Biocatalytic process for preparing compositions comprising rebaudioside m

Abstract

The present invention provides a biocatalytic process for preparing compositions comprising Rebaudioside M. The process uses a nucleotide diphosphate (NDP)-recycling enzyme, optionally a NDP synthesizing enzyme, and at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes.

Description

[0001] BIOCATALYTIC PROCESS FOR PREPARING COMPOSITIONS COMPRISING REBAUDIOSIDE M

[0002] TECHNICAL FIELD

[0003] The present invention relates to biocatalytic processes for preparing compositions comprising Rebaudioside M. In particular, this disclosure relates to the biocatalytic process of producing Rebaudioside M using a nucleotide diphosphate (NDP)-recycling enzyme, optionally a NDP-synthesizing enzyme, and at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes.

[0004] BACKGROUND OF THE INVENTION

[0005] High intensity sweeteners possess a sweetness level that is many times greater than the sweetness level of sucrose. They are essentially non-caloric and are commonly used in diet and reduced-calorie products, including foods and beverages. High intensity sweeteners do not elicit a glycemic response, making them suitable for use in products targeted at diabetics and others interested in controlling their carbohydrate intake.

[0006] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. They are characterized structurally by a single base, steviol, which differ by the presence of carbohydrate residues at positions C13 and C19. They accumulate in Stevia leaves, composing approximately 10% - 20% of the total dry weight. On a dry weight basis, the four major glycosides found in the leaves of Stevia typically include Stevioside (9.1%), Rebaudioside A (3.8%), Rebaudioside C (0.6-1.0%) and Dulcoside A (0.3%). Other known steviol glycosides include Rebaudioside, C, £>, E, F and M, Steviolbioside and Rubusoside.

[0007] Rebaudioside is recognized as the most potent of all known steviol glycosides, being approximately 300 times sweeter than sucrose and possessing the most favorable flavor profile. However, the stevia plant produces Rebaudioside M in very limited quantities, accounting for less than 1% of the total steviol glycoside content. This low concentration renders the isolation of Rebaudioside M from stevia leaves impractical.Although methods are known for preparing steviol glycosides from Stevia rebaudiana. many of these methods are unsuitable for use commercially. Accordingly, there remains a need for simple, efficient, and economical methods for preparing compositions comprising steviol glycosides, especially with higher yield of the desirable steviol glycosides such as Rebaudioside M. Biotransformation has a potential for steviol glycoside production because of its high efficiency. High-performing enzymes with high selectivity, activity and thermostability are required for an efficient biotransformation. Additionally, optimization of the biotransformation process is equally important to maximize the efficiency and cost-effectiveness of producing desirable steviol glycosides with high yield, including but not limited to Rebaudioside M.

[0008] The steviol glycosides are useful as non-caloric sweeteners, flavor enhancers, sweetness enhancers, flavor stabilizers, flavorings with modifying properties (FMP), foaming suppressors and solubility enhancing agents in consumable products such as any food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, and oral hygiene compositions.

[0009] SUMMARY OF THE INVENTION

[0010] As used herein, the abbreviation term “reb” refers to “rebaudioside”. Both terms have the same meaning and may be used interchangeably.

[0011] As used herein, the abbreviation term “SG” refers to “steviol glycoside(s)”. Both terms have the same meaning and may be used interchangeably.

[0012] As used herein, “biocatalysis” or “biocatalytic” refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells comprising one or more enzyme(s), capable of single or multiple step chemical transformations on organic compounds. Biocatalytic processes include fermentation, biosynthesis, bioconversion and biotransformation processes. The general methods of using isolated enzymes and wholecell biocatalysts are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins. As used herein, the terms “biocatalytic process”, “biocatalytic production”, “biocatalysis”, “biotransformation”, “bioconversion”, “enzymatic conversion”, “whole-cell bioconversion” and “biosynthesis” have the same meaning and may be used interchangeably.As used herein, the abbreviation term “UGT” refers to “UDP-glycosyltransferase” and “UDP -glucosyltransferase”. The terms have the same meaning and may be used interchangeably.

[0013] As used herein, the abbreviation term “NGT” refers to “NDP-glycosyltransferase” and “NDP -glucosyltransferase”. The terms have the same meaning and may be used interchangeably.

[0014] As used herein, the term “SuSy” refers to sucrose synthases.

[0015] The terms “wild type”, “native” or “endogenous” refer to components, such as nucleotide sequences or enzymes, that are present in the organism from which they are originated or are found in nature.

[0016] The present disclosure provides methods for producing Rebaudioside M via a biotransformation process that converts starting steviol glycosides in a starting steviol glycoside composition contained in a medium through enzymatic conversion by an enzyme preparation or a microorganism. The biotransformation method utilizes a NDP-recycling enzyme and at least two enzymes selected from the group of nucleotide diphosphatedependent glycosyltransferase (NGT)-enzymes, which have enhanced properties to improve the efficiency of producing Rebaudioside M. The medium is an aqueous phase containing the starting steviol glycoside composition, dissolved or partially dissolved, together with one or more cofactors, buffers, salts, or other components suitable for supporting enzymatic activity or microorganism functioning. The method may also involve the utilization of NDP-synthesizing enzyme(s) to synthesize NDP in situ for the production of NDP-sugar and / or the use of rhamnosidases or xylosidases. The Rebaudioside M is synthesized using UGT76G1, UGT74G1, UGTS12, and NDP-recycling enzyme(s) including sucrose synthase, and optionally NDP synthesizing enzyme(s) including UMP-kinase. In a preferred embodiment, UGT76G1 is a SrUGTl- and / or UGTSr-enzyme and / or UGT74G1 is a SrUGT2-enzyme.

[0017] In one embodiment, the enzymes UGT76G1, UGT74G1, UGTS12, the NDP-recycling enzymes and the NDP-synthesizing enzymes are produced using a genetically modified E. coli. In one preferred embodiment, the enzymes are produced using a genetically modified E. coli K-12 strain. In another embodiment, the enzymes aremanufactured via fermentation using genetically modified strains of E. coli K-12. In another preferred embodiment, the enzymes are manufactured via fermentation using genetically modified strains of E. coli K-12. In another embodiment, the enzymes are isolated for use in the enzymatic conversion process. The enzymes participate in the glycosylation of the starting steviol glycosides present in the starting steviol glycoside compositions, either directly or indirectly, to produce Rebaudioside M.

[0018] Starting steviol glycosides

[0019] Suitable starting steviol glycoside compositions refers to any composition containing one or more starting steviol glycosides, where one or more starting steviol glycosides serve as the substrate(s) for the biotransformation. Suitable starting steviol glycoside compositions include but are not limited to stevia extract, steviol glycosides, stevia manufacturing side-streams or by-products such as mother liquors or filtrates and highly purified steviol glycosides comprising at least one starting steviol glycoside selected from the group including but not limited to steviol, Dulcoside A, Dulcoside C, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside, steviolbioside D, steviolbioside C, Stevioside, Stevioside A, Stevioside B, Stevioside C, stevioside F, rubusoside, Rebaudioside O, Rebaudioside J, Rebaudioside V2, steviolbioside E, steviolbioside F, steviolbioside H, stevioside I, stevioside M, stevioside N, stevioside O, Rebaudioside C3, Rebaudioside C7, Rebaudioside C9, Rebaudioside C11, Dulcoside D, Dulcoside E, Rebaudioside B2, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside F, Rebaudioside G, Rebaudioside H, Rebaudioside N, Rebaudioside K, Rebaudioside L, Rebaudioside E, Rebaudioside E2, Rebaudioside E3, Rebaudioside E4, Rebaudioside E6, Rebaudioside D, Rebaudioside I, Rebaudioside D7, Rebaudioside AM, a synthetic steviol glycoside or combinations thereof, preferably Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside F, Rebaudioside F, Rebaudioside G, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside or combinations thereof, more preferably Rebaudioside B, Rebaudioside C, Rebaudioside F, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside, or mixtures thereof, and most preferably Rebaudioside B, Rebaudioside C, Dulcoside A, Stevioside, Steviolbioside, or mixtures thereof.In one embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside steviolmonoside.

[0020] In yet another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside steviolbioside.

[0021] In another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside Rubusoside.

[0022] In yet another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside Rebaudioside G.

[0023] In still another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside, Rebaudioside B.

[0024] In another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside stevioside.

[0025] In another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside Rebaudioside A.

[0026] In another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside Rebaudioside E.

[0027] In yet another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside Rebaudioside F.

[0028] In still another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside Rebaudioside C.

[0029] In yet another embodiment, the starting steviol glycoside composition comprises the starting steviol glycoside Dulcoside A.

[0030] In another embodiment, the starting steviol glycoside composition comprises two or more starting steviol glycosides.

[0031] In yet another embodiment, the starting steviol glycoside composition comprises a mixture of starting steviol glycosides.In one embodiment, the starting steviol glycoside composition may be synthetic or purified (partially or entirely), commercially available or prepared. In another embodiment, a starting steviol glycoside composition is an extract obtained from purification of Stevia rebaudiana plant material (e.g., leaves). In yet another embodiment, the starting steviol glycoside composition is a commercially available stevia extract brought into solution with a solvent. In still another embodiment, the starting steviol glycoside composition is a commercially available mixture of starting steviol glycosides brought into solution with a solvent. In still another embodiment, the starting steviol glycoside composition is a commercially available purified starting steviol glycoside brought into solution with a solvent.

[0032] In one embodiment, the starting steviol glycoside composition is an extract obtained from the purification of Stevia rebaudiana plant material, containing high level of Stevioside and purified to High Stevioside extracts (HSE). The material termed HSE contains mixture of starting steviol glycosides including Stevioside as the major molecule and lesser amounts of Dulcoside A, Rebaudioside E, Rebaudioside A, Rubusoside, Rebaudioside C and steviolbioside for the biotransformation to synthesize Rebaudioside M.

[0033] In another embodiment, the starting steviol glycoside composition is an extract obtained from the purification of Stevia rebaudiana plant material, containing high level of Rebaudioside E and purified to High Rebaudioside E extracts (HEE). The material termed HEE contains mixture of starting steviol glycosides including Rebaudioside E as the major molecule and lesser amounts of Stevioside, Dulcoside A, Rebaudioside, Rebaudioside A, Rubusoside and steviolbioside for the biotransformation to synthesize Rebaudioside M.

[0034] In another embodiment, the starting steviol glycoside composition is stevia manufacturing side-streams or by-products such as mother liquors or filtrates. In yet another embodiment, the starting steviol glycoside composition is the by-products of processes to isolate and purify the steviol glycosides.

[0035] In one specific embodiment, the starting steviol glycoside compositions are the byproducts of processes, including but not limited to the manufacturing of high purity Rebaudioside A. For example, the steviol glycosides are extracted from the leaves of S. rebaudiana and purified to various extracts, such as RA50 extracts containing high level ofRebaudioside A. The residual from the stevia leaf extraction, which may have lower total steviol glycoside content and contain a mixture of starting steviol glycosides including but not limited to steviolbioside and Rebaudioside B. A residual material termed SGB, containing high level of Rebaudioside B, can be used as a starting steviol glycoside composition for the biotransformation to synthesize Rebaudioside M.

[0036] The RA50 extract then goes through further refining which separates pure Rebaudioside A from a material termed mother liquor-02 (ML02). The ML02 is a starting steviol glycoside composition which contains mixture of starting steviol glycosides including Rebaudioside A and stevioside as the major molecules and lesser amounts of Rebaudioside C, Dulcoside A, Rebaudioside F, Rubusoside, Rebaudioside B, Rebaudioside £>, Rebaudioside G, and steviolbioside for the biotransformation to synthesize Rebaudioside M.

[0037] In another specific embodiment, S. rebaudiana leaves are placed in hot water at 50 to 60 °C for 1 to 2 hours in continuous countercurrent extractors. The filtrate is separated using mesh screens, collected in a holding tank, and treated with flocculants (calcium hydroxide, ferrous sulphate etc.) to remove the mechanical particles, proteins, polysaccharides, and coloring agents. A plate-and-frame filter press is used to separate the resulting precipitate from the filtrate, and the filtrate is fed to a column system packed with macroporous adsorption resin that retains the glycosides. The column is washed with deionized water to remove impurities that did not adsorb to the resin and then the glycosides are desorbed using aqueous ethanol. The desorbed solution (eluate) is deionized by ionexchange resins in (H+) and (OH-) form. The deionized eluate is evaporated to remove ethanol and concentrated using a nanofiltration membrane and the concentrated solution is spray dried to yield stevia extract powder containing >50% Rebaudioside A (RA50). The RA50 powder is further purified by dissolving in aqueous ethanol and incubating at low temperature for several hours to allow for Rebaudioside A to crystallize. The Rebaudioside A crystals containing >95% Rebaudioside A are separated by conventional centrifugation or filtration from a material termed mother liquor-02 (ML02) that contains a mixture of steviol glycosides with Rebaudioside A and stevioside as the major molecules with lesser amounts of Rebaudioside C, Dulcoside A, Rebaudioside F, Rubusoside, Rebaudioside B, Rebaudioside £>, Rebaudioside G and steviolbioside. The material, mother liquor-02(ML02) can also be used as a starting steviol glycoside composition for the biotransformation to synthesize Rebaudioside M.

[0038] In one embodiment, the starting steviol glycoside composition comprises a purified starting steviol glycoside, including but not limited to steviol, Dulcoside A, Dulcoside C, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside, steviolbioside D, steviolbioside C, Stevioside, Stevioside A, Stevioside B, Stevioside C, Stevioside F, rubusoside, Rebaudioside O, Rebaudioside J, Rebaudioside V2, steviolbioside E, steviolbioside F, steviolbioside H, stevioside I, stevioside M, stevioside N, stevioside O, Rebaudioside C3, Rebaudioside C7, Rebaudioside C9, Rebaudioside C11, Dulcoside D, Dulcoside E, Rebaudioside B2, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside F, Rebaudioside G, Rebaudioside H, Rebaudioside N, Rebaudioside K, Rebaudioside L, Rebaudioside E, Rebaudioside E2, Rebaudioside E3, Rebaudioside E4, Rebaudioside E6, Rebaudioside D, Rebaudioside I, Rebaudioside D7, Rebaudioside AM, a synthetic steviol glycoside or combinations thereof, preferably Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside F, Rebaudioside F, Rebaudioside G, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside or combinations thereof, more preferably Rebaudioside B, Rebaudioside C, Rebaudioside F, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside, or mixtures thereof, and most preferably Rebaudioside B, Rebaudioside C, Dulcoside A, Stevioside, Steviolbioside, or mixtures thereof. For example, the starting steviol glycoside composition may comprise greater than about 95%, about 96%, about 97%, about 98% or about 99% of a particular starting steviol glycoside by weight on a dry basis; preferably greater than about 97%, more preferably greater than about 96%, most preferably greater than about 95%.

[0039] In another embodiment, the starting steviol glycoside composition comprises a partially purified starting steviol glycoside, including but not limited to steviol, Dulcoside A, Dulcoside C, steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside, steviolbioside D, steviolbioside C, Stevioside, Stevioside A, Stevioside B, Stevioside C, Stevioside F, rubusoside, Rebaudioside O, Rebaudioside J, Rebaudioside V2, steviolbioside E, steviolbioside F, steviolbioside H, stevioside I, stevioside M, stevioside N, stevioside O, Rebaudioside C3, Rebaudioside C7, Rebaudioside C9, Rebaudioside C11, Dulcoside D, Dulcoside E, Rebaudioside B2, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside F, Rebaudioside G, Rebaudioside H, Rebaudioside N,Rebaudioside K, Rebaudioside L, Rebaudioside E, Rebaudioside E2, Rebaudioside E3, Rebaudioside E4, Rebaudioside E6, Rebaudioside D, Rebaudioside I, Rebaudioside D7, Rebaudioside AM, a synthetic steviol glycoside or combinations thereof, preferably Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside E, Rebaudioside F, Rebaudioside G, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside or combinations thereof, more preferably Rebaudioside B, Rebaudioside C, Rebaudioside F, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside, or mixtures thereof, and most preferably Rebaudioside B, Rebaudioside C, Dulcoside A, Stevioside, Steviolbioside, or mixtures thereof. For example, the starting steviol glycoside composition may comprise greater than about 50%, about 60%, about 70%, about 80% or about 90% of a particular starting steviol glycoside by weight on a dry basis; preferably greater than about 70%, more preferably greater than about 60%, most preferably greater than about 50% of a particular starting steviol glycoside by weight on a dry basis.

[0040] In one embodiment, the obtained Rebaudioside M is a part of a mixture. In yet another embodiment, the amount of Rebaudioside is increased compared to the amount of Rebaudioside M present in the starting steviol glycoside composition as a result of carrying out the method of the present invention.

[0041] Optionally, the method of the present invention further comprises separating the Rebaudioside M from the post-biotransformation medium to provide a highly purified Rebaudioside M composition. The Rebaudioside M can be separated by any suitable method, such as, for example, crystallization, membrane filtration, separation, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

[0042] In one embodiment, the process described herein results in a highly purified Rebaudioside M composition. The term “highly purified”, as used herein, refers to a composition having greater than about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% by weight of Rebaudioside M on an anhydrous (dried) basis (also referred to as purity); preferably greater than 90%, more preferably greater than 95%, most preferably greater than 98% by weight of the Rebaudioside M on an anhydrous (dried) basis.Biotransformation

[0043] In one embodiment, the biotransformation process of the present invention can be performed simultaneously in a “one-pot” reaction. The biotransformation method involves combining all the necessary enzymes, processing aids and starting steviol glycoside composition in a reaction medium at the beginning of the reaction to synthesize the final Rebaudioside M. The intermediate steviol glycoside is synthesized enzymatically in situ and then utilized as the starting steviol glycoside for the next intermediate steviol glycoside or the Rebaudioside M. This process continues, with each intermediate steviol glycoside undergoing further enzymatic biotransformation in situ, until the desirable Rebaudioside M is synthesized. This reaction requires enzymes with high substrate specificity to effectively and selectively synthesize the Rebaudioside M. A skilled artisan would understand the same steviol glycoside can serve as a starting steviol glycoside or an intermediate steviol glycoside in different embodiments.

[0044] In a particular embodiment, the present invention provides a “one-pot” biotransformation process for preparing Rebaudioside M by contacting the starting steviol glycoside composition with the enzyme preparation containing a NDP-recycling enzyme, and at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes in a medium. The biotransformation proceeds through one or more in situ glycosylation steps, synthesizing one or more intermediate steviol glycosides until the Rebaudioside M is synthesized. Multiple intermediate steviol glycosides may be produced, with subsequent glycosylation performed in situ until the Rebaudioside M is produced.

[0045] In another embodiment, the present invention provides a multi-step biotransformation process for preparing Rebaudioside M by contacting the starting steviol glycoside composition with the enzyme preparation containing a NDP-recycling enzyme, and at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes in a medium, thereby producing a composition comprising intermediate steviol glycoside(s) with one or more additional glucose unit(s) than the starting steviol glycoside(s); then contacting the composition comprising the intermediate steviol glycoside(s) with another enzyme preparation containing a NDP-recycling enzyme, and at least two enzymes selected from the group of nucleotidediphosphate-dependent glycosyltransferase (NGT)-enzymes, thereby producing Rebaudioside M. Depending on the number of times the method is carried out, there may be one or more intermediate steviol glycoside(s) (e.g., a first intermediate steviol glycoside, a second intermediate steviol glycoside, a third intermediate steviol glycoside, etc) involved in the production of the target steviol glycoside(s).

[0046] The method may also involve the utilization of NDP-synthesizing enzyme to synthesize NDP in situ for the production of NDP-sugar. In one specific embodiment, the Rebaudioside is synthesized using UGT76G1, UGT74G1, UGTSl2, and NDP-recycling enzyme(s) including sucrose synthase, and optionally NDP-synthesizing enzyme(s) including UMP -kinase. In a preferred embodiment, UGT76G1 is a SrUGTl- and / or UGTSr-enzyme and / or UGT74G1 is a SrUGT2-enzyme.

[0047] Various types of NGT-enzymes can participate in the glycosylation of starting steviol glycoside substrates. These include uridine diphosphate-dependent glycosyltransferase (UGT), adenosine diphosphate-dependent glycosyltransferase (AGT), guanosine diphosphate-dependent glycosyltransferase (GGT), cytidine diphosphate-dependent glycosyltransferase (CGT) and thymidine diphosphate-dependent glycosyltransferase (TGT). Each of these NGTs transfers monosaccharide units from their respective NDP-sugars to various substrates, including but not limited to terpenoids, such as steviol and / or steviol glycosides, catalyzing the glycosylation process. In one embodiment, the NGT-enzyme is a UGT-enzyme that transfers the monosaccharide units from uridine diphosphate (UDP)-sugars to the substrates. In another embodiment, the NGT-enzyme is an AGT-enzyme that transfers the monosaccharide units from adenosine diphosphate (ADP)-sugars to the substrates. In yet another embodiment, the NGT-enzyme is a GGT-enzyme that transfers the monosaccharide units from guanosine diphosphate (GDP)-sugars to the substrates. In still another embodiment, the NGT-enzyme is a CGT-enzyme that transfers the monosaccharide units from cytidine diphosphate (CDP)-sugars to the substrates. In another embodiment, the NGT-enzyme is a TGT-enzyme that transfers the monosaccharide units from thymidine diphosphate (TDP)-sugars to the substrates. Optionally, the NGT-enzymes may be used with their respective NDP-recy cling enzyme, including UDP -recycling enzyme, ADP-recycling enzyme, GDP -recycling enzyme, CDP-recycling enzyme, and / or TDP -recycling enzyme.In one embodiment, the nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are uridine diphosphate-dependent glycosyltransferase (UGT)-enzymes. In a particular embodiment, the UGT-enzymes are selected from the group consisting of UGT76G1 (SEQ ID NO: 1) and / or the variants thereof, UGT74G1 (SEQ ID NO: 2) and / or the variants thereof, UGTSl2 (SEQ ID NO: 3 or 7) and / or the variants thereof.

[0048] UGT76G1 preferably is a UGTSr (SEQ ID NO: 4) and / or the variants thereof and / or a SrUGTl-enzyme (SEQ ID NO: 5) and / or the variants thereof.

[0049] UGT74G1 preferably is a SrUGT2-enzyme (SEQ ID NO: 6) and / or the variants thereof.

[0050] In context of this application enzyme variants are to be understood as enzymes, which comprise or consist of an amino acid sequence that has at least 95% sequence identity or is 95% identical to the referring enzyme, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical. The amino acid sequence may have one or more amino acid modifications independently selected from translocation, inversion, substitution, insertion, deletion and / or duplication, with respect to the amino acid sequence of the referring enzyme. The amino acid modifications can be in various combinations and it is to be understood that any combination of these modifications is within the scope of the disclosure.

[0051] In a more preferred embodiment the UGTSr-enzyme is characterized in that the homology and / or identity of the amino acid sequence to SEQ ID NO: 4 is at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96.0%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97.0%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100%.In a further preferred embodiment the UGTSr-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 4, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical, with one or more amino acid substitution(s) at one or more amino acid position(s) within SEQ ID NO: 4, wherein these amino acid position(s) is / are each and independently selected from the group consisting of the amino acid positions L29, T82, G85, M88, H96, C154, P178, V179, F185, S195, H196, G197, V199, G200, 1203, V208, C321, 1383, W402, K440 and F458 of SEQ ID NO: 4.

[0052] In another more preferred embodiment the SrUGTl-enzyme is characterized in that the homology and / or identity of the amino acid sequence to SEQ ID NO: 5 is at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96.0%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97.0%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100%.

[0053] In a further more preferred embodiment the SrUGTl-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 5, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical, with one or more amino acid substitution(s) at one or more amino acid position(s) within SEQ ID NO: 5, wherein these amino acid position(s) is / are each and independently selected from the group consisting of the amino acid positions 129, L46, H96, L108, C119, S132, P140, A154, L155, Q178, A179, W197, 1199, 1200, 1203, D205, R212, 1234, H249, G250, S285, G348, S375, D376, V394, F402, A408, K440, 1455 and L458 of SEQ ID NO: 5.

[0054] In another more preferred embodiment the SrUGT2-enzyme is characterized in that the homology and / or identity of the amino acid sequence to SEQ ID NO: 6 is at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96.0%, at least 96.1%, at least 96.2%, at least 96.3%,at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97.0%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100%.

[0055] In a further more preferred embodiment the SrUGT2-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 6, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical, with one or more amino acid substitution(s) at one or more amino acid position(s) within SEQ ID NO: 6, wherein these amino acid position(s) is / are each and independently selected from the group consisting of the amino acid positions S20, M27, C73, L120, P121, M224, N252, E274, K286, K313, 1333, P338, F357, 1424 and V438 of SEQ ID NO: 6.

[0056] In another more preferred embodiment the UGTS12-enzyme is characterized in that the homology and / or identity of the amino acid sequence to SEQ ID NO: 7 is at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96.0%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97.0%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100%.

[0057] In a further more preferred embodiment the UGTS12-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 7, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical, with one or more amino acid substitution(s) at one or more amino acid position(s) within SEQ ID NO: 7, wherein these amino acid position(s) is / are each and independently selected from the group consisting of the amino acid positions W12, N56,H60, 162, N68, G134, L137, G139, S140, H148, S149, L150, G155, G185, M195, T202, T217, S255, D265, H268, 1333, Y358, V412, A427 and K440 of SEQ ID NO: 7.

[0058] In one embodiment, one or more further uridine diphosphate-dependent glycosyltransferase (UGT)-enzymes selected from the following list can be involved on the biocatalytic production of Rebaudioside M UGT76G1 (SEQ ID NO:1; Accession No. AAR06912.1), UGT74G1 (SEQ IDN0:2; Accession No. AAR06920.1), UGTS12 (SEQ ID NO:3; Accession No. XP_004250485.1), UGT91D2 (SEQ ID NO: 10; Accession No. BFR87189.1, also known as UGT91D2e or UGT91D or UGT91D-like), UGT85C2 (SEQ ID NO: 11; Accession No. AAR06916.1), UGT91D1 (Accession No. AAR06918.1), EUGT11 (SEQ ID NO: 12; Accession No. AAS07253.1), UGTS1 (Accession No. XP_004249992.1), UGT91A1 (Accession No. XP_004982059.1), UGT91Bl-like (Accession No. XP_044983991.1), and / or UGT91B1 (Accession No. XP_010230871.1), or mutant variant thereof having >85% amino-acid sequence identity with their respective amino acid sequence, preferably UGT91D2 (SEQ ID NO: 10), UGT85C2 (SEQ ID NO: 11), EUGT11 (SEQ ID NO: 12), variants or mixtures thereof.

[0059] In one embodiment, a variety of NDP-recycling enzymes can be involved in the biocatalytic production of Rebaudioside M. These include, but are not limited to, uridine diphosphate (UDP)-recycling enzyme, adenosine diphosphate (ADP)-recycling enzyme, guanosine diphosphate (GDP)-recycling enzyme, cytidine diphosphate (CDP)-recycling enzyme and thymidine diphosphate (TDP)-recycling enzyme. Each of these NDP-recycling enzymes catalyzes the synthesis of their respective NDP-sugar, which is used in the glycosylation of various substrates, including but not limited to terpenoids such as steviol and / or steviol glycosides. In one embodiment, the NDP-recycling enzyme is a UDP-recycling enzyme that catalyzes the synthesis of UDP-sugar. In another embodiment, the NDP-recycling enzyme is an ADP -recycling enzyme that catalyzes the synthesis of ADP-sugar. In yet another embodiment, the NDP-recycling enzyme is a GDP-recycling enzyme that catalyzes the synthesis of GDP-sugar. In yet another embodiment, the NDP-recycling enzyme is a CDP-recy cling enzyme that catalyzes the synthesis of CDP-sugar. In yet another embodiment, the NDP-recycling enzyme is a TDP-recycling enzyme that catalyzes the synthesis of TDP-sugar.

[0060] In another embodiment, various types of NDP-recycling enzymes involved in the biocatalytic production of Rebaudioside M include, but are not limited to, NDP-glucosesynthesizing enzyme, NDP -mannose synthesizing enzyme, NDP -galactose synthesizing enzyme, NDP-fructose synthesizing enzyme, NDP-rhamnose synthesizing enzyme, NDP-arabinose synthesizing enzyme, NDP -deoxy glucose synthesizing enzyme and NDP -xylose synthesizing enzyme. Each of these NDP-recycling enzyme catalyzes the synthesis of their respective NDP-sugar, which is used in the glycosylation of various substrates, including but not limited to terpenoids such as steviol and / or steviol glycosides. In one embodiment, the NDP-recycling enzyme is an NDP -glucose synthesizing enzyme that catalyzes the synthesis of NDP -glucose. In another embodiment, the NDP-recycling enzyme is an NDP-mannose synthesizing enzyme that catalyzes the synthesis of NDP -mannose. In another embodiment, the NDP-recycling enzyme is an NDP -galactose synthesizing enzyme that catalyzes the synthesis of NDP -galactose. In yet another embodiment, the NDP-recycling enzyme is an NDP-fructose synthesizing enzyme that catalyzes the synthesis of NDP-fructose. In yet another embodiment, the NDP-recycling enzyme is an NDP-rhamnose synthesizing enzyme that catalyzes the synthesis of NDP-rhamnose. In yet another embodiment, the NDP-recycling enzyme is an NDP-arabinose synthesizing enzyme that catalyzes the synthesis of NDP-arabinose. In yet another embodiment, the NDP-recycling enzyme is an NDP-deoxy glucose synthesizing enzyme that catalyzes the synthesis of NDP -deoxyglucose. In still another embodiment, the NDP-recycling enzyme is an NDP -xylose synthesizing enzyme that catalyzes the synthesis of NDP -xylose.

[0061] In one embodiment, the nucleotide diphosphate (NDP)-recycling enzyme is a nucleotide diphosphate (NDP)-glucose synthesizing enzyme. In another embodiment, the NDP-recycling enzyme is sucrose synthase (SuSy; e.g. SEQ ID NO: 8) originating from Arabidopsis thaliana and / or the variants thereof (including, but not limited to SEQ ID NO: 9).

[0062] In another more preferred embodiment the SuSy-enzyme is characterized in that the homology and / or identity of the amino acid sequence to SEQ ID NO: 9 is at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96.0%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97.0%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%,at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100%.

[0063] In a further more preferred embodiment the SuSy-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 9, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical, with one or more amino acid substitution(s) at one or more amino acid position(s) within SEQ ID NO: 9, wherein these amino acid position(s) is / are each and independently selected from the group consisting of the amino acid positions V30, 149, D205, S266, K317, K321, R492, T539, S548, 1564, S577, T588, V604, S615, Q635, A662, H696, L723 and E740 of SEQ ID NO: 9.

[0064] In one embodiment, a variety of NDP-synthesizing enzymes can be involved in the biocatalytic production of intermediate steviol glycosides and / or Rebaudioside M. These include, but not limited to, uridine diphosphate (UDP)-synthesizing enzyme, adenosine diphosphate (ADP)-synthesizing enzyme, guanosine diphosphate (GDP)-synthesizing enzyme, cytidine diphosphate (CDP)-synthesizing enzyme and thymidine diphosphate (TDP)-synthesizing enzyme. Each of these NDP-synthesizing enzymes catalyzes the synthesis of their respective NDP, which is used as a substrate to synthesize the respective NDP-sugar for the glycosylation of various substrates, including but not limited to terpenoids such as steviol and / or steviol glycosides. In one embodiment, the NDP-synthesizing enzyme is a UDP-synthesizing enzyme that catalyzes the synthesis of UDP. In another embodiment, the NDP-synthesizing enzyme is an ADP-synthesizing enzyme that catalyzes the synthesis of ADP. In yet another embodiment, the NDP-synthesizing enzyme is a GDP-synthesizing enzyme that catalyzes the synthesis of GDP. In yet another embodiment, the NDP-synthesizing enzyme is a CDP-synthesizing enzyme that catalyzes the synthesis of CDP. In yet another embodiment, the NDP-synthesizing enzyme is a TDP-synthesizing enzyme that catalyzes the synthesis of TDP. In one specific embodiment, the NDP-synthesizing enzyme is a UDP-synthesizing enzyme, such as UMP -kinase, that catalyzes the synthesis of UDP.

[0065] Specifically, the biotransformation process in this disclosure includes uridine diphosphate-dependent glycosyltransferase (UGT)-enzymes that can catalyze the transfer of a monosaccharide unit (e.g., glucose) from an NDP-sugar (e.g., UDP -glucose) to varioussubstrates, including but not limited to terpenoids, such as steviol and / or steviol glycosides to provide intermediate steviol glycosides and / or Rebaudioside M. The monosaccharide may be glucose, mannose, galactose, fructose, rhamnose, arabinose, deoxy-glucose or xylose. In one embodiment, the monosaccharide is glucose. These UGT-enzymes demonstrate glycosyltransferase activity on starting steviol glycosides.

[0066] In one embodiment, UGTSr facilitates the uridine diphosphate-dependent transfer of a monosaccharide moiety to the C3’ of the C 13 and / or C19 glycosyl group(s) of starting steviol glycosides. In another embodiment, SrUGTl facilitates the uridine diphosphatedependent transfer of a monosaccharide moiety to the C3’ of the C 13 and / or C19 glycosyl group(s) of starting steviol glycosides. In yet another embodiment, SrUGT2 facilitates the uridine diphosphate-dependent transfer of a monosaccharide moiety to the -COOH functional group at the C19 of a steviol or starting steviol glycoside. In also still another embodiment, UGTS12 facilitates the uridine diphosphate-dependent transfer of a monosaccharide moiety to the C2’, C4’ and / or C6’ of the C13 and / or C 19 glycosyl group(s) of starting steviol glycosides.

[0067] In another embodiment, the biotransformation process in this disclosure also includes nucleotide diphosphate (NDP)-recycling enzymes that can catalyze the synthesis of NDP-sugar (e.g., UDP-glucose) from nucleotide diphosphate (e.g., UDP) and a sugar donor (e.g., sucrose as glucose donor). The NDP-sugar, which contains a monosaccharide unit, is then used by nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes to transfer the monosaccharide unit for the glycosylation of various substrates, including but not limited to terpenoids, such as steviol and / or steviol glycosides, to provide intermediate steviol glycosides and / or Rebaudioside M. For example, sucrose synthase (SuSy) facilitates the synthesis of UDP-glucose from uridine diphosphate (UDP) and sucrose, with fructose as the side product. Uridine diphosphate-dependent glycosyltransferase (UGT)-enzymes then catalyze the transfer of glucose from the synthesized UDP-glucose to various substrates, including but not limited to terpenoids, such as steviol glycosides to provide intermediate steviol glycosides and / or Rebaudioside M. Optionally, the biotransformation of this invention also includes uridine diphosphate (UDP)-synthesizing enzyme, such as UMP-kinase that can catalyze the synthesis of UDP for the production of UDP-glucose.In one embodiment, the NDP-sugar can be generated in situ with NDP and a sugar donor, along with an NDP-sugar regeneration mechanism, NDP-sugar synthesizing or NDP-recycling enzyme system. In another embodiment, the NDP-sugar can be externally supplied for in vitro biocatalytic reactions, usually with an NDP-sugar regeneration mechanism, NDP-sugar synthesizing or NDP-recycling enzyme system. In still another embodiment, the NDP-sugar can be internally produced by a microorganism cell for microbial fermentation or whole cell reactions. NDP-sugar cofactor recycling could take place using the native cellular metabolism without requiring externally provided enzymes. In yet another embodiment, the microorganism cell can be modified in various ways to increase the availability of NDP-sugar to support the reactions.

[0068] In one embodiment, the NDP can be externally supplied for in vitro biocatalytic reactions. In another embodiment, the NDP can be synthesized in situ in a biocatalytic reaction by using NDP-synthesizing enzymes such as UMP -kinase, which synthesizes UDP from UMP and ATP. In yet another embodiment, the NDP can be internally produced by a microorganism cell for microbial fermentation or whole cell reactions. NDP cofactor synthesis and recycling could take place using the native cellular metabolism without requiring externally provided enzymes. In yet another embodiment, the microorganism cell can be modified in various ways to increase the availability of NDP to support the reactions.

[0069] For instance, the uridine diphosphate-dependent glycosyltransferase (UGT)-enzymes are capable of catalyzing the biosynthesis of Rebaudioside as shown in Fig. 1a – 1d. The biocatalytic process of Rebaudioside M involves the glycosylation of:

[0070] a) steviol to steviolmonoside;

[0071] b) steviol to steviolmonoside A;

[0072] c) steviolmonoside to steviolbioside D;

[0073] d) steviolmonoside to steviolbioside;

[0074] e) steviolmonoside to rubusoside;

[0075] f) steviolmonoside A to rubusoside;

[0076] g) steviolmonoside A to steviolbioside A;

[0077] h) steviolmonoside A to steviolbioside B

[0078] i) steviolbioside A to stevioside A;

[0079] j) steviolbioside A to stevioside C;

[0080] k) steviolbioside B to stevioside B;l) steviolbioside B to stevioside C;

[0081] m) steviolbioside D to Rebaudioside B; n) steviolbioside D to Rebaudioside G; o) steviolbioside to stevioside;

[0082] p) steviolbioside to Rebaudioside B;

[0083] q) rubusoside to Rebaudioside G;

[0084] r) rubusoside to stevioside A;

[0085] s) rubusoside to stevioside B;

[0086] t) rubusoside to stevioside;

[0087] u) Rebaudioside G to Rebaudioside A; v) Rebaudioside G to Rebaudioside E4; w) Rebaudioside G to Rebaudioside E6; x) Rebaudioside B to Rebaudioside A;

[0088] y) stevioside to Rebaudioside A;

[0089] z) stevioside to Rebaudioside E;

[0090] aa) stevioside to Rebaudioside E2

[0091] bb) stevioside A to Rebaudioside E;

[0092] cc) stevioside A to Rebaudioside E3;

[0093] dd) stevioside A to Rebaudioside E4

[0094] ee) stevioside B to Rebaudioside E2;

[0095] ff) stevioside B to Rebaudioside E3;

[0096] gg) stevioside B to Rebaudioside E6;

[0097] hh) stevioside C to Rebaudioside E3;

[0098] ii) Rebaudioside A to Rebaudioside I;

[0099] jj) Rebaudioside A to Rebaudioside D; kk) Rebaudioside E4 to Rebaudioside £>7; 11) Rebaudioside E4 to Rebaudioside Z); mm) Rebaudioside E6 to Rebaudioside £>7; nn) Rebaudioside E6 to Rebaudioside I; oo) Rebaudioside E to Rebaudioside D; pp) Rebaudioside E to Rebaudioside AM; qq) Rebaudioside E2 to Rebaudioside I; rr) Rebaudioside E2 to Rebaudioside AM; ss) Rebaudioside E3 to Rebaudioside AM;tt) Rebaudioside E3 to Rebaudioside £>7;

[0100] uu) Rebaudioside I to Rebaudioside M;

[0101] vv) Rebaudioside D7 to Rebaudioside M;

[0102] ww) Rebaudioside D to Rebaudioside M; and / or

[0103] xx) Rebaudioside AM to Rebaudioside M.

[0104] The UGT-enzymes may convert lower molecular weight starting steviol glycosides into higher molecular weight target steviol glycosides. The UGT-enzymes can be used individually, in combination or consecutively, as each UGT-enzyme is more selective to certain substrates. For example, the UGT74G1 might perform the glycosylation at the - COOH functional group of the C19 of a steviol or starting steviol glycoside while other UGT-enzymes including UGT76G1 and UGTSl2 might perform glycosylation at the C13 and / or C19 glycosyl group(s) of starting steviol glycosides.

[0105] The combinations of different UGT enzymes are capable of converting the starting steviol glycosides to various other steviol glycosides by carrying out the following reactions: (I) glucosylation of the -OH functional group at the C13 of a steviol or steviol glycoside, (II) glucosylation of the -COOH functional group at the C19 of a steviol or steviol glycoside, (III) beta 1,2-glucosylation at the C2’ of the C19 glycosyl group(s) of a steviol glycoside, (IV) beta 1,2-glucosylation at the C2’ of the C13 glycosyl group(s) of a steviol glycoside, (V) beta 1,3-glucosylation at the C3’ of the C19 glycosyl group(s) of a steviol glycoside, (VI) beta 1,3-glucosylation at the C3’ of the C13 glycosyl group(s) of a steviol glycoside, (VII) beta 1,4-glucosylation at the C4’ of the C19 glycosyl group(s) of a steviol glycoside, (VIII) beta 1,4-glucosylation at the C4’ of the C13 glycosyl group(s) of a steviol glycoside, (IX) beta 1,6-glucosylation at the C6’ of the C19 glycosyl group(s) of a steviol glycoside, and (X) beta 1,6-glucosylation at the C6’ of the C13 glycosyl group(s) of a steviol glycoside. For example, the UGT74G1 usually carries out (II); the UGTSl2 usually carries out (III), (IV), (VII), (VIII), (IX) and (X); the UGT76G1 usually carries out (V), (VI), (VII) and (VIII); UGT85C2 usually carries out (I); EUGT11 usually carries out (III), (IV), (VII), (VIII), (IX) and (X), UGT91D2 usually carries out (III), (IV), (VII), (VIII), (IX) and (X), while UGT91 Al carries out (III) and (IV).Those skilled in art will appreciate that enzyme activity and other properties such as thermo- and pH-stability, optimal temperature and pH, selectivity, solubility, protein folding, etc., can be influenced by changing its amino acid sequence. Non-conservative substitutions and significant insertions or deletions near active sites, substrate binding sites, substrate channel and at buried positions important for protein folding or conformations can significantly alter the enzyme properties. On the other hand, silent mutations, which are typically conservative substitutions or small insertions / deletions, occur near the nonfunctional parts of the enzyme may have relatively less effect on its properties, though the impacts cannot be entirely excluded.

[0106] A skilled artisans will recognize that a “conservative substitution” is the substitution of an amino acid residue with another amino acid residue having side chain (R group) with similar properties. Generally, a conservative amino acid substitution will not substantially change the functional properties of a protein. Amino acid residues in nature are divided into groups based on common side chain properties as listed in Table 1.

[0107] Table 1

[0108] Classification of amino acids based on side chain properties.

[0109] Side chain properties Amino acid

[0110] Basic Arginine (R), Histidine (H), Lysine (K) Acidic Aspartic Acid (D), Glutamic Acid (E) Neutral hydrophilic Cysteine (C), Serine (S), Threonine (T), Asparagine (N), Glutamine (Q)

[0111] Side chains that Glycine (G), Proline (P)

[0112] influence chain

[0113] orientation

[0114] Hydrophobic Alanine (A), Valine (V), Isoleucine (I), Leucine (L), (aliphatic) Methionine (M)

[0115] Hydrophobic Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0116]

[0117] (aromatic)

[0118] In one embodiment, the nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzyme is a functional homolog of any NGT enzyme discussed herein. The functional homologs comprise or consist of an amino acid sequence that has at least 95%sequence identity or is 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7. If the functional homologs can perform the same or equivalent activity as the NGT-enzymes discussed herein, it is obvious that the enzyme could also be used in this application. For instance, the functional homologs may include silent mutations and / or mutations, including deletions, substitutions, and insertions, that have minimal impact on the secondary structure or properties of the enzymes.

[0119] In one aspect, the nucleotide diphosphate (NDP)-recycling enzyme is a functional homolog of nucleotide diphosphate (NDP)-recycling enzyme discussed herein. The functional homologs comprise or consist of an amino acid sequence that has at least 95% sequence identity or is 95% identical to SEQ ID NO: 8 and / or SEQ ID NO: 9. If the functional homologs can perform the same or equivalent activity as the nucleotide diphosphate (NDP)-recycling enzymes discussed herein, it is obvious that the enzyme could also be used in this application. For instance, the functional homologs may include silent mutations and / or mutations, including deletions, substitutions, and insertions, that have minimal impact on the secondary structure or properties of the enzymes.

[0120] A functional homolog is a polypeptide, or specifically a protein, or more specifically an enzyme, that has sequence similarity to the reference enzyme, and that could perform one or more of the activities of the reference enzyme. The amino acid sequence of the functional homolog may have some deletions, insertions, substitutions, conservative substitutions, or modifications with respect to the amino acid sequence of the reference enzyme. A functional homolog may arise through domain swapping within a single polypeptide, specifically the polypeptide of the nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes and / or the nucleotide diphosphate (NDP)-recycling enzymes. Additionally, a functional homolog may also occur naturally or can also be created via mutagenesis or the combination of domains from other naturally occurring enzymes, specifically the NGT-enzymes and / or the NDP-recycling enzymes.

[0121] In yet another aspect, the functional homologs may have a signal sequence at their N-terminus, which helps in protein transfer either co-translationally or post-translationally. The functional homologs may also be linked to other polypeptides or linkers to facilitate the identification, purification, or synthesis.In another embodiment, any of the enzymes disclosed herein is a fusion enzyme, wherein the amino acid sequence of the nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzyme, the amino acid sequence of the nucleotide diphosphate (NDP)-recycling enzyme and / or the amino acid sequence of the NDP-synthesizing enzyme forms a part of said fusion enzyme’s complete amino acid sequence. For instance, the fusion enzyme could be a UGT-sucrose synthase fusion enzyme, with a UGT domain coupled to a sucrose synthase domain, allowing the enzyme to regenerate UDP-glucose from UDP and sucrose. A skilled person will understand that the fusion enzyme can be made from many different combinations of enzymes, containing several NGT-enzymes, NDP-recy cling enzymes, NDP-synthesizing enzymes and other enzymes.

[0122] In other aspects, the present disclosure provides polynucleotides that encode the enzymes disclosed herein, as well as a microorganism that comprises these polynucleotides.

[0123] The microorganism may be a fungal cell, an algal cell or a bacterial cell. The microorganism may be, for example, Escherichia spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., Pseudomonas spp., Saccharomyces spp., Aspergillus spp., Pichia spp., Bacillus spp., Penicillium spp., Trichoderma spp., and Yarrowia spp. etc. For instance, in some embodiments, the microorganism could be a species selected from Escherichia coli, Corynebacterium glutamicum, Rhodobacter capsulatus, Zymomonas mobilis. Vibrio natriegens, Pseudomonas putida, Saccharomyces cerevisiae, Aspergillus niger. Aspergillus oryzae, Pichia pastoris. Bacillus subtilis, Penicillium chrysogenum, Trichoderma reesei or Yarrowia lipolytica.

[0124] The microorganism may be a recombinant microorganism.

[0125] In another embodiment, the recombinant microorganism may also comprise the steviol biosynthesis enzymes selected from the group consisting of a mevalonate (MV A) pathway enzyme, a 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzyme, geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-l -deoxyxylulose 5-phosphate reductoisom erase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase or cytochrome P450 reductase.

[0126] The recombination microorganisms can be used as such for the biocatalytic process of producing Rebaudioside M (whole-cell or in vivo) or to express the required enzymes to use them as an enzyme preparation in vitro.

[0127] To optimize the cultivation and fermentation process of the recombinant microorganisms, a skilled artisan could select suitable culture medium and consider other factors such as pH levels, temperature, and oxygen concentration (aerobic, microaerobic or anaerobic).Various standard synthetic and molecular biology and biotechnology methods and techniques, including plasmid construction, protein and microorganism engineering, protein expression and purification, and other standard techniques, are generally performed according to well-established methods recognized by the scientific community. These methods are described in various reputable publications such as “Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986”, “Fermentation Microbiology and Biotechnology, fourth edition, CRC Press, Boca Raton, 2019”, “Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989”, “Current Protocols in Molecular Biology, Greene Publishing and Wiley -Interscience, New York, 1987” and “System Biology, Volumes 1 and 2, Oxford University Press, Oxford, 2006”, as well as other publications that are well recognized by the scientific community.

[0128] The usual fermentation conditions are a pH of between 6 to 8 and a temperature of between 25 to 37 °C. The fermentation process is continued until laboratory test data shows the desired enzyme production yield. Usually, after at least 15 hours, the fermentation is stopped. In a subsequent recovery process, the enzyme is isolated from the biomass. In a first solid / liquid separation, the biomass is separated from the culture broth by standard techniques (e.g., is centrifuged and / or filtered). The biomass is homogenized to disrupt the bacterial cells and treated with a nuclease (e.g., NuCLEANase, c-LEcta, Leipzig, Germany) to degrade the DNA / RNA nucleic acids released upon cell disruption. This is followed bysolid / liquid separation steps to further remove cell debris and other insoluble matter. The cell-free supernatant is filtered to obtain the purified enzyme preparation.

[0129] The invention also provides a whole-cell biocatalytic process of glycosylating the starting steviol glycoside. This method involves using the recombinant microorganism with the previously mentioned enzymes. The method involves contacting the recombinant microorganism and the starting steviol glycoside composition to produce Rebaudioside M. In one embodiment, the recombinant microorganism could be either a living or non-living cell. In another embodiment, the recombinant microorganism may have permeabilized cell membranes that could increase the permeability and provide easier access for specific molecules to move in and out of the cell more freely.

[0130] The whole-cell biocatalytic process may involve contacting the recombinant microorganism with NDP and sucrose to synthesize NDP -glucose for continuous NDP-glucose supply. The NDP may also be externally supplied or internally produced by the recombinant microorganism. For example, the biocatalytic process of converting the starting steviol glycosides to Rebaudioside M is carried out using catalytic amounts of uridine diphosphate (UDP)-glucose synthesized by recombinant microorganism containing UDP -recycling enzyme, such as sucrose synthase (SuSy), utilizing the externally supplied UDP or UDP produced internally by the recombinant microorganism.

[0131] The invention also provides an in vitro biocatalytic process of glycosylating the starting steviol glycoside. This method uses the previously mentioned enzymes as an enzyme preparation. The method involves contacting the enzyme(s) and the starting steviol glycoside composition to produce Rebaudioside M.

[0132] Optionally, the invention also provides a whole-cell biocatalytic process of producing uridine diphosphate (UDP). The method involves using a recombinant microorganism containing NDP-synthesizing enzyme, such as UMP-kinase. The method involves contacting the recombinant microorganism with ATP and UMP to produce UDP for the synthesis of UDP-glucose. For example, UDP production is carried out in situ by a recombinant microorganism containing an NDP-synthesizing enzyme, such as UMP-kinase, which is then used for UDP-glucose production in the biosynthesis of Rebaudioside M.In one embodiment of the present invention, the starting steviol glycoside composition and specifically the starting steviol glycoside(s) is fully dissolved in the reaction medium and is glycosylated by nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzyme to produce Rebaudioside M.

[0133] In another embodiment of the invention, the starting steviol glycoside composition and specifically the starting steviol glycoside(s) is only partially dissolved in the reaction medium and is glycosylated by nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzyme to Rebaudioside M.

[0134] In one embodiment of present invention, the recombinant microorganism, which comprises the previously mentioned enzymes, can be produced through a fermentation process. The recombinant microorganism can then be used in subsequent step of in vitro or whole-cell catalysis to synthesize Rebaudioside M.

[0135] In other embodiments, the method includes growing the recombinant microorganism in the presence of the starting steviol glycosides. The recombinant microorganism expresses the enzymes disclosed herein to produce Rebaudioside M from the starting steviol glycosides present in the growing media. The recombinant microorganism may also express NDP-recycling enzyme, which may include but is not limited to UDP-recy cling enzyme, such as sucrose synthase (SuSy), which is capable of synthesizing NDP-glucose for the glycosylation of the starting steviol glycosides. Optionally, the recombinant microorganism may also express NDP-synthesizing enzyme, such as UMP-kinase, to produce NDP for the synthesis of NDP-glucose.

[0136] In one embodiment, the enzymes disclosed herein can be overexpressed. The overexpressed enzymes can be isolated from the cell extract based on its physical and chemical properties, using techniques known in the art. Representative non-limiting techniques for isolating enzymes from a microorganism include cell lysis, centrifugation, membrane filtration, precipitation, flocculation, nuclease treatment, electrophoresis, liquid chromatography, ion exchange chromatography, gel filtration chromatography or affinity chromatography. The enzymes can be provided as a crude, semi-purified and purified enzyme preparation.

[0137] In some preferred embodiments, the enzyme preparation can be provided in the form of a whole cell suspension, a crude lysate, a supernatant, a filtrate, or a purified enzymepreparation or a combination thereof. The enzyme preparation can be located on the surface of the cell, inside the cell or located both on the surface of the cell and inside the cell.

[0138] In one embodiment, the enzyme preparation is cell-free. In another embodiment, the enzyme preparation is immobilized. For example, the enzyme preparation may be immobilized to a solid support made from inorganic or organic materials. Non-limiting examples of solid supports suitable to immobilize the enzyme preparation include derivatized cellulose or glass, ceramics, metal oxides, resins, gels, zeolites, metal-organic frameworks, covalent-organic frameworks, hydrogen bonded-organic frameworks, silica (including but not limited to mesoporous silica, sol -gel silica and fumed silica), chitosan, polyvinyl material such as polyvinyl alcohol (PVA), polyacrylic material, calcium alginate, nanotubes, hydroxyapatite or membranes. The enzyme may be immobilized onto the solid support for example by covalent attachment, adsorption, cross-linking, ionic bonding, entrapment or encapsulation.

[0139] In another embodiment, the recombinant microorganism is immobilized. For example, the recombinant microorganism may be immobilized to a solid support made from inorganic or organic materials. Non-limiting examples of solid supports suitable to immobilize the recombinant microorganism include derivatized cellulose or glass, ceramics, metal oxides, resins, gels, zeolites, metal-organic frameworks, covalent-organic frameworks, hydrogen bonded-organic frameworks, silica (including but not limited to mesoporous silica, sol-gel silica and fumed silica), chitosan, polyvinyl material such as polyvinyl alcohol (PVA), polyacrylic material, calcium alginate, nanotubes, hydroxyapatite or membranes. The recombinant microorganism may be immobilized onto the solid support for example by covalent attachment, adsorption, cross-linking, ionic bonding entrapment or encapsulation.

[0140] One embodiment of the present invention is cell-free methods wherein the enzyme preparation is in free form or immobilized to a solid support made from inorganic or organic materials. The enzyme preparation is located neither on the surface nor inside the cell(s). The enzyme preparation can be provided in the form of a crude lysate, a supernatant, a filtrate or as purified enzyme preparation.

[0141] In another embodiment, the enzyme preparation can be prepared from one cellular extract or combination of multiple cellular extracts from one recombinant microorganismor multiple recombinant microorganisms. Each recombinant microorganism could express one or a number of enzymes for the biocatalytic process of one or more of starting steviol glycosides to synthesize Rebaudioside M.

[0142] In one embodiment, the enzyme preparation, starting steviol glycoside compositions and processing aids are combined and incubated to produce Rebaudioside M. In one specific embodiment, the enzyme preparation, starting steviol glycoside compositions, processing aids such as sucrose and uridine-5'-diphosphate disodium salt (UDP-Na2) are combined, and the reaction medium is incubated at a temperature between 40 °C and 50 °C, preferably at 45°C. Optionally, the addition of UDP-Na2 could be replaced by the biosynthesis of UDP in situ using adenosine-5’ -triphosphate disodium salt (ATP-Na2) and uridine-5’-monophosphate disodium salt (UMP-Na2) with UMP -kinase, and the reaction medium is incubated at a temperature between 40 °C and 50 °C, preferably at 45°C. With ATP as the donor, UMP-kinase synthesizes UMP to UDP in situ, which is then used for the subsequent formation of UDP-glucose, catalyzed by sucrose synthase. Sucrose synthase transfers glucose residues from sucrose to UDP, yielding UDP-glucose. The nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes, including UGTSr, UGTSl2, SrUGT1 and SrUGT2, then transfer the glucose moiety from UDP-glucose (donor) to the starting steviol glycosides in the starting steviol glycoside compositions as acceptors. As a result, the starting steviol glycosides in the starting steviol glycoside compositions are transformed into Rebaudioside M.

[0143] In another embodiment, the reaction mixture is treated to inactivate the enzymes at the end of reaction. In yet another embodiment, the treated reaction mixture is purified by one or more techniques including but not limited to chromatographic separation, membrane filtration, separation, crystallization, precipitation, flocculation, trituration, membrane separation, centrifugation, extraction, or a combination of such techniques to yield Rebaudioside M. In one specific embodiment, the reaction mixture is heated to and held at 100 °C for 15 mins to inactivate the enzymes, then filtered through 0.1 μm filter, and subjected to crystallization to separate Rebaudioside M. The crystals are separated by centrifugation or press filtration, to yield highly purified Rebaudioside M crystals. The highly purified Rebaudioside AT crystals are then dried.

[0144] In another embodiment, the highly purified Rebaudioside M crystals are further dissolved and spray-dried to produce a highly purified Rebaudioside M powder.In one embodiment, the starting steviol glycoside compositions contain mixed sugar steviol glycosides. The mixed sugar steviol glycosides may contain a combination of one or more type of sugars, including but not limited to glucose, mannose, galactose, fructose, rhamnose, arabinose, deoxy-glucose or xylose. The mixed sugar steviol glycosides can be used as starting steviol glycosides for the synthesis of other steviol glycosides with mixed sugars via glycosylation with nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes. For example, the starting steviol glycoside Rebaudiosdie C, which contains both rhamnose and glucose, can be used as a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside N2, Rebaudioside N7, Rebaudioside N8, Rebaudioside O4, Rebaudioside O5, Rebaudioside O8 and Rebaudioside K, using NGT-enzymes. Similarly, the starting steviol glycoside Rebaudiosdie F, which contains both xylose and glucose, can be used as a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside U4, Rebaudioside U5 and Rebaudioside V3, using NGT-enzymes.

[0145] In another embodiment, the mixed sugar steviol glycoside can be hydrolyzed into glucose-only steviol glycoside which could be either be a starting steviol glycoside for the synthesis of another glucose-only target steviol glycoside. The hydrolysis can be performed using enzymes for the hydrolysis of their respective sugars, such as rhamnosidase for the hydrolysis of rhamnose and xylosidase for the hydrolysis of xylose. In one embodiment the starting steviol glycoside Rebaudioside C, which contains both rhamnose and glucose, can first be hydrolyzed with rhamosidase to produce glucose-only Rebaudioside G. The resulting Rebaudioside G can be a starting steviol glycoside for the synthesis of another steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes. Similarly, in another embodiment, the starting steviol glycoside Rebaudioside F, which contains both xylose and glucose can first be hydrolyzed with xylosidase to produce glucose-only Rebaudioside G, which can then be used as a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.

[0146] In yet another embodiment, the starting steviol glycoside Dulcoside A, which contains both rhamnose and glucose, can first be hydrolyzed with rhamosidase to produce glucose-only rubusoside. The resulting rubusoside can be a starting steviol glycoside for the synthesis of another steviol glycosides, including but not limited to Rebaudioside M, usingNGT-enzymes. In still another embodiment, the starting steviol glycoside stevioside F, which contains both xylose and glucose, can first be hydrolyzed with xylosidase to produce glucose-only rubusoside. The resulting rubusoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.

[0147] In yet another embodiment, the starting steviol glycoside steviolbioside C, which contains both rhamnose and glucose, can first be hydrolyzed with rhamnosidase to produce glucose-only steviolmonoside. The resulting steviolmonoside can be a starting steviol glycoside for the synthesis of another steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes. In still another embodiment, the starting steviol glycoside steviolbioside F, which contains both xylose and glucose, can first be hydrolyzed with xylosidase to produce glucose-only steviolmonoside. The resulting steviolmonoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.

[0148] In yet another embodiment, the starting steviol glycoside Dulcoside C, which contains both rhamnose and glucose, can first be hydrolyzed with rhamnosidase to produce glucose-only steviolbioside D. The resulting steviolbioside D can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes. In still another embodiment, the starting steviol glycoside stevioside F, which contains both xylose and glucose, can first be hydrolyzed with xylosidase to produce glucose-only steviolbioside D. The resulting steviolbioside D can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.

[0149] In one embodiment, the starting steviol glycosides can be hydrolyzed by enzyme(s), such as glucosidase, into lower molecular weight steviol glycosides which can be starting steviol glycosides for the synthesis of other steviol glycosides. For example, in one embodiment, the starting steviol glycoside Rebaudioside G can be hydrolyzed into Rubusoside which can be the starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes. Similarly, the starting steviol glycoside Rebaudioside C can be hydrolyzed into Dulcoside A and / or steviolbioside C which can be a starting steviol glycoside for other steviol glycosides, including but not limited to Rebaudioside F2, Rebaudioside F7, Rebaudioside TVS,Rebaudioside O4, Rebaudioside O5, Rebaudioside O8 and Rebaudioside K, using NGT-enzymes. In another embodiment, the starting steviol glycoside Rebaudioside F can be hydrolyzed into stevioside F and / or steviolbioside H which can be a starting steviol glycoside for other steviol glycosides, including but not limited to Rebaudioside U4, Rebaudioside U5 and Rebaudioside V3, using NGT-enzymes.

[0150] In one embodiment, a complex biosynthesis, including hydrolytic and glycosylation reactions, can be performed with different enzyme combinations, including but not limited to hydrolases, rhamnosidases, xylosidases, glucosidases and nucleotide diphosphatedependent glycosyltransferase (NGT)-enzymes. In another embodiment, the starting steviol glycosides can first be hydrolyzed, either once or multiple times, into other starting steviol glycosides (which could also be considered as intermediate steviol glycoside), followed by glycosylation to synthesise Rebaudioside M. In one embodiment, the complex biosynthesis process reactions can be performed consecutively. In another embodiment, the complex biosynthesis process reactions can be performed concurrently in a “one-pot” reaction. For instance, the complex biosynthesis process can be performed to synthesize Rebaudioside M from a mixed sugar starting steviol glycoside Rebaudioside C.

[0151] In one embodiment, the starting steviol glycoside Rebaudioside C can be hydrolyzed into Dulcoside A with a glucosidase. The resulting Dulcoside A can then be hydrolyzed into glucose-only rubusoside with a rhamnosidase. The resulting rubusoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes. In another embodiment, the starting steviol glycoside Rebaudioside C can be hydrolyzed into steviolbioside C with a glucosidase. The resulting steviolbioside C can then be hydrolyzed into glucose-only steviolmonoside with a rhamnosidase. The resulting steviolmonoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.

[0152] In one embodiment, the starting steviol glycoside Dulcoside A can be hydrolyzed into steviolbioside C with a glucosidase. The resulting steviolbioside C can then be hydrolyzed into glucose-only steviolmonoside with a rhamnosidase. The resulting steviolmonoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.In one embodiment, the starting steviol glycoside Rebaudioside F can be hydrolyzed into stevioside F with a glucosidase. The resulting stevioside F can then be hydrolyzed into glucose-only rubusoside with a xylosidase. The resulting rubusoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes. In one embodiment, the starting steviol glycoside Rebaudioside F can be hydrolyzed into steviolbioside H with a glucosidase. The resulting steviolbioside H can then be hydrolyzed into glucose-only steviolmonoside with a xylosidase. The resulting steviolmonoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.

[0153] In one embodiment, the starting steviol glycoside stevioside F can be hydrolyzed into steviolbioside H with a glucosidase. The resulting steviolbioside H can then be hydrolyzed into glucose-only steviolmonoside with a xylosidase. The resulting steviolmonoside can be a starting steviol glycoside for the synthesis of other steviol glycosides, including but not limited to Rebaudioside M, using NGT-enzymes.

[0154] To synthesize the intermediate steviol glycoside and / or Rebaudioside AT, a skilled artisan could use a combination of different enzymes, including but not limited to hydrolases, rhamnosidases, xylosidases, glucosidases and nucleotide diphosphatedependent glycosyltransferase (NGT)-enzymes in various sequences. Additionally, various mixed sugar steviol glycosides, including but not limited to Rebaudioside C, Rebaudioside F, Dulcoside A, stevioside, steviolbioside F, stevioside TV, Dulcoside C and steviolbioside C, can be used as starting steviol glycosides either individually or as mixtures for the biosynthesis of Rebaudioside AT using the methods described in this disclosure. The mixed sugar steviol glycosides may contain a combination of one or more type of sugars, including but not limited to glucose, mannose, galactose, fructose, rhamnose, arabinose, deoxy -glucose or xylose. The non-limiting examples illustrate various embodiments of the biocatalysts and methods described in this disclosure. These examples are provided for illustrative purposes only and are not intended to restrict the scope of the present disclosure.

[0155] The biocatalytic process of this invention could also be suitable for the glycosylation of various other substrates, including terpenoid, such as a monoterpenoid, sesquiterpenoid or triterpenoid, including but not limited to steviol or steviol glycosides and mogrol or mogrosides. In some embodiments, the biocatalytic process involves the glycosylation ofdifferent substrates, including glycosides, aliphatic and branched alcohols, substituted phenols, flavonoids and gallates. In another embodiment, the biocatalytic process involves the glycosylation of different substrates including terpenoids glycosides, such steviol glycosides, a type of diterpenoid glycosides and mogrosides, a type of triterpenoids.

[0156] In a preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTSr, UGTS12 and SrUGT2 and the starting composition comprises Rebaudioside B.

[0157] In another preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTSr, UGTS12 and SrUGTl and the starting composition comprises Stevioside, Rubusoside or a mixture thereof.

[0158] In another preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTSr, UGTS12, SrUGTl and SrUGT2 and the starting composition comprises Steviolbioside, Steviolmonoside, or a mixture thereof.

[0159] In another preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTSr, UGTS12, SrUGTl, the starting composition comprises Dulcoside A and rhamnosidase is added.

[0160] In another preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTS12, UGTSr, the starting composition comprises Rebaudioside C and a rhamnosidase is added.

[0161] In another preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTS12, UGTSr, the starting composition comprises Rebaudioside F and a xylosidase is added.

[0162] In another preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTSr and UGTS12 and the starting composition comprises Rebaudioside G, Rebaudioside A or a mixture thereof.In another preferred embodiment, the at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes are UGTSr and SrUGT1 and the starting composition comprises Rebaudioside E.

[0163] In a more preferred embodiment, the starting steviol glycoside composition comprises Rebaudioside A, Rebaudioside B, Stevioside and Steviolbioside and the NGT-enzymes are UGTSr, UGTSl2, SrUGT1 and SrUGT2.

[0164] In a most preferred embodiment, the starting steviol glycoside composition comprises 75 to 95% Rebaudioside B by weight on dry basis and the NGT-enzymes are UGTSr, UGTSl2, SrUGT1 and SrUGT2.

[0165] In another more preferred embodiment, the starting steviol glycoside composition comprises Rebaudioside^, Rebaudioside C, Rebaudioside E, Rebaudioside F, Dulcoside A, Stevioside, Rubusoside and Steviolbioside, the NGT-enzymes are UGTSr, UGTS12, SrUGTl and SrUGT2, and optionally a rhamnosidase, a xylosidase or a combination thereof is added.

[0166] In another most preferred embodiment, the starting steviol glycoside composition comprises 70 to 90% Stevioside by weight on dry basis and 4 to 15% Dulcoside A, the NGT-enzymes are UGTSr, UGTS12, SrUGTl, optionally SrUGT2, and a rhamnosidase, optionally a xylosidase or a combination thereof is added.

[0167] In another more preferred embodiment, the starting steviol glycoside composition comprises Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside F, Rebaudioside F, Dulcoside A, Stevioside, Rubusoside and Steviolbioside and the NGT-enzymes are UGTSr, UGTS12, SrUGTl and SrUGT2, and optionally a rhamnosidase, a xylosidase or a combination thereof is added.

[0168] In another most preferred embodiment, the starting steviol glycoside composition comprises 40 to 60% Rebaudioside A by weight on dry basis, 2 to 10% Rebaudioside B, 7 to 17% Rebaudioside C and 10 to 20% Stevioside, the NGT-enzymes are UGTSr, UGTS12, SrUGTl and SrUGT2, and optionally a rhamnosidase, a xylosidase or a combination thereof is added.In another more preferred embodiment, the starting steviol glycoside composition comprises and Rebaudioside B, Rebaudioside C, Rebaudioside F, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside and Steviolbioside, the NGT-enzymes are UGTSr, UGTS12, SrUGTl and SrUGT2, and optionally a rhamnosidase, a xylosidase or a combination thereof is added.

[0169] In another more preferred embodiment, the starting steviol glycoside composition comprises Rebaudioside B, Rebaudioside C, Dulcoside A, Stevioside, Steviolmonoside and Steviolbioside, the NGT-enzymes are UGTSr, UGTS12, SrUGTl and SrUGT2, and optionally a rhamnosidase is added.

[0170] In a further embodiment, the starting steviol glycoside composition used in the method described above is the by-product of processes to produce steviol glycosides.

[0171] In one embodiment, the invention provides a method for producing highly purified Rebaudioside M described above, wherein the purity of Rebaudioside M is greater than about 90% by weight on a dried basis, preferably greater than 95% and more preferably greater than 98%.

[0172] In one embodiment, the invention provides a method for, wherein the Rebaudioside M is obtained in a yield of greater than 85% (based on the percentage of the weight of isolated and purified Rebaudioside M relative to the weight of all the starting steviol glycosides used), preferably greater than 90%.

[0173] In one embodiment, the microorganism used in the method described above is selected from the from the group consisting of E. coli, Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., Pseudomonas spp., Saccharomyces spp., Aspergillus spp., Pichia spp., Bacillus spp., Penicillium spp., Trichoderma spp. and Yarrowia spp.

[0174] The following non-limiting examples illustrate various embodiments of the biocatalysts and methods described in this disclosure. These examples are provided for illustrative purposes only and are not intended to restrict the scope of the present disclosure. From the foregoing description and the following Examples, it should be apparent and understood by those skilled in the art that any modifications of the disclosure, in addition to those described herein, shall fall within the scope of the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0175] FIG. la-d show the pathways of producing Rebaudioside M and various steviol glycosides from steviol.

[0176] EXAMPLES

[0177] The examples that follow are illustrative of specific embodiments of the invention and various uses thereof. They are set forth for explanatory purposes only and are not to be taken as limiting to the invention.

[0178] EXAMPLE 1

[0179] Protein sequences of enzymes used in the biocatalytic process.

[0180] SEQ ID NO: 1

[0181] > UGT76G1 (UDP-glycosyltransferase; source ofWT gene: Stevia rebaudiana) MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPH FTFRFILDNDPQDERISNLPTHGPLAGMRIPI INEHGADELRRELELLMLASEEDEEVSC LITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQAS GFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAP SFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLV DSKQSFLWWRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWN STLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEG E Y I RQNARVLKQKADVS LMKGGS S YE S LE S LVS Y I SSL

[0182] SEQ ID NO: 2

[0183] > UGT74G1 (UDP-glycosyltransferase; source of WT gene: Stevia rebaudiana) MAEQQKIKKSPHVLLIPFPLQGHINPFIQFGKRLISKGVKTTLVTTIHTLNSTLNHSNTT TTSIEIQAISDGCDEGGFMSAGESYLETFKQVGSKSLADLIKKLQSEGTTIDAIIYDSMT EWVLDVAIEFGIDGGSFFTQACWNSLYYHVHKGLISLPLGETVSVPGFPVLQRWETPLI LQNHEQIQSPWSQMLFGQFANIDQARWVFTNSFYKLEEEVIEWTRKIWNLKVIGPTLPSM YLDKRLDDDKDNGFNLYKANHHECMNWLDDKPKESWYVAFGSLVKHGPEQVEEITRALI DSDVNFLWVIKHKEEGKLPENLSEVIKTGKGLIVAWCKQLDVLAHESVGCFVTHCGFNST LEAISLGVPWAMPQFSDQTTNAKLLDEILGVGVRVKADENGIVRRGNLASCIKMIMEEE RGVI I RKNAVKWKDLAKVAVHE GGS S DND I VE FVS E L I KA SEQ ID NO: 3

[0184] > UGTS12 (UDP-glycosyltransferase; source of WT gene: Solanum lycopersicum) MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYADSIH LIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKPDLLI YDVLQP WAEHVANEQNIPAGKLLTSCAAVFSYFFSFRKNPGVEFPFPAIHLPEVEKVKIREILAKE PEEGGRLDEGNKQMMLMCTSRTIEAKYIDYCTELCNWKWPVGPPFQDLITNDADNKELI DWLGTKHENSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSAMESIDFGVPIIAMPIHNDQ P INAKLMVELGVAVE I VRDDDGKIHRGE IAETLKS WTGETGE I LRAKVRE I SKNLKS IR DEEMDAVAEELIQLCRNSNKSK

[0185] SEQ ID NO: 4

[0186] > UGTSr-0588 (engineered glycosyltransferase; source of WT gene: Stevia rebaudiana) MENKTETTVRRRRRI ILFPVPFQGHINPLLQLANVLYSKGFAITILHTNFNKPKTSNYPH FTFRFILDNDPQDERISNLPTTGPGAGMRIPI INEHGADELRRELELLMLASEEDEEVSC LITDALWYFAQDVADSLNLRRLVLMTSSLFNFHCHVSLPQFDELGYLDPDDKTRLEEPVS GFPMFKVKDIKSAYSHGQVGKEILGKMVKQTKASSGVIWNSFKELEESELETVIREIPAP SFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLV DSGQSFLWWRPGFVKGSTWCEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWN STLESVCEGVPMIFSSFGGDQPINARYMSDVLRVGVYLENGWERGEWNAIRRVMVDEEG E Y I RQNARVLKQKADVS LMKGGS S YE S LE S LVS Y I S S F

[0187] SEQ ID NO: 5

[0188] > SrUGTl-0360 (engineered glycosyltransferase; source ofWT gene: Stevia rebaudiana) MENKTETTVRRRRRI ILFPVPFQGHINPILQLANVLYSKGFS I TILHTNFNKPKTSNYPH FTFRFILDNDPQDERISNLPTHGPLAGMRIPI INEHGADELRRELELLMLASEEDEEVCC LITDALWYFAQSVADSLNLPRLVLMTSSLFNFHALVSLPQFDELGYLDPDDKTRLEEQAS GFPMLKVKDIKSAYSNWQI IKEILDKMIKQTRASSGVIWNSFKELEESELETVIREIPAP SFLIPLPKHGTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLV DSKQSFLWWRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWN STLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGFERGEIANAIRRVMVDEEG E Y I RQNARVLKQKADVS LMKGGS S YE S LE S LVS Y I SSL

[0189] SEQ ID NO: 6

[0190] > SrUGT2-0125 (engineered glycosyltransferase; source of WT gene: Stevia rebaudiana) MAEQQKIKKSPHVLLIPFPSQGHINPMIQFGKRLISKGVKTTLVTTIHTLNSTLNHSNTT TTSIEIQAISDGCDEGGFMSAGESYLETFKQVGSKSLADLIKKLQSEGTTIDAIIYDSML PWVLDVAIEFGIDGGSFFTQACWNSLYYHVHKGLISLPLGETVSVPGFPVLQRWETPLI LQNHEQIQSPWSQMLFGQFANIDQARWVFTNSFYKLEEEVIEWMRKIWNLKVIGPTLPSM YLDKRLDDDKDNGFNLYKANHHECMNWLDDKPKESWYVAFGSLVKHGPEQVEEITRALI DSDVNFLWVIKHKEEGKLPENLSEVIKTGKGLIVAWCPQLDVLAHESVGCFVTHCGFNST LEAISLGVPWAMPQFSDQTTNAKLLDEILGVGVRVKADENGIVRRGNLASCIKMIMEEE RGVI I RKNAVKWKDLAKVAVHE GGS S DND I VE FVS E L I KA

[0191] SEQ ID NO: 7

[0192] > UGTS1-O728 (engineered glycosyltransferase; source of WT gene: Solanum lycopersicum) MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYNDSIH LIELQLPNLPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKPDLLIYDVLQP WAEHVANEQGIPAGKLLVGSAAVFSYFHSLRKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEEGNKQMMLMGTSRTIEAKYIDYCTELCNTKVVPVGPPFQDLITNDADNKELI DKLGTKPENSTVFVSFGSEYFLSKDDMHEIAFALEASNVNFIWVVRFPKGEERNLEDALP EGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPIHYDQ PIIAKLMVELGVVEIVRDDDGKIHRGEIAELKSVVTGETGEILRAKVREVSKNLKSIR DEEMDAAAEEL I QLCRNSNKSK

[0193] SEQ ID NO: 8

[0194] > SuSy (Sucrose synthase; source of WT gene: Arabidopsis thaliand)

[0195] MANAE RM I T RVH S QRE RLNE T L VS E RNE VLAL L S RVE AKGKG I L QQNQ 11 AE FE AL PE Q T RKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALWEELQPAEFLHF KEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPL LKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLER GWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNWILSPHGYFAQDNVLGYPD TGGQWYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSE YCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLI IGNYSDGNL VASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIIT STFQEIAGSKETVGQYESHTAFTLPGLYRWHGIDVFDPKFNIVSPGADMSIYFPYTEEK RRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRL RELANLVWGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRY ICDTKGAFVQPALYEAFGLTWEAMTCGLPTFATCKGGPAEI IVHGKSGFHIDPYHGDQA ADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDR LEARRYLEMFYALKYRPLAQAVPLAQDD

[0196] SEQ ID NO: 9

[0197] > SuSy-0362 (engineered sucrose synthase; source of WT gene: Arabidopsis ihaliana)

[0198] MANAERMITRVHSQRERLNETLVSERNEWALLSRVEAKGKGILQQNQI IAEFEALPEQT RKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALWEELQPAEFLHF KEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPL LDFLRLHSHQGKNLMLSEKIQNLNDLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLER GWGDNAERVLDMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD

[0199] TGGQVVYILDQVRALEKEMLKRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSE YCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLI IGNYSDGNL VASLLAHKLGVTQCTIAHALEKTKYPDSDIY WKKLDDKYHFSCQFTADIFAMNHTDFIIT STFQEIAGSKETVGQYESHTAFTLPGLYRWHGIDVFDPKFNIVSPGADMSIYFPYTETK RRLTKFHSEIEELLYSDVENDEHICVLKDKKKPILFTMARLDRVKNLTGLVEWYGKNTRL RELVNLVVVGGDRRSESKDNEEKAEMKKAYDLIEQYKLNGQFRWISSQMDRVRNGELYRY lADTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCHGGPAE I IVHGKSGFHIDPYHGDQA ADLLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDR LEHRRYLEMFYALKYRPLAQAVPLAQDD SEQ ID NO: 10

[0200] > UGT91D2 (UDP -glycosyltransferase; source of WT gene: Stevia rebaudiana) MYNVTYHQNSKAMATSDSIVDDRKQLHVATFPWLAFGHILPYLQLSKLIAEKGHKVSFLS TTRNIQRLSSHISPLINWQLTLPRVQELPEDAEATTDVHPEDIPYLKKASDGLQPEVTR FLEQHSPDWI IYDYTHYWLPSIAASLGISRAHFSVTTPWAIAYMGPSADAMINGSDGRTT VEDLTTPPKWFPFPTKVCWRKHDLARLVPYKAPGISDGYRMGLVLKGSDCLLSKCYHEFGTQWLPLLETLHQVPWPVGLLPPEIPGDEKDETWVSIKKWLDGKQKGSWYVALGSEVLV SQTEWELALGLELSGLPFVWAYRKPKGPAKSDSVELPDGFVERTRDRGLVWTSWAPQLR ILSHESVCGFLTHCGSGSIVEGLMFGHPLIMLPIFGDQPLNARLLEDKQVGIEIPRNEED GCLTKESVARSLRSVWEKEGEIYKANARELSKIYNDTKVEKEYVSQFVDYLEKNARAVA IDHES SEQ ID NO: 11

[0201] > UGT85C2 (UDP -glycosyltransferase; source ofWT gene: Stevia rebaudiana) MDAMATTEKKPHVIFIPFPAQSHIKAMLKLAQLLHHKGLQITFVNTDFIHNQFLESSGPH CLDGAPGFRFETIPDGVSHSPEASIPIRESLLRSIETNFLDRFIDLVTKLPDPPTCIISD GFLSVFTIDAAKKLGIPVMMYWTLAACGFMGFYHIHSLIEKGFAPLKDASYLTNGYLDTV IDWVPGMEGIRLKDFPLDWSTDLNDKVLMFTTEAPQRSHKVSHHI FHTFDELEPS I IKTL SLRYNHIYTIGPLQLLLDQIPEEKKQTGITSLHGYSLVKEEPECFQWLQSKEPNSWYVN FGSTTVMSLEDMTEFGWGLANSNHYFLWI IRSNLVIGENAVLPPELEEHIKKRGFIASWC SQEKVLKHPSVGGFLTHCGWGSTIESLSAGVPMICWPYSWDQLTNCRYICKEWEVGLEMG TKVKRDE VKRLVQE LMGE GGHKMRNKAKDWKEKAR I Al APNGS S S LN I DKMVKE I T VLAR N

[0202] SEQ ID NO: 12

[0203] > EUGT11 (glycosyltransferase; source of WT gene: Oryza sativa Japonica Group) MASWEEQLRDELAGRDLAVASVPGKGRGLFAARSFFPGEWISQEPYASTPNKISVGSNC DNCFASRNLRKCSVCRVAWYCGSACQREEWKLHQLECRAIAALTEDRKKMLTPTIRLMVR LVLRRKLQDDKAIPSSGTDNYNLVDALESHISEVDKNQLVLYAQMANLVQLILPSFELDL KEITHTFSKFACNAHTICDPELRPLGTGLYPVLSIINHSCVPNAVLIFEGRTAYVRALQP ISKNEEVSISYIETAATTMKRQDDLKHYYFTCTCPRCVKDSEEDALLEGYRCNDQKCDGF LLPNAGNKGYTCQKCSTSRDGEELQKMASDVLLLSDKVSSLVSSGNNSEVGSMYKTIEEL ERKLYHPLSITLLHTRETLLKIYMELQDWQTALMYCRLTIPVYERIYPPFHPMIGLQFYT CGKLEWLLEYTEDALMSLTRAADILRITHGTKSEFMKELLGKLEEVRAEASFRLSAGDEQ

[0204] EXAMPLE 2

[0205] Expression and formulation of SuSy-0362 variant (SEQ ID NO: 9)

[0206] The gene coding for the SuSy-0362 variant (SEQ ID NO: 9) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.

[0207] Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / 1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.2 mM) and carried out at 30°C and 200 rpm for 16-18 hours.

[0208] Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD600)) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl₂, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with distilled water, resulting in an enzymatic active preparation.

[0209] The activity of the SuSy-0362 variant is quantified in Units, which is defined as follows: 1 unit (U) of the enzyme produces 1 micromole (μmol) of fructose from 400 mM sucrose and 15 mM UDP at 30 °C.

[0210] EXAMPLE 3

[0211] Expression and formulation of UGTS1-0728 variant (SEQ ID NO: 7)

[0212] The gene coding for the UGTS1-0728 variant (SEQ ID NO: 7) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.

[0213] Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / 1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours.

[0214] Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD600)) with cell lysis buffer (100 mM Tris-HC1 pH 7.0; 2 mM MgCh, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

[0215] The activity of the UGTS1-0728 variant is quantified in Units, which is defined as follows: 1 milliunit (mU) of the enzyme converts 1 nanomole (nmol) of Rebaudioside A (Reb A) per minute to Rebaudioside D (Reb £>) at 30 °C. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 6.5, 10 mM Reb A at to, 440 mM sucrose, 2 mM MgCh, 0.25 mM uridine diphosphate (UDP) and 0.6 U / mL of SuSy.

[0216] EXAMPLE 4

[0217] Expression and formulation of UGTSr-0588 variant (SEQ ID NO: 4)

[0218] The gene coding for the UGTSr-0588 variant (SEQ ID NO: 4) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / 1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours.

[0219] Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD600)) with cell lysis buffer (100 mM Tris-HC1 pH 7.0; 2 mM MgCh, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

[0220] The activity of the UGTSr-0588 variant is quantified in Units, which is defined as follows: 1 milliunit (mU) of the enzyme converts 1 nanomole (nmol) of Rebaudioside D (Reb £>) per minute to Rebaudioside M (Reb M) at 30 °C. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 6.5, 0.5 mM Reb D at t₀, 440 mM sucrose, 2 mM MgCl₂, 0.25 mM uridine diphosphate (UDP) and 150 mU / mL of SuSy.

[0221] EXAMPLE 5

[0222] Expression and formulation of SrUGTl-0360 variant (SEQ ID NO: 5)

[0223] The gene coding for the SrUGTl-0360 variant (SEQ ID NO: 5) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.

[0224] Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / 1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours.

[0225] Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD600)) with cell lysis buffer (100 mM Tris-HC1 pH 7.0; 2 mM MgCh, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

[0226] The activity of the SrUGTl-0360 variant is quantified in Units, which is defined as follows: 1 milliunit (mU) of the enzyme converts 1 nanomole (nmol) of stevioside perminute to Rebaudioside A (Reb A) at 30 °C. Reaction conditions for the assay are 30 °C, 50 mM potassium phosphate buffer pH 6.5, 1 mM Stevioside at t₀, 440 mM sucrose, 2 mM MgCl₂, 0.25 mM uridine diphosphate (UDP) and 150 mU / mL of SuSy.

[0227] EXAMPLE 6

[0228] Expression and formulation of SrUGT2-0125 variant (SEQ ID NO: 6)

[0229] The gene coding for SrUGT2-0125 variant (SEQ ID NO: 6) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.

[0230] Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / 1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours.

[0231] Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD600)) with cell lysis buffer (100 mM Tris-HC1 pH 7.0; 2 mM MgCh, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

[0232] The activity of the SrUGT2-0125 variant is quantified in Units, which is defined as follows: 1 milliunit (mU) of the enzyme converts 1 nanomole (nmol) of Rebaudioside B (Reb B) per minute to Rebaudioside A (Reb A) at 30 °C. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 6.5, 0.5 mM Reb B at t₀, 440 mM sucrose, 2 mM MgCl₂, 0.25 mM uridine diphosphate (UDP) and 150 mU / mL of SuSy.

[0233] EXAMPLE 7

[0234] Synthesis of Rebaudioside M in a “one-pot” reaction with ML02, adding SuSy-0362, UGTS1-0728, SrUGTl-0360, SrUGT2-0125 and UGTSr-0588 at the same time.

[0235] Rebaudioside M was synthesized from mother liquor-02 (ML02) produced by PureCircle Malaysia (Table 2) in a “one-pot” reaction, utilizing the five enzymes (see EXAMPLES 2, 3, 4, 5 and 6): SuSy-0362 (SEQ ID NO: 9), UGTS1-0728 (SEQ ID NO: 7),SrUGTl-0360 (SEQ ID NO: 5), SrUGT2-0125 (SEQ ID NO: 6) and UGTSr-0588 (SEQ ID NO: 4).

[0236] Table 2

[0237] The steviol glycoside content (%wt / wt) in ML02

[0238] Steviol glycoside in

[0239] Content (%wt / wt)

[0240] ML02

[0241] Rebaudioside E 0.04

[0242] Rebaudioside D 1.30

[0243] Rebaudioside N 0.56

[0244] Rebaudioside M 0.94

[0245] Rebaudioside O 0.88

[0246] Rebaudioside A 58.94

[0247] Stevioside 15.32

[0248] Rebaudioside F 1.96

[0249] Rebaudioside C 10.06

[0250] Dulcoside A 0.23

[0251] Rubusoside 0.64

[0252] Rebaudioside B 7.65

[0253] Steviolbioside 1.50

[0254]

[0255] The final reaction solution contained approximately 1800 U / L UGTS1-0728, 2159 U / L SuSy-0362, 23 U / L UGTSr-0588, 286 U / L SrUGTl-0360, 407 U / L SrUGT2-0125, 100 g / L of ML02, 0.75 mM of uridine diphosphate (UDP), 600 mM sucrose, 0.50 mM MgCl₂ and 50 mM potassium phosphate buffer (pH 6.50). First, 700 mL of distilled water were mixed with 0.10 g MgCl₂•6H₂O, 205.38 g sucrose, 50 mL of 1 M potassium phosphate buffer (pH 6.5) and 100 g ML2. The final volume of the reaction mixture was adjusted to 1000 mL.

[0256] After dissolving the components, the temperature was adjusted to 45 °C and the pH was adjusted to pH 6.50 by 17% phosphoric acid / potassium hydroxide. Then, UGTS1-0728, SuSy-0362, UGTSr-0588, SrUGTl-0360, SrUGT2-0125 and 0.32 g of UDP-Na2were added. The reaction mixture was incubated at 45°C for 48 hours with agitation.

[0257] Once the reaction reached completion, the reaction mixture was adjusted to pH 5.5, heated to 100 °C for 5 mins to inactivate the enzymes, filtered through a cardboard filter,and subjected to crystallization to separate Rebaudioside M. The crystals were separated by filtration and dried in vacuum oven for 24 hours. The purity of the sample was evaluated by HPLC method described in EXAMPLE 11. Rebaudioside M was synthesized at 97% purity and 95% yield. The yield was calculated as a percentage based on the weight of isolated and purified Rebaudioside M crystals relative to the weight of all starting steviol glycosides used.

[0258] EXAMPLE 8

[0259] Synthesis of Rebaudioside M in a “one-pot” reaction with High Stevioside Extract (HSE), adding SuSy-0362, UGTS1-0728, SrUGTl-0360, SrUGT2-0125 and UGTSr-0588 at the same time.

[0260] Rebaudioside M was synthesized from High Stevioside Extract (HSE) produced by PureCircle Malaysia (Table 3) in a “one-pot” reaction, utilizing the five enzymes (see EXAMPLES 2, 3, 4, 5 and 6): SuSy-0362 (SEQ ID NO: 9), UGTS1-0728 (SEQ ID NO: 7), SrUGTl-0360 (SEQ ID NO: 5), SrUGT2-0125 (SEQ ID NO: 6) and UGTSr-0588 (SEQ ID NO: 4).

[0261] Table 3

[0262] The steviol glycoside content (%wt / wt) in HSE

[0263] Steviol glycoside in

[0264] Content (%wt / wt)

[0265] HSE

[0266] Rebaudioside E 1.57

[0267] Rebaudioside AM 0.22

[0268] Rebaudioside I 0.07

[0269] Rebaudioside A 0.59

[0270] Stevioside 86.15

[0271] Rebaudioside F 0.04

[0272] Rebaudioside C 0.33

[0273] Dulcoside A 9.42

[0274] Rubusoside 1.17

[0275] Steviolbioside 0.45

[0276]

[0277] The final reaction solution contained approximately 1800 U / L UGTS1-0728, 2159 U / L SuSy-0362, 23 U / L UGTSr-0588, 286 U / L SrUGTl-0360, 407 U / L SrUGT2-0125, 50 g / L of HSE, 0.75 mM uridine diphosphate (UDP), 600 mM sucrose, 0.5 mM MgCl₂ and 50 mM potassium phosphate buffer (pH 6.50). First, 700 mL of distilled water were mixed with 0.10 g MgCl₂•6H₂O, 205.38 g sucrose, 50 mL of 1 M potassium phosphate buffer (pH 6.5) and 50 g HSE. The final volume of the reaction mixture was adjusted to 1000 mL.

[0278] After dissolving the components, the temperature was adjusted to 45 °C and the pH was adjusted to pH 6.60 by 17% phosphoric acid / potassium hydroxide. Then, UGTS1-0728, SuSy-0362, UGTSr-0588, SrUGTl-0360, SrUGT2-0125 and 0.32 g of UDP were added. The reaction mixture was incubated at 45°C for 48 hours with agitation.

[0279] Once the reaction reached completion, the reaction mixture was adjusted to pH 5.5, heated to 100 °C for 5 mins to inactivate the enzymes, filtered through a cardboard filter, and subjected to crystallization to separate Rebaudioside M. The crystals were separated by filtration and dried in vacuum oven for 24 hours. The purity of the sample was evaluated by HPLC method described in EXAMPLE 11. Rebaudioside M was synthesized at 92% purity and 87% yield. The yield was calculated as a percentage based on the weight of isolated and purified Rebaudioside M crystals relative to the weight of all starting steviol glycosides used.

[0280] EXAMPLE 9

[0281] Synthesis of Rebaudioside M in a “one-pot” reaction with High Rebaudioside B material (SGB), adding SuSy-0362, UGTS1-0728, SrUGTl-0360, SrUGT2-0125 and UGTSr-0588 at the same time.

[0282] Rebaudioside M was synthesized from High Rebaudioside B material (SGB) produced by PureCircle Malaysia (Table 4) in a “one-pot” reaction, utilizing the five enzymes (see EXAMPLES 2, 3, 4, 5 and 6): SuSy-0362 (SEQ ID NO: 9), UGTS1-0728 (SEQ ID NO: 7), SrUGTl-0360 (SEQ ID NO: 5), SrUGT2-0125 (SEQ ID NO: 6) and UGTSr-0588 (SEQ ID NO: 4).

[0283] Table 4

[0284] The steviol glycoside content (%wt / wt) in SGB

[0285] Steviol glycoside in Content

[0286]

[0287] SGB (%wt / wt)Rebaudioside D 0.16

[0288] Rebaudioside M 0.12

[0289] Rebaudioside A 0.08

[0290] Stevioside 0.03

[0291] Rebaudioside B 88.59

[0292] Steviolbioside 1.04

[0293]

[0294] The final reaction solution contained approximately 1800 U / L UGTS1-0728, 2159 U / L SuSy-0362, 23 U / L UGTSr-0588, 550 U / L SrUGTl-0360, 407 U / L SrUGT2-0125, 50 g / L of SGB, 0.75 mM uridine diphosphate (UDP), 600 mM sucrose, 0.5 mM MgCl₂ and 50 mM potassium phosphate buffer (pH 6.50). First, 700 mL of distilled water were mixed with 0.10 g MgCl₂•6H₂O, 205.4 g sucrose, 50 mL of 1 M potassium phosphate buffer (pH 6.50) and 50 g SGB. The final volume of the reaction mixture was adjusted to 1000 mL.

[0295] After dissolving the components, the temperature was adjusted to 45 °C and the pH was adjusted to pH 6.6 by 17% phosphoric acid / potassium hydroxide. Then, UGTS1-0728, SuSy-0362, UGTSr-0588, SrUGTl-0360, SrUGT2-0125 and 0.32 g of UDP were added. The reaction mixture was incubated at 45 °C for 48 hours with agitation.

[0296] Once the reaction reached completion, the reaction mixture was adjusted to pH 6.6, heated to 100 °C for 5 mins to inactivate the enzymes, filtered through a cardboard filter, and subjected to crystallization to separate Rebaudioside M. The crystals were separated by filtration and dried in vacuum oven for 24 hours. The purity of the sample was evaluated by HPLC method described in EXAMPLE 11. Rebaudioside M was synthesized at 93% purity at 96% yield. The yield was calculated as a percentage based on the weight of isolated and purified Rebaudioside M crystals relative to the weight of all starting steviol glycosides used.

[0297] EXAMPLE 10

[0298] Synthesis of Rebaudioside M in a “one-pot” reaction with ML02, adding SuSy-0362, UGTS1-0728, SrUGTl-0360, SrUGT2-0125, UGTSr-0588, EcUMPK-001 at the same time.

[0299] Rebaudioside M was synthesized from mother liquor-02 (ML02) produced by PureCircle Malaysia (Table 5) in a “one-pot” reaction, utilizing the six enzymes (see EXAMPLES 2, 3, 4, 5 and 6): SuSy-0362 (SEQ ID NO: 9), UGTS1-0728 (SEQ ID NO: 7),SrUGTl-0360 (SEQ ID NO: 5), SrUGT2-0125 (SEQ ID NO: 6), UGTSr-0588 (SEQ ID NO: 4) and UMP-kinase (EcUMPK-001 produced by c-LEcta GmbH).

[0300] Table 5

[0301] The steviol glycoside content (%wt / wt) in ML02

[0302] Steviol glycoside in

[0303] Content (%wt / wt)

[0304] ML02

[0305] Rebaudioside E 0.04

[0306] Rebaudioside D 1.30

[0307] Rebaudioside N 0.56

[0308] Rebaudioside M 0.94

[0309] Rebaudioside O 0.88

[0310] Rebaudioside A 58.94

[0311] Stevioside 15.32

[0312] Rebaudioside F 1.96

[0313] Rebaudioside C 10.06

[0314] Dulcoside A 0.23

[0315] Rubusoside 0.64

[0316] Rebaudioside B 7.65

[0317] Steviolbioside 1.50

[0318]

[0319] The final reaction solution contained approximately 1800 U / L UGTS1-0728, 2159 U / L SuSy-0362, 23 U / L UGTSr-0588, 550 U / L SrUGTl-0360, 407 U / L SrUGT2-0125, 200 mU / mL EcUMPK-001, 100 g / L of ML02, 1.0 mM of uridine monophosphate (UMP), 1.0 mM adenosine triphosphate (ATP), 700 mM sucrose, 0.5 mM MgCl₂ and 50 mM potassium phosphate buffer (pH 6.5). First, 700 mL of distilled water were mixed with 101.66 mg MgCl₂•6H₂O, 239.61 g sucrose, 50 mL of 1 M potassium phosphate buffer (pH 6.5) and 100 g ML2. The final volume of the reaction mixture was adjusted to 1000 mL.

[0320] After dissolving the components, the temperature was adjusted to 45 °C and the pH was adjusted to pH 6.55 by 17% phosphoric acid / potassium hydroxide. Then, UGTS1-0728, SuSy-0362, UGTSr-0588, SrUGTl-0360, SrUGT2-0125, EcUMPK-001, 0.46 g of UMP-Na2 and 0.60 of ATP-Na2 were added. The reaction mixture was incubated at 45°C for 48 hours with agitation.Once the reaction reached completion, the reaction mixture was adjusted to pH 5.5, heated to 100 °C for 5 mins to inactivate the enzymes, filtered through a cardboard filter, and subjected to crystallization to separate Rebaudioside M. The crystals were separated by filtration and dried in vacuum oven for 24 hours. The purity of the sample was evaluated by HPLC method described in EXAMPLE 11. Rebaudioside M was synthesized at 91% purity and 94% yield. The yield was calculated as a percentage based on the weight of isolated and purified Rebaudioside M crystals relative to the weight of all starting steviol glycosides used.

[0321] EXAMPLE 11

[0322] HPLC analysis

[0323] For analysis, biotransformation samples were inactivated by adjusting the pH to pH5.5 using 17% H₃PO₄ and then boiled for 10 minutes. Resulting samples were filtered, the filtrates were diluted 10 times and used as samples for HPLC analysis. HPLC assay was carried out on Agilent HP 1200 HPLC system, comprised of a pump, a column thermostat, an auto sampler, a UV detector capable of background correction and a data acquisition system. Analytes were separated using Agilent Poroshell 120 SB- C18, 4.6 mm x 150 mm, 2.7 pm at 40°C. The mobile phase consisted of two premixes:

[0324] premix 1 containing 75% 10 mM phosphate buffer (pH2.6) and 25% acetonitrile, and

[0325] premix 2 containing 68% 10 mM phosphate buffer (pH2.6) and 32% acetonitrile. Elution gradient started with premix 1, changed to premix 2 to 50% at 12.5 minute, changed to premix 2 to 100% at 13 minutes. Total run time was 45 minutes. The column temperature was maintained at 40 °C. The injection volume was 5 pL. Steviol glycoside species were detected by UV at 210 nm.

[0326] Having described the invention and referenced specific embodiments, it is apparent that modifications and variations can be made without departing from the scope defined in the appended claims. Specifically, while certain aspects of the invention are highlighted, it is understood that the invention is not necessarily limited to these specific aspects.

Claims

CLAIMSWe claim:

1. A method for producing Rebaudioside M comprising the steps of:a) providing a starting steviol glycoside composition comprising at least one steviol glycoside selected from the list of Rebaudioside A, Rebaudioside B. Rebaudioside C, Rebaudioside E, Rebaudioside F, Rebaudioside G, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside or combinations thereof;b) providing an enzyme preparation or microorganism containing a NDP-recycling enzyme and, optionally a NDP-synthesizing enzyme, and at least two enzymes selected from the group of nucleotide diphosphate-dependent glycosyltransferase (NGT)-enzymes;c) contacting the enzyme preparation or microorganism with a medium containing the starting steviol glycoside composition to produce Rebaudioside M.

2. The method according to claim 1, wherein the NGT-enzymes are selected from the group of UGT74G1, UGT76G1, UGTS12 or mixtures thereof, wherein the UGT76G1 preferably is a SrUGTl -enzyme and / or UGTSr-enzyme and the UGT74G1 preferably is a SrUGT2-enzyme.

3. The method according to claim 1, wherein the NDP-recycling enzyme is sucrose synthase.

4. The method according to any one of the preceding claims, wherein the NDP- synthesizing enzyme is a UMP-kinase.

5. The method according to any one of the preceding claims, whereina) the at least two enzymes are UGTSr, UGTS12 and SrUGT2 and the starting composition comprises Rebaudioside B;b) the at least two enzymes are UGTSr, UGTS12 and SrUGTl and the starting composition comprises Stevioside, Rubusoside or a mixture thereof;c) the at least two enzymes are UGTSr, UGTS12, SrUGTl and SrUGT2 and the starting composition comprises Steviolbioside, Steviolmonoside, or a mixture thereof;d) the at least two enzymes are UGTSr, UGTS12, SrUGTl, the starting composition comprises Dulcoside A and a rhamnosidase is added;e) the at least two enzymes are UGTS12, UGTSr, the starting composition comprises Rebaudioside C and a rhamnosidase is added;f) the at least two enzymes are UGTS12, UGTSr, the starting composition comprises Rebaudioside F and a xylosidase is added;g) the at least two enzymes are UGTSr and UGTS12 and the starting composition comprises Rebaudioside G, Rebaudioside A or a mixture thereof; and / or h) the at least two enzymes are UGTSr and SrUGT1 and the starting composition comprises Rebaudioside E.

6. The method according to any one of the preceding claims, whereina) the SrUGTl-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 5, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical; b) the SrUGT2-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 6, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical; c) the UGTS12-enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 7, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical; d) the UGTSr-enzyme characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 4, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical; and / or e) the NDP-recycling enzyme is characterized in that it has at least 95% sequence identity or is 95% identical to SEQ ID NO: 9, preferably at least 98% sequence identity or 98% identical, or more preferably at least 99% sequence identity or 99% identical.

7. The method according to any one of the preceding claims, wherein starting composition comprises at least one steviol glycoside selected from the list of Rebaudioside B, Rebaudioside C, Rebaudioside F, Dulcoside A, Stevioside, Rubusoside, Steviolmonoside, Steviolbioside, or mixtures thereof, and more preferably Rebaudioside B, Rebaudioside C, Dulcoside A, Stevioside, Steviolbioside, or mixtures thereof.

8. The method according to any one of the preceding claims, wherein the enzyme preparation or microorganism further contains an enzyme selected from the group consisting of UGT91D2 (SEQ ID NO: 10), UGT85C2 (SEQ ID NO: 11), EUGT11 SEQ (ID NO: 15), or variants or mixtures thereof.

9. The method according to any one of the preceding claims, wherein the starting steviol glycoside composition is a by-product of processes to produce steviol glycosides.

10. The method according to any one of the preceding claims, wherein the obtained Rebaudioside M has a purity of greater than 90%, preferably greater than 95%, and more preferably greater than 98%.

11. The method according to any one of the preceding claims, wherein the Rebaudioside M is obtained in a yield of greater than 85%, preferably more than 90%.

12. The method according to any one of the preceding claims, wherein the microorganism is selected from the from the group consisting of E. coli, Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., Pseudomonas spp., Saccharomyces spp., Aspergillus spp., Pichia spp., Bacillus spp., Penicillium spp., Trichoderma spp. and Yarrowia spp.

13. The method according to any one of the preceding claims, wherein the microorganism is a recombination microorganism.

14. The method according to any one of the preceding claims 1-12, wherein the enzyme preparation is provided in the form of a whole cell suspension, a crude lysate, a supernatant, or a purified enzyme preparation or a combination thereof.

15. The method according to claim 14, wherein the enzyme preparation is in free form or immobilized to a solid support made from inorganic or organic materials.

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