UDP-glycosyltransferase

Abstract

The present invention relates to a polypeptide having UDP-glycosyltransferase activity, particularly UGT2 activity, and having at least 85% identity to an amino acid sequence set forth in one of SEQ ID NOs: 1, 3, 6, 9, 11, 14, 17, 20, 22, and 25. The present application also relates to a recombinant host comprising a recombinant nucleic acid sequence encoding the polypeptide and the use thereof in preparing glycosylated diterpenes such as steviol glycosides. The host cell may further comprise enzymes of the steviol glycoside biosynthesis pathway.

Description

[0001] This application is a divisional application of Chinese invention patent application 201680016548.2 (i.e. PCT / EP2016 / 055734) filed on March 16, 2016. Field of the Invention

[0002] The present invention relates to a recombinant host comprising a recombinant nucleic acid sequence encoding a variant UDP-glycosyltransferase (UGT) polypeptide. The present invention also relates to a method for preparing a glycosylated diterpene using such a recombinant host, and to a fermentation broth obtainable from such a method. The present invention also relates to a glycosylated diterpene obtained by such a method or obtainable from such a fermentation broth, and to a composition comprising two or more such glycosylated diterpenes. In addition, the present invention relates to a food, feed, or beverage comprising such a glycosylated diterpene or such a composition. The present invention also relates to a method for converting a first glycosylated diterpene into a second glycosylated diterpene using such a recombinant host. In addition, the present invention relates to variant UGT polypeptides, nucleic acid sequences encoding such polypeptides, nucleic acid constructs comprising such polynucleotide sequences, and a method for producing variant UGT polypeptides using such a recombinant host. Background of the Invention

[0003] The leaves of the perennial herb Stevia rebaudiana Bert. accumulate large amounts of intensely sweet compounds called steviol glycosides. Although the biological functions of these compounds are still unclear, they are of commercial interest as alternative high-potency sweeteners.

[0004] The functional and sensory properties of these sweet steviol glycosides appear to be superior to those of many high-potency sweeteners. In addition, studies have shown that steviosides can lower blood sugar levels in patients with type 2 diabetes and can lower blood pressure in patients with mild hypertension.

[0005] Steviol glycosides accumulate in stevia leaves, where they can comprise 10% to 20% of the leaf's dry weight. Both stevioside and rebaudioside A are heat- and pH-stable and suitable for use in carbonated beverages and many other foods. Stevioside is between 110 and 270 times sweeter than sucrose, and rebaudioside A is between 150 and 320 times sweeter than sucrose. Rebaudioside D is also a highly potent diterpene glycoside sweetener that accumulates in stevia leaves. It can be approximately 200 times sweeter than sucrose. Rebaudioside M is another highly potent diterpene glycoside sweetener. It is present in trace amounts in the leaves of some stevia varieties but has been shown to have an excellent taste profile.

[0006] Traditionally, steviol glycosides have been extracted from the stevia plant. In stevia, (-)-kaurenic acid (an intermediate in the biosynthesis of gibberellic acid (GA)) is converted into the tetracyclic diterpene steviol, which is then subjected to a multi-step glycosylation pathway to form various steviol glycosides. However, yields can be variable and are affected by agricultural and environmental conditions. In addition, stevia cultivation requires large amounts of land, long periods before harvest, intensive labor, and the additional costs of extracting and purifying glycosides.

[0007] Recently, there has been growing interest in the production of steviol glycosides using fermentation processes. WO2013 / 110673 and WO2015 / 007748 describe microorganisms that can be used to produce at least the steviol glycosides rebaudioside A and rebaudioside D.

[0008] Further improvement of such microorganisms is desirable so that higher amounts of steviol glycosides and / or additional or novel steviol glycosides and / or higher amounts of specific steviol glycosides and / or mixtures of steviol glycosides having desired ratios of different steviol glycosides can be produced. SUMMARY OF THE INVENTION

[0009] In Stevia, steviol is synthesized from GGPP, which is formed by the deoxyxylulose 5-phosphate pathway. The activity of two diterpene cyclases, (-)-copalyl diphosphate synthase (CPS) and (-)-kaurene synthase (KS), leads to the formation of (-)-kaurene, which is then oxidized by (-)-kaurene oxidase (KO) in a three-step reaction to form (-)-kaurenic acid.

[0010] In the Stevia leaf, (-)-kaurenoic acid is then hydroxylated by ent-kaurenoic acid 13-hydroxylase (KAH) to form steviol. Steviol is then glycosylated by a series of UDP-glycosyltransferases (UGTs) to form a number of steviol glycosides. Specifically, these molecules can be viewed as steviol molecules with their carboxyl hydrogen atoms replaced by glucose molecules to form esters, and their hydroxyl hydrogen atoms replaced by a combination of glucose and rhamnose to form acetals.

[0011] These pathways can be reconstituted in recombinant hosts such as yeast, eg, Saccharomyces and Yarrowia.

[0012] The present invention relates to the identification of novel variant UDP-glycosyltransferase (UGT) polypeptides that generally have improved properties compared to those currently known. These polypeptides can be used to generate recombinant hosts that produce higher amounts of steviol glycosides and / or additional or novel steviol glycosides and / or higher amounts of specific steviol glycosides and / or mixtures of steviol glycosides having desired ratios of different steviol glycosides.

[0013] Therefore, the present invention also relates to a recombinant host capable of producing glycosylated diterpenes (i.e., diterpene glycosides such as steviol glycosides), such as steviol monoside, steviol bioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rubusoside, dulcoside A, steviol-13-monoside, steviol-19-monoside or 13-[(β-D-glucopyranosyl)oxy) kauri-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester steviol-19-bioside,

[0014] Therefore, the present invention relates to a recombinant host comprising a recombinant nucleic acid sequence, typically having UDP-glycosyltransferase (UGT) activity, such as UGT2 activity, encoding a polypeptide, wherein:

[0015] a. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 1;

[0016] b. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 3;

[0017] c. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 6;

[0018] d. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 9;

[0019] e. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 11;

[0020] f. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 14;

[0021] g. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 17;

[0022] h. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 20;

[0023] i. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 22; or

[0024] j. is at least about 85% identical to the amino acid sequence set forth in SEQ ID NO: 25.

[0025] The present invention also relates to:

[0026] - a method for preparing a glycosylated diterpene, said method comprising fermenting a recombinant host of the present invention in a suitable fermentation medium and optionally recovering the glycosylated diterpene;

[0027] - a fermentation broth comprising the glycosylated diterpenes obtainable by the method of the present invention;

[0028] - a glycosylated diterpene obtained by this method or obtainable from such a fermentation broth;

[0029] - a composition comprising two or more such diterpenes;

[0030] – a food, feed or beverage comprising such a glycosylated diterpene;

[0031] A method for converting a first glycosylated diterpene into a second glycosylated diterpene, the method comprising:

[0032] - contacting the first glycosylated diterpene with a recombinant host of the present invention, a cell-free extract derived from such a recombinant host, or an enzyme preparation derived from either of the foregoing;

[0033] - thereby converting the first glycosylated diterpene into the second glycosylated diterpene.

[0034] - A polypeptide having UGT2 activity, wherein the polypeptide is selected from the group consisting of:

[0035] (a) a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22 or 25; or

[0036] (b) a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of any one of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22, or 25; or

[0037] (c) a polypeptide encoded by a polynucleotide comprising the polynucleotide sequence set forth in any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0038] (d) a polypeptide encoded by a polynucleotide comprising a polynucleotide sequence having at least 50% sequence identity to the polypeptide coding sequence in any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0039] (e) a polypeptide encoded by a polynucleotide that hybridizes, preferably under at least low stringency conditions, to the complementary strand of any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0040] (f) a polypeptide encoded by a polynucleotide that hybridizes, preferably under at least low stringency conditions, to the complementary strand of a polynucleotide having at least 50% sequence identity to any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0041] (g) A fragment of a polypeptide as defined in (a), (b), (c), (d), (e) or (f).

[0042] - a polynucleotide sequence encoding the polypeptide;

[0043] - a nucleic acid construct comprising such a polynucleotide sequence; and

[0044] - A method for producing a polypeptide of the present invention, the method comprising:

[0045] (a) cultivating the host cell under conditions conducive to production of the polypeptide by the recombinant host of the present invention, and optionally

[0046] (b) recovering the polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A schematic representation of plasmid pUG7-EcoRV is shown.

[0048] Figure 2 Schematic representation of the method for (A) design of ERG20, tHMG1 and BTS1 overexpression cassettes and (B) integration into the yeast genome. (C) The final situation after removal of the KANMX marker by Cre recombinase is shown.

[0049] Figure 3 A schematic diagram of the ERG9 knockdown construct is shown. The construct consists of a 500 bp 3' portion of ERG9, 98 bp of the TRP1 promoter, the TRP1 open reading frame and terminator, followed by a 400 bp downstream sequence of ERG9. Due to the introduction of an XbaI site at the end of the ERG9 open reading frame, the last amino acid is changed to Ser, and the stop codon is changed to Arg. The new stop codon is located in the TRP1 promoter, resulting in an 18-amino acid extension.

[0050] Figure 4Schematic representation showing how UGT2 is integrated into the genome. A. Different fragments used in transformation; B. Situation after integration; C. Situation after Cre recombinase expression).

[0051] Figure 5 Schematic representation showing how the pathway from GGPP to steviol is integrated into the genome. A. Different fragments used in transformation; B. Situation after integration.

[0052] Figure 6 Shown is the pSH65 plasmid carrying the CRE gene, which was used to remove the antibiotic marker.

[0053] Figure 7 A map of plasmid pUG7-NAT is shown.

[0054] Figure 8 Replacement of UGT2_1a from STV008 with the Nat selection marker is shown.

[0055] Figure 9 The removal of the NAT tag from STV008 is shown.

[0056] Figure 10 Integration of the UGT2 gene at the Chr09.01 locus is shown.

[0057] Figure 11 Shown is the production of rebaudioside A in Saccharomyces strains carrying different variants of UGT2.

[0058] Figure 12 The production of rebaudioside M in Saccharomyces strains expressing different variants of UGT2 is shown as a percentage of the rebaudioside M production in the Saccharomyces strain expressing UGT2_1a.

[0059] Figure 13 A map of plasmid MB6969 carrying the genes tHMG and UGT2_1a is shown.

[0060] Figure 14 A map of plasmid MB6856 carrying the gene tHMG is shown.

[0061] Figure 15 A map of plasmid MB6857 carrying the gene tHMG is shown.

[0062] Figure 16 A map of plasmid MB6948 carrying the gene GGS is shown.

[0063] Figure 17 A map of plasmid MB6958 carrying the gene GGS is shown.

[0064] Figure 18A map of plasmid MB7015 carrying the genes UGT1, UGT3 and UGT4 is shown.

[0065] Figure 19 A map of plasmid MB6986 carrying the genes tHMG and GGS is shown.

[0066] Figure 20 Shown is a map of plasmid MB7059 carrying the genes tCPS_SR, tKS_SR, KAH_4, KO_Gib and CPR_3.

[0067] Figure 21 A map of plasmid MB7100 carrying the genes tCPS_SR, tKS_SR, KAH_4, KO_Gib and CPR_3 is shown.

[0068] Figure 22 A map of plasmid MB6988 carrying the genes tHMG and GGS is shown.

[0069] Figure 23 A map of plasmid MB7044 carrying the genes tCPS_SR, tKS_SR, KAH_4, KO_Gib and CPR_3 is shown.

[0070] Figure 24 A map of plasmid MB7094 carrying the genes tCPS_SR, tKS_SR, KAH_4, KO_Gib and CPR_3 is shown.

[0071] Figure 25 Shown is a map of plasmid MB6128 carrying the CRE gene, which was used to remove the antibiotic marker.

[0072] Figure 26 The method is shown for assembly in a plasmid of the genes UGT2, KanMX, UGT1 and KAH_4 flanked by gsy1 integration flanks.

[0073] Figure 27 Shown Figure 26 Methods for amplifying and transforming plasmids into Yarrowia.

[0074] Figure 28 Shown is the production of rebaudioside A in Yarrowia strains expressing different variants of UGT2.

[0075] Figure 29 Shown is the production of rebaudioside M in Yarrowia strains expressing different variants of UGT2 as a percentage of the rebaudioside M production in the Yarrowia strain expressing UGT2_1a.

[0076] Figure 30Shown are the production of RebA (upper panel) and RebM (lower panel) in strains expressing UGT2_6b (left panel) or UGT2_7b (right panel).

[0077] Figure 31 A schematic representation of potential pathways leading to the biosynthesis of steviol glycosides is shown.

[0078] Figure 32 Shown is a schematic representation of the potential pathways leading to the biosynthesis of steviol glycosides. The compound shown with an asterisk is 13-[(β-D-glucopyranosyl)oxy]kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester.

[0079] Description of the Sequence Listing

[0080] Descriptions of the sequences are listed in Table 13. The sequences described herein may be defined by reference to the Sequence Listing or by reference to any of the database accession numbers listed herein, for example in Table 13. Detailed Description of the Invention

[0081] In this specification and the appended claims, the words "comprises," "comprising," and "having" and variations thereof should be construed as inclusive. That is, these words are intended to convey that other elements or integers not specifically recited may be included if the context permits.

[0082] When used without a quantifier, it is used herein to refer to one or more than one (ie, one or at least one) of the grammatical object. For example, "element" can mean one element or more than one element.

[0083] Herein, "rebaudioside" may be abbreviated as "reb." That is, for example, rebaudioside A and rebA are intended to refer to the same molecule.

[0084] The term "recombinant" when used in reference to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all. The term "recombinant" is synonymous with "genetically modified."

[0085] The present invention relates to novel variant polypeptides having UDP-glycosyltransferase (UGT) activity. For the purposes of the present invention, a polypeptide having UGT activity is a polypeptide having glycosyltransferase activity (EC 2.4), i.e., a polypeptide that acts as a catalyst for the transfer of a monosaccharide unit from an activated nucleotide sugar (also referred to as a "glycosyl donor") to a glycosyl acceptor molecule (typically an alcohol). The glycosyl donor for a UGT is typically the nucleotide sugar uridine diphosphate glucose (UDP-glucose). The polypeptides of the present invention typically have UGT activity, and the polynucleotide sequences of the present invention typically encode such polypeptides. Typically, the polypeptides of the present invention are variant polypeptides having UGT type 2 activity.

[0086] According to the present invention, there is therefore provided a polypeptide, typically a polypeptide having UGT activity, wherein the polypeptide is selected from the group consisting of:

[0087] (a) a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22 or 25; or

[0088] (b) a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of any one of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22, or 25; or

[0089] (c) a polypeptide encoded by a polynucleotide comprising the polynucleotide sequence set forth in any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0090] (d) a polypeptide encoded by a polynucleotide comprising a polynucleotide sequence having at least 50% sequence identity to the polypeptide coding sequence in any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0091] (e) a polypeptide encoded by a polynucleotide that hybridizes, preferably under at least low stringency conditions, to the complementary strand of any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0092] (f) a polypeptide encoded by a polynucleotide that hybridizes, preferably under at least low stringency conditions, to the complementary strand of a polynucleotide having at least 50% sequence identity to any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0093] (g) A fragment of a polypeptide as defined in (a), (b), (c), (d), (e) or (f).

[0094] Such a polypeptide may comprise an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22, or 25.

[0095] Therefore, the present invention relates to:

[0096] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 1;

[0097] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO 3;

[0098] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 6;

[0099] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 9;

[0100] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 11;

[0101] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 14;

[0102] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 17;

[0103] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 20;

[0104] - a polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO: 22; and

[0105] - A polypeptide generally having UGT activity, the polypeptide comprising an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 25.

[0106] As used herein, the term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. Amino acids are identified by single-letter or three-letter names. As used herein, the term "protein" is synonymous with the term "polypeptide" and may also refer to two or more polypeptides. Thus, the terms "protein," "peptide," and "polypeptide" are used interchangeably. A polypeptide may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, etc.) to increase functionality. A polypeptide that exhibits activity may be referred to as an enzyme. It will be understood that, as a result of the degeneracy of the genetic code, many nucleotide sequences encoding a given polypeptide may be generated.

[0107] The polypeptides of the present invention may comprise a signal peptide and / or a propeptide sequence. In the case where the polypeptides of the present invention comprise a signal peptide and / or a propeptide, sequence identity may be calculated on the mature polypeptide sequence.

[0108] The polypeptides of the present invention typically have UGT activity, and more preferably have UGT2 activity. Figure 31 and 32 A non-exhaustive list of reactions that can be catalyzed by polypeptides having UGT2 is shown.

[0109] A polypeptide having UGT2 activity is a polypeptide that functions as a uridine 5'-diphosphoglucosyl:steviol-13-O-glycosyltransferase (also known as steviol-13-monoglucosyl 1,2-transglucosylase), which transfers a glucose moiety to the C-2' of the 13-O-glucose of the acceptor molecule steviol-13-O-glycoside. Typically, a suitable UGT2 polypeptide also functions as a uridine 5'-diphosphoglucosyl:rubusoside transferase, which transfers a glucose moiety to the C-2' of the acceptor molecule rubusoside 13-O-glucose.

[0110] A polypeptide having UGT2 activity can also catalyze reactions that utilize steviol glycoside substrates other than steviol-13-O-glycoside and rubusoside. For example, a functional UGT2 polypeptide can utilize stevioside as a substrate, thereby transferring the glucose moiety to the C-2' of the 19-O-glucose residue to produce rebaudioside E. A functional UGT2 polypeptide can also utilize rebaudioside A as a substrate, thereby transferring the glucose moiety to the C-2' of the 19-O-glucose residue to produce rebaudioside D.

[0111] A polypeptide having UGT2 activity can also catalyze a reaction using steviol-19-glycoside or rubusoside as a substrate. For example, a functional UGT2 polypeptide can use steviol-19-glycoside or rubusoside as a substrate, thereby transferring the glucose moiety to the 19 position to produce steviol-19-bioside or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, respectively.

[0112] However, functional UGT2 polypeptides generally do not transfer a glucose moiety to steviol compounds having a 1,3-bound glucose at the C-13 position, i.e., transfer of a glucose moiety to steviol 1,3-bioside and 1,3-stevioside generally does not occur.

[0113] Polypeptides having UGT2 activity can also transfer sugar moieties from donors other than uridine diphosphate glucose. For example, a polypeptide having UGT2 activity acts as a uridine 5'-diphosphate D-xylosyl: steviol-13-O-glycoside transferase, which transfers the xylose moiety to the C-2' of the 13-O-glucose of the acceptor molecule steviol-13-O-glycoside. As another example, a polypeptide having UGT2 activity can act as a uridine 5'-diphosphate L-rhamnosyl: steviol-13-O-glycoside transferase, which transfers the rhamnose moiety to the C-2' of the 13-O-glucose of the acceptor molecule steviol.

[0114] One or more of the above activities can be used to define a polypeptide having UGT2 activity. Compared to the UGT2_1a polypeptide (SEQ ID NO: 27), the polypeptide of the present invention can have improved UGT2 activity in one or more of the above activities.

[0115] The polypeptides of the present invention can be used to direct the production of steviol glycosides in recombinant cells toward a desired steviol glycoside, such as rebaudioside A, rebaudioside D, or rebaudioside M. For example, a UGT2 polypeptide that preferentially catalyzes the conversion of steviol-13-monoside to steviolbioside and / or rubusoside to stevioside can favor the conversion of rebaudioside to rebaudioside, while a UGT2 polypeptide that preferentially catalyzes the conversion of stevioside to rebE or rubusoside to a compound having an additional sugar at position 19 can favor the conversion of rebaudioside to rebaudioside M. That is, a preference for the addition of a sugar moiety at position 13 can favor the conversion of rebaudioside A, while a preference for the addition of a sugar moiety at position 19 can favor the conversion of rebaudioside M.

[0116] The present invention also provides a polynucleotide sequence encoding the polypeptide as described herein.

[0117] Such a polynucleotide sequence may be selected from the group consisting of:

[0118] (a) a polynucleotide sequence comprising any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24, or 26, or comprising a polynucleotide sequence having at least 30% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24, or 26; or

[0119] (b) a polynucleotide sequence that hybridizes, preferably under at least low stringency conditions, to the complementary strand of any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0120] (c) a polynucleotide sequence that hybridizes, preferably under at least low stringency conditions, to the complementary strand of a polynucleotide having at least 30% sequence identity to any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26;

[0121] (d) a polynucleotide sequence that is a degenerate as a result of the degeneracy of the genetic code of a polynucleotide sequence as defined in any one of (a), (b) or (c); or

[0122] (e) A polynucleotide sequence that is the complement of a nucleotide sequence defined in (a), (b), (c) or (d).

[0123] The polynucleotide sequences of the present invention may have at least 40%, at least 50%, at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, most preferably at least 93%, most preferably at least about 95%, most preferably at least about 96%, most preferably at least about 97%, even most preferably at least about 98%, and even more preferably at least 99% sequence identity to any of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26.

[0124] As used in the present invention, the term "nucleic acid" refers to a nucleotide polymer comprising at least 5 nucleotide units. Nucleic acid refers to a ribonucleotide polymer (RNA), a deoxynucleotide polymer (DNA), or a modified form of any type of nucleic acid, or a synthetic form thereof, or a mixed polymer of any of the above. Nucleic acids can include either or both naturally occurring nucleic acids and modified nucleic acids linked together by naturally occurring and / or non-naturally occurring nucleic acid bonds. Nucleic acid molecules can be chemically or biochemically modified or can contain non-natural or derivatized nucleic acid bases, as readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, replacement of one or more naturally occurring nucleic acids with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelating agents, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). The term nucleic acid is also intended to include any topological conformation, including single-stranded (sense strand and antisense strand), double-stranded, partial duplex, triplex, hairpin, ring and padlock conformation. Also included are synthetic molecules that simulate nucleic acids in terms of the ability to be bound to a specified sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those molecules in which peptide linkages replace the phosphate bond in the molecular backbone. Mention that a nucleic acid sequence encompasses its complement, unless otherwise indicated. Therefore, mentioning a nucleic acid molecule with a specific sequence should be understood to encompass its complementary chain and its complementary sequence. Complementary chain is also applicable to, for example, antisense therapy, hybridization probes and PCR primers. The terms "nucleic acid," "polynucleotide," and "polynucleotide sequence" can be used interchangeably herein.

[0125] As used herein, the term "hybridization" refers to the pairing of substantially complementary strands of oligomeric compounds. One pairing mechanism involves hydrogen bonding between complementary nucleotide bases (nucleotides) of the oligomeric strands, which can be Watson-Crick, Hodgkin's, or other hybridization mechanisms. Or anti-Hodgkin's hydrogen bonding. For example, adenine and thymine are complementary nucleic acids that pair by forming hydrogen bonds. Hybridization can occur under different circumstances. "Stringent hybridization" or "hybridization under low stringency conditions, medium stringency conditions, high stringency conditions or very high stringency conditions" are used herein to describe the conditions for hybridization and washing, more specifically, the conditions under which the oligomeric compound will hybridize with its target sequence and with a minimum number of other sequences. Therefore, the oligomeric compound will hybridize with the target sequence to a higher degree than can be detected by hybridization with other sequences. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6:3.6. Aqueous and non-aqueous methods are described in the reference and any method can be used. Stringent conditions are sequence-dependent and will vary in different situations. Typically, for oligomeric compounds under defined ionic strength and pH, stringent conditions are selected to be about 5°C below the thermal melting point (Tm). The Tm is the temperature (under defined ionic strength and pH) at which 50% of the oligomeric compound hybridizes to a perfectly matched probe. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

[0126] Examples of specific hybridization conditions are as follows: 1) low stringency hybridization conditions of 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least two washes in 0.2X SSC, 0.1% SDS at 50°C (for low stringency conditions, the temperature of the washes can be increased to 55°C); 2) moderate stringency hybridization conditions of 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions of 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and 4) very high stringency hybridization conditions of 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2X SSC, 1% SDS at 65°C.

[0127] Generally, high stringency conditions, such as high hybridization temperature and optionally low salt concentration, allow hybridization only between highly similar sequences, while low stringency conditions, such as low hybridization temperature and optionally high salt concentration, allow hybridization when the sequences are less similar.

[0128] The present invention also provides a nucleic acid construct comprising the polynucleotide sequence of the present invention.

[0129] The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that has been isolated from a naturally occurring gene or has been modified to contain segments of nucleic acid combined and juxtaposed in a manner that would not otherwise exist in nature. The term nucleic acid construct has the same meaning as the term "expression cassette" when the nucleic acid construct contains all the control sequences required for expression of a coding sequence, wherein the control sequences are operably linked to the coding sequence.

[0130] The nucleic acid of the present invention can be an expression vector, in which the polynucleotide sequence of the present invention is operably linked to at least one control sequence for expressing the polynucleotide sequence in a host cell.

[0131] As used herein, the term "operably linked" refers to two or more nucleic acid sequence elements that are physically linked and in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence if the promoter is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control" of the promoter. Typically, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually be substantially contiguous, although this may not be required.

[0132] The expression vector comprises a polynucleotide encoding a polypeptide of the present invention operably linked to appropriate control sequences (such as a promoter and transcriptional and translational stop signals) for expressing and / or translating the polynucleotide in vitro or in a host cell.

[0133] The expression vector can be any vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and that can express the polynucleotide. The choice of vector will generally depend on the compatibility of the vector with the cell into which it is to be introduced. The vector can be a linear or closed circular plasmid. The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.

[0134] Alternatively, the vector can be a vector that is integrated into the genome when introduced into a host cell and replicated together with the chromosome into which it has been integrated. Integrative cloning vectors can be integrated into the chromosome of the host cell at random or at a predetermined target locus. Vectors of the present invention can comprise one or more selective markers that allow for easy selection of transformed cells.

[0135] The present invention also provides a recombinant host comprising a recombinant nucleic acid sequence encoding the polypeptide of the present invention.

[0136] That is, the recombinant host of the present invention may comprise, for example, a recombinant nucleic acid sequence encoding the following polypeptide:

[0137] a. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 1;

[0138] b. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 3;

[0139] c. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 6;

[0140] d. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 9;

[0141] e. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 11;

[0142] f. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 14;

[0143] g. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 17;

[0144] h. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 20;

[0145] i. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 22; or

[0146] j. is at least about 85% identical to the amino acid sequence set forth in SEQ ID NO: 25.

[0147] The recombinant host of the present invention may comprise any polynucleotide encoding the polypeptide of the present invention as described herein. The recombinant host of the present invention is generally capable of expressing the polypeptide of the present invention.

[0148] Typically, the recombinant host of the present invention is capable of producing glycosylated diterpenes, such as steviol glycosides. For example, the recombinant host of the present invention may be capable of producing, for example, steviol-13-monoside, steviol-19-monoside, 13-[(β-D-glucopyranosyl)oxy]kaur-16-ene-18-acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, rubusoside, stevioside, steviol-19-bioside, steviolbioside, rebA, rebE, rebD, or rebM. One or more of these.

[0149] The recombinant host according to the present invention may comprise one or more recombinant nucleotide sequences encoding one or more of the following:

[0150] a polypeptide having ent-copalyl pyrophosphate synthase activity;

[0151] a polypeptide having ent-kaurene synthase activity;

[0152] A polypeptide having ent-kaurene oxidase activity; and

[0153] A polypeptide having kaurenoate 13-hydroxylase activity.

[0154] For the purposes of the present invention, a polypeptide having ent-copalyl pyrophosphate synthase (EC 5.5.1.13) is capable of catalyzing the chemical reaction:

[0155]

[0156] The enzyme has a substrate, geranylgeranyl pyrophosphate, and a product, ent-copalyl pyrophosphate. The enzyme is involved in the biosynthesis of gibberellins. The enzyme belongs to the family of isomerases, particularly the class of intramolecular lyases. The systematic name for this class of enzymes is ent-copalyl-diphosphate lyase (decyclizing). Other commonly used names include ent-copalyl pyrophosphate synthase, ent-kaurene synthase A, and ent-kaurene synthase A.

[0157] Suitable nucleic acid sequences encoding ent-copalyl pyrophosphate synthase may, for example, comprise the sequences listed in SEQ ID. NO: 1, 3, 5, 7, 17, 19, 59, 61, 141, 142, 151, 152, 153, 154, 159, 160, 182 or 184 of WO2015 / 007748.

[0158] For the purposes of the present invention, a polypeptide having ent-kaurene synthase activity (EC 4.2.3.19) is a polypeptide capable of catalyzing the following chemical reaction:

[0159]

[0160] Thus, the enzyme has one substrate, ent-copalyl diphosphate, and two products, ent-kaurene and diphosphate.

[0161] The enzyme belongs to the lyase family, particularly acts on the carbon-oxygen lyase of phosphate / ester.The systematic name of the enzyme class is ent-copalyl diphosphate diphosphate-lyase (cyclization, ent-kaurene forms).Other names commonly used include ent-kaurene synthase B, ent-kaurene synthase B, ent-copalyl-diphosphate diphosphate-lyase and (cyclization).The enzyme participates in the biosynthesis of diterpenoids.

[0162] Suitable nucleic acid sequences encoding ent-kaurene synthase may, for example, comprise the sequences listed in SEQ ID. NO: 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, 157, 158, 159, 160, 183 or 184 of WO 2015 / 007748.

[0163] Et-copalyl diphosphate synthases can also have different ent-kaurene synthase activities associated with the same protein molecule. The reaction catalyzed by ent-kaurene synthase is the next step in the biosynthetic pathway of gibberellins. The two types of enzyme activities are different, and site-directed mutagenesis to inhibit the ent-kaurene synthase activity of the protein leads to the accumulation of ent-copalyl diphosphate.

[0164] Thus, a single nucleotide sequence used in the present invention may encode a polypeptide having both ent-copalyl pyrophosphate synthase activity and ent-kaurene synthase activity. Alternatively, the two activities may be encoded by two different, separate nucleotide sequences.

[0165] For the purposes of the present invention, a polypeptide having ent-kaurene oxidase activity (EC 1.14.13.78) is a polypeptide capable of catalyzing the three consecutive oxidations of the 4-methyl group of ent-kaurene to produce kaurenic acid. This activity generally requires the presence of cytochrome P450.

[0166] Suitable nucleic acid sequences encoding ent-kaurene oxidase may, for example, comprise the sequences listed in SEQ ID. NO: 21, 23, 25, 67, 85, 145, 161, 162, 163, 180 or 186 of WO 2015 / 007748.

[0167] For the purposes of the present invention, a polypeptide having kaurenoate 13-hydroxylase activity (EC 1.14.13) is a polypeptide that can catalyze the formation of steviol (ent-kaure-16-ene-13-ol-19-oic acid) using NADPH and O. This activity may also be referred to as ent-kaurenoate 13-hydroxylase activity.

[0168] Suitable nucleic acid sequences encoding kaurenoate 13-hydroxylase may, for example, comprise the sequences listed in SEQ ID. NO: 27, 29, 31, 33, 69, 89, 91, 93, 95, 97, 146, 164, 165, 166, 167 or 185 of WO2015 / 007748.

[0169] The recombinant host of the present invention may comprise a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity. That is, the recombinant host of the present invention may be capable of expressing a nucleotide sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity. For purposes of the present invention, a polypeptide having NADPH-cytochrome P450 reductase activity (EC 1.6.2.4; also referred to as NADPH: methemoglobin oxidoreductase, NADPH: heme protein oxidoreductase, NADPH: P450 oxidoreductase, P450 reductase, POR, CPR, CYPOR) is typically a polypeptide that is a membrane-bound enzyme that allows electrons to be transferred from the enzyme NADPH: cytochrome P450 reductase (POR; EC 1.6.2.4), which contains FAD and FMN, to the cytochrome P450 in the microsomes of eukaryotic cells.

[0170] The recombinant host according to any one of the preceding claims, comprising one or more recombinant nucleic acid sequences encoding one or more of:

[0171] (i) a polypeptide having UGT74G1 activity (UGT3 activity);

[0172] (ii) a polypeptide having UGT85C2 activity (UGT1 activity); and

[0173] (iii) A polypeptide having UGT76G1 activity (UGT4 activity).

[0174] Figure 31 and 32 A schematic representation of potential pathways leading to the biosynthesis of steviol glycosides is shown.

[0175] The recombinant hosts of the present invention will typically contain at least one recombinant nucleic acid encoding a polypeptide having UGT1 activity, at least one recombinant nucleic acid encoding a polypeptide having UGT2 activity, at least one recombinant nucleic acid encoding a polypeptide having UGT3 activity, and at least one recombinant nucleic acid encoding a polypeptide having UGT4 activity. A single nucleic acid may encode two or more such polypeptides.

[0176] Nucleic acids encoding polypeptides of the present invention can be used to direct the production of steviol glycosides in recombinant cells toward a desired steviol glycoside, such as rebaudioside A, rebaudioside D, or rebaudioside M. For example, a recombinant nucleic acid encoding a UGT2 polypeptide that preferentially catalyzes the conversion of steviol-13-monoside to steviolbioside and / or rubusoside to stevioside can favor the conversion of rebaudioside to rebaudioside, while a recombinant nucleic acid encoding a UGT2 polypeptide that preferentially catalyzes the conversion of stevioside to rebE or rubusoside to a compound having an additional sugar at position 19 can favor the conversion of rebaudioside to rebaudioside M. That is, a preference for adding a sugar moiety at position 13 can favor the conversion of rebaudioside A, while a preference for adding a sugar moiety at position 19 can favor the conversion of rebaudioside M.

[0177] The recombinant host of the present invention may comprise a nucleotide sequence encoding a polypeptide that can catalyze the addition of C-13-glucose to steviol. That is, the recombinant host of the present invention may comprise a UGT that can catalyze the reaction of converting steviol into steviol monoglycosides.

[0178] The recombinant host of the present invention may comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT85C2, whereby the nucleotide sequence after transformation of the host confers the ability of the host to convert steviol into steviol monosides.

[0179] The activity of UGT85C2 is to transfer a glucose unit to the 13-OH of steviol. Thus, a suitable UGT85C2 can function as a uridine 5'-diphosphoglucosyl:steviol 13-OH transferase and a uridine 5'-diphosphoglucosyl:steviol-19-O-glycoside 13-OH transferase. Functional UGT85C2 polypeptides can also catalyze glucosyltransferase reactions that utilize steviol glycoside substrates other than steviol and steviol-19-O-glycoside. Such sequences may be referred to herein as UGT1 sequences.

[0180] The recombinant host of the present invention may contain a nucleotide sequence encoding a polypeptide having UGT activity, and may contain a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of C-13-glucose to steviol or steviol monoside. In other words, the recombinant host of the present invention may contain a UGT capable of catalyzing the reaction of converting steviol monoside into steviolbioside. Therefore, such a recombinant host may be capable of converting steviol monoside into steviolbioside. Expression of such a nucleotide sequence may confer upon the host the ability to produce at least steviolbioside.

[0181] The recombinant microorganisms of the present invention also contain a nucleotide sequence encoding a polypeptide having UGT activity, and may contain a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of C-19-glucose to steviolbioside. That is, the microorganisms of the present invention may contain a UGT capable of catalyzing the reaction of converting steviolbioside into stevioside. Thus, such microorganisms may be capable of converting steviolbioside into stevioside. Expression of such a nucleotide sequence may confer upon the microorganism the ability to produce at least stevioside.

[0182] Therefore, the microorganism of the present invention may further comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT74G1, whereby the nucleotide sequence after transformation of the microorganism confers the cell the ability to convert steviolbioside into stevioside.

[0183] Suitable UGT74G1 polypeptides can be capable of transferring a glucose unit to the 13-OH or 19-COOH of steviol, respectively. Suitable UGT74G1 polypeptides can function as uridine 5'-diphosphoglucosyl:steviol 19-COOH transferases and uridine 5'-diphosphoglucosyl:steviol-13-O-glycoside 19-COOH transferases. Functional UGT74G1 polypeptides can also catalyze glycosyltransferase reactions that use steviol glycoside substrates other than steviol and steviol-13-O-glycoside, or transfer sugar moieties from donors other than uridine diphosphoglucose. Such sequences may be referred to herein as UGT3 sequences.

[0184] The recombinant host of the present invention may comprise a nucleotide sequence encoding a polypeptide capable of catalyzing the glucosylation of the C-3' portion of the glucose at the C-13 position of stevioside. That is, the recombinant host of the present invention may comprise a UGT capable of catalyzing the reaction of stevioside to rebaudioside A. Thus, such a recombinant host may be capable of converting stevioside to rebaudioside A. Expression of such a nucleotide sequence may confer upon the host the ability to produce at least rebaudioside A.

[0185] Therefore, the recombinant microorganism of the present invention may further comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT76G1, whereby the nucleotide sequence after transformation of the microorganism confers the ability to convert stevioside into rebaudioside A to the cell.

[0186] Suitable UGT76G1 adds a glucose moiety to the C-3' of the C-13-O-glucose of the acceptor molecule steviol 1,2-glycoside. Thus, UGT76G1 acts, for example, as a uridine 5'-diphosphoglucosyl:steviol 13-O-1,2-glucosidase C-3' glucosyltransferase and a uridine 5'-diphosphoglucosyl:steviol-19-O-glucose, 13-O-1,2-disaccharide C-3' glucosyltransferase. Functional UGT76G1 polypeptides can also catalyze glucosyltransferase reactions using steviol glycoside substrates containing sugars other than glucose, such as steviol rhamnoside and steviol xyloside. Such sequences may be referred to herein as UGT4 sequences.

[0187] The recombinant hosts of the present invention typically comprise a nucleotide sequence encoding a polypeptide having all four of the above-described UGT activities. A given nucleic acid may encode a polypeptide having one or more of the above-described activities. For example, the nucleic acid encodes a polypeptide having two, three, or four of the above-described activities. Preferably, the recombinant hosts of the present invention comprise UGT1, UGT2, and UGT3, as well as UGT4 activities. Suitable UGT1, UGT3, and UGT4 sequences are described in Table 1 of WO2015 / 007748.

[0188] The recombinant hosts of the present invention may comprise recombinant nucleic acid sequences encoding additional polypeptides having UGT2 activity. That is, the recombinant hosts of the present invention may comprise nucleic acid sequences encoding a variant UGT2 of the present invention and one or more additional different variants of the present invention or any other different UGT2.

[0189] Use of nucleic acid sequences encoding UGT2_lb, UGT2_2b, UGT2_3b, UGT2_4b, UGT2_5b, UGT2_6b, UGT2_7b, UGT2_8b, UGT2_9b, or UGT2_10b polypeptides (or related polypeptides as described herein) may be useful for improving rebA production.

[0190] Use of nucleic acid sequences encoding UGT2-7b polypeptides (or related polypeptides as described herein) may be useful for improving the production of rebM.

[0191] In the recombinant host of the present invention, the host's ability to produce geranylgeranyl diphosphate (GGPP) can be upregulated. Upregulated in the context of the present invention means that the recombinant host produces more GGPP than an equivalent non-recombinant host.

[0192] Thus, the recombinant host of the present invention may comprise one or more nucleotide sequences encoding hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthase, and geranylgeranyl diphosphate synthase, whereby said nucleotide sequences, after transformation of the microorganism, confer upon the microorganism the ability to produce increased levels of GGPP. Thus, the recombinant host according to the present invention may comprise one or more recombinant nucleic acid sequences encoding one or more of hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthase, and geranylgeranyl diphosphate synthase.

[0193] Thus, the recombinant host of the present invention may comprise a nucleic acid sequence encoding one or more of the following:

[0194] A polypeptide having hydroxymethylglutaryl-CoA reductase activity;

[0195] a polypeptide having farnesyl-pyrophosphate synthetase activity;

[0196] A polypeptide having geranylgeranyl diphosphate synthase activity.

[0197] As defined herein, a host or host cell is an organism suitable for genetic manipulation and an organism that can be cultured at a cell density suitable for industrial production of a target product. Suitable hosts can be microorganisms, such as microorganisms that can be maintained in a fermentation unit. The host cell can be a host cell found in nature or a host cell derived from a parent host cell after genetic manipulation or traditional mutagenesis.

[0198] As used herein, recombinant host is the host that carries out genetic modification or conversion / transfection with one or more nucleotide sequences as defined herein.The existence of described one or more such nucleotide sequences changes the ability of microorganism to produce diterpene glucosides, particularly one or more steviol glycosides.Non-recombinant host, promptly the host of unconverted / transfection or genetic modification does not comprise one or more nucleotide sequences that make cell produce diterpene glucosides usually.Therefore, non-recombinant host is normally the host that does not produce diterpene glucosides naturally, but natural generation diterpene or diterpene glucosides and the host that has modified (and therefore has the ability of generation diterpene glucosides that changes) according to the present invention are considered to recombinant host according to the present invention.

[0199] In particular, it is feasible that the enzyme selected from the group consisting of ent-copalyl pyrophosphate synthase, ent-kaurene synthase, ent-kaurene oxidase and kaurenoate 13-hydroxylase, UGT, hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthase, geranylgeranyl diphosphate synthase and NADPH-cytochrome p450 reductase is native to the host, and transformation with one or more nucleotide sequences encoding these enzymes may not be necessary to confer the host cell the ability to produce diterpene glycosides. Preferred hosts according to the present invention may be recombinant hosts capable of naturally producing GGPP (i.e. in its non-recombinant form).

[0200] Further improvements in diterpene glycoside production by host microorganisms can be achieved through traditional strain improvement.

[0201] The host cell can be a prokaryotic, archaebacterial or eukaryotic host cell.

[0202] Prokaryotic host cells may be, but are not limited to, bacterial host cells. Eukaryotic host cells may be, but are not limited to, yeast, fungi, amoeba, algae, animals, or insect host cells.

[0203] The eukaryotic host cell can be a fungal host cell. "Fungi" includes all species of the subdivision Eumycotina (Alexopoulos, CJ, 1962, in Introductory Mycology, John Wiley & Sons, Inc., New York). Thus, the term fungi includes filamentous fungi and yeast, etc.

[0204] "Filamentous fungi" are defined herein as eukaryotic microorganisms that include all filamentous forms of the subdivision Fungi and Oomycetes (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon metabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of the following: Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimast x), Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasmsonia, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.Preferred filamentous fungal strains that can serve as host cells belong to the following species: Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla. Reference host cells for comparison of fermentation characteristics of transformed and untransformed cells include, for example, Aspergillus niger CBS 120.49, CBS 513.88; Aspergillus oryzae ATCC 16868, ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC 14488-14491, ATCC 11601, ATCC 12892; Aspergillus fumigatus AF293 (CBS 101355); Penicillium chrysogenum CBS 455.95; Penicillium citrinum ATCC 38065; Penicillium chrysogenum P2; Acremonium clade 36225, ATCC 48272; Trichoderma reesei ATCC 26921, ATCC 56765, ATCC26921; Aspergillus sojae ATCC11906; Chinostoma rukvenica ATCC44006 and derivatives thereof. Aspergillus niger CBS 513.88 and its derivatives are particularly preferred as filamentous fungal host cells.

[0205] The eukaryotic host cell can be a yeast cell. Preferred yeast host cells can be selected from the following genera: Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), Brettanomyces, Kluyveromyces, Candida (e.g., C. krusei, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis), I. ssampeytes (e.g., C. truncatula ... ssatchenkia) (e.g., I. orientalis), Pichia (e.g., P. pastoris), Schizosaccharomyces, Hansenula, Kloeckera, Pachysolen, Schwanniomyces, Trichosporon, Yarrowia (e.g., Y. lipolytica (formerly classified as Candidalipolytica)), Yamadazyma.

[0206] The prokaryotic host cell can be a bacterial host cell. The bacterial host cell can be a Gram-negative or Gram-positive bacterium. Examples of bacteria include, but are not limited to, bacteria belonging to the genus Bacillus (e.g., Bacillus subtilis (B.subtilis), Bacillus amyloliquefaciens (B.amyloliquefaciens), Bacillus licheniformis (B.licheniformis), Bacillus puntis (B.puntis), Bacillus megaterium (B.megaterium), alkaliphilic Bacillus (B.halodurans), Bacillus pumilus (B.pumilus)), Acinetobacter (Acinetobacter), Nocardia (Nocardia), Xanthobacterium (Xanthobacter), Escherichia (Escherichia) (e.g., Escherichia coli (e.g., strain DH 10B, Stbl2, DH5-α, DB3, DB3.1), DB4, DB5, JDP682, and ccdA-over (e.g., U.S. Application No. 09 / 518,188), Streptomyces, Erwinia, Klebsiella, Serratia (S. marcessans), Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhimurium, S. typhi). Bacteria also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum)), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum), purple sulfur bacteria bacteria (e.g., Chromatium (e.g., C. okenii)) and purple non-sulfur bacteria (e.g., Rhodospirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii)).

[0207] The host cell can be a host cell from a non-microorganism. Examples of such cells include, but are not limited to, insect cells (e.g., Drosophila (Drosophila) (e.g., Drosophila melanogaster (D.melanogaster)), Spodoptera (Spodoptera) (e.g., Spodoptera frugiperda (S.frugiperda) Sf9 or Sf21 cells) and Trichoplusa (Trichoplusa) (e.g., High-Five cells)); nematode cells (e.g., C.elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis cells); reptile cells; and mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma, and HeLa cells).

[0208] The present invention also provides a method for producing the polypeptide of the present invention, comprising:

[0209] (a) culturing the recombinant host cell of the present invention under conditions conducive to production of the polypeptide by the recombinant host cell, and optionally

[0210] (b) recovering the polypeptide.

[0211] Recombinant host according to the present invention can be grown on any suitable carbon source as known in the art, and is converted into glycosylated diterpenes, for example, steviol glycosides.Recombinant host can be directly converted into plant biomass, Mierocrystalline cellulose, hemicellulose, pectin, rhamnose, galactose, fucose, maltose, maltodextrin, ribose, ribulose or starch, starch derivatives, sucrose, lactose and glycerol.Therefore, preferred host expression enzyme is as for being used for Mierocrystalline cellulose conversion into glucose monomer and hemicellulose conversion into the required cellulase (endo-cellulase and exo-cellulase) and hemicellulase (for example endo- and exo-xylanase, arabinase) of wood sugar and arabinose monomer, can be converted into the pectinase of glucuronic acid and galacturonic acid or the amylase of starch conversion into glucose monomer.Preferably, the host can convert the carbon source selected from the group consisting of the following: glucose, wood sugar, arabinose, sucrose, lactose and glycerol. The host cell may for example be a eukaryotic host cell as described in WO03 / 062430, WO06 / 009434, EP1499708B1, WO2006096130 or WO04 / 099381.

[0212] Therefore, in another aspect, the present invention also provides a method for preparing glycosylated diterpenes such as steviol glycosides, the method comprising fermenting a recombinant host of the present invention, the recombinant host being capable of producing at least one glycosylated diterpene in a suitable fermentation medium; and optionally recovering the glycosylated diterpene.

[0213] The glycosylated terpene (e.g., steviol glycoside) can be steviol-19-monoside, steviol-19-bioside, steviol-19-trioside, steviol-13-monoside, rubusoside, 13-[(β-D-glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, steviolbioside, stevioside, rebaudioside, rebaudioside E, rebaudioside B, rebaudioside A, rebaudioside D, or rebaudioside M. Therefore, the present invention provides a method for producing one or more such steviol glycosides.

[0214] The fermentation medium used in the method for producing glycosylated diterpenes can be any suitable fermentation medium that allows the growth of a particular eukaryotic host cell. The basic elements of the fermentation medium are known to those skilled in the art and can be adapted to the selected host cell.

[0215] Preferably, the fermentation medium comprises a carbon source selected from the group consisting of plant biomass, cellulose, hemicellulose, pectin, rhamnose, galactose, fucose, fructose, maltose, maltodextrin, ribose, ribulose or starch, starch derivatives, sucrose, lactose, fatty acids, triglycerides and glycerol. Preferably, the fermentation medium further comprises a nitrogen source, such as urea; or an ammonium salt, such as ammonium sulfate, ammonium chloride, ammonium nitrate or ammonium phosphate.

[0216] Fermentation process according to the present invention can be carried out in batches, batch feeding or continuous mode.Also can apply independent hydrolysis and fermentation (SHF) method or saccharification and fermentation (SSF) method simultaneously.The combination of these fermentation process patterns also can be feasible for optimal productivity.If starch, cellulose, hemicellulose or pectin are used as carbon source in the fermentation process, then the SSF method can be particularly attractive, wherein can need to add lytic enzyme such as cellulase, hemicellulase or pectinase to hydrolyze substrate.

[0217] The recombinant host used in the method for preparing glycosylated diterpenes may be any suitable recombinant host as defined above. The use of a recombinant eukaryotic host according to the present invention in the method may be advantageous because most eukaryotic cells do not require sterile conditions for propagation and are not susceptible to phage infection. In addition, eukaryotic host cells can be grown at low pH to prevent bacterial contamination.

[0218] The recombinant host according to the present invention can be a facultative anaerobic microorganism. The facultative anaerobic recombinant host can be propagated aerobically to a high cell density. The anaerobic stage can then be carried out at a high cell density, which significantly reduces the required fermentation volume and minimizes the risk of contamination by aerobic microorganisms.

[0219] The fermentation process for producing the glycosylated diterpenes according to the present invention may be an aerobic or anaerobic fermentation process.

[0220] Anaerobic fermentation processes may be defined herein as fermentation processes that operate in the absence of oxygen or that consume substantially no oxygen (preferably less than 5, 2.5, or 1 mmol / L / h), and in which organic molecules act as both electron donors and electron acceptors. The fermentation process according to the present invention may also be run first under aerobic conditions and subsequently under anaerobic conditions.

[0221] Fermentation processes can also be carried out under oxygen-limited or microaerobic conditions. Alternatively, the fermentation process can be run first under aerobic conditions and then under oxygen-limited conditions. Oxygen-limited fermentation processes are processes in which oxygen consumption is limited by oxygen transfer from gas to liquid. The degree of oxygen limitation is determined by the amount and composition of the incoming gas stream and the actual mixing / mass transfer characteristics of the fermentation equipment used.

[0222] The production of glycosylated diterpenes in the process according to the invention may take place during the growth phase of the host cells, during the stationary (steady state) phase or during both phases.It may be feasible to run the fermentation process at different temperatures.

[0223] The method for producing glycosylated diterpenes can be performed at a temperature that is optimal for the recombinant host. The optimal growth temperature can be different for each transformed recombinant host and is known to those skilled in the art. The optimal temperature can be higher than the optimum temperature of the wild-type organism to effectively grow the organism under non-sterile conditions with minimal infection susceptibility and minimal cooling costs. Alternatively, the method can be performed at a temperature that is not optimal for the growth of the recombinant host.

[0224] The method for producing glycosylated diterpenes according to the present invention can be carried out at any suitable pH value. If the recombinant host is a yeast, the pH in the fermentation medium preferably has a value below 6, preferably below 5.5, preferably below 5, preferably below 4.5, preferably below 4, preferably below pH 3.5 or below pH 3.0 or below pH 2.5, preferably above pH 2. The advantage of carrying out the fermentation at these low pH values ​​is that the growth of contaminating bacteria in the fermentation medium can be prevented.

[0225] This method can be carried out on an industrial scale. The product of this method is one or more glycosylated diterpenes, such as one or more steviol glycosides, for example, one or more of 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, steviolbioside, stevioside, rebaudioside E, rebaudioside B, rebaudioside A, rebaudioside D, or rebaudioside M.

[0226] Recovery of the glycosylated diterpenes from the fermentation medium can be carried out by methods known in the art, such as by distillation, vacuum extraction, solvent extraction or evaporation.

[0227] In the process for producing glycosylated diterpenes according to the present invention, it is possible to achieve a concentration of more than 5 mg / l fermentation broth, preferably more than 10 mg / l, preferably more than 20 mg / l, preferably more than 30 mg / l fermentation broth, preferably more than 40 mg / l, more preferably more than 50 mg / l, preferably more than 60 mg / l, preferably more than 70, preferably more than 80 mg / l, preferably more than 100 mg / l, preferably more than 1 g / l, preferably more than 5 g / l, preferably more than 10 g / l, for example more than 20 g / l, but typically up to about 200 g / l, such as up to about 150 g / l, such as up to about 100 g / l, for example up to about 70 g / l. Such concentrations may be the concentration of the total fermentation broth or the supernatant.

[0228] The present invention also provides a fermentation broth containing the glycosylated diterpene obtainable by the method for preparing the glycosylated diterpene of the present invention.

[0229] Where one or more glycosylated diterpenes are expressed in a microorganism, it may be desirable to treat such cells to release them. Preferably, at least one glycosylated diterpene, such as a steviol glycoside, for example rebA or rebM, is produced extracellularly.

[0230] The present invention also provides a glycosylated diterpene obtained by the method for preparing a glycosylated diterpene according to the present invention or obtainable from the fermentation broth of the present invention. The glycosylated diterpene may be a non-naturally occurring glycosylated diterpene, that is, a glycosylated diterpene that is not produced in plants.

[0231] The present invention also provides a composition comprising two or more glycosylated diterpenes obtainable by the method for preparing a glycosylated diterpene of the present invention or obtainable from the fermentation broth of the present invention. In this composition, one or more glycosylated diterpenes may be non-naturally occurring glycosylated diterpenes, that is, glycosylated diterpenes that do not occur in plants.

[0232] Furthermore, the present invention provides a method for converting a first glycosylated diterpene into a second glycosylated diterpene, the method comprising:

[0233] - contacting the first glycosylated diterpene with a recombinant host of the present invention, a cell-free extract derived from such a recombinant host, or an enzyme preparation derived from either of the foregoing;

[0234] - thereby converting the first glycosylated diterpene into the second glycosylated diterpene.

[0235] In this method, the second glycosylated diterpene can be steviol-19-bioside, steviolbioside, stevioside, RebE, RebD, or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester.

[0236] In this method, the first glycosylated diterpene can be steviol-13-monoside, steviol-19-monoside, rubusoside, stevioside, rebaudioside A or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, and the second glycosylated diterpene is steviol-19-bioside, steviolbioside, stevioside, RebE, RebD or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester.

[0237] These are the first and second steviol glycosides involved in the reaction catalyzed by the polypeptide having UGT2 activity of the present invention.

[0238] That is, the present invention relates to a bioconversion or biotransformation method.

[0239] The steviol glycosides or compositions produced by the fermentation method according to the present invention can be used in any application known for such compounds. In particular, they can be used as sweeteners, for example, in foods or beverages. Therefore, according to the present invention, there is provided a food, feed or beverage comprising a glycosylated diterpene, such as a steviol glycoside or composition according to the present invention.

[0240] For example, the glycosylated diterpenes or compositions of the present invention can be formulated into soft drinks, as tabletop sweeteners, chewing gum, dairy products such as yogurt (e.g., plain yogurt), cakes, cereals or cereal-based foods, nutritional foods, pharmaceuticals, edible gels, confectionery products, cosmetics, toothpaste or other oral compositions, etc. Furthermore, the glycosylated diterpenes or compositions of the present invention can be used as sweeteners not only for beverages, foods and other products intended for human consumption, but also for animal feed and fodder with improved properties.

[0241] Therefore, the present invention particularly provides a food, feed or beverage comprising a diterpene or a glycosylated diterpene prepared according to the method of the present invention.

[0242] In the manufacturing process of food, beverage, medicine, cosmetic, tabletop product, chewing gum, conventional methods such as mixing, kneading, dissolving, pickling, infiltration, percolation, spraying, atomizing, pouring and other methods can be used.

[0243] The glycosylated diterpenes, such as steviol glycosides, or compositions of the present invention can be used in dry or liquid form. They can be added to the food before or after heat treatment. The amount of sweetener depends on the intended use. They can be added alone or in combination with other compounds.

[0244] The compounds produced according to the methods of the present invention can be blended with one or more other non-caloric or caloric sweeteners. Such blending can be used to improve flavor or temporal characteristics or stability. A wide range of both non-caloric and caloric sweeteners are suitable for blending with the glycosylated diterpenes or compositions of the present invention. For example, non-caloric sweeteners include mogroside, monatin, aspartame, acesulfame potassium salts, cyclamate salts, sucralose, saccharin salts, or erythritol. Caloric sweeteners suitable for blending with the glycosylated diterpenes or compositions of the present invention include sugar alcohols and carbohydrates such as sucrose, glucose, fructose, and HFCS. Sweet amino acids such as glycine, alanine, or serine can also be used.

[0245] The glycosylated diterpenes or compositions of the present invention can be used in combination with sweetener inhibitors such as natural sweetener inhibitors. It can be combined with umami enhancers such as amino acids or salts thereof.

[0246] The glycosylated diterpenes or compositions of the present invention may be combined with polyols or sugar alcohols, carbohydrates, physiologically active substances or functional ingredients (e.g., carotenoids, dietary fibers, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols or stanols (phytosterols and phytostanols), polyols, prebiotics, probiotics, phytoestrogens, soy proteins, sulfides / thiols, amino acids, proteins, vitamins, minerals and / or substances classified based on health benefits such as cardiovascular, cholesterol-lowering or anti-inflammatory.

[0247] Compositions having glycosylated diterpenes or compositions of the present invention may include flavorings, aroma components, nucleotides, organic acids, organic acid salts, inorganic acids, bitter compounds, proteins or protein hydrolysates, surfactants, flavonoids, astringent compounds, vitamins, dietary fiber, antioxidants, fatty acids and / or salts.

[0248] The glycosylated diterpenes or compositions of the present invention can be used as high-intensity sweeteners to produce zero-calorie, low-calorie or diabetic beverages and foods with improved taste characteristics. They can also be used in beverages, foods, pharmaceuticals and other products where sugar cannot be used.

[0249] Furthermore, the glycosylated diterpenes or compositions of the present invention can be used as sweeteners not only in beverages, foods and other products intended for human consumption, but also in animal feed and fodder with improved properties.

[0250] Examples of products in which the glycosylated diterpenes or compositions of the present invention can be used as sweetening compounds include alcoholic beverages such as vodka, wine, beer, spirits, sake, etc.; natural fruit juices, energy drinks, carbonated soft drinks, diet drinks, zero-calorie drinks, low-calorie drinks and foods, yogurt drinks, instant fruit juices, instant coffee, powdered instant drinks, canned products, syrups, fermented soybean paste, soy sauce, vinegar, condiments, mayonnaise, ketchup, curry, soups, instant broths, soy sauce powder, vinegar powder, Various types of biscuits, rice crackers, salty crackers, bread, chocolate, caramel, candy, chewing gum, jelly, pudding, preserves and pickles, fresh cream, jam, marmalade, sugar flower paste, milk powder, ice cream, sorbet, packaged vegetables and fruits, canned and cooked beans, meat and food cooked in sweet sauces, agricultural vegetable food, seafood, ham, sausage, fish ham, fish sausage, fish sauce, fried fish products, dried seafood, frozen food, pickled kelp, bacon, tobacco, pharmaceutical products, etc. In principle, it has unlimited applications.

[0251] Sweet compositions include beverages, non-limiting examples of which include non-carbonated and carbonated beverages such as cola, ginger ale, root beer, apple juice, fruit-flavored soft drinks (e.g., citrus-flavored soft drinks such as lemon-lime or orange juice), soft drink powders, etc.; fruit juices from fruits or vegetables, including squeezed juices, etc., fruit juices containing fruit pulp, fruit drinks, fruit juice drinks, fruit juice-containing drinks, drinks with fruit flavorings, vegetable juices, juices containing vegetables, and mixed juices containing fruits and vegetables; sports drinks, energy drinks, drinks close to water, etc. (e.g., water with natural or synthetic flavorings); tea or favorite beverages such as coffee, cocoa, black tea, green tea, oolong tea, etc.; beverages containing milk ingredients such as milk drinks, milk-containing coffee, milk coffee, milk tea, fruit milk drinks, drinking yogurt, lactic acid bacteria drinks, etc.; and dairy products.

[0252] Typically, the amount of sweetener present in a sweetening composition varies widely depending on the specific type of sweetening composition and its desired sweetness. One of ordinary skill in the art can readily determine the appropriate amount of sweetener to add to a sweetening composition.

[0253] The glycosylated diterpenes or compositions of the present invention can be used in dry or liquid form. They can be added to the food before or after heat treatment. The amount of sweetener depends on the intended use. They can be added alone or in combination with other compounds.

[0254] In the manufacturing process of food, beverage, medicine, cosmetic, tabletop product, chewing gum, conventional methods such as mixing, kneading, dissolving, pickling, infiltration, percolation, spraying, atomizing, pouring and other methods can be used.

[0255] Thus, the compositions of the present invention may be prepared by any method known to those skilled in the art that provides a uniform or homogeneous mixture of the ingredients, including dry blending, spray drying, agglomeration, wet granulation, compaction, co-crystallization, and the like.

[0256] When in solid form, the glycosylated diterpenes or compositions of the present invention can be provided to the consumer in any form suitable for delivery to the food to be sweetened, including sachets, packets, bulk bags or boxes, cubes, tablets, sprays or dissolvable strips. The composition can be delivered in unit dose or bulk form.

[0257] For liquid sweetener systems and compositions, a convenient range of fluid, semi-fluid, pasty and paste forms, suitable packaging using suitable packaging materials in any shape or form, should be developed that is convenient for carrying or dispensing or storing or transporting any combination of the above sweetener products or combinations of the above products.

[0258] The composition may contain various fillers, functional ingredients, colorants, flavoring agents.

[0259] The terms "sequence homology" or "sequence identity" are used interchangeably herein. For the purposes of the present invention, this is defined as the percentage of sequence homology or sequence identity between two amino acid sequences or two nucleic acid sequences that is aligned for optimal comparison purposes. In order to optimize the alignment between two sequences, spaces can be introduced in either of the two sequences being compared. This alignment can be performed over the full length of the sequences being compared. Alternatively, the alignment can be performed over a shorter length, for example, over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

[0260] The sequence comparison between two sequences and the determination of sequence identity percentage can be completed using a mathematical algorithm. Those skilled in the art will appreciate the following fact: several different computer programs can be used to compare two sequences and determine the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (editor), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The sequence identity percentage between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for the comparison of two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be compared by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ) was used. For protein sequences, EBLOSUM62 was used for the substitution matrix. For nucleotide sequences, EDNAFULL was used. The optional parameters used were a gap opening penalty of 10 and a gap extension penalty of 0.5. It will be appreciated by those skilled in the art that all of these different parameters will produce slightly different results, but the overall percent identity of the two sequences does not change significantly when different algorithms are used.

[0261] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and the sequence of the invention is calculated as follows: the number of corresponding positions in the alignment showing identical amino acids or identical nucleotides in the two sequences divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is labeled "longest identity" in the program's output.

[0262] The nucleic acid and protein sequences of the present invention can be further used as "query sequences" to perform searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402 can be utilized. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0263] Some embodiments of the present invention:

[0264] 1. A recombinant host comprising a recombinant nucleic acid sequence encoding a polypeptide, wherein the polypeptide:

[0265] a. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 1;

[0266] b. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 3;

[0267] c. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 6;

[0268] d. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 9;

[0269] e. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 11;

[0270] f. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 14;

[0271] g. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 17;

[0272] h. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 20;

[0273] i. having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 22; or

[0274] j. is at least about 85% identical to the amino acid sequence set forth in SEQ ID NO: 25.

[0275] 2. The recombinant host according to embodiment 1, which is capable of producing glycosylated diterpenes, such as steviol glycosides.

[0276] 3. The recombinant host according to embodiment 1 or 2, comprising one or more recombinant nucleotide sequences encoding:

[0277] a polypeptide having ent-copalyl pyrophosphate synthase activity;

[0278] a polypeptide having ent-kaurene synthase activity;

[0279] A polypeptide having ent-kaurene oxidase activity; and

[0280] A polypeptide having kaurenoate 13-hydroxylase activity.

[0281] 4. The recombinant host according to any one of the preceding embodiments, comprising a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity.

[0282] 5. The recombinant host according to any one of the preceding embodiments, comprising a recombinant nucleic acid sequence encoding one or more of:

[0283] (i) a polypeptide having UGT74G1 (UGT3) activity;

[0284] (ii) a polypeptide having UGT85C2 (UGT1) activity; and

[0285] (iii) A polypeptide having UGT76G1 (UGT4) activity.

[0286] 6. The recombinant host of any one of the preceding embodiments, comprising a recombinant nucleic acid sequence encoding an additional polypeptide having UGT2 activity.

[0287] 7. The recombinant host of any one of the preceding embodiments, wherein the host belongs to one of the genera Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issaffine, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadzyma, or Escherichia.

[0288] 8. The recombinant host of embodiment 7, wherein the recombinant host is a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an I. orientalis cell, or an Escherichia coli cell.

[0289] 9. The recombinant host of any preceding embodiment, wherein the host's ability to produce geranylgeranyl diphosphate (GGPP) is upregulated.

[0290] 10. The recombinant host of any one of the preceding embodiments, comprising one or more recombinant nucleic acid sequences encoding hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthase, and geranylgeranyl diphosphate synthase.

[0291] 11. The recombinant host of any one of the preceding embodiments, comprising a nucleic acid sequence encoding one or more of:

[0292] A polypeptide having hydroxymethylglutaryl-CoA reductase activity;

[0293] a polypeptide having farnesyl-pyrophosphate synthetase activity;

[0294] A polypeptide having geranylgeranyl diphosphate synthase activity.

[0295] 12. A method for preparing a glycosylated diterpene, the method comprising fermenting the recombinant host according to any one of embodiments 2 to 11 in a suitable fermentation medium, and optionally recovering the glycosylated diterpene.

[0296] 13. The method according to any one of embodiment 12, for preparing glycosylated diterpenes, wherein the method is performed on an industrial scale.

[0297] 14. A fermentation broth comprising a glycosylated diterpene obtainable by the method according to embodiment 12 or 13.

[0298] 15. A glycosylated diterpene obtained by the method according to embodiment 12 or 13 or obtainable from the fermentation broth according to embodiment 14.

[0299] 16. A composition comprising two or more glycosylated diterpenes obtained by the method according to embodiment 12 or 13 or obtainable from the fermentation broth according to embodiment 14.

[0300] 17. A food, feed or beverage comprising the glycosylated diterpene according to embodiment 15 or the composition according to embodiment 16.

[0301] 18. A method for converting a first glycosylated diterpene into a second glycosylated diterpene, the method comprising:

[0302] - contacting the first glycosylated diterpene with a recombinant host according to any one of embodiments 1 to 11, a cell-free extract derived from such a recombinant host, or an enzyme preparation derived from any of the foregoing;

[0303] - thereby converting the first glycosylated diterpene into the second glycosylated diterpene.

[0304] 19. The method of embodiment 18, wherein the second glycosylated diterpene is steviol-19-bioside, steviolbioside, stevioside, RebE, RebD, or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester.

[0305] 20. The method of embodiment 19, wherein the first glycosylated diterpene is steviol-13-monoside, steviol-19-monoside, rubusoside, stevioside, rebaudioside A, or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, and the second glycosylated diterpene is steviol-19-bioside, steviolbioside, stevioside, RebE, RebD, or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester.

[0306] 21. A polypeptide having UGT2 activity, wherein the polypeptide is selected from the group consisting of:

[0307] (a) a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22 or 25; or

[0308] (b) a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of any one of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22, or 25; or

[0309] (c) a polypeptide encoded by a polynucleotide comprising the polynucleotide sequence set forth in any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0310] (d) a polypeptide encoded by a polynucleotide comprising a polynucleotide sequence having at least 30% sequence identity to the polypeptide coding sequence in any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0311] (e) a polypeptide encoded by a polynucleotide that hybridizes, preferably under at least low stringency conditions, to the complementary strand of any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0312] (f) a polypeptide encoded by a polynucleotide that hybridizes, preferably under at least low stringency conditions, to the complementary strand of a polynucleotide having at least 30% sequence identity to any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0313] (g) A fragment of a polypeptide as defined in (a), (b), (c), (d), (e) or (f).

[0314] 22. The polypeptide of embodiment 21, comprising a polypeptide having an amino acid sequence having at least about 86% sequence identity, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any one of SEQ ID NO: 1, 3, 6, 9, 11, 14, 17, 20, 22, or 25.

[0315] 23. A polynucleotide sequence encoding the polypeptide according to embodiment 21 or 22.

[0316] 24. The polynucleotide sequence of embodiment 23, wherein the polynucleotide sequence is selected from the group consisting of:

[0317] (a) a polynucleotide sequence comprising any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24, or 26, or comprising a polynucleotide sequence having at least 30% sequence identity to the polynucleotide sequence of any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24, or 26; or

[0318] (b) a polynucleotide sequence that hybridizes under conditions of preferably at least low stringency to the complementary strand of any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26; or

[0319] (c) a polynucleotide sequence that hybridizes, under conditions of preferably at least low stringency, to the complementary strand of a polynucleotide having at least 30% sequence identity to any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24 or 26;

[0320] (d) a polynucleotide sequence that is a degenerate as a result of the degeneracy of the genetic code of a polynucleotide sequence as defined in any one of (a), (b) or (c); or

[0321] (e) A polynucleotide sequence that is the complement of a nucleotide sequence defined in (a), (b), (c) or (d).

[0322] 25. The polynucleotide sequence of embodiment 5, which has at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, most preferably at least 93%, most preferably at least about 95%, most preferably at least about 96%, most preferably at least about 97%, even most preferably at least about 98%, and even more preferably at least 99% sequence identity to any one of SEQ ID NO: 2, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 21, 23, 24, or 26.

[0323] 26. A nucleic acid construct comprising the polynucleotide sequence of any one of embodiments 23 to 25.

[0324] 27. The nucleic acid construct of embodiment 26, which is an expression vector, wherein the polynucleotide sequence of any one of embodiments 23 to 25 is operably linked to at least one control sequence for expressing the polynucleotide sequence in a host cell.

[0325] 28. A method of producing the polypeptide of embodiment 21 or 22, the method comprising:

[0326] (a) cultivating the host cell according to embodiment 1 under conditions conducive to production of the polypeptide by the host cell, and optionally

[0327] (b) recovering the polypeptide.

[0328] The reference herein to a patent document or other matter cited as prior art is not to be taken as an admission that the document or matter is known or that the information it contains was part of the common general knowledge at the priority date of any claim.

[0329] The disclosure of each reference listed herein is incorporated by reference in its entirety.

[0330] The present invention is further illustrated by the following examples: Example

[0331] Example 1: Overexpression of ERG20, BTS1 and tHMG in Saccharomyces cerevisiae

[0332] For overexpression of ERG20, BTS1tHMG1, the expression cassette was designed to be integrated into a locus using the technology described in WO2013 / 076280. In order to amplify the 5' and 3' integration flanks of the integration locus, suitable primers and genomic DNA from the CEN.PK yeast strain (van Dijken et al. Enzyme and Microbial Technology 26 (2000) 706-714) were used. Different genes were ordered as cassettes (containing homologous sequences, promoters, genes, terminators, homologous sequences) in DNA2.0. The genes in these cassettes were flanked by constitutive promoters and terminators. See Table 1. Plasmid DNA containing ERG20, tHMG1, and BTS1 cassettes from DNA2.0 was dissolved to a concentration of 100 ng / μl. In a 50 μl PCR mixture, 20 ng of template was used together with 20 pmol of primers. The material was dissolved to a concentration of 0.5 μg / μl.

[0333] Table 1: Composition of overexpression constructs

[0334] promoter ORF terminator Eno2 (SEQ ID NO: 30) ERG20 (SEQ ID NO: 31) Adh1 (SEQ ID NO: 32) Fba1 (SEQ ID NO: 33) tHMG1 (SEQ ID NO: 34) Adh2 (SEQ ID NO: 35) Tef1 (SEQ ID NO: 36) BTS1 (SEQ ID NO: 37) Gmp1 (SEQ ID NO: 38)

[0335] To amplify the selectable marker, the pUG7-EcoRV construct was used ( Figure 1 ) and appropriate primers. The KanMX fragment was purified from the gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research). The yeast strain Cen.PK113-3C was transformed with the fragments listed in Table 2.

[0336] Table 2: DNA fragments used for transformation of ERG20, tHMG1, and BTS1

[0337] snippet 5'YPRcTau3 ERG20 box tHMG1 box KanMX Box BTS1 Box 3'YPRcTau3

[0338] After transformation and recovery for 2.5 hours at 30°C in YEPhD (yeast extract phytone dextrose; BBL phytone from BD), cells were plated on YEPhD agar with 200 μg / ml G418 (Sigma). Plates were incubated at 30°C for 4 days. Correct integration was confirmed by diagnostic PCR and sequencing. Overexpression was confirmed by LC / MS on the protein. Figure 2 A schematic diagram of the assembly of ERG20, tHMG1, and BTS1 is shown in Figure 1. This strain was named STV002.

[0339] Expression of the CRE-recombinase in this strain results in epi-recombination of the KanMX marker. Diagnostic PCR was used to confirm the presence and correct epi-recombination of ERG20, tHMG and BTS1.

[0340] Example 2. Knockdown of Erg9

[0341] To reduce the expression of Erg9, an Erg9 knockdown construct was designed and used, which contained a modified 3' end that continued into the TRP1 promoter driving TRP1 expression.

[0342] The construct containing the Erg9-KD fragment was transformed into E. coli TOP10 cells. The transformants were grown in 2PY (2 times plant peptone yeast extract), sAMP medium. The plasmid DNA was isolated using a QIAprep spin miniprep kit (Qiagen) and digested with SalI-HF (New England Biolabs). For concentration, the DNA was precipitated with ethanol. The fragment was transformed into Saccharomyces cerevisiae and the colonies were plated on agar plates containing tryptophan-free inorganic medium (Verduyn et al., 1992. Yeast 8:501-517). The correct integration of the Erg9-KD construct was confirmed by diagnostic PCR and sequencing. A schematic diagram of the transformation of the Erg9-KD construct is shown in Figure 3 The strain was named STV003.

[0343] Example 3. Overexpression of UGT2 1a

[0344] For overexpression of UGT2_1a, techniques as described in patent application numbers WO2013 / 076280 and WO2013 / 144257 were used. UGT2_1a was ordered as a cassette (containing homologous sequences, promoter, gene, terminator, homologous sequences) from DNA2.0. For details, see Table 3. To obtain a fragment containing the marker and Cre recombinase, techniques as described in patent application number WO2013 / 135728 were used. A NAT marker, which confers resistance to nourseothricin, was used for selection.

[0345] Table 3: Composition of overexpression constructs

[0346]

[0347] Appropriate primers were used for amplification. To amplify the 5' and 3' integration flanks of the integration locus, appropriate primers and genomic DNA from the CEN.PK yeast strain were used.

[0348] The Saccharomyces cerevisiae yeast strain STV003 was transformed with the fragments listed in Table 4, and the transformation mixture was plated on YEPhD agar plates containing 50 μg / ml nourseothricin (Lexy NTC from Jena Bioscience).

[0349] Table 4: DNA fragments used for transformation of UGT2 1a

[0350] snippet 5'Chr09.01 UGT2_1a box NAT-CR RE 3'Chr09.01

[0351] The expression of the CRE recombinase was activated by the presence of galactose. To induce the expression of the CRE recombinase, the transformants were restreaked on YEPh galactose medium. This resulted in exo-recombination of the marker located between the lox sites. Correct integration of UGT2_1a and exo-recombination of the NAT marker were confirmed by diagnostic PCR. The resulting strain was named STV004. A schematic diagram of the transformation of the UGT2_1a construct is shown in Figure 4 Shown in.

[0352] Example 4. Overexpression of RebA production pathway : CPS, KS, KO, KAH, CPR, UGT1, UGT3 and UGT4.

[0353] All pathway genes leading to RebA production were designed to be integrated into one locus using the technology described in patent application numbers WO2013 / 076280 and WO2013 / 144257. To amplify the 5' and 3' integration flanks of the integration locus, appropriate primers and genomic DNA from the CEN.PK yeast strain were used. Different genes were ordered as cassettes (containing homologous sequences, promoters, genes, terminators, homologous sequences) in DNA2.0 (see Table 5 for an overview). DNA from DNA2.0 was dissolved to 100 ng / μl. This stock solution was further diluted to 5 ng / μl, with 1 μl used in a 50 μl-PCR mixture. The reaction contained 25 pmol of each primer. After amplification, the DNA was purified using a NucleoSpin 96 PCR cleanup kit (Macherey-Nagel) or concentrated using ethanol precipitation.

[0354] Table 5 Sequences for the production pathway of RebA

[0355]

[0356] All fragments (tags and flanks) of the pathway to RebA (see Table 6 for an overview) were transformed into Saccharomyces cerevisiae strain STV004. After overnight recovery in YEPhD at 20°C, the transformation mixture was plated on YEPhD agar containing 200 μg / ml G418. These were incubated at 25°C for 3 days and at room temperature overnight.

[0357] Table 6. DNA fragments used for transformation of CPS, KS, KO, KanMX, KAH, CPR, UGT1, UGT3, and UGT4

[0358] snippet 5'INT1 CPS Box KS Box KO box KanMX Box KAH Box CPR Box UGT1 box UGT3 Box UGT4 Box 3'INT1

[0359] Correct integration was confirmed by diagnostic PCR and sequence analysis (3500 Genetic Analyzer, Applied Biosystems). Sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies). Each reaction (10 μl) contained 50 ng of template and 3.2 pmol of primer. The product was purified by ethanol / EDTA precipitation, dissolved in 10 μl of HiDi formamide, and applied to the device. The strain was designated STV006. A schematic diagram of how the pathway from GGPP to RebA integrates into the genome is shown in Figure 5 Table 7 lists the strains used in Examples 1 to 5.

[0360] Table 7. List of strains

[0361]

[0362]

[0363] Example 5. Removal of the KanMX selection marker from STV006

[0364] In order to remove the KanMX marker present in the strain, the plasmid pSH65 ( Figure 6 ) was transformed into STV006. Transformants were first selected on YEPD medium containing 20 μg / ml phleomycin (Invitrogen) and then restreaked on YEP galactose medium to induce CRE recombinase expression. Correct epi-recombination of the marker was confirmed by diagnostic PCR. RebA production by this marker-free strain was confirmed in production experiments. The untagged version of STV006 was designated STV008.

[0365] Example 6. Removal of UGT2 1a in STV008 by NAT selection marker

[0366] To remove UGT2_1a located at the Chr09.01 locus of STV008, a genomic DNA fragment containing the Chr09.01 integration flank ( Figure 8 ) were isolated from plasmid pUG7-NAT ( Figure 7 ) amplified the nourseothricin selection (NAT) marker and surrounding lox sites. The PCR product was purified using a NucleoSpin gel and PCR cleanup kit (Macherey-Nagel) and transformed into STV008. Transformants were selected on YEPD containing 50 μg / ml nourseothricin (LexyNTC from Jena Bioscience). Correct integration of the NAT marker and the absence of UGT2_1a were confirmed by diagnostic PCR. This new strain was designated STV009.

[0367] Example 7. Removal of the Nat selection marker of STV009

[0368] To be able to use the same integration locus to test UGT2 variants, the NAT marker had to be removed from strain STV009 ( Figure 9 Therefore, the CRE recombinase located on plasmid pSH65 was transformed into STV009, and transformants were selected on YEPD containing 20 μg / ml phleomycin. Colonies were restreaked on YEP galactose agar plates. The plates were incubated at 30°C. Diagnostic PCR confirmed the removal of the NAT marker by the CRE recombinase. In production experiments, the STV009ΔNAT strain was shown to accumulate the same amount of rubusoside as its parent, STV009. This new strain was designated STV053.

[0369] Example 8. Integration of UGT2 gene variants at the Chr09.01 locus

[0370] Different gene variants encoding UGT2 activity (SEQ ID NOs: 4, 7, 10, 12, 15, 18, 21, 23, and 28) were individually integrated into the Chr09.01 locus using several separate DNA fragments containing 50 bp flanking homology segments for recombination ( Figure 10 ).

[0371] The 5'- and 3'-Chr09.01 integration flanks were amplified using appropriate primers from genomic DNA of the CEN.PK yeast strain (van Dijken et al. Enzyme and Microbial Technology 26 (2000) 706-714). For the 5'-flank, the reverse primer contained an extended 50 bp sequence homologous to the promoter sequence to be used, i.e., the ScENO1 promoter (SEQ ID NO: 60). The forward primer for the 3'-flank contained a 50 bp linker extension.

[0372] The KanMX selection marker was amplified from the pUG7-EcoRV construct. The forward primer contained an additional 50 bp sequence homologous to the KIGAP1 (SEQ ID NO: 61) terminator. The reverse primer also had a 50 bp linker extension.

[0373] Different UGT2 gene variants were ordered from SGI-DNA. Their open reading frames were flanked by 50 bp of the pScENO1 promoter (SEQ ID NO: 60) upstream and 50 bp of the Klgap1T terminator (SEQ ID NO: 61) downstream. The genes were amplified from the SGI-DNA construct using primers that anneal to these promoter and terminator sequences.

[0374] PCR products were purified using the NucleoSpin 96 PCR Clean-Up Kit (Macherey-Nagel). For each UGT2 variant to be tested, equimolar amounts of the 5'-Chr09.01 flank, the ENO1 promoter, the UGT2 gene, the KIGAP1 terminator, the KanMX selectable marker, and the 3'-Chr09.01 flank were combined. A separate mixture containing UGT2_1a was prepared. These mixtures were transformed into STV053 and plated on YEPD containing 200 μg / ml G418.

[0375] For each UGT2 variant, several replicate transformants were tested in production experiments.

[0376] Example 9. Production of rebaudioside A using Saccharomyces cerevisiae

[0377] The preculture was inoculated with colony material from YEPD agar. The preculture was grown in 200 μl of inorganic medium with glucose as carbon source. The preculture was incubated in an Infors incubator at 27° C., 750 rpm and 80% humidity for 72 hours.

[0378] 40 μl of pre-culture was used to inoculate 2.5 ml of inorganic medium with glucose as the carbon source. The main culture was incubated in an Infors incubator at 27°C, 550 rpm, and 80% humidity for 120 hours. The culture was well homogenized by pipetting back and forth, and 1 ml of the culture was transferred to a 96-well plate. The 96-well plate was incubated in a water bath at 95°C for 15 minutes and cooled to room temperature. 0.5 ml of acetonitrile was added to each well and homogenized by pipetting back and forth. Cell debris was precipitated by centrifugation at 3000 x g for 10 minutes. The supernatant was diluted 200-fold in 33% acetonitrile.

[0379] The samples were analyzed for RebA using LC / MS. RebA (RV0141-94, DAE Pyung Co. Ltd.) was used as a standard. It was found that strains with specific UGT2 gene variants as described produced higher RebA titers compared to strains containing UGT2_1a, as shown in Tables 8 and Figure 11 As shown in .

[0380] Table 8: Rebaudioside A production in Saccharomyces strains expressing UGT2 variant enzymes

[0381] UGT2 variants RebA (mg / L) UGT2_1b 30,8 UGT2_2b 38,4 UGT2_3b 40,6 UGT2_4b 26,5 UGT2_5b 41,8 UGT2_6b 28,1 UGT2_7b 33,3 UGT2_8b 25,2 UGT2_9b 26,7 UGT2_1a 25,0

[0382] Example 10: Production of rebaudioside M using Saccharomyces cerevisiae

[0383] The preculture was inoculated with colony material from YEPD agar. The preculture was grown in 200 μl of inorganic medium with glucose as carbon source. The preculture was incubated in an Infors incubator at 27° C., 750 rpm and 80% humidity for 72 hours.

[0384] 40 μl of pre-culture was used to inoculate 2.5 ml of inorganic medium with glucose as the carbon source. The main culture was incubated in an Infors incubator at 27°C, 550 rpm, and 80% humidity for 120 hours. The culture was well homogenized by pipetting back and forth, and 1 ml of the culture was transferred to a 96-well plate. The 96-well plate was incubated in a water bath at 95°C for 15 minutes and cooled to room temperature. 0.5 ml of acetonitrile was added to each well and homogenized by pipetting back and forth. Cell debris was precipitated by centrifugation at 3000 x g for 10 minutes. The supernatant was diluted 200-fold in 33% acetonitrile.

[0385] The presence of Reb M was confirmed by LC and MS analysis using an LTQ Orbitrap (Thermo) equipped with an Acella LC and a Waters Acquity UPLC BEH Amide 1.7 μm 2.1*150 mm column. The eluents used for the separation were A: 10 mM ammonium acetate in MilliQ water, B: acetonitrile, and the gradient started at 65% A and held for 1.5 minutes, then increased to 95% B over 0.5 minutes and held for 0.5 minutes, followed by regeneration at 65% A for 1.5 minutes. The flow rate was 0.6 ml / min, and the column temperature was maintained at 50°C. Mass spectrometry analysis was performed in negative electrospray ionization mode, scanning from m / z 100-1800 at a resolution of 7500. Reb M eluted at tr = 0.72 minutes, just after reb D at tr = 0.63. Reb M is characterized by a deprotonated molecule at m / z 1289.5286. Accurate mass analysis can be used to estimate elemental composition.

[0386] It was found that strains with specific UGT2 gene variants as described produced higher RebM titers compared to strains containing UGT2_1a, such as Figure 12 and as shown in Table 9.

[0387] Table 9. Rebaudioside M production in Saccharomyces strains expressing UGT2 variant enzymes as a percentage of UGT2 1a

[0388] UGT2 variants RebM (relative to UGT2_1a) UGT2_7b 555 UGT2_9b 256 UGT2_1a 100

[0389] Example 11: Description of steviol glycoside producing strain ML14094 (MAT-A lineage)

[0390] Two strains of Yarrowia lipolytica, mating types MATA and MATB, were engineered for the production of steviol glycosides. These strains were mated, diploid spores formed, and spores with steviol glycoside production were selected. One of these spores was further developed for the production of steviol glycosides, including rebaudioside A.

[0391] Step 1: Transform strain ML10371 (MAT-A, lys1-, ura3-, leu2-) with the five defined DNA fragments. All transformations were performed using a lithium acetate / PEG fungal transformation protocol and selected on appropriate minimal media YPD+100 μg / ml nourseothricin or YPD+100 μg / ml hygromycin.

[0392] 1) In plasmid MB6969 ( Figure 13) was isolated by gel purification after HindIII / NotI digestion of 1% WT and 1% WT. This construct encodes a synthetic construct for overexpression of UGT2-1a (SEQ ID NO: 29) linked to the pPGM promoter (SEQ ID NO: 62) and xprT terminator (SEQ ID NO: 69) flanked by lox sites (Guldener et al., 1996, Lambert et al., 2007) and the HPH hygromycin resistance gene, as well as a synthetic construct for overexpression of the codon-optimized Yarrowia lipolytica hydroxymethylglutaryl-CoA reductase open reading frame lacking a 5' membrane anchor sequence (tHMGopt: SEQ ID NO: 75) linked to the pHSP promoter (SEQ ID NO: 63) and cwpT terminator (SEQ ID NO: 70).

[0393] 2) In MB6856( Figure 14 A 2.7 kb DNA fragment was isolated by gel purification after HindIII / SspI digestion of 5-11a. This construct encodes tHMGopt (SEQ ID NO: 75) linked to the pHYPO promoter (SEQ ID NO: 64) and the gpdT terminator (SEQ ID NO: 71).

[0394] 3) In MB6857( Figure 15 A 2.5 kb DNA fragment was isolated by gel purification after SspI digestion of 1% tHMGopt (SEQ ID NO: 75) linked to the pHSP promoter (SEQ ID NO: 63) and the cwpT terminator (SEQ ID NO: 70).

[0395] 4) In MB6948( Figure 16 ) was isolated by gel purification following SspI digestion of a 2.0 kb DNA fragment. This construct encodes a synthetic construct for overexpression of codon-optimized Yarrowia lipolytica geranyl-geranyl-pyrophosphate synthase (GGSopt: SEQ ID NO: 76) linked to the pHSP promoter (SEQ ID NO: 63) and cwpT terminator (SEQ ID NO: 70).

[0396] 5) In MB6958( Figure 17 A 2.2 kb DNA fragment was isolated by gel purification after HindIII / SspI digestion of 5-10 μg / mL of 1% β-lactamase (5-10 μg / mL). This construct encodes GGSopt (SEQ ID NO: 76) linked to the pHYPO promoter (SEQ ID NO: 64) and the gpdT terminator (SEQ ID NO: 71). The resulting strain was designated ML13462.

[0397] Step 2. Use plasmid MB7015 ( Figure 18 ) was digested with Sfil and isolated by gel purification to transform strain ML13462. This construct encodes a synthetic construct for overexpression of UGT1 (SEQ ID NO: 77) linked to the pENO (SEQ ID NO: 65) promoter and gpdT terminator (SEQ ID NO: 71), UGT3 (SEQ ID NO: 78) linked to the pHSP promoter (SEQ ID NO: 63) and pgmT terminator (SEQ ID NO: 72), UGT4 (SEQ ID NO: 79) linked to the pCWP (SEQ ID NO: 66) promoter and pgkT terminator (SEQ ID NO: 73), and a lox-flanked nourseothricin resistance marker (NAT). Note that the placement of the lox sites allows for subsequent removal of nourseothricin resistance by CRE recombinase-mediated recombination. The nourseothricin-resistant isolate was designated ML13500.

[0398] Step 3. Use plasmid MB6986 ( Figure 19 The 9.1 kb fragment isolated by gel purification after PvuI / SapI digestion of the 5-amino-3-pyrimidine antagonist (5-amino-3-pyrimidine antagonist) was transformed into strain ML13500. This construct encodes tHMGopt (SEQ ID NO: 75) linked to the pHSP promoter (SEQ ID NO: 63) and the cwpT terminator (SEQ ID NO: 70), the lox-flanked URA3 impact (URA3blaster) prototrophy marker, and GGSopt (SEQ ID NO: 76) linked to the pHYPO promoter (SEQ ID NO: 64) and the gpdT terminator (SEQ ID NO: 71). Transformants were selected on minimal medium lacking uracil. One selected uracil prototroph was designated ML13723.

[0399] Step 4. Use plasmid MB7059 ( Figure 20The 18.1 kb fragment isolated by gel purification after Sfil digestion of 5-nitro-1-nitro-1-phosphate was transformed into strain ML13723. MB7059 encodes tCPS_SR (SEQ ID NO:80) linked to the pCWP promoter (SEQ ID NO:66) and cwpT terminator (SEQ ID NO:70), tKS_SR (SEQ ID NO:81) linked to the pHYPO promoter (SEQ ID NO:64) and gpdT terminator (SEQ ID NO:71), KAH_4 (SEQ ID NO:92) linked to the pHSP promoter (SEQ ID NO:63) and pgmT terminator (SEQ ID NO:72), KO_Gib (SEQ ID NO:83) linked to the pTPI promoter (SEQ ID NO:67) and pgkT terminator (SEQ ID NO:73), CPR_3 (SEQ ID NO:84) linked to the pENO promoter (SEQ ID NO:65) and xprT terminator (SEQ ID NO:69), and the native Yarrowia lipolytica LEU2 locus. One of the selected rebaudioside A-producing transformants was designated ML14032.

[0400] Step 5. Strain ML14032 was streaked onto YPD and grown overnight, then streaked onto 5-FOA plates to allow recombination-mediated loss of the URA3 marker introduced in step 3. One selected 5-FOA-resistant transformant was designated ML14093.

[0401] Step 6. Use plasmid MB7100 ( Figure 21The 19.0 kb fragment isolated by gel purification after Sfil digestion of 5-nitropropene was transformed into strain ML14093. MB7100 encodes tCPS_SR (SEQ ID NO: 80) connected to the pHYPO promoter (SEQ ID NO: 64) and cwpT terminator (SEQ ID NO: 70), tKS_SR (SEQ ID NO: 81) connected to the pCWP promoter (SEQ ID NO: 66) and gpdT terminator (SEQ ID NO: 71), KAH_4 (SEQ ID NO: 82) connected to the pHSP promoter (SEQ ID NO: 63) and pgmT terminator (SEQ ID NO: 72), KO_Gib (SEQ ID NO: 83) connected to the pENO promoter (SEQ ID NO: 65) and pgkT terminator (SEQ ID NO: 73), CPR_3 (SEQ ID NO: 84) connected to the pTPI promoter (SEQ ID NO: 67) and xprT terminator (SEQ ID NO: 69), and the URA3 impact prototrophy marker. Transformants were selected on minimal medium lacking uracil. One selected rebaudioside A-producing uracil prototroph was designated ML14094.

[0402] Example 12 . Description of steviol glycoside-producing strain ML14087 (MAT-B lineage) :

[0403] Step 1. Transform strain ML13206 (MAT-B, ade1-, ure2-, leu2-) with the five defined DNA fragments. All transformations were performed using the lithium acetate / PEG fungal transformation protocol and selected on appropriate minimal media YPD + 100 μg / ml nourseothricin or YPD + 100 μg / ml hygromycin.

[0404] 1) In plasmid MB6969 ( Figure 13) was isolated by gel purification after HindIII / NotI digestion of 1% dapoxetine (1% dapoxetine). This construct encodes a synthetic construct for overexpression of a codon-pair-optimized (CpO) ORF of UGT2-1a (SEQ ID NO: 29) linked to the pPGM (SEQ ID NO: 62) promoter and xprT terminator (SEQ ID NO: 69) flanked by lox sites (Guldener et al., 1996, Lambert et al., 2007) and the HPH hygromycin resistance gene, as well as a synthetic construct for overexpression of a codon-optimized Yarrowia lipolytica hydroxymethylglutaryl-CoA reductase open reading frame lacking a 5' membrane anchor sequence (tHMGopt: SEQ ID NO: 75) linked to the pHSP promoter (SEQ ID NO: 63) and cwpT terminator (SEQ ID NO: 70).

[0405] 2) In MB6856( Figure 14 A 2.7 kb DNA fragment was isolated by gel purification after HindIII / SspI digestion of 5-11a. This construct encodes tHMGopt (SEQ ID NO: 75) linked to the pHYPO promoter (SEQ ID NO: 64) and the gpdT terminator (SEQ ID NO: 71).

[0406] 3) In MB6857( Figure 15 A 2.5 kb DNA fragment was isolated by gel purification after SspI digestion of 1% tHMGopt (SEQ ID NO: 75) linked to the pHSP promoter (SEQ ID NO: 63) and the cwpT terminator (SEQ ID NO: 70).

[0407] 4) A 2.0 kb DNA fragment was isolated by gel purification after SspI digestion of MB6948 ( Figure 16 This construct encodes a synthetic construct for overexpression of codon-optimized Yarrowia lipolytica geranyl-geranyl-pyrophosphate synthase (GGSopt: SEQ ID NO: 76) linked to the pHSP promoter (SEQ ID NO: 63) and cwpT terminator (SEQ ID NO: 70).

[0408] 5) In MB6958( Figure 17A 2.2 kb DNA fragment was isolated by gel purification after HindIII / SspI digestion of 5-10 μg / mL of 1% β-lactamase (5-10 μg / mL). This construct encodes GGSopt (SEQ ID NO: 76) linked to the pHYPO (SEQ ID NO: 64) promoter and the gpdT terminator (SEQ ID NO: 71). The resulting strain was designated ML13465.

[0409] Step 2. Transformation of strain ML13465 with two defined DNA fragments:

[0410] 1). In plasmid MB7015 ( Figure 18 ) was digested with Sfil and isolated by gel purification of a 9.7 kb fragment. This construct encodes a synthetic construct for overexpression of UGT1 (SEQ ID NO: 77) linked to the pENO promoter (SEQ ID NO: 65) and gpdT (SEQ ID NO: 71) terminator, UGT3 (SEQ ID NO: 78) linked to the pHSP promoter (SEQ ID NO: 63) and pgmT terminator (SEQ ID NO: 72), UGT4 (SEQ ID NO: 79) linked to the pCWP promoter (SEQ NO: 66) and pgkT terminator (SEQ ID NO: 73), and a lox-flanked nourseothricin resistance marker (NAT). Note that the placement of the lox sites allows for subsequent removal of nourseothricin resistance by CRE recombinase-mediated recombination.

[0411] 2). In plasmid MB6988 ( Figure 22 ) was isolated by gel purification after PvuI / SapI digestion of the 9.1 kb fragment. This construct encodes tHMGopt (SEQ ID NO: 75) linked to the pHSP promoter (SEQ ID NO: 63) and cwpT terminator (SEQ ID NO: 70), a lox-flanked URA2 impact prototrophy marker, and GGSopt (SEQ ID NO: 76) linked to the pHYPO promoter (SEQ ID NO: 64) and gpdT terminator (SEQ ID NO: 71). Strains were selected on YPD + 100 μg / ml nourseothricin, and replicates were plated on minimal medium lacking uracil. A nourseothricin-resistant uracil prototrophic isolate was designated ML13490.

[0412] Step 3. Strain ML13490 was streaked onto YPD and grown overnight, then streaked onto 5-FOA plates to allow recombination-mediated loss of the URA2 marker introduced above in step 3. One selected 5-FOA-resistant transformant was designated ML13501.

[0413] Step 4. Use plasmid MB6988 ( Figure 22 The 9.1 kb fragment isolated by gel purification after PvuI / SapI digestion of the 5-mer was transformed into strain ML13501. Transformants were selected on minimal medium lacking uracil. One selected uracil prototrophic microorganism was designated ML13724.

[0414] Step 5. Use plasmid MB7044 ( Figure 23 The 18.1 kb fragment isolated by gel purification after Sfil digestion of 5-nitropropene was transformed into strain ML13724. MB7044 encodes tCPS_SR (SEQ ID NO:80) linked to a pHYPO promoter (SEQ ID NO:64) and a cwpT terminator (SEQ ID NO:70), tKS_SR (SEQ ID NO:81) linked to a pCWP promoter (SEQ ID NO:66) and a gpdT terminator (SEQ ID NO:70), KAH_4 (SEQ ID NO:82) linked to a pHSP promoter (SEQ ID NO:63) and a pgmT terminator (SEQ ID NO:72), KO_Gib (SEQ ID NO:83) linked to a pENO promoter (SEQ ID NO:65) and a pgkT terminator (SEQ ID NO:73), CPR_3 (SEQ ID NO:84) linked to a pTPI promoter (SEQ ID NO:67) and an xprT terminator (SEQ ID NO:69), and the LEU2 locus. One of the selected rebaudioside A-producing transformants was designated ML14044.

[0415] Step 6. Strain ML14044 was streaked onto YPD and grown overnight, and then streaked onto 5-FOA plates to allow for recombination-mediated loss of the URA2 marker introduced above in step 4. One selected 5'-FOA-resistant transformant was designated ML14076.

[0416] Step 7. Use plasmid MB7094 ( Figure 24The 19.0 kb fragment isolated by gel purification after Sfil digestion of 5-nitropropene was transformed into strain ML14076. MB7094 encodes tCPS_SR (SEQ ID NO: 80) connected to the pHYPO promoter (SEQ ID NO: 64) and cwpT terminator (SEQ ID NO: 70), tKS_SR (SEQ ID NO: 81) connected to the pCWP promoter (SEQ ID NO: 66) and gpdT terminator (SEQ ID NO: 71), KAH_4 (SEQ ID NO: 82) connected to the pHSP promoter (SEQ ID NO: 63) and pgmT terminator (SEQ ID NO: 72), KO_Gib (SEQ ID NO: 83) connected to the pENO promoter (SEQ ID NO: 65) and pgkT terminator (SEQ ID NO: 73), CPR_3 (SEQ ID NO: 84) connected to the pTPI promoter (SEQ ID NO: 67) and xprT terminator (SEQ ID NO: 69), and the URA2 impact prototrophy marker. Transformants were selected on minimal medium lacking uracil. One selected rebaudioside A-producing uracil prototroph was designated ML14087.

[0417] Example 13. Joining MATA and MATB lines and selecting progeny for steviol glycoside production

[0418] Strains of opposite mating morphs (ML14094 and ML14087) with complementary auxotrophic deficiencies (ADE1+lys1- and ade1-LYS1+) were allowed to mate and then plated on a selective medium (minimal medium lacking adenine and lysine) that only allows diploid growth. Diploid cells (ML14143) were then induced to undergo meiosis and sporulation by starvation, and the resulting haploid progeny were replicate plated to identify prototrophic isolates with hygromycin and nourseothricin resistance. One selected rebaudioside A-producing strain was designated ML14737.

[0419] Example 14. Making the strain free of UGT2 1a

[0420] In use MB6128( Figure 25) After transformation, the hygromycin and nourseothricin antibiotic markers were removed from strain ML14737, and the MB6128 encoding construct was used to constitutively overexpress the CRE recombinase. The CRE recombinase deletes the antibiotic marker by recombination at the Lox66 and Lox71 sites. The inactive Lox72 site remains in the genome (Guldener et al., 1996, Lambert et al., 2007). Plasmid MB6128 is a CEN plasmid that replicates as an episome in Yarrowia lipolytica and contains the CRE recombinase coding region under the control of the native Yarrowia lipolytica pHHF promoter and hhfT terminator, and neoR (encoding G418 resistance) under the control of the native Yarrowia lipolytica pTEF1 promoter and xprT terminator. After selection of MB6128 transformants on YPD+G418 and selection of transformants that lost hygromycin and nourseothricin resistance through successful Cre-Lox recombination, sensitive colonies were grown on non-selective medium to remove the MB6128 CEN plasmid (the CEN plasmid had been lost spontaneously). The resulting antibiotic marker-free variant was designated ML14869. Due to the loss of UGT2_1a along with hygromycin resistance, this strain no longer produces rebaudioside A, but instead produces the intermediate rubusoside.

[0421] Example 15. Transformation of UGT2 gene

[0422] The UGT2 gene variants and the control UGT2_1a were placed behind the Yarrowia lipolytica pHSP promoter (SEQ ID NO: 63) and combined with the Yarrowia lipolytica terminator gpdT (SEQ ID NO: 71). Each UGT2 was assembled in Saccharomyces cerevisiae into a construct on the CEN plasmid p417[5-3] (see Figure 26 ).

[0423] Table 10. Promoters, ORFs, and terminators used in the construction of strains with UGT2 variants

[0424]

[0425] These constructs (one for each UGT2) were used as templates in PCR to amplify the 5'- and 3'-parts (see Figure 27 The 5' portion consists of everything between the start of the 3'-GSY1 integration flank and the end of the KanMX open reading frame. The 3' portion consists of everything between the second codon of the KanMX open reading frame and the end of the 5'-GSY1 integration flank.

[0426] For UGT2 testing, each 5'-part and 3'-part combination was transformed into strain ML14869. Transformants were selected on YPD medium containing G418. Twelve colonies were selected from each transformation for production experiments.

[0427] Example 16. Production of RebA using Yarrowia lipolytica

[0428] The pre-culture was inoculated with the colony material from YEPh-D agar. The pre-culture was grown in 200 μl YEP with glucose as a carbon source. The pre-culture was hatched for 72 hours in an Infors incubator at 27°C, 750rpm and 80% humidity. 40 μl of the pre-culture was inoculated with 2.5 ml YEP with glucose as a carbon source. The main culture was hatched for 120 hours in an Infors incubator at 27°C, 550rpm and 80% humidity. After 120 hours, the main culture was centrifuged at 2750rpm for 10 minutes. 100 μl was taken out from the supernatant and diluted 2.5 times in 55% acetonitrile. This was further diluted in 33% acetonitrile.

[0429] The results are Figure 28 and are shown in Table 11. As can be seen, strains expressing variant UGT2 produced higher RebA titers.

[0430] Table 11: Rebaudioside A production in Yarrowia strains expressing UGT2 variant enzymes

[0431] sample RebA (mg / L) UGT2_2b 254 UGT2_3b 396 UGT2_5b 422 UGT2_9b 246 UGT2_10b 249 UGT2_1a 198

[0432] Example 17. Production of RebM using Yarrowia lipolytica

[0433] The pre-culture was inoculated with the colony material from YEPh-D agar. The pre-culture was grown in 200 μl YEP with glucose as a carbon source. The pre-culture was hatched for 72 hours in an Infors incubator at 27°C, 750rpm and 80% humidity. 40 μl of the pre-culture was inoculated with 2.5 ml YEP with glucose as a carbon source. The main culture was hatched for 120 hours in an Infors incubator at 27°C, 550rpm and 80% humidity. After 120 hours, the main culture was centrifuged at 2750rpm for 10 minutes. 100 μl was taken out from the supernatant and diluted 2.5 times in 55% acetonitrile. This was further diluted in 33% acetonitrile.

[0434] The results are Figure 29 and are shown in Table 12. As can be seen, strains expressing variant UGT2 produced higher RebM titers.

[0435] Table 12: Production of rebaudioside M in Yarrowia strains expressing UGT2 variant enzymes

[0436] sample RebM (mg / L) UGT2_7b 37,5 UGT2_1a 23,3

[0437] Example 18:

[0438] To evaluate the effects of different UGT2 variants on steviol glycoside production in a bioreactor, two strains described in Example 15 were selected. One strain expressed UGT2_6b and the other expressed UGT2_7b. The fermentation protocol employed was a fed-batch fermentation, and whole broth samples were collected daily for analysis of steviol glycosides using LC / MS.

[0439] If you can Figure 30 As can be seen in Figure 4, the strain expressing UGT2_6b produced more RebA than the strain expressing UGT2_7b. However, the strain expressing UGT2_7b produced significantly more RebM than the strain expressing UGT2_6b. Both strains produced more RebA than RebM. At the end of the fermentation, the RebA concentration was 20 times the RebM concentration in the strain expressing UGT2_6b, while the RebA concentration was 4 times the RebM concentration in the strain expressing UGT2_7b. The different product ratios reflect the intrinsic differences in the properties of UGT2, where UGT2_7b has a higher glucose glycosylation activity at position 19 compared to UGT2_6b. Products such as RebE and RebD, derived from the glycosylation reaction at position 19 of stevioside and RebA, respectively, are further converted to RebM by the activity of UGT4, see Figure 32 .

[0440] This demonstrates that by using the different variants of UGT2 described here, production can be efficiently directed towards the target product.

[0441] Table 13: Description of Sequence Listing

[0442]

[0443]

[0444]

Claims

1. A recombinant host comprising a recombinant nucleic acid sequence encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:

17.

2. The recombinant host according to claim 1, which is capable of producing glycosylated diterpenes.

3. The recombinant host according to claim 1 or 2, comprising one or more recombinant nucleotide sequences encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-kaurene synthase activity; A polypeptide having ent-kaurene oxidase activity; and A polypeptide having kaurenoate 13-hydroxylase activity. The recombinant host according to claim 1 or 2, comprising a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity.

5. The recombinant host according to claim 1 or 2, comprising a recombinant nucleic acid sequence encoding one or more of the following: (i) a polypeptide having UGT74G1 activity; (ii) a polypeptide having UGT85C2 activity; and (iii) A polypeptide having UGT76G1 activity.

6. The recombinant host of claim 1 or 2, comprising a recombinant nucleic acid sequence encoding an additional polypeptide having UGT2 activity, the polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:

29.

7. The recombinant host according to claim 1 or 2, wherein the host belongs to one of the following genera: Saccharomyces ( Saccharomyces ), Aspergillus ( Aspergillus ), Pichia ( Pichia ), Kluyveromyces ( Kluyveromyces ), Candida spp. ( Candida ), Hansenula ( Hansenula ), Humicola ( Humicola ), Issaffine ( Issatchenkia ), Trichosporon spp. ( Trichosporon ), Brettanomyces spp. Brettanomyces ), Pachysolensis ( Pachysolen ), Yarrowia ( Yarrowia )、 Yamadazyma or Escherichia ( Escherichia ).

8. The recombinant host according to claim 7, wherein the recombinant host is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) cells, Yarrowia lipolytica ( Yarrowia lipolytica ) cells, Candida krusei ( Candida krusei ) cells, Ississa officinalis ( Issatchenkia orientalis ) cells or Escherichia coli ( Escherichia coli )cell.

9. The recombinant host according to claim 1 or 2, wherein the host's ability to produce geranylgeranyl diphosphate (GGPP) is upregulated.

10. The recombinant host of claim 1 or 2, comprising one or more recombinant nucleic acid sequences encoding hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthase, and geranylgeranyl diphosphate synthase.

11. The recombinant host according to claim 1 or 2, comprising a nucleic acid sequence encoding one or more of: A polypeptide having hydroxymethylglutaryl-CoA reductase activity; a polypeptide having farnesyl-pyrophosphate synthetase activity; A polypeptide having geranylgeranyl diphosphate synthase activity.

12. A method for preparing a glycosylated diterpene, the method comprising fermenting the recombinant host according to any one of claims 2 to 11 in a suitable fermentation medium, and optionally recovering the glycosylated diterpene.

13. The method according to claim 12, for preparing glycosylated diterpenes, wherein the method is carried out on an industrial scale.

14. A method for converting a first glycosylated diterpene into a second glycosylated diterpene, wherein The first glycosylated diterpene is steviol-13-monoside, steviol-19-monoside, rubusoside, stevioside, rebaudioside A or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, and The second glycosylated diterpene is steviol-19-bioside, steviolbioside, stevioside, RebE, RebD or 13-[(β-D-glucopyranosyl)oxy)kaur-16-ene-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, The method comprises: - contacting the first glycosylated diterpene with a recombinant host according to any one of claims 1 to 11, a cell-free extract derived from such a recombinant host, or an enzyme preparation derived from any one of the foregoing; - thereby converting the first glycosylated diterpene into the second glycosylated diterpene.

15. A polypeptide having UGT2 activity, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 17; or (b) a polypeptide encoded by a polynucleotide consisting of the polynucleotide sequence set forth in any one of SEQ ID NO: 18 or 19.

16. A polynucleotide sequence encoding the polypeptide according to claim 15. A nucleic acid construct comprising the polynucleotide sequence of claim 16 .

18. The nucleic acid construct according to claim 17, which is an expression vector, wherein the polynucleotide sequence according to claim 16 is operably linked to at least one control sequence for expressing the polynucleotide sequence in a host cell.

19. A method for producing the polypeptide of claim 15, comprising: (a) cultivating the host cell according to claim 1 under conditions conducive to production of the polypeptide by the host cell, and optionally (b) recovering the polypeptide.

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