Cells and methods for producing glycosylated compounds

Abstract

The present disclosure relates generally to the production of glycosylated compounds. A recombinant cell and a method for enzymatic modification of a glycosyl acceptor are provided. The method comprises: contacting a glycosyl donor and a glycosyl acceptor with a recombinant cell, the 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 are compositions obtainable by the methods of the present disclosure and uses thereof.

Description

Technical Field

[0001] This disclosure generally relates to the production of glycosylated compounds. The present invention provides a method for the enzymatic glycosylation of compounds (e.g., terpenoids) and recombinant cells. Background Technology

[0002] Secondary metabolic pathways in plants and microorganisms produce a large number of small molecules, such as terpenoids, flavonoids, and polyketides. The vast majority of these molecules exist in nature in the form of glycosides, where sugar residues play a decisive role in molecular structural complexity and biological activity.

[0003] For example, terpenoids (also known as isoprene derivatives) are one of the largest classes of secondary metabolites and are ubiquitous in plants. They exist primarily as glycosides, with the sugar moiety attached to their core skeleton. They are recognized for their many industrially desirable properties, such as sweeteners, flavoring agents, aromatizers, coloring agents, pharmaceuticals, and nutritional supplements.

[0004] Terpenoids are derived from two isomers of five-carbon compounds, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In plants, DMAPP and IPP are synthesized via two distinct and independent biochemical pathways: the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in the plastids. In the downstream terpenoid pathway, DMAPP and IPP condense to produce geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and geranylfarnesyl diphosphate (GFPP). These isopentenyl diphosphate intermediates are converted by specific enzymes, including terpene synthases, to produce various terpenoids. Based on the number of isopentenyl units, terpenoids are classified into C5 semiterpenoids, C5 hemi ... 10 Monoterpenoids, C 15 Sesquiterpenoids, C 20 Diterpenoids, C 25 Disesquiterpenoids, C 30 Triterpenoids, C 40 Tetraterpenoids and C >40 Polyterpenoids.

[0005] Examples of terpenoids include mogroside, which is a C16 compound. 30 Triterpenoids have recently gained significant commercial importance as alternative high-intensity sweeteners. These compounds exist in... Siraitia grosvenorii (Also known as monk fruit, luohan fruit or) luohan guo The fruit of this plant belongs to the Cucurbitaceae family (Cucurbitaceae). CurcubitaceaeThe mogroside family, and almost exclusively found in Guangxi Province, China. All mogrosides share the same mogrol skeleton, but differ primarily in their glycosylation patterns (including the amount of glucose moiety). In plants, mogroside glycosylation is mainly mediated by uridine-5'-bisphosphate (UDP)-glycosyltransferase (UGT), which uses nucleotide-activated sugars as donors (e.g., UDP-glucose) and typically catalyzes the formation of β-glycosidic bonds. Depending on the glycosylation pattern, the sweetness level and taste characteristics can vary among different mogroside compounds. Mogroside V is the most abundant found in mogroside. It is estimated to be hundreds of 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 mogroside, but these are generally less sweet than mogroside V, and their sweetness may be accompanied by off-flavors (e.g., aftertaste, bitterness). Recently, siamansiside I (SI) was isolated from monk fruit and identified as having a sweetness even superior to that of mogroside V. Manufacturers often use combinations of mogrosides to achieve a balanced sweetness profile while minimizing any potential off-flavors, thus taking advantage of the strength of each compound.

[0006] With the increasing demand for developing novel glycosylated compounds with customized properties, the engineering modification of the glycosylation patterns of such compounds has attracted considerable interest. For example, in the context of terpenoids used as sweeteners, engineered glycosylation of these compounds has been explored to alter their flavor characteristics (e.g., making them more palatable and closer to the taste of sugar) to improve their sweetness and solubility and / or contribute to their stability and shelf life. Recent examples include WO2018204483 A2, WO2020096905 A1, WO2020096907 A1, and Xu et al. (Xu...) et al Modified mogrosides described in (2021), Food Chemistry 359:129938 。 The compound, referred to as α-Symanosin I or α-SI, is obtained by enzymatic α-glycosylation of mogroside IIIE in vitro, wherein mogroside IIIE is reacted with soluble starch (as a glucose donor) and *E. coli* containing recombinant cyclic maltodextrin glucan transferase (CGTase). E. coli The cell lysate was not exposed. Compared with mogroside IIIE, α-SI showed increased sweetness intensity and improved taste quality, thus promoting α-SI as another potential low-calorie sweetener. Xu et al. also concluded that α-glycosylation, like β-glycosylation which typically occurs during the formation of mogroside in plants, can also enhance the sweetness of mogroside.

[0007] Two types of in vitro and in vivo biocatalytic glycosylation tools have been developed for the engineering of compounds, such as terpenes. Purified biocatalysts are a core component of in vitro methods. However, obtaining biocatalysts in pure form is laborious and expensive, and sometimes immobilization is required to improve their activity and stability. Furthermore, in some cases, such as with UGT for the glycosylation of mogrosides, expensive activation donor molecules (e.g., UDP-glucose) are required. Alternatively, in vivo biocatalytic glycosylation tools for the production and glycosylation of terpenes have also emerged. For example, WO2014 / 086842, WO2016 / 038617, and WO2016 / 050890 describe recombinant cells and methods capable of producing mogrosides and / or mogrosides. However, while these recombinant microbial production systems appear suitable for the production of a wide variety of β-glycosylated terpenes, they are not suitable for the production of α-glycosylated terpenes.

[0008] From an industrial perspective, given the growing interest in the global industrial market, there remains a need for more robust microbial production systems capable of producing a variety of glycosylated compounds (including α-glycosylated and / or β-glycosylated terpenes) in a sustainable, controlled and commercially viable manner.

[0009] Attached Figure Figure 1 The chemical structures of several mogroside compounds were described.

[0010] Figure 2 The biosynthetic pathway from acetyl-CoA to squalene was described.

[0011] Figure 3 The biosynthetic pathway from squalene to mogroside was described.

[0012] Figure 4 The biosynthetic pathway from mogroside to mogroside was described.

[0013] Figure 5 The chemical structures of several mogroside precursors in the biosynthetic pathway for the production of mogrosides from squalene are described.

[0014] Figure 6 A schematic diagram depicts one or more reactions that may occur in recombinant cells according to this disclosure.

[0015] Figure 7 Compound (I) was formed in vitro using M3E as a substrate via various DexT methods. A portion of the supplemented M3E appeared to be converted into other components that were not further analyzed, resulting in a mass gap.

[0016] Figure 8Compound (I) was described in vitro using M3E as a substrate via various truncated DexT. The production level of compound (I) with reference DexT (i.e., the least truncated DexT variant in each species) was set at 100%.

[0017] Figure 9 The formation of compound (I) in vitro via various DexT methods in the presence and absence of α-glucosidase was depicted. Samples were treated with (-) and (+) α-glucosidase (dextran) to remove any α-glycosylated compounds (I). Non-specific M3E glycosylation and poly-α-glycosylation compounds (I) were not measured, which explains the quality gap in the analysis.

[0018] Figure 10 Compound (I) was formed in vitro via engineered DexT. Non-specific α-glycosylation and poly-α-glycosylation of compound (I) were not analyzed, which explains the mass gap in the analysis.

[0019] Figure 11 Depicting through Yarrowia lipolytica SIR001 (without Yl-BGL2 overexpression) and SIR002 (with Yl-BGL2 overexpression) produce M3E and siamansiol.

[0020] Figure 12 The production of compound (I) in vivo by a DexT mutant using intracellular (A), extracellular membrane anchoring (B), and extracellular (C) localization was depicted. The strain was cultured in the absence (-) or presence (+) of sucrose. The level of compound (I) production is relative to the level of M3E produced in a culture without sucrose supplementation (which was set to 100%).

[0021] Figure 13 The production of compound (I) in vivo using extracellularly localized DexT variants in combination with (+) overexpressed α-glucosidase (AGL) or without (-) overexpressed α-glucosidase (AGL) was described. The compound (I) production level of strains containing the AGL gene is relative to the compound (I) formation level produced in strains without AGL overexpression (which was set to 100%).

[0022] Figure 14 The study describes in situ sucrose production by MOG-producing strains with and without heterosucrose synthesis gene overexpression.

[0023] Figure 15The production of Mog3E and compound (I) in DexT overexpressing strains lacking (-) or containing (+) heterologous sucrose synthesis genes was depicted. Numerical values ​​are presented relative to M3E production levels in strains without in situ sucrose synthesis genes. Non-specific M3E glycosylation and poly-α-glycosylation of compound (I) were not measured, which explains the quality gap in the analysis.

[0024] Sequence Description The sequence descriptions are listed in Table 1.

[0025] Summary of the Invention

[0026] This disclosure provides a method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor, comprising: contacting the glycosyl donor and the glycosyl acceptor with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (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 the contact under suitable conditions enables said at least one (heterologous) enzyme to catalyze a glycosidic bond (e.g., glycosidic bond) between the transferred glycosyl group and the glycosyl acceptor. The formation of glycosidic bonds; thereby producing modified glycosyl receptors. Typically, such contact under suitable conditions allows the recombinant cells to express at least one heterologous enzyme.

[0027] Another aspect of this disclosure provides a method for producing a modified glycosyl receptor, comprising: contacting a glycosyl donor and a glycosyl receptor with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (over)expressing a polynucleotide encoding at least one (heterologous) enzyme, said (heterologous) enzyme being capable of catalyzing the transfer of a glycosyl from said glycosyl donor to said glycosyl receptor and / or its precursor, wherein said contact under suitable conditions comprises culturing said recombinant cells in a suitable culture medium in the presence of said glycosyl donor and said glycosyl receptor and / or its precursor, optionally wherein said contact under suitable conditions allows said recombinant cells to express at least one (heterologous) enzyme, wherein said contact under suitable conditions enables said at least one (heterologous) enzyme to catalyze a glycosidic bond (preferably) between the transferred glycosyl and said glycosyl receptor. The formation of glycosidic bonds; thereby producing modified glycosyl receptors, and optionally, isolating the resulting modified glycosyl receptors.

[0028] Another aspect of this disclosure provides a method for producing a modified glycosyl acceptor, comprising: contacting a glycosyl donor and a glycosyl acceptor with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (over)expressing a polynucleotide encoding at least one heterozyme, said (hetero)enzyme being capable of catalyzing the transfer of a glycosyl group from said glycosyl donor to said glycosyl acceptor, wherein said contact under suitable conditions comprises contacting said recombinant cells, said glycosyl donor, and said glycosyl acceptor and / or its precursor in a reaction mixture, wherein said contact under suitable conditions enables said at least one (hetero)enzyme to catalyze a glycosidic bond (preferably) between the transferred glycosyl group and said glycosyl acceptor. The formation of a glycosidic bond; thereby producing a modified glycosyl receptor, and optionally, isolating the thus produced modified glycosyl receptor. Optionally, the contact under suitable conditions allows the recombinant cells to express the at least one heterozyme.

[0029] Glycosidic bonds are typically α-glycosidic bonds. Modified glycosyl receptors (e.g., modified terpenoids) are typically α-glycosylated glycosyl receptors (e.g., α-glycosylated terpenoids).

[0030] The methods disclosed herein for generating modified glycosyl receptors can be fermentation, biotransformation, or a combination thereof.

[0031] In the method, at least one heterologous enzyme may be an intracellular enzyme and / or an extracellular enzyme.

[0032] In the method described, the recombinant cells are typically live cells or intact cells.

[0033] Typically, in the method according to this disclosure, "contacting the glycosyl donor and glycosyl acceptor with the recombinant cell under suitable conditions" can occur as follows: a) The glycosyl donor and the glycosyl acceptor come into contact inside the recombinant cell (intracellular); and / or b) The glycosyl donor and the glycosyl acceptor come into contact outside the recombinant cell (extracellularly).

[0034] It was also made public: - Recombinant cells used in the methods according to this disclosure, such as recombinant cells comprising at least one heteropolynucleotide encoding at least one enzyme capable of catalyzing a transglycosylation (e.g., transglucosylation) reaction, the transglycosylation reaction being, for example, the transfer of a glycosyl group from a glycosyl donor (e.g., sucrose) to a glycosyl acceptor (e.g., a terpenoid compound, such as mogroside), wherein the recombinant cells are deficient in enzymes (e.g., invertase) capable of hydrolyzing the glycosyl donor, and optionally wherein the cells are deficient in β-glucanase, and optionally wherein the cells comprise at least one polynucleotide sequence encoding α-glucosidase and / or at least one polynucleotide encoding β-glucosidase. Typically, the at least one enzyme capable of catalyzing the transglycosylation reaction is an enzyme capable of catalyzing an α-glycosidic bond between a glycosyl group from a glycosyl donor (e.g., sucrose) and a glycosyl acceptor (e.g., a terpenoid compound, such as mogroside).

[0035] - A reaction mixture comprising recombinant cells according to the present disclosure.

[0036] - A culture medium containing recombinant cells according to the present disclosure.

[0037] - A fermentation broth containing recombinant cells according to the present disclosure.

[0038] - A composition that is obtained or can be obtained by the method according to this disclosure.

[0039] - A food product, beverage, pet food, feed, oral composition or pharmaceutical composition comprising the composition according to this disclosure.

[0040] - The use of recombinant cells according to this disclosure for producing modified glycosyl receptors (e.g., modified terpenoids, such as modified mogrosides), such as α-glycosylated glycosyl receptors (e.g., α-glycosylated terpenoids, such as α-glycosylated mogrosides).

[0041] In one embodiment of this disclosure, the α-glycosylation receptor is α-glycosylated mogroside, and more particularly α-1,6-glycosylated mogroside IIIE.

[0042] Preferably, α-glycosylated mogroside is compound (I) having the following structure (CAS 2419125-32-7): General definition To facilitate a clearer understanding of this disclosure, certain terms and methods are first defined. As used herein, unless otherwise expressly provided, each of the following terms shall have the meaning set forth below. Additional definitions are listed throughout this application. In case of conflict, this application (including the definitions) shall prevail. Unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. For all purposes, all publications, patents, and other references mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0043] Unless otherwise defined, 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 relates.

[0044] Although methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not limiting. Other features and advantages of this disclosure will become apparent from the detailed description and claims.

[0045] Throughout this disclosure, the words “comprising,” “including,” and “having,” as well as variations such as “containing,” should be interpreted as inclusive. That is, where the context permits, these words are intended to express the possibility of including other elements or integers not specifically stated.

[0046] As used in this disclosure and claims, the singular forms “an,” “a,” and “the,” as well as cases where no quantifier is used, include the plural forms, unless the context clearly indicates otherwise. As an example, “an element” can refer to one element or more than one element, i.e., “at least one element.”

[0047] The term "about" refers to a value or composition that is within an acceptable range of error for a 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, according to practice in the art, "about" may mean within one or more standard deviations. Or, "about" may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, these terms may represent values ​​up to an order of magnitude or up to five times. When a particular value or composition is provided in the application and claims, unless otherwise stated, the meaning of "about" or "substantially comprises" should be assumed to be within the acceptable range of error for that particular value or composition.

[0048] Unless specifically stated or clearly apparent from the context, the term "or" as used herein should be understood as inclusive. The term "and / or" as used in phrases such as "A and / or B" is intended to include both "A and B," "A or B," and "A and B." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover 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).

[0049] As defined herein, a “cell” is an organism suitable for genetic manipulation and capable of being cultured at cell densities suitable for the industrial production of a target product. Suitable organisms may be microorganisms, such as those that can be maintained in a fermentation apparatus. With respect to this disclosure, it should be understood that “cell” (e.g., microorganisms, fungi, algae, or plants) also includes synonyms or essentially synonyms for such species having the same physiological characteristics, 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.

[0050] Based on the various aspects and embodiments disclosed herein in a cellular context, the present invention preferably does not include methods for cloning humans, methods for modifying the germline genetic identity of humans, and / or the use of human embryos for industrial or commercial purposes.

[0051] A “nucleic acid molecule” or “polynucleotide” (the terms are used interchangeably in this document) is represented by a nucleotide sequence.

[0052] A polypeptide is represented by an amino acid sequence.

[0053] When used to refer to polynucleotides or polypeptides, the term "expression" refers to any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. When used to refer to genes, the term "constitutive expression" refers to a situation where gene expression is controlled by a constitutive promoter that allows for continuous gene transcription. When used to refer to genes, the term "inducible expression" refers to a mode of regulating gene expression, where molecules called inducers regulate gene expression by: a) binding to gene promoter repressor proteins or b) binding to gene promoter activator molecules, thus allowing RNA polymerase to transcribe the gene.

[0054] "Gene expression" includes "heterologous expression" and "overexpression," and involves gene transcription and the translation of mRNA into polypeptides (e.g., enzymes). Overexpression refers to the production of gene products, measured by the levels of mRNA, polypeptide, and / or enzyme activity in transgenic cells or organisms, exceeding the levels produced in non-transformed cells or organisms with similar genetic backgrounds.

[0055] In this article, "gene" is defined as a polynucleotide containing an open reading frame (ORF) and its transcriptional control sequence. An ORF is a polynucleotide region on a gene that encodes a polypeptide (e.g., an enzyme) and will be transcribed and translated into said polypeptide.

[0056] As used herein, the term "control sequence" refers to a component that participates in regulating the expression of a coding sequence in a particular organism or in vitro. Examples of control sequences are transcription initiation sequences, termination sequences, promoters, leader sequences, signal peptides, propeptides, prepropeptides, or enhancer sequences; Shine-Delgarno sequences, repressor or activator sequences; effective 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 sequences that enhance protein secretion when needed.

[0057] As used herein, the term "operably linked" refers to the connection of polynucleotide elements (containing, for example, a coding sequence or another polynucleotide sequence) in a functional relationship. A polynucleotide is "operably linked" when it is placed in a functional relationship with another polynucleotide. For example, if a promoter sequence or enhancer sequence affects the transcription of a coding sequence, then the promoter sequence or enhancer sequence is operably linked to that coding sequence.

[0058] As used herein, the term "promoter" refers to a polynucleotide segment that functions to control the transcription of one or more genes, located upstream of the transcription start site of the gene in the transcriptional direction, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other polynucleotide segment, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that directly or indirectly regulates 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 that can be regulated.

[0059] When used to indicate the relationship between a given (recombinant) polynucleotide (such as DNA or RNA) or polypeptide and a given host organism or host cell (such as the cells disclosed herein), the terms “homologous” or “endogenous” should be understood to mean that the polynucleotide or polypeptide molecule is produced in nature by a host cell or organism of the same species (such as the same variety or strain).

[0060] When used with respect to polynucleotides (such as DNA or RNA) or polypeptides, the term "heterogeneous" refers to a polynucleotide or polypeptide that is not naturally present as part of a host cell (such as the cell disclosed herein). In other words, a heterologous polynucleotide or polypeptide is not endogenous to the cell in which it is introduced, but has been acquired from another cell or synthesized or recombined.

[0061] As used herein, the term "naturally occurring" refers to a process, event, or product that occurs or exists in nature in its relevant form. In contrast, "non-naturally occurring" refers to a process, event, or product whose existence or formation involves human intervention. The term "non-naturally occurring" is synonymous with "man-made" in this document. Generally, the term "naturally occurring" for polypeptides or nucleic acids can be used interchangeably with the terms "wild-type" or "natural." It refers to a polypeptide or nucleic acid encoding a polypeptide that has the same amino acid sequence or polynucleotide sequence as those found in nature. Naturally occurring polypeptides include natural polypeptides, such as those naturally expressed or found in specific cells. Naturally occurring polynucleotides include natural polynucleotides, such as those naturally found in the genome of specific cells. Additionally, wild-type or naturally occurring sequences can refer to sequences from which variants or synthetic sequences are derived.

[0062] The term “derived from” also includes the terms “originating from,” “obtained from,” “available from,” “isolated from,” and “produced from,” and generally indicates that a specified material finds its origin in another specified material or has features that can be described with reference to the other specified material. As used herein, “derived from” a cell (e.g., a nucleic acid molecule or polypeptide) preferably means that the material is native to the microorganism.

[0063] The term "recombinant" is synonymous with "genetically modified." When used in relation to polynucleotides or peptides, "recombinant" indicates that the polynucleotide or peptide has been modified in its sequence through human intervention compared to its native form. When referring to cells, the term "recombinant" indicates that the cell has been genetically modified in its sequence through human intervention or transformed / transfected with one or more polynucleotides compared to its native form. The presence of one or more polynucleotides alters a microorganism's ability to produce one or more products.

[0064] As used herein, a “recombinant cell” is defined as a cell that has preferably been genetically modified or transformed / transfected with one or more polynucleotides as defined elsewhere herein. The presence of one or more such polynucleotides alters the ability of a microorganism to produce one or more products. Cells that have not been transformed / transfected or genetically modified are not recombinant cells and typically do not contain one or more polynucleotides that enable the cell to produce products. Therefore, untransformed / untransfected cells are generally cells that do not naturally produce products; however, cells that naturally produce products and have been modified as disclosed herein are considered recombinant cells as disclosed herein.

[0065] When used to refer to cells, the term "deficient in the production of polypeptides (e.g., enzymes)" typically means that a cell contains a modification (preferably in its genome) that, when analyzed under the same conditions, results in reduced or no production of polypeptides (e.g., enzymes) compared to an unmodified parental cell. Alternatively, or in addition, a cell contains a modification that, when analyzed under the same conditions, results in reduced or no activity (which may be enzymatic activity or other biological activity) of polypeptides derived from those described herein compared to an unmodified parental cell. In this context, a cell is deficient in the production of polypeptides as described herein when: when analyzed under the same conditions, a) it produces fewer polypeptides as defined herein or does not produce polypeptides as defined herein; and / or b) the mRNA transcribed from the gene encoding the polypeptide has a reduced expression level or a reduced translation level; and / or c) it produces polypeptides with reduced or no activity.

[0066] Defects in the production of peptides in cells, as defined herein, can be measured by determining the amount and / or (specific) activity of the relevant peptide produced by cells modified in their genome, compared to parental cells not modified in their genome; and / or by determining the amount of (free) mRNA transcribed from the gene encoding the peptide; and / or by determining the amount of product produced by the peptide from cells modified in their genome; and / or by gene or genome sequencing. Defects in the production of said peptides can be measured using any assay available to a person skilled in the art, such as transcriptomic profiling, Northern blotting, RT-PCR, Q-PCR, and Western blotting.

[0067] In the context of this disclosure, when used to refer to cells, the term "deficient in the production of polypeptides (e.g., enzymes)" can also mean that the cell is naturally deficient in said polypeptides (e.g., enzymes). For example, Yarrowia lipolytica It cannot metabolize sucrose naturally because it lacks the invertase required to cleave disaccharides (or is deficient in the invertase required to cleave disaccharides).

[0068] The phrase "measured under the same conditions" or "analyzed under the same conditions" means that modified and unmodified cells are cultured under the same conditions, and the amount and / or activity of peptides lacking or overexpressed in the modified and unmodified cells are measured in the modified and unmodified cells respectively using the same conditions (preferably by using the same assay and / or method, more preferably within the same experiment) compared to the unmodified cells.

[0069] As defined herein, an enzyme capable of catalyzing the transfer of glycosyl groups from a glycosyl donor (e.g., sucrose) to a glycosyl acceptor (e.g., a terpene) is an enzyme capable of catalyzing transglycosylation reactions. The enzyme preferably belongs to the glycoside hydrolase family 70 (GH70), the glycoside hydrolase family 13 (GH13), and / or the glycoside hydrolase family 77 (GH77). GH70 enzymes are involved in the synthesis of α-glucans, polysaccharides composed of α-linked glucose units. Examples of GH70 enzymes include glucan sucrase (EC 2.4.1.5), which is an enzyme that synthesizes various glucans from sucrose. GH13 enzymes include transglycosidases. Examples of GH13 enzymes include cyclomaltodextrin glucan transferase or CGTase (EC 2.4.1.19), which typically cleaves α-1,4-glucosidic linkages in a substrate (e.g., starch) to create new α-1,4-glucosidic bonds in the acceptor molecule (e.g., a terpene). The GH77 family is related to the GH13 family.

[0070] In the context of this disclosure, the term "glycosylation" or "glycosylation reaction" refers to an enzyme-catalyzed reaction in which a glycosyl group (typically via an anomeric carbon in such a glycosyl group) is covalently attached to a hydroxyl group or other functional group of another molecule (i.e., a glycosyl acceptor); thereby resulting in the formation of a glycosidic bond. Depending on the functional group, different types of glycosylation exist, including N-glycosylation, S-glycosylation, and O-glycosylation. In the context of this disclosure, glycosylation is typically O-glycosylation, and the glycosidic bond is typically an O-glycosidic bond.

[0071] The term "transglycosylation" or "transglycosylation reaction" refers to a specific type of glycosylation reaction involving the transfer of a glycosyl group from one molecule (i.e., a glycosyl donor, such as sucrose) to another molecule containing a hydroxyl group or other functional groups (i.e., a glycosyl acceptor, such as terpenoids); resulting in the formation of a glycosidic bond. Enzymatic transglycosylation can be catalyzed by glycosyltransferases and glycosidases (also known as glycosidases).

[0072] The term "glycosyl" is used in this document to refer to a sugar molecule (e.g., a glucose molecule) or a sugar moiety (e.g., a glucose moiety).

[0073] In the context of this disclosure, the terms “glycosyl donor,” “glycosyl donor molecule,” “donor molecule,” “sugar donor,” “glucose donor,” “sugar donor molecule,” and “glucose donor molecule” are used interchangeably. “Glycosyl donor” is used herein to refer to a molecule that provides a glycosyl group in a glycosylation reaction (e.g., transglycosylation). A glycosyl donor can be an activated donor (i.e., a glyconucleotide, such as UDP-glucose or GDP-mannose) or an inactivated sugar (i.e., a sugar or polysaccharide having one or more glycosidic bonds, such as sucrose, lactose, or starch).

[0074] The terms "glycosyl acceptor," "glycosyl acceptor molecule," or "receptor molecule" (e.g., terpenoids or glycosylated terpenoids) are used herein to refer to the molecule that receives a glycosyl group during a glycosylation reaction (e.g., transglycosylation). In glycosylation, the glycosyl acceptor typically contains a functional group (e.g., a hydroxyl group) capable of reacting with the glycosyl group. The resulting reaction forms a glycosidic bond between the glycosyl donor and the glycosyl acceptor.

[0075] The term "glycosidic bond," particularly "O-glycosidic bond," is used herein to refer to a type of covalent bond formed between the anodic carbon atom of a glycosyl donor (e.g., C1 of the glucose moiety) and the oxygen atom of a glycosyl acceptor molecule (typically another sugar (e.g., glucose) or a non-sugar moiety (e.g., the monk fruit skeleton)). This glycosidic bond is typically formed through a condensation reaction when the hydroxyl group on the anodic carbon of the sugar molecule reacts with the hydroxyl or carboxyl group of the acceptor molecule (resulting in the elimination of water molecules).

[0076] The specific type of glycosidic bond formed depends on which hydroxyl (-OH) group and the orientation of the anodic carbon are involved in the linkage. Two common types of glycosidic bonds are α-glycosidic bonds and β-glycosidic bonds. In an α-glycosidic bond, the -OH group on the anodic carbon of the sugar molecule is oriented below the plane of the ring. This causes the linked substituent to protrude downwards. In a β-glycosidic bond, the -OH group on the anodic carbon of the sugar molecule is oriented above the plane of the ring. This causes the linked substituent to protrude upwards.

[0077] In α-x,y-glycosidic bonds, the numbers x and y refer to the carbon atoms involved in the glycosidic bond. For example, in an α-1,6-glycosidic bond, the bond can be formed between the anomeric carbon at position 1 (C1; x=1) of one sugar molecule and a hydroxyl group attached to the carbon at position 6 (C6; y=6) of another sugar molecule, where the bond is in the α configuration.

[0078] In the context of this disclosure, the terms “terpenoids,” “terpenoid-like compounds,” “isoprene-like compounds,” “isoprene-like compounds,” “terpenes,” and “terpenoid compounds” are used interchangeably herein. “Terpenoids” refers to compounds derived from two isomers of five-carbon compounds (dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP)). In plants, DMAPP and IPP are synthesized via two distinct and independent biochemical pathways: the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in the plastids. In the downstream terpenoid pathway, DMAPP and IPP condense to produce isopentenyl diphosphate substrates, such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and geranylfarnesyl diphosphate (GFPP). These isopentenyl diphosphate intermediates are utilized by terpene synthases to produce various terpenoids. Based on the number of isoprene units, terpenoids are classified into C5 sesquiterpenes, C6 sesquiterpenes, C7 sesquiterpenes, C8 sesquiterpenes, C9 ... 10 Monoterpenoids, C 15 Sesquiterpenoids, C 20 Diterpenoids, C 25 Disesquiterpenoids, C 30 Triterpenoids, C 40 Tetraterpenoids and C >40 Polyterpenoids. Examples of terpenoids include mogroside, which is a C16 compound. 30 Triterpenoids have recently gained significant commercial importance as alternative high-intensity sweeteners.

[0079] The terms "monkhorinoside" and "monkhorinoside compound" are used interchangeably in this document and refer to C 30The triterpenoid glycoside family. Non-limiting exemplary examples of mogrosides include mogroside V, sarmandin I, mogroside IVE, isomogroside V, mogroside IIIE, 11-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 IIIE, 11-oxo-mogroside IVE, mogroside IE, mogroside alcohol, 11-oxo-mogroside alcohol, mogroside IIE, mogroside IIIA2, and mogroside III, which have been derived from mogroside (Monk Fruit). Siraitia grosvenorii It was identified in the fruit of Swingle that it is responsible for the fruit's sweetness.

[0080] In the context of this disclosure, the term "modified glycosyl receptor" (e.g., "modified terpene" or "modified mogroside") refers to a glycosyl receptor (e.g., a terpene or mogroside) modified with at least one additional glycosyl group. The glycosyl receptor can be modified at multiple sites. Modification can involve different types of glycosidic bonds (e.g., α-glycosidic bonds and / or β-glycosidic bonds).

[0081] As used herein, the terms “isoprenediphosphoric acid” and “isoprenediphosphoric acid” are used interchangeably.

[0082] The mevalonate pathway (also known as the isoprenoid pathway or HMG-CoA reductase pathway) is an essential metabolic pathway found in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon structural units called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The key enzyme is acetoacetyl-CoA sulfonase (AJ). Figure 2 The enzyme is represented as AACT in Chinese, and HMG-CoA synthase (HMG-CoA synthase) is also called AACT. Figure 2 HMGS and HMG-CoA reductase (represented in Chinese) Figure 2 The Chinese text refers to HMGR, mevalonate kinase (...). Figure 2 The Chinese character for MVK is used to represent mevalonate kinase (MVK). Figure 2 The enzyme is represented as PMVK in Chinese, and mevalonate diphosphate decarboxylase (PMVK) is also known as mevalonate diphosphate decarboxylase. Figure 2 (represented as MDD) and isopentenyl diphosphate isomerase ( Figure 2 (represented as IPI in Chinese).

[0083] The term "terpene synthase" refers to an enzyme that catalyzes the cyclization of isoprenyl diphosphate substrates (such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and geranylfarnesyl diphosphate (GFPP)), resulting in the formation of various (poly)cyclic terpene structures. Terpene synthases are classified into different types based on the type of product they produce. For example, monoterpene synthases produce monoterpenes, sesquiterpene synthases produce sesquiterpenes, and diterpene synthases produce diterpenes. Based on their structure and catalytic properties, terpene synthases can also be divided into two classes: Type I and Type II. Type I enzymes possess the highly conserved DDXXD motif (α-domain) and the less conserved NSE / DTE motif metal (Mg2+ or Mn2+)-binding domain required for catalysis. Type II enzymes have a DXDD motif (β-domain) near the N-terminus, which is crucial for the protonation and cyclization of GGPP to convert to copper diphosphate / cyclic diterpene diphosphates. Another conserved motif, R(R)X8W, was found downstream of the N-terminus of many TPS genes. Terpene synthases are also classified according to their gene structure, with class I containing 12–14 introns, class II containing 9 introns, and class III containing 6 introns. Based on recent classifications based on sequence similarity and functional characteristics, the TPS gene family can be further classified into eight subfamilies: 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 the others belong to type I.

[0084] When referring to compounds, the term "precursor" is a compound that precedes another compound in a metabolic pathway or synthetic process. In other words, a precursor molecule can be converted into another molecule through one or more chemical reaction steps and / or one or more biological (e.g., enzymatic) process steps. For example, mogroside IIE is a precursor of mogroside IIIE.

[0085] In the context of this disclosure, the term "contact" refers to the interaction or association between a biocatalyst (e.g., an enzyme, a cell), a glycosyl donor, and a glycosyl acceptor. The contact process involves the physical association of the biocatalyst with the glycosyl donor and glycosyl acceptor in a manner that allows a catalytic reaction to occur.

[0086] In the context of this disclosure, the term "living cell" (as disclosed herein) refers to a unit of life defined by a selectively permeable lipid bilayer called the cell membrane, which separates its internal ("intracellular") environment from its external ("extracellular") environment. Cells contain genetic material, typically DNA, which encodes instructions for cellular structure and function. In eukaryotic cells, this genetic material is organized into chromosomes within the membrane-bound nucleus, or in nucleoid regions in prokaryotic cells. The ability to perform basic cellular functions, respond to stimuli, and maintain a certain level of metabolic activity is considered an indicator of a living state. For clarity, "living cell" is not a lysed cell, not a dead cell, not a crude cell extract, and not a cell-free extract. "Living cell" can be a "proliferating cell" or a "resting cell." "Living cell" can be an "undamaged cell" or a "permeable cell."

[0087] The term "proliferating cell" in this article refers to a cell undergoing cell division, in which a single parent cell divides to produce two or more daughter cells.

[0088] The term "resting cell" in this article refers to a cell that is in a non-dividing or quiescent state. Resting cells are living cells, meaning they are alive and capable of resuming growth (i.e., a proliferative state) when conditions become more favorable.

[0089] The term "undamaged cell" in this article refers to a cell that has an undisturbed and fully functional cell membrane, maintaining its natural barrier properties. In other words, an undamaged cell maintains the integrity of its natural membrane.

[0090] In the context of this disclosure, when referring to cells, the terms "permeability," "osmosis," "alteration of the cell membrane," etc., refer to cells whose cell membranes have been intentionally or unintentionally altered to allow certain substances that would not normally pass through the membrane to pass through. In the context of this disclosure, cell permeability can facilitate the entry or exit of specific molecules (e.g., glycosyl donors such as sucrose) into or out of the cell interior or exterior. In the context of this disclosure, a "permeable cell" is a living cell. Methods for permeating cells are well known in the art.

[0091] In the context of this disclosure, the terms “extracellular” and “extracellular” are used interchangeably. When referring to polypeptides (e.g., enzymes) and / or any other molecules (e.g., glycosyl donors, such as sucrose; glycosyl acceptors, such as terpenoids), the term “extracellular” indicates that the polypeptide and / or molecule is located outside the cell, and the cell is an intact cell. This space is generally considered to be outside the cell’s plasma membrane and / or cell wall and is occupied by a fluid (e.g., culture medium, fermentation broth, reaction buffer). The extracellular polypeptide and / or molecule can be provided (i.e., fed) to the cell extracellularly. For example, the extracellular polypeptide and / or molecule can be added to a fluid (or liquid) in which cells are present, such as a culture medium, fermentation broth, or reaction buffer. Alternatively or additionally, the extracellular polypeptide and / or molecule can be produced by the cell and subsequently secreted or transported outside the cell. For example, when “extracellular” refers to a polypeptide expressed by a cell, the polypeptide can be “secreted in a free (i.e., completely released) manner outside the cell” or “displayed on the surface of the cell.” The term "extracellular" contrasts with "intracellular" or "inside the cell."

[0092] When referring to peptides (such as enzymes), the term "secretion" refers to the process by which a cell releases the peptide into the extracellular environment or transports it to a specific cellular compartment. Proteins can be secreted outside the cell through various mechanisms. In the context of this disclosure, peptides can be secreted "in free outside the cell" or "displayed on the surface of the cell."

[0093] When referring to peptides (such as enzymes), the term "displayed on the cell surface" or "surface display" indicates that the peptide is presented and anchored on the extracellular surface of the cell. For this purpose, a gene encoding the target peptide is fused with a gene encoding a surface-display anchoring protein. This anchoring protein is typically a component of the cell membrane that facilitates protein attachment to the cell surface. The engineered fusion gene is introduced into cells (e.g., bacterial, yeast, or mammalian cells) using transformation (for bacteria and yeast) or transfection (for mammalian cells). The cell expresses the fusion protein, resulting in the simultaneous expression of both the anchoring protein and the target peptide. The anchoring protein guides the display of the target peptide on the cell surface.

[0094] When referring to peptides (such as enzymes), the terms "extracellularly free" and "displayed on the surface of the cell" are distinctly different. The peptide does not contain surface-displayed anchoring proteins. The peptide may interact with the extracellular surface of the cell, but is not anchored to the surface as defined above.

[0095] In the context of this disclosure, the terms "intracellular" and "inside the cell" are used interchangeably. When referring to polypeptides (e.g., enzymes) and / or any other molecules (e.g., glycosyl donors, such as sucrose; glycosyl acceptors, such as terpenoids), the term "intracellular" indicates that the polypeptide and / or molecule is located inside a cell, which is an intact cell. The term "intracellular" is distinctly different from "extracellular."

[0096] In the context of this disclosure, when referring to cells expressing enzymes (such as the cells disclosed herein), the terms "intact cell" and "intact cell biocatalyst" are used interchangeably and refer to a living cell, typically a microorganism such as bacteria or yeast, used as a catalyst to carry out a specific biochemical reaction. Generally, an intact cell biocatalyst contains one or more enzymes for converting a substrate into a desired product. The intact cell biocatalyst may be genetically modified or selected to carry out a specific reaction, such as producing a desired product or precursor. Intact cells can be used in fermentation processes and / or biotransformation processes.

[0097] As used herein, the terms “biotransformation”, “biotransformation process”, etc., refer to the process in which a compound (e.g., a terpene) is modified by a living organism. This can involve introducing, modifying, or removing specific groups (e.g., glycosyl groups) from a compound to produce a modified compound (e.g., a modified terpene).

[0098] As used herein, the terms “fermentation,” “fermentation,” and “fermenting” refer to the process by which microorganisms (e.g., bacteria, yeast, or fungi) convert a carbon source (e.g., carbohydrates) into a desired product (e.g., modified terpenoids). Carbon sources typically serve as substrates for microbial 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 because it is a simple sugar that can be readily metabolized by many microorganisms.

[0099] "Batch fermentation" is defined in this paper as fermentation in which all nutrients are added at the start of fermentation and the fermentation process continues until the desired product is obtained or fermentation is stopped.

[0100] "Feed-on-batch fermentation" or "semi-batch fermentation" is a fermentation in which an initial growth medium is added to the bioreactor at the beginning (similar to a batch process), but additional nutrients, substrates or feedstocks are introduced into the bioreactor intermittently or continuously during the culture period, and the desired product is retained in the bioreactor until the end of the run.

[0101] "Continuous fermentation" is a fermentation in which nutrients are continuously added to the fermentation process and in which the desired product or fermentation broth is continuously removed from the bioreactor.

[0102] As used herein, the terms "culture medium" and "growth medium" are used interchangeably to refer to a liquid or solid medium that supports cell growth. Typically, a culture medium may contain one or more of the following: carbon source (e.g., glucose, fructose, sucrose, xylose, glycerol, plant biomass, cellulose, hemicellulose, pectin, rhamnose, galactose, fucose, maltose, maltodextrin, ribose, ribulose, or starch, starch derivatives, lactose, fatty acids, and triglycerides). Typically, the medium may also contain a nitrogen source (e.g., urea) or an ammonium salt (e.g., ammonium sulfate, ammonium chloride, ammonium nitrate, or ammonium phosphate). The medium may further contain trace metals, vitamins, salts, amino acids, etc. Trace metals can be divalent cations, including but not limited to Mn²⁺, Mg²⁺, Fe²⁺, Cu²⁺, etc.

[0103] The term "fermentation broth" refers to a liquid culture medium containing nutrients and microorganisms used in the fermentation process.

[0104] As used herein, in the context of enzymatic reactions (using, for example, enzymes, intact cells, cell lysates, cell extracts), the term "reaction mixture" refers to the combination of all components involved in the reaction. Among other things, the reaction typically involves enzymes, substrates, buffer solutions, salts and ions, and products.

[0105] When referring to products as used herein, the terms “isolated” or “recovered” mean products that have been removed or isolated from the cells, fermentation broth, culture medium, crude cell-free extract and / or reaction mixture from which the product was produced.

[0106] As used herein, when referring to a product, “purified” means that the product is present in a higher amount relative to other compounds that are usually found with the indicated compound (e.g., in their natural environment).

[0107] As used herein, “purification” can refer to a method of extracting products from cell lysates and / or supernatants, wherein the cells are secreting products. “Lysates” as used herein comprise the cellular contents of cells after the cell walls and cell membranes have ruptured, and may include, for example, proteins, sugars, and mogrosides. Purification may include ammonium sulfate precipitation to remove proteins, salting out to remove proteins, hydrophobic separation (HPLC), and the use of affinity columns. Given the products produced by the methods described herein, affinity media are considered for the removal of specific mogrosides using adsorption resins.

[0108] As described herein, "HPLC" is a form of liquid chromatography used to separate compounds dissolved in solution. Without limitation, an HPLC instrument may include a mobile phase reservoir, a pump, a syringe, a separation column, and a detector. The compounds can then be separated by injecting a sample mixture onto the column. Due to the difference in partitioning behavior between the mobile liquid phase and the stationary phase, different components in the mixture channel can pass through the column at different rates. Several types of columns can be used. Without limitation, the column can be a normal-phase column, a reversed-phase column, a size exclusion column, or an ion-exchange column.

[0109] As used herein, “sweetener,” “sweet flavoring agent,” “sweet flavor entity,” “sweet compound,” or “sweetness compound” means a compound or a physiologically acceptable salt thereof that induces a detectable sweetness in a subject. As used herein, “sweetness modifier” means a compound or a physiologically acceptable salt thereof that enhances, modifies, or improves the perception of sweetness.

[0110] In this paper, “genomic modification” of a cell is defined as any event that results in a change in a polynucleotide in the cell’s genome. A modification is interpreted as one or more modifications. Modifications can be introduced, for example, through classical strain modification (such as random mutagenesis followed by modification). Modifications can be accomplished by introducing (inserting), substituting (or replacing amino acids), or removing (deleting) one or more nucleotides in a polynucleotide. Such modifications can, for example, occur in regulatory elements or coding sequences required for the transcription or translation of polynucleotides. For example, nucleotides can be inserted or removed to result in the introduction of a stop codon, the removal of a start codon, or a change or frameshift in the open reading frame of a coding sequence. Modification of coding sequences or their regulatory elements can be accomplished by site-directed or random mutagenesis, DNA shuffling methods, DNA recombination methods, gene synthesis (see, for example, Young and Dong (2004), Nucleic Acids Research 32, (7) electronic access http: / / nar.oupjournals.org / cgi / reprint / 32 / 7 / e59, or Gupta et al. (1968), Proc. Natl. Acad. Sci USA, 60: 1338-1344; Scarpulla et al. (1982), Anal. Biochem. 121: 356-365; Stemmer et al. (1995), Gene 164:49-53) or by mutagenesis produced by PCR according to methods known in the art. Examples of random mutagenesis procedures are well known in the art, such as chemical (e.g., NTG) mutagenesis or physical (e.g., UV) mutagenesis.Examples of directed mutagenesis procedures are the QuickChange® Site-Directed Mutagenesis Kit (Stratagene Cloning Systems, La Jolla, CA), 'The Altered Sites® II in vitro Mutagenesis Systems' (Promega Corporation), or PCR using the method described in Gene. 1989 Apr15;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 PCR using overlap extension as described in "Molecular Biology: Current Innovations and Future Trends." (Eds. AM Griffin and HG Griffin. ISBN 1-898486-01-8;1995 Horizon Scientific Press, PO Box 1, Wymondham, Norfolk, UK).

[0111] Modifications in the genome can be determined by comparing the polynucleotide sequences of modified cells with those of unmodified cells. Polynucleotide sequencing and genome sequencing can be performed using standard methods known to those skilled in the art, such as Sanger sequencing technology and / or next-generation sequencing technologies like Illumina GA2, Roche 454, etc., as reviewed in Elaine R. Mardis (2008), Next-Generation DNA Sequencing Methods, Annual Review of Genomics and Human Genetics, 9: 387-402. (doi:10.1146 / annurev.genom.9.081307.164359).

[0112] Exemplary modification methods are based on techniques of gene substitution, gene deletion, or gene disruption.

[0113] For example, in the case of replacing a polynucleotide, polynucleotide construct, or expression cassette, a suitable polynucleotide can be introduced at the target locus to be replaced. The suitable polynucleotide can be present on the cloning vector. An exemplary integration cloning vector contains a DNA fragment homologous to the polynucleotide and / or homologous to the polynucleotide flanking the locus to be replaced, to target the integration of the cloning vector into the predetermined locus. To facilitate targeted integration, the cloning vector can be linearized prior to transformation of microorganisms. In some embodiments, linearization is performed such that at least one or both ends of the cloning vector are flanked by a polynucleotide sequence homologous to the polynucleotide (or flanking sequence) to be replaced. This process is called homologous recombination, and this technique can also be used to achieve (partial) gene deletion or gene disruption.

[0114] For example, in the case of gene disruption, a polynucleotide corresponding to an endogenous polynucleotide can be replaced with a defective polynucleotide, i.e., a polynucleotide that cannot produce a (fully functional) protein. Through homologous recombination, the defective polynucleotide replaces the endogenous polynucleotide. It may be expected that the defective polynucleotide also encodes a marker that can be used to select transformants in which the polynucleotide has been modified.

[0115] Alternatively, modification can be performed using established antisense techniques with polynucleotides complementary to the polynucleotide encoding the disclosed polypeptide, thereby creating cells with defects in the polypeptide as disclosed herein. More specifically, the expression of the polynucleotide encoding the disclosed polypeptide in recombinant cells can be reduced or eliminated by introducing a polynucleotide having a sequence complementary to the sequence of the polynucleotide encoding the disclosed polypeptide, which can be transcribed in the recombinant cells and is capable of hybridizing with the mRNA encoding the polypeptide produced in the recombinant cells. Under conditions that allow the complementary antisense polynucleotide to hybridize with the polypeptide, the amount of translated protein is thus reduced or eliminated. Examples of antisense RNA expression are 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 potatouridinediphosphate-glucose pyrophosphorylase and its inhibition by antisenseRNA. Planta. (1993); 190(2):247-52).

[0116] Furthermore, the modification, downregulation, or inactivation of peptides can be achieved using RNA interference (RNAi) technology (FEMS Microb. Lett. 237 (2004): 317-324). In this method, the same sense and antisense moieties of a peptide encoding a polynucleotide to be affected (e.g., an invertase) are sequentially cloned, separated by a nucleotide spacer, and inserted into an expression vector. After transcription, the formation of small nucleotide fragments leads to the targeted degradation of the mRNA, which is then affected. The elimination of specific peptide mRNAs can be of varying degrees. RNA interference techniques described in WO2008 / 053019, WO2005 / 05672A1, WO2005 / 026356A1, Oliveira et al., “Efficient cloning system for construction of genesilencing 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, can be used to downregulate, modify, or inactivate polynucleotides.

[0117] To increase the likelihood that the introduced enzyme is expressed in its active form in cells as disclosed herein, the corresponding encoding polynucleotide can be adapted to optimize codon usage to the codon usage of the selected modified cells. The adaptability of the polynucleotide encoding the enzyme to the codon usage of the selected cells can be expressed as the codon fitness index (CAI). The codon fitness index is defined herein as a measure of the relative fitness of a gene’s codon usage relative to the codon usage of a highly expressed gene. The relative fitness (w) of each codon is the ratio of the usage of each codon to the usage of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative fitness values. Nonsynonymous codons and stop codons (depending on the 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; see also: Jansen et al. (2003), Nucleic Acids Res.31(8):2242-51). The adapted polynucleotide may have a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.

[0118] The cells disclosed herein can be genetically modified with polynucleotides, which are adapted to the codon usage of the cells using codon pair optimization techniques well known to those skilled in the art. Codon pair optimization is a method for producing polypeptides in cells, wherein the polynucleotide encoding the polypeptide has been modified relative to its codon usage, particularly the codon pair used, to obtain increased expression levels of the polynucleotide encoding the polypeptide and / or increased polypeptide yield. A codon pair is defined as a set of two consecutive triplets (codons) in a coding sequence.

[0119] Further enhancements in in vivo enzyme activity as disclosed herein can be achieved through well-known methods such as error-prone PCR or directed evolution. An exemplary method of directed evolution is described in WO03010183 and WO03010311.

[0120] As used herein, the term "marker" refers to a gene encoding a trait or phenotype that allows selection or screening of recombinant microorganisms containing the marker. The marker gene may be an antibiotic resistance gene, thereby allowing selection of transformed cells from untransformed cells using an appropriate antibiotic. Alternatively, non-antibiotic resistance markers, such as auxotrophic markers (URA3, TRP1, LEU2), may also be used. Recombinant cells transformed with a polynucleotide construct may be marker-free. A method for constructing recombinant cells without recombinant marker genes is disclosed in EP-A-0 635 574, based on the use of bidirectional markers. Alternatively, selectable markers (e.g., Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, β-glucuronidase) may be incorporated into the polynucleotide constructs disclosed herein, thereby allowing screening of transformed cells. An exemplary marker-free method for introducing heterologous polynucleotides is described in WO0540186.

[0121] "Sequence identity" is defined herein as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences determined by sequence comparison. Typically, sequence identity or similarity is compared across the entire length of the sequences being compared. For the purposes of this disclosure, sequences are aligned to achieve optimal comparison in order to determine the percentage of sequence homology or sequence identity between two amino acid sequences or two nucleic acid sequences. To optimize the alignment between two sequences, a gap may be introduced in either of the two sequences being compared. This alignment may be performed across the entire length of the sequences being compared. Alternatively, it may be performed over a shorter length, such as about 20, about 50, about 100, or more nucleic acid / base or amino acid sequences. Sequence identity is the percentage of identical matches between two sequences in the reported aligned region.

[0122] Sequence comparison and determination of the percentage of sequence identity between two sequences can be achieved using mathematical algorithms. Those skilled in the art will recognize that several different computer programs can be used to align two sequences and determine their identity (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percentage of sequence identity between two amino acid sequences or two nucleotide sequences can be determined using the Needleman-Wunsch algorithm used for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid and nucleotide sequences can be aligned using algorithms. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of this disclosure, the NEEDLE program from the EMBOSS software package (version 2.8.0 or later, 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 / ) was used. For protein sequences, EBLOSUM62 was used for the substitution matrix. For nucleotide sequences, EDNAFULL was used. Optional parameters used were a gap opening penalty of 10 and a gap extension penalty of 0.5. Those skilled in the art will understand that all these different parameters will produce slightly different results, but the overall percentage of identity between the two sequences will not change significantly when different algorithms are used.

[0123] After alignment using the NEEDLE procedure as described above, the percentage of sequence identity between the query sequence and the sequence of this disclosure is calculated as follows: the number of corresponding positions showing the same amino acid or nucleotide in both sequences divided by (the total length of the alignment minus the total number of gaps in the alignment). Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "Longest Identity" in the program's output.

[0124] The nucleic acid and protein sequences disclosed herein can also be used as "query sequences" to perform searches against public databases, such as to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTX programs (version 2.0) of Altschul et al. (1990) J. Mol. (1990) Biol. 215:403-10. BLAST nucleotide searches can be performed using the BLASTN program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of this disclosure. BLAST protein searches can be performed using the BLASTX program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of this disclosure. For gapped alignments for comparative purposes, gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters for the respective programs (e.g., BLASTX and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / .

[0125] In the context of this disclosure, the terms "functional homolog," "functional equivalent," and "functional variant" are used interchangeably. A functional homolog of a polypeptide is at least one polypeptide that is identical to the polypeptide in biological function and / or one activity. Typically, a functional homolog has a certain level of sequence similarity or identity with the amino acid sequence of the polypeptide, typically having at least 50% sequence identity, 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. Compared to the amino acid sequence of the polypeptide, the amino acid sequence of a functional homolog of the polypeptide may contain one or more amino acid substitutions, deletions, or additions.

[0126] Functional homologs of peptides (also referred to as reference peptides) can be naturally occurring peptides, such as homologs, orthologs, or paralogs of the peptide. Functional homologs can be identified by analyzing nucleotide and peptide sequence alignments. For example, searching databases of nucleotide or peptide sequences can identify homologs of the reference peptide. Sequence analysis can involve basic local alignment search tools, such as protein BLAST, nucleotide BLAST, and SmartBLAST analyses using non-redundant databases of the reference peptide's amino acid sequences. In some cases, the amino acid sequence is deduced from the nucleotide sequence, and in such cases, BLASTX can be used. Peptides in the database that have greater than 40% sequence identity with the reference peptide are candidates for further evaluation of their suitability as functional homologs of the peptide. Amino acid sequence similarity allows for conserved amino acid substitutions, such as substituting one hydrophobic residue for another or one polar residue for another. If necessary, such candidates can be manually examined to narrow down the number of candidates to be further evaluated. Manual examination can be performed by selecting candidates that appear to possess domains (e.g., conserved functional domains) present in the reference peptide. In some implementations, nucleic acids and peptides are identified based on expression levels rather than from transcriptome data using BLAST analysis. Conserved regions can be identified by locating regions within the primary amino acid sequence of a reference peptide that are repetitive sequences, form some secondary structure (e.g., helices and β-sheets), establish positively or negatively charged domains, or represent protein motifs or domains. For example, the InterPro database (see Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, Bileschi ML, Bork P, Bridge A, Colwell L, Gough J, Haft DH, Letun) I, Marchler-Bauer A, Mi H, Natale DA, Orengo CA, Pandurangan AP, Rivoire C, Sigerst 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 identify conserved regions in reference peptides. Conserved regions can also be identified by comparing sequences of the same or related peptides from closely related species. Closely related species are preferably from the same family. In some embodiments, comparison of sequences from two different species is sufficient to identify such homologs.

[0127] Alternatively, functional homologs can be synthesized using a variety of techniques known to those skilled in the art. For example, the amino acid sequences of potential functional homologs of peptides can be generated using protein engineering tools known to those skilled in the art, such as directed evolution (Arnold, FH 2001. "Combinatorial and computational challenges for biocatalyst design." Nature 409:253–257; Powell, KA, SWRamer, SB del Cardayre, WPC Stemmer, MB Tobin, PF Longchamp, and GW Huisman. 2001. "Directed evolution and biocatalysis." Angewandte Chemie-International Edition 40:3948–3959; Rohlin, L., MK Oh, and JCLiao. 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, QS, U. Schwaneberg, M. Fischer, J. Schmitt, J. Pleiss, S. Lutz-Wahl, and RD Schmid. 2001. “Rationalevolution of a medium chain-specific cytochrome P-450 BM-3 variant.” Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology1545:114–121; Looger, LL, MA Dwyer, JJ Smith, and HW Hellinga.2003.“Computational design of receptor and sensor proteins with novel functions.” Nature 423:185–190; Voigt, CA, SL Mayo, FH Arnold, and ZG 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., TE Ferrin, and JD Keasling. 2006. “Designed divergent evolution of enzyme function.” Nature 440:1078–1082). Techniques for modifying genes encoding the functional peptides described herein are known and include, in particular, directed evolution, site-directed mutagenesis, and random mutagenesis, and can be used to increase the specific activity of peptides, alter substrate specificity, change expression levels, alter subcellular locations, or modify peptide-peptide interactions in desired ways. Such modified peptides are considered functional homologs. The term "functional homolog" is sometimes applied to nucleic acids that encode functional homologous polypeptides. Detailed Implementation

[0128] In the context of this disclosure, it has been surprisingly discovered that heterologous enzymes capable of transferring glycosyl groups from glycosyl donors to glycosyl receptors can be functionally expressed in recombinant cells, whether intracellularly and / or extracellularly (free outside the cell or displayed on the outer surface of the cell), thereby enabling robust cellular production platforms and methods for the intracellular (in vivo) and / or extracellular production of modified glycosyl receptors, particularly α-glycosylated glycosyl receptors (e.g., α-glycosylated terpenoids, such as α-glycosylated mogroside).

[0129] Therefore, the object of this disclosure is to provide a method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor (and / or its precursor), the method comprising: contacting the glycosyl donor and the glycosyl acceptor (and / or its precursor) with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (over)expressing a polynucleotide encoding at least one heterozyme, said (hetero)enzyme being capable of catalyzing the transfer of a glycosyl group from said glycosyl donor to said glycosyl acceptor (and / or its precursor), wherein said contact under suitable conditions enables said at least one (hetero)enzyme to catalyze a glycosidic bond (e.g., between the transferred glycosyl group and said glycosyl acceptor (and / or its precursor)) between the glycosyl group and said glycosyl acceptor (and / or its precursor). The formation of glycosidic bonds; thereby producing modified glycosyl receptors. Typically, under suitable conditions, this contact allows the recombinant cells to express at least one (heterologous) enzyme.

[0130] Another object of this disclosure is to provide a method for producing a modified glycosyl receptor, comprising: contacting a glycosyl donor and a glycosyl receptor (and / or its precursor) with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (over)expressing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl receptor (and / or its precursor), wherein said contact under suitable conditions comprises culturing the recombinant cells in a suitable culture medium in the presence of the glycosyl donor and the glycosyl receptor and / or its precursor, wherein said contact under suitable conditions enables said at least one heterozyme to catalyze the formation of a glycosidic bond between the transferred glycosyl group and the glycosyl receptor (and / or its precursor), thereby producing a modified glycosyl receptor, and optionally, isolating the thereby produced modified glycosyl receptor. Typically, said contact under suitable conditions allows the recombinant cells to express said at least one heterozyme.

[0131] Another aspect of this disclosure provides a method for producing a modified glycosyl receptor, comprising: contacting a glycosyl donor and a glycosyl receptor (and / or its precursor) with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (over)expressing at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl receptor (and / or its precursor), wherein the contact under suitable conditions comprises contacting the recombinant cells, the glycosyl donor, and the glycosyl receptor (and / or its precursor) in a reaction mixture, and wherein the contact under suitable conditions enables said at least one heterozyme to catalyze the formation of a glycosidic bond between the transferred glycosyl group and the glycosyl receptor (and / or its precursor), thereby producing a modified glycosyl receptor, and optionally, isolating the thereby produced modified glycosyl receptor. Typically, the contact under suitable conditions allows the recombinant cells to express said at least one heterozyme.

[0132] Typically, in the method according to this disclosure, "contacting the glycosyl donor and glycosyl acceptor with the recombinant cell under suitable conditions" can mean: a) The glycosyl donor and glycosyl acceptor come into contact within the recombinant cell (intracellular); typically, at least one heterologous enzyme capable of catalyzing the transfer of glycosyl groups from the glycosyl donor to the glycosyl acceptor is also present intracellularly (i.e., the heterologous enzyme is an intracellular enzyme), and / or b) The glycosyl donor and glycosyl acceptor are in contact outside the recombinant cell (extracellular); typically, at least one heterologous enzyme capable of catalyzing the transfer of glycosyl groups from the glycosyl donor to the glycosyl acceptor is also present outside the cell (i.e., the heterologous enzyme is an extracellular enzyme).

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

[0134] The methods disclosed herein for generating modified glycosyl receptors can be fermentation, biotransformation, or a combination thereof.

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

[0136] In the method, the recombinant cells can be live cells or intact cells.

[0137] Figure 6 The reactions that occur in the methods and cells according to this disclosure are described.

[0138] Enzymes used to transfer glycosyl groups from glycosyl donors to glycosyl acceptors In the method according to this disclosure, the recombinant cell contains, (over)expresses, or is capable of (over)expressing a polynucleotide encoding at least one heterozyme, said heterozyme being capable of catalyzing the transfer of glycosyl groups from a glycosyl donor to a glycosyl acceptor.

[0139] In one embodiment of this disclosure, the heterologous enzyme capable of catalyzing the transfer of glycosyl groups to glycosyl acceptors is an enzyme capable of catalyzing glycosylation reactions, more specifically transglycosylation reactions. The enzyme may be a glycosyltransferase and / or a glycosidase (i.e., a glycosidase). Preferably, the enzyme belongs to the glycosidase family 70 (GH70), the glycosidase family 13 (GH13), and / or the glycosidase family 77 (GH77).

[0140] In another embodiment, the heterologous enzyme capable of catalyzing the transfer of glycosyl groups to glycosyl acceptors is preferably a glycosyltransferase, more preferably a non-Leloir glycosyltransferase. One advantage of non-Leloir glycosyltransferases is that they use commercially available donors, such as inactive sugars, as donors when catalyzing transglycosylation reactions.

[0141] Examples of non-Leloir glycosyltransferases include, but are not limited to, glucan sucrase belonging to the GH70 family and cyclic maltodextrin glucan transferase (CGTase) belonging to the GH13 family. These enzymes, together with enzymes from the GH77 family, form the GH-H group, sharing common mechanisms, structural features, and evolutionary characteristics.

[0142] Dextran sucrase typically uses sucrose as a glycosyl donor and catalyzes reactions via an α-retention double substitution mechanism. First, the α-glycosidic bond of the donor sucrose is broken, leading to the formation of a β-glycosyl-enzyme intermediate. Second, a glycosyl acceptor (e.g., a terpene compound) attacks the β-glycosyl-enzyme intermediate, and subsequently, the glycosyl moiety is transferred to the acceptor while retaining the α-anomeric conformation; thereby resulting in the formation of an α-glycosidic bond.

[0143] Examples of dextran sucrases include, but are not limited to, dextran sucrase (EC 2.4.1.5), alternosaccharide sucrase (EC 2.4.1.140), polysaccharide sucrase (EC 2.4.1.5), and reuteransucrase (EC 2.4.1.5). Therefore, in another embodiment of this disclosure, the heterozyme capable of catalyzing glycosyl transfer to the glycosyl acceptor is an enzyme selected from the group consisting of dextran sucrase, alternosaccharide sucrase, polysaccharide sucrase, and reuteransucrase.

[0144] CGTase (EC 2.4.1.19) typically uses starch as a glycosyl donor and, similar to dextran sucrase, catalyzes α-glycosidic bonds during glycosylation reactions.

[0145] Therefore, in another embodiment, the heterozyme capable of catalyzing the transfer of glycosyl groups to glycosyl receptors is an enzyme capable of catalyzing the α-glycosidic bond between the glycosyl group and the glycosyl receptor.

[0146] In the receptor reaction described above, a glycosyl group can be transferred to a hydroxyl group present in the backbone of the glycosyl receptor (e.g., the mogroside backbone) and / or to a hydroxyl group of a sugar moiety attached to the backbone of the glycosyl receptor; thereby producing a modified glycosyl receptor containing a glycosyl group. When the reaction occurs between the glycosyl group and the sugar moiety in the backbone of the glycosyl receptor, the heterozyme of this disclosure can catalyze α-1,3-glycosidic bonds, α-1,6-glycosidic bonds, and / or α-1,4-glycosidic bonds. In one example, the enzyme is dextran sucrase and is capable of catalyzing α-1,6-glycosidic bonds and / or α-1,3-glycosidic bonds. In another example, the enzyme is CGTase and is capable of catalyzing α-1,4-glycosidic bonds.

[0147] Enzymes used to transfer glycosyl groups from glycosyl donors to glycosyl acceptors: Dextran sucrase (DexT) In a preferred embodiment of this disclosure, the heterologous enzyme capable of catalyzing glycosyl transfer to glycosyl acceptors is a dextran sucrase, preferably dextran sucrase (EC 2.4.1.5). Any dextran sucrase can be used in the methods and recombinant cells according to this disclosure. Suitable non-limiting examples of dextran sucrase that can be used in the methods and recombinant cells according to this disclosure are those listed 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 dextran sucrase variant disclosed in Table 2 below.

[0148] According to all aspects and embodiments herein, at least one DexT enzyme disclosed herein, its truncated or engineered variants, can be used alone or in combination in an in vivo (i.e., at least partially via fermentation) or in vitro (biocatalysis outside living cells, i.e., in vivo or after synthesization of the synthase) glycosylation reaction, or in a combination of at least one in vivo process and at least one in vitro process (see, for example, Example 5), to obtain compound (I) molecules with particularly higher yields and / or quality compared to prior art methods.

[0149] Some dextran sucrase enzymes (alone or in combination) exhibit progressive activity, resulting in compound (I) molecules having one or more additional α1-6 glycosides linked to the first α1-6 glucose already present in compound (I) (poly-α-glycosylated compound (I)); or exhibit nonspecific activity, resulting in mogrosides containing different α-glycosidic linkages that lead to compounds different from compound (I), such as enzymes as shown in Table 16 and detailed in Example 1.

[0150] The inventors have surprisingly discovered that reducing the size of DexT by truncation can improve the functional expression and production of compound (I). Advantageous truncated variants of DexT are shown, but not limited to, those detailed in Table 17 (see Example 2), which can be used alone or in combination for even further improvements in the production of compound (I).

[0151] In some embodiments, DexT or its mutants or variants according to various aspects of this disclosure (e.g., as shown in Table 18, but not limited thereto) can be advantageously combined with α-glycosidase (AGL) / dextranase to achieve even higher levels of compound (I) in various biocatalytic processes compared to samples without AGL. The inventors have surprisingly discovered that α-glycosidase is capable of catalyzing the hydrolysis of poly-α-glycosylated compound (I) formed by the progressive activity of DexT to improve the yield of compound (I).

[0152] In yet another embodiment, the engineered DexT mutants / variants described below may be used alone or in combination, for example, but not limited to, as shown in Tables 19, 20 and 21, to achieve improved production of compound (I).

[0153] In yet another embodiment, at least one DexT enzyme, mutant, or variant disclosed herein can be expressed in cells capable of producing mogroside, and these cells are deficient in or lack certain β-glucanase activities to achieve even higher amounts of the Mog3E substrate and subsequent compound (I).

[0154] In some preferred embodiments, the strains according to this disclosure can be engineered to produce sucrose in situ, which can be advantageously used with at least one DexT enzyme, mutant or variant disclosed herein, for example, but not limited to, as shown in Tables 20-21, alone or in combination, optionally with the presence of AGL enzyme activity.

[0155] In yet another preferred embodiment, at least one DexT enzyme, mutant or variant disclosed herein, preferably at least one of mutants ID 18, 226, 232, 251 and 291, can be used in combination with in situ sucrose production during in vivo fermentation of compound (I) using DexT to conveniently obtain significant amounts of compound (I) in such a suitable production strain in a simplified, reliable and therefore cost-effective manner.

[0156] For example, the dextran sucrase can be derived from... Lactobacillus reuteri,For example, Lreu180gtf180 (SEQ ID NO: 1). Truncated versions of the N-end and / or C-end of Lreu180 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). Alternatively, versions containing the N-end and / or C-end of Lreu180 gtf180 can be used. Lactobacillus reuteri One or more amino acid-modified variants of DexT, such as any DexT variant of Lreu-DexT.orf (SEQ ID NO: 2), namely mutants _0014 to _0197 in Table 2.

[0157] Derived from Oenococcus Other suitable examples of dextran sucrase from species include Oesp_DexT.orf (SEQ ID NO: 28), truncated versions of which at the N-terminus and / or C-terminus include 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 mutants _0198 to _0223 in Table 2.

[0158] Other dextran sucrase are derived from Streptococcus downeiThose, such as the full-length wild-type enzymes GTFS_STRDO dextran sucrase (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), Sdow_DexT.orf_003 (SEQ ID NO: 44), Sdow_DexT.orf_004 (SEQ ID NO: 45), Sdow_DexT.orf_005 (SEQ ID NO: 46), and mutants of Sdow_DexT.orf_001, namely mutants _0224 to _0291 in Table 2.

[0159] Other dextran sucrase derivatives Leuconostoc citreum For example, mutants of 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 Lcit_DexT.orf_0003, namely mutants _0001 to _0013 in Table 2; or derived from Streptococcus mutans Dextran sucrase, such as GTFD_STRMU glucan sucrase (SEQ ID NO: 35), Smut_DexT.orf (SEQ ID NO: 36) and Smut_DexT.orf_0001 (SEQ ID NO: 37).

[0160] The functional homologs of the DexT enzyme disclosed above can also be used in recombinant cells according to the methods of this disclosure. For example, the 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% or at least 100% sequence identity with any of the following SEQ ID NOs: 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 any of the mutants (i.e., mutants _0001 to _0291) described in Table 2.

[0161] Enzymes used to transfer glycosyl groups from glycosyl donors to glycosyl acceptors: Dextran sucrase variant According to one aspect of this disclosure, the dextran sucrase to be used in recombinant cells according to the methods of this disclosure is a dextran sucrase variant of the parent polypeptide. As used herein, the terms "variant" or "mutant" are used interchangeably. They can refer to polypeptides or nucleic acids. Variants include substitutions, insertions, deletions, truncations, transversions, and / or inversions at one or more positions relative to the parent sequence. Variants can be prepared, for example, by site-saturation mutagenesis, scan mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as various other recombinant or synthetic methods. Variant polypeptides may differ from the parent polypeptide by a few amino acid residues and can be defined by their level of primary amino acid sequence homology / identity with a reference polypeptide (e.g., the parent polypeptide). Preferably, the variant polypeptide has 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., the parent polypeptide). Methods for determining percentage identity are known in the art and described herein. Typically, the variant retains the characteristic properties of the parent polypeptide but has altered properties in certain specific aspects.

[0162] According to one embodiment, the variant comprises an amino acid sequence that, when compared with the amino acid sequence of a parent polypeptide (or any reference polypeptide, such as a 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), contains at least one amino acid residue modification corresponding to any amino acid in the amino acid sequence of the parent polypeptide.

[0163] The phrase “amino acid sequence aligned with the amino acid sequence listed in the sequence of the parent polypeptide” (or aligned with the amino acid sequence listed in the sequence of the reference polypeptide, such as 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 amino acid sequence of the variant and the amino acid sequence of the parent polypeptide (or, for example, listed in SEQ ID NO: 21, SEQ ID NO: 2, SEQ ID NO: 34 or SEQ ID NO: 26) are aligned by a suitable method that allows: a) the sequences to be compared with each other, and b) if compared with the amino acid sequence of the parent polypeptide or the reference polypeptide (e.g., listed in SEQ ID NO: 21, SEQ ID NO: 2, SEQ ID NO: 26), to identify the position in the amino acid sequence of the variant where the same amino acid is present (same position), or another amino acid is present (substitution), or one or more additional amino acids are present (insertion or extension), or no amino acid is present (deletion or truncation). Therefore, in the context of this disclosure, modification refers to the substitution, addition, or deletion of any amino acid residues corresponding to any amino acid present in the parent or reference polypeptide when aligned using a suitable method. A suitable method for comparing two amino acid sequences can be any suitable pairwise sequence alignment method known to those skilled in the art, preferably a global pairwise sequence alignment method. The preferred global pairwise sequence alignment method is the EMBOSS Needle method (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453) based on the Needleman-Wunsch alignment algorithm described herein (aimed at finding the optimal alignment of two sequences along their entire length (including gaps)). In one implementation, the NEEDLE procedure from the EMBOSS software package is used, with EBLOSUM62 as the substitution matrix, a 10-point nick opening penalty, and a 0.5-point nick extension penalty, to align the amino acid sequence with the amino acid sequence of the parent polypeptide or a reference polypeptide (e.g., as shown in SEQ ID NO: 21, SEQ ID NO: 2, SEQ ID NO: 34, or SEQ ID NO: 26).

[0164] In one aspect, a dextran sucrase variant (of the parental polypeptide) is disclosed, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 21, contains at least one modification of an amino acid residue corresponding to any of the following amino acids: 274, 275, 276, 319, 322, 406, 478, 481 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 21.

[0165] In one embodiment, the modification is the substitution, addition, or deletion of amino acid residues corresponding to any of the following amino acids: 274, 275, 276, 319, 322, 406, 478, 481 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 21.

[0166] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 21, contains one or more of the following: The value at position 274 is F or L; at position 275 it is Y, F, K, or S; at position 276 it is W; at position 319 it is P; at position 322 it is A; at position 406 it is Q; at position 478 it is Y; and at position 481 it is A. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 21.

[0167] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 21, contains amino acid substitutions or combinations of amino acid substitutions at the following positions: -481, preferably the substitution present in mutant _0001 in Table 2; -319 and 322, preferably the substitutions indicated in mutant _0002 in Table 2; -276 and 319, preferably the substitutions present in mutant _0003 in Table 2; -274 and 319, preferably substitutions present in mutant _0004 or mutant -0005 in Table 2; -322 and 478, preferably the substitutions present in mutant _0006 in Table 2; -406 and 478, preferably the substitutions present in mutant _0007 in Table 2; -319, 406 and 478, preferably the substitutions present in mutant _0008 in Table 2; -275 and 276, preferably substitutions present in mutants _0009, _0010 or _0011 in Table 2; -275, 276 and 319, preferably substitutions present in mutant _0012 or mutant _0013 in Table 2.

[0168] In one embodiment, the parent polypeptide of the dextran sucrase variant comprises either a polypeptide having the amino acid sequence shown in SEQ ID NO: 21 or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21.

[0169] In one embodiment, the dextran sucrase 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 listed in SEQ ID NO: 21.

[0170] According to another aspect of this disclosure, the dextran sucrase used in the methods and recombinant cells according to this disclosure is a dextran sucrase variant of the parent polypeptide, said variant comprising an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 2, contains at least one modification of an amino acid residue corresponding to any of the following amino acids: 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, The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 2.

[0171] In one embodiment, the modification is the substitution, addition, or deletion of amino acid residues corresponding to any of the following amino acids: 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, The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 2.

[0172] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 2, contains one or more of the following: Position 196 is F, position 197 is S, position 199 is F, position 201 is W or F, position 215 is P, position 227 is M or Q, position 228 is P, position 239 is P, position 242 is A, position 282 is S, position 289 is Y or W, position 290 is M, position 326 is L, F, Y or Q, position 328 is F, L, S, M or H, position 334 is F, and position... Position 346 is F, position 349 is H, position 398 is Y, position 399 is E or D, position 400 is V, S or L, position 401 is E, A, D, F, G, H, L, M, N, S, T, V, W or Y, position 402 is T or S, position 417 is H, position 715 is R, position 721 is T or Q, position 726 is W, position 763 is M, position 788 is R, and position 867 is S. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 2.

[0173] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 2, contains amino acid substitutions or combinations of amino acid substitutions at the following positions: -239, preferably the substitution present in mutant _0014 in Table 2; -401, preferably the substitution present in mutant _0015 in Table 2; -398, preferably the substitution present in mutant _0016 in Table 2; -239 and 401, preferably the substitutions present in mutant _0017 in Table 2; -239 and 398, preferably the substitutions present in mutant _0018 in Table 2; -398 and 401, preferably the substitutions present in mutant _0019 in Table 2; -242, preferably the substitution present in mutant _0020 in Table 2; -326, preferably the substitution present in mutant _0021 or mutant _0022 in Table 2; -242 and 401, preferably the substitutions present in mutant _0023 in Table 2; -326 and 401, preferably substitutions present in mutant _0024 or mutant _0025 in Table 2; -289, preferably the substitution present in mutant _0026 or mutant _0027 in Table 2; -201, preferably the substitution present in mutant _00208 in Table 2; -201 and 401, preferably the substitutions present in mutant _0029 in Table 2; -201, 242 and 401, preferably the substitutions present in mutant _0030 in Table 2; -201, 326 and 401, preferably the substitutions present in mutant _0031 in Table 2; -239 and 242, preferably the substitutions present in mutant _0032 in Table 2; -239 and 326, preferably substitutions present in mutant _0033 or mutant _0034 in Table 2; -239, 242 and 326, preferably substitutions present in mutant _0035 or mutant _0036 in Table 2; -239 and 201, preferably substitutions present in mutant _0037 or mutant _0112 in Table 2; -239, 201 and 242, preferably the substitutions present in mutant _0038 in Table 2; -239, 201 and 326, preferably substitutions present in mutant _0039 or mutant _0040 in Table 2; -239, 201, 242 and 326, preferably substitutions present in mutant _0041 in Table 2; -239, 398 and 242, preferably the substitutions present in mutant _0042 in Table 2; -239, 398 and 326, preferably substitutions present in mutant _0043 or mutant _0044 in Table 2; -239, 398, 242 and 326, preferably substitutions present in mutant _0045 or mutant _0046 in Table 2; -239, 398 and 201, preferably substitutions present in mutant _0047 or mutant _0115 in Table 2; -239, 398, 201 and 242, preferably the substitutions present in mutant _0048 in Table 2; -239, 398, 201 and 326, preferably substitutions present in mutant _0049 or mutant _0050 in Table 2; -239, 398, 201, 242 and 326, preferably the substitutions present in mutant _0051 in Table 2; -239, 401, 398 and 242, preferably the substitutions present in mutant _0052 in Table 2; -239, 401, 398 and 326, preferably substitutions present in mutant _0053 or mutant _0054 in Table 2; -239, 401, 398, 242 and 326, preferably substitutions present in mutant _0055 or mutant _0056 in Table 2; -239, 401, 398 and 201, preferably substitutions present in mutants _0057, _0051 or _0114 in Table 2; -239, 401, 398, 201 and 242, preferably the substitutions present in mutant _0058 in Table 2; -239, 401, 398, 201 and 326, preferably substitutions present in mutant _0059 or mutant _0060 in Table 2; -239, 401, 398, 201, 242 and 326, preferably the substitutions present in mutant _0061 in Table 2; -239 and 401, preferably substitutions present in mutants _0062, _0063, _0064, _0065, _0066, _0067, _0068, _0069, _0070, _0071, _0072, _0073 or _0074 in Table 2; -239, 398 and 401, preferably substitutions present in mutants _0075, _0076, _0077, _0078, _0079, _0080, _0081, _0082, _0083, _0084, _0085, _0086, _0087 or _110 in Table 2; -199 and 239, preferably substitutions present in mutant _0088 or mutant _0113 in Table 2; -199 and 398, preferably the substitutions present in mutant _0089 in Table 2; -199, 239 and 398, preferably substitutions present in mutant _0090 or mutant _0116 in Table 2; -199, 242 and 239, preferably the substitutions present in mutant _0091 in Table 2; -199, 239 and 326, preferably substitutions present in mutant _0092 or mutant _0093 in Table 2; -199, 201 and 239, preferably the substitutions present in mutant _0094 in Table 2; -199, 242, 239 and 398, preferably the substitutions present in mutant _0095 in Table 2; -199, 239, 326 and 398, preferably substitutions present in mutants _0096, _0097 or _0098 in Table 2; -239 and 290, preferably the substitutions present in mutant _0099 in Table 2; -398 and 290, preferably the substitutions present in mutant _0100 in Table 2; -239, 398 and 290, preferably the substitutions present in mutant _0101 in Table 2; -242, 239 and 290, preferably the substitutions present in mutant _0102 in Table 2; -239, 326 and 290, preferably substitutions present in mutant _0103 or mutant _0104 in Table 2; -201, 239 and 290, preferably the substitutions present in mutant _0105 in Table 2; -242, 239, 398 and 290, preferably the substitutions present in mutant _0106 in Table 2; -239, 326, 398 and 290, preferably substitutions present in mutant _0107 or mutant _0108 in Table 2; -201, 239, 398 and 290, preferably substitutions present in mutant _0109 in Table 2; -239 and 215, preferably the substitutions present in mutant _0117 in Table 2; -239, 199 and 215, preferably the substitutions present in mutant _0118 in Table 2; -239, 398 and 215, preferably the substitutions present in mutant _0119 in Table 2; -239 and 715, preferably the substitutions present in mutant _0120 in Table 2; -239, 401 and 417, preferably the substitutions present in mutant _0121 in Table 2; -239, 398 and 417, preferably the substitutions present in mutant _0122 in Table 2; -239 and 417, preferably the substitutions present in mutant _0123 in Table 2; -239 and 721, preferably substitutions present in mutant _0124 or mutant _0125 in Table 2; -239, 398, 715 and 721, preferably substitutions present in mutant _0126 or mutant _0127 in Table 2; -239, 398, 201 and 721, preferably substitutions present in mutant _0128 or mutant _0129 in Table 2; -239, 398 and 196, preferably the substitutions present in mutant _0130 in Table 2; -239, 398, 196 and 197, preferably the substitutions present in mutant _0131 in Table 2; -239, 398 and 197, preferably the substitutions present in mutant _0132 in Table 2; -239, 398, 201 and 196, preferably the substitutions present in mutant _0133 in Table 2; -239, 398, 201, 215 and 196, preferably the substitutions present in mutant _0134 in Table 2; -239, 227 and 228, preferably substitutions present in mutant _0135 or mutant _0136 in Table 2; -239 and 227, preferably substitutions present in mutant _0137 or mutant _0138 in Table 2; -239 and 788, preferably the substitutions present in mutant _0139 in Table 2; -239, 788 and 288, preferably the substitutions present in mutant _0140 in Table 2; -239, 228, 788 and 227, preferably the substitutions present in mutant _0141 in Table 2; -239, 398 and 282, preferably the substitutions present in mutant _0142 in Table 2; -239 and 282, preferably the substitutions present in mutant _0143 in Table 2; -239, 398 and 334, preferably the substitutions present in mutant _0144 in Table 2; -239 and 334, preferably the substitutions present in mutant _0145 in Table 2; -239, 398 and 328, preferably substitutions present in mutants _0146, _0147, _0148, _0149 or _0150 in Table 2; -239 and 328, preferably substitutions present in mutants _0151, _0152, _0153, _0154 or _0155 in Table 2; -239, 401 and 328, preferably substitutions present in mutants _0156, _0157, _0158, _0159, _0160 or _0161 in Table 2; -239, 401, 328 and 346, preferably substitutions present in mutant _0162 or mutant _0163 in Table 2; -239, 398, 328 and 346, preferably the substitutions present in mutant _0164 in Table 2; -239, 401 and 346, preferably the substitutions present in mutant _0165 or mutant _0166; -239, 398 and 346, preferably the substitutions present in mutant _0167 in Table 2; -239, 398 and 726, preferably the substitutions present in mutant _0168 in Table 2; -239, 401 and 763, preferably the substitutions present in mutant _0169 or mutant _0170; -239, 398 and 763, preferably the substitutions present in mutant _0171 in Table 2; -239 and 763, preferably the substitutions present in mutant _0172 in Table 2; -239 and 867, preferably the substitutions present in mutant _0173 in Table 2; -239, 867 and 401, preferably the substitutions present in mutant _0174 in Table 2; -239, 867 and 398, preferably the substitutions present in mutant _0175 in Table 2; -239 and 349, preferably the substitutions present in mutant _0176 in Table 2; -239, 349 and 401, preferably the substitutions present in mutant _0177 in Table 2; -239, 349 and 398, preferably the substitutions present in mutant _0178 in Table 2; -239 and 326, preferably the substitutions present in mutant _0179 in Table 2; -239, 398 and 326, preferably substitutions present in mutants _0180 and _0181 in Table 2; -239 and 400, preferably substitutions present in mutants _0182, _0183 or _0184 in Table 2; -239, 398 and 400, preferably substitutions present in mutants _0185, _0186 or _0187 in Table 2; -239 and 399, preferably substitutions present in mutant _0188 or mutant _0189 in Table 2; -239, 398 and 399, preferably substitutions present in mutant _0190 or mutant _0191 in Table 2; -239 and 402, preferably substitutions present in mutant _0192 or mutant _0193 in Table 2; -239, 402 and 399, preferably substitutions present in mutant _0194 or mutant _0195 in Table 2; -239, 398 and 402, preferably substitutions present in mutant _0196 or mutant _0197 in Table 2.

[0174] In one embodiment, the parent polypeptide of the dextran sucrase variant comprises either a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0175] In one embodiment, the dextran sucrase 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 listed in SEQ ID NO: 2.

[0176] According to one aspect of this disclosure, the dextran sucrase used in the methods and recombinant cells according to this disclosure is a dextran sucrase variant of the parent polypeptide, said variant comprising an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 34, contains at least one modification of an amino acid residue corresponding to any of the following amino acids: 200, 203, 204, 249, 252, 336, 413, 416 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 34.

[0177] In one embodiment, the modification is the substitution, addition, or deletion of amino acid residues corresponding to any of the following amino acids: 200, 203, 204, 249, 252, 336, 413, 416 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 34.

[0178] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 34, contains one or more of the following: The value at position 200 is F or L, at position 203 is S or F, at position 204 is W, at position 249 is P, at position 252 is A, at position 336 is Q or L, at position 413 is Y, and at position 416 is E, A, or H. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 34.

[0179] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 34, contains amino acid substitutions or combinations of amino acid substitutions at the following positions: -249, preferably the substitution present in mutant _0198 in Table 2; -416, preferably the substitution present in mutants _0199, _0200 or _0201 in Table 2; -413, preferably the substitution present in mutant _0202 in Table 2; -249 and 413, preferably the substitutions present in mutant _0203 in Table 2; -249 and 416, preferably the substitutions present in mutant _0204 in Table 2; -413 and 416, preferably the substitutions present in mutant _0205 in Table 2; -249, 413 and 416, preferably the substitutions present in mutant _0206 in Table 2; -249 and 252, preferably the substitutions present in mutant _0207 in Table 2; -249 and 336, preferably substitutions present in mutant _0208 or mutant _0209 in Table 2; -249 and 204, preferably the substitutions present in mutant _0210 in Table 2; -249 and 200, preferably substitutions present in mutant _0211 or mutant _0212 in Table 2; -413 and 252, preferably the substitutions present in mutant _0213 in Table 2; -413 and 336, preferably substitutions present in mutant _0214 or mutant _0215 in Table 2; -413 and 204, preferably the substitutions present in mutant _0216 in Table 2; -413 and 200, preferably the substitutions present in mutant _0217 in Table 2; -249, 413 and 252, preferably the substitutions present in mutant _0218 in Table 2; -249, 413 and 200, preferably the substitutions present in mutant _0219 in Table 2; -203 and 204, preferably substitutions present in mutant _0220 or mutant _0221 in Table 2; -203, 204 and 249, preferably substitutions present in mutant _0222 or mutant _0223 in Table 2.

[0180] In one embodiment, the parent polypeptide of the dextran sucrase variant comprises either a polypeptide having the amino acid sequence shown in SEQ ID NO: 34 or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34.

[0181] In one embodiment, the dextran sucrase 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 listed in SEQ ID NO: 34.

[0182] According to another aspect of this disclosure, the dextran sucrase used in the methods and recombinant cells according to this disclosure is a dextran sucrase variant of the parent polypeptide, said variant comprising an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 26, contains at least one modification of an amino acid residue corresponding to any of the following amino acids: 212, 253, 256, 259, 302, 303, 412, 414, 419, 422, 425, 433, 436, 688, 691, 722, 733, 739, 743, 745, 782, 787 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 26.

[0183] In one embodiment, the modification is the substitution, addition, or deletion of amino acid residues corresponding to any of the following amino acids: 212, 253, 256, 259, 302, 303, 412, 414, 419, 422, 425, 433, 436, 688, 691, 722, 733, 739, 743, 745, 782, 787 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 26.

[0184] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 26, contains one or more of the following: The position is 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; and M or I at position 787. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 26.

[0185] In one embodiment, the dextran sucrase variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 26, contains amino acid substitutions or combinations of amino acid substitutions at the following positions: -256, preferably the substitution present in mutant _0224 in Table 2; -414, preferably the substitution present in mutant _0225 in Table 2; -256 and 414, preferably the substitutions present in mutant _0226 in Table 2; -256 and 212, preferably substitutions present in mutant _0227 or mutant _0228 in Table 2; -256, 414 and 212, preferably the substitutions present in mutant _0229 in Table 2; -256, 414, 212 and 302, preferably the substitutions present in mutant _0230 in Table 2; -256, 414 and 259, preferably substitutions present in mutant _0231 or mutant _0232 in Table 2; -256 and 253, preferably substitutions present in mutants _0233, _0234 or _0235 in Table 2; -414 and 253, preferably substitutions present in mutants _0236, _0237 or _0238 in Table 2; -256, 259 and 253, preferably substitutions present in mutants _0239, _0240 or _0241 in Table 2; -414, 259 and 253, preferably substitutions present in mutants _0242, _0243 or _0244 in Table 2; -256, 414 and 412, preferably substitutions present in mutants _0245, _0246 or _0247 in Table 2; -256, 414 and 782, preferably the substitutions present in mutant _0248 in Table 2; -256, 414 and 733, preferably substitutions present in mutants _0249, _0250 or _0251; -256 and 733, preferably the substitutions present in mutant _0252 in Table 2; -414, 253 and 733, preferably substitutions present in mutants _0253, _0254, _0255, _0256, _0257, _0258, _0259, _0260, _0261, _0262, _0263 or _0264 in Table 2; -256, 414 and 422, preferably substitutions present in mutants _0265, _0267, _0268 or _0269 in Table 2; -256, 414 and 425, preferably substitutions present in mutants _0270, _0271 or _0272 in Table 2; -256, 414 and 436, preferably substitutions present in mutant _0273 or mutant _0274 in Table 2; -256, 414 and 433, preferably substitutions present in mutant _0275 or mutant _0276 in Table 2; -256, 414 and 302, preferably the substitutions present in mutant _0277 in Table 2; -256, 414 and 419, preferably substitutions present in mutant _0278 or mutant _0279 in Table 2; -256, 414 and 691, preferably substitutions present in mutant _0280 or mutant _0281 in Table 2; -256, 414 and 739, preferably substitutions present in mutant _0282 or mutant _0283 in Table 2; -256, 414 and 743, preferably the substitutions present in mutant _0284 in Table 2; -256, 414 and 745, preferably the substitutions present in mutant _0285 in Table 2; -256, 414 and 303, preferably substitutions present in mutant _0286 or mutant _0287 in Table 2; -256, 414 and 787, preferably substitutions present in mutant _0288 or mutant _0289 in Table 2; -256, 414 and 722, preferably the substitutions present in mutant _0290 in Table 2; -256, 414 and 688, preferably the substitutions present in mutant _0291 in Table 2.

[0186] In one embodiment, the parent polypeptide of the dextran sucrase variant comprises either a polypeptide having the amino acid sequence shown in SEQ ID NO: 26 or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 26.

[0187] In one embodiment, the dextran sucrase 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 listed in SEQ ID NO: 26.

[0188] Table 2: Dextran sucrase variants according to this disclosure. In the table, M1 (SEQ ID NO: 5)* indicates a specific In the mutant, compared to the parental DexT, the Met at position 1 of the parental DexT is replaced by the mutant according to SEQ ID NO: 5. Signal peptide substitution Glycosyl donor The term "glycosyl donor" is used herein to refer to the molecule that provides a glycosyl group in a glycosylation reaction. Most commonly, the glycosyl donor is an activated donor (i.e., a glyconucleotide, such as UDP-glucose or GDP-mannose). However, in the context of this disclosure, the glycosyl donor is preferably an inactivated sugar. Examples of inactivated sugars include disaccharides or polysaccharides, such as sucrose, maltose, lactose, amylase, and (soluble) starch. Using inactivated sugars in glycosylation reactions is advantageous because they are generally readily available and less expensive than activated sugars.

[0189] In one embodiment of this disclosure, when the heterologous enzyme capable of catalyzing the transfer of glycosyl groups to glycosyl acceptors is a dextran sucrase (e.g., dextran sucrase), the glycosyl donor is preferably sucrose.

[0190] In another embodiment of this disclosure, when the heterozyme capable of catalyzing glycosyl transfer to the glycosyl acceptor is CGTase, the glycosyl donor is preferably (soluble) starch. Methods for producing soluble starch are known to those skilled in the art.

[0191] Glycosyl receptors and / or their precursors The term "glycosyl acceptor" is used herein to refer to a molecule that accepts a glycosyl group in a glycosylation reaction. In the context of this invention, a glycosyl acceptor can be a terpenoid compound, such as a glycosylated terpenoid compound.

[0192] Terpenoids can be selected from hemiterpenoids, monoterpenoids, sesquiterpenoids, diterpenoids, disesquiterpenoids, triterpenoids, tetraterpenoids, and polyterpenoids. Preferably, terpenoids can be selected from triterpenoids or their glycosylated forms (e.g., mogroside) and diterpenoids or their glycosylated forms (e.g., steviol glycosides). More preferably, terpenoids may comprise mogrosides and / or steviol glycosides.

[0193] In one embodiment, the terpene compound is a (glycosylated)terpene compound, such as mogroside. Mogroside can be of any type. For example, mogroside can be selected from the group consisting of mogroside V, symmonoside I, mogroside IV, isomogroside V, mogroside IIIE, 11-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 IIIE, 11-oxo-mogroside IV, mogroside IE1, mogroside alcohol, 11-oxo-mogroside alcohol, mogroside IIE, mogroside IIIA2, and mogroside III.

[0194] Mogrosides are glycosylated forms of the triterpenoid mogroside. The mogroside skeleton (i.e., mogroside aglycone) contains a hydroxyl (-OH) group, which can form glycosidic bonds at the 3-carbon (C3), 11-carbon (C11), 24-carbon (C24), and / or 25-carbon (C25) positions. Typically, most relevant mogrosides contain primary and / or secondary glycosylations at C3 and / or C24 of the mogroside skeleton. For example, mogroside IE1 contains a β-glycosidic bond between the oxygen atom of the hydroxyl group at C1 of the glucose moiety and C3 of the mogroside skeleton. Mogroside IA1 contains a β-glycosidic bond between the oxygen atom of the hydroxyl group at C1 of the glucose moiety and C24 of the mogroside skeleton. This glycosylation of a glucose moiety at the C3 and / or C24 positions of the mogroside skeleton is called primary glycosylation. Mogroside IIE contains β-glycosidic bonds at the oxygen atoms of the hydroxyl groups at both C3 and C24 of the mogroside skeleton. The glucose moiety at C3 and / or C24 of the primary glycosylated mogroside can be further glycosylated via a β-1,6-glycosidic bond between the oxygen atom of the hydroxyl group at C6 of the secondary glucose moiety and / or via a β-1,2-glycosidic bond between the oxygen atom of the hydroxyl group at C2 of the secondary glucose moiety and the oxygen atom of the hydroxyl group at C2 of the primary glucose moiety. For example, mogroside IIIE (CAS 88901-37-5), which contains primary glucose moieties at both C3 and C24, further contains secondary glucose moieties via a β-1,2-glycosidic bond between the oxygen atom of the hydroxyl group at C2 of the primary glucose moiety linked at C1 of the secondary glucose moiety and C2 of the primary glucose moiety linked at C24 of the mogroside skeleton. Some mogrosides may contain a β-1,4-glycosidic bond between the oxygen atom of the hydroxyl group at C4 of the primary glucose moiety attached to the C3 position of the mogroside backbone. Isomogroside IVE contains such a β-1,4-glycosidic bond. The β-1,2-glycosylation, β-1,4-glycosylation, β-1,6-glycosylation, and α-1,6-glycosylation in the mogroside molecule are referred to as secondary glycosylation. Figure 1 The structures of several known mogroside molecules are shown.

[0195] In one specific embodiment, within the context of this disclosure, the terpene compound comprises mogroside IIIE (CAS 88901-37-5) (abbreviated as M3E) having the following structure: The starting substrate for the glycosylation reaction in the methods of this disclosure can be the glycosyl receptor itself and / or its precursor. The term "precursor" herein refers to a compound preceding another compound in a (bio)synthetic pathway. In the context of this disclosure, the precursor can be converted to a glycosyl receptor (e.g., a terpene compound) through one or more chemical reactions and / or one or more enzymatic reactions. For example, when the glycosyl receptor is a terpene compound, the precursor can be converted to the glycosyl receptor through one or more glycosylation (e.g., β-glycosylation) reactions and / or one or more deglycosylation (e.g., β-deglycosylation) reactions.

[0196] In one embodiment of this disclosure, the glycosyl acceptor is mogroside IIIE. Mogroside IIIE can be obtained by β-glycosylation of a precursor selected from, but not limited to, mogroside IE1, mogroside IA1, and mogroside IIE using one or more UGTs.

[0197] Alternatively or additionally, mogroside IIIE can be obtained by deglycosylation of highly glycosylated mogrosides by one or more hydrolytic enzymes (e.g., β-glucosidase), such as, but not limited to, mogroside V, symmancoside I, mogroside IVE, isomogroside V, 11-deoxy-mogroside V, 11-oxo-mogroside V, mogroside VI, mogroside IVA, 11-oxo-mogroside VI, 11-oxo-mogroside IVE, and combinations thereof. Such methods for deglycosylating highly glycosylated mogrosides are disclosed, for example, in WO2018 / 229283A1.

[0198] Therefore, in this disclosure, the precursors of mogroside IIIE may include, but are not limited to: mogroside alcohol, mogroside IE1, mogroside IA1 and mogroside IIE, mogroside V, symmonoside I, mogroside IVE, isomogroside V, 11-deoxy-mogroside V, 11-oxo-mogroside V, mogroside VI, mogroside IVA, 11-oxo-mogroside VI, 11-oxo-mogroside IVE, and combinations thereof.

[0199] Modified glycosyl receptors The term "modified glycosyl receptor" is used herein to refer to a glycosyl receptor modified with at least one additional glycosyl group according to the method of this disclosure.

[0200] In the context of this invention, the modified glycosyl acceptor can be a modified (glycosylated) terpene compound, such as modified mogroside. Terpene compounds can be modified at multiple positions. Modifications can involve different types of glycosidic bonds (e.g., α-glycosidic bonds and / or β-glycosidic bonds).

[0201] In one embodiment of the invention, the terpenoid compound is modified with at least one glycosyl group via an α-glycosidic bond. Therefore, the modified terpenoid compound can be an α-glycosylated terpenoid compound. In a further embodiment, the modification comprises adding a glycosyl group to glucose (e.g., primary glucose) present in the glycosylated terpenoid compound. Thus, one or more glycosyl groups can be provided to the modified terpenoid compound via α-1,3-glycosidic bonds, α-1,6-glycosidic bonds, and / or α-1,4-glycosidic bonds. Preferably, one or more glycosyl groups are provided to the modified terpenoid compound via an α-1,6-glycosidic bond.

[0202] In one specific embodiment, the glycosyl acceptor is a (glycosylated) terpene compound, preferably mogroside, and the modification occurs at the C3 and / or C24 positions of mogroside. Preferably, mogroside is modified at least at the C24 position. More preferably, mogroside is selectively modified at the C24 position. For example, in one embodiment, the present invention provides a method for preparing modified mogroside, wherein the modified mogroside is selectively modified at the C24 position with a single glycosyl residue via an α-glycosidic bond. In particular, the modification comprises adding a single glycosyl residue to the primary glucose moiety linked to the C24 position of the mogroside backbone via an α-glycosidic bond.

[0203] In a more specific embodiment, the terpene compound is mogroside IIIE, and the modification comprises adding a single glycosyl group to the primary glucose moiety attached to the C24 position of the mogroside skeleton. More specifically, the modified mogroside IIIE comprises an α-1,6-glycosidic bond between the oxygen atom of the hydroxyl group at C1 of the glycosyl group and at C6 of the primary glucose moiety attached to the C24 position of the mogroside skeleton. The modified mogroside IIIE (CAS 2419125-32-7) is referred to herein as compound (I) of this disclosure and has the following structure: Compound (I) (sometimes referred to in the literature as α-Symanosin I or α-SI) is a high-intensity sweetener that can be used in a variety of products where sweetness is desired. Compound (I) offers the advantage of low calories compared to other sweeteners such as sucrose or fructose.

[0204] Recombinant cells According to this disclosure, the recombinant cell contains, is overexpressed, or is capable of overexpressing a polynucleotide encoding at least one heterozyme capable of catalyzing glycosyl transfer to a glycosyl receptor (e.g., a terpene compound).

[0205] The cells can be prokaryotic cells, archaea cells, or eukaryotic cells.

[0206] Prokaryotic cells can be, but are not limited to, bacterial cells. Bacterial cells can be Gram-negative or Gram-positive bacteria. Examples of bacteria include, but are not limited to, bacteria belonging to the following genera: Bacillus (For example, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus ), Acinetobacter,Nocardia,Xanthobacter,Escherichia (For example, E. coli ), Streptomyces,Erwinia,Klebsiella,Serratia (For example, S. marcessans ), Pseudomonas (For example, P. aeruginosa, P. fluorescens ), Salmonella (For example, 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 non-sulfur bacteria).

[0207] Eukaryotic cells can be, but are not limited to, fungi (such as yeast or filamentous fungi), algae, plant cells, and cell lines.

[0208] Eukaryotic cells can be fungi, such as filamentous fungi or yeast. Filamentous fungal strains include, but are not limited to, the following strains: Acremonium, Aspergillus (For example, A. niger、A rice、A. nestling ), Agaricus, Aureobasidium, Coprinus, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium、Humicola、Magnaporthe、Monascus、Mucor、Myceliophthora、Mortierella、 Neocallimastix、Neurospora、Paecilomyces、Penicillium (For example, P. chrysogenum, P. Camembert ), Piromyces、Phanerochaete Pleurotus、Podospora、Pycnoporus、Rhizopus、 Schizophyllum、Sordaria、Talaromyces、Rasamsonia (For example, Rasamsonia emersonii ), Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma .

[0209] Yeast cells can be selected from the following genera: Saccharomyces (For example, S. cerevisiae, S. bayanus, S. pastorianus、S. carlsbergensis ), Kluyveromyces, Candida (For example, C. rugosa, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis) , Peach (For example, Pastor's P. ), Schizosaccharomyces、Issatchenkia、Zygosaccharomyces、Hansenula、Kloeckera、 Schwanniomyces and Yarrow (For example, Y. lipolytica Originally categorized as Candida lipolytica ).

[0210] Cells can be algae, microalgae, or marine eukaryotes. Labyrinthulomycetes Cells, preferably ThraustochytrialesTarget cells, more preferably Thraustochytriaceae Cells of the family, more preferably members of the genera selected from the group consisting of: Aurantiochytrium, Oblongichytrium, Schizochytrium、Thraustochytrium and Ulkenya Even more preferably Schizochytrium sp.ATCC# 20888.

[0211] The recombinant cells disclosed in this article may belong to one of the following genera: Saccharomyces、Aspergillus、 Pichia、Kluyveromyces、Candida、Hansenula、Humicola、Issatchenkia、Trichosporon、 Brettanomyces, Pachysolen, Yarrowia, Yamadazyma or Escherichia ,For example Saccharomyces cerevisiae cell, Yarrowia lipolytica cell, Candida krusei cell, Issatchenkia orientalis Cells or Escherichia coli cell.

[0212] Therefore, in one embodiment, the recombinant cell according to this disclosure may be a prokaryotic cell, a eukaryotic cell, or an archaea cell, particularly a plant cell or a cell selected from the following: Saccharomyces cerevisiae cell, Yarrowia lipolytica cell, Candida krusei cell, Issatchenkia orientalis cell, Pichia pastoris Cells or Escherichia coli cell.

[0213] For clarity, in the methods disclosed herein, the recombinant cells can be live cells or intact cells. The recombinant cells can be proliferating cells or resting cells. The recombinant cells can be undamaged cells or permeable cells. Preferably, the recombinant cells are undamaged cells.

[0214] The heterozyme disclosed herein can be a large enzyme with a complex multi-domain structure and containing 10 or more putative N-glycosylation sites. In the context of this invention, it has been surprisingly found that said heterozyme can be functionally expressed in the recombinant cells of this disclosure. Equally surprising is that the inventors of this invention have further demonstrated that said heterozyme can be functionally expressed intracellularly and / or extracellularly.

[0215] Therefore, the recombinant cells of this disclosure contain at least one heterologous enzyme capable of catalyzing the transfer of glycosyl groups from glycosyl donors to glycosyl acceptors. The enzyme is preferably dextran sucrase and / or CGTase as described above. More preferably, the enzyme is dextran sucrase as described above.

[0216] In one embodiment, the recombinant cell contains, (over)expresses, or is capable of (over)expressing a polynucleotide encoding at least one heterologous enzyme as described above, and said enzyme is an intracellular enzyme. In other words, said enzyme can be expressed intracellularly. More specifically, said heterologous enzyme can be expressed intracellularly and is active inside the cell after expression. Specifically, the 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 any mutant (i.e., mutant_0001 to mutant_0291) as described in Table 2. The heterozyme can be any dextran sucrase variant described above according to this disclosure. More specifically, the recombinant cells of this disclosure are genetically modified with any suitable heteropolynucleotide encoding the intracellular enzyme, which is associated with the enzyme according to SEQ ID NO: The amino acid sequences of 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 any mutant (i.e., mutants _0001 to _0291) listed in Table 2, have an amino acid sequence identity of 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%. The enzyme and / or the polynucleotide encoding the enzyme may be a heterozyme and / or a heteropolynucleotide. The enzyme and / or the polynucleotide encoding the enzyme can be (over)expressed. Typically, the polynucleotide can be constitutively expressed. Alternatively, the expression of the polynucleotide can be induced. The recombinant cells of this disclosure can be genetically modified with any suitable heterologous polynucleotide encoding the intracellular enzyme, which is any dextran sucrase variant according to this disclosure.

[0217] In another embodiment, the recombinant cell contains, (over)expresses, or is capable of (over)expressing a polynucleotide encoding at least one heterozyme as described above, and said enzyme is an extracellular enzyme. In other words, said enzyme can be expressed extracellularly and has extracellular activity. Furthermore, said heterozyme can be an extracellular enzyme that is freely secreted outside the recombinant cell and / or an extracellular enzyme displayed (anchored) on the external surface of the recombinant cell. In the specific case where the heterozyme is freely secreted outside the recombinant cell, said enzyme may include a signal peptide at its N-terminus (linked to said enzyme via its C-terminus) to promote the secretion of the heterozyme outside the cell membrane. Any signal peptide known in the art can be used for this purpose. A suitable signal peptide according to this 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 specifically, the recombinant cells of this disclosure are genetically modified with any suitable heteropolynucleotide encoding the enzyme, the enzyme having an amino acid sequence with 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 specific case where the heterozyme is displayed (anchored) on the outer surface of the recombinant cell, the enzyme may contain an anchoring protein at its N-terminus, which in turn is linked to the C-terminus of a signal peptide. Alternatively, the anchoring protein may be linked to the C-terminus of the heterozyme via its N-terminus. Any anchoring protein known in the art may be used for this purpose. Suitable examples of anchoring proteins that may 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 specifically, the recombinant cells of this disclosure are genetically modified with any suitable heteropolynucleotide encoding the enzyme, the enzyme having an amino acid sequence identity of 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% according to the amino acid sequence of SEQ ID NO: 6, 7, 103, or 104.

[0218] Enzymes as described above and / or polynucleotides encoding said enzymes can be overexpressed. Typically, polynucleotides can be constitutively expressed. Alternatively, the expression of polynucleotides can be induced.

[0219] Enzymes capable of catalyzing the transfer of glycosyl groups to glycosyl acceptors (such as the aforementioned glucan sucrases (e.g., dextran sucrase) or CGTases) possess high continuous processing activity, meaning they can add multiple glucose moieties to a growing polysaccharide chain without releasing products between each addition. The formation of these oligosaccharides and / or polysaccharides is undesirable, and another object of this disclosure is the hydrolysis of these back into single glucose molecules, which can be used as a carbon source for recombinant cells. This can be accomplished by applying α-glucosidases; for example, dextran anhydrase (EC 3.2.1.11). Therefore, in another embodiment, recombinant cells may contain, may (over) express, or may be (over) express polynucleotides encoding enzymes capable of hydrolyzing oligosaccharides and / or polysaccharides. In particular, recombinant cells may contain, may (over) express, or may be (over) express dextran anhydrase (EC 3.2.1.11). Any dextran anhydrase known in the art may be used in the context of this disclosure. Dextranase is an enzyme that hydrolyzes α-1,6-linked polydextrose chains present in dextran back to their monomers. Any other enzyme capable of performing the same function can be used as a substitute for dextranase. The enzyme and / or the polynucleotide encoding the enzyme can be a heterozyme and / or a heteropolynucleotide. The enzyme and / or polynucleotide can be (over)expressed. Typically, the polynucleotide can be constitutively expressed. On the other hand, the expression of the polynucleotide can be induced. Examples of such enzymes are shown in SEQ ID NO: 101 or α-dextranase (GenBank accession number AGM20695.1) or the enzyme dextranase L (from Amano) as disclosed in the examples.

[0220] In another further embodiment, the recombinant cell may contain, be able to overexpress, or be able to overexpress a polynucleotide encoding an enzyme capable of deglycosylating a highly glycosylated glycosyl receptor (e.g., mogroside V) to produce a desired glycosyl receptor (e.g., mogroside IIIE). The highly glycosylated glycosyl receptor may be mogroside. The highly glycosylated mogroside may be at least a trisaccharidated mogroside product or a higher glycosylated mogroside product, at least a tetrasaccharidated mogroside product, at least a pentasaccharidated mogroside product, at least a hexasaccharidated mogroside product, or a higher glycosylated mogroside product. The highly glycosylated mogroside may contain one or more of the following bonds: (a) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton; (b) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton; (c) The β1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton of mogroside; (d) The β1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton; (e) The β1,4 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton of mogroside.

[0221] Therefore, peptides capable of deglycosylating highly glycosylated glycosyl receptors (e.g., highly glycosylated mogroside products) can typically hydrolyze at least trisaccharilated, tetrasaccharilated, pentasaccharilated, hexasaccharilated mogroside products, or even more glycosylated ones, into mogrosides containing lower levels of glycosylation. Typically, peptides capable of deglycosylating highly glycosylated glycosyl receptors (e.g., highly glycosylated mogroside products) can hydrolyze one or more of the following bonds: (a) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton; (b) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton; (c) The β1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton of mogroside; (d) The β1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton; (e) The β1,4 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton of mogroside.

[0222] Therefore, recombinant cells may contain, may (over)express, or may (over)express polynucleotides encoding polypeptides capable of deglycosylating highly glycosylated glycosyl receptors (e.g., highly glycosylated mogroside products). For the purposes of this disclosure, the term "polypeptide capable of deglycosylating highly glycosylated glycosyl receptors (e.g., highly glycosylated mogroside products)" may be interchangeably referred to as "beta-glucosidase," "β-glucosidase," "beta-glucosidase," or "β-glucosidase." An example of such enzymes may be Yl BGL2 according to SEQ ID NO: 9. Such enzymes are particularly useful in cases where it is desired to produce Mog III E (M3E) as a glycosyl receptor. Suitable enzymes for deglycosylating mogroside products to contain mogrosides with lower glycosylation levels are described in WO2018 / 229283 A1 or WO2024 / 220717 A1.

[0223] In another embodiment, the recombinant cells disclosed herein used in the methods disclosed herein are defective in terms of peptides capable of deglycosylating mogroside products. Typically, the peptides may be β-glucosidase (EC 3.2.1.21), dextran 1,3-β-glucosidase (EC 3.2.1.58), or dextran 1,4-β-glucosidase (EC 3.2.1.74). For example, the recombinant cells disclosed herein used in the methods disclosed herein have been modified to result in defects in peptides capable of deglycosylating mogroside products, optionally wherein the peptides capable of deglycosylating mogroside products can hydrolyze at least one of the following: (a) The glycosidic bond between the carbon (C24 atom) at position 24 of the mogroside skeleton and the β-1-glucose bonded at that position; and / or (b) The glycosidic bond between the carbon (C3 atom) at position 3 of the mogroside skeleton and the β-1-glucose bonded at that position.

[0224] Optionally, the polypeptide capable of deglycosylation of mogroside products can be further hydrolyzed by one or more of the following bonds: (a) The glycosidic bond between the carbon (C24 atom) at position 24 of the mogroside skeleton and the β-1-glucose moiety bonded at that position; (b) The glycosidic bond between the carbon (C3 atom) at position 3 of the mogroside skeleton and the β-1-glucose moiety partially bonded at that position; (c) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton; (d) The β1,2 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton; (e) The β1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C24 atom of the mogroside skeleton of mogroside. (f) The β1,6 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton; (g) The β1,4 glycosidic bond between the second glucose molecule and the β-1-glucose bonded at the C3 atom of the mogroside skeleton.

[0225] In one implementation, when the recombinant cells are Yarrowia lipolytica, The polypeptides capable of deglycosylation of mogroside products can be YALI0F01672g (SEQ ID NO: 8) and YALI0B14289g. （ SEQ ID NO: 9) or YALI0F05390g (SEQ ID NO: 10). WO2024 / 218244 A1 describes recombinant cells with defects in peptides capable of deglycosylation of mogroside products.

[0226] In another embodiment, the polypeptide capable of deglycosylation of the mogroside product may comprise or consist of the amino acid sequence listed in SEQ ID NO: 8, 9, or 10. The polypeptide may contain amino acids 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.

[0227] As defined herein, the "polypeptide capable of deglycosylation of mogroside products" according to this disclosure can be a glycoside hydrolase (EC: 3.2.1.-), such as an exogenous glycoside hydrolase. Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic bond of a glycoside, resulting in the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycone. Glycoside hydrolases are also called glycosidases, and sometimes glycosylated enzymes. Typically, the glycoside hydrolases according to this disclosure are capable of exocleaving the glucose moiety, i.e., capable of breaking the glycosidic bond and releasing the glucose molecule. Typically, the glycoside hydrolases according to this disclosure can be β-glucosidase (EC 3.2.1.21), glucan 1,3-β-glucosidase (EC 3.2.1.58), or glucan 1,4-β-glucosidase (EC 3.2.1.74). For the purposes of this disclosure, the terms "polypeptide capable of deglycosylation mogroside products", "beta-glucanase", "β-glucanase", "beta-glucanase" and "β-glucanase" are used interchangeably (abbreviated as BG).

[0228] In another embodiment of this disclosure, the recombinant cells of this disclosure may be deficient in enzymes capable of hydrolyzing glycosyl donors (e.g., sucrose, starch). Therefore, the recombinant cells of this disclosure may lack one or more enzymes selected from invertase (EC 3.2.1.26), maltase (EC 3.2.1.20), isomaltase (EC 3.2.1.10), and α-amylase (EC 3.2.1.1).

[0229] In one specific embodiment, the recombinant cells express at least one heteroglucan sucrase (which uses sucrose as a glycosyl donor), such as dextran sucrase, and the recombinant cells may be deficient in invertase (EC 3.2.1.26). Invertase, also known as β-fructofuranosidase, is an enzyme capable of catalyzing the hydrolysis of sucrose into glucose and fructose.

[0230] Recombinant cells may naturally lack the enzyme or enzyme activity described herein. For example, Yarrowia lipolytica It cannot metabolize sucrose naturally because it lacks the invertase required to cleave disaccharides (it is a defective form of the invertase required to cleave disaccharides).

[0231] Recombinant cells may naturally possess the enzyme or enzyme activity described above, and the cells may be made deficient in relation to the enzyme by any method known in the art and / or as described above. For example, SaccharomycesNaturally occurring invertases. To make the cells defective in terms of the enzyme or enzyme activity, recombinant cells may (preferably in their genome) contain modifications that, when analyzed under the same conditions, result in reduced or no enzyme production compared to unmodified parental cells. Alternatively, or in addition, cells may contain modifications that, when analyzed under the same conditions, result in reduced or no enzyme activity compared to unmodified parental cells.

[0232] In the context of this disclosure, the inventors have also discovered that recombinant cells of this disclosure, comprising and expressing a heterologous sucrose biosynthesis pathway, produce unexpectedly high sucrose titers, ranging in the order of g / L. For example, recombinant cells of this disclosure expressing a heterologous sucrose biosynthesis pathway can produce or generate sucrose at concentrations greater than 0.5 g / L, greater than 1 g / L, 2 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, or 50 g / L. Furthermore, the inventors have surprisingly discovered that the sucrose produced by these cells successfully achieves glycosylation of glycosyl receptors, thereby reducing or even completely eliminating the need for extracellular sucrose feeding or supplementation. Finally, when the method of this disclosure is carried out via fermentation as described below, no separate sucrose feed is required in addition to the glucose (or other carbon source) feed supporting cell growth and energy production. Therefore, this embodiment enables the production of modified glycosyl receptors from any commercial and simple carbon source (e.g., glucose) where sucrose is absent or unavailable. The advantages of the described implementation include, but are not limited to, reducing costs associated with sugar and its transportation, reducing costs associated with equipment (e.g., feed lines, storage containers), and reducing the complexity of the production process.

[0233] Therefore, in another embodiment of this disclosure, the recombinant cells produce or are capable of producing sucrose. Specifically, the cells capable of producing sucrose may contain, be capable of (over)expressing, or be able to (over)express one or more of the following: (a) Capable of phosphorylating glucose to form glucose- Phosphorylase, preferably hexokinase (EC2.7.1.1). (b) An enzyme capable of converting glucose-6-phosphate to fructose-6-phosphate, preferably a glucose-6-phosphate isomerase (EC 5.3.1.9). (c) An enzyme capable of catalyzing the transfer of hexose from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6-phosphate, preferably sucrose phosphate synthase (EC 2.4.1.14). (d) An enzyme capable of converting sucrose-6-phosphate into sucrose, preferably sucrose-6-phosphate phosphatase (EC3.1.3.24).

[0234] More specifically, cells capable of producing sucrose contain one or more of the following enzymes or their functional homologs: from Saccharomyces cerevisiae Hexokinase (SEQ ID NO: 11), from Yarrowia lipolytica Hexokinase (SEQ ID NO: 12), from Yarrowia lipolytica The glucose-6-phosphate isomerase (also known as phosphoglucose isomerase) (SEQ ID NO: 13), from Synechocystis sp. PCC6803 sucrose phosphate synthase (SEQ ID NO: 14) or from Saccharum officinarum Sucrose phosphate synthase (SEQ ID NO: 15), from Zea mays sucrose-6-phosphate phosphatase (SEQ ID NO: 16) or from Nicotiana tabacum Sucrose-6-phosphate phosphatase (SEQ ID NO: 17).

[0235] In one embodiment, the (heterologous) enzyme (preferably sucrose phosphate synthase (EC 2.4.1.14)) capable of catalyzing the transfer of a hexose group from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6-phosphate may comprise or be composed of the amino acid sequence listed in SEQ ID NO: 14 or 15. The polypeptide may contain amino acids 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.

[0236] In one embodiment, the (heterologous) enzyme (preferably sucrose-6-phosphate phosphatase (EC 3.1.3.24)) capable of converting sucrose-6-phosphate to sucrose may comprise or be composed of the amino acid sequence listed in SEQ ID NO: 16 or 17. The polypeptide may contain amino acids 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.

[0237] In one embodiment, the (heterologous) enzyme (preferably hexokinase (EC 2.7.1.1)) capable of phosphorylating glucose to form glucose-6-phosphate may comprise or consist of the amino acid sequence listed in SEQ ID NO: 11 or 12. The polypeptide may contain amino acids 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.

[0238] In one embodiment, the (heterologous) enzyme (preferably glucose-6-phosphate isomerase (EC 5.3.1.9)) capable of converting glucose-6-phosphate to fructose-6-phosphate may comprise or consist of the amino acid sequence listed in SEQ ID NO: 13. The polypeptide may contain amino acids 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.

[0239] The enzyme and / or the polynucleotide encoding the enzyme can be a heterozyme and / or a heteropolynucleotide. The enzyme and / or polynucleotide can be overexpressed. Typically, the polynucleotide encoding the enzyme can be constitutively expressed. Alternatively, the expression of the polynucleotide can be induced.

[0240] Optionally, recombinant cells comprising the sucrose biosynthesis pathway may also comprise, be able to (over)express, or may (over)express a transport system that promotes the transport of glucose from the extracellular environment into the recombinant cell. Any glucose ingress transport system known in the art may be used in the context of this disclosure. Suitable non-limiting examples of glucose ingress transport systems include the HXT (hexose transporter) family, facilitated diffusion, phosphotransferase systems (PTS), and ABC transporters. The transport system and / or the polynucleotide encoding the transport system may be heterologous. The transport system and / or the polynucleotide encoding the transport system may be overexpressed. Typically, the polynucleotide may be constitutively expressed. On the other hand, the expression of the polynucleotide may be induced. Alternatively or additionally, the recombinant cell may comprise cell membrane alterations (e.g., permeabilization) that promote glucose transport into the recombinant cell interior.

[0241] Alternatively, the recombinant cells comprising the sucrose biosynthesis pathway may also comprise, be able to (over)express, or may (over)express a transport system capable of facilitating sucrose transport to the outside of the recombinant cells. Any sucrose export transport system known in the art may be used in the context of this disclosure. The transport system and / or the polynucleotide encoding the transport system may be heterologous. The transport system and / or the polynucleotide encoding the 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 cells may comprise cell membrane alterations (e.g., permeabilization) capable of facilitating sucrose transport to the outside of the recombinant cells.

[0242] In a separate implementation, such as in a method of providing (feeding) a glycosyl donor (e.g., sucrose) to a recombinant cell as described below, the recombinant cell of this disclosure may also include, be capable of (over)expressing, or be (over)expressing a transport system that facilitates the transport of the glycosyl donor (e.g., sucrose) into the recombinant cell. When the glycosyl donor is sucrose, any sucrose ingestion transport system known in the art may be used in the context of this disclosure. The transport system and / or the polynucleotide encoding the transport system may be heterologous. The transport system and / or the polynucleotide encoding the transport system may be overexpressed. Typically, the polynucleotide may be constitutively expressed. On the other hand, the expression of the polynucleotide may be induced. Alternatively or additionally, the recombinant cell may include cell membrane alterations (e.g., permeabilization) that facilitate the transport of the glycosyl donor (e.g., sucrose) into the recombinant cell.

[0243] In yet another further embodiment, the recombinant cells may produce or be able to produce glycosyl receptors and / or their precursors, particularly terpenoids and / or their precursors, and more particularly mogrosides and / or their precursors. Thus, the recombinant cells may contain, may (over)express, or be able to (over)express glycosyl receptor biosynthetic pathways, such as terpenoid biosynthetic pathways, such as the mogroside biosynthetic pathway.

[0244] In one respect, recombinant cells contain, (over)express, or are capable of (over)expressing at least one polynucleotide encoding a UGT polypeptide. The “UGT” polypeptide is a polypeptide that catalyzes or is capable of catalyzing the transfer of glycosyl groups from UDP-glycans (glycosyl donors) to receptor molecules (particularly to mogrosides or mongholic acid). For example... Figure 4As shown, the synthesis of mogrosides begins with the primary glycosylation of mogroside at the C3 and / or C24 carbon positions, resulting in mogroside IA1, mogroside IE1, and / or mogroside IIE through the action of one or more UGT polypeptides capable of catalyzing the β-1 glycosylation of mogroside at the C24, C3, and C3 and C24 positions, respectively. Alternatively, primary glycosylation can occur at the C11 or C25 position of the mogroside backbone. UGT polypeptides capable of primary glycosylation are referred to herein as primary UGT polypeptides. Therefore, primary UGT polypeptides can be capable of glycosylating mogroside or mogroside compounds at their C3, C24, C11, and / or C25 hydroxyl groups. In other words, primary UGT polypeptides can catalyze the glycosylation of the mogroside backbone or the hydroxyl groups at the C3, C24, C11, and / or C25 positions of mogroside. Figure 4 As shown, mogroside IA1, mogroside IIE, and / or mogroside IE1 can be converted into more glycosylated mogroside compounds through the activity of one or more UGT peptides capable of catalyzing β-1,2-glycosylation and / or β-1,6-glycosylation (via β-1 glycosidic bond) of the hydroxyl group at the C2' position of the glucose moiety (C24-O-glucose) attached to the C24 position of the mogroside backbone and / or β-1,6-glycosylation (via β-1 glycosidic bond) of the glucose moiety (C3-O-glucose) attached to the C3 position of the mogroside backbone. Starting from mogroside IA1, mogroside IIE and / or mogroside IE1, through the action of one or more secondary UGT peptides, one or more of the following can be obtained: mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIIA1, mogroside IIIA2, mogroside IIIE, mogroside III, mogroside IVA, mogroside IVE, mogroside V, mogroside VI, mogroside VIA, mogroside VIa1, mogroside VIB, symmonoside I, isomogroside IVE, and isomogroside V.

[0245] Therefore, in another aspect of this disclosure, recombinant cells that produce or are capable of producing mogroside and / or its precursors are cells that contain, (over)express, or are capable of (over)expressing one or more of the following polynucleotides: (a) A polynucleotide encoding a polypeptide capable of glycosylation of mogroside or mogroside compounds at its C3 hydroxyl, C11 hydroxyl, C24 hydroxyl and / or C25 hydroxyl (typically at its C3 hydroxyl and / or C24 hydroxyl); (b) Polynucleotides encoding polypeptides that can β-1,2-glycosylate the C2' hydroxyl group of the glucose moiety at C24 (C24-O-glucose) of mogroside and / or β-1,2-glycosylate the C2' hydroxyl group of the glucose moiety at C3 (C3-O-glucose) of mogroside. (c) A polynucleotide encoding a polypeptide capable of β-1,6-glycosylation of the C6' hydroxyl group of the glucose moiety at C3 position of mogroside and / or capable of β-1,6-glycosylation of the C6' hydroxyl group of the glucose moiety at C24 position of mogroside. Specifically, the polypeptide according to (a), (b), or (c) is a uridine 5'-bisphosphate-dependent glycosyltransferase polypeptide (UGT polypeptide). Typically, one or more polypeptides and / or polynucleotides according to (a), (b), or (c) can be heterologous polypeptides and / or heterologous polynucleotides. Typically, one or more polynucleotides according to (a), (b), or (c) can be constitutively expressed. On the other hand, the expression of one or more polynucleotides according to (a), (b), or (c) can be induced.

[0246] The polypeptides (especially UGT polypeptides) according to (a), (b) or (c) can be any UGT polypeptide known in the art, such as any polypeptide having the amino acid sequences depicted in Tables 3a to 3e.

[0247] In one aspect of this disclosure, the UGT polypeptide according to this disclosure may be any polypeptide having the amino acid sequence according to SEQ ID NO: 57 to 61 herein or any amino acid sequence shown in Tables 3a to 3e; or alternatively, it may be a UGT polypeptide (typically a functional homolog thereof) having an amino acid sequence identity of 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% with the amino acid sequence of any polypeptide having the amino acid sequence according to SEQ ID NO: 57 to 61 herein or with the amino acid sequence shown in Tables 3a to 3e.

[0248] In one aspect of this disclosure, the polypeptide (especially a UGT polypeptide) capable of glycosylation of mogroside or mogroside compounds at their C3 hydroxyl group can be any polypeptide disclosed in the art, such as a UGT polypeptide having the amino acid sequence shown in Table 3a; or alternatively, it can be a UGT polypeptide (e.g., its functional homologs) having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 3a.

[0249] In another aspect of this disclosure, the polypeptide (especially a UGT polypeptide) capable of glycosylating mogroside or mogroside compounds at its C24 hydroxyl group can be any polypeptide disclosed in the art, such as a UGT polypeptide having the amino acid sequence shown in Table 3b; or alternatively, it can be a UGT polypeptide (e.g., its functional homologs) having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 3b.

[0250] In another aspect of this disclosure, a polypeptide (particularly a UGT polypeptide) capable of β-1,2-glycosylation of the 'C2' hydroxyl group of the glucose moiety (C24-O-glucose) at the C24 position of mogroside and / or capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety (C3-O-glucose) at the C3 position of the mogroside compound can be any polypeptide disclosed in the art, such as a UGT polypeptide having the amino acid sequence shown in Table 3c, such as its functional homologs; or alternatively, it can be a UGT polypeptide whose amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 3c.

[0251] In another aspect of this disclosure, the polypeptide (especially the UGT polypeptide) capable of β-1,6-glycosylation of the C6' hydroxyl group of the glucose moiety at the C3 position of the mogroside compound and / or capable of β-1,6-glycosylation of the C6' hydroxyl group of the glucose moiety at the C24 position of the mogroside compound can be any polypeptide disclosed in the art, such as the UGT polypeptide having the amino acid sequence shown in Table 3d; or the amino acid sequence of the UGT polypeptide can have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 3d.

[0252] Tables 3a to 3e provide non-limiting examples of the amino acid sequences of UGT peptides disclosed in patent documents and their ability to undergo specific glycosylation according to the patent documents.

[0253] Table 3a reports the amino acid sequences of UGT peptides, which, according to their respective patent documents, possess the ability to catalyze β-1 glycosylation at the C3 carbon of the mogroside backbone in mogroside or mogroside. Table 3b reports the amino acid sequences of UGT peptides, which, according to their respective patent documents, possess the ability to catalyze β-1 glycosylation at the C24 carbon of the mogroside backbone in mogroside or mogroside. Similarly, Table 3c reports the amino acid sequences of UGT peptides exhibiting β-1,2 glycosylation activity at glucose at C3 and / or C24 of the mogroside or mogroside backbone, and Table 3d reports the amino acid sequences of UGT peptides exhibiting β-1,6 glycosylation activity at glucose at C3 and / or C24 of the mogroside or mogroside backbone. Finally, Table 3e reports the amino acid sequences of those UGT peptides for which the specific type of UGT activity is not indicated in the corresponding patent documents. When known from the relevant patent documents, the specific substrates are given in the table. For example, UGT94C9, which can use MogIVE as a substrate to generate MoGV, is indicated as "UGT94C9(MogIVE)" in Table 3d. When known from the patent documents, the products of the reaction catalyzed by UGT are also indicated. For example, UGT98, which can convert MogIIE to MogIIIA2, is indicated as "UGT98 (MogIIE→IIIA2)". The SEQ ID NOs in the table are their numbers as disclosed in the relevant patent documents.

[0254] Table 3a Table 3b Table 3c Table 3d Table 3e Depending on the desired modified mogroside and the mogroside in the method of this disclosure, the cell will contain, will express, or will be able to express one or more polynucleotides encoding a UGT polypeptide.

[0255] For example, a recombinant cell according to this disclosure capable of generating mogroside IIIE from mogroside may contain at least one polynucleotide encoding a polypeptide (e.g., one or more suitable polypeptides disclosed in Tables 3a and 3b) capable of glycosylating mogroside or mogroside at its C3 hydroxyl and / or its C24 hydroxyl group, and at least one polynucleotide encoding a polypeptide (e.g., one or more suitable polypeptides disclosed in Table 3c) capable of β-1,2-glycosylating the C2' hydroxyl group of the glucose moiety at the C24 position of the mogroside compound.

[0256] For example, a recombinant cell capable of generating symmenidine I from mogroside according to this disclosure may contain at least one polynucleotide encoding a polypeptide capable of glycosylating mogroside or mogroside at its C3 and C24 hydroxyl groups (e.g., one or more suitable polypeptides disclosed in Tables 3a and 3b, respectively), at least one polynucleotide encoding a polypeptide capable of β-1,2-glycosylating the C2' hydroxyl group of the glucose moiety at the C24 position of the mogroside compound (e.g., one or more suitable polypeptides disclosed in Table 3c), and at least one polynucleotide encoding a polypeptide capable of β-1,6-glycosylating the C2' hydroxyl group of the glucose moiety at the C24 position of the mogroside compound (e.g., one or more suitable polypeptides disclosed in Table 3d, respectively).

[0257] Figure 4 The putative biosynthetic pathway from mogroside to mogroside was described, including the types of UGT peptides required to produce each mogroside.

[0258] Research has been conducted Siraitia grosvenoriiThe estimated biosynthesis of mogrosides and some enzymes involved (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 biosyntheticgenes by RNA-seq and digital gene expression analysis.” BMC Genomics (2011)12:343). Subsequently, methods for producing mogrosides using recombinant cells have been described (see, for example, WO2014 / 086842A1, WO2016 / 038617 A1, WO2016 / 050890 A2).

[0259] Mogroside possesses a triterpenoid skeleton. Triterpenoids (such as mogroside) are synthesized via the isoprene pathway, resulting from the cyclization of 2,3-epoxysqualene to form a cucurbitane skeleton. The triterpenoid skeleton subsequently undergoes various transformations (e.g., oxidation, substitution), mediated by cytochrome P450-dependent monooxygenases and other enzymes, as well as glycosylation mediated by UGT peptides as described above.

[0260] Figure 3 A putative biosynthetic pathway for the synthesis of mogroside from squalene is described. Squalene can be formed from acetyl-CoA via the mevalonate pathway (MVA pathway). The biosynthesis of squalene from acetyl-CoA is well-known and has been described in [the study / analysis]. Figure 2 Alternatively, synthesis can proceed from 1-deoxy-D-xylitol 5-phosphate via the (MEP / DOXP) pathway, an alternative metabolic pathway for the biosynthesis of isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (W. Eisenreich, A. Bacher, D. Arigoni and F. Rohdich. “Biosynthesis of isoprenoids via the non-mevalonate pathway”). Cell. Mol. Life Sci. (2004) 61:1401–1426).

[0261] Squalene is a biochemical precursor to steroids, which are essential components of all cells. Therefore, squalene is synthesized in all cells. To enable recombinant cells to produce mogroside, a catalyst capable of catalyzing multiple steps (e.g., Figure 3One or more polypeptides (those described in the text) are used to transform cells, and the steps lead to the synthesis of mogroside.

[0262] The synthesis of mogroside from squalene can involve one or more steps as described below.

[0263] 2,3-Epoxysqualene can be obtained from squalene through the action of peptides with squalene epoxygenase activity. Squalene epoxygenase can also catalyze the conversion of 2,3-epoxysqualene to 2,3,22,23-diepoxysqualene.

[0264] Cucurbitadienol can be obtained from 2,3-epoxysqualene through the action of cucurbitadienol synthase. Cucurbitadienol synthase can also catalyze the cyclization of 2,3,22,23-diepoxysqualene to 24,25-epoxy-cucurbitadienol.

[0265] Cucurbitadienol, 24,25-epoxy-cucurbitadienol, and 24,25-dihydroxy-cucurbitadienol can be converted into 11-hydroxycucurbitadienol, 11-hydroxy-24,25-epoxy-cucurbitadienol, and mogroside, respectively, by combination of one or more cytochrome P450 enzymes (CYP) with at least one CYP activator (e.g., cytochrome P450 reductase (CPR)).

[0266] Cucurbitadienol and 11-hydroxycucurbitadienol can be converted into 24,25-epoxycucurbitadienol and 11-hydroxy-24,25-epoxycucurbitadienol, respectively, by combining one or more cytochrome P450 enzymes with at least one CYP activator.

[0267] 11-hydroxy-24,25-epoxy-cucurbitadienol and 24,25-epoxy-cucurbitadienol can be converted into mogroside and 24,25-dihydroxy-cucurbitadienol, respectively, by one or more epoxyhydrolases (EPH).

[0268] WO2022 / 192688 A1 describes recombinant cells capable of producing mogroside precursors, mogroside, and / or mogrosides, wherein said recombinant cells contain a heterologous polynucleotide encoding a cytochrome b5 (CB5) polypeptide. The recombinant cells are capable of producing more mogroside than control cells that do not contain said heterologous polynucleotide.

[0269] Therefore, the synthesis of mogroside from squalene may involve one or more steps in which cytochrome b5 (CB5) peptides, such as the CB5 peptide disclosed in WO2022 / 192688 A1, can be used.

[0270] Therefore, in one embodiment of this disclosure, the recombinant cell according to this disclosure further comprises, expresses, or is capable of expressing one or more of the following recombinant polynucleotides: (a) A polynucleotide encoding a polypeptide capable of synthesizing 2,3-epoxysqualene from squalene or 2,3,22,23-diepoxysqualene from 2,3-epoxysqualene, wherein said polypeptide is squalene epoxyenase (SQE). (b) A polynucleotide encoding a polypeptide capable of synthesizing cucurbitadienol from 2,3-epoxysqualene or 24,25-epoxy-cucurbitadienol from 2,3,22,23-diepoxysqualene, wherein the polypeptide is, in particular, a cucurbitadienol synthase (CDS). (c) One or more polynucleotides encoding a polypeptide capable of synthesizing 11-hydroxycucurbita alcohol from cucurbita alcohol, 11-hydroxy-24,25-epoxy-cucurbita alcohol from 24,25-epoxy-cucurbita alcohol, mogroside from 24,25-dihydroxy-cucurbita alcohol, or 24,25-epoxy-cucurbita alcohol from cucurbita alcohol, particularly wherein said polypeptide is a cytochrome P450 enzyme (CYP450). (d) A polynucleotide encoding a polypeptide capable of reducing the cytochrome P450 complex, particularly a cytochrome P450 reductase (CPR). (e) A polynucleotide encoding a polypeptide capable of synthesizing mogroside from 11-hydroxy-24,25-epoxy-cucurbitadienol, or 24,25-dihydroxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol, wherein the polypeptide is an epoxyhydrolase (EPH). (f) Polynucleotides encoding cytochrome b5 polypeptide (CB5).

[0271] Typically, one or more polypeptides and / or polynucleotides according to (a), (b), (c), (d), (e), or (f) can be heterologous polypeptides and / or heterologous polynucleotides. Typically, one or more polynucleotides according to (a), (b), (c), (d), (e), or (f) can be constitutively expressed. On the other hand, the expression of one or more polynucleotides according to (a), (b), (c), (d), (e), or (f) can be induced.

[0272] Typically, the recombinant cells according to this disclosure will contain at least one polynucleotide selected from (a), (b), (c), (d), (e), or (f), or will contain at least two or more polynucleotides selected from (a), (b), (c), (d), (e), or (f) (preferably each from a different class of enzymes), such as at least three polynucleotides selected from (a), (b), (c), (d), (e), or (f), such as at least four polynucleotides selected from (a), (b), (c), (d), (e), or (f) (preferably each from a different class of enzymes), such as at least five polynucleotides selected from (a), (b), (c), (d), (e), or (f) (preferably each from a different class of enzymes), such as at least six polynucleotides selected from (a), (b), (c), (d), (e), or (f) (preferably each from a different class of enzymes), typically all polynucleotides (a), (b), (c), (d), (e), and (f).

[0273] In the methods and recombinant cells according to this disclosure, a polypeptide capable of synthesizing 2,3-epoxysqualene from squalene or 2,3,22,23-diepoxysqualene, squalene epoxysqualene (SQE), can be used.

[0274] Squalene epoxygenase (EC 1.14.99.7) catalyzes the production of 2,3-epoxysqualene (also known as oxidized squalene) from squalene or 2,3,22,23-diepoxysqualene (also known as squalene dioxide) from oxidized squalene, typically in the presence of NADPH. Therefore, recombinant cells capable of producing mogroside and / or its precursors according to this disclosure may contain, express, or be able to express polynucleotides encoding a polypeptide capable of synthesizing 2,3-epoxysqualene from squalene or 2,3,22,23-diepoxysqualene from 2,3-epoxysqualene, particularly wherein said polypeptide is a squalene epoxygenase. Some recombinant cells may contain endogenous squalene epoxygenase, in which case the endogenous enzyme may be sufficient. Endogenous oxidized squalene and squalene dioxide production pathways exist in eukaryotic metabolism (e.g., yeast metabolism), therefore, if the recombinant host is eukaryotic, the steps may be endogenous to the recombinant host. Nevertheless, enhancing the expression of endogenous squalene epoxygenase by any method known to those skilled in the art may be advantageous.

[0275] Therefore, in one embodiment, the recombinant cells according to this disclosure capable of producing mogroside and / or its precursors may comprise, express, or be able to express at least one polynucleotide encoding a polypeptide capable of synthesizing 2,3-epoxysqualene from squalene or 2,3,22,23-diepoxysqualene from 2,3-epoxysqualene, particularly said polypeptide being squalene epoxyenase (SQE). Table 4 provides non-limiting examples of SQE polypeptides that can be used in the recombinant cells and methods according to this disclosure. 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 (title “Squalene epoxidase amino acid sequence”), or the SQE polypeptide may have an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence comprising SEQ ID NO: 67 herein or with the amino acid sequence comprising the amino acid sequence shown in Table 4.

[0276] Table 4 provides non-limiting examples of amino acid sequences of SQE peptides disclosed in various patent documents. The SEQ ID NO in the table is the number of the peptide as disclosed in the corresponding patent document.

[0277] Table 4 In the methods and recombinant cells according to this disclosure, polypeptides capable of synthesizing cucurbitacinol from 2,3-epoxysqualene or 24,25-epoxy-cucurbitacinol from 2,3,22,23-diepoxysqualene, such as cucurbitacinol synthase (CDS), can be used. Cucurbitacinol synthase (EC 5.4.99.8) is a triterpenoid cyclase, also known as oxidized squalene cyclase or triterpenoid cyclase, which has been derived from... Cucurbita Isolation from plants (Shibuya M., Adachi S, and Ebizuka Y. "Cucurbitadienol synthase, the rst committed enzyme for cucurbitacinbiosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterolbiosynthesis” Tetrahedron(2004) 60:6995–7003). Cucurbita dienol synthases disclosed herein can catalyze the conversion of oxidized squalene (e.g., 2,3-epoxysqualene or 2,3,22,23-diepoxysqualene) into cucurbita dienol compounds (e.g., cucurbita dienol and 24,25-epoxy-cucurbita dienol). The activity of cucurbita dienol synthases can be measured using any method known in the art. For example, the activity of CDS can be measured from the amount of cucurbita dienol compound produced per unit of enzyme. CDS enzymes can be identified by those skilled in the art by their ability to convert oxidized squalene into cucurbita dienol compounds. Alternatively, novel CDS enzymes can also be identified based on a comparison of the amino acid sequence with known CDS, for example, based on the presence of one or more domains in the protein associated with CDS activity. Furthermore, novel CDS enzymes can also be identified based on a comparison of the three-dimensional structure with known CDS.

[0278] Therefore, in one embodiment, the recombinant cells according to this disclosure may contain, express, or be able to express at least one polynucleotide encoding a polypeptide capable of synthesizing cucurbitacinol from 2,3-epoxysqualene or 24,25-epoxy-cucurbitacinol from 2,3,22,23-diepoxysqualene, particularly wherein said polypeptide is cucurbitacinol synthase (CDS). Table 5 provides non-limiting examples of CDS polypeptides that can be used in the recombinant cells and methods according to this disclosure. Therefore, the CDS polypeptide may have an amino acid sequence according to the amino acid sequence of SEQ ID NO: 52 herein or any sequence disclosed in Table 5, or the CDS polypeptide may have an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 52 or with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 5.

[0279] Table 5 provides a non-limiting list of amino acid sequences of CDS peptides disclosed in multiple patent documents. The SEQ ID NO in the table is their number when they were disclosed in the corresponding patent documents.

[0280] Table 5 In the methods and recombinant cells according to this disclosure, cytochrome P450 polypeptide (abbreviated CYP or CYP450) can be used. CYP is a heme-β-containing monooxygenase.

[0281] As disclosed herein, cytochrome P450 polypeptide is defined as a heme-b-containing enzyme that functions as a monooxygenase and catalyzes the insertion of one oxygen atom into the organic substrate RH, while simultaneously using the reducing power of NAD(P)(H) to reduce the other oxygen atom to water, as shown below: RH + O2 + H + + NAD(P)H → ROH + H2O + NAD(P) + In cytochrome P450 peptides, this mechanism involves two consecutive single-electron transfer steps, in which electrons derived from NAD(P)(H) are transferred to the CYP heme center via one or more redox chaperones. Different systems have been classified and described in the literature, for example in Hanneman et al., Biochimica et Biophysica Acta (2007), 1770: 330-344; Urlacher and Girhard, Trends in Biotechnology (2012) 30(1):26-36; Sadeghi and Gilardi, Biotechnology and Applied Biochemistry (2013) 60(1): 102-110; Roberts et al. (2002) Journal of Bacteriology 184(14):3898-3908.

[0282] Based on the number and organization of redox chaperones, the cytochrome P450 system can be classified into three types of topological structures: a) A three-component system in which electrons derived from NAD(P)(H) are transferred to the P450 protein via two separate redox chaperones (e.g., flavin reductase containing FAD or FMN and ferric reductin bound to a sulfur-iron cluster). b) A two-component system, such as a system in which electrons derived from NAD(P)(H) are transferred to the P450 protein via a single redox chaperone (i.e., a biflavin reductase containing FMN / FAD), wherein the biflavin reductase containing FMN / FAD is also called cytochrome P450 reductase (abbreviated CPR). c) A 1-component system in which electrons derived from NAD(P)(H) are transferred to the P450 domain via a reductase domain fused to the P450 domain, such as an FMN-containing flavin reductase domain and a ferric reductin domain bound to a sulfur-iron cluster (abbreviated Fdx), or, as an alternative to an FMN-containing reductase and Fdx, a reductase domain of an FMN / FAD-containing biflavin reductase (e.g., as described in the aforementioned Sadeghi et al. literature). Figure 1 (C) According to this disclosure, the CYP polypeptide can belong to a two-component cytochrome P450 system (usually referred to as the class II P450 system, as described in the literature by Hanneman et al. and Sadeghi et al.). It requires cytochrome P450 protein on the one hand, and may require a flavin P450 reductase (also known as cytochrome P450 reductase or CPR) containing FAD / FMN on the other hand to support the monooxygenase activity of cytochrome P450 protein.

[0283] Alternatively, the CYP polypeptides according to this disclosure may belong to the cytochrome P450 system, which may require electron transfer protein flavin oxoreductase (FPR) and / or ferroreductase (FDXR).

[0284] In the methods and recombinant cells according to this disclosure, CYP polypeptides can participate in the hydroxylation of the C11 carbon of the mogroside precursor. These CYP enzymes may also be referred to as C11-hydroxylases. As used herein, the C11-hydroxylase polypeptide can be a CYP polypeptide capable of introducing a hydroxyl group at the 11th carbon (i.e., C11) of the mogroside precursor. In some embodiments, the C11-hydroxylase can catalyze the conversion of cucurbitacinol to 11-hydroxycucurbitacinol. In some other embodiments, the C11-hydroxylase can catalyze the conversion of 24,25-epoxy-cucurbitacinol to 11-hydroxy-24,25-epoxy-cucurbitacinol. In still other embodiments, the C11-hydroxylase can catalyze the conversion of 24,25-dihydroxy-cucurbitacinol to mogroside. The CYP polypeptide disclosed herein can catalyze the oxidation of mogroside precursors, for example, oxidizing cucurbitacin (cyclic) to 24,25-epoxy-cucurbitacin or oxidizing 11-hydroxycucurbitacin (cyclic) to 11-hydroxy-24,25-epoxy-cucurbitacin.

[0285] Therefore, in one embodiment, the recombinant cells according to this disclosure may contain, be able to express, or be able to express at least one polynucleotide encoding a polypeptide capable of synthesizing 11-hydroxycucurbita alcohol from cucurbita alcohol, 11-hydroxy-24,25-epoxy-cucurbita alcohol from 24,25-epoxy-cucurbita alcohol, or mogroside from 24,25-dihydroxy-cucurbita alcohol, or a polynucleotide encoding a polypeptide capable of synthesizing 24,25-epoxy-cucurbita alcohol from cucurbita alcohol or 11-hydroxy-24,25-epoxy-cucurbita alcohol from 11-hydroxy-cucurbita alcohol, particularly wherein said polypeptide is a cytochrome P450 enzyme (CYP450). Table 6 provides non-limiting examples of CYP polypeptides that can be used in the recombinant cells and methods of this disclosure. Therefore, a CYP polypeptide may have an amino acid sequence according to any sequence disclosed in Table 6 or an amino acid sequence listed in SEQ ID NO: 53 to 55 herein, or a CYP polypeptide may have an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 53 to 55 herein or with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 6.

[0286] Table 6 provides non-limiting examples of amino acid sequences of CYP peptides disclosed in various patent documents. The SEQ ID NO in the table is their number when they were disclosed in the respective patent documents.

[0287] Table 6 In the methods and recombinant cells according to this disclosure, cytochrome P450 polypeptide (CYP) can be used in combination with cytochrome P450 reductase (CPR) to support monooxygenase activity.

[0288] Therefore, in one embodiment, the recombinant cells according to this disclosure may comprise, express, or be able to express at least one polynucleotide encoding a polypeptide (particularly cytochrome P450 reductase (CPR)) capable of reducing the cytochrome P450 complex. Table 7 provides non-limiting examples of CPR polypeptides that may be used in recombinant cells and methods according to this disclosure. Thus, the CPR polypeptide may have an amino acid sequence of any sequence disclosed in Table 7 or an amino acid sequence listed herein in SEQ ID NO: 47, 51, or 105, or the CPR polypeptide may have an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown herein in SEQ ID NO: 47, 51, or 105 or with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 7.

[0289] Table 7 provides non-limiting examples of amino acid sequences of CPR peptides disclosed in various patent documents. The SEQ ID NO in the table is their number when they were disclosed in the respective patent documents.

[0290] Table 7 Epoxyhydrolase (EPH) can be used in recombinant cells according to the methods of this disclosure.

[0291] Epoxyhydrolase is an enzyme of the EC 3.3.xx family that catalyzes the hydrolysis of epoxides to produce diols.

[0292] In the context of this disclosure, the epoxidase polypeptide disclosed herein can catalyze the conversion of 11-hydroxy-24,25-epoxid-cucurbitadienol to mogroside. Alternatively or additionally, the epoxidase disclosed herein can catalyze the conversion of 24,25-epoxid-cucurbitadienol to 24,25-dihydroxy-cucurbitadienol.

[0293] Therefore, in one embodiment, the recombinant cells according to this disclosure may contain, express, or be able to express at least one polynucleotide encoding a polypeptide capable of synthesizing mogroside from 11-hydroxy-24,25-epoxy-cucurbitadienol or 24,25-dihydroxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol, particularly wherein said polypeptide is an epoxyhydrolase (EPH). Table 8 provides non-limiting examples of EPH polypeptides that can be used in the recombinant cells and methods of this disclosure. Therefore, the EPH polypeptide may have an amino acid sequence according to SEQ ID NO: 56 herein or any sequence disclosed in Table 8, or the polypeptide may have an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 56 herein or with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 8.

[0294] Table 8 provides non-limiting examples of amino acid sequences of EPH peptides disclosed in various patent documents. The SEQ ID NO in the table is their number when they were disclosed in the respective patent documents.

[0295] Table 8 WO2022 / 192688 A1 discloses host cells that can be used to produce mogroside precursors and / or mogroside, wherein the host cells contain a heteropolynucleotide encoding cytochrome b5, and wherein the host cells are capable of producing more mogroside than control cells that do not contain the heteropolynucleotide. Therefore, cytochrome b5 polypeptide (CB5) can be used in the methods and recombinant cells according to this disclosure.

[0296] Therefore, in one embodiment of the method or recombinant cell according to this disclosure, recombinant cells capable of producing mogroside and / or its precursors can express or may be able to express at least one polynucleotide encoding a cytochrome b5 polypeptide (CB5). Table 9 provides non-limiting examples of CB5 polypeptides that can be used in recombinant cells or methods according to this disclosure. Thus, the CB5 polypeptide may have an amino acid sequence of any sequence disclosed in Table 8 or an amino acid sequence listed in SEQ ID NO: 50 herein, or the polypeptide may have an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to SEQ ID NO: 50 herein.

[0297] Table 9 provides non-limiting examples of amino acid sequences of the CB5 polypeptide disclosed in WO2022 / 192688 A1. The SEQ ID NO in the table is their number as disclosed in the respective patent documents.

[0298] Table 9 In the method and recombinant cells according to this disclosure, recombinant cells capable of producing mogroside or its precursors may contain, express, or be able to express one or more polynucleotides encoding squalene epoxygenase, cucurbitacin synthase, cytochrome P450 polypeptide, C11-hydroxylase, cytochrome P450 reductase, epoxyhydrolase, cytochrome b5 polypeptide, and / or UGT polypeptide.

[0299] Therefore, in one aspect of this disclosure, recombinant cells capable of producing mogroside and / or its precursors are disclosed, which express or are able to express one or more of the following polynucleotides: (a) A polynucleotide encoding squalene epoxygenase polypeptide (SQE); (b) Polynucleotides encoding cucurbitacin synthase polypeptide (CDS); (c) Polynucleotides encoding cytochrome P450 polypeptide (CYP); (d) Polynucleotides encoding cytochrome P450 reductase polypeptide (CPR); (e) Polynucleotides encoding epoxide hydrolase (EPH); (f) Polynucleotides encoding cytochrome b5 polypeptide (CB5); (g) Polynucleotides encoding UGT polypeptide.

[0300] Typically, one or more 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 polynucleotides according to (a), (b), (c), (d), (e), (f), or (g) can be constitutively expressed. On the other hand, the expression of one or more polynucleotides according to (a), (b), (c), (d), (e), or (f) can be induced.

[0301] Typically, the recombinant cells according to this disclosure will contain at least one polynucleotide selected from (a), (b), (c), (d), (e), (f), or (g), or will contain at least two or more polynucleotides selected from (a), (b), (c), (d), (e), (f), or (g) (preferably each derived from a different class of enzymes), such as at least three polynucleotides selected from (a), (b), (c), (d), (e), (f), or (g), such as selected from (a), (b), (c), (d), and (e). At least four polynucleotides (preferably each from a different class of enzymes) selected from (a), (b), (c), (d), (e), (f), or (g), for example, at least five polynucleotides (preferably each from a different class of enzymes) selected from (a), (b), (c), (d), (e), (f), or (g), for example, at least six polynucleotides (preferably each from a different class of enzymes) selected from (a), (b), (c), (d), (e), (f), or (g), typically all polynucleotides (a), (b), (c), (d), (e), (f), and (g).

[0302] Typically, recombinant cells capable of producing mogrosides and / or their precursors, as described herein, can produce one or more of the following: 2,3-epoxysqualene, 2,3,22,23-diepoxysqualene, cucurbitadienol, 11-hydroxycucurbitadienol, 24,25-epoxy-cucurbitadienol, 11-hydroxy-24,25-epoxy-cucurbitadienol and / or 24,25-dihydroxy-cucurbitadienol. Figure 5 The chemical structures of some mogroside precursors and intermediates in the mogroside biosynthesis pathway are shown.

[0303] As described above, squalene is a biochemical precursor of mogroside. Squalene is synthesized from farnesyl diphosphate (FPP) in a reaction catalyzed by squalene synthase (SQS), which is believed to occur in the presence of NADPH as a co-substrate. Recombinant cells may contain endogenous squalene synthase, in which case the endogenous enzyme may be sufficient. Endogenous squalene production pathways exist in eukaryotic metabolism (e.g., yeast metabolism), therefore, if the recombinant host is eukaryotic, the steps can be endogenous to the recombinant host. Nevertheless, enhancing the expression of endogenous squalene synthase by any method known to those skilled in the art may be advantageous.

[0304] Therefore, in one embodiment of the methods and recombinant cells according to this disclosure, recombinant cells capable of producing mogroside and / or its precursors can express or express polynucleotides encoding polypeptides capable of synthesizing squalene from farnesyl diphosphate (FPP), particularly said polypeptide being squalene synthase (EC 2.5.1.21) (SQS). Table 10 provides non-limiting examples of SQS polypeptides that can be used in the recombinant cells and methods of this disclosure. Thus, SQS polypeptides may have an amino acid sequence according to any sequence disclosed in Table 10 or according to the amino acid sequence of SEQ ID NO: 68 herein, or SQS polypeptides may have an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 68 herein or with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 10.

[0305] Table 10 provides non-limiting examples of amino acid sequences of SQS peptides disclosed in various patent documents. The SEQ ID NO in the table is the number of the peptide as disclosed in the corresponding patent document.

[0306] Table 10 Farnesyl diphosphate (FPP) and geranyl diphosphate (GPP) can be substrates in the production of squalene from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Therefore, in one embodiment, recombinant cells can be modified to increase the production of farnesyl diphosphate and / or geranyl diphosphate.

[0307] Farnesyl diphosphate (FPP) can be synthesized from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP) by farnesyl diphosphate synthase (FPPS). Correspondingly, geranyl diphosphate (GPP) can be synthesized from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) by geranyl diphosphate synthase (GPPS) and / or farnesyl diphosphate synthase (FPPS).

[0308] Therefore, in one embodiment of the method or recombinant cell according to this disclosure, recombinant cells capable of producing mogroside and / or its precursors express or express polynucleotides encoding polypeptides capable of synthesizing farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP), particularly said polypeptide being farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). Table 11 provides non-limiting examples of FPPS polypeptides that can be used in the recombinant cells and methods of this disclosure. Therefore, the FPPS polypeptide may have an amino acid sequence listed in SEQ ID NO: 63 herein or an amino acid sequence disclosed according to Table 11, or the FPPS polypeptide may have an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence listed in SEQ ID NO: 63 herein or with the amino acid sequence of any polypeptide having the amino acid sequence shown in Table 11.

[0309] Table 11 provides non-limiting examples of amino acid sequences of FPPS peptides disclosed in various patent documents. The SEQ ID NO in the table is the number of the peptide as disclosed in the corresponding patent document.

[0310] Table 11 In one embodiment, recombinant cells capable of producing mogroside and its precursors express or express polynucleotides encoding polypeptides capable of synthesizing gerany diphosphate (GPP) from dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is gerany diphosphate synthase (EC 2.5.1.1) (GPPS) and / or farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). Non-limiting examples of FPPS polypeptides that can be used in the recombinant cells and methods of this disclosure have been provided above. Non-limiting examples of GGPS polypeptides can be any suitable GGPS known to those skilled in the art and can be, for example, derived from prokaryotic or eukaryotic sources. Table 12 provides non-limiting examples of GPPS polypeptides that can be used in the recombinant cells and methods of this disclosure. 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 that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any polypeptide having an amino acid sequence shown in Table 12.

[0311] Table 12 provides non-limiting examples of amino acid sequences of GPPS peptides disclosed in various patent documents. The SEQ ID NO in the table is the number of the peptide when it was disclosed in the corresponding patent document.

[0312] Table 12 Therefore, according to one aspect of the method and recombinant cells of this disclosure, recombinant cells capable of producing mogroside and / or its precursors can express or express one or more of the following polynucleotides: (a) A polynucleotide encoding a polypeptide capable of synthesizing geraniol diphosphate (GPP) from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), particularly wherein said polypeptide is geraniol diphosphate synthase (EC2.5.1.1) (GPPS) and / or farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). (b) A polynucleotide encoding a polypeptide capable of synthesizing farnesyl diphosphate (FPP) from gerany diphosphate (GPP) and isopentenyl diphosphate (IPP), particularly wherein said polypeptide is farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). (c) A polynucleotide encoding a polypeptide capable of synthesizing squalene from farnesyl diphosphate (FPP), wherein said polypeptide is squalene synthase (EC 2.5.1.21) (SQS).

[0313] Typically, one or more polypeptides and / or polynucleotides according to (a), (b), or (c) are heterologous polypeptides and / or heterologous polynucleotides. Typically, one or more polynucleotides according to (a), (b), or (c) can be constitutively expressed. On the other hand, the expression of one or more polynucleotides according to (a), (b), or (c) can be induced.

[0314] Typically, a cell may contain, be able to express, or be able to express all of the polynucleotides according to (a), (b), and (c), or the polynucleotides according to (a) and (b), or the polynucleotides according to (a) and (c), or the polynucleotides according to (b) and (c).

[0315] In some embodiments of this disclosure, recombinant cells capable of producing mogroside and / or its precursors may contain or (over)express one or more polynucleotides encoding a polypeptide belonging to the mevalonate pathway.

[0316] The mevalonic acid pathway refers to Figure 2 The well-known biosynthetic pathway shown converts acetyl-CoA into isopentenyl diphosphate. Such biosynthetic pathways are commonly found in eukaryotic cells, while prokaryotic cells produce isopentenyl diphosphate via the MEP pathway (a mevalonate-independent pathway that converts glyceraldehyde-3-phosphate and pyruvate into IPP and DMAPP).

[0317] The mevalonate pathway includes the following steps: (a) Synthesis of acetyl-CoA (AACoA) from acetyl-CoA (AcCoA) catalyzed by acetyl-CoA acetyltransferase (EC 2.3.1.9) (AACT); (b) Synthesis of hydroxymethylglutaryl-CoA (HMGCoA) from acetyl-CoA (AACoA) catalyzed by hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) (HMGS); (c) Synthesis of mevalonic acid (MVA) from hydroxymethylglutaryl-CoA (HMGCoA) catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) (HMGR); (d) Synthesis of mevalonate-5-phosphate (MVA-P) from mevalonate (MVA) catalyzed by mevalonate kinase (EC 2.7.1.36) (MK); (e) Synthesis of mevalonate-5-bisphosphate (MVA-PP) from mevalonate-5-phosphate (MVA-P) catalyzed by phosphate mevalonate kinase (EC 2.7.4.2) (PMK). (f) Synthesis of isopentenyl diphosphate (IPP) from mevalonate-5-bisphosphate (MVA-PP) catalyzed by mevalonate decarboxylase (EC 4.1.1.33) (MDD) and / or isopentenyl / dimethylallyl diphosphate synthase (EC 1.17.1.2) (IPPS); (g) A polynucleotide encoding a polypeptide capable of synthesizing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP), particularly wherein said polypeptide is an isopentenyl diphosphate δ-isomerase (EC 5.3.3.2) (IPI).

[0318] In one embodiment of the method and recombinant cells according to this disclosure, recombinant cells capable of producing mogroside and / or its precursors may contain, be able to (over) express, or be able to (over) express one or more of the following polynucleotides: (a) A polynucleotide encoding a polypeptide capable of synthesizing acetyl-CoA (AACoA) from acetyl-CoA, particularly wherein said polypeptide is an acetyl-CoA acetyltransferase (also known as acetyl-CoA thiolyte) (EC2.3.1.9) (AACT). (b) A polynucleotide encoding a polypeptide capable of synthesizing hydroxymethylglutaryl-CoA (HMGCoA) from acetyl-CoA, particularly wherein said polypeptide is hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) (HMGS). (c) A polynucleotide encoding a polypeptide capable of synthesizing mevalonic acid (MVA) from hydroxymethylglutaryl-CoA (HMGCoA), particularly wherein said polypeptide is 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) (HMGR). (d) A polynucleotide encoding a polypeptide capable of synthesizing mevalonate-5-phosphate (MVA-P) from mevalonate (MVA), particularly wherein said polypeptide is mevalonate kinase (EC 2.7.1.36) (MK). (e) A polynucleotide encoding a polypeptide capable of synthesizing mevalonate-5-bisphosphate (MVA-PP) from mevalonate-5-phosphate (MVA-P), particularly wherein said polypeptide is phosphate mevalonate kinase (EC 2.7.4.2) (PMK). (f) A polynucleotide encoding a polypeptide capable of synthesizing isopentenyl diphosphate (IPP) from mevalonate-5-bisphosphate (MVA-PP), particularly wherein said polypeptide is mevalonate diphosphate decarboxylase (EC 4.1.1.33) (MDD) and / or isopentenyl / dimethylallyl diphosphate synthase (EC 1.17.1.2) (IPPS). (g) A polynucleotide encoding a polypeptide capable of synthesizing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP), particularly wherein said polypeptide is an isopentenyl diphosphate δ-isomerase (EC 5.3.3.2) (IPI).

[0319] Typically, one or more 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 polynucleotides according to (a), (b), (c), (d), (e), (f), or (g) can be constitutively expressed. On the other hand, the expression of one or more polynucleotides according to (a), (b), (c), (d), (e), (f), or (g) can be induced. Typically, the recombinant cells according to this disclosure will contain at least one polynucleotide selected from (a), (b), (c), (d), (e), (f), or (g), or will contain at least two or more polynucleotides selected from (a), (b), (c), (d), (e), (f), or (g) (preferably each derived from a different class of enzymes), such as at least three polynucleotides selected from (a), (b), (c), (d), (e), (f), or (g), such as selected from (a), (b), (c), (d), and (e). At least four polynucleotides (preferably each from a different class of enzymes) selected from (a), (b), (c), (d), (e), (f), or (g), for example, at least five polynucleotides (preferably each from a different class of enzymes) selected from (a), (b), (c), (d), (e), (f), or (g), for example, at least six polynucleotides (preferably each from a different class of enzymes) selected from (a), (b), (c), (d), (e), (f), or (g), typically all polynucleotides (a), (b), (c), (d), (e), (f), and (g).

[0320] Any acetyl-CoA acetyltransferase known in the art may be used in the context of this disclosure. Suitable non-limiting examples of usable acetyl-CoA thiolytic enzymes are enzymes of ERG10 (UniProtKB accession number P41338) or UniProtKB accession number P10551, or enzymes of UniProtKB / Swiss-Prot: Q6L8K7.1, or any acetyl-CoA acetyltransferase having sequence identity with its amino acid sequence of 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%.

[0321] Hydroxymethylglutaryl-CoA synthases known in the art can be used to convert acetyl-CoA to hydroxymethylglutaryl-CoA. Suitable non-limiting examples include protein ERG13 UniProtKB accession number P54839 or YALI0F30481p NCBI number XP_506052.1 or SEQ ID NO: 64, or any HMG-CoA synthase polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with its amino acid sequence.

[0322] Suitable 3-hydroxy-3-methylglutaryl-CoA reductase polypeptides for use in recombinant cells and methods according to this disclosure are known to those skilled in the art. Suitable non-limiting examples are the HMG1 polypeptide of SEQ ID NO: 69, UniProtKB accession number P12683 or the HMG2 gene of UniProtKB accession number P12684, provided as non-limiting examples of HMG-CoA reductase or HMG-CoA reductase polypeptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with their amino acid sequences.

[0323] Any mevalonate kinase known in the art may be used in the context of this disclosure. Suitable non-limiting examples of mevalonate kinase are the ERG12 peptide of UniProtKB accession number P07277 or the mevalonate kinase of GenBank accession number QNP96798.1, or any MK peptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with its amino acid sequence.

[0324] Non-limiting examples of phosphovalerate kinase that can be used in the methods of this disclosure and in recombinant cells are peptides of GenBank accession number GFP66625 or GenBank accession number VBB79048.1, peptides of UniProtKB / Swiss-Prot number D4GXZ3.1, or any PMK peptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with its amino acid sequence.

[0325] Non-limiting examples of mevalonate diphosphate decarboxylases that can be used in the methods of this disclosure and in recombinant cells are peptides with GenBank accession number CAA66158.1 or GenBank accession number QNQ00565.1 or KAG5364843.1 or AAT93171.1; non-limiting examples of isopentenyl diphosphate delta-isomerases are peptides with UniProtKB / Swiss-Prot number P15496.2 or GenBank accession number QNQ The polypeptide of 01368.1 or AOW06629.1, or any mevalonate diphosphate decarboxylase or isopentenyl diphosphate delta-isomerase having sequence identity with its amino acid sequence of 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%.

[0326] like Figure 3As shown, 2,3-epoxysqualene (oxidized squalene) can be converted into lanosterol by the enzyme lanosterol synthase (ERG7). In fungal cells, lanosterol is a precursor in sterol production. Sterols are tetracyclic triterpenoid lipids, essential for all eukaryotes to maintain key cellular functions such as membrane fluidity, phagocytosis, stress tolerance, and cell signaling.

[0327] In one embodiment of the method and recombinant cells according to this disclosure, when recombinant cells produce endogenous lanosterol synthase, the level of 2,3-epoxysqualene can be increased, for example, by reducing the activity or expression of endogenous lanosterol synthase. In recombinant hosts expressing endogenous lanosterol synthase, this can be achieved by making the cells defective or partially defective in the production of lanosterol synthase. Therefore, in one embodiment of this disclosure, recombinant cells capable of producing mogroside and / or its precursors may include defects in the production of lanosterol synthase polypeptides.

[0328] Since lanosterol is an essential metabolite in eukaryotes, cells are preferably deficient in lanosterol synthase production by any method known in the art that does not completely eliminate lanosterol synthase activity. For example, recombinant cells can be modified to produce less lanosterol synthase, for example, by reducing the expression level of mRNA transcribed from the gene encoding lanosterol synthase or reducing its translation level. In one embodiment, the expression level of the gene encoding lanosterol synthase can be reduced by replacing the endogenous promoter of lanosterol synthase with a promoter weaker than the endogenous promoter or other suitable methods. Alternatively, the gene encoding native lanosterol synthase can be replaced with a gene that, while still encoding native lanosterol synthase, is more difficult to translate into a protein by recombinant cells due to different codon usage. Alternatively, recombinant cells can be modified to produce polypeptides with reduced lanosterol synthase activity. For example, recombinant cells can be modified by replacing the gene encoding native lanosterol synthase with a modified lanosterol synthase gene that has reduced lanosterol synthase activity compared to native or homologous lanosterol synthase. Non-restrictive examples of suitable lanosterol synthases with reduced activity are described in WO2022 / 212917 and WO2022 / 212924.

[0329] In one embodiment, recombinant cells capable of producing mogrosides and / or their precursors, as disclosed herein, can produce one or more mogrosides selected from the group consisting of: mogroside IA, mogroside IE1, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIIA1, mogroside IIIA2, mogroside IIIE, mogroside III, and mogroside IV. A Monk fruit glycoside IV EMogroside V, mogroside VI, mogroside VIA, mogroside VIa1, mogroside VIB, sarcoside I, isomogroside IV, isomogroside V, or sarcoside I. Typically, recombinant cells may contain, can (over) express, or are able to (over) express one or more of the following: (a) A polynucleotide encoding squalene epoxygenase polypeptide (SQE); (b) Polynucleotides encoding cucurbitacin synthase polypeptide (CDS); (c) Polynucleotides encoding cytochrome P450 polypeptides (CYP), such as C11-hydroxylase polypeptides and / or cyclooxygenases; (d) Polynucleotides encoding cytochrome P450 reductase polypeptide (CPR); (e) Polynucleotides encoding epoxide hydrolase (EPH); (f) Polynucleotides encoding cytochrome b5 polypeptide (CB5); (g) Polynucleotides encoding UGT polypeptides, particularly one or more of the following UGT polypeptides: i. A UGT capable of glycosylation mogrosides or mogrosides at their C3 hydroxyl, C11 hydroxyl, C24 hydroxyl and / or C25 hydroxyl; ii. UGTs capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety at C24 (C24-O-glucose) of mogroside compounds and / or capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety at C3 (C3-O-glucose) of mogroside compounds; iii. A UGT capable of β-1,6-glycosylation of the C6' hydroxyl group of the glucose moiety at the C3 position of a mogroside compound and / or capable of β-1,6-glycosylation of the C6' hydroxyl group of the sugar moiety at the C24 position of a mogroside compound. And optionally, one or more of the following: A. Geraniyl diphosphate synthase (GPPS) and / or farnesyl diphosphate synthase (FPPS); B. Farnesyl diphosphate synthase (FPPS); C. Squalene synthase (SQS); D. Acetyl-CoA acetyltransferase (AACT); E. Hydroxymethylglutaryl-CoA synthase (HMGS); F. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR); G. Mevalonate kinase (MK); H. phosphate mevalonate kinase (PMK); I. Mevalonate diphosphate decarboxylase (MDD) and / or isopentenyl / dimethylallyl diphosphate synthase (IPPS); J. isopentenyl diphosphate δ-isomerase (IPI).

[0330] Typically, regarding the above, the polypeptide and / or the polynucleotide encoding the polypeptide can be a heterozyme and / or a heteropolynucleotide. The enzyme and / or polynucleotide can be overexpressed. Typically, the polynucleotide can be constitutively expressed. On the other hand, the expression of the polynucleotide can be induced.

[0331] Optionally, recombinant cells capable of producing glycosyl receptors and / or their precursors (e.g., terpenoids and / or their precursors, such as mogroside and / or their precursors) as disclosed herein may also contain, may (over) express, or may be (over) express transport proteins capable of facilitating the transport of said glycosyl receptors and / or their precursors (e.g., mogroside and / or their precursors) to the exterior of the recombinant cells. Any transport proteins known in the art may be used in the context of this disclosure.

[0332] Intracellular (in vivo) and extracellular glycosylation One aspect of this disclosure provides a method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor, comprising: contacting a glycosyl donor and a glycosyl acceptor with recombinant cells under suitable conditions, said recombinant cells comprising, over-expressing, or capable of over-expressing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein the contact under suitable conditions enables said at least one heterozyme 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 transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor that can be used in the method according to this disclosure have been described above.

[0333] The glycosidic bond can be an α-glycosidic bond. The glycosyl acceptor can be a (glycosylated) terpene, such as mogroside. The modified glycosyl acceptor (e.g., a modified (glycosylated) terpene) can be an α-glycosylated glycosyl acceptor (e.g., an α-glycosylated terpene, such as α-glycosylated mogroside).

[0334] In one specific example, the method may include: contacting sucrose (a glycosyl donor) and mogroside IIIE (a glycosyl acceptor) under suitable conditions with a recombinant cell containing at least one heterologous dextran sucrase according to the present disclosure, thereby producing compound (I). Mogroside IIIE is converted to compound (I) by dextran sucrase (the enzyme) according to the following glycosylation reaction: Glycosylation can occur inside recombinant cells (in vivo) and / or outside recombinant cells.

[0335] In one embodiment, the glycosylation reaction occurs within the recombinant cell. In this embodiment, at least one heterologous enzyme capable of catalyzing the transfer of glycosyl groups to glycosyl receptors is an intracellular enzyme. More specifically, the recombinant cell contains, (over)expresses, or is capable of (over)expressing the enzyme intracellularly, and the enzyme is active within the recombinant cell. In this embodiment, the recombinant cell may also produce or be capable of producing a glycosyl donor (e.g., sucrose), as described above. Alternatively or additionally, the glycosyl donor (e.g., sucrose) may be extracellularly provided (feeded) to the recombinant cell. When the glycosyl donor is extracellularly provided (feeded), the recombinant cell may also contain or (over) express a transport protein capable of facilitating the transport of the glycosyl donor (e.g., sucrose) into the recombinant cell, as described above. In this embodiment, the recombinant cell may also produce or be capable of producing a glycosyl receptor and / or its precursor (e.g., mogrosides such as mogroside IIIE), as described above. Alternatively, glycosyl receptors and / or their precursors (e.g., mogrosides, such as mogroside IIIE) can be provided extracellularly (feed). When glycosyl receptors and / or their precursors are provided extracellularly, the recombinant cells may also contain transport systems and / or cell membrane alterations (e.g., permeabilization) that facilitate the transport of glycosyl receptors and / or their precursors into the cell interior.

[0336] In one embodiment, the glycosylation reaction occurs outside the recombinant cell. In this embodiment, at least one heterologous enzyme capable of catalyzing the transfer of glycosyl groups to glycosyl receptors is an extracellular enzyme. More specifically, according to this embodiment, the recombinant cell contains, (over)expresses, or is capable of (over)expressing the enzyme extracellularly, and the enzyme is active outside the recombinant cell after expression and secretion into the extracellular environment. The extracellular enzyme may be freely secreted outside the recombinant cell and / or displayed on the surface of the recombinant cell, as described above. In this embodiment, the recombinant cell may also produce or be capable of producing glycosyl donors (e.g., sucrose), as described above. Alternatively or additionally, glycosyl donors (e.g., sucrose) may be extracellularly provided (feeded) to the recombinant cell. When the recombinant cell produces glycosyl donors, the recombinant cell may also express transport proteins capable of facilitating the transport of glycosyl donors (e.g., sucrose) to the outside of the recombinant cell, as described above. In this embodiment, the recombinant cell may also produce or be capable of producing glycosyl receptors and / or their precursors (e.g., mogrosides, such as mogroside IIIE), as described above. Alternatively, glycosyl receptors and / or their precursors (e.g., mogrosides, such as mogroside IIIE) can be provided extracellularly (feed). When recombinant cells produce glycosyl receptors and / or their precursors, the recombinant cells may also express transport proteins capable of facilitating the transport of glycosyl receptors and / or their precursors to the outside of the recombinant cells, as described above. In the described embodiments, the at least one heterologous enzyme expressed by the recombinant cells of this disclosure may also be extracellularly supplemented with one or more additional enzymes capable of catalyzing the transfer of glycosyl groups to glycosyl receptors. Examples of such additional enzymes (e.g., CGTase) have been described above.

[0337] Production methods Another aspect of this disclosure provides a method for generating a modified glycosyl receptor, comprising: contacting a glycosyl donor and a glycosyl receptor with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (over)expressing a polynucleotide encoding at least one heterozyme, said at least one heterozyme being capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl receptor, wherein said contact under suitable conditions comprises culturing said recombinant cells in a suitable culture medium in the presence of the glycosyl donor and the glycosyl receptor, wherein said contact under suitable conditions enables said at least one heterozyme to catalyze the formation of a glycosidic bond between the transferred glycosyl group and the glycosyl receptor, thereby generating a modified glycosyl receptor; and optionally, isolating the thereby generated modified glycosyl receptor. Optionally, said contact under suitable conditions enables said recombinant cells to express said at least one heterozyme.

[0338] Another aspect of this disclosure provides a method for generating a modified glycosyl receptor, comprising contacting a glycosyl donor and a glycosyl receptor with recombinant cells under suitable conditions, said recombinant cells comprising, (over)expressing, or capable of (over)expressing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl receptor, wherein said contact under suitable conditions comprises contacting the recombinant cells, the glycosyl donor, and the glycosyl receptor in a reaction mixture, wherein said contact under suitable conditions enables the at least one heterozyme to catalyze the formation of a glycosidic bond between the transferred glycosyl group and the glycosyl receptor; thereby generating a modified glycosyl receptor, and optionally, isolating the thereby generated modified glycosyl receptor. Optionally, contact under suitable conditions enables the recombinant cells to express said at least one heterozyme.

[0339] The glycosidic bond is preferably an α-glycosidic bond, and the modified glycosyl receptor (e.g., a modified terpene compound) is preferably an α-glycosylated glycosyl receptor (e.g., an α-glycosylated terpene compound).

[0340] Methods for generating modified glycosyl acceptors may include fermentation, biotransformation, or a combination thereof.

[0341] As used herein, the term "fermentation" refers to a method in which cells convert a carbon source (e.g., glucose) into a desired compound (e.g., a modified glycosyl receptor). The carbon source also serves as a substrate for cell growth. In the context of this disclosure, "fermentation" specifically refers to the enzymatic modification of a glycosyl receptor in the presence of cells and a glycosyl donor, wherein neither the glycosyl donor nor the glycosyl receptor needs to be provided to the cells. In other words, the cells are able to produce the modified glycosyl receptor solely from a carbon source (e.g., glucose) used for cell growth. As those skilled in the art will understand, "fermentation" includes culturing cells in a culture medium.

[0342] As used herein, the term "biotransformation" refers to a method in which a compound (e.g., a glycosyl receptor) is modified by a cell, thereby producing a modified compound (e.g., a modified glycosyl receptor). In the context of this disclosure, "biotransformation" specifically refers to the enzymatic modification of a glycosyl receptor in the presence of a cell and a glycosyl donor, wherein the glycosyl donor and / or glycosyl receptor must be extracellularly provided to the cell for the enzymatic modification to proceed. The "biotransformation" is typically carried out in a reaction mixture, and the cells are typically non-proliferating cells.

[0343] As used herein, the term "fermentation and biotransformation combination" refers to a combination of the two methods described above. In the context of this disclosure, "fermentation and biotransformation combination" specifically refers to the enzymatic modification of glycosyl acceptors in the presence of cells and a glycosyl donor, wherein the method involves culturing cells in a medium containing a carbon source (e.g., glucose) for growth (fermentation), and wherein the glycosyl donor and / or glycosyl acceptor must still be extracellularly supplied to the cells for enzymatic modification (biotransformation). In this case, the "fermentation and biotransformation combination" is typically carried out in a medium used for cell growth.

[0344] For clarity, in the methods described in this disclosure, the recombinant cells are intact cells or live cells. Depending on the method, the live cells can be proliferating cells or non-proliferating cells (e.g., resting cells). The live cells can be undamaged cells or permeable cells.

[0345] In the production methods (i.e., fermentation, fermentation combined with biotransformation or biotransformation) further described below, the recombinant cells contain, express, or are able to express at least one heterologous enzyme capable of catalyzing glycosyl transfer to glycosyl receptors, and the enzyme may be an intracellular enzyme and / or an extracellular enzyme.

[0346] The inventors have surprisingly demonstrated that recombinant cells that express and secrete at least one of the heterologous enzymes extracellularly can also achieve an efficient and robust method for producing modified glycosyl receptors (e.g., α-glycosylated mogrosides, such as compound (I)).

[0347] In particular, when the production method involves fermentation, the inventors have surprisingly discovered that recombinant cells expressing heterologous enzymes extracellularly are able to produce glycosylated glycosyl receptors in the culture medium. In other words, it is surprising to find that the heterologous enzymes are functionally expressed and secreted extracellularly, and even more surprisingly, they are active in the culture medium.

[0348] The inventors have surprisingly demonstrated that recombinant cells expressing and secreting at least one heterologous enzyme extracellularly also provide an efficient and robust method for producing modified glycosyl receptors (e.g., α-glycosylated mogrosides such as compound (I)).

[0349] In particular, when the production method involves fermentation, the inventors have surprisingly discovered that recombinant cells expressing the heterozyme extracellularly are able to produce glycosylated glycosyl receptors in the culture medium. In other words, it has been surprising to find that the heterozyme is functionally expressed and secreted extracellularly, and even more surprisingly, it is active in the culture medium.

[0350] Therefore, methods for producing (fermenting) at least one heterologous enzyme as an extracellular enzyme are also considered relevant to the industrial production of modified glycosyl receptors. Furthermore, these production methods offer the following benefits compared to methods involving the intracellular expression of at least one heterologous enzyme (leading to intracellular production of modified glycosyl receptors), including but not limited to: ease of product harvesting, reduced downstream processing, scalability, reduced cellular stress, and continuous production; these are accompanied by associated cost savings and operational efficiency. Additionally, when at least one heterologous enzyme is displayed (anchored) on the outer surface of recombinant cells, these methods offer further benefits, including but not limited to: enhanced enzyme stability, facilitated enzyme recovery (e.g., harvesting intact cells), and cell recycling; these also involve associated cost savings and operational efficiency.

[0351] Fermentation The inventors have surprisingly discovered that recombinant cells of this disclosure, overexpressing the sucrose biosynthesis pathway, produce unexpectedly high titers of sucrose, ranging in the order of g / L, as disclosed above herein. It has also been surprisingly discovered that when said recombinant cells producing sucrose also produce glycosyl receptors and / or their precursors, said cells are able to produce modified glycosyl receptors from simple carbon sources (e.g., glucose). In particular, recombinant cells that simultaneously produce sucrose and mogroside IIIE and / or their precursors are able to produce compound (I) from glucose.

[0352] Therefore, the embodiments described below reduce or even completely eliminate the need to provide (feed) glycosyl donors and glycosyl acceptors and / or their precursors to the extracellular environment of recombinant cells. In other words, the embodiments enable pure fermentation. Therefore, the method can be implemented by fermentation using any commercially available and simple carbon source (e.g., glucose). Advantages include, but are not limited to: reduced costs associated with sucrose and its transportation, reduced costs associated with equipment (e.g., feed lines, storage containers), and reduced complexity of the production process.

[0353] In one embodiment, the production method may be solely a fermentation method, and the recombinant cells may express at least one heterologous enzyme of the present disclosure, and said enzyme may be an intracellular enzyme (i.e., active within the recombinant cells after expression, for example, in the cytosol). In other words, the glycosylation reaction can occur intracellularly (in vivo). In addition to expressing at least one heterologous enzyme of the present disclosure, the recombinant cells may also produce glycosyl donors and glycosyl acceptors and / or their precursors. Specifically, the recombinant cells may intracellularly (over)express heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), and may also produce sucrose and mogroside and / or their precursors, as described above. Preferably, the recombinant cells also produce mogroside IIIE and / or its precursor. The recombinant cells may further be deficient in convertase (EC 3.2.1.26) and / or (over) express β-glucosidase, and / or (over) express α-glucosidase and / or may be deficient in β-glucanase, as disclosed above.

[0354] In another embodiment, the production method may be solely a fermentation method, and the recombinant cells may express at least one heterologous enzyme of the present disclosure, wherein the enzyme may be an extracellular enzyme secreted outside the recombinant cells and / or an extracellular enzyme displayed on the surface of the recombinant cells. In other words, the glycosylation reaction occurs extracellularly. In addition to expressing at least one heterologous enzyme of the present disclosure, the recombinant cells also produce glycosyl donors and glycosyl acceptors and / or their precursors. Specifically, the recombinant cells may extracellularly (over)express heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), and may also produce sucrose and mogroside and / or their precursors, as described above. Preferably, the recombinant cells also produce mogroside IIIE and / or its precursor. The recombinant cells may further be deficient in convertase (EC 3.2.1.26) and / or (over) express β-glucosidase, and / or (over) express α-glucosidase and / or be deficient in β-glucanase, as disclosed above.

[0355] Fermentation and biotransformation combination In a further embodiment, the recombinant cells must be supplemented with exogenous glycosyl donors and / or exogenous glycosyl acceptors and / or their precursors. In these embodiments (such as one described below), the method comprises a combination of fermentation and biotransformation.

[0356] In one embodiment, the production method may be a combination of fermentation and biotransformation, wherein recombinant cells are cultured in a suitable culture medium and intracellularly express at least one heterologous enzyme of the present disclosure. The enzyme is an intracellular enzyme (i.e., active within the recombinant cells after expression, for example, in the cytosol). In other words, the glycosylation reaction occurs intracellularly (in vivo).

[0357] In one specific embodiment, the recombinant cells neither produce glycosyl donors nor glycosyl acceptors and / or their precursors, but instead provide (feed) both glycosyl donors and glycosyl acceptors and / or their precursors extracellularly. In one example, the recombinant cells intracellularly (over)express heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), and extracellularly provide (feed) sucrose and mogroside and / or its precursors (e.g., mogroside IIIE and / or its precursors). In the aforementioned embodiments, the recombinant cells may also contain, express, or be able to express one or more transport proteins capable of facilitating the transport of the compound into the recombinant cells, as described above. Alternatively or additionally, the recombinant cells may also contain cell membrane alterations (e.g., permeabilization) capable of facilitating the transport of the compound into the cell. The recombinant cells may further be deficient in convertase (EC 3.2.1.26) and / or β-glucanase, and / or (over) express β-glucosidase, and / or (over) express α-glucosidase, as disclosed above.

[0358] In one specific embodiment, the recombinant cells produce glycosyl donors but not glycosyl acceptors and / or their precursors, and provide (feed) glycosyl acceptors and / or their precursors extracellularly to the recombinant cells. For example, the recombinant cells intracellularly (over)express heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), and provide (feed) mogroside and / or its precursors (e.g., mogroside IIIE and / or its precursors) extracellularly to the recombinant cells. In the aforementioned embodiment, the recombinant cells may also contain, express, or be able to express one or more transport proteins capable of facilitating the transport of said compounds (e.g., mogroside and / or their precursors) into the recombinant cells. Alternatively or additionally, the recombinant cells may also contain cell membrane alterations (e.g., permeabilization) capable of facilitating the transport of said compounds into the cell interior. The recombinant cells may further be deficient in convertase (EC 3.2.1.26) and / or β-glucanase, and / or (over) express β-glucosidase, and / or (over) express α-glucosidase, as disclosed above.

[0359] In one specific embodiment, the recombinant cells produce glycosyl acceptors and / or their precursors but not glycosyl donors, and provide (feed) glycosyl donors extracellularly to the recombinant cells. For example, the recombinant cells intracellularly (over)express heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), and provide (feed) sucrose extracellularly to the recombinant cells. In the aforementioned embodiment, the recombinant cells may also contain, express, or be able to express one or more transport proteins capable of facilitating the transport of said compounds (e.g., sucrose) into the recombinant cells. Alternatively or additionally, the recombinant cells may also contain cell membrane alterations (e.g., permeabilization) capable of facilitating the transport of said compounds into the cell interior. The recombinant cells may further be deficient in invertase (EC 3.2.1.26) and / or (over)express β-glucosidase, and / or (over)express α-glucosidase, as disclosed above.

[0360] In one embodiment, the production method may be a combination of fermentation and biotransformation, wherein recombinant cells are cultured in a suitable culture medium and express at least one heterologous enzyme of the present disclosure extracellularly. The enzyme is an extracellular enzyme that is free outside the recombinant cells and / or displayed on the surface of the recombinant cells. In other words, the glycosylation reaction occurs extracellularly.

[0361] In one specific embodiment, the recombinant cells neither produce glycosyl donors nor glycosyl acceptors and / or their precursors, but are supplied extracellularly with both glycosyl donors and glycosyl acceptors and / or their precursors. In one example, the recombinant cells extracellularly (over)express heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), and are extracellularly supplied with sucrose and mogroside and / or its precursors (e.g., mogroside IIIE and / or its precursors). The recombinant cells may further be deficient in invertase (EC 3.2.1.26) and / or β-glucanase, and / or (over)express β-glucosidase, and / or (over)express α-glucosidase, as disclosed above.

[0362] In one specific embodiment, the recombinant cells produce glycosyl donors but not glycosyl acceptors and / or their precursors, and provide (feed) glycosyl acceptors and / or their precursors extracellularly to the recombinant cells. For example, the recombinant cells extracellularly (over)express and secrete heteroglucan sucrases, such as dextran sucrase (EC 2.4.1.5), and extracellularly provide (feed) mogrosides and / or their precursors (e.g., mogroside IIIE and / or their precursors) to the recombinant cells. In the aforementioned embodiment, the recombinant cells may also contain, express, or be able to express one or more transport proteins capable of facilitating the transport of the produced glycosyl donors (e.g., sucrose) to the exterior of the recombinant cells. The recombinant cells may further be deficient in invertases (EC 3.2.1.26) and / or (over)express β-glucosidase and / or (over)express α-glucosidase, as disclosed above.

[0363] In one specific embodiment, the recombinant cells produce glycosyl acceptors and / or their precursors but not glycosyl donors, and provide (feed) glycosyl donors extracellularly to the recombinant cells. For example, the recombinant cells extracellularly (over)express heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), and extracellularly provide (feed) sucrose to the recombinant cells. In the aforementioned embodiment, the recombinant cells may also contain, express, or be able to express one or more transport proteins capable of facilitating the transport of the produced glycosyl acceptors and / or their precursors (e.g., mogrosides and / or their precursors, such as mogroside IIIE and / or their precursors) to the outside of the recombinant cells. The recombinant cells may further be deficient in convertase (EC 3.2.1.26) and / or (over)express β-glucosidase and / or (over)express α-glucosidase, as disclosed above.

[0364] Biotransformation In cases where the production method may be solely a biotransformation method, the recombinant cells contain, (over)express, or are capable of (over)expressing at least one heterologous enzyme capable of catalyzing glycosyl transfer to a glycosyl receptor, and the recombinant cells are used as an intact-cell biocatalyst for the transfer in a reaction mixture under suitable conditions. Specifically, the method includes contacting recombinant cells expressing at least one heterologous enzyme of this disclosure with a glycosyl donor and a glycosyl receptor and / or their precursors in a reaction mixture under suitable conditions for glycosyl transfer to the glycosyl receptor to occur. More specifically, the at least one heterologous enzyme is intracellular and / or extracellularly displayed on the surface of the recombinant cells.

[0365] The components of the reaction mixture may include, but are not limited to: recombinant cells containing at least one heterologous enzyme of this disclosure, glycosyl donors (e.g., sucrose), glycosyl acceptors (e.g., terpenoids and / or their precursors), buffer solutions that promote enzyme activity, salts, and ions. The components (e.g., buffers, salts, ions) and conditions of the reaction mixture can be selected and optimized through routine experiments, as will be understood by those skilled in the art.

[0366] In one embodiment, the recombinant cell contains at least one heterologous enzyme of the present disclosure, and said enzyme is an intracellular enzyme (i.e., active within the recombinant cell). In other words, the glycosylation reaction occurs intracellularly (in vivo). In this embodiment, a glycosyl donor and / or glycosyl acceptor and / or its precursor is provided extracellularly to the recombinant cell. In one example, the recombinant cell contains a heteroglucan sucrase, such as dextran sucrase (EC 2.4.1.5), intracellularly, and sucrose and mogroside and / or its precursor (e.g., mogroside IIIE and / or its precursor) are provided extracellularly to the recombinant cell. In this embodiment, the recombinant cell may also contain one or more transport proteins capable of facilitating the transport of said compounds into the recombinant cell, as described above. Alternatively or additionally, the recombinant cell may also contain cell membrane alterations (e.g., permeabilization) capable of facilitating the transport of said compounds into the cell. The recombinant cells may further be defective in terms of the convertase (EC3.2.1.26) and / or (over) express β-glucosidase and / or (over) express α-glucosidase, as disclosed above.

[0367] In a preferred embodiment, the recombinant cell contains at least one heterologous enzyme of the present disclosure, and said enzyme is an extracellular enzyme displayed on the surface of the recombinant cell (i.e., active outside the recombinant cell). In other words, the glycosylation reaction occurs extracellularly. In the embodiment, a glycosyl donor and / or glycosyl acceptor and / or its precursor is provided extracellularly to the recombinant cell. In one example, the recombinant cell contains a heterologous glucan sucrase, such as dextran sucrase (EC 2.4.1.5), displayed extracellularly on the surface of the recombinant cell, and sucrose and mogroside and / or its precursor (e.g., mogroside IIIE and / or its precursor) are provided extracellularly to the recombinant cell. The recombinant cell may further be deficient in invertase (EC 3.2.1.26) and / or (over)express β-glucosidase and / or (over)express α-glucosidase, as disclosed above.

[0368] In the above embodiments, prior to actual biotransformation (i.e., the contact step in the reaction mixture), recombinant cells can be prepared by culturing them in a suitable culture medium under conditions where at least one heterologous enzyme capable of catalyzing glycosyl transfer to a glycosyl acceptor is expressed. After a suitable culture time, the recombinant cells are harvested from the culture medium, optionally washed with water or a buffer solution, and suspended in a desired buffer solution favorable to the activity of the heterologous enzyme. Biotransformation is then initiated by contacting the recombinant cells with a glycosyl donor, a glycosyl acceptor, and / or its precursor.

[0369] In all the above embodiments of the production methods (i.e., fermentation, fermentation combined with biotransformation, and biotransformation), the recombinant cells contain, (over)express, or are capable of (over)expressing the heterologous enzymes of this disclosure, producing glycosyl receptors and / or their precursors, or producing glycosyl donors. The recombinant cells may be extracellularly supplemented with one or more additional enzymes, one or more additional glycosyl receptors and / or their precursors, or one or more additional glycosyl donors capable of catalyzing glycosyl transfer to glycosyl receptors. The supplemented enzymes, glycosyl receptors and / or their precursors and / or glycosyl donors may be the same as and / or different from those produced by the recombinant cells. In a specific case, said at least one heterologous enzyme is an intracellular enzyme, and the recombinant cells may also contain, be capable of expressing, or be able to express one or more transport proteins capable of facilitating the transport of said compounds / enzymes into the recombinant cells. Alternatively or additionally, the recombinant cells may also contain cell membrane alterations (e.g., permeabilization) capable of facilitating the transport of said compounds / enzymes into the cell interior.

[0370] Typically, in the methods of this disclosure, recombinant cells are cultured in a bioreactor or fermenter at defined temperatures and pH for a desired period of time, typically under conditions in which genes involved in the production of modified glycosyl receptors, as disclosed herein, are expressed. Recombinant cells can be grown in various operating modes, such as batch, (repeated) fed-batch, or continuous processes.

[0371] Culture media for various recombinant cells are well known in the art. The culture medium used to culture recombinant cells will depend on the identity of the recombinant cells. Culture media typically contain inorganic salts and compounds, amino acids, carbohydrates, vitamins, and other compounds necessary for or improving the health or growth of the recombinant cells, or both. Optimization of bacterial and yeast cell growth can also be achieved by adding nutrients and supplements to the culture medium. Alternatively, cultures can be grown in fermenters designed for temperature, pH control, and controlled aeration rates. Culture conditions are typically selected from aerobic, microaerobic, and anaerobic conditions. Therefore, oxygen and nitrogen can be introduced into the culture medium as needed.

[0372] Any recombinant cells described in this disclosure can be cultured in any type of culture medium. The conditions for culture or the culture process can be optimized through routine experiments, as will be understood by those skilled in the art.

[0373] In the methods of this disclosure, recombinant cells are extracellularly supplied with (feed) glycosyl donors and / or the recombinant cells are extracellularly supplemented with one or more additional glycosyl donors, the source of which can vary. For example, the glycosyl donor can be derived from plants. In the case of sucrose as an additional glycosyl donor, sucrose can be derived, for example, from sugarcane and / or sugar beets. Thus, one or more additional glycosyl donors in this disclosure can be plant extracts or purified forms thereof. Alternatively or additionally, in the context of this disclosure, one or more additional glycosyl donors can be produced and extracellularly secreted by cells other than the recombinant cells of this disclosure. In the context of this invention, one or more additional glycosyl donors can be or may comprise inactive disaccharides and / or inactive polysaccharides, such as sucrose or (soluble) starch.

[0374] In the methods of this disclosure, glycosyl receptors and / or their precursors are extracellularly provided (feeded) to recombinant cells and / or one or more glycosyl receptors are extracellularly supplemented to recombinant cells, and the source of the glycosyl receptors and / or their precursors can vary. For example, the glycosyl receptors and / or their precursors can be derived from plants. In the case of mogrosides and / or their precursors, the mogrosides and / or their precursors can be derived, for example, from *Siraitia grosvenorii* (monk fruit). Therefore, the glycosyl receptors and / or their precursors can be plant (e.g., *Siraitia grosvenorii*) extracts or their purified forms. Alternatively or additionally, in the context of this disclosure, the glycosyl receptors and / or their precursors can be produced and extracellularly secreted by cells other than the recombinant cells of this disclosure. Among other things, one or more glycosyl receptors can be or may contain mogrosides (e.g., mogroside IIIE), terpenoids other than mogrosides (e.g., steviol glycosides), and / or flavonoids.

[0375] In the methods disclosed herein, recombinant cells are extracellularly supplemented with one or more additional enzymes capable of catalyzing glycosyl transfer to glycosyl receptors, the source of which may vary. For example, the enzyme may be a pure enzyme, a cell (cell-free) lysate containing the enzyme, its purified form, and / or an enzyme produced by cells other than the recombinant cells. In the context of this invention, one or more additional enzymes capable of catalyzing glycosyl transfer to glycosyl receptors may be or may include enzymes belonging to the glycoside hydrolase family 70 (GH70), the glycoside hydrolase family 13 (GH13), and / or the glycoside hydrolase family 77 (GH77); for example, cyclomaltodextrin dextran transferase (EC 2.4.1.19), dextran sucrases, such as alternosaccharide sucrase (EC 2.4.1.140), dextran sucrase (EC 2.4.1.5), polysaccharide sucrase (EC 2.4.1.5), and rougelan sucrase (EC 2.4.1.5).

[0376] In methods involving fermentation (e.g., fermentation alone or in combination with the biotransformation disclosed above), where recombinant cells are extracellularly supplied (feeded) or supplemented with glycosyl donors (e.g., sucrose) and / or glycosyl acceptors and / or their precursors (e.g., mogroside and / or their precursors), the feeding of said compounds can begin at the moment the recombinant cells are inoculated into the culture medium. Alternatively, the feeding of said compounds can begin during or after cell growth; that is, after the recombinant cells are inoculated into the culture medium. Thus, said compounds can be fed 1 hour before inoculation, during inoculation, or 3, 6, 12, 24, 48, 72, 96, or 120 hours after the recombinant cells are inoculated into the culture medium. Feeding can continue until the end of fermentation and / or until the desired (level) of modified glycosyl acceptors is obtained.

[0377] In further methods involving fermentation (e.g., fermentation alone or in combination with the biotransformations disclosed above), wherein recombinant cells are extracellularly fed (feeded) or supplemented with a glycosyl donor (e.g., sucrose) and the recombinant cells also produce glycosyl acceptors and / or their precursors, the extracellular feeding of the glycosyl donor (e.g., sucrose) can begin before, at, or after the production of the glycosyl acceptor and / or its precursor (i.e., after the expression of the glycosyl acceptor biosynthetic pathway as described above). Therefore, the extracellular feeding of the glycosyl donor can begin 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours after the recombinant cells begin producing the glycosyl acceptor and / or its precursor (i.e., after the expression of the glycosyl acceptor biosynthetic pathway). Preferably, the extracellular feeding of the glycosyl donor begins after the production of the glycosyl acceptor and / or its precursor has begun; more preferably, it begins 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours later. Feeding can continue until the end of fermentation and / or until the desired (level) of modified glycosyl acceptors is obtained.

[0378] In one example, where recombinant cells express a heterodecanosucrase (e.g., dextran sucrase) intracellularly and produce glycosyl receptors and / or their precursors, extracellular feeding of sucrose can begin before, during, or after the production of glycosyl receptors and / or their precursors (i.e., after the expression of the glycosyl receptor biosynthetic pathway as described above). Therefore, extracellular feeding of sucrose can begin 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours after the recombinant cells begin producing glycosyl receptors and / or their precursors (i.e., after the expression of the glycosyl receptor biosynthetic pathway). Preferably, extracellular feeding of sucrose begins after the production of glycosyl receptors and / or their precursors; more preferably, it begins 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours later. Feeding can continue until fermentation is complete and / or until the desired (level) of modified glycosyl receptors is obtained.

[0379] In a further example, where recombinant cells express heterodecanosucrase (e.g., dextran sucrase) extracellularly (free outside the recombinant cells and / or displayed on the surface of the recombinant cells) and produce glycosyl receptors and / or their precursors, extracellular sucrose feeding can begin before, during, or after the production of glycosyl receptors and / or their precursors (i.e., after the expression of the glycosyl receptor biosynthetic pathway as described above). Therefore, extracellular sucrose feeding can begin 0 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours after the recombinant cells begin producing glycosyl receptors and / or their precursors (i.e., after the expression of the glycosyl receptor biosynthetic pathway begins). Preferably, extracellular sucrose feeding begins after the production of glycosyl receptors and / or their precursors has begun; more preferably, it begins 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours later. Feeding can continue until the end of fermentation and / or until the desired (level) of modified glycosyl acceptors is obtained.

[0380] In the above embodiments, the inventors have surprisingly discovered that delaying the start of sucrose feed (relative to the start of production of the glycosyl acceptor and / or its precursor) increases the yield of the modified glycosyl acceptor to the consumed glycosyl donor.

[0381] Feeding strategies (e.g., the amount of glycosyl donor and feeding time) can be further optimized through routine experiments, as will be understood by those skilled in the art. For example, when sucrose is fed extracellularly or supplemented into the recombinant cells of this disclosure, its concentration in the culture medium or fermentation broth can be maintained between 0.5 g / l and 50 g / l, 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, and 1 g / l-5 g / l.

[0382] Separation and purification In the methods of this disclosure, modified glycosyl receptors (e.g., modified mogroside compounds, such as compound (I)) can optionally be isolated or recovered from the fermentation broth or reaction mixture, and optionally further purified using various techniques known to those skilled in the art. For example, after fermentation, the fermentation broth can be treated to kill recombinant cells and remove cells before or after cell wall rupture. Cell-free lysates can be obtained, for example, by mechanical or enzymatic rupture of host cells, followed by additional centrifugation to remove cell debris. The dried fermentation broth material can also be mechanically ruptured, for example by sonication. The dissolved or suspended fermentation broth material can be filtered. The fermentation medium or cell-free lysate can optionally be treated to remove low molecular weight compounds (e.g., salts); and can optionally be dried and redissolved in a mixture of water and solvent prior to purification.

[0383] The supernatant or cell-free lysate can be purified, for example, by adsorption chromatography using different types of resins and elution solvents. The levels of (modified) glycosyl receptors (e.g., (modified) mogrosides) in each fraction (including the flow-through) can then be analyzed by LC-MS. Fractions can then be combined and the volume reduced using a vacuum evaporator. Additional purification steps, such as additional chromatographic steps and crystallization, can be used if desired. For example, (modified) mogrosides can be separated by methods not limited to ion-exchange chromatography, reversed-phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon column and / or decolorization steps.

[0384] This disclosure also provides recombinant cells as disclosed above in the methods of this disclosure. Specifically, the recombinant cells comprise at least one heteropolynucleotide encoding at least one enzyme (e.g., dextran sucrase, such as dextran sucrase) capable of catalyzing an α-glycosidic bond between a glycosyl donor (e.g., sucrose) and a glycosyl acceptor (e.g., a terpenoid compound, such as mogroside), wherein the cell is deficient in enzymes capable of hydrolyzing glycosyl donors (e.g., the cell is deficient in invertases), and optionally comprises at least one polynucleotide sequence encoding an α-glucosidase and / or at least one polynucleotide encoding a β-glucosidase. The enzyme and / or the polynucleotide encoding the α-glucosidase or the β-glucosidase can be heteroenzymes and / or heteropolynucleotides. The enzyme and / or polynucleotide can be (over)expressed. Typically, the polynucleotide can be constitutively expressed. Alternatively, the expression of the polynucleotide can be induced.

[0385] This disclosure also provides a reaction mixture comprising the recombinant cells disclosed above.

[0386] This disclosure also provides a culture medium comprising the recombinant cells disclosed above.

[0387] This disclosure also provides a fermentation broth containing the recombinant cells disclosed above.

[0388] This disclosure also provides compositions, such as terpenoid compositions, such as mogroside compositions, obtained or obtainable by the methods disclosed above. In particular, this disclosure also provides mogroside compositions or sweetener compositions obtained or obtainable by the methods of this disclosure. Preferably, the mogroside or sweetener composition comprises α-glycosylated mogroside. More preferably, the mogroside or sweetener composition comprises α-glycosylated mogroside IIIE. Even more preferably, the mogroside or sweetener composition comprises compound (I). Furthermore, food products, beverages, pet foods, animal feeds, oral compositions, bioactive compositions, or pharmaceutical compositions comprising the mogroside or sweetener compositions are also provided.

[0389] Mogrosides, which are obtained or obtainable by the methods disclosed herein, are also provided. Mogrosides or compositions thereof can be used in any known application of such compounds. In particular, mogrosides or compositions thereof can be used, for example, as sweeteners, such as in foods or beverages. For example, mogrosides or compositions thereof can be formulated in soft drinks, as table-top sweeteners, chewing gum, dairy products such as yogurt (e.g., plain yogurt), cakes, cereals or cereal-based foods, nutritional supplements, pharmaceuticals, edible gels, confectionery products, cosmetics, toothpaste, or other oral compositions. Furthermore, mogrosides or compositions thereof can be used not only as sweeteners in beverages, foods, and other products intended for human consumption, but also as animal feed and forage with improving properties. Therefore, such foods, feeds, or beverages containing said mogrosides or compositions thereof are also provided. Conventional methods, such as mixing, kneading, dissolving, marinating, permeating, filtering, spraying, atomizing, impregnating, and other methods, can be used in the manufacture of foods, beverages, pharmaceuticals, cosmetics, tableware, and chewing gum.

[0390] Mogrosides or combinations thereof, as disclosed herein, can be used in dry or liquid form. They can be added before or after food heat treatment. The amount of sweetener depends on the intended use. They can be added alone or in combination with other compounds.

[0391] Mogrosides or compositions thereof obtained or obtainable according to the methods disclosed herein can be blended with one or more other non-caloric or caloric sweeteners. Such blending can be used to improve flavor, time characteristics, or stability. A variety of non-caloric and caloric sweeteners are suitable for blending with mogrosides. For example, non-caloric sweeteners include steviol glycosides, monadine, aspartame, acesulfame potassium salt, cyclamate, sucralose, saccharin salt, or erythritol. Caloric sweeteners suitable for blending with mogrosides include sugar alcohols and carbohydrates such as sucrose, glucose, fructose, and HFCS. Sweet amino acids such as glycine, alanine, or serine may also be used.

[0392] Mogroside or combinations thereof can be used in combination with sweetness inhibitors (e.g., natural sweetness inhibitors). It can also be combined with umami enhancers (e.g., amino acids or their salts).

[0393] Monk fruit glycosides or combinations thereof may be combined with polyols or sugar alcohols, carbohydrates, physiologically active substances or functional ingredients (e.g., carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutritional supplements, flavonoids, isothiocyanates, phenols, phytosterols or sterols (phytosterols and phytosterols), polyols, prebiotics, probiotics, post-biotics, phytoestrogens, soy protein, sulfides / thiols, amino acids, proteins, vitamins, minerals and / or substances classified based on health benefits, such as cardiovascular, cholesterol-lowering or anti-inflammatory substances).

[0394] Compositions containing mogrosides as disclosed above may include flavoring agents, aroma components, nucleotides, organic acids, organic acid salts, inorganic acids, bitter compounds, proteins or protein hydrolysates, surfactants, flavonoids, astringent compounds, vitamins, dietary fiber, antioxidants, fatty acids and / or salts.

[0395] As disclosed herein, mogrosides or combinations thereof can be used as high-intensity sweeteners to produce zero-calorie, low-calorie, or diabetic beverages and foods with improved flavor characteristics. Furthermore, they can be used in beverages, foods, pharmaceuticals, and other products where sugar cannot be used.

[0396] In addition, mogrosides or combinations thereof, as disclosed herein, can be used not only as sweeteners in beverages, foods and other products intended for human consumption, but also as animal feed and fodder with improving properties.

[0397] Examples of products for which mogrosides disclosed in this article can be used as sweeteners include: alcoholic beverages such as vodka, wine, beer, spirits, and sake; natural fruit juices, soft drinks, including carbonated soft drinks, diet drinks, zero-calorie drinks, and low-calorie drinks; as well as foods, yogurt drinks, instant fruit juices, instant coffee, powdered instant beverages, canned products, syrups, fermented soybean pastes, soy sauce, vinegar, condiments, mayonnaise, ketchup, curry, soups, instant clear broths, and powdered sauces. Oils, powdered vinegar, various biscuits, rice cakes, crisp biscuits, bread, chocolate, caramel, candy, chewing gum, jelly, pudding, candied fruits and vegetables, whipped cream, jam, orange marmalade, flower jam, milk powder, ice cream, popsicles, bottled vegetables and fruits, canned and boiled beans, meat and sweet sauce-cooked foods, agricultural vegetable products, seafood, ham, sausages, fish ham, fish sausages, fish cakes, fried fish products, dried seafood products, frozen foods, pickled seaweed, cured meats, tobacco, pharmaceuticals, and many other products. In principle, their application is unrestricted.

[0398] Sweetening compositions comprise beverages, non-limiting examples of which include: non-carbonated and carbonated beverages such as cola, ginger beer, root beer, cider, fruit-flavored soft drinks (e.g., citrus-flavored soft drinks, such as lemon-lime or orange), powdered soft drinks, etc.; fruit juices derived from fruits or vegetables, including juice extracted from fruit pulp, fruit juices containing fruit pieces, fruit drinks, fruit juice drinks, fruit juice-containing drinks, fruit-flavored drinks, vegetable juices, vegetable juices, and mixed fruit and vegetable juices; sports drinks, energy drinks, near-water drinks, etc. (e.g., water containing natural or synthetic flavorings); tea or flavored beverages, such as coffee, cocoa, black tea, green tea, oolong tea, etc.; beverages containing dairy components, such as milk drinks, milk-based coffee, milk coffee, milk tea, fruit milk drinks, drinkable yogurt, lactic acid bacteria drinks, etc.; and dairy products.

[0399] Typically, the amount of sweetener in a sweetening composition varies considerably depending on the specific type of sweetening composition and the desired sweetness. Those skilled in the art can readily determine the appropriate amount of sweetener to be added to the sweetening composition, which can be used in dry or liquid form. It can be added before or after heat treatment of the food product. The amount of sweetener depends on the intended use. It can be added alone or in combination with other compounds.

[0400] In the manufacturing processes of food, beverages, pharmaceuticals, cosmetics, tableware, and chewing gum, conventional methods can be used, such as mixing, kneading, dissolving, marinating, permeating, filtering, spraying, atomizing, impregnating, and other methods.

[0401] Therefore, the sweetening compositions disclosed herein can be prepared by any method known to those skilled in the art. These methods include dry mixing, spray drying, agglomeration, wet granulation, compaction, co-crystallization, etc.

[0402] In solid form, mogrosides or compositions thereof prepared as disclosed herein can be provided to consumers in any form suitable for delivery to an edible product to be sweetened, including sachets, packages, bulk bags or boxes, cubes, tablets, sprays, or soluble strips. The compositions can be delivered in unit doses or in bulk form.

[0403] In liquid form, mogrosides or compositions thereof prepared as disclosed herein may be provided in any form suitable for consumer use, including fluid, semi-fluid, paste and cream forms, and in any suitable packaging using any suitable packaging material in any shape or form that facilitates carrying, dispensing, storage and / or transport.

[0404] The composition may contain a variety of fillers, functional ingredients, colorants and / or flavorings.

[0405] This disclosure also provides a method for producing modified glycosyl receptors (e.g., modified terpenoids such as modified mogroside) using recombinant cells as disclosed above. In particular, this disclosure also provides a method for producing α-glycosylated glycosyl receptors (e.g., α-glycosylated terpenoids such as α-glycosylated mogroside) using recombinant cells as disclosed above. More specifically, this disclosure also provides a method for producing α-glycosylated mogroside IIIE (such as compound (I)) using recombinant cells as disclosed above.

[0406] References to patent documents or other matters given in this document as background art should not be construed as an admission that such document or matter was known at the priority date of any claim or that the information contained therein was part of common general knowledge.

[0407] The following is a list of embodiments according to this disclosure, but it is not intended to be limiting: 1. A dextran sucrase variant, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 21, contains at least one modification of an amino acid residue corresponding to any of the following amino acids: 274, 275, 276, 319, 322, 406, 478, 481 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 21, optionally wherein the 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%, or at least 99% sequence identity with the amino acid sequence listed in SEQ ID NO: 21.

[0408] 2. The dextran sucrase variant according to embodiment 1, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 21, contains one or more of the following: The value at position 274 is F or L; at position 275 it is Y, F, K, or S; at position 276 it is W; at position 319 it is P; at position 322 it is A; at position 406 it is Q; at position 478 it is Y; and at position 481 it is A. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 21.

[0409] 3. A dextran sucrase variant, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 2, contains at least one modification of an amino acid residue corresponding to any of the following amino acids: 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, The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 21, optionally wherein the 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%, or at least 99% sequence identity with the amino acid sequence listed in SEQ ID NO: 2.

[0410] 4. The dextran sucrase variant according to embodiment 3, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 2, contains one or more of the following: Position 196 is F, position 197 is S, position 199 is F, position 201 is W or F, position 215 is P, position 227 is M or Q, position 228 is P, position 239 is P, position 242 is A, position 282 is S, position 289 is Y or W, position 290 is M, position 326 is L, F, Y or Q, position 328 is F, L, S, M or H, position 334 is F, and position... Position 346 is F, position 349 is H, position 398 is Y, position 399 is E or D, position 400 is V, S or L, position 401 is E, A, D, F, G, H, L, M, N, S, T, V, W or Y, position 402 is T or S, position 417 is H, position 715 is R, position 721 is T or Q, position 726 is W, position 763 is M, position 788 is R, and position 867 is S. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 2.

[0411] 5. A dextran sucrase variant, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 34, contains at least one modification of an amino acid residue corresponding to any of the following amino acids: 200, 203, 204, 249, 252, 336, 413, 416 The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 34, optionally wherein the 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%, or at least 99% sequence identity with the amino acid sequence listed in SEQ ID NO: 34.

[0412] 6. The dextran sucrase variant according to embodiment 5, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 34, contains one or more of the following: The value at position 200 is F or L, at position 203 is S or F, at position 204 is W, at position 249 is P, at position 252 is A, at position 336 is Q or L, at position 413 is Y, and at position 416 is E, A, or H. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 34.

[0413] 7. A dextran sucrase variant, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 26, contains at least one amino acid residue modification corresponding to any of the following amino acids: 212, 253, 256, 259, 302, 303, 412, 414, 419, 422, 425, 433, 436, 688, 691, 722, 733, 739, 743, 745, 782, 787, The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 26, optionally wherein the 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%, or at least 99% sequence identity with the amino acid sequence listed in SEQ ID NO: 26.

[0414] 8. The dextran sucrase variant according to embodiment 7, wherein the variant comprises an amino acid sequence that, when compared with the amino acid sequence listed in SEQ ID NO: 26, contains one or more of the following: The position is 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; and M or I at position 787. The position is defined with reference to the amino acid sequence listed in SEQ ID NO: 26.

[0415] 9. A method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor, comprising: contacting the glycosyl donor and the glycosyl acceptor with a recombinant cell under suitable conditions, the recombinant cell containing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor, wherein the contact under suitable conditions enables the at least one heterozyme to catalyze the formation of an α-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor.

[0416] 10. A method for producing a modified glycosyl receptor, comprising: contacting a glycosyl donor and a glycosyl receptor with recombinant cells under suitable conditions, the recombinant cells containing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl from the glycosyl donor to the glycosyl receptor, wherein the contact under suitable conditions comprises culturing the recombinant cells in a suitable culture medium in the presence of the glycosyl donor and the glycosyl receptor and / or their precursors, optionally wherein the contact under suitable conditions allows the recombinant cells to express the at least one heterozyme, wherein the contact under suitable conditions enables the at least one heterozyme to catalyze the formation of an α-glycosidic bond between the transferred glycosyl and the glycosyl receptor, thereby producing a modified glycosyl receptor; and optionally, isolating the thereby produced modified glycosyl receptor.

[0417] 11. A method for producing a modified glycosyl receptor, comprising: contacting a glycosyl donor and a glycosyl receptor with a recombinant cell under suitable conditions, the recombinant cell containing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl receptor; optionally, wherein the contact under suitable conditions allows the recombinant cell to express the at least one heterozyme; wherein the contact under suitable conditions includes contacting the recombinant cell, the glycosyl donor, and the glycosyl receptor in a reaction mixture; wherein the contact under suitable conditions enables the at least one heterozyme to catalyze the formation of an α-glycosidic bond between the transferred glycosyl group and the glycosyl receptor, thereby producing a modified glycosyl receptor; and optionally, isolating the resulting modified terpene compound.

[0418] 12. The method according to any one of embodiments 9 to 11, wherein contacting the glycosyl donor and glycosyl acceptor with the recombinant cell under suitable conditions means that: a) the glycosyl donor and glycosyl acceptor are in contact inside the recombinant cell (intracellular); and / or b) the glycosyl donor and glycosyl acceptor are in contact outside the recombinant cell (extracellular).

[0419] 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 transglycosylation, typically an enzyme belonging to the glycoside hydrolase family 70 (GH70), the glycoside hydrolase family 13 (GH13), and / or the glycoside hydrolase family 77 (GH77).

[0420] 14. The method according to any one of embodiments 9 to 13, wherein the at least one heterologous enzyme is an enzyme selected from the group consisting of: cyclomaltodextrin dextran transferase (EC 2.4.1.19), dextran sucrase, such as alternan sucrase (EC 2.4.1.140), dextran sucrase (EC 2.4.1.5), polysaccharide sucrase (EC 2.4.1.5), and roi polysaccharide sucrase (EC 2.4.1.5); preferably, the at least one heterologous enzyme is dextran sucrase (EC 2.4.1.5).

[0421] 15. The method according to embodiment 14, wherein the at least one heterologous enzyme is dextran sucrase (EC 2.4.1.5), optionally dextran sucrase according to any one of embodiments 1 to 8, or dextran sucrase comprising an amino acid sequence having at least 50% sequence identity with any of the following amino acid sequences: 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 dextran sucrase having at least 50% sequence identity with any of mutants _0001 to _0291 in Table 2.

[0422] 16. The method according to any one of embodiments 9 to 15, wherein the glycosyl donor is an inactive disaccharide and / or an inactive polysaccharide, such as sucrose and / or (soluble) starch; preferably, the glycosyl donor comprises sucrose.

[0423] 17. The method according to any one of embodiments 9 to 16, wherein the glycosyl receptor is a terpenoid or a glycosylated terpenoid, such as mogroside, preferably mogroside IIIE.

[0424] 18. The method according to any one of embodiments 9 to 17, wherein the modified glycosyl receptor (e.g., a modified (glycosylated) terpene compound) is α-glycosylated mogroside, preferably α-glycosylated mogroside IIIE.

[0425] 19. The method according to any one of embodiments 9 to 18, wherein the glycosyl acceptor comprises mogroside IIIE, the glycosyl donor comprises sucrose, and the at least one heterozyme comprises dextran sucrase; wherein the dextran sucrase catalyzes an α-1,6-glycosidic bond between the C1 of the transferred glycosyl group and the C6 hydroxyl group of glucose linked at the C24 position of mogroside IIIE; thereby producing compound (I). .

[0426] 20. The method according to any one of embodiments 9 to 19, wherein the recombinant cell is a living cell or an intact cell.

[0427] 21. The method according to any one of embodiments 9 to 20, wherein the recombinant cells are defective in terms of being able to deglycosylate mogroside products and hydrolyze polypeptides selected from at least one of the following glycosidic bonds: (a) The glycosidic bond between the carbon (C24 atom) at position 24 of the mogroside skeleton and the β-1-glucose bonded at that position; and / or (b) The glycosidic bond between the carbon (C3 atom) at position 3 of the mogroside skeleton and the β-1-glucose bonded at that position.

[0428] 22. The method according to any one of embodiments 9 to 21, wherein the recombinant cells are deficient in enzymes capable of hydrolyzing the glycosyl donor; preferably the recombinant cells are deficient in invertase (EC 3.2.1.26).

[0429] 23. The method according to any one of embodiments 9 to 22, wherein the recombinant cells further comprise α-glucosidase, such as dextranase (EC 3.2.1.11).

[0430] 24. The method according to any one of embodiments 9 to 23, wherein the recombinant cell further comprises a polypeptide capable of deglycosylating a highly glycosylated glycosyl receptor, optionally wherein the polypeptide is a β-glucosidase.

[0431] 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 extracellularly provided to the recombinant cell.

[0432] 26. The method according to embodiment 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.

[0433] 27. The method according to embodiment 25 or 26, wherein the glycosyl donor extracellularly provided to the recombinant cells or the additional glycosyl donor extracellularly supplemented to the recombinant cells is a plant extract, its purified form, and / or produced by cells other than the recombinant cells.

[0434] 28. The method according to any one of embodiments 25 to 27, wherein the recombinant cells produce or are capable of producing the glycosyl donor, and comprise one or more of the following: (a) Capable of phosphorylating glucose to form glucose- Phosphorylase, preferably hexokinase (EC2.7.1.1). (b) An enzyme capable of converting glucose-6-phosphate to fructose-6-phosphate, preferably a glucose-6-phosphate isomerase (EC 5.3.1.9). (c) An enzyme capable of catalyzing the transfer of hexose from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6-phosphate, preferably sucrose phosphate synthase (EC 2.4.1.14). (d) An enzyme capable of converting sucrose-6-phosphate to sucrose, preferably sucrose-6-phosphate phosphatase (EC 3.1.3.24); and, optionally... (e) Changes in transport systems and / or cell membranes that can promote glucose transport into the recombinant cells; (f) Changes in transport systems and / or cell membranes that can promote the transport of sucrose to the outside of the recombinant cells.

[0435] 29. The method according to any one of embodiments 9 to 28, wherein the recombinant cell further produces the glycosyl receptor and / or its precursor, and / or wherein the glycosyl receptor and / or its precursor is extracellularly provided to the recombinant cell.

[0436] 30. The method according to embodiment 29, wherein the recombinant cells further produce the glycosyl receptor and / or its precursor, and the recombinant cells are further extracellularly supplemented with one or more additional glycosyl receptors and / or their precursors.

[0437] 31. The method according to embodiment 29 or 30, wherein the glycosyl receptor and / or its precursor provided extracellularly to the recombinant cells and / or the additional glycosyl receptor and / or its precursor provided extracellularly to the recombinant cells are plant extracts, their purified forms, and / or produced by cells other than the recombinant cells.

[0438] 32. The method according to any one of embodiments 29 to 31, wherein the recombinant cells produce or are capable of producing the glycosyl receptor (e.g., a terpene compound) and / or its precursor, and comprise one or more of the following: (a) A polypeptide capable of synthesizing geraniol diphosphate (GPP) from dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is geraniol diphosphate synthase (EC 2.5.1.1) (GPPS) and / or farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). (b) A polypeptide capable of synthesizing farnesyl diphosphate (FPP) from gerany diphosphate (GPP), particularly wherein said polypeptide is farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). (c) A polypeptide capable of synthesizing geraniol diphosphate (GGPP) from farnesyl diphosphate (FPP), particularly wherein said polypeptide is geraniol diphosphate synthase (EC 2.5.1.29) (GGPPS). (d) A polypeptide capable of synthesizing gerany-farnesyl diphosphate (GFPP) from gerany-gerany-farnesyl diphosphate (GGPP), particularly wherein said polypeptide is gerany-farnesyl diphosphate synthase (EC 2.5.1.81) (GFPPS). (e) A polypeptide encoding terpene synthase.

[0439] 33. The method according to any one of embodiments 29 to 32, wherein the recombinant cells produce or are capable of producing terpenoid compounds (e.g., mogroside) and / or their precursors, and comprise one or more of the following: (a) Squalene synthase (SQS); (b) Squalene epoxidase (SQE); (c) Cucurbita dienol synthase (CDS); (d) Cytochrome P450 (CYP); (e) Cytochrome P450 reductase (CPR); (f) Epoxide hydrolase (EPH); (g) Cytochrome b5 (CB5).

[0440] 34. The method according to any one of embodiments 29 to 33, wherein the recombinant cells produce or are capable of producing terpenoid compounds (e.g., mogroside) and / or their precursors, and comprise one or more of the following uridine diphosphate-dependent glycosyltransferases (UGTs): (a) A polypeptide capable of glycosylation of mogroside or mogroside at its C3 hydroxyl, C11 hydroxyl, C24 hydroxyl and / or C25 hydroxyl; (b) A polypeptide capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety at C24 (C24-O-glucose) of mogroside and / or capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety at C3 (C3-O-glucose) of mogroside. (c) A polypeptide capable of β-1,6-glycosylation of the C6'-hydroxyl group of the glucose moiety at C3 position of mogroside and / or capable of β-1,6-glycosylation of the C6'-hydroxyl group of the glucose moiety at C24 position of mogroside.

[0441] 35. The method according to any one of embodiments 29 to 34, wherein the recombinant cells produce or are capable of producing terpenoid compounds (e.g., mogroside) and / or their precursors, and comprise one or more of the following: (a) A polypeptide capable of synthesizing acetyl-CoA (AACoA) from acetyl-CoA, particularly wherein said polypeptide is acetyl-CoA acetyltransferase (EC 2.3.1.9) (AACT). (b) A polypeptide capable of synthesizing hydroxymethylglutaryl-CoA (HMGCoA) from acetyl-CoA, particularly wherein said polypeptide is hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) (HMGS). (c) A polypeptide capable of synthesizing mevalonic acid (MVA) from hydroxymethylglutaryl-CoA (HMGCoA), particularly wherein said polypeptide is hydroxyl Methylglutaryl-CoA reductase (EC 1.1.1.34) (HMGR); (d) A polypeptide capable of synthesizing mevalonate-5-phosphate (MVA-P) from mevalonate (MVA), particularly wherein said polypeptide is mevalonate kinase (EC 2.7.1.36) (MK). (e) A polypeptide capable of synthesizing mevalonate-5-bisphosphate (MVA-PP) from mevalonate-5-phosphate (MVA-P), particularly wherein said polypeptide is mevalonate phosphate kinase (EC 2.7.4.2) (PMK). (f) A polypeptide capable of synthesizing isopentenyl diphosphate (IPP) from mevalonate-5-bisphosphate (MVA-PP), particularly wherein said polypeptide is mevalonate diphosphate decarboxylase (EC 4.1.1.33) (MDD) and / or isopentenyl / dimethylallyl diphosphate synthase (EC 1.17.1.2) (IPPS). (g) A polypeptide capable of synthesizing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP), particularly wherein said polypeptide is an isopentenyl diphosphate δ-isomerase (EC 5.3.3.2) (IPI).

[0442] 36. The method according to any one of embodiments 9 to 35, wherein the at least one heterologous enzyme is an intracellular enzyme and / or an extracellular enzyme.

[0443] 37. The method according to embodiment 36, wherein the at least one heterologous enzyme is an extracellular enzyme, and optionally, wherein the enzyme is secreted and released outside the recombinant cell and / or displayed on the surface of the recombinant cell.

[0444] 38. The method according to embodiment 36, wherein the at least one heterologous enzyme is an intracellular enzyme.

[0445] 39. The method according to any one of embodiments 25 to 38, wherein the glycosyl donor is provided extracellularly to the recombinant cell, and the recombinant cell further comprises a transport system and / or cell membrane alterations capable of facilitating the transport of the glycosyl donor into the cell interior.

[0446] 40. The method according to any one of embodiments 36 or 37, wherein the at least one heterologous enzyme is an extracellular enzyme.

[0447] 41. The method according to embodiment 40, wherein the recombinant cell further produces the glycosyl receptor and / or its precursor, and / or wherein the glycosyl receptor and / or its precursor is extracellularly provided to the recombinant cell.

[0448] 42. The method according to an...

Claims

1. A method for transferring a glycosyl group from a glycosyl donor to a glycosyl acceptor, comprising: Under suitable conditions, a glycosyl donor and a glycosyl acceptor are contacted with recombinant cells containing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of glycosyl groups from the glycosyl donor to the glycosyl acceptor. Under suitable conditions, the contact enables the at least one heterozyme to catalyze the formation of an α-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor.

2. A method for producing modified glycosyl receptors, comprising: Under suitable conditions, a glycosyl donor and a glycosyl acceptor are contacted with recombinant cells containing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor. The contact under suitable conditions comprises culturing the recombinant cells in a suitable culture medium in the presence of the glycosyl donor and the glycosyl acceptor and / or their precursors. Optionally, the contact under suitable conditions allows the recombinant cells to express the at least one heterozyme. The contact under suitable conditions enables the at least one heterozyme to catalyze the formation of an α-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor. Optionally, the resulting modified glycosyl acceptor is isolated.

3. A method for producing modified glycosyl receptors, comprising: Under suitable conditions, a glycosyl donor and a glycosyl acceptor are contacted with recombinant cells containing a polynucleotide encoding at least one heterozyme capable of catalyzing the transfer of a glycosyl group from the glycosyl donor to the glycosyl acceptor. Optionally, the contact under suitable conditions allows the recombinant cells to express the at least one heterozyme. The contact under suitable conditions includes contacting the recombinant cells, the glycosyl donor, and the glycosyl acceptor in a reaction mixture. The contact under suitable conditions enables the at least one heterozyme to catalyze the formation of an α-glycosidic bond between the transferred glycosyl group and the glycosyl acceptor, thereby producing a modified glycosyl acceptor. Optionally, the resulting modified terpene compound is isolated.

4. The method according to any one of the preceding claims, wherein contacting the glycosyl donor and the glycosyl acceptor with the recombinant cell under suitable conditions means that: a) the glycosyl donor and the glycosyl acceptor are in contact inside the recombinant cell (intracellular); and / or b) the glycosyl donor and the glycosyl acceptor are in contact outside the recombinant cell (extracellular).

5. The method according to any one of the preceding claims, wherein the at least one heterologous enzyme is an enzyme capable of catalyzing transglycosylation, typically an enzyme belonging to the glycosidase family 70 (GH70), the glycosidase family 13 (GH13), and / or the glycosidase 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 dextrantransferase (EC 2.4.1.19), dextranase sucrase, such as alternanase (EC 2.4.1.140), dextranase (EC 2.4.1.5), polysaccharide sucrase (EC 2.4.1.5), and roi polysaccharide sucrase (EC 2.4.1.5); preferably, the at least one heterologous enzyme is dextranase (EC 2.4.1.5).

7. The method according to any one of the preceding claims, wherein the glycosyl donor is an unactivated disaccharide and / or an unactivated 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 receptor is a terpenoid or a glycosylated terpenoid, such as mogroside, preferably mogroside IIIE.

9. The method according to any one of the preceding claims, wherein the modified glycosyl receptor (e.g., a modified (glycosylated) terpene compound) is α-glycosylated mogroside, preferably α-glycosylated mogroside IIIE.

10. The method according to any one of the preceding claims, wherein the glycosyl acceptor comprises mogroside IIIE, the glycosyl donor comprises sucrose, and the at least one heterozyme comprises dextran sucrase; wherein the dextran sucrase catalyzes an α-1,6-glycosidic bond between the C1 of the transferred glycosyl group and the C6 hydroxyl group of glucose linked at the C24 position of mogroside IIIE; thereby producing compound (I). 。 11. The method according to any one of the preceding claims, wherein the recombinant cell is a living cell or an intact cell.

12. The method according to any one of the preceding claims, wherein the recombinant cells are defective in terms of being able to deglycosylate mogroside products and hydrolyze polypeptides selected from at least one of the following glycosidic bonds: (a) The glycosidic bond between the carbon (C24 atom) at position 24 of the mogroside skeleton and the β-1-glucose bonded at that position; and / or (b) The glycosidic bond between the carbon (C3 atom) at position 3 of the mogroside skeleton and the β-1-glucose bonded at that position.

13. The method according to any one of the preceding claims, wherein the recombinant cells are deficient in enzymes capable of hydrolyzing the glycosyl donor; preferably, the recombinant cells are deficient in invertase (EC 3.2.1.26).

14. The method according to any one of the preceding claims, wherein the recombinant cells further comprise α-glucosidase, such as dextranase (EC 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 highly glycosylated glycosyl receptor, optionally wherein the polypeptide is a β-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 extracellularly provided to the recombinant cell.

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

19. The method according to any one of claims 16 to 18, wherein the recombinant cell produces or is capable of producing the glycosyl donor, and comprises one or more of the following: a. Able to phosphorylate glucose to form glucose- Phosphorylase, preferably hexokinase (EC 2.7.1.1). b. An enzyme capable of converting glucose-6-phosphate to fructose-6-phosphate, preferably a glucose-6-phosphate isomerase (EC5.3.1.9). c. An enzyme capable of catalyzing the transfer of hexose from uridine diphosphate glucose (UDP-glucose) to fructose-6-phosphate to form sucrose-6-phosphate, preferably sucrose phosphate synthase (EC 2.4.1.14). d. An enzyme capable of converting sucrose-6-phosphate to sucrose, preferably sucrose-6-phosphate phosphatase (EC 3.1.3.24); and, optionally... e. Changes in the transport system and / or cell membrane that can promote glucose transport into the recombinant cells; f. Changes in transport systems and / or cell membranes that can promote the transport of sucrose to the outside of the recombinant cells.

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

21. The method of claim 20, wherein the recombinant cells further produce the glycosyl receptor and / or its precursor, and the recombinant cells are further extracellularly supplemented with one or more additional glycosyl receptors and / or their precursors.

22. The method according to claim 20 or 21, wherein the glycosyl receptor and / or its precursor provided extracellularly to the recombinant cells and / or the additional glycosyl receptor and / or its precursor provided extracellularly to the recombinant cells are plant extracts, their purified forms, and / or produced by cells other than the recombinant cells.

23. The method according to any one of claims 20 to 22, wherein the recombinant cells produce or are capable of producing the glycosyl receptor (e.g., a terpene compound) and / or its precursor, and comprise one or more of the following: a. A polypeptide capable of synthesizing geraniol diphosphate (GPP) from dimethylallyl diphosphate (DMAPP), particularly wherein said polypeptide is geraniol diphosphate synthase (EC 2.5.1.1) (GPPS) and / or farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). b. A polypeptide capable of synthesizing farnesyl diphosphate (FPP) from gerany diphosphate (GPP), particularly wherein said polypeptide is farnesyl diphosphate synthase (EC 2.5.1.10) (FPPS). c. A polypeptide capable of synthesizing geraniol diphosphate (GGPP) from farnesyl diphosphate (FPP), particularly wherein said polypeptide is geraniol diphosphate synthase (EC 2.5.1.29) (GGPPS). d. A polypeptide capable of synthesizing gerany-farnesyl diphosphate (GFPP) from gerany-gerany-farnesyl diphosphate (GGPP), particularly wherein said polypeptide is gerany-farnesyl diphosphate synthase (EC 2.5.1.81) (GFPPS). e. A polypeptide encoding terpene synthase.

24. The method according to any one of claims 20 to 23, wherein the recombinant cells produce or are capable of producing terpenoid compounds (e.g., mogroside) and / or their precursors, and comprise one or more of the following: a. Squalene synthase (SQS); b. Squalene epoxidase (SQE); c. Cucurbita dienol synthase (CDS); d. Cytochrome P450 (CYP); e. Cytochrome P450 reductase (CPR); f. Epoxide hydrolase (EPH); g. Cytochrome b5 (CB5).

25. The method according to any one of claims 20 to 24, wherein the recombinant cells produce or are capable of producing terpenoid compounds (e.g., mogroside) and / or their precursors, and comprise one or more of the following uridine diphosphate-dependent glycosyltransferases (UGTs): (a) A polypeptide capable of glycosylation of mogroside or mogroside at its C3 hydroxyl, C11 hydroxyl, C24 hydroxyl and / or C25 hydroxyl; (b) A polypeptide capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety at C24 (C24-O-glucose) of mogroside and / or capable of β-1,2-glycosylation of the C2' hydroxyl group of the glucose moiety at C3 (C3-O-glucose) of mogroside. (c) A polypeptide capable of β-1,6-glycosylation of the C6'-hydroxyl group of the glucose moiety at C3 position of mogroside and / or capable of β-1,6-glycosylation of the C6'-hydroxyl group of the glucose moiety at C24 position of mogroside.

26. The method according to any one of claims 20 to 25, wherein the recombinant cells produce or are capable of producing terpenoids (e.g., mogroside) and / or their precursors, and comprise one or more of the following: a. A polypeptide capable of synthesizing acetyl-CoA (AACoA) from acetyl-CoA, particularly wherein said polypeptide is acetyl-CoA acetyltransferase (EC 2.3.1.9) (AACT). b. A polypeptide capable of synthesizing hydroxymethylglutaryl-CoA (HMGCoA) from acetyl-CoA, particularly wherein said polypeptide is hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) (HMGS). c. A polypeptide capable of synthesizing mevalonic acid (MVA) from hydroxymethylglutaryl-CoA (HMGCoA), particularly wherein said polypeptide is hydroxyl Methylglutaryl-CoA reductase (EC 1.1.1.34) (HMGR); d. A polypeptide capable of synthesizing mevalonate-5-phosphate (MVA-P) from mevalonate (MVA), particularly wherein said polypeptide is mevalonate kinase (EC 2.7.1.36) (MK). e. A polypeptide capable of synthesizing mevalonate-5-bisphosphate (MVA-PP) from mevalonate-5-phosphate (MVA-P), particularly wherein said polypeptide is phosphate mevalonate kinase (EC 2.7.4.2) (PMK). f. A polypeptide capable of synthesizing isopentenyl diphosphate (IPP) from mevalonate-5-bisphosphate (MVA-PP), particularly wherein said polypeptide is mevalonate diphosphate decarboxylase (EC 4.1.1.33) (MDD) and / or isopentenyl / dimethylallyl diphosphate synthase (EC 1.17.1.2) (IPPS). g. A polypeptide capable of synthesizing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP), particularly wherein said polypeptide is an isopentenyl diphosphate δ-isomerase (EC 5.3.3.2) (IPI).

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 of claim 27, wherein the at least one heterologous enzyme is an extracellular enzyme, optionally wherein the enzyme is secreted and released outside the recombinant cell and / or displayed on the surface of the recombinant cell.

29. The method of 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 extracellularly to the recombinant cell, and the recombinant cell further comprises a transport system and / or cell membrane alterations capable of facilitating the transport of the glycosyl donor into the cell interior.

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 of claim 31, wherein the recombinant cell further produces the glycosyl receptor and / or its precursor, and / or wherein the glycosyl receptor and / or its precursor is provided extracellularly to the recombinant cell.

33. The method according to any one of claims 31 or 32, wherein the recombinant cells further produce the glycosyl receptor and / or its precursor, and the recombinant cells further comprise a transport system and / or cell membrane alterations capable of facilitating the transport of the glycosyl receptor and / or its precursor to the outside of the 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 extracellularly to the recombinant cell.

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

36. The method according to any one of the preceding claims further comprises: One or more additional enzymes capable of catalyzing the transfer of the glycosyl group to the glycosyl receptor are extracellularly supplemented into the recombinant cells.

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 cells also produce the glycosyl receptor and / or the terpene compound and / or its precursor.

39. The method according to any one of claims 2 to 38, wherein the glycosyl receptor and / or its precursor are provided extracellularly to the recombinant cells, and / or wherein the glycosyl donor is provided extracellularly to the recombinant cells.

40. The method of claim 39, wherein the glycosyl donor is provided extracellularly to the recombinant cells, and wherein the glycosyl donor is provided 1 hour before, during, or 3, 6, 12, 24, 48, 72, 96, or 120 hours after the recombinant cells are inoculated into the culture medium.

41. The method according to any one of claims 2 to 40, wherein the recombinant cell expresses a polynucleotide encoding the at least one heterozyme, and the recombinant cell also 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 its precursor are extracellularly provided to the recombinant cells.

43. The method of claim 42, wherein the method comprises: a. The recombinant cells are cultured in a suitable culture medium under conditions in which at least one heterologous enzyme is expressed, wherein the at least one heterologous enzyme is intracellular and / or extracellularly displayed on the surface of the recombinant cells; b. Harvest the recombinant cells from the culture medium, and c. Contact the recombinant cells obtained from (b), the glycosyl donor, the glycosyl acceptor and / or its precursor, and optionally one or more other enzymes capable of catalyzing the transfer of the glycosyl group to the glycosyl acceptor in a reaction mixture; thereby generating a modified glycosyl acceptor in the reaction mixture.

44. A recombinant cell as described in any of the preceding claims.

45. A recombinant cell comprising at least one heteropolynucleotide encoding at least one enzyme capable of catalyzing a transglucosylation reaction, said transglucosylation reaction being, for example, the transfer of a glycosyl group from a glycosyl donor (e.g., sucrose) to a glycosyl acceptor (e.g., a terpenoid compound, such as mogroside), said recombinant cell being deficient in an enzyme capable of hydrolyzing said glycosyl donor (e.g., invertase), and optionally comprising at least one polynucleotide sequence encoding α-glucosidase and / or at least one polynucleotide encoding β-glucosidase.

46. ​​The recombinant cell of claim 45, wherein the enzyme capable of catalyzing transglycosylation between a glycosyl donor and a glycosyl acceptor is a dextran sucrase, such as dextran sucrase, and 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 coli*; or a eukaryotic cell, particularly a plant cell or a fungal cell, more particularly a yeast cell selected from: Saccharomyces sp. ,For example Saccharomyces cerevisiae ; Yarrowia sp. ,For example Yarrowia lipolytica ; Candida sp .,For example Candida krusei ; Issatchenkia sp .,For example Issatchenkia orientalis ; Pichia sp. ,For example Pichia pastoris .

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

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

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

51. A terpenoid composition, which is obtained or can be obtained by the method according to any one of claims 1 to 43, 47 or by using recombinant cells according to any one of claims 44 to 47.

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

53. Use of the recombinant cells according to any one of claims 44 to 47 for the production of modified terpenoid compounds.

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