GERANYLGERANYL PYROPHOSPHATE SYNTHASE

Abstract

The present invention relates to a recombinant host capable of producing a steviol glycoside comprising a recombinant nucleic acid sequence encoding a polypeptide having geranylgeranyl pyrophosphate (GGPP) synthase activity which comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 45% sequence identity thereto.

Description

FIELD OF THE INVENTION

The present invention relates to a recombinant host capable of producing a steviol glycoside. The invention also relates to a process for the preparation of a steviol glycoside using such a recombinant host and to a fermentation broth which may be the result of such a process. The invention further relates to a steviol glycoside obtained by such a process or obtainable from such a fermentation broth and to a composition comprising two or more such steviol glycosides. In addition the invention relates to a foodstuff, feed or beverage which comprises such a steviol glycoside or a such composition.

BACKGROUND TO THE INVENTION

The leaves of the perennial herb, Stevia rebaudiana Bert., accumulate quantities of intensely sweet compounds known as steviol glycosides. Whilst the biological function of these compounds is unclear, they have commercial significance as alternative high potency sweeteners.

These sweet steviol glycosides have functional and sensory properties that appear to be superior to those of many high potency sweeteners. In addition, studies suggest that stevioside can reduce blood glucose levels in Type II diabetics and can reduce blood pressure in mildly hypertensive patients.

Steviol glycosides accumulate in Stevia leaves where they may comprise from 10 to 20% of the leaf dry weight. Stevioside and rebaudioside A are both 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, rebaudioside A between 150 and 320 times sweeter than sucrose. In addition, rebaudioside D is also a high-potency diterpene glycoside sweetener which accumulates in Stevia leaves. It may be about 200 times sweeter than sucrose. Rebaudioside M is a further high-potency diterpene glycoside sweetener. It is present in trace amounts in certain stevia variety leaves, but has been suggested to have a superior taste profile.

Steviol glycosides have traditionally been extracted from the Stevia plant. In Stevia, (−)-kaurenoic acid, an intermediate in gibberellic acid (GA) biosynthesis, is converted into the tetracyclic diterpene steviol, which then proceeds through a multi-step glycosylation pathway to form the various steviol glycosides. However, yields may be variable and affected by agriculture and environmental conditions. Also, Stevia cultivation requires substantial land area, a long time prior to harvest, intensive labour and additional costs for the extraction and purification of the glycosides.

More recently, interest has grown in producing steviol glycosides using fermentative processes. WO2013/110673 and WO2015/007748 describe microorganisms that may be used to produce at least the steviol glycosides rebaudioside A, rebaudioside M and rebaudioside D.

Further improvement of such microoganisms is desirable in order that higher amounts of steviol glycosides may be produced and/or additional or new steviol glycosides and/or higher amounts of specific steviol glycosides and/or mixtures of steviol glycosides having desired ratios of different steviol glycosides.

SUMMARY OF THE INVENTION

In Stevia rebaudiana, 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) results in the formation of (−)-Kaurene which is then oxidized in a three step reaction by (−)-kaurene oxidase (KO) to form (−)-kaurenoic acid.

In Stevia rebaudiana leaves, (−)-kaurenoic acid is then hydroxylated, by ent-kaurenoic acid 13-hydroxylase (KAH) to form steviol. Steviol is then glucosylated by a series of UDP-glucosyltransferases (UGTs) leading to the formation of a number of steviol glycosides. Specifically, these molecules can be viewed as a steviol molecule, with its carboxyl hydrogen atom replaced by a glucose molecule to form an ester, and an hydroxyl hydrogen with combinations of glucose and rhamnose to form an acetal.

These pathways may be reconstructed in recombinant hosts, for example yeasts such as yeasts of the genera Saccharomyces and Yarrowia.

Accordingly, the invention relates to a recombinant host capable of producing a steviol glycoside and which comprises a recombinant nucleic acid sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 45% sequence identity thereto. Typically, the polypeptide has geranylgeranyl pyrophosphate synthase activity.

The invention also relates to:

• • a process for the preparation of a steviol glycoside which comprises fermenting a recombinant host of the invention in a suitable fermentation medium and, optionally, recovering the steviol glycoside;
  • a fermentation broth comprising a steviol glycoside obtainable by such a process;
  • a steviol glycoside obtainable by such a process or obtainable from such a fermentation broth;
  • a composition comprising two or more such diterpenes; and
  • a foodstuff, feed or beverage which comprises such a steviol glycoside or such a composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets out sets out sets out a schematic diagram of the potential pathways leading to biosynthesis of steviol glycosides.

FIG. 2 sets out a schematic representation of the plasmid MB6969, encoding tHMG, UGT2_1a, HPH

FIG. 3 sets out a schematic representation of the plasmid MB6856, encoding tHMG.

FIG. 4 sets out a schematic representation of the plasmid MB6857, encoding tHMG

FIG. 5 sets out a schematic representation of the plasmid MB6948, encoding GGS

FIG. 6 sets out a schematic representation of the plasmid MB6958, encoding GGS

FIG. 7 sets out a schematic representation of the plasmid MB7015, encoding UGT1, UGT3, UGT4, NAT

FIG. 8 sets out a schematic representation of the plasmid MB6986, encoding tHMG, URA3, GGS

FIG. 9 sets out a schematic representation of the plasmid MB7059, encoding tCPS_SR, tKS_SR, KAH_4, KO_Gib, CPR_3, LEU2

FIG. 10 sets out a schematic representation of the plasmid MB7100, encoding tCPS_SR, tKS_SR, KAH_4, KO_Gib, CPR_3, URA3

FIG. 11 sets out a schematic representation of the plasmid MB6988, encoding tHMG, URA2, GGS

FIG. 12 sets out a schematic representation of the plasmid MB7044, encoding tCPS_SR, tKS_SR, KAH_4, KO_Gib, CPR_3, LEU2

FIG. 13 sets out a schematic representation of the plasmid MB7094, encoding tCPS_SR, tKS_SR, KAH_4, KO_Gib, CPR_3, URA2

FIG. 14 sets out a schematic representation of the plasmid MB6128, encoding CRE, neoR

FIG. 15 sets out a schematic representation of the construct containing KAH and HPH

FIG. 16 sets out a schematic representation of the construct containing tCPS_SR

FIG. 17 sets out a schematic representation of the plasmid MB6986, encoding tHMG, URA3, GGS.

FIG. 18 sets out rebaudioside A production in a batch fermentation assay in ML15087 pHSP-carG and pHSP-GGS1: n=11.

FIG. 19 sets out rebaudioside A production in a batch fermentation assay in ML15186 TEF-GGS1 and HSP-carG: n=12; ML15186 HYPO-carG: n=6; ML15187 TEF-GGS1, HSP-carG, HYPO-carG: n=12.

FIG. 20 sets rebaudioside A production in STV2119 HSP-CarG, STV2119 TPI-CarG, STV2121 HSP-CarG and STV2121 TPI-CarG.

FIG. 21 sets out a schematic diagram of the potential pathways leading to biosynthesis of steviol glycosides. The compound shown with an asterisk is 13-[(β-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester.

DESCRIPTION OF THE SEQUENCE LISTING

A description of the sequences is set out in Table 2. Sequences described herein may be defined with reference to the sequence listing or with reference to any database accession numbers set out herein. letter abbreviation for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.

DETAILED DESCRIPTION OF THE INVENTION

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

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element.

Herein, “rebaudioside” may be shortened to “reb”. That is to say, rebaudioside A and reb A, for example, are intended to indicate the same molecule.

Previously, we have increased flux to the isoprenoid precursor geranyl-geranyl pyrophosphate by overexpression of the native geranylgeranyl pyrophosphate (GGPP) synthase, for example in Saccharomyces cerevisiae and in Yarrowia lipolitica, in order to increase production of steviol glycosides in recombinant host cells which are capable of producing such steviol glycosides.

We have now identified a heterologous enzyme from Mucor circenelloides, the product of the carG gene, capable of greatly increasing flux to GGPP in a background where further overexpression of the native enzyme was not effective. Expression of a heterologous protein potentially has several advantages over expression of the native enzyme. Post-translational modification or degradation of a native enzyme may limit its ability to be upregulated. Similarly, it may be subject to allosteric regulation by pathway intermediates.

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, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. The term “recombinant” is synonymous with “genetically modified”.

The invention concerns recombinant hosts expressing polypeptides identified as having geranylgeranyl pyrophosphate (GGPP) synthase activity: typically, the host is one which may be used for the production of steviol glycosides. The ability of a given recombinant host to produce a steviol glycoside may be a property of the host in non-recombinant form or may be a result of the introduction of one or more recombinant nucleic acid sequences (i.e. encoding enzymes leading to the production of a steviol glycoside).

The steviol glycoside may be steviolmonoside, steviolbioside, 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)kaur-16-en-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester steviol-19-diside. A recombinant host of the invention may be capable of producing two or more of steviol glycosides, for example two or more of the steviol glycosides mentioned above,

GGPP synthase activity is a term well known to the skilled person. For the purpose of this invention, a polypeptide having GGPP synthase (or synthetase) activity is typically one which catalyzes the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. GGPP synthase activity may also be referred to as GGPPS activity, GGS activity, GGS1 activity, GGPS1 activity or GGPPS1 activity. GGPP synthase activity may also be defined in terms of activity of the product of the carG gene of Mucor circinelloides. The product of the carG gene of Mucor circinelloides may catalyze one or more of:

dimethylallyl diphosphate+isopentenyl diphosphate=diphosphate+geranyl diphosphate; geranyl diphosphate+isopentenyl diphosphate=diphosphate+(2E,6E)-farnesyl diphosphate; or(2E,6E)-farnesyl diphosphate+isopentenyl diphosphate=diphosphate+geranylgeranyl diphosphate

Any of these catalytic activites may be used to define a GGPP synthase of the invention.

The invention thus provides a recombinant host capable of producing a steviol glycoside comprising a recombinant nucleic acid sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 45% sequence identity thereto. Such an amino acid sequence typically has GGPP synthase activity.

The recombinant nucleic acid sequence may also be defined as one which encodes a polypeptide comprising the following amino acid sequence (or an amino acid sequence having at least about 45% sequence identity thereto):

MLNSHNRTEERSTEDIILEPYTYLISQPGKDIRAKLISAFDLWLHVPKD
VLCVINKIIGMLHNASLMIDDVQDDSDLRRGVPVAHHIYGVPQTINTAN
YVIFLALQEVMKLNIPSMMQVCTEELINLHRGQGIELYWRDSLTCPTEE
EYIDMVNNKTSGLLRLAVRLMQAASESDIDYTPLVNIIGIHFQVRDDYM
NLQSTSYTNNKGFCEDLTEGKFSFPIIHAIRKDPSNRQLLNIISQKPTS
IEVKKYALEVIRKAGSFEYVREFLRQKEAESLKEIKRLGGNPLLEKYIE
TIRVEATND

The sequence may also be defined with reference to UniProtKB-Q9P885.

A polypeptide, typically having GGPP synthase activity, encoded by a recombinant nucleic acid present in a recombinant host of the invention may comprise an amino acid sequence having at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, 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: 1 or the sequences set out above.

A polypeptide, typically having GGPP synthase activity, encoded by a recombinant nucleic acid present in a recombinant host of the invention may comprise an amino acid sequence which is a fragment of an amino acid sequence described herein, for example a truncated version of such an amino acid sequence.

A recombinant host of the invention may comprise recombinant nucleic acid sequences encoding more than one such polypeptide, for example two, three, four or more such polypeptides. The polypeptides thus encoded may be the same of different.

A polypeptide encoded by a recombinant nucleic acid present in a recombinant host may be one which is obtainable from or derived from or found in an organism of the genus Mucor, for example one which is obtainable from or derived from or found in a Mucor circinelloides.

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

A recombinant host of the invention may thus have increased GGPP synthase activity as compared to a form of the host which does not express a said polypeptide.

A recombinant host of the invention typically produces an enhanced amount of a steviol glycoside as compared to a form of the host which does not express a said polypeptide.

In the recombinant host of the invention, one or more steviol glycosides may be produced which are derived from GGPP.

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. The amino acids are identified by either the single-letter or three-letter designations. The term “protein” as used herein is synonymous with the term “polypeptide” and may also refer to two or more polypeptides. Thus, the terms “protein”, “peptide” and “polypeptide” can be used interchangeably. Polypeptides may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, and the like) to add functionality. Polypeptides exhibiting activity may be referred to as enzymes. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given polypeptide may be produced.

A polypeptide encoded by a recombinant nucleic acid for use in a recombinant host of the invention may comprise a signal peptide and/or a propeptide sequence. In the event that a polypeptide expressed by a recombinant host of the invention comprises a signal peptide and/or a propeptide, sequence identity may be calculated over the mature polypeptide sequence.

A recombinant nucleic acid sequence for use in a recombinant host of the invention may be provided in the form of a nucleic acid construct. The term “nucleic acid construct” refers to as a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains all the control sequences required for expression of a coding sequence, wherein said control sequences are operably linked to said coding sequence.

A recombinant nucleic acid sequence for use in a recombinant host of the invention may be provided in the form of an expression vector, wherein the polynucleotide sequence is operably linked to at least one control sequence for the expression of the polynucleotide sequence in a recombinant host cell.

The term “operably linked” as used herein refers to two or more nucleic acid sequence elements that are physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being “under the control of” the promoter. Generally, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They usually will be essentially contiguous, although this may not be required.

An expression vector comprises a polynucleotide coding for a polypeptide as described herein, operably linked to the appropriate control sequences (such as a promoter, and transcriptional and translational stop signals) for expression and/or translation in vitro, or in the host cell of the polynucleotide.

The expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector, which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.

Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The integrative cloning vector may integrate at random or at a predetermined target locus in the chromosomes of the host cell. A vector may comprise one or more selectable markers, which permit easy selection of transformed cells.

A recombinant host of the invention may comprise any polypeptide as described herein. Typically, a recombinant host of the invention is capable of producing a steviol glycoside. A recombinant host of the invention may be capable of producing two or more steviol glycosides. For example, a recombinant host of the invention may be capable of producing one or more of, or two or more of, for example, steviol-13-monoside, steviol-19-monoside, 13-[(β-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester, rubusoside, dulcoside A, stevioside, steviol-19-diside, steviolbioside, rebA, rebB, rebC rebE, rebF, rebD or rebM.

A recombinant host of the invention may comprise one or more recombinant nucleic acid sequences encoding one or more polypeptides having UDP-glycosyltransferase (UGT) activity.

For the purposes of this invention, a polypeptide having UGT activity is one which has glycosyltransferase activity (EC 2.4), i.e. that can act as a catalyst for the transfer of a monosaccharide unit from an activated nucleotide sugar (also known as the “glycosyl donor”) to a glycosyl acceptor molecule, usually an alcohol. The glycosyl donor for a UGT is typically the nucleotide sugar uridine diphosphate glucose (uracil-diphosphate glucose, UDP-glucose).

Such additional UGTs may be selected so as to produce a desired steviol glycoside. Schematic diagrams of steviol glycoside formation are set out in Humphrey et al., Plant Molecular Biology (2006) 61: 47-62 and Mohamed et al., J. Plant Physiology 168 (2011) 1136-1141. In addition, FIGS. 1 and 21 set out schematic diagrams of steviol glycoside formation.

A recombinant host of the invention may thus comprise one or more recombinant nucleic acid sequences encoding one or more of:

(i) a polypeptide having UGT74G1 activity (UGT3 activity);

(ii) a polypeptide having UGT2 activity;

(ii) a polypeptide having UGT85C2 activity (UGT1 activity); and

(iii) a polypeptide having UGT76G1 activity (UGT4 activity).

A recombinant host of the invention will typically comprise 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. One nucleic acid may encode two or more of such polypeptides.

A recombinant host of the invention typically comprises polynucleotides expressing at least one of each of a UGT1, UGT2, UGT3 and UGT4 polypeptide and 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 kaurenoic acid 13-hydroxylase activity. In such a recombinant host, some or all polynucleotides encoding such polypeptides may be recombinant.

The choice of nucleic acid encoding polypeptides having UGT1, 2, 3 or 4 activity may be used to steer production of steviol glycosides in a recombinant cell to a desired steviol glycoside, such as rebaudioside A, rebaudioside D or rebaudioside M.

A recombinant yeast suitable for use in the invention may comprise a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-13-glucose to steviol. That is to say, a recombinant yeast suitable for use in a method of the invention may comprise a UGT which is capable of catalyzing a reaction in which steviol is converted to steviolmonoside.

Such a recombinant yeast suitable for use in a method of the invention may comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT85C2, whereby the nucleotide sequence upon transformation of the yeast confers on that yeast the ability to convert steviol to steviolmonoside.

UGT85C2 activity is transfer of a glucose unit to the 13-OH of steviol. Thus, a suitable UGT85C2 may function as a uridine 5′-diphospho glucosyl: steviol 13-OH transferase, and a uridine 5′-diphospho glucosyl: steviol-19-0-glucoside 13-OH transferase. A functional UGT85C2 polypeptides may also catalyze glucosyl transferase reactions that utilize steviol glycoside substrates other than steviol and steviol-19-0-glucoside. Such sequences may be referred to as UGT1 sequences herein.

A recombinant yeast suitable for use in the invention may comprise a nucleotide o sequence encoding a polypeptide which has UGT2 activity.

A polypeptide having UGT2 activity is one which functions as a uridine 5′-diphospho glucosyl: steviol-13-O-glucoside transferase (also referred to as a steviol-13-monoglucoside 1,2-glucosylase), transferring a glucose moiety to the C-2′ of the 13-O-glucose of the acceptor molecule, steviol-13-O-glucoside. Typically, a suitable UGT2 polypeptide also functions as a uridine 5′-diphospho glucosyl: rubusoside transferase transferring a glucose moiety to the C-2′ of the 13-O-glucose of the acceptor molecule, rubusoside. That is to say be capable of converting steviol-13-monoside to steviolbioside and/or capable of converting rubusoside to stevioside.

A polypeptide having UGT2 activity may also catalyze reactions that utilize steviol glycoside substrates other than steviol-13-O-glucoside and rubusoside, e.g., functional UGT2 polypeptides may utilize stevioside as a substrate, transferring a glucose moiety to the C-2′ of the 19-O-glucose residue to produce rebaudioside E. A functional UGT2 polypeptides may also utilize rebaudioside A as a substrate, transferring a glucose moiety to the C-2′ of the 19-O-glucose residue to produce rebaudioside D.

A polypeptide having UGT2 activity may also catalyze reactions that utilize steviol-19-glucoside or rubusoside as a substrate, e.g., a functional UGT2 polypeptide may utilize steviol-19-glucoside or rubusoside as a substrate, transferring a glucose moiety to the 19 position to produce steviol-19-2side or 13-[(β-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester respectively.

However, a functional UGT2 polypeptide typically does 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 typically does not occur.

A polypeptide having UGT2 activity may also transfer sugar moieties from donors other than uridine diphosphate glucose. For example, a polypeptide having UGT2 activity act as a uridine 5′-diphospho D-xylosyl: steviol-13-O-glucoside transferase, transferring a xylose moiety to the C-2′ of the 13-O-glucose of the acceptor molecule, steviol-13-O-glucoside. As another example, a polypeptide having UGT2 activity may act as a uridine 5′-diphospho L-rhamnosyl: teviol-13-O-glucoside transferase, transferring a rhamnose moiety to the C-2′ of the 13-O-glucose of the acceptor molecule, steviol.

A polynucleotide encoding a UGT2 polypeptide for use in a recombinant host of the invention may be used to steer production of steviol glycosides in a recombinant cell to a desired steviol glycoside, such as rebaudioside A, rebaudioside D or rebaudioside M. For example, a UGT2 polypeptide which preferentially catalyzes conversion of steviol-13-monoside to steviolbioside and/or conversion of rubusoside to stevioside may help to steer production towards rebaudioside A, whereas a UGT2 polypeptide which preferentially catalyzes conversion of stevioside to rebE or rubusoside to a compound with an additional sugar at the 19 position may help to steer production towards rebaudioside M. That is to say preference for addition of a sugar moiety at the 13 position may help steer production towards rebaudioside A, whereas preference for addition of a sugar moiety at the 19 position may help steer production towards rebaudioside M.

A recombinant yeast suitable for use in the method of the invention may comprise a nucleotide sequence encoding a polypeptide having UGT activity may comprise a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-19-glucose to steviolbioside. That is to say, a recombinant yeast of the invention may comprise a UGT which is capable of catalyzing a reaction in which steviolbioside is converted to stevioside. Accordingly, such a recombinant yeast may be capable of converting steviolbioside to stevioside. Expression of such a nucleotide sequence may confer on the recombinant yeast the ability to produce at least stevioside.

A recombinant yeast suitable for use in a method of the invention may thus also comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT74G1, whereby the nucleotide sequence upon transformation of the yeast confers on the cell the ability to convert steviolbioside to stevioside.

Suitable UGT74G1 polypeptides may be capable of transferring a glucose unit to the 13-OH or the 19-COOH, respectively, of steviol. A suitable UGT74G1 polypeptide may function as a uridine 5′-diphospho glucosyl: steviol 19-COOH transferase and a uridine 5′-diphospho glucosyl: steviol-13-O-glucoside 19-COOH transferase. Functional UGT74G1 polypeptides also may catalyze glycosyl transferase reactions that utilize steviol glycoside substrates other than steviol and steviol-13-O-glucoside, or that transfer sugar moieties from donors other than uridine diphosphate glucose. Such sequences may be referred to herein as UGT3 sequences.

A recombinant yeast suitable for use in a method the invention may comprise a nucleotide sequence encoding a polypeptide capable of catalyzing glucosylation of the C-3′ of the glucose at the C-13 position of stevioside. That is to say, a recombinant yeast suitable for use in a method of the invention may comprise a UGT which is capable of catalyzing a reaction in which stevioside is converted to rebaudioside A. Accordingly, such a recombinant yeast may be capable of converting stevioside to rebaudioside A. Expression of such a nucleotide sequence may confer on the yeast the ability to produce at least rebaudioside A.

A recombinant yeast suitable for use in a method of the invention may thus also comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT76G1, whereby the nucleotide sequence upon transformation of a yeast confers on that yeast the ability to convert stevioside to rebaudioside A.

A suitable UGT76G1 adds a glucose moiety to the C-3′of the C-13-O-glucose of the acceptor molecule, a steviol 1,2 glycoside. Thus, UGT76G1 functions, for example, as a uridine 5′-diphospho glucosyl: steviol 13-0-1,2 glucoside C-3′ glucosyl transferase and a uridine 5′-diphospho glucosyl: steviol-19-0-glucose, 13-0-1,2 bioside C-3′ glucosyl transferase. Functional UGT76G1 polypeptides may also catalyze glucosyl transferase reactions that utilize steviol glycoside substrates that contain sugars other than glucose, e.g., steviol rhamnosides and steviol xylosides. Such sequences may be referred to herein as UGT4 sequences. A UGT4 may alternatively or in addition be capable of converting RebD to RebM.

A recombinant yeast suitable for use in a method of the invention typically comprises nucleotide sequences encoding at least one polypeptide having UGT1 activity, at least one polypeptide having UGT2 activity, least one polypeptide having UGT3 activity and at least one polypeptide having UGT4 activity. One or more of these nucleic acid sequences may be recombinant. A given nucleic acid may encode a polypeptide having one or more of the above activities. For example, a nucleic acid encode for a polypeptide which has two, three or four of the activities set out above. Preferably, a recombinant yeast for use in the method of the invention comprises UGT1, UGT2 and UGT3 and UGT4 activity. Suitable UGT1, UGT2, UGT3 and UGT4 sequences are described in Table 1 of WO2015/007748.

A recombinant host of the invention may comprise two or more nucleic acid sequences encoding a polypeptide having any one UGT activity, for example UGT1, 2, 3 or 4, activity. Where a recombinant host of the invention comprises two or more nucleic acid sequence encoding a polypeptide having any one UGT activity, those nucleic acid sequences may be the same or different and/or may encode the same or different polypeptides. In particular, a recombinant host of the invention may comprise a nucleic acid sequence encoding a two different UGT2 polypeptides.

A recombinant host according to the invention may comprise one or more recombinant nucleotide sequence(s) encoding one of more of:

• • 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 kaurenoic acid 13-hydroxylase activity.

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

This enzyme has one substrate, geranylgeranyl pyrophosphate, and one product, ent-copalyl pyrophosphate. This enzyme participates in gibberellin biosynthesis. This enzyme belongs to the family of isomerase, specifically the class of intramolecular lyases. The systematic name of this enzyme class is ent-copalyl-diphosphate lyase (decyclizing). Other names in common use include having ent-copalyl pyrophosphate synthase, ent-kaurene synthase A, and ent-kaurene synthetase A.

Suitable nucleic acid sequences encoding an ent-copalyl pyrophosphate synthase may for instance comprise a sequence as set out 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.

For the purposes of this invention, a polypeptide having ent-kaurene synthase activity (EC 4.2.3.19) is a polypeptide that is capable of catalyzing the chemical reaction:

ent-copalyl diphosphateent-kaurene+diphosphate

Hence, this enzyme has one substrate, ent-copalyl diphosphate, and two products, ent-kaurene and diphosphate.

This enzyme belongs to the family of lyases, specifically those carbon-oxygen lyases acting on phosphates. The systematic name of this enzyme class is ent-copalyl-diphosphate diphosphate-lyase (cyclizing, ent-kaurene-forming). Other names in common use include ent-kaurene synthase B, ent-kaurene synthetase B, ent-copalyl-diphosphate diphosphate-lyase, and (cyclizing). This enzyme participates in diterpenoid biosynthesis.

Suitable nucleic acid sequences encoding an ent-Kaurene synthase may for instance comprise a sequence as set out in SEQ ID. NO: 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, 157, 158, 159, 160, 183 or 184 of WO2015/007748.

ent-copalyl diphosphate synthases may also have a distinct ent-kaurene synthase activity associated with the same protein molecule. The reaction catalyzed by ent-kaurene synthase is the next step in the biosynthetic pathway to gibberellins. The two types of enzymic activity are distinct, and site-directed mutagenesis to suppress the ent-kaurene synthase activity of the protein leads to build up of ent-copalyl pyrophosphate.

Accordingly, a single nucleotide sequence used in a recombinant host of the invention may encode a polypeptide having ent-copalyl pyrophosphate synthase activity and ent-kaurene synthase activity. Alternatively, the two activities may be encoded two distinct, separate nucleotide sequences.

For the purposes of this invention, a polypeptide having ent-kaurene oxidase activity (EC 1.14.13.78) is a polypeptide which is capable of catalysing three successive oxidations of the 4-methyl group of ent-kaurene to give kaurenoic acid. Such activity typically requires the presence of a cytochrome P450.

Suitable nucleic acid sequences encoding an ent-Kaurene oxidase may for instance comprise a sequence as set out in SEQ ID. NO: 21, 23, 25, 67, 85, 145, 161, 162, 163, 180 or 186 of WO2015/007748.

For the purposes of the invention, a polypeptide having kaurenoic acid 13-hydroxylase activity (EC 1.14.13) is one which is capable of catalyzing the formation of steviol (ent-kaur-16-en-13-ol-19-oic acid) using NADPH and O₂. Such activity may also be referred to as ent-ka 13-hydroxylase activity.

Suitable nucleic acid sequences encoding a kaurenoic acid 13-hydroxylase may for instance comprise a sequence as set out in SEQ ID. NO: 27, 29, 31, 33, 69, 89, 91, 93, 95, 97, 146, 164, 165, 166, 167 or 185 of WO2015/007748.

A recombinant host of the invention may comprise a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity. That is to say, a recombinant host of the invention may be capable of expressing a nucleotide sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity. For the purposes of the invention, a polypeptide having NADPH-Cytochrome P450 reductase activity (EC 1.6.2.4; also known as NADPH:ferrihemoprotein oxidoreductase, NADPH:hemoprotein oxidoreductase, NADPH:P450 oxidoreductase, P450 reductase, POR, CPR, CYPOR) is typically one which is a membrane-bound enzyme allowing electron transfer to cytochrome P450 in the microsome of the eukaryotic cell from a FAD- and FMN-containing enzyme NADPH:cytochrome P450 reductase (POR; EC 1.6.2.4).

In a recombinant host of the invention, the ability of the host to produce geranylgeranyl diphosphate (GGPP) may be upregulated. Upregulated in the context of this invention implies that the recombinant host produces more GGPP than an equivalent non-recombinant host.

Accordingly, a recombinant host of the invention may comprise one or more nucleotide sequence(s) encoding hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase and geranylgeranyl diphosphate synthase, whereby the nucleotide sequence(s) upon transformation of a host confer(s) on that host the ability to produce elevated levels of GGPP. Thus, a recombinant host according to the invention may comprise one or more recombinant nucleic acid sequence(s) encoding one or more of hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase and geranylgeranyl diphosphate synthase.

Accordingly, a recombinant host of the invention may comprise nucleic acid sequences encoding one or more of:

a polypeptide having hydroxymethylglutaryl-CoA reductase activity;

a polypeptide having farnesyl-pyrophosphate synthetase activity; and

A recombinant host of the invention may be, for example, an multicellular organism or a cell thereof or a unicellular organism. A host of the invention may be a prokaryotic, archaebacterial or eukaryotic host cell.

A prokaryotic host cell may, but is not limited to, a bacterial host cell. An eukaryotic host cell may be, but is not limited to, a yeast, a fungus, an amoeba, an algae, an animal, a plant or an insect host cell.

An eukaryotic host cell may be a fungal host cell. “Fungi” include all species of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc., New York). The term fungus thus includes among others filamentous fungi and yeast.

“Filamentous fungi” are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligatory aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasmsonia, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma. Preferred filamentous fungal strains that may serve as host cells belong to the 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 the comparison of fermentation characteristics of transformed and untransformed cells, include e.g. Aspergillus niger CBS120.49, CBS 513.88, Aspergillus oryzae ATCC16868, ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, Aspergillus fumigatus AF293 (CBS101355), P. chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Acremonium chrysogenum ATCC 36225, ATCC 48272, Trichoderma reesei ATCC 26921, ATCC 56765, ATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense ATCC44006 and derivatives of all of these strains. Particularly preferred as filamentous fungal host cell are Aspergillus niger CBS 513.88 and derivatives thereof.

An eukaryotic host cell may be a yeast cell. Preferred yeast host cells may be selected from the 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), Issatchenkia (e.g. I. orientalis) Pichia (e.g., P. pastoris), Schizosaccharomyces, Hansenula, Kloeckera, Pachysolen, Schwanniomyces, Trichosporon, Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)), Yamadazyma.

A eukaryotic host cell may be a plant cell, for example a Stevia rebaudiana cell,

Prokaryotic host cells may be bacterial host cells. Bacterial host cell may be Gram negative or Gram positive bacteria. Examples of bacteria include, but are not limited to, bacteria belonging to the genus Bacillus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus,), Acinetobacter, Nocardia, Xanthobacter, Escherichia (e.g., E. coli (e.g., strains DH 1 OB, Stbl2, DH5-alpha, DB3, DB3.1), DB4, DB5, JDP682 and ccdA-over (e.g., U.S. application Ser. No. 09/518,188))), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., 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 (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)).

Host Cells may be host cells from non-microbial organisms. Examples of such cells, include, but are not limited to, insect cells (e.g., Drosophila (e.g., D. melanogaster), Spodoptera (e.g., S. frugiperda Sf9 or Sf21 cells) and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C. elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis cells); reptilian cells; and mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells).

A recombinant host according to the present invention may be able to grow on any suitable carbon source known in the art and convert it to a steviol glycoside. The recombinant host may be able to convert directly plant biomass, celluloses, hemicelluloses, pectines, rhamnose, galactose, fucose, maltose, maltodextrines, ribose, ribulose, or starch, starch derivatives, sucrose, lactose and glycerol. Hence, a preferred host expresses enzymes such as cellulases (endocellulases and exocellulases) and hemicellulases (e.g. endo- and exo-xylanases, arabinases) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, pectinases able to convert pectines into glucuronic acid and galacturonic acid or amylases to convert starch into glucose monomers. Preferably, the host is able to convert a carbon source selected from the group consisting of glucose, xylose, arabinose, sucrose, lactose and glycerol. The host cell may for instance be a eukaryotic host cell as described in WO03/062430, WO06/009434, EP1499708B1, WO2006096130 or WO04/099381.

Thus, in a further aspect, the invention also provides a process for the preparation of a steviol glycoside which comprises fermenting a recombinant host of the invention which is capable of producing at least one steviol glycoside in a suitable fermentation medium, and optionally recovering the steviol glycoside.

The fermentation medium used in the process for the production of a steviol glycoside may be any suitable fermentation medium which allows growth of a particular eukaryotic host cell. The essential elements of the fermentation medium are known to the person skilled in the art and may be adapted to the host cell selected.

Preferably, the fermentation medium comprises a carbon source selected from the group consisting of plant biomass, celluloses, hemicelluloses, pectines, rhamnose, galactose, fucose, fructose, maltose, maltodextrines, ribose, ribulose, or starch, starch derivatives, sucrose, lactose, fatty acids, triglycerides and glycerol. Preferably, the fermentation medium also comprises a nitrogen source such as ureum, or an ammonium salt such as ammonium sulphate, ammonium chloride, ammoniumnitrate or ammonium phosphate.

The fermentation process according to the present invention may be carried out in batch, fed-batch or continuous mode. A separate hydrolysis and fermentation (SHF) process or a simultaneous saccharification and fermentation (SSF) process may also be applied. A combination of these fermentation process modes may also be possible for optimal productivity. A SSF process may be particularly attractive if starch, cellulose, hemicelluose or pectin is used as a carbon source in the fermentation process, where it may be necessary to add hydrolytic enzymes, such as cellulases, hemicellulases or pectinases to hydrolyse the substrate.

The recombinant host used in the process for the preparation of a steviol glycoside may be any suitable recombinant host as defined herein above. It may be advantageous to use a recombinant eukaryotic recombinant host according to the invention in the process since most eukaryotic cells do not require sterile conditions for propagation and are insensitive to bacteriophage infections. In addition, eukaryotic host cells may be grown at low pH to prevent bacterial contamination.

The recombinant host according to the present invention may be a facultative anaerobic microorganism. A facultative anaerobic recombinant host can be propagated aerobically to a high cell concentration. This anaerobic phase can then be carried out at high cell density which reduces the fermentation volume required substantially, and may minimize the risk of contamination with aerobic microorganisms.

The fermentation process for the production of a steviol glycoside according to the present invention may be an aerobic or an anaerobic fermentation process.

An anaerobic fermentation process may be herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, and wherein organic molecules serve as both electron donor and electron acceptors. The fermentation process according to the present invention may also first be run under aerobic conditions and subsequently under anaerobic conditions.

The fermentation process may also be run under oxygen-limited, or micro-aerobical, conditions. Alternatively, the fermentation process may first be run under aerobic conditions and subsequently under oxygen-limited conditions. An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used.

The production of a steviol glycoside in the process according to the present invention may occur during the growth phase of the host cell, during the stationary (steady state) phase or during both phases. It may be possible to run the fermentation process at different temperatures.

The process for the production of a steviol glycoside may be run at a temperature which is optimal for the recombinant host. The optimum growth temperature may differ for each transformed recombinant host and is known to the person skilled in the art. The optimum temperature might be higher than optimal for wild type organisms to grow the organism efficiently under non-sterile conditions under minimal infection sensitivity and lowest cooling cost. Alternatively, the process may be carried out at a temperature which is not optimal for growth of the recombinant host.

The process for the production of a steviol glycoside according to the present invention may 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 of 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. An advantage of carrying out the fermentation at these low pH values is that growth of contaminant bacteria in the fermentation medium may be prevented.

Such a process may be carried out on an industrial scale. The product of such a process is one or more steviol glycosides.

Recovery of steviol glycoside(s) from the fermentation medium may be performed by known methods in the art, for instance by distillation, vacuum extraction, solvent extraction, or evaporation.

In the process for the production of a steviol glycoside according to the invention, it may be possible to achieve a concentration of above 5 mg/I fermentation broth, preferably above 10 mg/I, preferably above 20 mg/I, preferably above 30 mg/I fermentation broth, preferably above 40 mg/I, more preferably above 50 mg/I, preferably above 60 mg/I, preferably above 70 mg/ml, preferably above 80 mg/I, preferably above 100 mg/I, preferably above 1 g/I, preferably above 5 g/I, preferably above 10 g/I, for example above 20 g/I, but usually up to a concentration of about 200 g/I, such as up to about 150 g/I, such as up to about 100 g/I, for example up to about 70 g/I. Such concentrations may be concentration of the total broth or of the supernatant.

The invention further provides a fermentation broth comprising a steviol glycoside obtainable by the process of the invention for the preparation of a steviol glycoside.

In the event that one or more steviol glycosides is expressed within a recombinant host of the invention, such cells may need to be treated so as to release them. Preferentially, at least one steviol glycoside, for example rebA or rebM, is produced extracellularly

The invention also provides a steviol glycoside obtained by a process according to the invention for the preparation of a steviol glycoside or obtainable from a fermentation broth of the invention. Such a steviol glycoside may be a non-naturally occurring steviol glycoside, that is to say one which is not produced in plants.

Also provided is a composition comprising two or more steviol glycosides obtainable by a process of the invention for the preparation of a steviol glycoside or obtainable from a fermentation broth of the invention. In such a composition, one or more of the steviol glycosides may be a non-naturally occurring steviol glycoside, that is to say one which is not produced in plants.

A steviol glycoside or composition produced by the fermentation process according to the present invention may be used in any application known for such compounds. In particular, they may for instance be used as a sweetener, for example in a food or a beverage. According to the invention therefore, there is provided a foodstuff, feed or beverage which comprises a steviol glycoside or a composition of the invention.

For example a steviol glycoside or a composition of the invention may be formulated in soft drinks, such as a carbonated beverage, as a tabletop sweetener, chewing gum, dairy product such as yoghurt (e.g. plain yoghurt), cake, cereal or cereal-based food, nutraceutical, pharmaceutical, edible gel, confectionery product, cosmetic, toothpastes or other oral cavity composition, etc. In addition, a steviol glycoside or a composition of the invention can be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics.

Accordingly, the invention provides, inter alia, a foodstuff, feed or beverage which comprises a steviol glycoside prepared according to a process of the invention.

The terms “sequence homology” or “sequence identity” or “homology” or “identity” are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison o purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

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

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

Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671, WO90/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No. 6,265,186.

Embodiments of the invention

• 1. A recombinant host capable of producing a steviol glycoside comprising a recombinant nucleic acid sequence encoding a polypeptide having geranylgeranyl pyrophosphate (GGPP) synthase activity which comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 45% sequence identity thereto.
• 2. A recombinant host according to embodiment 1, wherein the recombinant nucleic acid is one derived from Mucor circinelloides.
• 3. A recombinant host according to embodiment 1 or 2 which comprises one or more recombinant nucleotide sequence(s) 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 kaurenoic acid 13-hydroxylase activity.
• 4. A recombinant host according to any one of the preceding embodiments, which comprises a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity.
• 5. A recombinant host according to any one of the preceding embodiments which comprises a recombinant nucleic acid sequence encoding one or more of:
  • (i) a polypeptide having UGT74G1 activity;
  • (ii) a polypeptide having UGT2 activity;
  • (iii) a polypeptide having UGT85C2 activity; and
  • (iv) a polypeptide having UGT76G1 activity.
• 6. A recombinant host according to any one of the preceding embodiments, wherein the host belongs to one of the genera Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadazyma or Escherichia.
• 7. A recombinant host according to embodiment 6, wherein the recombinant host is a Saccharomyces cerevisiae cell, a Yarrowia lipolitica cell, a Candida krusei cell, an Issatchenkia orientalis or an Escherichia coli cell.
• 8. A recombinant host according to any one of the preceding embodiments, wherein the ability of the host to produce geranylgeranyl diphosphate (GGPP) is upregulated.
• 9. A recombinant host according to any one of the preceding embodiments which comprises a nucleic acid sequence encoding one or more of:
  • a polypeptide having hydroxymethylglutaryl-CoA reductase activity; or
  • a polypeptide having farnesyl-pyrophosphate synthetase activity.
• 10. A process for the preparation of a steviol glycoside which comprises fermenting a recombinant host according to any one of the preceding embodiments in a suitable fermentation medium and, optionally, recovering the steviol glycoside.
• 11. A process according to any one of embodiment 10 for the preparation of a steviol glyocisde, wherein the process is carried out on an industrial scale.
• 12. A fermentation broth comprising a steviol glycoside obtainable by the process according to embodiment 10 or 11.
• 13. A steviol glycoside obtained by a process according to embodiment 10 or 11 or obtained from a fermentation broth according to embodiment 12.
• 14. A composition comprising two or more steviol glycosides obtained by a process according to embodiment 10 or 11 or obtained from a fermentation broth according to embodiment 12.
• 15. A foodstuff, feed or beverage which comprises a steviol glycoside according to embodiment 13 or a composition according to embodiment 14.

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

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

The present invention is further illustrated by the following Examples:

EXAMPLES

General

Standard genetic techniques, such as overexpression of enzymes in the host cells, as well as for additional genetic modification of host cells, are known methods in the art, such as described in Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671.

The genotype of the strains described in the Examples are listed in Table 1 below.

Example 1

Description of Steviol Glycoside Production Strain ML14094 (MAT-A Lineage)

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

Step 1: Strain ML10371 (MAT-A, lys1-, ura3-, leu2-) was transformed with 5 defined DNA fragments. All transformations were carried out via a lithium acetate/PEG fungal transformation protocol method and transformants were selected on minimal medium, YPD+100 ug/ml nourseothricin or YPD+100 ug/ml hygromycin, as appropriate.

1) a 7.0 kb DNA fragment isolated by gel purification following HindIII/NotI digestion of plasmid MB6969 (FIG. 2). This construct encodes a synthetic construct for the overexpression of UGT2_1a (SEQ ID NO: 8) linked to the pPGM promoter (SEQ ID NO: 19) and xprT terminator (SEQ ID NO: 26) and the HPH hygromycin resistance gene, together flanked by lox sites (Guldener et al, 1996, Lambert et al, 2007), and a synthetic construct for the overexpression of the codon optimized Y. lipolytica hydroxymethylglutaryl-coenzyme A reductase open reading frame lacking the 5′ membrane anchor sequence (tHMGopt: SEQ ID NO: 9) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27).

2) a 2.7 kb DNA fragment isolated by gel purification following HindIII/SspI digestion of MB6856 (FIG. 3). This construct encodes tHMGopt (SEQ ID NO: 9) linked to the pHYPO promoter (SEQ ID NO: 21) and gpdT terminator (SEQ ID NO: 28).

3) a 2.5 kb DNA fragment isolated by gel purification following SspI digestion of MB6857 (FIG. 4). This construct encodes tHMGopt (SEQ ID NO: 9) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27).

4) a 2.0 kb DNA fragment isolated by gel purification following SspI digestion of MB6948 (FIG. 5). This construct encodes a synthetic construct for the overexpression of the codon optimized Y. lipolytica geranyl-geranyl-pyrophosphate synthetase (GGSopt: SEQ ID NO: 7) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27).

5) a 2.2 kb DNA fragment isolated by gel purification following HindIII/SspI digestion of MB6958 (FIG. 6). This construct encodes GGSopt (SEQ ID NO: 7) linked to the pHYPO promoter (SEQ ID NO: 21) and gpdT terminator (SEQ ID NO: 28). The resulting strain was denoted ML13462.

Step 2. Strain ML13462 was transformed with a 9.7 kb fragment isolated by gel purification following SfiI digestion of plasmid MB7015 (FIG. 7). This construct encodes a synthetic construct for the overexpression of UGT1 (SEQ ID NO: 10) linked to the pENO (SEQ ID NO: 22) promoter and gpdT terminator (SEQ ID NO: 28), UGT3 (SEQ ID NO: 11) linked to the pHSP promoter (SEQ ID NO: 20) and pgmT terminator (SEQ ID NO: 29), UGT4 (SEQ ID NO: 12) linked to the pCWP (SEQ NO: 23) promoter and pgkT terminator (SEQ ID NO: 30), and the lox-flanked nourseothricin resistance marker (NAT). Note that placement of lox sites allows for subsequent removal of nourseothricin resistance via CRE recombinase mediated recombination. A nourseothricin resistant isolate was denoted ML13500.

Step 3. Strain ML13500 was transformed with a 9.1 kb fragment isolated by gel purification following PvuI/SapI digestion of plasmid MB6986 (FIG. 8). This construct encodes tHMGopt (SEQ ID NO: 9) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27), the lox-flanked URA3blaster prototrophic marker, and GGSopt (SEQ ID NO: 7) linked to the pHYPO promoter (SEQ ID NO: 21) and gpdT terminator (SEQ ID NO: 28). Transformants were selected on minimal medium lacking uracil. One selected uracil prototroph was denoted ML13723.

Step 4. Strain ML13723 was transformed with an 18.1 kb fragment isolated by gel purification following SfiI digestion of plasmid MB7059 (FIG. 9). MB7059 encodes the tCPS_SR (SEQ ID NO: 13) linked to pCWP promoter (SEQ ID NO: 23) and cwpT terminator (SEQ ID NO: 27), the tKS_SR (SEQ ID NO: 14) linked to the pHYPO promoter (SEQ ID NO: 21) and gpdT terminator (SEQ ID NO: 28), the KAH_4 (SEQ ID NO: 15) linked to the pHSP promoter (SEQ ID NO: 20) and pgmT terminator (SEQ ID NO: 29), the KO_Gib (SEQ ID NO: 16) linked to the pTPI promoter (SEQ ID NO: 24) and pgkT terminator (SEQ ID NO: 30), the CPR_3 (SEQ ID NO: 17) linked to the pENO promoter (SEQ ID NO: 22) and xprT terminator (SEQ ID NO: 26) and the native Y. lipolytica LEU2 locus. One selected rebaudioside A-producing transformant was denoted ML14032.

Step 5. Strain ML14032 was struck to YPD and grown overnight and then struck to 5-FOA plates to allow for recombination mediated loss of the URA3 marker introduced previously. One selected 5-FOA resistant transformant was denoted ML14093.

Step 6. Strain ML14093 was transformed with a 19.0 kb fragment isolated by gel purification following SfiI digestion of plasmid MB7100 (FIG. 10). MB7100 encodes the tCPS_SR (SEQ ID NO: 13) linked to the pHYPO promoter (SEQ ID NO: 21) and cwpT terminator (SEQ ID NO: 27), the tKS_SR (SEQ ID NO: 14) linked to the pCWP promoter (SEQ ID NO: 23) and gpdT terminator (SEQ ID NO: 28), the KAH_4 (SEQ ID NO: 15) linked to the pHSP promoter (SEQ ID NO: 20) and pgmT terminator (SEQ ID NO: 29), the KO_Gib (SEQ ID NO: 16) linked to the pENO promoter (SEQ ID NO: 22) and pgkT terminator (SEQ ID NO: 30), the CPR_3 (SEQ ID NO: 17) linked to the pTPI promoter (SEQ ID NO: 24) and xprT terminator (SEQ ID NO: 26) and URA3blaster prototrophic marker. Transformants were selected on minimal medium lacking uracil. One selected rebaudioside A producing uracil prototroph was denoted ML14094.

Example 2

Description of Steviol Glycoside Production Strain ML14087 (MAT-B Lineage)

Step 1. Strain ML13206 (MAT-B, ade1-, ure2-, leu2-) was transformed with 5 defined DNA fragments. All transformations were carried out via a lithium acetate/PEG fungal transformation protocol method and transformants were selected on minimal medium, YPD+100 ug/ml nourseothricin or YPD+100 ug/ml hygromycin, as appropriate.

1) a 7.0 kb DNA fragment isolated by gel purification following HindIII/NotI digestion of plasmid MB6969 (FIG. 2). This construct encodes a synthetic construct for the overexpression of the codon pair optimized (CpO) ORF of UGT2_1a (SEQ ID NO: 8) linked to the pPGM (SEQ ID NO: 19) promoter and xprT terminator (SEQ ID NO: 26) and the HPH hygromycin resistance gene, together flanked by lox sites (Guldener et al, 1996, Lambert et al, 2007), and a synthetic construct for the overexpression of the codon optimized Y. lipolytica hydroxymethylglutaryl-coenzyme A reductase open reading frame lacking the 5′ membrane anchor sequence (tHMGopt: SEQ ID NO: 9) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27).

2) a 2.7 kb DNA fragment isolated by gel purification following HindIII/SspI digestion of MB6856 (FIG. 3). This construct encodes tHMGopt (SEQ ID NO: 9) linked to the pHYPO promoter (SEQ ID NO: 21) and gpdT terminator (SEQ ID NO: 28).

3) a 2.5 kb DNA fragment isolated by gel purification following SspI digestion of MB6857 (FIG. 4). This construct encodes tHMGopt (SEQ ID NO: 9) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27).

4) a 2.0 kb DNA fragment isolated by gel purification following SspI digestion of MB6948 (FIG. 5). This construct encodes a synthetic construct for the overexpression of the codon optimized Y. lipolytica geranyl-geranyl-pyrophosphate synthetase (GGSopt: SEQ ID NO: 7) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27). 5) a 2.2 kb DNA fragment isolated by gel purification following HindIII/SspI digestion of MB6958 (FIG. 6). This construct encodes GGSopt (SEQ ID NO: 7) linked to the pHYPO (SEQ ID NO: 21) promoter and gpdT terminator (SEQ ID NO: 28). The resulting strain was denoted ML13465.

Step 2. Strain ML13465 was transformed with 2 defined DNA fragments:

1). a 9.7 kb fragment isolated by gel purification following SfiI digestion of plasmid MB7015 (FIG. 7). This construct encodes a synthetic construct for the overexpression of UGT1 (SEQ ID NO: 10) linked to the pENO promoter (SEQ ID NO: 22) and gpdT (SEQ ID NO: 28) terminator, UGT3 (SEQ ID NO: 11) linked to the pHSP promoter (SEQ ID NO: 20) and pgmT terminator (SEQ ID NO: 29), UGT4 (SEQ ID NO: 12) linked to the pCWP promoter (SEQ ID NO: 23) and pgkT terminator (SEQ ID NO: 30), and the lox-flanked nourseothricin resistance marker (NAT). Note that placement of lox sites allows for subsequent removal of nourseothricin resistance via CRE recombinase mediated recombination.

2). a 9.1 kb fragment isolated by gel purification following PvuI/SapI digestion of plasmid MB6988 (FIG. 11). This construct encodes tHMGopt (SEQ ID NO: 9) linked to the pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27), the lox-flanked URA2blaster prototrophic marker, and GGSopt (SEQ ID NO: 7) linked to the pHYPO promoter (SEQ ID NO: 21) and gpdT terminator (SEQ ID NO: 28). Strains were selected on YPD+100 ug/ml nourseothricin and replica plated to minimal medium lacking uracil. A nourseothricin resistant, uracil prototrophic isolate was denoted ML13490

Step 3. Strain ML13490 was struck to YPD and grown overnight and then struck to 5-FOA plates to allow for recombination mediated loss of the URA2 marker introduced previously. One selected 5-FOA resistant transformant was denoted ML13501.

Step 4. Strain ML13501 was transformed with a 9.1 kb fragment isolated by gel purification following PvuI/SapI digestion of plasmid MB6988 (FIG. 11). Transformants were selected on minimal medium lacking uracil. One selected uracil prototroph was denoted ML13724.

Step 5. Strain ML13724 was transformed with an 18.1 kb fragment isolated by gel purification following SfiI digestion of plasmid MB7044 (FIG. 12). MB7044 encodes the tCPS_SR (SEQ ID NO: 13) linked to the pHYPO promoter (SEQ ID NO: 21) and cwpT terminator (SEQ ID NO: 27), the tKS_SR (SEQ ID NO: 14) linked to the pCWP promoter (SEQ ID NO: 23) and gpdT terminator (SEQ ID NO: 28), the KAH_4 (SEQ ID NO: 15) linked to the pHSP promoter (SEQ ID NO: 20) and pgmT terminator (SEQ ID NO: 29), the KO_Gib (SEQ ID NO: 16) linked to the pENO promoter (SEQ ID NO: 22) and pgkT terminator (SEQ ID NO: 30), the CPR_3 (SEQ ID NO: 17) linked to the pTPI promoter (SEQ ID NO: 24) and xprT terminator (SEQ ID NO: 26) and the LEU2 locus. One selected rebaudioside A-producing transformant was denoted ML14044.

Step 6. Strain ML14044 was struck to YPD and grown overnight and then struck to 5-FOA plates to allow for recombination mediated loss of the URA2 marker introduced previously. One selected 5′-FOA resistant transformant was denoted ML14076.

Step 7. Strain ML14076 was transformed with a 19.0 kb fragment isolated by gel purification following SfiI digestion of plasmid MB7094 (FIG. 13). MB7094 encodes the tCPS_SR (SEQ ID NO: 13) linked to the pHYPO promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27), the tKS_SR (SEQ ID NO: 14) linked to the pCWP promoter (SEQ ID NO: 23) and gpdT terminator (SEQ ID NO: 28), the KAH_4 (SEQ ID NO: 14) linked to the pHSP promoter (SEQ ID NO: 20) and pgmT terminator (SEQ ID NO: 29), the KO_Gib (SEQ ID NO: 16) linked to the pENO promoter (SEQ ID NO: 22) and pgkT terminator (SEQ ID NO: 30), the CPR_3 (SEQ ID NO: 17) linked to the pTPI promoter (SEQ ID NO: 24) and xprT terminator (SEQ ID NO: 26) and URA2blaster prototrophic marker. Transformants were selected on minimal medium lacking uracil. One selected rebaudioside A producing uracil prototroph was denoted ML14087.

Example 3

Mating MATA and MATB Lineage and Selecting Steviol Glycoside-Producing Progeny

Strains of opposite mating types (ML14094 and ML14087) with complementary nutritional deficiencies (ADE1+ lys1- and ade1-LYS1+) were allowed to mate and then plated on selective media that would allow only diploids to grow (minimal media lacking both adenine and lysine). Diploid cells (ML14143) were then induced to undergo meiosis and sporulation by starvation, and the resulting haploid progenies were replica-plated to identify prototrophic isolates with hygromycin and nourseothricin resistance. One selected rebaudioside A-producing strain was denoted ML14737

Example 4

Making the Strain UGT2 1a-Free

The hygromycin antibiotic marker and the nourseothricin antibiotic marker were removed from strain ML14737 after transformation with MB6128 (FIG. 14) which encodes a construct for constitutive overexpression of the CRE recombinase. CRE recombinase deletes the antibiotics markers by recombination over the Lox66 and Lox71 sites. An inactive Lox72 site is left in the genome (Guldener et al, 1996, Lambert et al, 2007). Plasmid MB6128 is a CEN plasmid which replicates episomally in Yarrowia lipolytica and which contains the CRE recombinase coding region under control of the native Yarrowia lipolytica pHHF promoter and hhfT terminator, and a neoR (encoding for G418 resistance) under the control of the native Yarrowia lipolytica pTEF1 promoter and xprT terminator. After selection of MB6128 transformants on YPD+G418 and screening for transformants that lost hygromycin and nourseothricin resistance by successful Cre-Lox recombination, the sensitive colonies were grown on non-selective medium to remove the MB6128 CEN plasmid (spontaneous loss of the CEN plasmid). The resulting antibiotic marker-free variant is denoted ML14869. This strain no longer produces rebaudioside A due to the loss of UGT2_1a along with the hygromycin resistance and produces the intermediate rubusoside instead.

Example 5

Introduction of UGT2 10b

ML14869 was transformed with a 4.2 kb DNA fragment produced by PCR and purified following gel electrophoresis. The fragment encoded a sequence optimized variant of UGT2_10b and hygromycin resistance. The DNA fragment was generated by fusion PCR as follows. UGT2_10b was codon pair optimized for expression in Y. lipolytica and synthesized by DNA2.0 (SEQ ID NO: 18), linked to the native Yarrowia lipolytica pHSP promoter (SEQ ID NO: 20) and gpdT terminator (SEQ ID NO: 28) and flanked by connector sequences. This 1.4 kb DNA fragment was amplified using appropriate oligos and purified by gel electrophoresis. The HPH marker was flanked by lox sites, and linked to the Ashbya gossypii pTEF1 promoter and tef1T terminator and flanked by connector sequences. This 1.8 kb DNA fragment was amplified using appropriate oligos and purified by gel electrophoresis. A 4.2 kb DNA fragment was obtained by PCR using these two DNA fragments with followed by gel electrophoresis and purification. Transformation of ML14869 with this defined DNA fragment and selection on YPD+100 ug/ml hygromycin yielded the rebaudioside A producing strain ML14937.

Example 6

Making Strain ML14937 Marker-Free

The hygromycin antibiotic marker was removed from strain ML14937 after transformation with MB6128 (FIG. 14) which encodes a construct for constitutive overexpression of the CRE recombinase. CRE recombinase deletes the antibiotics markers by recombination over the Lox66 and Lox71 sites. An inactive Lox72 site is left in the genome (Guldener et al, 1996, Lambert et al, 2007). Plasmid MB6128 is a CEN plasmid which replicates episomally in Yarrowia lipolytica and which contains the CRE recombinase coding region under control of the native Yarrowia lipolytica pHHF promoter and hhfT terminator and a neoR (encoding for G418 resistance) under the control of the native Yarrowia lipolytica pTEF1 promoter and xprT terminator. After selection of MB6128 transformants on YPD+G418 and screening for transformants that lost hygromycin and nourseothricin resistance by successful Cre-Lox recombination, the sensitive colonies were grown on non-selective medium to remove the MB6128 CEN plasmid (spontaneous loss of the CEN plasmid). The resulting antibiotic marker-free variant is denoted ML14958.

Example 7

Transformation with Extra Gene Copies

Strain ML14958 was struck to YPD and grown overnight and then struck to 5-FOA plates to allow for recombination-mediated loss of the URA2 marker. One selected 5′-FOA resistant transformant was denoted ML15075. Strain ML15075 was transformed with 3 defined DNA fragments and selected for transformation on YPD with 100 ug/ml hygromycin. The three fragments were as follows:

1) a 4.6 kb DNA fragment encoding the KAH open reading frame (SEQ ID NO: 15) linked to the native Y. lipolytica pHYPO promoter (SEQ ID NO: 21) and the xprT terminator (SEQ ID NO: 26) and also encoding the HPH hygromycin resistance gene flanked by lox sites, produced by PCR and purified following gel electrophoresis. Sequences were assembled in Saccharomyces cerevisiae, and DNA from this S. cerevisiae strain was used as template for PCR yielding the 4.6 kb DNA fragment (see FIG. 15) used to transform ML15075.

2) a 3.3 kb DNA fragment encoding the tCPS open reading frame (SEQ ID NO: 13) linked to the native Y. lipolytica pHSP promoter (SEQ ID NO: 20) and xprT terminator (SEQ ID NO: 26), produced by PCR and purified following gel electrophoresis. Sequences were assembled in Saccharomyces cerevisiae, and DNA from this S. cerevisiae strain was used as template for PCR yielding the 3.3 kb DNA fragment (FIG. 16) used to transform ML15075.

3) a 9.1 kb fragment isolated by gel purification following PvuI/SapI digestion of plasmid MB6986 (FIG. 17). This construct encodes tHMG (SEQ ID NO: 9) linked to the native Y. lipolytica HSP promoter (SEQ ID NO: 20) and CWP terminator (SEQ ID NO: 27), the lox-flanked URA3blaster prototrophic marker, and GGS1 (SEQ ID NO: 7) linked to the native Y. lipolytica HYPO promoter (SEQ ID NO: 21) and GPD terminator (SEQ ID NO: 28). ML15075 is auxotrophic due to a mutation in ura2, so this fragment was not selected for.

One selected hygromycin-resistant transformant was denoted ML15085.

Example 8

Transformation of Extra Copies of tHMG and GGS

Strain ML15085 was transformed with a 8.4 kb fragment isolated by gel purification following PvuI/SapI digestion of plasmid MB6988 (FIG. 11). This construct encodes tHMGopt (SEQ ID NO: 9) linked to the native Y. lipolytica pHSP promoter (SEQ ID NO: 20) and cwpT terminator (SEQ ID NO: 27), the lox-flanked URA2blaster prototrophic marker, and GGSopt (SEQ ID NO: 7) linked to the native Y. lipolytica pHYPO promoter (SEQ ID NO: 21) and gpdT terminator (SEQ ID NO: 28). Transformants were selected on minimal medium lacking uracil. One selected uracil prototroph was denoted ML15086.

Example 9

Making Strain ML15086 Marker-Free

The hygromycin antibiotic marker was removed from strain ML15086 after transformation with MB6128 (FIG. 14) which encodes a construct for constitutive overexpression of the CRE recombinase. CRE recombinase deletes the antibiotics markers by recombination over the Lox66 and Lox71 sites. An inactive Lox72 site is left in the genome (Guldener et al, 1996, Lambert et al, 2007). Plasmid MB6128 is a CEN plasmid which replicates episomally in Yarrowia lipolytica and which contains the CRE recombinase coding region under control of the native Yarrowia lipolytica pHHF promoter and hhfT terminator and a neoR (encoding for G418 resistance) under the control of the native Yarrowia lipolytica pTEF1 promoter and xprT terminator.

After selection of MB6128 transformants on YPD+G418 and screening for transformants that lost hygromycin and nourseothricin resistance by successful Cre-Lox recombination, the sensitive colonies were grown on non-selective medium to remove the MB6128 CEN plasmid (spontaneous loss of the CEN plasmid). One prototrophic, antibiotic marker-free variant is denoted ML15087.

Example 10

Making Strain ML15086 Marker-Free

The hygromycin antibiotic marker and the URA2 prototrophic marker were removed from strain ML15086 after transformation with MB6128 which encodes a construct for constitutive overexpression of the CRE recombinase. CRE recombinase deletes the antibiotic markers by recombination over the Lox66 and Lox71 sites. An inactive Lox72 site is left in the genome. Plasmid MB6128 is a CEN plasmid which replicates episomally in Yarrowia lipolytica and which contains the CRE recombinase coding region under control of the native Yarrowia lipolytica HHF promoter and a neo G418 under the control of the native Yarrowia lipolytica TEF1 promoter. After selection of MB6128 transformants on YPD+G418 and screening for transformants that lost hygromycin resistance and URA2 by successful Cre-Lox recombination, the sensitive colonies were grown on non-selective medium to remove the MB6128 CEN plasmid (spontaneous loss of the CEN plasmid). The resulting antiobiotic marker-free, uracil auxotroph variant is denoted ML15184.

Strain ML15184 was transformed with a 19.0 kb fragment isolated by gel purification following SfiI digestion of plasmid MB7094. MB7094 encodes the CPS (SEQ ID NO: 13) linked to the native Y. lipolytica HYPO promoter (SEQ ID NO: 21) and CWP terminator (SEQ ID NO: 27), the KS (SEQ ID NO: 14) linked to the native Y. lipolytica CWP promoter (SEQ ID NO: 23) and GPD terminator (SEQ ID NO: 28), the KAH (SEQ ID NO: 15) linked to the native Y. lipolytica HSP promoter (SEQ ID NO: 20) and PGM terminator (SEQ ID NO: 29), the KO (SEQ ID NO: 16) linked to the native Y. lipolytica ENO promoter (SEQ ID NO: 22) and PGK terminator (SEQ ID NO: 30), the CPR (SEQ ID NO: 17) linked to the native Y. lipolytica TPI promoter (SEQ ID NO: 24) and XPR terminator (SEQ ID NO: 26) and URA2blaster prototrophic marker. Transformants were selected on minimal medium lacking uracil. One selected rebaudioside producing uracil prototroph was denoted ML15186 and another was denoted ML15187.

Example 11

Comparison of Expression of Yarrowia lipolitica GGS1 Gene and Mucor cirinelloides carG Gene

Yarrowia lipolytica strain ML15087 was transformed with a linearized plasmid for chromosomal integration of plasmid containing hygromycin resistance for chromosomal integration and the HSP promoter and GPD terminator for expression of DNA encoding two geranylgeranylsynthases, the Yarrowia lipolytica GGS1 gene and the Mucor circinelloides carG gene. Eleven individual transformants were isolated from each transformation reaction and tested for rebaudioside A production in a batch fermentation assay. Isolates were inoculated in duplicate alongside the parent (ML15087) to 0.8 ml YPD in 24 well shake plates and grown 3 days at 30 C with shaking at 180 rpm. 40 ul of these cultures were used to inoculate 0.76 ml of ¼ X YP with 5% dextrose which was grown as above for four days. Broth was diluted 200 fold into 33% acetonitrile prior to centrifugation, and clarified supernatant was submitted to analysis by LC-MS as described in WO213/110673.

The results are set out in FIG. 18. Expression of the carG gene leads to greater accumulation of rebA than expression of the GGS1 gene.

Yarrowia lipolytica strains ML15186 and ML15187 were transformed with linearized plasmid for chromosomal integration of plasmid containing hygromycin resistance for chromosomal integration of the Yarrowia lipolytica GGS1 gene expressed behind the strong constitutive TEF promoter and the Mucor circinelloides CarG gene expressed behind either the strong constitutive HSP promoter or the strong constitutive HYPO promoter. Individual transformants were isolated from each transformation reaction [ML15186 TEF-GGS1 and HSP-CarG: n=12; ML15186 HYPO-CarG n=6; ML15187 TEF-GGS1, HSP-CarG, HYPO-CarG: n=12] and tested for rebaudioside A production in a batch fermentation assay. Isolates were inoculated alongside the parents (ML15186 and ML15187) to 0.8 ml YPD in 24 well shake plates and grown 3 days at 30 C with shaking at 180 rpm. 40 ul of these cultures were used to inoculate 0.76 ml of ¼ X YP with 5% dextrose which was grown as above for four days. Broth was diluted 200 fold into 33% acetonitrile prior to centrifugation, and clarified supernatant was submitted to analysis by LC-MS as described in WO213/110673.

The results are set out in FIG. 19. Expression of the carG gene leads to greater accumulation of rebA than expression of the GGS1 gene.

Example 12

Use of CarG to Increase Steviol Glycoside Production in Mutagenized Derivatives of Strain ML15186

This example shows the general utility of the CarG gene in increasing production of steviol glycosides in a genetically diverse population of steviol glycoside producing Y. lipolytica.

ML15186 was grown in 500 ml shake flasks containing 100 ml YEPD with 2% glucose at 280 rpm and 30° C. At an optical density (OD) of 2, cells were spun down and washed twice with milliQ and suspended in 25 ml 0.1M sodium citrate buffer pH 5.5. To 4 ml of the cell suspension NTG was added to a final concentration of 0.150 mg/ml. The cell suspension was then incubated for 45 minutes in a water bath at 25° C. and 75 rpm. The reaction was stopped by addition of 1 ml 10% Na2S2O3.5H2O. The suspension was poured directly into 50 ml Greiner tubes, filled up to 50 ml with sterile 0.85% physiological salt solution, mixed and centrifuged. The pellets were washed once and re-suspended in 10 ml sterile physiological salt solution. Cells were plated for single colonies, and tested for the production of steviol glycosides. One of these colonies had acquired a chromosomal mutation which resulted in higher production of RebA at a higher purity and was named STV2119.

ML15186 was grown in 500 ml shake flasks containing 100 ml YEPD with 2% glucose at 280 rpm and 30° C. At an OD of 2, cells were spun down and washed twice and re-suspended in milliQ to a final OD of 1.0. 10 ml of this cell suspension was transferred to a plastic petri dish and exposed to UV using the DARK TOP UV (Osram HQV 125\N) for 300 sec with gentle shaking at 6 rpm. After UV mutagenesis the suspension was kept in the dark for 2 hours to avoid photo reactivation. Cells were plated for single colonies, and tested for the production of steviol glycosides. One of these colonies had acquired a chromosomal mutation which resulted in the production of more RebA with a higher purity profile was named STV2121.

Yarrowia lipolytica strains STV2119 and STV2121 were transformed with linearized plasmid for chromosomal integration of plasmid containing hygromycin resistance for chromosomal integration of the Mucor circinelloides CarG gene expressed behind either the constitutive TPI promoter (MB7351; SEQ ID NO: 24) or the strong constitutive HSP promoter (MB7282; SEQ ID NO: 20). Individual transformants were isolated from each transformation reaction [STV2119 TPI-CarG and HSP-CarG: n=11; STV2121 TPI-CarG and HSP-CarG: n=11] and tested for rebaudioside A production in a batch fermentation assay. Isolates were inoculated in duplicate alongside the parents (STV2119, STV2121; 4 replicates each) to 0.8 ml YPD in 24 well shake plates and grown 3 days at 30 C with shaking at 180 rpm. 40 ul of these cultures were used to inoculate 0.76 ml of ¼ X YP with 5% dextrose which was grown as above for four days. Broth was diluted 200 fold into 33% acetonitrile prior to centrifugation, and clarified supernatant was submitted to analysis by LC-MS as described in WO213/110673. In the case of STV2121, two transformants lost the ability to produce RebA and were not considered in the analysis

The results are set out in FIG. 20. Expression of the carG gene leads to greater accumulation of rebA in both genetic backgrounds with p-values <0.01, <0.025, <0.03 and <0.001 for STV2119 HSP-CarG, STV2119 TPI-CarG, STV2121 HSP-CarG and STV2121 TPI-CarG as determined by two-tailed student's t-test.

TABLE 1
Genotype of strains
Strain name Genotype
ML10371     MAT-A, lys1-, ura3-, leu2-,
ML13462F    MAT-A, lys1-, ura3-, leu2-, tHMG, GGS1, UGT2, HPH
ML13500     MAT-A, lys1-, ura3-, leu2-, tHMG, GGS1,
            UGT2, HPH, UGT1, UGT3, UGT4, NAT
ML13723     MAT-A, lys1-, ura3-, leu2-, tHMG, GGS1, UGT2,
            HPH, UGT1, UGT3, UGT4, NAT, URA3
ML14032     MAT-A, lys1-, ura3-, leu2-, tHMG, GGS1, UGT2,
            HPH, UGT1, UGT3, UGT4, NAT, CPS, KS, KAH, KO,
            CPR, URA3, LEU2
ML14093     MAT-A, lys1-, ura3-, leu2-, tHMG, GGS1, UGT2,
            HPH, UGT1, UGT3, UGT4, NAT, CPS, KS, KAH, KO,
            CPR, LEU2
ML14094*    MAT-A, lys1-, ura3-, leu2-, tHMG, GGS1, UGT2,
            HPH, UGT1, UGT3, UGT4, NAT, CPS, KS, KAH, KO,
            CPR, LEU2, URA3
ML350       MAT-B, ade1-, ura2-, leu2-
ML13206     MAT-B, ade1-, ura2-, leu2-
ML13465     MAT-B, ade1-, ura2-, leu2-, tHMG, GGS1, UGT2, HPH
ML13490     MAT-B, ade1-, ura2-, leu2-, tHMG, GGS1, UGT2, HPH,
            UGT1, UGT3, UGT4, NAT, URA2
ML13501     MAT-B, ade1-, ura2-, leu2-, tHMG, GGS1, UGT2, HPH,
            UGT1, UGT3, UGT4, NAT
ML13724     MAT-B, ade1-, ura2-, leu2-, tHMG, GGS1, UGT2, HPH,
            UGT1, UGT3, UGT4, NAT, URA2
ML14044     MAT-B, ade1-, ura2-, leu2-, tHMG, GGS1, UGT2, HPH,
            UGT1, UGT3, UGT4, NAT, CPS, KS, KAH, KO, CPR,
            URA2, LEU2
ML14076     MAT-B, ade1-, ura2-, leu2-, tHMG, GGS1, UGT2,
            HPH, UGT1, UGT3, UGT4, NAT, CPS, KS, KAH, KO,
            CPR, LEU2
ML14087*    MAT-B, ade1-, ura2-, leu2-, tHMG, GGS1, UGT2,
            HPH, UGT1, UGT3, UGT4, NAT, CPS, KS, KAH, KO,
            CPR, LEU2, URA2
ML14737**   MAT-B ura2-, leu2-,tHMG, GGS1, UGT2, HPH, UGT1,
            UGT3, UGT4, NAT, CPS, KS, KAH, KO, CPR, LEU2,
            URA2, URA3
ML14869**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3,
            UGT4, CPS, KS, KAH, KO, CPR, LEU2, URA2, URA3
ML14937**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3,
            UGT4, CPS, KS, KAH, KO, CPR, LEU2, URA2, URA3,
            UGT2_10b, HPH
ML14958**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA2, URA3,
            UGT2_10b
ML15075**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA3, UGT2_10b
ML15085**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA3, UGT21_10b,
            HPH
ML15086**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA2, URA3,
            UGT2_10b, HPH
ML15087**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA2, URA3,
            UGT2_10b
ML15184**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA3, UGT2_10b
ML15186**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA2, URA3,
            UGT2_10b
ML15187**   MAT-B, ura2-, leu2-, tHMG, GGS1, UGT1, UGT3, UGT4,
            CPS, KS, KAH, KO, CPR, LEU2, URA2, URA3,
            UGT2_10b
STV2119     Identical to ML15186 except for uncharacterized
            random mutations
STV2121     Identical to ML15186 except for uncharacterized
            random mutations
All strains except ML10371, ML350 and ML13206 contain one or more copies of tHMG and GGS1
*contains two copies of CPS, KS, KAH, KO and CPR.
***contains 1, 2, 3 or 4 copies of CPS, KS, KAH, KO and CPR

TABLE 2
Description of the sequence listing
SEQ ID NO     Description
SEQ ID NO: 1  carG amino acid
              from Mucor circinelloides
SEQ ID NO: 2  carG nucleic acid
              from Mucor circinelloides
              (incl. introns)
SEQ ID NO: 3  carG nucleic acid
              from Mucor circinelloides
              (excl. introns)
SEQ ID NO: 4  carG nucleic acid codon
              optimized for Y. lipolitica
              Mucor circinelloides
SEQ ID NO: 5  GGS amino acid
              from Y. lipolitica
SEQ ID NO: 6  GGS nucleic acid
              from Y. lipolitica
SEQ ID NO: 7  GGS codon optimised for
              Y. lipolitica
SEQ ID NO: 8  UGT2_1a CpO for
              Y. lipolitica
SEQ ID NO: 9  tHMG CpO for
              Y. lipolitica
SEQ ID NO: 10 UGT1 CpO for
              Y. lipolitica
SEQ ID NO: 11 UGT3 CpO for
              Y. lipolitica
SEQ ID NO: 12 UGT4 CpO for
              Y. lipolitica
SEQ ID NO: 13 tCPS from
              S. rebaudiana
              CpO for Y. lipolitica
SEQ ID NO: 14 tKS from
              S. rebaudiana
              CpO for Y. lipolitica
SEQ ID NO: 15 KAH_4 CpO for
              Y. lipolitica
SEQ ID NO: 16 KO from
              Gibberella fujikori
              CpO for
              Y. lipolitica
SEQ ID NO: 17 CPR_3 CpO for
              Y. lipolitica
SEQ ID NO: 18 UGT2_10b CpO
              for Y. lipolitica
SEQ ID NO: 19 PGM promoter
              from Y. lipolitica
SEQ ID NO: 20 HSP promoter
              from Y. lipolitica
SEQ ID NO: 21 HYPO promoter
              from Y. lipolitica
SEQ ID NO: 22 ENO promoter
              from
              Y. lipolitica
SEQ ID NO: 23 CWP promoter
              from
              Y. lipolitica
SEQ ID NO: 24 TPI promoter
              from
              Y. lipolitica
SEQ ID NO: 25 YP001
              promoter from
              Y. lipolitica
SEQ ID NO: 26 Xpr terminator
              from
              Y. lipolitica
SEQ ID NO: 27 Cwp terminator
              from
              Y. lipolitica
SEQ ID NO: 28 Gpd terminator
              from
              Y. lipolitica
SEQ ID NO: 29 Pgm terminator
              from
              Y. lipolitica
SEQ ID NO: 30 Pgk terminator
              from
              Y. lipolitica
SEQ ID NO: 31 act1T
              terminator
              from
              Y. lipolitica

Claims

1. A recombinant host capable of producing a steviol glycoside comprising a recombinant nucleic acid sequence encoding a polypeptide having geranylgeranyl pyrophosphate (GGPP) synthase activity which comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least about 45% sequence identity thereto.

2. A recombinant host according to claim 1, wherein the recombinant nucleic acid encodes a polypeptide derived from Mucor circinelloides.

3. A recombinant host according to claim 1 which comprises one or more recombinant nucleotide sequence(s) 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 kaurenoic acid 13-hydroxylase activity.

4. A recombinant host according to claim 1, which comprises a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity.

5. A recombinant host according to claim 1 which comprises a recombinant nucleic acid sequence encoding one or more of:

(i) a polypeptide having UGT74G1 activity;

(ii) a polypeptide having UGT2 activity;

(iii) a polypeptide having UGT85C2 activity; and

(iv) a polypeptide having UGT76G1 activity.

6. A recombinant host according to claim 1, wherein the host belongs to one of the genera Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadazyma or Escherichia.

7. A recombinant host according to claim 6, wherein the recombinant host is a Saccharomyces cerevisiae cell, a Yarrowia lipolitica cell, a Candida krusei cell, an Issatchenkia orientalis or an Escherichia coli cell.

8. A recombinant host according to claim 1, wherein the ability of the host to produce geranylgeranyl diphosphate (GGPP) is upregulated.

9. A recombinant host according to claim 1 which comprises a nucleic acid sequence encoding one or more of:

a polypeptide having hydroxymethylglutaryl-CoA reductase activity; or

a polypeptide having farnesyl-pyrophosphate synthetase activity.

10. A process for preparation of a steviol glycoside which comprises fermenting a recombinant host according to claim 1 in a suitable fermentation medium and, optionally, recovering the steviol glycoside.

11. A process according to any one of claim 10 for preparation of a steviol glycoside, wherein the process is carried out on an industrial scale.

12. A fermentation broth comprising a steviol glycoside obtainable by the process according to claim 10.

13. A steviol glycoside obtained by a process according to claim 10 or obtained from a fermentation broth obtainable from said process.

14. A composition comprising two or more steviol glycosides obtained by a process according to claim 10 or obtained from a fermentation obtainable from said process.

15. A foodstuff, feed or beverage which comprises a steviol glycoside according to claim 13 or a composition comprising two or more of said steviol glycosides.