055 or mutant_0056 of Table 2;

[0212] -239, 401 , 398 and 201 , preferably the substitutions present in mutant_0057, mutant_01 11 or mutant_0114 of Table 2;

[0213] -239, 401 , 398, 201 and 242, preferably the substitutions present in mutant_0058 of Table 2;

[0214] -239, 401 , 398, 201 and 326, preferably the substitutions present in mutant_0059 or mutant_0060 of Table 2;

[0215] -239, 401 , 398, 201 , 242, and 326, preferably the substitutions present in mutant_0061 of Table 2; -239 and 401 , preferably the substitutions present in mutant_0062, mutant_0063, mutant_0064, mutant_0065, mutant_0066, mutant_0067, mutant_0068, mutant_0069, mutant_0070, mutant_0071 , mutant_0072, mutant_0073, or mutant_0074 of Table 2;

[0216] -239, 398 and 401 , preferably the substitutions present in mutant_0075, mutant_0076, mutant_0077, mutant_0078, mutant_0079, mutant_0080, mutant_0081 , mutant_0082, mutant_0083, mutant_0084, mutant_0085, mutant_0086, mutant_0087 or mutant_110 of Table 2;

[0217] -199 and 239, preferably the substitutions present in mutant_0088 or mutant_01 13 of Table 2;

[0218] -199 and 398, preferably the substitutions present in mutant_0089 of Table 2;

[0219] -199, 239 and 398, preferably the substitutions present in mutant_0090 or mutant_0116 of Table 2;

[0220] -199, 242 and 239, preferably the substitutions present in mutant_0091 of Table 2;

[0221] -199, 239 and 326, preferably the substitutions present in mutant_0092 or mutant_0093 of Table 2;

[0222] -199, 201 and 239, preferably the substitutions present in mutant_0094 of Table 2;

[0223] -199, 242, 239 and 398, preferably the substitutions present in mutant_0095 of Table 2;

[0224] -199, 239, 326 and 398, preferably the substitutions present in mutant_0096, mutant_0097, or mutant_0098 of Table 2;

[0225] -239 and 290, preferably the substitutions present in mutant_0099 of Table 2;

[0226] -398 and 290, preferably the substitutions present in mutant_0100 of Table 2;

[0227] -239, 398 and 290, preferably the substitutions present in mutant_0101 of Table 2;

[0228] -242, 239 and 290, preferably the substitutions present in mutant_0102 of Table 2;

[0229] -239, 326 and 290, preferably the substitutions present in mutant_0103 or mutant_0104 of Table 2;

[0230] -201 , 239 and 290, preferably the substitutions present in mutant_0105 of Table 2;

[0231] -242, 239, 398 and 290, preferably the substitutions present in mutant_0106 of Table 2; -239, 326, 398 and 290, preferably the substitutions present in mutant_0107 or mutant_0108 of Table 2;

[0232] -201 , 239, 398 and 290, preferably the substitutions present in mutant_0109 of Table 2;

[0233] -239 and 215, preferably the substitutions present in mutant_0117 of Table 2;

[0234] -239, 199 and 215, preferably the substitutions present in mutant_0118 of Table 2;

[0235] -239, 398 and 215, preferably the substitutions present in mutant_0119 of Table 2;

[0236] -239 and 715, preferably the substitutions present in mutant_0120 of Table 2;

[0237] -239, 401 and 417, preferably the substitutions present in mutant_0121 of Table 2;

[0238] -239, 398 and 417, preferably the substitutions present in mutant_0122 of Table 2;

[0239] -239 and 417, preferably the substitutions present in mutant_0123 of Table 2;

[0240] -239 and 721 , preferably the substitutions present in mutant_0124 or mutant_0125 of Table 2;

[0241] -239, 398, 715 and 721 , preferably the substitutions present in mutant_0126 or mutant_0127 of Table 2;

[0242] -239, 398, 201 and 721 , preferably the substitutions present in mutant_0128 or mutant_0129 of Table 2;

[0243] -239, 398 and 196, preferably the substitutions present in mutant_0130 of Table 2;

[0244] -239, 398, 196 and 197, preferably the substitutions present in mutant_0131 of Table 2;

[0245] -239, 398 and 197, preferably the substitutions present in mutant_0132 of Table 2;

[0246] -239, 398, 201 and 196, preferably the substitutions present in mutant_0133 of Table 2;

[0247] -239, 398, 201 , 215 and 196, preferably the substitutions present in mutant_0134 of Table 2;

[0248] -239, 227 and 228, preferably the substitutions present in mutant_0135 or mutant_0136 of Table 2;

[0249] -239 and 227, preferably the substitutions present in mutant_0137 or mutant_0138 of Table 2;

[0250] -239 and 788, preferably the substitutions present in mutant_0139 of Table 2;

[0251] -239, 788 and 288, preferably the substitutions present in mutant_0140 of Table 2;

[0252] -239, 228, 788 and 227, preferably the substitutions present in mutant_0141 of Table 2;

[0253] -239, 398 and 282, preferably the substitutions present in mutant_0142 of Table 2;

[0254] -239 and 282, preferably the substitutions present in mutant_0143 of Table 2;

[0255] -239, 398 and 334, preferably the substitutions present in mutant_0144 of Table 2;

[0256] -239 and 334, preferably the substitutions present in mutant_0145 of Table 2;

[0257] -239, 398 and 328, preferably the substitutions present in mutant_0146, mutant_0147, mutant_0148, mutant_0149 or mutant_0150 of Table 2;

[0258] -239 and 328, preferably the substitutions present in mutant_0151 , mutant_0152, mutant_0153, mutant_0154 or mutant_0155 of Table 2;

[0259] -239, 401 and 328, preferably the substitutions present in mutant_0156, mutant_0157, mutant_0158, mutant_0159, mutant_0160 or mutant_0161 of Table 2;

[0260] -239, 401 , 328 and 346, preferably the substitutions present in mutant_0162 or mutant_0163 of Table 2;

[0261] -239, 398, 328 and 346, preferably the substitutions present in mutant_0164 of Table 2; -239, 401 and 346, preferably the substitutions present in mutant_0165 or mutant_0166;

[0262] -239, 398 and 346, preferably the substitutions present in mutant_0167 of Table 2;

[0263] -239, 398 and 726, preferably the substitutions present in mutant_0168 of Table 2;

[0264] -239, 401 and 763, preferably the substitutions present in mutant_0169 or mutant_0170;

[0265] -239, 398 and 763, preferably the substitutions present in mutant_0171 of Table 2;

[0266] -239 and 763, preferably the substitutions present in mutant_0172 of Table 2;

[0267] -239 and 867, preferably the substitutions present in mutant_0173 of Table 2;

[0268] -239, 867 and 401 , preferably the substitutions present in mutant_0174 of Table 2;

[0269] -239, 867 and 398, preferably the substitutions present in mutant_0175 of Table 2;

[0270] -239 and 349, preferably the substitutions present in mutant_0176 of Table 2;

[0271] -239, 349 and 401 , preferably the substitutions present in mutant_0177 of Table 2;

[0272] -239, 349 and 398, preferably the substitutions present in mutant_0178 of Table 2;

[0273] -239 and 326, preferably the substitutions present in mutant_0179 of Table 2;

[0274] -239, 398 and 326, preferably the substitutions present in mutant_0180 and mutant_0181 of Table 2;

[0275] -239 and 400, preferably the substitutions present in mutant_0182, mutant_0183 or mutant_0184 of Table 2;

[0276] -239, 398 and 400, preferably the substitutions present in mutant_0185, mutant_0186 or mutant_0187 of Table 2;

[0277] -239 and 399, preferably the substitutions present in mutant_0188, or mutant_0189 of Table 2;

[0278] -239, 398 and 399, preferably the substitutions present in mutant_0190 or mutant_0191 of Table 2;

[0279] -239 and 402, preferably the substitutions present in mutant_0192 or mutant_0193 of Table 2;

[0280] -239, 402 and 399, preferably the substitutions present in mutant_0194 or mutant_0195 of Table 2;

[0281] -239, 398 and 402, preferably the substitutions present in mutant_0196 or mutant_0197 of Table 2;

[0282] In one embodiment the parent polypeptide of said dextransucrase variant comprises or is the polypeptide with the amino acid sequence set out in SEQ ID NO: 2 or a polypeptide which 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 set out in SEQ ID NO: 2.

[0283] In one embodiment the dextransucrase variant 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 set out in SEQ ID NO: 2. According to one aspect of the disclosure, the dextransucrase to be used in the methods and the recombinant cells according to the disclosure is a dextransucrase variant of a parent polypeptide, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 34, comprises at least one modification of an amino acid residue corresponding to any of amino acids: 200, 203, 204, 249, 252, 336, 413, 416, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 34. In one embodiment said modification is a substitution, an addition or a deletion, of an amino acid residue corresponding to any of amino acids:

[0284] 200, 203, 204, 249, 252, 336, 413, 416, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 34. In one embodiment, said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 34, comprises one or more of

[0285] F or L at position 200, S or F at position 203, W at position 204, P at position 249, A at position 252, Q or L at position 336, Y at position 413, E, A or H at position 416, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 34. In one embodiment said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 34, comprises the following amino acid substitutions or combinations of amino acid substitutions at positions:

[0286] -249, preferably the substitution present in mutant_0198 of Table 2;

[0287] -416, preferably the substitution present in mutant_0199, mutant_0200 or mutant_0201 of Table 2;

[0288] -413, preferably the substitution present in mutant_0202 of Table 2;

[0289] -249 and 413, preferably the substitution present in mutant_0203 of Table 2;

[0290] -249 and 416, preferably the substitution present in mutant_0204 of Table 2;

[0291] -413 and 416, preferably the substitution present in mutant_0205 of Table 2;

[0292] -249, 413 and 416, preferably the substitution present in mutant_0206 of Table 2;

[0293] -249 and 252, preferably the substitution present in mutant_0207 of Table 2;

[0294] -249 and 336, preferably the substitution present in mutant_0208 or mutant_0209 of Table 2;

[0295] -249 and 204, preferably the substitution present in mutant_0210 of Table 2;

[0296] -249 and 200, preferably the substitution present in mutant_021 1 or mutant_0212 of Table 2;

[0297] -413 and 252, preferably the substitution present in mutant_0213 of Table 2;

[0298] -413 and 336, preferably the substitution present in mutant_0214 or mutant_0215 of Table 2;

[0299] -413 and 204, preferably the substitution present in mutant_0216 of Table 2;

[0300] -413 and 200, preferably the substitution present in mutant_0217 of Table 2;

[0301] -249, 413 and 252, preferably the substitution present in mutant_0218 of Table 2;

[0302] -249, 413 and 200, preferably the substitution present in mutant_0219 of Table 2;

[0303] -203 and 204, preferably the substitution present in mutant_0220 or mutant_0221 of Table 2; -203, 204 and 249, preferably the substitution present in mutant_0222 or mutant_0223 of Table 2. In one embodiment the parent polypeptide of said dextransucrase variant comprises or is the polypeptide with the amino acid sequence set out in SEQ ID NO: 34 or a polypeptide which 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 set out in SEQ ID NO: 34.

[0304] In one embodiment the dextransucrase variant 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 set out in SEQ ID NO: 34.

[0305] According to yet another aspect of the disclosure, the dextransucrase to be used in the methods and the recombinant cells according to the disclosure is a dextransucrase variant of a parent polypeptide, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 26, comprises at least one modification of an amino acid residue corresponding to any of amino acids:

[0306] 212, 253, 256, 259, 302, 303, 412, 414, 419, 422, 425, 433, 436, 688, 691 , 722, 733, 739, 743, 745, 782, 787, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 26. In one embodiment said modification is a substitution, an addition or a deletion, of an amino acid residue corresponding to any of amino acids:

[0307] 212, 253, 256, 259, 302, 303, 412, 414, 419, 422, 425, 433, 436, 688, 691 , 722, 733, 739, 743, 745, 782, 787, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 26. In one embodiment, said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 26, comprises one or more of

[0308] W, L, or F at position 212, S, D or P at position 253, P at position 256, M or P at position 259, I or E at position 302, E or N at position 303, Y, R or W at position 412, Y at position 414, V or Y at position 419, R, Y, W, T or Q at position 422, K, N or Y at position 425, V or T at position 433, M or F at position 436, D at position 688, D or P at position 691 , P at position 722, Y, M, L or W at position 733, F or Q at position 739, S at position 743, E at position 745, W at position 782, M or I at position 787, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 26. In one embodiment said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 26, comprises the following amino acid substitutions or combinations of amino acid substitutions at positions:

[0309] -256, preferably the substitution present in mutant_0224 of Table 2;

[0310] -414, preferably the substitution present in mutant_0225 of Table 2;

[0311] -256 and 414, preferably the substitutions present in mutant_0226 of Table 2; -256 and 212, preferably the substitutions present in mutant_0227or mutant_0228 of Table 2;

[0312] -256, 414 and 212, preferably the substitutions present in mutant_0229 of Table 2;

[0313] -256, 414, 212 and 302, preferably the substitutions present in mutant_0230 of Table 2;

[0314] -256, 414 and 259, preferably the substitutions present in mutant_0231 or mutant_0232 of Table 2;

[0315] -256 and 253, preferably the substitutions present in mutant_0233, mutant_0234 or mutant_0235 of Table 2;

[0316] -414 and 253, preferably the substitutions present in mutant_0236, mutant_0237 or mutant_0238 of Table 2;

[0317] -256, 259 and 253, preferably the substitutions present in mutant_0239, mutant_0240 or mutant_0241 of Table 2;

[0318] -414, 259 and 253, preferably the substitutions present in mutant_0242, mutant_0243 or mutant_0244 of Table 2;

[0319] -256, 414 and 412, preferably the substitutions present in mutant_0245, mutant_0246, or mutant_0247 of Table 2;

[0320] -256, 414 and 782, preferably the substitutions present in mutant_0248 of Table 2;

[0321] -256, 414 and 733, preferably the substitutions present in mutant_0249, mutant_0250, or mutant_0251 ;

[0322] -256 and 733, preferably the substitutions present in mutant_0252 of Table 2;

[0323] -414, 253 and 733, preferably the substitutions present in mutant_0253, mutant_0254, mutant_0255, mutant_0256, mutant_0257, mutant_0258, mutant_0259, mutant_0260, mutant_0261 , mutant_0262, mutant_0263, or mutant_0264 of Table 2;

[0324] -256, 414 and 422, preferably the substitutions present in mutant_0265, mutant_0266, mutant_0267, mutant_0268, or mutant_0269 of Table 2;

[0325] -256, 414 and 425, preferably the substitutions present in mutant_0270, mutant_0271 , or mutant_0272 of Table 2;

[0326] -256, 414 and 436, preferably the substitutions present in mutant_0273, or mutant_0274 of Table 2;

[0327] -256, 414 and 433, preferably the substitutions present in mutant_0275, or mutant_0276 of Table 2;

[0328] -256, 414 and 302, preferably the substitutions present in mutant_0277 of Table 2;

[0329] -256, 414 and 419, preferably the substitutions present in mutant_0278, or mutant_0279 of Table 2;

[0330] -256, 414 and 691 , preferably the substitutions present in mutant_0280, or mutant_0281 of Table 2;

[0331] -256, 414 and 739, preferably the substitutions present in mutant_0282, or mutant_0283 of Table 2;

[0332] -256, 414 and 743, preferably the substitutions present in mutant_0284 of Table 2;

[0333] -256, 414 and 745, preferably the substitutions present in mutant_0285 of Table 2; -256, 414 and 303, preferably the substitutions present in mutant_0286 or mutant_0287 of Table 2;

[0334] -256, 414 and 787, preferably the substitutions present in mutant_0288 or mutant_0289 of Table 2; -256, 414 and 722, preferably the substitutions present in mutant_0290 of Table 2;

[0335] -256, 414 and 688, preferably the substitutions present in mutant_0291 of Table 2.

[0336] In one embodiment the parent polypeptide of said dextransucrase variant comprises or is the polypeptide with the amino acid sequence set out in SEQ ID NO: 26 or a polypeptide which 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 set out in SEQ ID NO: 26.

[0337] In one embodiment the dextransucrase variant 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 set out in SEQ ID NO: 26.

[0338] Table 2: Dextransucrase variants according to the disclosure. In the table, M1 (SEQ ID NO: 5)* means that in the specific mutant, if compared to the parent

[0339] DexT, the Met at position 1 of the parent is replaced by the signal peptide according to SEQ ID NO: 5 in the mutant

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] Glycosyl donor

[0355] The glycosyl donor is used herein to refer to a molecule that donates a glycosyl group in a glycosylation reaction. Most commonly, the glycosyl donor is an activated donor (i.e. a sugar nucleotide e.g. UDP-glucose or GDP-mannose). However, in the context of the present disclosure, the glycosyl donor is preferably a non-activated sugar. Examples of non-activated sugars include disaccharides or polysaccharides, such as sucrose, maltose, lactose, amylase and (soluble) starch. The use of non-activated sugars in a glycosylation reaction is advantageous as these are typically readily available and less expensive than activated sugars.

[0356] In one embodiment ofthe disclosure, when the heterologous enzyme capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor is a glucansucrase (e.g. a dextransucrase), the glycosyl donor is preferably sucrose.

[0357] In another embodiment of the disclosure, when the heterologous enzyme capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor is a CGTase, the glycosyl donor is preferably (soluble) starch. Methods to produce soluble starch are known to those in the art.

[0358] Glycosyl acceptor and / or precursor thereof

[0359] The term “glycosyl acceptor” is used herein to refer to a molecule that receives a glycosyl group in a glycosylation reaction. In the context of the invention, the glycosyl acceptor may be a terpenoid, such as a glycosylated terpenoid.

[0360] The terpenoid may be selected from the group consisting of hemiterpenoids, monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids and polyterpenoids. Preferably, the terpenoid may be selected from the group of triterpenoids or glycosylated forms thereof (e.g., mogrosides) and diterpenoids or glycosylated forms thereof (e.g. steviol glycosides). More preferably, the terpenoid may comprise a mogroside and / or a steviol glycoside.

[0361] In one embodiment, the terpenoid is a (glycosylated) terpenoid such as a mogroside. The mogroside may be of any type. For example, the mogroside may be selected from the group of Mogroside V, Siamenoside I, Mogroside IVE, Iso-mogroside V, Mogroside HIE, 1 1-Deoxy- mogroside V, 11-Oxo-mogroside V, Mogroside VI, Mogroside IVA, Mogroside IIA, Mogroside IIA1 , Mogroside IIA2, Mogroside IA, 11-oxo-Mogroside VI, 11-oxo-Mogroside HIE, 1 1-oxo-Mogroside IVE, Mogroside IE1 , Mogrol, 1 1-oxo-mogrol, Mogroside HE, Mogroside IIIA2 and Mogroside III. Mogrosides are glycosylated forms of the triterpene Mogrol. The mogrol backbone (i.e. the mogrol aglycone) comprises hydroxylic (-OH) 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 C3 and / or C24 of the mogrol backbone. For example, Mogroside I E1 comprises a p-glycosylic bond between the C1 of a glucose moiety and the oxygen atom of the hydroxyl group at C3 of the mogrol backbone. Mogroside IA1 comprises a p-glycosylic bond between the C1 of a glucose moiety and the oxygen atom of the hydroxyl group at C24 of the mogrol backbone. This glycosylation of one glucose moiety at position C3 and / or C24 of the mogrol backbone is called primary glycosylation. Mogroside HE comprises a p-glycosylic bond both at the oxygen atom of the hydroxyl group at C3 and at 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 p-1 ,6-glycosidic bond between the C1 of a secondary glucose moiety and the oxygen atom of the hydroxyl group at C6 of the primary glucose and / or through a p-1 ,2-glycosidic bond between the C1 of a secondary glucose moiety and the oxygen atom of the hydroxyl group at C2 of the primary glucose. For example, Mogroside HIE (CAS 88901-37-5) which comprises a primary glucose moiety at both position C3 and C24, further comprises a secondary glucose moiety through a p-1 ,2-glycosidic bond between the C1 of a secondary glucose moiety and the oxygen atom of the hydroxyl group at C2 of the primary glucose moiety linked at position C24 of the mogrol backbone. Some mogrosides may comprise a p-1 ,4- glycosidic bond between the C1 of a secondary glucose moiety and the oxygen of the hydroxyl group at C4 of the primary glucose moiety linked at position C3 of the mogrol backbone. Isomogroside IVE comprises such a p-1 ,4-glycosidic bond. Said p-1 ,2-, p-1 ,4-, p-1 ,6-, a-1 ,6- glycosylation in a mogroside molecule are known as secondary glycosylation. Figure 1 shows the structure of several known mogroside molecules.

[0362] In one specific embodiment, in the context of the present disclosure, the terpenoid comprises Mogroside HIE (CAS 88901-37-5) (abbreviated as M3E) with the structure of:

[0363] Mogroside HIE

[0364] The starting substrate for the glycosylation reaction in the method of the present disclosure may be the glycosyl acceptor as such and / or a precursor thereof. The term “precursor” refers herein to a compound that precedes another compound in a (bio)synthetic pathway. In the context of the disclosure, a precursor may be transformed into a glycosyl acceptor (e.g. a terpenoid) through one or more chemical reactions and / or one or more enzymatic reactions. For example, when the glycosyl acceptor is a terpenoid, the precursor may be transformed into the glycosyl acceptor through one or more glycosylation (e.g. p-glycosylation) reactions and / or one or more deglycosylation (e.g. p-deglycosylation) reactions.

[0365] In one embodiment of the present disclosure, the glycosyl acceptor is Mogroside HIE. Mogroside HIE may be obtained through the p-glycosylation by one or more UGTs of a precursor selected from, but not limited to, Mogrol, Mogroside IEi , Mogroside IAi and Mogroside HE.

[0366] Alternatively, or additionally, Mogroside HIE may be obtained through the deglycosylation by one or more hydrolases (e.g. p-glucosidases) of hyper-glycosylated mogrosides such as, but not limited to, Mogroside V, Siamenoside I, Mogroside IVE, Iso-mogroside V, 11-Deoxy-mogroside V, 11-Oxo-mogroside V, Mogroside VI, Mogroside IVA, 11-oxo-Mogroside VI, 1 1-oxo-Mogroside IVE, and combinations thereof. Such methods for deglycosylating hyper-glycosylated mogrosides are for example disclosed in WO2018 / 229283A1 .

[0367] Accordingly, in the present disclosure, a precursor of Mogroside HIE may comprise, but is not limited to, Mogrol, Mogroside I Ei , Mogroside I Ai and Mogroside HE, Mogroside V, Siamenoside I, Mogroside IVE, Iso-mogroside V, 11-Deoxy-mogroside V, 11-Oxo-mogroside V, Mogroside VI, Mogroside IVA, 11-oxo-Mogroside VI, 11-oxo-Mogroside IVE, and combinations thereof.

[0368] Modified glycosyl acceptor

[0369] The term “modified glycosyl acceptor” is used herein to refer to a glycosyl acceptor that is modified according to the method of the disclosure with at least one additional glycosyl group.

[0370] In the context of the invention, the modified glycosyl acceptor may be a modified (glycosylated) terpenoid, such as a modified mogroside. The terpenoid may be modified at multiple positions. The modification may involve different types of glycosidic bond (e.g. a- and / or p- glycosidic bond).

[0371] In one embodiment of the invention, the terpenoid is modified with at least one glycosyl group via an a-glycosidic bond. Accordingly, the modified terpenoid may be an a-glycosylated terpenoid. In one further embodiment, the modification comprises the addition of a glycosyl group to a glucose, e.g. a primary glucose, present in the glycosylated terpenoid. Accordingly, the modified terpenoid may be provided with one or more glycosyl groups via an a-1 ,3-glycosidic bonds, a-1 ,6-glycosidic bonds and / or a-1 ,4-glycosidic bonds. Preferably, the modified terpenoid is provided with one or more glycosyl groups via an a-1 ,6-glycosidic bonds.

[0372] In one specific embodiment, the glycosyl acceptor is a (glycosylated) terpenoid, preferably a mogroside, and the modifications occur at the C3 and / or C24 positions of the mogroside. Preferably, the mogroside is at least modified at the C24 position of the mogroside. More preferably, the mogroside is selectively modified at the C24 position of the mogroside. For example, in one embodiment, the invention provides a method for the production of a modified mogroside which is selectively modified with a single glycosyl residue at the C24 position via an a-glycosidic bond. In particular, the modification comprises the addition of a single glycosyl group to the primary glucose moiety linked at position C24 of the mogrol backbone via an a-glycosidic bond.

[0373] In one more specific embodiment, the terpenoid is Mogroside HIE and the modification comprises the addition of a single glycosyl group to the primary glucose moiety linked at position C24 of the mogrol backbone. More specifically, the modified Mogroside HIE comprises an a-1 ,6- glycosidic bond between the C1 of the glycosyl group and the oxygen atom of the hydroxyl group at C6 of the primary glucose moiety linked at position C24 of the mogrol backbone. Said modified Mogroside HIE (CAS 2419125-32-7) is referred herein as Compound (I) in the present disclosure and has the structure of:

[0374] Compound (I)

[0375] Compound (I) (also sometimes referred as a-Siamenoside I or a-SI in the literature) is a high-intensity sweetener that can be used in a wide variety of products in which a sweet taste is desired. Compound (I) provides a low-calorie advantage to other sweeteners such as sucrose or fructose.

[0376] Recombinant cell

[0377] In the present disclosure, a recombinant cell comprises, is capable of (over)expressing or (over)expresses a polynucleotide encoding at least one heterologous enzyme (capable of) catalyzing the transfer of a glycosyl group to a glycosyl acceptor (e.g. a terpenoid).

[0378] The cell may be a prokaryotic, archaebacterial or eukaryotic cell.

[0379] A prokaryotic cell may, but is not limited to, a bacterial cell. Bacterial cell may be Gramnegative 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. fluorescens), 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.

[0380] 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.

[0381] 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.

[0382] 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. tropicalis, 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).

[0383] The cell may be an algae, a microalgae or a marine eukaryote. The cell may be a Labyrinth ulomycetes 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.

[0384] 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 coli cell.

[0385] Accordingly, in one embodiment, the recombinant cell according to the present disclosure may be a prokaryote, a eukaryote or an 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, a Pichia pastoris cell or an Escherichia coli cell.

[0386] For the sake of clarity, in the methods of the present disclosure, the recombinant cell may be a living cell or a whole cell. The recombinant cell may be a proliferating cell or a resting cell. The recombinant cell may be an intact cell or a permeabilized cell. Preferably, the recombinant cell is an intact cell.

[0387] The heterologous enzyme of the present disclosure may be a large enzyme having a complex multi-domain structure and containing 10 or more putative N-glycosylation sites. In the context of the present invention, it has been surprisingly found that said heterologous enzyme can be functionally expressed in the recombinant cell of the present disclosure. Also surprisingly, the inventors of the present invention have further shown that it was possible to functionally express said heterologous enzyme intracellularly and / or extracellularly.

[0388] Accordingly, the recombinant cell of the present disclosure comprises at least one heterologous enzyme capable of catalyzing the transfer of a glycosyl group from a glycosyl donor to a glycosyl acceptor. Said enzyme is preferably a glucansucrase and / or a CGTase as described herein above. More preferably, said enzyme is a dextransucrase as described hereinabove.

[0389] In one embodiment, the recombinant cell comprises, is capable of (over)expressing or (over)expresses a polynucleotide encoding at least one heterologous enzyme as described herein above and said enzyme is an intracellular enzyme. In other words, said enzyme may be expressed intracellularly. More specifically, said heterologous enzyme may be expressed intracellularly and be active inside the cell upon expression. In particular, said intracellular enzyme may comprise an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 1 , 2, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, or 46 or to any of mutants described in Table 2, i.e. mutant_0001 to mutant_0291. Said heterologous enzyme may be any of the dextransucrase variants according to the disclosure described hereinabove More in particular, the recombinant cell of the present disclosure is genetically modified with any suitable heterologous polynucleotide encoding said intracellular enzyme that has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 1 , 2, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, or 46 or to any of mutants described in Table 2, i.e. mutant_0001 to mutant_0291. The enzyme and / or the polynucleotide encoding said enzyme may be a heterologous enzyme and / or a heterologous polynucleotide. The enzyme and / or the polynucleotide encoding said enzyme may be (over)expressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced. The recombinant cell of the present disclosure may be genetically modified with any suitable heterologous polynucleotide encoding said intracellular enzyme being any of the dextransucrase variants according to the disclosure. In another embodiment, the recombinant cell comprises, is capable of (over)expressing or (over)expresses a polynucleotide encoding at least one heterologous enzyme as described herein above and said enzyme is an extracellular enzyme. In other words, said enzyme may be expressed extracellularly and be active extracellularly. Furthermore, said heterologous enzyme may be an extracellular enzyme secreted free outside the recombinant cell and / or an extracellular enzyme displayed (anchored) at the outside surface of the recombinant cell. In the particular case, wherein the heterologous enzyme is secreted free outside the recombinant cell, said enzyme may comprise at the N-terminus a signal peptide (linked through its C-terminus to said enzyme) to facilitate secretion of the heterologous enzyme outside the cell membrane. Any signal peptide known in the art may be used for this purpose. A suitable signal peptide according to the disclosure may comprise an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 3 to 5, or SEQ ID NO: 102. More in particular, the recombinant cell of the present disclosure is genetically modified with any suitable heterologous polynucleotide encoding said enzyme that has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 3 to 5, or SEQ ID NO: 102. In the particular case, wherein the heterologous enzyme is displayed (anchored) at the outside surface of the recombinant cell, said enzyme may comprise at the N-terminus an anchor protein which in turn is linked at its N-terminus with the C-terminus of a signal peptide. Alternatively, the anchor protein may be linked to the C-terminus of the heterologous enzyme through its N-terminus. Any anchor protein known in the art could be used at this purpose. Suitable examples of anchor proteins which can be used comprise an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 6, 7, 103 or 104. More in particular, the recombinant cell of the present disclosure is genetically modified with any suitable heterologous polynucleotide encoding said enzyme that has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 6, 7, 103 or 104.

[0390] The enzyme and / or the polynucleotide encoding said enzyme as described herein above may be overexpressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced. The enzymes capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor, such as glucansucrases (e.g. dextransucrases) or CGTases as described herein above, have a high degree of processivity, which means they can add multiple glucose moieties in a growing polysaccharide chain without releasing the product between each addition. The formation of these oligo- and / or polysaccharides is not desirable, and it is further an object of the disclosure to hydrolyze these back into single glucose molecules which might serve as a carbon source for the recombinant cell. This can be done by the application of a-glucosidases; for example, dextranases (E.C. 3.2.1.1 1). Accordingly, in one further embodiment, the recombinant cell may comprise, may be capable of (over)expressing or may (over)express a polynucleotide encoding an enzyme capable of hydrolyzing oligo- and / or polysaccharides. In particular, the recombinant cell may comprise, may be capable of (over)expressing or may (over)express a dextranase (E.C. 3.2.1.11). Any dextranase enzyme known in the art may be used in the context of this disclosure. A dextranase is an enzyme that can hydrolyse the alpha-1 ,6 linked poly-glucose chains present in dextran back to their monomers. Any other enzyme able to perform the same function can be used in alternative to dextranase. The enzyme and / or the polynucleotide encoding said enzyme may be a heterologous enzyme and / or a heterologous polynucleotide. The enzyme and / or the polynucleotide may be (over)expressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced. An example of such an enzyme is shown in SEQ ID NO: 101 or a-dextranase (GenBank accession number AGM20695.1) or the enzyme Dextranase L (from Amano) as disclosed in the examples..

[0391] In another further embodiment, the recombinant cell may comprise, may be capable of

[0392] (over)expressing or may (over)express a polynucleotide encoding an enzyme capable of deglycosylating a hyper-glycosylated glycosyl acceptor (e.g. Mogroside V) to produce the desired glycosyl acceptor (e.g. Mogroside HIE). The hyperglycosylated glycosyl acceptor may be a mogroside. The hyperglycosylated mogroside, may be at least a tri-glycosylated mogroside product or more highly glycosylated mogroside product, at least tetra-glycosylated mogroside product, at least a penta-glycosylated mogroside product, at least an exa-glycosylated or more highly glycosylated mogroside product. The hyperglycosylated mogroside may comprise one or more of the following bonds:

[0393] (a) a 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;

[0394] (b) a 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;

[0395] (c) a 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;

[0396] (d) a 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; (e) a 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.

[0397] Therefore, such polypeptide capable of deglycosylating a hyper-glycosylated glycosyl acceptor, such as a hyper glycosylated mogroside product, may typically hydrolyze at least a triglycosylated, tetra-glycosylated, penta-glycosylated, exa-glycosylated mogroside product or more highly glycosylated mogroside product to a mogroside comprising a lower level of glycosylation. Typically, the polypeptide capable of deglycosylating a hyper-glycosylated glycosyl acceptor, such as a hyper glycosylated mogroside product, may hydrolyze one or more of the following bonds:

[0398] (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;

[0399] (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;

[0400] (c) 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;

[0401] (d) 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;

[0402] (e) 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.

[0403] Accordingly, the recombinant cell may comprise, may be capable of (over)expressing or may (over)express a polynucleotide encoding a polypeptide capable of deglycosylating a hyperglycosylated glycosyl acceptor, such as a hyper glycosylated mogroside product. For the purpose of this disclosure the terms “polypeptide capable of deglycosylating a hyper-glycosylated glycosyl acceptor, such as a hyper glycosylated mogroside product”, may be interchangeably indicated as “beta-glucosidase”, “p-glucosidase”, “beta glucosidase”, “p glucosidase”. An example of these enzyme may be Yl BGL2 according to SEQ ID NO: 9. This enzyme is particularly useful in case one wishes to produce Mog III E (M3E) as glycosyl acceptor. Suitable enzymes to be used in the deglycosylation of a mogroside product to a mogroside comprising a lower level of glycosylation are described in WO2018 / 229283 A1 or WO2024 / 220717 A1 .

[0404] In another embodiment, the recombinant cell as disclosed herein, used in the methods as disclosed herein, is deficient in a polypeptide capable of deglycosylating a mogroside product. Typically, the polypeptide 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 example, the recombinant cell as disclosed herein, used in the methods as disclosed herein, has been modified to result in a deficiency of a polypeptide capable of deglycosylating a mogroside product, optionally wherein the polypeptide capable of deglycosylating a mogroside product is capable of hydrolyzing at least one of:

[0405] (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.

[0406] Optionally, the polypeptide capable of deglycosylating the mogroside product can further hydrolyze one or more of the bonds selected from:

[0407] (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;

[0408] (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;

[0409] (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;

[0410] (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;

[0411] (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;

[0412] (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;

[0413] (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.

[0414] In one embodiment, when the recombinant cell is Yarrowia lipolytica, the polypeptide capable of deglycosylating a mogroside product may be YALI0F01672g (SEQ ID NO: 8), YALI0B14289g (SEQ ID NO: 9) or YALI0F05390g (SEQ ID NO: 10). Recombinant cells deficient in polypeptide capable of deglycosylating a mogroside product are described in WO2024 / 218244 A1 .

[0415] In one other embodiment, the polypeptide capable of deglycosylating a mogroside product may comprises, or consists of, an amino acid sequence set forth in SEQ ID NOs: SEQ ID NO: 8, 9 or 10. Said polypeptide may comprise an amino acid having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 8, 9, or 10.

[0416] 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).

[0417] In one further embodiment of the present disclosure, the recombinant cell of the present disclosure may be deficient in an enzyme capable of hydrolyzing the glycosyl donor (e.g. sucrose, starch). Accordingly, the recombinant cell of the present disclosure may be deficient in one or more enzymes selected from an invertase (E.C. 3.2.1 .26), a maltase (E.C. 3.2.1 .20), an isomaltase (E.C. 3.2.1.10), an a-amylase (E.C. 3.2.1.1).

[0418] In one specific embodiment, the recombinant cell expresses at least one heterologous glucansucrase (which uses sucrose as glycosyl donor) such as for example a dextransucrase, and said recombinant cell may be deficient in an invertase (E.C. 3.2.1 .26). Invertase, also known as p- fructofuranosidase, is an enzyme that is capable of catalyzing the hydrolysis of sucrose into glucose and fructose.

[0419] The recombinant cell may naturally lack said enzyme or enzyme activity. For example, Yarrowia lipolytica is naturally unable to metabolize sucrose because it lacks (is deficient in) an invertase enzyme required for the cleavage of the disaccharide.

[0420] The recombinant cell may naturally have said enzyme or enzyme activity and said cell may be made deficient in said enzyme by any method known in the art and / or as described herein before. For example, Saccharomyces naturally produces an invertase. To render the cell deficient in said enzyme or enzyme activity, the recombinant cell may comprise a modification, preferably in its genome, which results in a reduced or no production of the enzyme if compared to a parent cell that has not been modified, when analyzed under the same conditions. Alternatively, or in addition thereto, the cell may comprise a modification which results in an enzyme with decreased or no activity if compared to the parent cell that has not been modified, when analyzed under the same conditions.

[0421] In the context of the disclosure, the inventors have further found that a recombinant cell of the present disclosure comprising and expressing a heterologous sucrose biosynthetic pathway produces sucrose titers that are unexpectedly high, ranging in the order of grams per liter (g / l). For example, a recombinant cell of the present disclosure expressing a heterologous sucrose biosynthetic pathway may be capable of producing or produces more than 0.5 g / l of sucrose, more than 1 g / l, 2 g / l, 5g / l, 10 g / l, 20g / l, 30 g / l, 40g / l or 50 g / l of sucrose. Additionally, the inventors have surprisingly found that said sucrose production by the cell successfully enables the glycosylation of the glycosyl acceptor; thereby alleviating or even completely eliminating the need for sucrose feed or supplementation extracellularly. Ultimately, when the method of the disclosure is performed by fermentation as described herein below, a separate sucrose feed is not required next to a glucose (or other carbon source) feed which supports growth and energy production of the cells. Consequently, this embodiment enables the production of a modified glycosyl acceptor from any commodity and simple carbon source (e.g. glucose) where sucrose is not present or not available. Advantages of said embodiment include, but are not limited to, reduction of costs linked to sucrose and its transportation, reduction of costs linked to equipment (e.g. feeding lines, storage vessels) and decreased complexity of the production process.

[0422] Accordingly, in one further embodiment of the disclosure, the recombinant cell is capable of producing or produces sucrose. In particular, the cell capable of producing sucrose may comprise, may be capable of (over)expressing or may (over)express one or more of the following:

[0423] (a) an enzyme capable of phosphorylating glucose to form glucose-6-phosphate, preferably a hexokinase (E.C. 2.7.1.1);

[0424] (b) an enzyme capable of converting glucose-6-phosphate to fructose-6-phosphate, preferably a glucose-6-phosphate isomerase (E.C. 5.3.1.9);

[0425] (c) an enzyme capable of catalyzing the transfer of a hexosyl group from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6- phosphate, preferably a sucrose-phosphate synthase (E.C. 2.4.1 .14);

[0426] (d) an enzyme capable of converting sucrose-6-phosphate to sucrose, preferably a sucrose-6-phosphate phosphatase (E.C. 3.1.3.24).

[0427] More in particular, the cell capable of producing sucrose comprises one of more of the following enzymes or functional homologs thereof: Hexokinase from Saccharomyces cerevisiae (SEQ ID NO: 1 1) hexokinase from Yarrowia lipolytica (SEQ ID NO: 12), Glucose-6-phosphate isomerase (also known as phosphoglucose isomerase) from Yarrowia lipolytica (SEQ ID NO: 13), sucrose phosphate synthase from Synechocystis sp. PCC6803 (SEQ ID NO: 14) or sucrose phosphate synthase from Saccharum officinarum (SEQ ID NO: 15), sucrose-6-phosphate phosphatase from Zea mays (SEQ ID NO: 16), or sucrose-6-phosphate phosphatase from Nicotiana tabacum (SEQ ID NO: 17).

[0428] In one embodiment, the (heterologous) enzyme capable of catalyzing the transfer of a hexosyl group from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6-phosphate, preferably a sucrose-phosphate synthase (E.C. 2.4.1.14) may comprise, or consists of, an amino acid sequence set forth in SEQ ID NOs: 14 or 15. Said polypeptide may comprise an amino acid having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 14 or 15.

[0429] In one embodiment, the (heterologous) enzyme capable of converting sucrose-6- phosphate to sucrose, preferably a sucrose-6-phosphate phosphatase (E.C. 3.1.3.24) may comprise, or consists of, an amino acid sequence set forth in SEQ ID NOs: 16 or 17. Said polypeptide may comprise an amino acid having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 16 or 17. In one embodiment, the (heterologous) enzyme capable of phosphorylating glucose to form glucose-6-phosphate, preferably a hexokinase (E.C. 2.7.1.1), may comprise, or consists of, an amino acid sequence set forth in SEQ ID NOs: 1 1 or 12. Said polypeptide may comprise an amino acid having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 11 or 12.

[0430] In one embodiment, the (heterologous) enzyme capable of converting glucose-6- phosphate to fructose-6-phosphate, preferably a glucose-6-phosphate isomerase (E.C. 5.3.1.9), may comprise, or consists of, an amino acid sequence set forth in SEQ ID NOs: 13. Said polypeptide may comprise an amino acid having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 13.

[0431] The enzyme and / or the polynucleotide encoding said enzyme may be a heterologous enzyme and / or a heterologous polynucleotide. The enzyme and / or the polynucleotide may be overexpressed. Typically, the polynucleotide encoding for the enzyme may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced.

[0432] Optionally, the recombinant cell comprising a sucrose biosynthetic pathway may further comprise, may further be capable of (over)expressing or may further (over)express a transport system facilitating the transport of glucose from the extracellular environment into the recombinant cell. Any glucose import transport system known in the art may be used in the context of this disclosure. Suitable, non-limiting examples of glucose import transport system include the HXT (hexose transporter) family, facilitated diffusion, Phosphotransferase System (PTS), and ABC transporters. Said transport system and / or the polynucleotide encoding said transport system may be heterologous. Said transport system and / or the polynucleotide encoding said transport system may be overexpressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced. Alternatively, or additionally, the recombinant cell may comprise an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport of glucose inside the recombinant cell.

[0433] Also optionally, the recombinant cell comprising a sucrose biosynthetic pathway may further comprise, may further be capable of (over)expressing or may further (over)express a transport system capable of facilitating the transport of the sucrose outside the recombinant cell. Any sucrose export transport system known in the art may be used in the context of this disclosure. Said transport system and / or the polynucleotide encoding said transport system may be heterologous. Said transport system and / or the polynucleotide encoding said transport system may be overexpressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced. Alternatively, or additionally, the recombinant cell may comprise an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport of sucrose outside the recombinant cell.

[0434] In a separate embodiment, e.g. in a method wherein the glycosyl donor (e.g. sucrose) is provided (fed) to the recombinant cell extracellularly as described in herein below, the recombinant cell of the disclosure may further comprise, may further be capable of (over)expressing or may further (over)express a transport system capable of facilitating the transport of the glycosyl donor (e.g. sucrose) inside the recombinant cell. When the glycosyl donor is sucrose, any sucrose import transport system known in the art may be used in the context of this disclosure. Said transport system and / or the polynucleotide encoding said transport system may be heterologous. Said transport system and / or the polynucleotide encoding said transport system may be overexpressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced. Alternatively, or additionally, the recombinant cell may comprise an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport of glycosyl donor (e.g. sucrose) inside the recombinant cell.

[0435] In yet one further embodiment, the recombinant cell may be capable of producing or may produce a glycosyl acceptor and / or a precursor thereof, in particular a terpenoid and / or a precursor thereof, more in particular a mogroside and / or a precursor thereof. Accordingly, the recombinant cell may comprise, may be capable of (over)expressing or may (over)express a glycosyl acceptor biosynthetic pathway, e.g. a terpenoid biosynthetic pathway such as a mogroside biosynthetic pathway.

[0436] In one aspect, the recombinant cell 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 or catalyzes the transfer of a glycosyl group from a UDP-sugar (glycosyl donor) to an acceptor molecule, in particular to or a 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 C24, leading to Mogroside IAi , Mogroside IEi , 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 both C3 and C24, respectively. Alternatively primary glycosylation may occur at position C1 1 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 C3 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 IEi 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 C24-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 C3 of the mogrol backbone (a C3-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 IEi, 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 may be obtained.

[0437] Hence, in one further aspect of the disclosure, the recombinant cell which is capable of producing or produces a mogroside and / or a precursor thereof, is a cell comprising, capable of (over)expressing, or (over)expressing, one or more of the following polynucleotides:

[0438] (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, typically at it C3 hydroxyl group and / or C24 hydroxyl group;

[0439] (b) a polynucleotide encoding a polypeptide capable of p-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 (a C24-O-glucose) of a mogroside compound and / or or capable of p-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C3 (a C3-O-glucose) of a mogroside compound;

[0440] (c) a polynucleotide encoding a polypeptide capable of p-1 ,6-glycosylation of the C6' hydroxyl group of a glucose moiety at position C3 of a mogroside compound and / or capable of p-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 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.

[0441] 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 Tables 3a to 3e.

[0442] 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: 57 to 61 herein, or any of the amino acid sequences indicated in Tables 3a to 3e 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: 57 to 61 herein, or with amino acid sequence as indicated in Tables 3a to 3e.

[0443] 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 3a 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 3a.

[0444] 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 3b 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 3b.

[0445] In yet another aspect of the disclosure, a polypeptide, particularly a UGT polypeptide capable of p-1 ,2-glycosylation ofthe’C2' hydroxyl group of a glucose moiety at position C24 (a C24- O-glucose) of a mogroside and / or or capable of p-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C3 (a C3-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 3c 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 3c.

[0446] In yet another aspect of the disclosure, a polypeptide, particularly a UGT polypeptide capable of p-1 ,6-glycosylation of the C6' hydroxyl group of a glucose moiety at position C3 of a mogroside compound and / or capable of p-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 3d 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 3d.

[0447] Tables 3a to 3e 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.

[0448] Table 3a 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 3b 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 3c 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 3d 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 3e 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 3d. 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). The SEQ ID NOs in the table are those in the corresponding patent documents in which they are disclosed.

[0449] Table 3a

[0450] Table 3b Table 3c

[0451] Table 3d

[0452] Table 3e

[0453] Depending on the modified mogroside that one wishes to produce and depending on the mogroside in the method of the disclosure, the cell will comprise, will be capable to express or will express one or more polynucleotides encoding a UGT polypeptide. For example, a recombinant cell according to the disclosure which is capable of producing

[0454] Mogroside HIE starting from mogrol, may comprise at least one polynucleotide encoding a polypeptide capable of glycosylating mogrol or mogroside at its C3 hydroxyl group and / or at its C24 hydroxyl group, such as one or more of the suitable polypeptides disclosed in Table 3a and 3b, and at least a polynucleotide encoding a polypeptide capable of p-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 3c.

[0455] For example, a recombinant cell according to the disclosure which is capable of producing Siamenoside I 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 Tables 3a and 3b, respectively, at least a polynucleotide encoding a polypeptide capable of p-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 3c, and at least a polynucleotide encoding a polypeptide capable of p-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 3d.

[0456] Figure 4 depicts a putative mogroside biosynthetic pathway from Mogrol to mogrosides, including the types of UGT polypeptides needed to produce each mogroside.

[0457] 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 (201 1) 12:343). Subsequently, methods to use recombinant cells to produce mogrosides have been described (see for example WO2014 / 086842 A1 , WO2016 / 038617 A1 , WO2016 / 050890 A2).

[0458] 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.

[0459] 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).

[0460] 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.

[0461] The synthesis of mogrol starting from squalene may comprise one or more steps described hereafter.

[0462] 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.

[0463] Cucurbitadienol, 24,25-epoxy-curcubitadienol and 24,25-dihydroxy-cucurbitadienol respectively, may be converted to 1 1 -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).

[0464] 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.

[0465] 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).

[0466] 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.

[0467] 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 .

[0468] Thus, in one embodiment of the disclosure, the recombinant cell according to the disclosure further comprises, is capable of expressing or expresses one or more of the following recombinant polynucleotides:

[0469] (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);

[0470] (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);

[0471] (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);

[0472] (d) a polynucleotide encoding a polypeptide capable of reducing a cytochrome P450 complex, particularly a cytochrome P450 reductase (CPR);

[0473] (e) 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) (f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5).

[0474] Typically, one or more of the polypeptides and / or polynucleotides according to (a), (b), (c),

[0475] (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.

[0476] Typically, a recombinant cell 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),

[0477] (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).

[0478] In the method and recombinant cell 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.

[0479] Squalene epoxidase (E.C. 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 di-epoxy squalene (also known as dioxido-squalene) from oxido squalene, typically in the presence of NADPH. Therefore, a recombinant cell capable of producing a mogroside and / or a precursor thereof 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.

[0480] Therefore, in one embodiment the recombinant cell capable of producing a mogroside and / or a precursor thereof according to the disclosure may comprise, may be capable to express or may 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 cell and method according to the disclosure are provided in Table 4. Therefore, the SQE polypeptide may have an amino acid sequence according to SEQ ID NO: 67 herein, or an amino acid sequence according to any sequence disclosed in column 2 of Table 4 (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 4 or with the amino acid sequence according to SEQ ID NO: 67 herein.

[0481] Table 4 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.

[0482] Table 4 In the method and the recombinant cell 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 di-epoxy squalene may be used, such as a cucurbitadienol synthase (CDS). Cucurbitadienol synthase (E.C. 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.

[0483] Accordingly, in one embodiment, the recombinant cell according to the disclosure may comprise, may be capable of expressing or may 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 di-epoxy squalene, particularly wherein said polypeptide is a cucurbitadienol synthase (CDS). Non limiting examples of CDS polypeptides which can be used in the recombinant cell and the method according to the disclosure are provided in Table 5. Therefore, the CDS polypeptide may have an amino acid sequence according to SEQ ID NO: 52 herein or according to any sequence disclosed in Table 5, 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: 52 or of any polypeptide with amino acid sequence as indicated in Table 5.

[0484] Table 5 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.

[0485] Table 5

[0486] 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:

[0487] RH + 02 + H++ NAD(P)H -> ROH + H20 + NAD(P)+

[0488] 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.

[0489] 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);

[0490] 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.

[0491] 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).

[0492] In the method and the recombinant cell 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 C1 1 -hydroxylases. As used herein a C1 1 -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.

[0493] Accordingly, in one embodiment the recombinant according to the disclosure may comprise, may be capable to express or may 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 6. Therefore the CYP polypeptide may have an amino acid sequence according to any sequence disclosed in Table 6 or according to the amino acid sequences set out in SEQ ID NO: 53 to 55 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: 53 to 55 herewith or with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 6.

[0494] Table 6 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.

[0495] Table 6

[0496] In the method and recombinant cell 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 according to the disclosure may comprise, may be capable to express or may 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 cell and method according to the disclosure are provided in Table 7. Therefore the CPR polypeptide may have an amino acid sequence according to any sequence disclosed in Table 7 or according to the amino acid sequence set out in SEQ ID NO: 47, 51 or 105 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: 47, 51 or 105 herewith or the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 7.

[0497] Table 7 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.

[0498] Table 7

[0499] In the method and recombinant cell according to the disclosure epoxide hydrolase (EPH), may be used.

[0500] Epoxide hydrolases are enzymes of the family EC 3.3.x.x. which catalyze the hydrolysis of an epoxide yielding a glycol.

[0501] 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.

[0502] Accordingly, in one embodiment the recombinant cell according to the disclosure may comprise, may be capable to express or may express at least 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). Non limiting examples of EPH polypeptides which can be used in the recombinant cells and methods according to the disclosure are provided in Table 8. Therefore the EPH polypeptide may have an amino acid sequence according to SEQ ID NO: 56 herein or according to any sequence disclosed in Table 8, 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: 56 herewith or with an amino acid sequence according to any polypeptide with amino acid sequence as indicated in Table 8.

[0503] Table 8 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.

[0504] Table 8

[0505] 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 method and recombinant cell according to the disclosure, cytochrome b5 polypeptides (CB5) may be used.

[0506] Accordingly, in one embodiment according to the method or the recombinant cell of the disclosure, the recombinant cell capable of producing a mogroside and / or a precursor thereof may be capable of expressing or may 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 9. 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: 50 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 9 or according to SEQ ID NO: 50 herein.

[0507] Table 9 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.

[0508] Table 9

[0509] In the method and recombinant cell according to the disclosure, a recombinant cell capable of producing a mogroside or a precursor thereof may comprise, may be capable of expressing or may express one or more polynucleotides coding for a squalene epoxidase, a cucurbitadienol synthase a cytochrome P450 polypeptide, a C1 1 -hydroxylase a cytochrome P450 reductase, an epoxide hydrolase a cytochrome b5 polypeptide and or a UGT polypeptide.

[0510] Therefore, in one aspect of the present disclosure, it is herewith disclosed a recombinant cell capable of producing a mogroside and / or a precursor thereof which is capable of expressing or expresses one or more of the following polynucleotides:

[0511] (a) a polynucleotide encoding a squalene epoxidase polypeptide (SQE);

[0512] (b) a polynucleotide encoding a cucurbitadienol synthase polypeptide (CDS);

[0513] (c) a polynucleotide encoding a cytochrome P450 polypeptide (CYP); (d) a polynucleotide encoding a cytochrome P450 reductase polypeptide (CPR);

[0514] (e) a polynucleotide encoding an epoxide hydrolase (EPH);

[0515] (f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5);

[0516] (g) a polynucleotide encoding a UGT polypeptide.

[0517] 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.

[0518] Typically a recombinant cell 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),

[0519] (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),

[0520] (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).

[0521] Typically, a recombinant cell capable of producing a mogroside and / or a precursor thereof 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, 1 1 -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.

[0522] 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.

[0523] Therefore, in one embodiment of the method and recombinant cell according to the disclosure, the recombinant cell capable of producing a mogroside and / or a precursor thereof is capable of expressing or expresses a polynucleotide encoding a polypeptide capable of synthesizing Squalene from Farnesyl diphosphate (FPP), particularly wherein said polypeptide is a squalene synthase (E.C. 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 10. Therefore the SQS polypeptide may have an amino acid sequence according to any sequence disclosed in Table 10 or according to SEQ ID NO: 68 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: 68 herewith or with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 10.

[0524] Table 10 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.

[0525] Table 10

[0526] 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.

[0527] 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).

[0528] Therefore, in one embodiment according to the method or recombinant cell according to the disclosure, the recombinant cell capable of producing a mogroside and / or a precursor thereof is capable of expressing or 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 (E.C. 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 1 1 . Therefore the FPPS polypeptide may have an amino acid sequence according to the amino acid sequence set out in SEQ ID NO: 63 herewith or according to any sequence disclosed in Table 11 , 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: 63 herein or with the amino acid sequence of any polypeptide with amino acid sequence as indicated in Table 11.

[0529] Table 1 1 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.

[0530] Table 11

[0531] In one embodiment, the recombinant cell capable of producing a mogroside and a precursor thereof is capable of expressing or 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 (E.C. 2.5.1.1) (GPPS) and / or a farnesyl diphosphate synthase (E.C. 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 12. Therefore the GPPS polypeptide may have an amino acid sequence according to any sequence disclosed in Table 12, 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 12.

[0532] Table 12 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 12

[0533] Therefore, in one aspect of the method and recombinant cell according to the disclosure the recombinant cell capable of producing a mogroside and / or a precursor thereof is capable of expressing or expresses one or more of the following polynucleotides:

[0534] (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 (E.C. 2.5.1.1) (GPPS) and / or a farnesyl diphosphate synthase (E.C. 2.5.1 .10) (FPPS);

[0535] (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 (E.C. 2.5.1.10) (FPPS);

[0536] (c) a polynucleotide encoding a polypeptide capable of synthesizing Squalene from Farnesyl diphosphate (FPP), particularly wherein said polypeptide is a squalene synthase (E.C. 2.5.1.21) (SQS).

[0537] 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.

[0538] Typically, the cell may comprise, may be capable to express or may 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).

[0539] In some embodiments according to the disclosure the recombinant cell capable of producing a mogroside and / or a precursor thereof may comprise or (over)express one or more polynucleotides coding for polypeptides belonging to the mevalonate pathway.

[0540] 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.

[0541] 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 (E.C. 2.3.1.9) (AACT);

[0542] (b) the synthesis of Hydroxymethylglutaryl-Coenzyme A (HMGCoA) from Acetoacetyl- Coenzyme A (AACoA) catalyzed by an Hydroxymethylglutaryl-Coenzyme A synthase (E.C. 2.3.3.10) (HMGS);

[0543] (c) the synthesis of mevalonic acid (MVA) from Hydroxymethylglutaryl-Coenzyme A (HMGCoA) catalysed by a 3-hydroxy-3- methylglutaryl-coenzyme A reductase (E.C. 1.1.1.34) (HMGR);

[0544] (d) the synthesis of Mevalonate-5-phosphate (MVA-P) from Mevalonic acid (MVA) catalyzed by a mevalonate kinase (E.C. 2.7.1 .36) (MK);

[0545] (e) the synthesis of Mevalonate-5-diphosphate (MVA-PP) from Mevalonate-5- phosphate (MVA-P) catalyzed by a phosphomevalonate kinase (E.C. 2.7.4.2) (PMK);

[0546] (f) the synthesis of Isopentenyl diphosphate (IPP) from Mevalonate-5-diphosphate (MVA-PP), catalyzed by a diphosphomevalonate decarboxylase (E.C. 4.1.1.33) (MDD) and / or an isopentenyl / dimethylallyl diphosphate synthase (E.C. 1 .17.1 .2) (IPPS);

[0547] (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 (E.C. 5.3.3.2) (I PI) .

[0548] In one embodiment of the method and recombinant cell according to the disclosure, the recombinant cell capable of producing a mogroside and / or a precursor thereof may comprise, may be capable of (over)expressing, may (over)express one or more of the following polynucleotides:

[0549] (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) (E.C. 2.3.1 .9) (AACT);

[0550] (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 (E.C. 2.3.3.10) (HMGS);

[0551] (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 (E.C. 1.1.1.34) (HMGR);

[0552] (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 (E.C. 2.7.1.36) (MK);

[0553] (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 (E.C. 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 (E.C. 4.1.1.33) (MDD) and / or an isopentenyl / dimethylallyl diphosphate synthase (E.C. 1.17.1.2) (IPPS);

[0554] (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 (E.C. 5.3.3.2) (I PI).

[0555] 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 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).

[0556] 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.

[0557] 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: 64 or any HMG-CoA synthase polypeptide with 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 thereto.

[0558] 3-Hydroxy-3- methylglutaryl-coenzyme A reductase polypeptides suitable to be applied in the recombinant cell and method according to the disclosure are known to those skilled in the art. Suitable, non-limiting examples are SEQ ID NO: 69 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.

[0559] 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.

[0560] Non-limiting examples of phosphomevalonate kinase which can be used in the method and recombinant cell 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.

[0561] Non limiting examples of diphosphomevalonate decarboxylase which can be used in the method and recombinant cell 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.

[0562] 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.

[0563] In one embodiment of the method and recombinant cell 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 a mogroside and / or a precursor thereof may comprise a deficiency in the production of a lanosterol synthase polypeptide.

[0564] 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.

[0565] In one embodiment the recombinant cell capable of producing a mogroside and / or a precursor thereof as disclosed herewith may be capable of producing one or more mogrosides selected from Mogroside IA, Mogroside IEi, Mogroside HA, 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 Siamenoside I. Typically, the recombinant cell may comprise, may be capable of (over)expressing or may (over)express one or more of:

[0566] (a) a polynucleotide encoding a squalene epoxidase polypeptide (SQE);

[0567] (b) a polynucleotide encoding a cucurbitadienol synthase polypeptide (CDS);

[0568] (c) a polynucleotide encoding a cytochrome P450 polypeptide (CYP), such as a C11 - hydroxylase polypeptide and / or an epoxidase;

[0569] (d) a polynucleotide encoding a cytochrome P450 reductase polypeptide (CPR);

[0570] (e) a polynucleotide encoding an epoxide hydrolase (EPH)

[0571] (f) a polynucleotide encoding for a cytochrome b5 polypeptide (CB5).

[0572] (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 C24-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 C3-O-glucose) of a mogroside compound;

[0573] Hi. a UGT 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, and optionally, one or more of the following

[0574] A. a geranyl diphosphate synthase (GPPS) and / or a farnesyl diphosphate synthase (FPPS);

[0575] B. a farnesyl diphosphate synthase (FPPS);

[0576] C. a squalene synthase (SQS);

[0577] D. an acetyl-CoA acetyltransferase (AACT);

[0578] E. an Hydroxymethylglutaryl-Coenzyme A synthase (HMGS);

[0579] F. a 3-hydroxy-3- methylglutaryl-coenzyme A reductase (HMGR);

[0580] G. a mevalonate kinase (MK);

[0581] H. a phosphomevalonate kinase (PMK);

[0582] I. a diphosphomevalonate decarboxylase (MDD) and / or an isopentenyl / dimethylallyl diphosphate synthase (IPPS);

[0583] J. an isopentenyl-diphosphate delta-isomerase (I PI).

[0584] Typically, with reference to the above, the polypeptide and / or the polynucleotide encoding said polypeptide may be a heterologous enzyme and / or a heterologous polynucleotide. The enzyme and / or the polynucleotide may be overexpressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced.

[0585] Optionally, the recombinant cell capable of producing a glycosyl acceptor and / or precursor thereof, e.g. a terpenoid and / or a precursor thereof such as a mogroside and / or a precursor thereof, as disclosed herewith may further comprise, may further be capable of (over)expressing or may further (over)express a transporter capable of facilitating the transport of said glycosyl acceptor and / or precursor thereof (e.g. the mogroside and / or precursor thereof) outside the recombinant cell. Any transporter known in the art may be used in the context of this disclosure.

[0586] Intracellular (in vivo) and extracellular glycosylation One aspect of the disclosure provides herein a method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor comprising contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with a recombinant cell comprising, capable of (over)expressing or (over)expressing a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze the formation of a glycosidic bond between the transferred glycosyl group and the glycosyl acceptor; thereby, producing a modified glycosyl acceptor. Suitable enzymes for the transfer of a glycosyl group from a glycosyl donor to a glycosyl acceptor which can be used in said method according to the disclosure have been described herein above

[0587] The glycosidic bond may be an a-glycosidic bond. The glycosyl acceptor may be a (glycosylated) terpenoid, such as a mogroside. The modified glycosyl acceptor (e.g. modified (glycosylated) terpenoid) may be an a-glycosylated glycosyl acceptor (e.g. a-glycosylated terpenoid, such as a-glycosylated mogroside).

[0588] In one specific example, the method may comprise contacting sucrose (glycosyl donor) and Mogroside HIE (glycosyl acceptor) with a recombinant cell comprising at least one heterologous dextransucrase according to the disclosure under suitable conditions, thereby producing Compound (I). Mogroside HIE is converted into Compound (I) with a dextransucrase (Enzyme) according to the following glycosylation reaction:

[0589] The glycosylation reaction may take place inside the recombinant cell ( / n vivo) and / or outside the recombinant cell.

[0590] In one embodiment, the glycosylation reaction takes place inside the recombinant cell. In said embodiment, the at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group to the glycosyl acceptor is an intracellular enzyme. More in particular, the recombinant cell comprises, is capable of (over)expressing or (over)expresses said enzyme intracellularly and said enzyme is active inside the recombinant cell. In said embodiment, the recombinant cell may further be capable of producing or may produce the glycosyl donor (e.g. sucrose) such as described herein above. Alternatively, or additionally, the glycosyl donor (e.g. sucrose) may be provided (fed) to the recombinant cell extracellularly. When the glycosyl donor is provided (fed) extracellularly, the recombinant cell may further comprise or (over)express a transporter capable of facilitating the transport of the glycosyl donor (e.g. sucrose) inside the recombinant cell as described herein above. In said embodiment, the recombinant cell may further be capable of producing or may produce the glycosyl acceptor and / or precursor thereof (e.g. mogroside such as Mogroside HIE) such as described herein above. Alternatively, the glycosyl acceptor and / or precursor thereof (e.g. mogroside such as Mogroside HIE) may be provided (fed) extracellularly. When the glycosyl acceptor and / or precursor thereof is provided extracellularly, the recombinant cell may further comprise a transport system and / or an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport of the glycosyl acceptor and / or precursor thereof inside the cell.

[0591] In one embodiment, the glycosylation reaction takes place outside the recombinant cell. In said embodiment, the at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group to the glycosyl acceptor is an extracellular enzyme. More in particular, according to said embodiment the recombinant cell comprises, is capable of (over)expressing or (over)expresses said enzyme extracellularly and said enzyme is active outside the recombinant cell upon expression and secretion in the extracellular environment. The extracellular enzyme may be secreted free outside the recombinant cell and / or displayed at the surface of the recombinant cell as described herein above. In said embodiment, the recombinant cell may further be capable of producing or may produce the glycosyl donor (e.g. sucrose) such as described herein above. Alternatively, or additionally, the glycosyl donor (e.g. sucrose) may be provided (fed) to the recombinant cell extracellularly. When the recombinant cell produces the glycosyl donor, the recombinant cell may further express a transporter capable of facilitating the transport of the glycosyl donor (e.g. sucrose) outside the recombinant cell as described herein above. In said embodiment, the recombinant cell may further be capable of producing or may produce the glycosyl acceptor and / or precursor thereof (e.g. mogroside such as Mogroside HIE) such as described herein above. Alternatively, the glycosyl acceptor and / or precursor thereof (e.g. mogroside such as Mogroside HIE) may be provided (fed) extracellularly. When the recombinant cell produces the glycosyl acceptor and / or precursor thereof, the recombinant cell may further express a transporter capable of facilitating the transport of the glycosyl acceptor and / or precursor thereof outside the recombinant cell as described herein above. In said embodiment, the at least one heterologous enzyme expressed by the recombinant cell of the disclosure may be further supplemented extracellularly with one or more additional enzymes capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor. Examples of said additional enzymes (e.g. a CGTase) are described herein above.

[0592] Production methods Another aspect of the disclosure provides herein a method for producing a modified glycosyl acceptor comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising, capable of (over)expressing, or (over)expressing a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions comprises culturing the recombinant cell in a suitable culture medium in the presence of the glycosyl donor and the glycosyl acceptor, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyzes the formation of a glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby, producing the modified glycosyl acceptor,; and optionally, isolating the modified glycosyl acceptor produced therefrom. Optionally said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme,

[0593] Another aspect of the disclosure provides herein a method for producing a modified glycosyl acceptor comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising, capable of (over)expressing or (over)expressing a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions comprises contacting the recombinant cell, the glycosyl donor and the glycosyl acceptor in a reaction mixture, wherein said contacting under suitable conditions allow the at least one heterologous enzyme to catalyze the formation of a glycosidic bond between the transferred glycosyl group and the glycosyl acceptor; thereby, producing a modified glycosyl acceptor, and optionally, isolating the modified glycosyl acceptor produced therefrom. Optionally said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme,

[0594] The glycosidic bond is preferably an a-glycosidic bond and the modified glycosyl acceptor (e.g. modified terpenoid) is preferably an a-glycosylated glycosyl acceptor (e.g. a-glycosylated terpenoid).

[0595] The method for producing a modified glycosyl acceptor may be a fermentation, a biotransformation or a combination thereof.

[0596] The term “fermentation” as used herein refers to a method wherein a cell is used to convert a carbon source (e.g. glucose) into a desired compound (e.g. a modified glycosyl acceptor). The carbon source also serves as the substrate that the cell uses for growth. In the context of the disclosure, a “fermentation” specifically refers to an enzymatic modification of a glycosyl acceptor in the presence of a cell and a glycosyl donor, wherein both the glycosyl donor and the glycosyl acceptor do not need to be provided to the cell. In other words, the cell is able to produce the modified glycosyl acceptor solely from the carbon source used for cell growth (e.g. glucose). As would be understood by one of skill in the art, the “fermentation” comprises growing a cell in a culture medium.

[0597] The term “biotransformation” as used herein refers to a method wherein a compound (e.g. a glycosyl acceptor) is modified by a cell; thereby producing a modified compound (e.g. a modified glycosyl acceptor). In the context of the disclosure, a “biotransformation” specifically refers to an enzymatic modification of a glycosyl acceptor in the presence of a cell and a glycosyl donor, wherein the glycosyl donor and / or the glycosyl acceptor must be provided to the cell extracellularly for the enzymatic modification to proceed. Said “biotransformation” is typically performed in a reaction mixture and the cell is typically a non-proliferating cell.

[0598] The term “fermentation combined with biotransformation” as used herein refers to the combination of both above methods. In the context of the disclosure, a “fermentation combined with biotransformation” specifically refers to an enzymatic modification of a glycosyl acceptor in the presence of a cell and a glycosyl donor, wherein the method comprises growing the cell in a culture medium comprising a carbon source for growth such as glucose (fermentation) and wherein the glycosyl donor and / or the glycosyl acceptor must still be provided to the cell extracellularly for the enzymatic modification to proceed (biotransformation). In this case, said “fermentation combined with biotransformation” is typically performed in a culture medium that is used for cell growth.

[0599] For the sake of clarity, in the methods described in the present disclosure, the recombinant cell is a whole cell or a living cell. Depending on the method, the living cell may be a proliferating cell or a non-proliferating cell (e.g. a resting cell). The living cell may be an intact cell or a permeabilized cell.

[0600] In said production methods as further described herein below (i.e. fermentation, fermentation combined with biotransformation or biotransformation), the recombinant cell comprises, is able to express or expresses the at least one heterologous enzyme capable of transferring the transfer of the glycosyl group to glycosyl acceptor and said enzyme may be an intracellular enzyme and / or an extracellular enzyme.

[0601] The inventors have surprisingly shown that a recombinant cell expressing and secreting the at least one heterologous enzyme extracellularly also enables an efficient and robust process for the production of a modified glycosyl acceptor (e.g. a a-glycosylated mogroside such as Compound (I)).

[0602] In particular, when the production method involves a fermentation, the inventors have surprisingly found that a recombinant cell expressing the heterologous enzyme extracellularly is able to produce a glycosylated glycosyl acceptor in the culture medium. In other words, the heterologous enzyme was surprisingly found to be functionally expressed and secreted extracellularly, and even more surprisingly active in the culture medium.

[0603] Accordingly, a production (fermentation) method wherein the at least one heterologous enzyme is an extracellular enzyme, is also considered of relevance for the industrial production of modified glycosyl acceptors. Furthermore, compared to a method wherein the at least one heterologous enzyme is expressed intracellularly (resulting in intracellular production of the modified glycosyl acceptor), said production method offers the following benefits including, but not limited to, ease of harvesting of the product, reduced downstream processing, scalability, reduced cellular stress, continuous production; these with associated cost savings and operational efficiency. Furthermore, when the at least heterologous enzyme is displayed (anchored) at the external surface of the recombinant cell, said method offers further benefits including, but not limited to, enhanced enzyme stability, facilitated recovery of enzyme (e.g. harvest of the whole cell), cell recycling; these with associated cost saving and operational efficiency as well.

[0604] Fermentation

[0605] The inventors have surprisingly found that a recombinant cell of the disclosure (over)expressing a sucrose biosynthetic pathway produces unexpected titers of sucrose, ranging in the order of grams / liter such as disclosed herein above. It was also surprisingly found that, when said recombinant cell producing sucrose further produces a glycosyl acceptor and / or a precursor thereof, said cell is capable of producing a modified glycosyl acceptor from a simple carbon source, such as glucose. In particular, the recombinant cell producing both sucrose and Mogroside HIE and / or a precursor thereof is capable of producing Compound (I) from glucose.

[0606] Accordingly, the embodiments as described herein below alleviate or even completely eliminate the need for providing (feeding) the recombinant cell with extracellular sources of glycosyl donor and glycosyl acceptor and / or precursor thereof. In other words, said embodiments enable a pure fermentation. Accordingly, the method may be performed by fermentation using any commodity and simple carbon sources such as glucose. Advantages include, but are not limited to, reduction of costs linked to sucrose and its transportation, reduction of costs linked to equipment (e.g. feeding lines, storage vessels) and decreased complexity of the production process.

[0607] In one embodiment, wherein the production method may be solely a fermentation method, the recombinant cell may express the at least one heterologous enzyme of the present disclosure and said enzyme may be an intracellular enzyme (i.e. active inside the recombinant cell, e.g. in the cytosol, upon expression). In other words, the glycosylation reaction may take place intracellularly ( / n vivo). In addition to expressing the at least one heterologous enzyme of the disclosure, the recombinant cell may further produce the glycosyl donor and the glycosyl acceptor and / or precursor thereof. Specifically, the recombinant cell may (over)express a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1 .5) intracellularly and may further produce sucrose and a mogroside and / or a precursor thereof such as described herein above. Preferably, the recombinant cell further produces Mogroside HIE and / or a precursor thereof. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1.26) and / or (over)expresses a p-glucosidase, and / or (over)expresses an a-glucosidase and / or may be deficient in a p-glucanase such as disclosed herein above.

[0608] In another embodiment, wherein the production method may be solely a fermentation method, the recombinant cell may express the at least one heterologous enzyme of the present disclosure and said enzyme may be an extracellular enzyme secreted outside the recombinant cell and / or displayed at the surface of the recombinant cell. In other words, the glycosylation reaction takes place extracellularly. In addition to expressing the at least one heterologous enzyme of the disclosure, the recombinant cell further produces the glycosyl donor and the glycosyl acceptor and / or a precursor thereof. Specifically, the recombinant cell may (over)express a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1 .5) extracellularly and further may produce sucrose and a mogroside and / or precursor thereof such as described herein above. Preferably, the recombinant cell further produces Mogroside HIE and / or a precursor thereof. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1.26) and / or (over)expresses a p-glucosidase, and / or (over)expresses an a-glucosidase and / or is deficient in a p-glucanase such as disclosed herein above.

[0609] Fermentation in combination with biotransformation

[0610] In further embodiments, the recombinant cell must be supplemented with an external source of glycosyl donor and / or an external source of glycosyl acceptor and / or precursor thereof. In these embodiments, such as the one described herein below, the method comprises a fermentation combined with a biotransformation.

[0611] In one embodiment, wherein the production method may be a fermentation combined with a biotransformation method, the recombinant cell is cultured in a suitable culture medium and expresses the at least one heterologous enzyme of the present disclosure intracellularly. Said enzyme is an intracellular enzyme (i.e. active inside the recombinant cell, e.g. in the cytosol, upon expression). In other words, the glycosylation reaction takes place intracellularly ( / n vivo).

[0612] In one specific embodiment, wherein the recombinant cell does not produce the glycosyl donor nor the glycosyl acceptor and / or a precursor thereof, the recombinant cell is provided (fed) extracellularly both with a glycosyl donor and a glycosyl acceptor and / or a precursor thereof. In one example, the recombinant cell (over)expresses a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1.5) intracellularly and the recombinant cell is provided (fed) extracellularly with sucrose and a mogroside and / or a precursor thereof (e.g. Mogroside HIE and / or a precursor thereof). In said embodiment, the recombinant cell may further comprise, be capable of expressing, or expresses one or more transporters capable of facilitating the transport of said compounds inside the recombinant cell such as described herein above. Alternatively, or additionally, the recombinant cell may further comprise an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport of said compounds inside the cell. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1.26) and / or a p-glucanase and / or (over)expresses a p-glucosidase, and / or (over)expresses an a-glucosidase such as disclosed herein above.

[0613] In one specific embodiment, wherein the recombinant cell produces the glycosyl donor but does not produce the glycosyl acceptor and / or a precursor thereof, the recombinant cell is provided (fed) extracellularly with the glycosyl acceptor and / or a precursor thereof. For example, the recombinant cell (over)expresses a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1.5) intracellularly and the recombinant cell is provided (fed) extracellularly with a mogroside and / or a precursor thereof (e.g. Mogroside HIE and / or a precursor thereof). In said embodiment, the recombinant cell may further comprise, be capable of expressing, or expresses one or more transporters capable of facilitating the transport of said compounds (e.g. a mogroside and / or a precursor thereof) inside the recombinant cell. Alternatively, or additionally, the recombinant cell may further comprise an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport of said compounds inside the cell. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1 .26) and or a p-glucanase and / or (over)expresses a p- glucosidase, and / or (over)expresses an a-glucosidase such as disclosed herein above.

[0614] In one specific embodiment, wherein the recombinant cell produces the glycosyl acceptor and / or precursor thereof but does not produce the glycosyl acceptor, the recombinant cell is provided (fed) extracellularly with the glycosyl donor. For example, the recombinant cell (over)expresses a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1.5) intracellularly and the recombinant cell is provided (fed) extracellularly with sucrose. In said embodiment, the recombinant cell may further comprise, be capable of expressing, or expresses one or more transporters capable of facilitating the transport of said compound (e.g., sucrose) inside the recombinant cell. Alternatively, or additionally, the recombinant cell may further comprise an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport of said compound inside the cell. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1.26) and / or (over)expresses a p-glucosidase, and / or (over)expresses an a-glucosidase such as disclosed herein above.

[0615] In one embodiment, wherein the production method may be a fermentation combined with a biotransformation method, the recombinant cell is cultured in a suitable culture medium and expresses the at least one heterologous enzyme of the present disclosure extracellularly. Said enzyme is an extracellular enzyme free outside the recombinant cell and / or displayed at the surface of the recombinant cell. In other words, the glycosylation reaction takes place extracellularly.

[0616] In one specific embodiment, wherein the recombinant cell does not produce the glycosyl donor nor the glycosyl acceptor and / or a precursor thereof, the recombinant cell is provided (fed) extracellularly both with a glycosyl donor and a glycosyl acceptor and / or a precursor thereof. In one example, the recombinant cell (over)expresses a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1.5) extracellularly and the recombinant cell is provided (fed) extracellularly with sucrose and a mogroside and / or a precursor thereof (e.g., Mogroside HIE and / or a precursor thereof). Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1.26) and / or a p- glucanase, and / or (over)expresses a p-glucosidase, and / or (over)expresses an a- glucosidase such as disclosed herein above.

[0617] In one specific embodiment, wherein the recombinant cell produces the glycosyl donor but does not produce the glycosyl acceptor and / or a precursor thereof, the recombinant cell is provided (fed) extracellularly with the glycosyl acceptor and / or a precursor thereof. For example, the recombinant cell (over)expresses and secretes a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1.5) extracellularly and the recombinant cell is provided (fed) extracellularly with a mogroside and / or a precursor thereof (e.g. Mogroside HIE and / or a precursor thereof). In said embodiment, the recombinant cell may further comprise, be capable of expressing, or expresses one or more transporters capable of facilitating the transport of the produced glycosyl donor (e.g. sucrose) outside the recombinant cell. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1 .26) and / or (over)expresses a p-glucosidase, and / or (over)expresses an a- glucosidase such as disclosed herein above.

[0618] In one specific embodiment, wherein the recombinant cell produces the glycosyl acceptor and / or precursor thereof but does not produce the glycosyl acceptor, the recombinant cell is provided (fed) extracellularly with the glycosyl donor. For example, the recombinant cell (over)expresses a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1.5) extracellularly and the recombinant cell is provided (fed) extracellularly with sucrose. In said embodiment, the recombinant cell may further comprise, be capable of expressing, or expresses one or more transporters capable of facilitating the transport of the produced glycosyl acceptor and / or a precursor thereof (e.g., the mogroside and / or a precursor thereof such as Mogroside HIE and / or a precursor thereof) outside the recombinant cell. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1.26) and / or (over)expresses a p-glucosidase, and / or (over)expresses an a-glucosidase such as disclosed herein above.

[0619] Biotransform ation

[0620] In the context wherein the production method may be solely a biotransformation method, the recombinant cell comprises, is capable of (over)expressing or (over)expresses the at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group to the glycosyl acceptor, and said recombinant cell is used as a whole cell biocatalyst in a reaction mixture under suitable conditions for the transfer to occur. In particular, the method comprises contacting the recombinant cell expressing at least one heterologous enzyme of the disclosure with a glycosyl donor and the glycosyl acceptor and / or precursor thereof in a reaction mixture under suitable conditions for the transfer of the glycosyl group to the glycosyl acceptor to occur. More in particular, the at least one heterologous enzyme is intracellular and / or extracellular displayed at the surface of the recombinant cell.

[0621] The components of the reaction mixture may include, but are not limited to, the recombinant cell comprising the at least one heterologous enzyme of the disclosure, the glycosyl donor (e.g. sucrose), the glycosyl acceptor (e.g. a terpenoid and / or a precursor thereof), a buffer solution conducive to the enzymatic activity, salts and ions. The components of the reaction mixture (e.g buffer, salts, ions) and the conditions may be selected and optimized through routine experimentation as would be understood by one of ordinary skill in the art.

[0622] In one embodiment, the recombinant cell comprises the at least one heterologous enzyme of the present disclosure and said enzyme is an intracellular enzyme (i.e. active inside the recombinant cell). In other words, the glycosylation reaction takes place intracellularly ( / n vivo). In said embodiment, the recombinant cell is provided (fed) extracellularly with a glycosyl donor and / or a glycosyl acceptor and / or a precursor thereof. In one example, the recombinant cell comprises a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1 .5) intracellularly and the recombinant cell is provided (fed) extracellularly with sucrose and a mogroside and / or a precursor thereof (e.g. Mogroside HIE and / or a precursor thereof). In said embodiment, the recombinant cell may further comprise one or more transporters capable of facilitating the transport of said compounds inside the recombinant cell such as described herein above. Alternatively, or additionally, the recombinant cell may further comprise an alteration of the cell membrane (e.g. permeabilization) capable of facilitating the transport said compounds inside the cell. Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1.26) and / or (over)expresses a p-glucosidase, and / or (over)expresses an a-glucosidase such as disclosed herein above.

[0623] In one preferred embodiment, the recombinant cell comprises the at least one heterologous enzyme of the present disclosure and said enzyme is an extracellular enzyme displayed at the surface of the recombinant cell (i.e. active outside the recombinant cell). In other words, the glycosylation reaction takes place extracellularly. In said embodiment, the recombinant cell is provided (fed) extracellularly with a glycosyl donor and / or a glycosyl acceptor and / or a precursor thereof. In one example, the recombinant cell comprises a heterologous glucansucrase such as a dextransucrase (E.C. 2.4.1.5) extracellularly displayed at the surface of the recombinant cell and the recombinant cell is provided (fed) extracellularly with sucrose and a mogroside and / or a precursor thereof (e.g. Mogroside HIE and / or a precursor thereof). Said recombinant cell may further be deficient in an invertase (E.C. 3.2.1 .26) and / or (over)expresses a p-glucosidase, and / or (over)expresses an a-glucosidase such as disclosed herein above. In the above embodiments, prior to the actual biotransformation (i.e. contacting step in the reaction mixture), the recombinant cell may be prepared by culturing it in a suitable culture medium, under conditions in which the at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group to the glycosyl acceptor is expressed. After a suitable culturing time, the recombinant cell is harvested from the culture medium, optionally washed with water or a buffer solution, and suspended in a desired buffer conducive to the activity of the heterologous enzyme. The biotransformation is then initiated by contacting the recombinant cell with the glycosyl donor, the glycosyl acceptor and / or precursor thereof.

[0624] In all the above embodiments of the production method (i.e. fermentation, fermentation combined with biotransformation and biotransformation), wherein a recombinant cell comprises, is capable of (over)expressing or (over)expresses a heterologous enzyme of the disclosure, produces a glycosyl acceptor and / or a precursor thereof, or produces a glycosyl donor, said recombinant cell may be further supplemented extracellularly with one or more additional enzymes capable of catalyzing the transfer of a glycosyl group to a glycosyl acceptor, with one or more additional glycosyl acceptors and / or precursors thereof, or with one or more additional glycosyl donors, respectively. The supplemented enzyme(s), glycosyl acceptor(s) and / or precursor(s) thereof, and / or glycol donor(s) may be the same and / or may be different from the one(s) produced by the recombinant cell. In the specific cases, wherein the at least one heterologous enzyme is an intracellular enzyme, the recombinant cell may further comprise, may be capable of expressing, or may express one or more transporters capable of facilitating the transport of said compounds / enzymes inside the recombinant cell. Alternatively, or additionally, the recombinant cell may further comprise an alteration of the cell membrane (e.g., permeabilization) capable of facilitating the transport of said compounds / enzymes inside the cell.

[0625] Typically, in a method of the disclosure, the recombinant cell is grown in a bioreactor or a fermenter at a defined temperature(s), pH and for a desired period of time, typically under conditions in which the genes involved in the production of the modified glycosyl acceptor as disclosed herewith are expressed. The recombinant cell may be grown under different operation modes such batch, (repeated) fed batch, or continuous process.

[0626] Culture media used for various recombinant cells are well known in the art. Culture media used to culture recombinant 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.

[0627] Any of the recombinant cells described in this disclosure 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.

[0628] In a method of the disclosure, wherein the recombinant cell is provided (fed) with a glycosyl donor extracellularly and / or wherein the recombinant cell is further supplemented with one or more additional glycosyl donors extracellularly, the source of the glycosyl donor may vary. For example, the glycosyl donor may be sourced from a plant. In the case of sucrose as additional glycosyl donor, the sucrose may be sourced for example from sugar cane and / or sugar beet. Accordingly, the one or more additional glycosyl donors in the present disclosure may be a plant extract or a purified form thereof. Alternatively, or additionally, in the context of the disclosure, the one or more additional glycosyl donors may be produced and secreted extracellularly by a cell other than the recombinant cell of the disclosure. In the context of the present invention, the one or more additional glycosyl donors may be or may comprise a non-activated disaccharide and / or a non-activated polysaccharide, such as sucrose or (soluble) starch.

[0629] In a method of the disclosure, wherein the glycosyl acceptor and / or precursor thereof is provided (fed) to the recombinant cell extracellularly and / or the one or more glycosyl acceptors are supplemented to the recombinant cell extracellularly, the source of the glycosyl acceptor and / or precursor thereof may vary. For example, the glycosyl acceptor and / or precursor thereof may be sourced from a plant. In the case of a mogroside and / or precursor thereof, said mogroside and / or precursor thereof may be sourced for example from monk fruit. Accordingly, said glycosyl acceptor and / or precursor thereof may be a plant (e.g. monk fruit) extract or a purified from thereof. Alternatively, or additionally, in the context of the disclosure, the glycosyl acceptor and / or precursor thereof may be produced and secreted extracellularly by a cell other than the recombinant cell of the disclosure. Among others, the one or more glycosyl acceptors may be or may comprise morgrosides (e.g. Mogroside HIE), terpenoids other than mogrosides (e.g. steviol glycosides) and / or flavonoids.

[0630] In a method of the disclosure, wherein the recombinant cell is further supplemented extracellularly with one or more additional enzymes capable of catalyzing the transfer of a glycosyl group to the glycosyl acceptor, the source of said enzyme may vary. For example, said enzyme may be pure enzyme, a cell (free) lysate comprising said enzyme, a purified form therefrom and / or an enzyme produced by a cell other than the recombinant cell. In the context of the present invention, the one or more additional enzymes capable of catalyzing the transfer of a glycosyl group to the glycosyl acceptor may be or may comprise an enzyme belonging to glycoside hydrolase family 70 (GH70), glycoside hydrolase family 13 (GH13) and / or glycoside hydrolase family 77 (GH77); for example, a cyclomaltodextrin glucanotransferase (E.C. 2.4.1 .19), a glucansucrase such as an alternansucrase (E.C. 2.4.1.140), a dextransucrase (E.C. 2.4.1.5), a mutansucrase (E.C.

[0631] 2.4.1.5) and a reuteransucrase (EC 2.4.1.5).

[0632] In a method involving fermentation (e.g. fermentation alone or in combination with biotransformation as disclosed herein above) wherein the recombinant cell is provided (fed) or supplemented extracellularly with the glycosyl donor (e.g. sucrose) and / or the glycosyl acceptor and / or a precursor thereof (e.g. mogroside and / or a precursor thereof), the feeding of said compounds may start at the moment the recombinant cell is inoculated in the culture medium. Alternatively, the feeding of said compounds may start during cell growth or after cell growth; i.e. after the inoculation of the recombinant cell in the culture medium. Accordingly, said compounds may be fed 1 hour before inoculation, during inoculation, or after 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h following the inoculation of the recombinant cell in the culture medium. The feeding may be performed till end of fermentation and / or till the desired (level of) modified glycosyl acceptor is obtained.

[0633] In a further method involving fermentation (e.g. fermentation alone or in combination with biotransformation as disclosed herein above), wherein the recombinant cell is provided (fed) or supplemented extracellularly with the glycosyl donor (e.g. sucrose) and the recombinant cell further produces the glycosyl acceptor and / or precursor thereof, the extracellular feeding of the glycosyl donor (e.g. sucrose) may start before, when or after the start of the production of the glycosyl acceptor and / or precursor thereof (i.e. after the start of the expression of the glycosyl acceptor biosynthetic pathway as described herein above). Accordingly, the extracellular feeding of the glycosyl donor may start before or after 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h following the start of the production of the glycosyl acceptor and / or precursor thereof by the recombinant cell (i.e. following the start of the expression of the glycosyl acceptor biosynthetic pathway). Preferably, the extracellular feeding of the glycosyl donor starts after the start of the production of the glycosyl acceptor and / or precursor thereof; preferably after 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h. The feeding may be performed till end of fermentation and / or till the desired (level of) modified glycosyl acceptor is obtained.

[0634] In one example, wherein the recombinant cell expresses a heterologous glucansucrase (e.g. a dextransucrase) intracellularly and produces the glycosyl acceptor and / or precursor thereof, the extracellular feeding of sucrose may start before, when or after the start of the production of the glycosyl acceptor and / or precursor thereof (i.e. after the start of the expression of the glycosyl acceptor biosynthetic pathway as described herein above). Accordingly, the extracellular feeding of sucrose may start before or after 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h following the start of the production of the glycosyl acceptor and / or precursor thereof by the recombinant cell (i.e. following the start of the expression of the glycosyl acceptor biosynthetic pathway). Preferably, the extracellular feeding of sucrose starts after the start of the production of the glycosyl acceptor and / or precursor thereof; preferably after 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h. The feeding may be performed till end of fermentation and / or till the desired (level of) modified glycosyl acceptor is obtained.

[0635] In a further example, wherein the recombinant cell expresses a heterologous glucansucrase (e.g. a dextransucrase) extracellularly (free outside the recombinant cell and / or displayed at the surface of the recombinant cell) and produces the glycosyl acceptor and / or precursor thereof, the extracellular feeding of sucrose may start before, when or after the start of the production of the glycosyl acceptor and / or precursor thereof (i.e. after the start of the expression of the glycosyl acceptor biosynthetic pathway as described herein above). Accordingly, the extracellular feeding of sucrose may start before or after 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h following the start of the production of the glycosyl acceptor and / or precursor thereof by the recombinant cell (i.e. following the start of the expression of the glycosyl acceptor biosynthetic pathway). Preferably, the extracellular feeding of sucrose starts after the start of the production of the glycosyl acceptor and / or precursor thereof; preferably after 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h. The feeding may be performed till end of fermentation and / or till the desired (level of) modified glycosyl acceptor is obtained.

[0636] In the above embodiments, the inventors have surprisingly found that the delayed start of sucrose feed (relative to the start of the production of the glycosyl acceptor and / or precursor thereof) increases the yield of modified glycosyl acceptor production on consumed glycosyl donor.

[0637] The feeding strategy (e.g., amount of glycosyl donor, feeding period) may be further optimized through routine experimentation as would be understood by one of ordinary skill in the art. For example, when sucrose is fed or supplemented to a recombinant cell of the disclosure extracellularly, it may be maintained at a concentration between 0.5 g / l and 50 g / l in the culture medium or the fermentation broth, preferably between 1 g / l - 40 g / l, 1 g / l - 35 g / l, 1 g / l - 30 g / l, 1 g / l - 25 g / l, 1— g / l - 20 g / l, 1 g / l - 15 g / l, 1 g / l - 10 g / l, 1 g / l - 7.5 g / l, 1 g / l - 5 g / l.

[0638] Isolation and purification

[0639] In the methods according to the disclosure, the modified glycosyl acceptors (e.g. a modified mogroside compounds such as Compound (I)) can be optionally isolated or recovered from the fermentation broth or the reaction mixture, and optionally further purified using various techniques known to those skilled in the art. For example, following fermentation, a fermentation 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 (modified) glycosyl acceptors, e.g. (modified) mogrosides, 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, (modified) mogrosides 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.

[0640] The disclosure further provides a recombinant cell as disclosed herein above in a method of the disclosure. In particular, the recombinant cell comprises at least one heterologous polynucleotide encoding at least one enzyme capable of catalyzing an a-glycosidic bond (e.g. a glucansucrase such as a dextransucrase) between a glycosyl group from a glycosyl donor (e.g. sucrose) and a glycosyl acceptor (e.g. a terpenoid such as a mogroside), wherein the cell is deficient in an enzyme capable of hydrolyzing the glycosyl donor (e.g. the cell is deficient in an invertase) and, optionally comprises at least one polynucleotide sequence encoding an a- glycosidase and / or at least one polynucleotide encoding a p-glucosidase. The enzyme and / or the polynucleotide encoding said a-glycosidase or said p-glucosidase may be a heterologous enzyme and / or a heterologous polynucleotide. The enzyme and / or the polynucleotide may be (over)expressed. Typically, the polynucleotide may be constitutively expressed. In another aspect, the expression of the polynucleotide may be induced.

[0641] The disclosure further provides a reaction mixture comprising the recombinant cell as disclosed herein above.

[0642] The disclosure further provides a culture medium comprising the recombinant cell as disclosed herein above.

[0643] The disclosure further provides a fermentation broth comprising the recombinant cell as disclosed herein above.

[0644] The disclosure further provides a composition, e.g. a terpenoid composition such as a mogroside composition, obtained or obtainable by a method as disclosed herein above. In particular, the disclosure further provides a mogroside composition or sweetener composition obtained or obtainable by a method of the disclosure. Preferably, said mogroside or sweetener composition comprises an a-glycosylated mogroside. More preferably, said mogroside or sweetener composition comprises an a-glycosylated Mogroside HIE. Even more preferably, said mogroside or sweetener composition comprises Compound (I). Furthermore, a food product, a beverage, a pet-food, a feed, an oral, a bioactive or a pharmaceutical composition comprising said mogroside or sweetener composition are also provided.

[0645] Further provided is a mogroside obtainable or obtained by a method as disclosed herein. The mogroside or composition thereof, may be used in any application known for such compounds. In particular, the mogroside or composition thereof may for instance be used as a sweetener, such as in a food or a beverage. For example, the mogroside or composition thereof 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, the mogroside or composition thereof 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 or composition thereof. 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.

[0646] The mogroside or composition thereof obtainable or 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.

[0647] The mogroside or composition thereof obtainable or obtained according to a 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.

[0648] The mogroside or composition thereof 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.

[0649] The mogroside or composition thereof 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 stanol (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 as disclosed herein above 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.

[0650] A mogroside or composition thereof 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.

[0651] In addition, a mogroside or composition thereof 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.

[0652] 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.

[0653] 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.

[0654] 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.

[0655] 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.

[0656] Thus, the sweetened 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.

[0657] In solid form, a mogroside or composition thereof 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.

[0658] In liquid form, a mogroside or composition thereof 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.

[0659] The composition may include various bulking agents, functional ingredients, colorants, and / or flavors.

[0660] The disclosure further provides a method of using the recombinant cell as disclosed herein above for producing a modified glycosyl acceptor, e.g. a modified terpenoid such as a modified mogroside. In particular, the disclosure further provides a method of using the recombinant cell as disclosed herein above for producing an a-glycosylated glycosyl acceptor, e.g. an a-glycosylated terpenoid such as an a-glycosylated mogroside. More in particular, the disclosure further provides a method of using the recombinant cell as disclosed herein above for producing an a-glycosylated Mogroside HIE, such as Compound (I).

[0661] 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.

[0662] Hereafter follows a list of embodiments according to the disclosure which is however not intended to be limiting:

[0663] 1. A dextransucrase variant wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 21 , comprises at least one modification of an amino acid residue corresponding to any of amino acids:

[0664] 274, 275, 276, 319, 322, 406, 478, 481 , said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 21 , optionally wherein said variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set out in SEQ ID NO: 21.

[0665] 2. The dextransucrase variant according to embodiment 1 , wherein said variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 21 , comprises one or more of:

[0666] F or L at position 274, Y, F, K, or S at position 275, W at position 276, P at position 319, A at position 322, Q at position 406, Y at position 478, A at position 481 , said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 21 .

[0667] 3. A dextransucrase variant wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one modification of an amino acid residue corresponding to any of amino acids:

[0668] 196, 197, 199, 201 , 215, 227, 228, 239, 242, 282, 289, 290, 326, 328, 334, 346, 349, 398, 399, 400, 401 , 402, 417, 715, 721 , 726, 763, 788, 867, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 2, optionally wherein said variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set out in SEQ ID NO: 2.

[0669] 4. The dextransucrase variant according to embodiment 3, wherein said variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises one or more of

[0670] F at position 196, S at position 197, F at position 199, W or F at position 201 , P at position 215, M or Q at position 227, P at position 228, P at position 239, A at position 242, S at position 282, Y or W at position 289, M at position 290, L, F, Y or Q at position 326, F, L, S, M or H at position 328, F at position 334, F at position 346, H at position 349, Y at position 398, E or D at position 399, V, S or L at position 400, E, A, D, F, G, H, L, M, N, S, T, V, W or Y at position 401 , T or S at position 402, H at position 417, R at position 715, T or Q at position 721 , W at position 726, M at position 763, R at position 788, S at position 867, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 2.

[0671] 5. A dextransucrase variant wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 34, comprises at least one modification of an amino acid residue corresponding to any of amino acids: 200, 203, 204, 249, 252, 336, 413, 416, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 34, optionally wherein said variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set out in SEQ ID NO: 34.

[0672] 6. The dextransucrase variant according to embodiment 5, wherein said variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 34, comprises one or more of

[0673] F or L at position 200, S or F at position 203, W at position 204, P at position 249, A at position 252, Q or L at position 336, Y at position 413, E, A or H at position 416, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 34.

[0674] 7. A dextransucrase variant wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 26, comprises at least one modification of an amino acid residue corresponding to any of amino acids:

[0675] 212, 253, 256, 259, 302, 303, 412, 414, 419, 422, 425, 433, 436, 688, 691 , 722, 733, 739, 743, 745, 782, 787, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 26, optionally wherein said variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence set out in SEQ ID NO: 26.

[0676] 8. The dextransucrase variant according to embodiment 7 wherein said dextransucrase variant comprises an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 26, comprises one or more of

[0677] W, L, or F at position 212, S, D or P at position 253, P at position 256, M or P at position 259, 1 or E at position 302, E or N at position 303, Y, R or W at position 412, Y at position 414, V or Y at position 419, R, Y, W, T or Q at position 422, K, N or Y at position 425, V or T at position 433, M or F at position 436, D at position 688, D or P at position 691 , P at position 722, Y, M, L or W at position 733, F or Q at position 739, S at position 743, E at position 745, W at position 782, M or I at position 787, said positions being defined with reference to the amino acid sequence set out in SEQ ID NO: 26.

[0678] 9. A method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor, comprising contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with a recombinant cell comprising a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze the formation of an a-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor.

[0679] 10. A method for producing a modified glycosyl acceptor, comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions comprises culturing the recombinant cell in a suitable culture medium in the presence of the glycosyl donor and the glycosyl acceptor and / or a precursor thereof, optionally wherein said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze the formation of an a-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor; and optionally, isolating the modified glycosyl acceptor produced therefrom.

[0680] 11. A method for producing a modified glycosyl acceptor, comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, optionally wherein said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme, wherein said contacting under suitable conditions comprises contacting the recombinant cell, the glycosyl donor and the glycosyl acceptor in a reaction mixture, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze the formation of an a-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor; and optionally, isolating the modified terpenoid produced therefrom.

[0681] 12. The method according to any one of embodiments 9 to 11 wherein contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with the recombinant cell implies that: a) the glycosyl donor and the glycosyl acceptor come into contact inside (intracellularly to) the recombinant cell; and / or that b) the glycosyl donor and the glycosyl acceptor come into contact outside (extracellularly to) the recombinant cell.

[0682] 13. The method according to any one of embodiments 9 to 12, wherein the at least one heterologous enzyme is an enzyme capable of catalyzing a transglycosylation reaction, typically an enzyme belonging to glycoside hydrolase family 70 (GH70), glycoside hydrolase family 13 (GH13) and / or glycoside hydrolase family 77 (GH77). 14. The method according to any one of the embodiments 9 to 13, wherein the at least one heterologous enzyme is an enzyme selected from the group consisting of cyclomaltodextrin glucanotransferases (E.C. 2.4.1 .19), glucansucrases such as alternansucrases (E.C. 2.4.1.140), dextransucrases (E.C. 2.4.1.5), mutansucrases (E.C. 2.4.1 .5) and reuteransucrases (EC 2.4.1.5); preferably, the at least one heterologous enzyme is a dextransucrase (E.C. 2.4.1 .5).

[0683] 15. The method according to embodiment 14 wherein the at least one heterologous enzyme is a dextransucrases (E.C. 2.4.1.5), optionally a dextransucrases according to any one of embodiments 1 to 8 or a dextransucrase enzyme comprising an amino acid sequence having at least 50% sequence identity with the amino acid sequence according to any one of the following: SEQ ID NO: 1 , 2, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, or 46 or to any of mutants mutant_0001 to mutant_0291 in Table 2.

[0684] 16. The method according to any one of embodiments 9 to 15, wherein the glycosyl donor is a non-activated disaccharide and / or a non-activated polysaccharide such as sucrose and / or (soluble) starch; preferably the glycosyl donor comprises sucrose.

[0685] 17. The method according to any one of embodiments 9 to 16, wherein the glycosyl acceptor is a terpenoid or glycosylated terpenoid such as a mogroside, preferably Mogroside HIE.

[0686] 18. The method according to any one of embodiments 9 to 17, wherein the modified glycosyl acceptor, such as a modified (glycosylated) terpenoid, is an a-glycosylated mogroside, preferably an a-glycosylated Mogroside HIE.

[0687] 19. The method according to any one of embodiments 9 to 18, wherein the glycosyl acceptor comprises Mogroside HIE, the glycosyl donor comprises sucrose and the at least one heterologous enzyme comprises a dextransucrase, wherein the dextransucrase catalyzes an a-1 ,6-glycosidic bond between C1 of the transferred glycosyl group and the hydroxyl group at C6 of the glucose linked at position C24 of Mogroside HIE; thereby producing Compound (I).

[0688]

[0689] Compound (I)

[0690] 20. The method according to any one of embodiments 9 to 19, wherein the recombinant cell is a living cell or a whole cell.

[0691] 21 . The method according to any one of embodiments 9 to 20 wherein the recombinant cell is deficient in a polypeptide capable of deglycosylating a mogroside product and capable of hydrolyzing at least one of:

[0692] (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

[0693] (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.

[0694] 22. The method according to any one of embodiments 9 to 21 , wherein the recombinant cell is deficient in an enzyme capable of hydrolyzing the glycosyl donor; preferably the recombinant cell is deficient in an invertase (E.C. 3.2.1 .26).

[0695] 23. The method according to any one of embodiments 9 to 22, wherein the recombinant cell further comprises an a-glucosidase such as a dextranase (E.C. 3.2.1 .11).

[0696] 24. The method according to any one of embodiments 9 to 23, wherein the recombinant cell further comprises a polypeptide capable of deglycosylating a hyper-glycosylated glycosyl acceptor, optionally wherein said polypeptide is a p-glucosidase.

[0697] 25. The method according to any one of embodiments 9 to 24, wherein the recombinant cell further produces the glycosyl donor and / or the glycosyl donor is provided to the recombinant cell extracellularly. 26. The method according to embodiments 25, wherein the recombinant cell further produces the glycosyl donor and the recombinant cell is further supplemented extracellularly with one or more additional glycosyl donors.

[0698] 27. The method according to embodiment 25 or 26, wherein the glycosyl donor provided to the recombinant cell extracellularly or the additional glycosyl donor supplemented to the recombinant cell extracellularly is a plant extract, a purified form thereof and / or is produced by a cell other than the recombinant cell.

[0699] 28. The method according to any one of embodiments 25 to 27, wherein the recombinant cell is capable of producing or produces the glycosyl donor and comprises one or more of the following:

[0700] (a) an enzyme capable of phosphorylating glucose to form glucose-6-phosphate, preferably a hexokinase (E.C. 2.7.1.1);

[0701] (b) an enzyme capable of converting glucose-6-phosphate to fructose-6-phosphate, preferably a glucose-6-phosphate isomerase (E.C. 5.3.1.9);

[0702] (c) an enzyme capable of catalyzing the transfer of a hexosyl group from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6- phosphate, preferably a sucrose-phosphate synthase (E.C. 2.4.1 .14);

[0703] (d) an enzyme capable of converting sucrose-6-phosphate to sucrose, preferably a sucrose-6-phosphate phosphatase (E.C. 3.1.3.24); and, optionally

[0704] (e) a transport system and / or an alteration of the cell membrane capable of facilitating the transport of glucose inside the recombinant cell

[0705] (f) a transport system and / or an alteration of the cell membrane capable of facilitating the transport of sucrose outside the recombinant cell.

[0706] 29. The method according to any one of embodiments 9 to 28, wherein the recombinant cell further produces the glycosyl acceptor, and / or a precursor thereof and / or wherein the glycosyl acceptor and / or a precursor thereof, is provided to the recombinant cell extracellularly.

[0707] 30. The method according to embodiment 29, wherein the recombinant cell further produces the glycosyl acceptor, and / or a precursor thereof and the recombinant cell is further supplemented extracellularly with one or more additional glycosyl acceptors, and / or a precursors thereof.

[0708] 31. The method according to embodiment 29 or 30, wherein the glycosyl acceptor, and / or a precursor thereof provided to the recombinant cell extracellularly and / or the additional glycosyl acceptor, and / or a precursor thereof supplemented to the recombinant cell extracellularly is a plant extract, a purified form thereof and / or is produced by a cell other than the recombinant cell. 32. The method according to any one of embodiments 29 to 31 , wherein the recombinant cell is capable of producing or produces the glycosyl acceptor, such as the terpenoid and / or a precursor thereof and comprises one or more of the following:

[0709] (a) a polypeptide capable of synthesizing Geranyl diphosphate (GPP) from Dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is a geranyl diphosphate synthase (E.C. 2.5.1.1) (GPPS) and / or a farnesyl diphosphate synthase (E.C. 2.5.1.10) (FPPS);

[0710] (b) a polypeptide capable of synthesizing Farnesyl diphosphate (FPP) from Geranyl diphosphate (GPP), particularly wherein said polypeptide is a farnesyl diphosphate synthase (E.C. 2.5.1.10) (FPPS);

[0711] (c) a polypeptide capable of synthesizing Geranylgeranyl di...

Claims

CLAIMS1. A method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor, comprising contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with a recombinant cell comprising a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze the formation of an a-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor.

2. A method for producing a modified glycosyl acceptor, comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein said contacting under suitable conditions comprises culturing the recombinant cell in a suitable culture medium in the presence of the glycosyl donor and the glycosyl acceptor and / or a precursor thereof, optionally wherein said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze the formation of an a-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor; and optionally, isolating the modified glycosyl acceptor produced therefrom.

3. A method for producing a modified glycosyl acceptor, comprising contacting under suitable conditions a glycosyl donor and a glycosyl acceptor with a recombinant cell comprising a polynucleotide encoding at least one heterologous enzyme capable of catalyzing the transfer of the glycosyl group from the glycosyl donor to the glycosyl acceptor, optionally wherein said contacting under suitable conditions allows the recombinant cell to express the at least one heterologous enzyme, wherein said contacting under suitable conditions comprises contacting the recombinant cell, the glycosyl donor and the glycosyl acceptor in a reaction mixture, wherein said contacting under suitable conditions allows the at least one heterologous enzyme to catalyze formation of an a-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor; and optionally, isolating the modified terpenoid produced therefrom.

4. The method according to any one of the preceding claims wherein contacting under suitable conditions the glycosyl donor and the glycosyl acceptor with the recombinant cell implies that: a) the glycosyl donor and the glycosyl acceptor come into contact inside (intracellularly to) the recombinant cell; and / or that b) the glycosyl donor and the glycosyl acceptor come into contact outside (extracellularly to) the recombinant cell.

5. The method according to any one of the preceding claims, wherein the at least one heterologous enzyme is an enzyme capable of catalyzing a transglycosylation reaction, typically an enzyme belonging to glycoside hydrolase family 70 (GH70), glycoside hydrolase family 13 (GH13) and / or glycoside hydrolase family 77 (GH77).

6. The method according to any one of the preceding claims, wherein the at least one heterologous enzyme is an enzyme selected from the group consisting of cyclomaltodextrin glucanotransferases (E.C. 2.4.1.19), glucansucrases such as alternansucrases (E.C. 2.4.1.140), dextransucrases (E.C. 2.4.1.5), mutansucrases (E.C. 2.4.1 .5) and reuteransucrases (EC 2.4.1.5); preferably, the at least one heterologous enzyme is a dextransucrase (E.C. 2.4.1 .5).

7. The method according to any one of the preceding claims, wherein the glycosyl donor is a non-activated disaccharide and / or a non-activated polysaccharide such as sucrose and / or (soluble) starch; preferably the glycosyl donor comprises sucrose.

8. The method according to any one of the preceding claims, wherein the glycosyl acceptor is a terpenoid or glycosylated terpenoid such as a mogroside, preferably Mogroside HIE.

9. The method according to any one of the preceding claims, wherein the modified glycosyl acceptor, such as a modified (glycosylated) terpenoid, is an a-glycosylated mogroside, preferably an a-glycosylated Mogroside HIE.

10. The method according to any one of the preceding claims, wherein the glycosyl acceptor comprises Mogroside HIE, the glycosyl donor comprises sucrose and the at least one heterologous enzyme comprises a dextransucrase, wherein the dextransucrase catalyzes an a-1 ,6-glycosidic bond between C1 of the transferred glycosyl group and the hydroxyl group at C6 of the glucose linked at position C24 of Mogroside HIE; thereby producing Compound (I).11 . The method according to any one of the preceding claims, wherein the recombinant cell is a living cell or a whole cell.

12. The method according to any one of the preceding claims wherein the recombinant cell is deficient in a polypeptide capable of deglycosylating a mogroside product and 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.

13. The method according to any one of the preceding claims, wherein the recombinant cell is deficient in an enzyme capable of hydrolyzing the glycosyl donor; preferably the recombinant cell is deficient in an invertase (E.C. 3.2.1 .26).

14. The method according to any one of the preceding claims, wherein the recombinant cell further comprises an a-glucosidase such as a dextranase (E.C. 3.2.1 .11).

15. The method according to any one of the preceding claims, wherein the recombinant cell further comprises a polypeptide capable of deglycosylating a hyper-glycosylated glycosyl acceptor, optionally wherein said polypeptide is a p-glucosidase.

16. The method according to any one of the preceding claims, wherein the recombinant cell further produces the glycosyl donor and / or the glycosyl donor is provided to the recombinant cell extracellularly.

17. The method according to claims 16, wherein the recombinant cell further produces the glycosyl donor and the recombinant cell is further supplemented extracellularly with one or more additional glycosyl donors.

18. The method according to claim 16 or 17, wherein the glycosyl donor provided to the recombinant cell extracellularly or the additional glycosyl donor supplemented to the recombinant cell extracellularly is a plant extract, a purified form thereof and / or is produced by a cell other than the recombinant cell.

19. The method according to any one of claims 16 to 18, wherein the recombinant cell is capable of producing or produces the glycosyl donor and comprises one or more of the following:a. an enzyme capable of phosphorylating glucose to form glucose-6-phosphate, preferably a hexokinase (E.C. 2.7.1.1); b. an enzyme capable of converting glucose-6-phosphate to fructose-6-phosphate, preferably a glucose-6-phosphate isomerase (E.C. 5.3.1.9); c. an enzyme capable of catalyzing the transfer of a hexosyl group from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6- phosphate, preferably a sucrose-phosphate synthase (E.C. 2.4.1 .14); d. an enzyme capable of converting sucrose-6-phosphate to sucrose, preferably a sucrose-6-phosphate phosphatase (E.C. 3.1.3.24); and, optionally e. a transport system and / or an alteration of the cell membrane capable of facilitating the transport of glucose inside the recombinant cell f. a transport system and / or an alteration of the cell membrane capable of facilitating the transport of sucrose outside the recombinant cell.

20. The method according to any one of the preceding claims, wherein the recombinant cell further produces the glycosyl acceptor, and / or a precursor thereof and / or wherein the glycosyl acceptor and / or a precursor thereof, is provided to the recombinant cell extracellularly.21 . The method according to claim 20, wherein the recombinant cell further produces the glycosyl acceptor, and / or a precursor thereof and the recombinant cell is further supplemented extracellularly with one or more additional glycosyl acceptors, and / or a precursors thereof.

22. The method according to claim 20 or 21 , wherein the glycosyl acceptor, and / or a precursor thereof provided to the recombinant cell extracellularly and / or the additional glycosyl acceptor, and / or a precursor thereof supplemented to the recombinant cell extracellularly is a plant extract, a purified form thereof and / or is produced by a cell other than the recombinant cell.

23. The method according to any one of claims 20 to 22, wherein the recombinant cell is capable of producing or produces the glycosyl acceptor, such as the terpenoid and / or a precursor thereof and comprises one or more of the following: a. a polypeptide capable of synthesizing Geranyl diphosphate (GPP) from Dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is a geranyl diphosphate synthase (E.C. 2.5.1.1) (GPPS) and / or a farnesyl diphosphate synthase (E.C. 2.5.1.10) (FPPS); b. a polypeptide capable of synthesizing Farnesyl diphosphate (FPP) from Geranyl diphosphate (GPP), particularly wherein said polypeptide is a farnesyl diphosphate synthase (E.C. 2.5.1.10) (FPPS);c. a polypeptide capable of synthesizing Geranylgeranyl diphosphate (GGPP) from Farnesyl diphosphate (FPP), particularly wherein said polypeptide is a geranylgeranyl diphosphate synthase (E.C. 2.5.1.29) (GGPPS); d. a polypeptide capable of synthesizing Geranylfarnesyl diphosphate (GFPP) from Geranylgeranyl diphosphate (GGPP), particularly wherein said polypeptide is a geranylfarnesyl diphosphate synthase (E.C. 2.5.1 .81) (GFPPS); e. a polypeptide encoding a terpene synthase.

24. The method according to any one of claims 20 to 23, wherein the recombinant cell is capable of producing or produces a terpenoid, such as a mogroside and / or a precursor thereof and comprises one or more of the following: a. a squalene synthase (SQS); b. a squalene epoxidase (SQE); c. a cucurbitadienol synthase (CDS); d. a cytochrome P450 (CYP); e. a cytochrome P450 reductase (CPR); f. an epoxide hydrolase (EPH); g. a cytochrome b5 (CB5).

25. The method according to any one of claims 20 to 24, wherein the recombinant cell is capable of producing or produces the terpenoid, such as a mogroside and / or a precursor thereof and comprises one or more of the following uridine diphosphate dependent glycosyl transferase (UGT) enzymes:(a) a polypeptide capable of glycosylating mogrol or a mogroside at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group, and / or C25 hydroxyl group;(b) a polypeptide capable of beta-1 ,2-glycosylation of the C2' hydroxyl group of a glucose moiety at position C24 (a C24-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;(c) a polypeptide capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glucose moiety at position 03 of a mogroside and / or capable of beta-1 ,6-glycosylation of the C6' hydroxyl group of a glycose moiety at position C24 of a mogroside.

26. The method according to any one of claims 20 to 25, wherein the recombinant cell is capable of producing or produces the terpenoid, such as a mogroside and / or a precursor thereof and comprises one or more of the following: a. a polypeptide capable of synthesizing Acetoacetyl-Coenzyme A (AACoA) from Acetyl Coenzyme A (AcCoA), particularly wherein said polypeptide is an acetyl-CoA acetyltransferase (E.C. 2.3.1.9) (AACT);b. a polypeptide capable of synthesizing Hydroxymethylglutaryl-Coenzyme A (HMGCoA) from Acetoacetyl-Coenzyme A (AACoA), particularly wherein said polypeptide is an Hydroxymethylglutaryl-Coenzyme A synthase (E.C. 2.3.3.10) (HMGS); c. 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 (E.C. 1.1.1.34) (HMGR); d. a polypeptide capable of synthesizing Mevalonate-5-phosphate (MVA-P) from Mevalonic acid (MVA), particularly wherein said polypeptide is a mevalonate kinase (E.C. 2.7.1.36) (MK); e. a polypeptide capable of synthesizing Mevalonate-5-diphosphate (MVA-PP) from Mevalonate-5-phosphate (MVA-P), particularly wherein said polypeptide is a phosphomevalonate kinase (E.C. 2.7.4.2) (PMK); f. a polypeptide capable of synthesizing Isopentenyl diphosphate (IPP) from Mevalonate-5-diphosphate) (MVA-PP), particularly wherein said polypeptide is a diphosphomevalonate decarboxylase (E.C. 4.1.1 .33) (MDD) and / or an isopentenyl / dimethylallyl diphosphate synthase (E.C. 1 .17.1 .2) (IPPS); g. a polypeptide capable of synthesizing Dimethylallyl diphosphate (DMAPP) from Isopentenyl diphosphate (IPP), particularly wherein said polypeptide is an isopentenyl- diphosphate delta-isomerase (E.C. 5.3.3.2) (I PI).

27. The method according to any one of the preceding claims, wherein the at least one heterologous enzyme is an intracellular enzyme and / or an extracellular enzyme.

28. The method according to claim 27, wherein the at least one heterologous enzyme is an extracellular enzyme, optionally wherein said enzyme is secreted and released outside the recombinant cell and / or displayed at the surface of the recombinant cell.

29. The method according to claim 27, wherein the at least one heterologous enzyme is an intracellular enzyme.

30. The method according to any one of claims 16 to 29, wherein the glycosyl donor is provided to the recombinant cell extracellularly and the recombinant cell further comprises a transport system and / or an alteration of the cell membrane capable of facilitating the transport of the glycosyl donor inside said cell.31 . The method according to any one of claims 27 or 28, wherein the at least one heterologous enzyme is an extracellular enzyme.

32. The method according to claim 31 , wherein the recombinant cell further produces the glycosyl acceptor and / or a precursor thereof and / or wherein the glycosyl acceptor and / or a precursor thereof is provided to the recombinant cell extracellularly.

33. The method according to any one of claims 31 or 32, wherein the recombinant cell further produces the glycosyl acceptor and / or a precursor thereof and the recombinant cell further comprises a transport system and / or an alteration of the cell membrane capable of facilitating the transport of the glycosyl acceptor and / or a precursor thereof outside said cell.

34. The method according to any one of claims 31 to 33, wherein the recombinant cell further produces the glycosyl donor and / or wherein the glycosyl donor is provided to the recombinant cell extracellularly.

35. The method according to claim 34, wherein the recombinant cell further produces the glycosyl donor and the recombinant cell further comprises a transport system and / or an alteration of the cell membrane capable of facilitating the transport of the glycosyl donor outside said cell.

36. The method according to any one of the preceding claims, comprising further supplementing the recombinant cell extracellularly with one or more additional enzymes capable of catalyzing the transfer of the glycosyl group to the glycosyl acceptor.

37. The method according to any one of claims 2 to 36, wherein the at least one heterologous enzyme is an intracellular enzyme and / or an extracellular enzyme.

38. The method according to any one of claims 2 to 37, wherein the recombinant cell further produces the glycosyl acceptor and further produces the terpenoid and / or a precursor thereof.

39. The method according to any one of claims 2 to 38, wherein the glycosyl acceptor and / or a precursor thereof is provided to the recombinant cell extracellularly and / or wherein the glycosyl donor is provided to the recombinant cell extracellularly.

40. The method according to claim 39, wherein the glycosyl donor is provided to the recombinant cell extracellularly and wherein the glycosyl donor is provided 1 hour before inoculation, during inoculation, or after 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h or 120 h following inoculation ofthe recombinant cell in the culture medium.41 . The method according to any one of claims 2 to 40, wherein the recombinant cell expresses the polynucleotide encoding the at least one heterologous enzyme, and the recombinant cell further produces the glycosyl donor and the glycosyl acceptor.

42. The method according to any one of claims 3 to 36, wherein the glycosyl donor and / or the glycosyl acceptor and / or a precursor thereof are provided to the recombinant cell extracellularly.

43. The method according to claim 42, wherein the method comprises: a. growing the recombinant cell in a suitable culture medium under conditions in which the at least one heterologous enzyme is expressed and wherein the at least one heterologous enzyme is intracellular and / or extracellular displayed at the surface of the recombinant cell, b. harvesting the recombinant cell from the culture medium, and c. contacting in a reaction mixture the recombinant cell obtained from (b), the glycosyl donor, the glycosyl acceptor and / or a precursor thereof and optionally, one or more additional enzymes capable of catalyzing the transfer of the glycosyl group to the glycosyl acceptor; thereby, producing a modified glycosyl acceptor in the reaction mixture.

44. A recombinant cell as described in any of the methods according to any one of the preceding claims.

45. A recombinant cell comprising at least one heterologous polynucleotide encoding at least one enzyme capable of catalyzing a transglucosylation reaction, such as the transfer of a glycosyl group from a glycosyl donor (e.g. sucrose) to a glycosyl acceptor (e.g. a terpenoid such as a mogroside), wherein the recombinant cell is deficient in an enzyme capable of hydrolyzing the glycosyl donor (such as an invertase) and, optionally comprises at least one polynucleotide sequence encoding an a-glycosidase and / or at least one polynucleotide encoding a p-glucosidase.

46. A recombinant cell according to claim 45, wherein the enzyme capable of catalyzing the transglycosylation between a glycosyl group from a glycosyl donor and a glycosyl acceptor is a glucansucrase, such as a dextransucrase, wherein the enzyme capable of hydrolyzing the glycosyl donor is an invertase.

47. The method according to any one of claims 1 to 43 or the recombinant cell according to any one of claims 44 to 46, wherein the recombinant cell is a prokaryotic cell, preferably a bacterial cell such as Escherichia coir, or an eukaryotic cell, particularly a plant cell or a fungal cell, more particularly a yeast cell selected from a Saccharomyces sp., such as Saccharomyces cerevisiae, a Yarrowia sp., such as Yarrowia lipolytica, a Candida sp., such as Candida krusei, an Issatchenkia sp., such as Issatchenkia orientalis, a Pichia sp., such as Pichia pastoris.

48. A reaction mixture comprising the recombinant cell according to any one of claims 44 to 47.

49. A culture medium comprising the recombinant cell according to any one of claims 44 to 48.

50. A fermentation broth comprising the recombinant cell according to any one of claims 44 to 49.

51. A terpenoid composition obtained or obtainable by the method according to any one of claim 1 to 43, 47 or using the recombinant cell according to any one of claims 44 to 47.

52. A food product, a beverage, a pet-food, a feed, an oral, a bioactive or a pharmaceutical composition comprising the terpenoid composition according to claim 51 .

53. Use of the recombinant cell according to any one of claims 44 to 47 to produce a modified terpenoid.

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