Patent Description:
The worldwide demand for high potency sweeteners is increasing and, with blending of different artificial sweeteners, becoming a standard practice. However, the demand for alternatives is expected to increase. 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, with the added advantage that Stevia sweeteners are natural plant products.

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 <NUM> to <NUM>% 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 <NUM> and <NUM> times sweeter than sucrose, rebaudioside A between <NUM> and <NUM> 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 <NUM> times sweeter than sucrose. Rebaudioside M s also a high-potency diterpene glycoside sweetener present in trace amount in certain stevia variety leaves but has been suggested to have best taste profile.

Currently, steviol glycosides are extracted from the Stevia plant. In Stevia, (-)-kaurenoic acid, an intermediate in gibberellic acid (GA) biosynthesis, is converted into the tetracyclic dipterepene steviol, which then proceeds through a multi-step glucosylation 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.

<CIT> describes a method to purify Rebaudioside X (also known as Rebaudioside M) as derived from Stevia rebaudiana plant material. Such methd may comprise passing a solution of steviol glycosides through a multi-column system including a plurality of columns packed with an adsorbent resin to provide at least one column having adsorbed steviol glycosides; and subeseqeuntly eluting fractions with high Reb X content from the at least one column having adsorbed steviol glycosides to provide an eluted solution with high Reb X content. Alternatively, the Reb X may be purified by a crystallisation method from alcoholic solvent. This document further discloses Reb X compositions obtaind by said purification methods.

New, more standardized, clean single composition, no after-taste, sources of glycosides, such as rebaudioside M, are required to meet growing commercial demand for high potency, natural sweeteners.

Development of fermentation technologies for production of high-value steviol glycosides that may be lower-cost, more abundant, and currently perceived as trace by-products is desired.

There are more than <NUM> different steviol glycosides found within the stevia leaf, including Reb A, and next-generation sweeteners such as Reb D and Reb M, which have superior taste profiles but which are found in much lower quantities within the stevia leaf. Because the most desirable steviol glycosides are rare within the stevia leaf, fermentation processes offer attractive commercial advantages for large-scale production.

In Stevia, steviol is synthesized from GGPP, which is formed by the deoxyxylulose <NUM>-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 leaves, (-)-kaurenoic acid is then hydroxylated, by ent-kaurenoic acid <NUM>-hydroxylase (KAH) to form steviol. Steviol is then glucosylated by a series of UDP-glucosyltransferases (UGTs).

Microorganism capable of producing a diterpene, such as steviol, or a glycosylated diterpene (i.e. a diterpene glycoside), such as steviolmonoside, steviolbioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rubusoside or dulcoside A are used in the invention.

A recombinant microorganism used in the invention comprises one or more nucleotide sequence(s) encoding:.

Typically, a recombinant microorganism used in the invention will comprise all of the above nucleotide sequences.

The invention provides a process for the preparation of rebaudioside M by fermentation which comprises fermenting a microorganism of the genus Yarrowia in a suitable fermentation medium at a pH of below <NUM>, and recovering rebaudioside M,.

A description of the sequences is set out in Table <NUM>. Sequences described herein may be defined with reference to the sequence listing or with reference to the database accession numbers also set out in Table <NUM>.

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.

A recombinant microorganism that is capable of producing rebaudioside M (RebM) is used in the invention. The structure of RebM is set out in <NPL>.

RebM is a glycosylated diterpene. For the purposes of this disclosure, a diterpene typically means an organic compound composed of four isoprene units. Such a compound may be derived from geranylgeranyl pyrophosphate. A glycosylated diterpene or diterpene glycoside is a diterpene in which a sugar is bound, typically to a non-carbohydrate moiety. Typically, in a diterpene glycoside, the sugar group may be bonded through its anomeric carbon to another group via a glycosidic bond.

A recombinant microorganism is used in the invention. The recombinant microorganism comprises one or more nucleotide sequence(s) encoding:.

A polypeptide having ent-copalyl pyrophosphate synthase (EC <NUM>. <NUM>) is capable of catalyzing the chemical reation:
<CHM>.

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.

For the purposes of this disclosure, a polypeptide having ent-kaurene synthase activity (EC <NUM>. <NUM>) is a polypeptide that is capable of catalyzing the chemical reaction:
<CHM>.

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.

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 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.

A polypeptide having ent-kaurene oxidase activity (EC <NUM>. <NUM>) is a polypeptide which is capable of catalysing three successive oxidations of the <NUM>-methyl group of ent-kaurene to give kaurenoic acid. Such activity typically requires the presence of a cytochrome P450.

A polypeptide having kaurenoic acid <NUM>-hydroxylase activity (EC <NUM>. <NUM>) is one which is capable of catalyzing the formation of steviol (ent-kaur-<NUM>-en-<NUM>-ol-<NUM>-oic acid) using NADPH and O<NUM>. Such activity may also be referred to as ent-ka <NUM>-hydroxylase activity.

A recombinant microorganism used in the invention also comprises nucleotide sequences encoding polypeptides having UDP-glucosyltransferase (UGT) activity, whereby expression of the nucleotide sequences confers on the microorganism the ability to produce at least rebaudioside M.

A polypeptide having UGT activity is one which has glycosyltransferase activity (EC <NUM>), 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).

The UGTs used are selected so as to produce a desired diterpene glycoside, such as a steviol glycoside. Schematic diagrams of steviol glycoside formation are set out in <NPL> and<NPL>. In addition, <FIG> sets out a schematic diagram of steviol glycoside formation.

The biosynthesis of rebaudioside A involves glucosylation of the aglycone steviol. Specifically, rebaudioside A can be formed by glucosylation of the <NUM>-OH of steviol which forms the <NUM>-O-steviolmonoside, glucosylation of the C-<NUM>' of the <NUM>-O-glucose of steviolmonoside which forms steviol-<NUM>,<NUM>-bioside, glucosylation of the C-<NUM> carboxyl of steviol-<NUM>,<NUM>-bioside which forms stevioside, and glucosylation of the C-<NUM>' of the C-<NUM>-O-glucose of stevioside. The order in which each glucosylation reaction occurs can vary-see <FIG>. One UGT may be capable of catalyzing more than one conversion as set out in this scheme.

We have shown that conversion of steviol to rebaudioside M may be accomplished in a recombinant host by the expression of gene(s) encoding the following functional UGTs: UGT74G1, UGT85C2, UGT76G1 and UGT2.

Thus, a recombinant microorganism expressing these four UGTs can make rebaudioside M if it produces steviol or when fed steviol in the medium. Typically, one or more of these genes are recombinant genes that have been transformed into a microorganism that does not naturally possess them.

Examples of all of these enzmyes are set out in Table <NUM>. A microorganism used in the invention may comprise any combination of a UGT74G1, UGT85C2, UGT76G1 and UGT2. In Table <NUM> UGT64G1 sequences are indicated as UGT1 sequences, UGT74G1 sequences are indicated as UGT3 sequences and UGT76G1 sequences are indicated as UGT4 sequences. UGT2 sequences are indicated as UGT2 sequences in Table <NUM>.

A recombinant microorganism used in the invention comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-<NUM>-glucose to steviol. That is to say, a microorganism used in the invention may comprise a UGT which is capable of catalyzing a reaction in which steviol is converted to steviolmonoside.

Such a microorganism 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 microorganism confers on the cell the ability to convert steviol to steviolmonoside.

UGT85C2 activity is transfer of a glucose unit to the <NUM>-OH of steviol. Thus, a suitable UGT85C2 may function as a uridine <NUM>'-diphospho glucosyl: steviol <NUM>-OH transferase, and a uridine <NUM>'-diphospho glucosyl: steviol- <NUM>-O-glucoside <NUM>-OH transferase. A functional UGT85C2 polypeptides may also catalyze glucosyl transferase reactions that utilize steviol glycoside substrates other than steviol and steviol- <NUM>-O-glucoside. Such sequences are indicated as UGT1 sequences in Table <NUM>.

A recombinant microorganism used in the invention also comprises 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-<NUM>-glucose to steviol or steviolmonoside. That is to say, a microorganism used in the invention may comprise a UGT which is capable of catalyzing a reaction in which steviolmonoside is converted to steviolbioside. Accordingly, such a microorganism may be capable of converting steviolmonoside to steviolbioside. Expression of such a nucleotide sequence may confer on the microorganism the ability to produce at least steviolbioside.

A microorganism used in the invention may thus comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT2, whereby the nucleotide sequence upon transformation of the microorganism confers on the cell the ability to convert steviolmonoside to steviolbioside.

A suitable UGT2 polypeptide functions as a uridine <NUM>'-diphospho glucosyl: steviol- <NUM>-<NUM>-glucoside transferase (also referred to as a steviol-<NUM>-monoglucoside <NUM>,<NUM>-glucosylase), transferring a glucose moiety to the C-<NUM>' of the <NUM>-O-glucose of the acceptor molecule, steviol- <NUM>-O-glucoside. Typically, a suitable UGT2 polypeptide also functions as a uridine <NUM>'-diphospho glucosyl: rubusoside transferase transferring a glucose moiety to the C-<NUM>' of the <NUM>-O-glucose of the acceptor molecule, rubusoside.

Functional UGT2 polypeptides may also catalyze reactions that utilize steviol glycoside substrates other than steviol- <NUM>-O-glucoside and rubusoside, e.g., functional UGT2 polypeptides may utilize stevioside as a substrate, transferring a glucose moiety to the C-<NUM>' of the <NUM>-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-<NUM>' of the <NUM>-O-glucose residue to produce Rebaudioside D. However, a functional UGT2 polypeptide typically does not transfer a glucose moiety to steviol compounds having a <NUM> ,<NUM>-bound glucose at the C-<NUM> position, i.e., transfer of a glucose moiety to steviol <NUM>,<NUM>-bioside and <NUM>,<NUM>-stevioside does not occur. Functional UGT2 polypeptides may also transfer sugar moieties from donors other than uridine diphosphate glucose. For example, a functional UGT2 polypeptide may act as a uridine <NUM>'-diphospho D-xylosyl: steviol-<NUM>-O-glucoside transferase, transferring a xylose moiety to the C-<NUM>' of the <NUM>-O-glucose of the acceptor molecule, steviol-<NUM>-O-glucoside. As another example, a functional UGT2 polypeptide can act as a uridine <NUM>'-diphospho L-rhamnosyl: steviol- <NUM>-O-glucoside transferase, transferring a rhamnose moiety to the C-<NUM>' of the <NUM>-O-glucose of the acceptor molecule, steviol-<NUM>-O-glucoside. Such sequences are indicated as UGT2 sequences in Table <NUM>.

A recombinant microorganism used in the invention also comprises 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-<NUM>-glucose to steviolbioside. That is to say, a microorganism used in the invention may comprise a UGT which is capable of catalyzing a reaction in which steviolbioside is converted to stevioside. Accordingly, such a microorganism may be capable of converting steviolbioside to stevioside. Expression of such a nucleotide sequence may confer on the microorganism the ability to produce at least stevioside.

A microorganism used inthe 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 microorganism confers on the cell the ability to convert steviolbioside to stevioside.

Suitable UGT74G1 polypeptides may be capable of transferring a glucose unit to the <NUM>-OH or the <NUM>-COOH, respectively, of steviol. A suitable UGT74G1 polypeptide may function as a uridine <NUM>'-diphospho glucosyl: steviol <NUM>-COOH transferase and a uridine <NUM>'-diphospho glucosyl: steviol-<NUM>-O-glucoside <NUM>-COOH transferase. Functional UGT74G1 polypeptides also may catalyze glycosyl transferase reactions that utilize steviol glycoside substrates other than steviol and steviol-<NUM>-O-glucoside, or that transfer sugar moieties from donors other than uridine diphosphate glucose. Such sequences are indicated as UGT1 sequences in Table <NUM>.

A recombinant microorganism used inthe invention also comprises a nucleotide sequence encoding a polypeptide capable of catalyzing glucosylation of the C-<NUM>' of the glucose at the C-<NUM> position of stevioside. That is to say, a microorganism used in the invention may comprise a UGT which is capable of catalyzing a reaction in which stevioside to rebaudioside A. Accordingly, such a microorganism may be capable of converting stevioside to rebaudioside A. Expression of such a nucleotide sequence may confer on the microorganism the ability to produce at least rebaudioside A.

A microorganism used in 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 the microorganism confers on the cell the ability to convert stevioside to rebaudioside A.

A suitable UGT76G1 adds a glucose moiety to the C-<NUM>' of the C-<NUM>-O-glucose of the acceptor molecule, a steviol <NUM>,<NUM> glycoside. Thus, UGT76G1 functions, for example, as a uridine <NUM>'-diphospho glucosyl: steviol <NUM>-<NUM>-<NUM>,<NUM> glucoside C-<NUM>' glucosyl transferase and a uridine <NUM>'-diphospho glucosyl: steviol-<NUM>-O-glucose, <NUM>-<NUM>-<NUM>,<NUM> bioside C-<NUM>' 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 are indicated as UGT4 sequences in Table <NUM>.

A microorganism used in the invention comprises nucleotide sequences encoding polypeptides having all four UGT activities described above. A given nucleic acid may encode a polypeptide having one or more of the above activities. For example, a nucleic acid encodes for a polypeptide which has two, three or four of the activities set out above. Preferably, a recombinant microorganism used in the invention comprises UGT1, UGT2 and UGT3 and UGT4 activity.

A microorganism used in the invention comprises a nucleotide sequence encoding a polypeptide having UGT activity capable of catalyzing the glucosylation of stevioside or rebaudioside A. That is to say, a microorganism used in the invention may comprise a UGT which is capable of catalyzing a reaction in which stevioside or rebaudioside A is converted to rebaudioside D. Accordingly, such a microorganism may be capable of converting stevioside or rebaudioside A to rebaudioside D. Expression of such a nucleotide sequence may confer on the microorganism the ability to produce at least rebaudioside D.

We have shown that a microorganism expression a combination of UGT85C2, UGT2, UGT74G1 and UGT76G1 polypeptides is capable of rebaudioside M production.

A microorganism used in the invention which comprises a nucleotide sequence encoding a polypeptide having UGT activity may comprise a nucleotide sequence encoding a polypeptide capable of catalyzing the glucosylation of stevioside. That is to say, a microorganism used in the invention may comprise a UGT which is capable of catalyzing a reaction in which stevioside is converted to rebaudioside E. Accordingly, such a microorganism may be capable of converting stevioside to rebaudioside E. Expression of such a nucleotide sequence may confer on the microorganism the ability to produce at least rebaudioside E.

A microorganism used in the invention which comprises a nucleotide sequence encoding a polypeptide having UGT activity may comprise a nucleotide sequence encoding a polypeptide capable of catalyzing the glucosylation of rebaudioside E. That is to say, a microorganism used in the invention may comprise a UGT which is capable of catalyzing a reaction in which rebaudioside E is converted to rebaudioside D. Accordingly, such a microorganism may be capable of converting stevioside or rebaudioside A to rebaudioside D. Expression of such a nucleotide sequence may confer on the microorganism the ability to produce at least rebaudioside D.

A recombinant microorganism used in the invention may be capable of expressing a nucleotide sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity. That is to say, a recombinant microorganism used in the invention may comprise sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity.

For the purposes of the disclosure, a polypeptide having NADPH-Cytochrome P450 reductase activity (EC <NUM>. <NUM>; 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 <NUM>.

Preferably, a recombinant microorganism disclosed but not part of the invention is capable of expressing one or more of:.

Preferably, a recombinant microorganism used in the invention is one which is capable of expressing one or more of:.

In a recombinant microorganism used in the invention, which is capable of expressing a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-<NUM>-glucose to steviol (addition of glucose to the C-<NUM> position of steviol), said nucleotide may comprise:.

In a recombinant microorganism used in the invention, which is capable of expressing a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-<NUM> position of steviolmonoside (this typically indicates glucosylation of the C-<NUM>' of the C-<NUM>-glucose/<NUM>-O-glucose of steviolmonoside), said nucleotide sequence may comprise:.

In a recombinant microorganism used in the invention, which is capable of expressing a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-<NUM> position of steviolbioside, said nucleotide sequence may comprise:.

In a recombinant microorganism used in the invention, which expresses a nucleotide sequence encoding a polypeptide capable of catalyzing glucosylation of the C-<NUM>' of the glucose at the C-<NUM> position of stevioside, said nucleotide sequence may comprise:.

In a recombinant microorganism used in the invention, which expresses a nucleotide sequence encoding a polypeptide capable of catalysing one or more of: the glucosylation of stevioside or rebaudioside A to rebaudioside D; the glucosylation of stevioside to rebaudioside E; the glucosylation of rebaudioside E to rebaudioside D; or the glucosylation of rebaudioside D to rebaudioside M, said nucleotide sequence may comprise:.

A microorganism used in the invention, may be one in which the ability of the microorganism to produce geranylgeranyl pyrophosphate (GGPP) is upregulated. Upregulated in the context of this dislcosure implies that the microorganism produces more GGPP than an equivalent non-transformed strain.

Accordingly, a microorganism used in 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 the microorganism confer(s) on the microorganism the ability to produce elevated levels of GGPP.

Preferably, a microorganism used in the invention is one which is capable of expressing one or more of:.

A microorganism or microbe, is typically an organism that is not visible to the human eye (i.e. microscopic). A microorganism may be from bacteria, fungi, archaea or protists. Typically a microorganism will be a single-celled or unicellular organism.

As used herein a recombinant microorganism is defined as a microorganism which is genetically modified or transformed/transfected with one or more of the nucleotide sequences as defined herein. The presence of the one or more such nucleotide sequences alters the ability of the microorganism to produce a diterpene or diterpene glycoside, in particular steviol or steviol glycoside. A microorganism that is not transformed/transfected or genetically modified, is not a recombinant microorganism and does typically not comprise one or more of the nucleotide sequences enabling the cell to produce a diterpene or diterpene glycoside. Hence, a non-transformed/non-transfected microorganism is typically a microorganism that does not naturally produce a diterpene, although a microorganism which naturally produces a diterpene or diterpene glycoside and which has been modified as disclosed herein (and which thus has an altered ability to produce a diterpene/diterpene gylcoside) is considered a recombinant microorganism according to the dislcosure.

Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by various methods, known to those skilled in the art. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Typically then, identities and similarities are calculated over the entire length of the sequences being compared. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the BestFit, BLASTP, BLASTN, and FASTA (<NPL>), publicly available from NCBI and other sources (<NPL>). Preferred parameters for amino acid sequences comparison using BLASTP are gap open <NUM>, gap extend <NUM>, Blosum <NUM> matrix. Preferred parameters for nucleic acid sequences comparison using BLASTP are gap open <NUM>, gap extend <NUM>, DNA full matrix (DNA identity matrix).

Nucleotide sequences encoding the enzymes expressed in the cell used in the invention may also be defined by their capability to hybridize with the nucleotide sequences of SEQ ID NO. 's <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> ir any other sequence mentioned herein respectively, under moderate, or preferably under stringent hybridisation conditions. Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of at least about <NUM>, preferably about <NUM> nucleotides, <NUM> or <NUM> and most preferably of about <NUM> or more nucleotides, to hybridise at a temperature of about <NUM> in a solution comprising about <NUM> salt, preferably <NUM> x SSC or any other solution having a comparable ionic strength, and washing at <NUM> in a solution comprising about <NUM> salt, or less, preferably <NUM> x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for <NUM> hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having about <NUM>% or more sequence identity.

Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least <NUM> nucleotides, preferably of about <NUM> or more nucleotides, to hybridise at a temperature of about <NUM> in a solution comprising about <NUM> salt, preferably <NUM> x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about <NUM> salt, preferably <NUM> x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for <NUM> hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having up to <NUM>% sequence identity. The person skilled in the art will be able to modify these hybridisation conditions in order to specifically identify sequences varying in identity between <NUM>% and <NUM>%.

The nucleotide sequences encoding an ent-copalyl pyrophosphate synthase; ent-Kaurene synthase; ent-Kaurene oxidase; kaurenoic acid <NUM>-hydroxylase; UGT; hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase; geranylgeranyl diphosphate synthase; NADPH-cytochrome p450 reductase, may be from prokaryotic or eukaryotic origin.

A nucleotide sequence encoding an ent-copalyl pyrophosphate synthase may for instance comprise a sequence as set out in SEQ ID. NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

A nucleotide sequence encoding an ent-Kaurene synthase may for instance comprise a sequence as set out in SEQ ID. NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

A nucleotide sequence encoding an ent-Kaurene oxidase may for instance comprise a sequence as set out in SEQ ID. NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. A preferred KO is the polypeptide encoded by the nucleic acid set out in SEQ ID NO: <NUM>.

A nucleotide sequence encoding a kaurenoic acid <NUM>-hydroxylase may for instance comprise a sequence as set out in SEQ ID. NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. A preferred KAH sequence is the polypeptide encoded by the nucleic acid set out in SEQ ID NO: <NUM>.

A further preferred recombinant microorganism used in the invention may express a combination of the polypeptides encoded by SEQ ID NO: <NUM> and SEQ ID NO: <NUM> or a variant of either thereof as herein described. A preferred recombinant microorganism used in the invention may expression the combination of sequences set out in Table <NUM> (in combination with any UGT2, but in particular that encoded by SEQ ID NO: <NUM>).

A nucleotide sequence encoding a UGT may for instance comprise a sequence as set out in SEQ ID. NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

A nucleotide sequence encoding a hydroxymethylglutaryl-CoA reductase may for instance comprise a sequence as set out in SEQ ID.

A nucleotide sequence encoding a farnesyl-pyrophosphate synthetase may for instance comprise a sequence as set out in SEQ ID.

A nucleotide sequence encoding a geranylgeranyl diphosphate synthase may for instance comprise a sequence as set out in SEQ ID.

A nucleotide sequence encoding a NADPH-cytochrome p450 reductase may for instance comprise a sequence as set out in SEQ ID. NO: <NUM>, <NUM>, <NUM> or <NUM>.

In the case of the UGT sequences, combinations of at least one from each of: (i) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>; (ii) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>; (iii) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>; and (iv) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> may be preferred. Typically, at least one UGT from group (i) may be used. If at least one UGT from group (iii) is used, generally at least one UGT from group (i) is also used. If at least one UGT from group (iv) is used, generally at least one UGT from group (i) and at least one UGT from group (iii) is used. Typically, at least one UGT form group (ii) is used.

A sequence which has at least about <NUM>%, about <NUM>%, about <NUM>%, preferably at least about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, or about <NUM>% sequence identity with a sequence as mentioned may be used in the invention.

To increase the likelihood that the introduced enzymes are expressed in active form in a eukaryotic cell used in the invention, the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage to that of the chosen eukaryote host cell. The adaptiveness of the nucleotide sequences encoding the enzymes to the codon usage of the chosen host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from <NUM> to <NUM>, with higher values indicating a higher proportion of the most abundant codons (see <NPL>; also see: <NPL>). An adapted nucleotide sequence preferably has a CAI of at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

The eukaryotic cell according to the disclosure is genetically modified with (a) nucleotide sequence(s) which is (are) adapted to the codon usage of the eukaryotic cell using codon pair optimisation technology as disclosed in <CIT>. Codon-pair optimisation is a method for producing a polypeptide in a host cell, wherein the nucleotide sequences encoding the polypeptide have been modified with respect to their codon-usage, in particular the codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.

Further improvement of the activity of the enzymes in vivo in a eukaryotic host cell used in the invention, can be obtained by well-known methods like error prone PCR or directed evolution. A preferred method of directed evolution is described in <CIT> and <CIT>.

The microorganism used in the present invention may be any suitable host cell from microbial origin. Preferably, the host cell is a yeast or a filamentous fungus. More preferably, the host cell belongs to one of the genera Saccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces, Yarrowia, Candida, Hansenula, Humicola, Torulaspora, Trichosporon, Brettanomyces, Pachysolen or Yamadazyma or Zygosaccharomyces.

A more preferred microorganism belongs to the species Aspergillus niger, Penicillium chrysogenum, Pichia stipidis, Kluyveromyces marxianus, K. thermotolerans, Yarrowia lipolytica, Candida sonorensis, C. glabrata, Hansenula polymorpha, Torulaspora delbrueckii, Brettanomyces bruxellensis, Zygosaccharomyces bailii, Saccharomyces uvarum, Saccharomyces bayanus or Saccharomyces cerevisiae species. Preferably, the eukaryotic cell is a Saccharomyces cerevisiae.

A recombinant yeast cell used in the invention may be modified so that the ERG9 gene is down-regulated and or the ERG5/ERG6 genes are deleted. Corresponding genes may be modified in this way in other microorganisms.

Such a microorganism may be transformed as set out herein, whereby the nucleotide sequence(s) with which the microorganism is transformed confer(s) on the cell the ability to produce RebM.

A preferred microorganism used in the invention is a Yarrowia lipolytica cell. A recombinant microorganism used in the invention, such as a recombinant Yarrowia lipolytica cell may comprise one or more nucleotide sequence(s) from each of the following groups;.

Such a microorganism will typically also comprise one or more nucleotide sequence(s) as set out in SEQ ID. NO: <NUM>, <NUM>, <NUM> or <NUM>.

Such a microorganism may also comprise one or more nucleotide sequences as set out in <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. In the case of these sequences, combinations of at least one from each of (i) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>; (ii) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>; (iii) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>; and (iv) SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> may be preferred. Typically, at least one UGT from group (i) may be used. If at least one UGT from group (iii) is used, generally at least one UGT from group (i) is also used. If at least one UGT from group (iv) is used, generally at least one UGT from group (i) and at least one UGT from group (iii) is used. Typically, at least one UGT form group (ii) is used.

Such a microorganism may also comprise the following nucleotide sequences: SEQ ID. NO: <NUM>; SEQ ID. NO: <NUM>; and SEQ ID.

For each sequence set out above (or any sequence mentioned herein), a variant having at least about <NUM>%, preferably at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>%, sequence identity with the stated sequence may be used.

The nucleotide sequences encoding the ent-copalyl pyrophosphate synthase, ent-Kaurene synthase, ent-Kaurene oxidase, kaurenoic acid <NUM>-hydroxylase, UGTs, hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase, geranylgeranyl diphosphate synthase and NADPH-cytochrome p450 reductase may be ligated into one or more nucleic acid constructs to facilitate the transformation of the microorganism used in the present invention.

A nucleic acid construct may be a plasmid carrying the genes encoding enzymes of the RebM pathway as described above, or a nucleic acid construct may comprise two or three plasmids carrying each three or two genes, respectively, encoding the enzymes of the diterpene pathway distributed in any appropriate way.

Any suitable plasmid may be used, for instance a low copy plasmid or a high copy plasmid.

It may be possible that the enzymes selected from the group consisting of ent-copalyl pyrophosphate synthase, ent-Kaurene synthase, ent-Kaurene oxidase, and kaurenoic acid <NUM>-hydroxylase, UGTs, hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase, geranylgeranyl diphosphate synthase and NADPH-cytochrome p450 reductase are native to the host microorganism and that transformation with one or more of the nucleotide sequences encoding these enzymes may not be required to confer the host cell the ability to produce a diterpene or diterpene glycosidase. Further improvement of diterpene/diterpene glycosidase production by the host microorganism may be obtained by classical strain improvement.

The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence sequence. If the host cell is of fungal origin, a suitable episomal nucleic acid construct may e.g. be based on the yeast 2µ or pKD1 plasmids (<NPL>), or the AMA plasmids (<NPL>).

Alternatively, each nucleic acid construct may be integrated in one or more copies into the genome of the host cell. Integration into the host cell's genome may occur at random by non-homologous recombination but preferably the nucleic acid construct may be integrated into the host cell's genome by homologous recombination as is well known in the art (see e.g. <CIT>, <CIT>, <CIT> and <CIT>).

Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a microorganism containing the marker. The marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Alternatively or also, non-antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1, LEU2). The host cells transformed with the nucleic acid constructs may be marker gene free. Methods for constructing recombinant marker gene free microbial host cells are disclosed in <CIT> and are based on the use of bidirectional markers. Alternatively, a screenable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into nucleic acid constructs allowing to screen for transformed cells. A preferred marker-free method for the introduction of heterologous polynucleotides is described in <CIT>.

In a preferred embodiment, the nucleotide sequences encoding ent-copalyl pyrophosphate synthase, ent-Kaurene synthase, ent-Kaurene oxidase, and kaurenoic acid <NUM>-hydroxylase, UGTs, hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase geranylgeranyl diphosphate synthase and NADPH-cytochrome p450 reductase, are each operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequences in eukaryotic cell to confer to the cell the ability to produce RebM.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

The promoter that could be used to achieve the expression of the nucleotide sequences coding for an enzyme as defined herein above, may be not native to the nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

Suitable promoters in microorganisms may be GAL7, GAL10, or GAL <NUM>, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and AOX1. Other suitable promoters include PDC, GPD1, PGK1, TEF1, and TDH. Further suitable promoters are set out in the Examples.

Any terminator, which is functional in the cell, may be used. Preferred terminators are obtained from natural genes of the host cell. Suitable terminator sequences are well known in the art. Preferably, such terminators are combined with mutations that prevent nonsense mediated mRNA decay in the host cell (see for example:<NPL>).

Nucleotide sequences may include sequences which target them to desired compartments of the microorganism. For example, in a preferred microorganism used in the invention, all nucleotide sequences, except for ent-Kaurene oxidase, kaurenoic acid <NUM>-hydroxylase and NADPH-cytochrome p450 reductase encoding sequences may be targeted to the cytosol. This approach may be used in a yeast cell.

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain.

The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced.

Typically, recombinant microorganism used in the invention will comprise heterologous nucleotide sequences. Alternatively, a recombinant microorganism used in the invention may comprise entirely homologous sequence which has been modified as set out herein so that the microorganism produces increased amounts of RebM in comparison to a non-modified version of the same microorganism.

One or more enzymes of the diterpene pathway as described herein may be overexpressed to achieve a sufficient diterpene production by the cell.

There are various means available in the art for overexpression of enzymes in the host cells used in the invention. In particular, an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the host cell, e.g. by integrating additional copies of the gene in the host cell's genome.

A preferred host cell used in the invention may be a recombinant cell which is naturally capable of producing GGPP.

A recombinant microorganism used in the present invention may be able to grow on any suitable carbon source known in the art and convert it to RebM. The recombinant microorganism 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 organism 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 cell 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 <CIT>, <CIT>, <CIT>, <CIT> or <CIT>.

The present invention relates to a process for the preparation of rebaudioside M by fermentation which comprises fermenting a microorganism of the genus Yarrowia in a suitable fermentation medium at a pH of below <NUM>, and recovering rebaudioside M,.

The fermentation medium used in the process for the production of RebM 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 microorganism used in the process for the preparation of RebM may be any suitable microorganism as defined herein above. It may be advantageous to use a recombinant eukaryotic microorganism in the process for the production of RebM, because 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 microorganism used in the present invention may be a facultative anaerobic microorganism. A facultative anaerobic microorganism 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 Reb M 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 <NUM>, <NUM> or <NUM> 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 diterpene 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 RebM may be run at a temperature which is optimal for the eukaryotic cell. The optimum growth temperature may differ for each transformed eukaryotic cell 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 microorganism.

The process for the production of RebM according to the present invention may be carried out at any suitable pH value. If the recombinant microorganism is yeast, the pH in the fermentation medium preferably has a value of below <NUM>, preferably below <NUM>,<NUM>, preferably below <NUM>, preferably below <NUM>,<NUM>, preferably below <NUM>, preferably below pH <NUM>,<NUM> or below pH <NUM>,<NUM>, or below pH <NUM>,<NUM>, preferably above pH <NUM>. 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 rebaudioside M.

Recovery of RebM 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 RebM according to the invention, it may be possible to achieve a concentration of above <NUM>/l fermentation broth, preferably above <NUM>/l, preferably above <NUM>/l, preferably above <NUM>/l fermentation broth, preferably above <NUM>/l, more preferably above <NUM>/l, preferably above <NUM>/l, preferably above <NUM>, preferably above <NUM>/l, preferably above <NUM>/l, preferably above <NUM>/l, preferably above <NUM>/l, preferably above <NUM>/l, but usually below <NUM>/l.

In the event that RebM is expressed within the microorganism, such cells may need to be treated so as to release RebM. Preferentially, RebM is produced extracellularly.

RebM 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. For example RebM may be formulated in soft drinks, as a tabletop sweetener, chewing gum, dairy product such as yoghurt (eg. plain yoghurt), cake, cereal or cereal-based food, nutraceutical, pharmaceutical, edible gel, confectionery product, cosmetic, toothpastes or other oral cavity composition, etc. In addition, RebM 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.

A foodstuff, feed or beverage which comprises Reb M is disclosed but not part of the invention.

During the manufacturing of foodstuffs, drinks, pharmaceuticals, cosmetics, table top products, chewing gum the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods can be used.

The RebM obtained in the process according to the invention can be used in dry or liquid forms. It can be added before or after heat treatment of food products. The amount of the sweetener depends on the purpose of usage. It can be added alone or in the combination with other compounds.

Compounds produced according to the method of the invention may be blended with one or more further non-calorific or calorific sweeteners. Such blending may be used to improve flavour or temporal profile or stability. A wide range of both non-calorific and calorific sweeteners may be suitable for blending with RebM. For example, non-calorific sweeteners such as mogroside, monatin, aspartame, acesulfame salts, cyclamate, sucralose, saccharin salts or erythritol. Calorific sweeteners suitable for blending with RebM include sugar alcohols and carbohydrates such as sucrose, glucose, fructose and HFCS. Sweet tasting amino acids such as glycine, alanine or serine may also be used.

The RebM can be used in the combination with a sweetener suppressor, such as a natural sweetener suppressor. It may be combined with an umami taste enhancer, such as an amino acid or a salt thereof.

RebM can be combined with a polyol or sugar alcohol, a carbohydrate, a physiologically active substance or functional ingredient (for example a carotenoid, dietary fiber, fatty acid, saponin, antioxidant, nutraceutical, flavonoid, isothiocyanate, phenol, plant sterol or stanol (phytosterols and phytostanols), a polyols, a prebiotic, a probiotic, a phytoestrogen, soy protein, sulfides/thiols, amino acids, a protein, a vitamin, a mineral, and/or a substance classified based on a health benefits, such as cardiovascular, cholesterol-reducing or anti-inflammatory.

A composition with RebM may include a flavoring agent, an aroma component, a nucleotide, an organic acid, an organic acid salt, an inorganic acid, a bitter compound, a protein or protein hydrolyzate, a surfactant, a flavonoid, an astringent compound, a vitamin, a dietary fiber, an antioxidant, a fatty acid and/or a salt.

RebM may be applied as a high intensity sweetener to produce zero calorie, reduced calorie or diabetic beverages and food products with improved taste characteristics. Also it can be used in drinks, foodstuffs, pharmaceuticals, and other products in which sugar cannot be used.

In addition, RebM may be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics.

The examples of products where RebM can be used as a sweetening compound can be as alcoholic beverages such as vodka, wine, beer, liquor, sake, etc; natural juices, refreshing drinks, carbonated soft drinks, diet drinks, zero calorie drinks, reduced calorie drinks and foods, yogurt drinks, instant juices, instant coffee, powdered types of instant beverages, canned products, syrups, fermented soybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchups, curry, soup, instant bouillon, powdered soy sauce, powdered vinegar, types of biscuits, rice biscuit, crackers, bread, chocolates, caramel, candy, chewing gum, jelly, pudding, preserved fruits and vegetables, fresh cream, jam, marmalade, flower paste, powdered milk, ice cream, sorbet, vegetables and fruits packed in bottles, canned and boiled beans, meat and foods boiled in sweetened sauce, agricultural vegetable food products, seafood, ham, sausage, fish ham, fish sausage, fish paste, deep fried fish products, dried seafood products, frozen food products, preserved seaweed, preserved meat, tobacco, medicinal products, and many others. In principle it can have unlimited applications.

The sweetened composition comprises a beverage, non-limiting examples of which include non-carbonated and carbonated beverages such as colas, ginger ales, root beers, ciders, fruit-flavored soft drinks (e.g., citrus-flavored soft drinks such as lemon-lime or orange), powdered soft drinks, and the like; fruit juices originating in fruits or vegetables, fruit juices including squeezed juices or the like, fruit juices containing fruit particles, fruit beverages, fruit juice beverages, beverages containing fruit juices, beverages with fruit flavorings, vegetable juices, juices containing vegetables, and mixed juices containing fruits and vegetables; sport drinks, energy drinks, near water and the like drinks (e.g., water with natural or synthetic flavorants); tea type or favorite type beverages such as coffee, cocoa, black tea, green tea, oolong tea and the like; beverages containing milk components such as milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages, drinkable yogurt, lactic acid bacteria beverages or the like; and dairy products.

Generally, the amount of sweetener present in a sweetened composition varies widely depending on the particular type of sweetened composition and its desired sweetness. Those of ordinary skill in the art can readily discern the appropriate amount of sweetener to put in the sweetened composition.

RebM obtained in the process according to the invention can be used in dry or liquid forms. It can be added before or after heat treatment of food products. The amount of the sweetener depends on the purpose of usage. It can be added alone or in the combination with other compounds.

During the manufacturing of foodstuffs, drinks, pharmaceuticals, cosmetics, tabletop products, chewing gum the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods can be used.

Thus, compositions disclosed but not part of the invention can be made by any method known to those skilled in the art that provide homogenous even or homogeneous mixtures of the ingredients. These methods include dry blending, spray drying, agglomeration, wet granulation, compaction, co-crystallization and the like.

In solid form RebM can be provided to consumers in any form suitable for delivery into the comestible to be sweetened, including sachets, packets, bulk bags or boxes, cubes, tablets, mists, or dissolvable strips. The composition can be delivered as a unit dose or in bulk form.

For liquid sweetener systems and compositions convenient ranges of fluid, semi-fluid, paste and cream forms, appropriate packing using appropriate packing material in any shape or form shall be invented which is convenient to carry or dispense or store or transport any combination containing any of the above sweetener products or combination of product produced above.

The composition may include various bulking agents, functional ingredients, colorants, flavors.

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 present invention is further illustrated by the following Examples:.

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 <NPL>, or <NPL>). Methods for transformation and genetic modification of fungal host cells are known from e.g. <CIT>, <CIT>, <CIT> and <CIT>.

For over-expression of ERG20, BTS1 tHMG1, expression cassettes were designed to be integrated in one locus using technology described in co-pending patent application no. To amplify the <NUM>' and <NUM>' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN. PK yeast strain (<NPL>) was used. The different genes were ordered as cassettes (containing homologous sequence, promoter, gene, terminator, homologous sequence) at DNA2. The genes in these cassettes were flanked by constitutive promoters and terminators. See Table <NUM>. Plasmid DNA from DNA2. <NUM> containing the ERG20, tHMG1 and BTS1 cassettes were dissolved to a concentration of <NUM> ng/µl. In a <NUM>µl PCR mix <NUM> ng template was used together with <NUM> pmol of the primers. The material was dissolved to a concentration of <NUM>µg/µl.

For amplification of the selection marker, the pUG7-EcoRV construct (<FIG>) and suitable primers were used. The KanMX fragment was purified from gel using the Zymoclean Gel DNA Recovery kit (ZymoResearch). Yeast strain Cen. PK113-3C was transformed with the fragments listed in Table <NUM>.

After transformation and recovery for <NUM> hours in YEPhD (yeast extract phytone peptone glucose; BBL Phytone Peptone from BD) at <NUM> the cells were plated on YEPhD agar with <NUM>µg/ml G418 (Sigma). The plates were incubated at <NUM> for <NUM> days. Correct integration was established with diagnostic PCR and sequencing. Over-expression was confirmed with LC/MS on the proteins. The schematic of the assembly of ERG20, tHMG1 and BTS1 is illustrated in <FIG>. This strain is named STV002.

Expression of the CRE-recombinase in this strain led to out-recombination of the KanMX marker. Correct out-recombination, and presence of ERG20, tHMG and BTS1 was established with diagnostic PCR.

For reducing the expression of Erg9, an Erg9 knock down construct was designed and used that contains a modified <NUM>' end, that continues into the TRP1 promoter driving TRP1 expression.

The construct containing the Erg9-KD fragment was transformed to E. coli TOP10 cells. Transformants were grown in 2PY(<NUM> times Phytone peptone Yeast extract), sAMP medium. Plasmid DNA was isolated with the QIAprep Spin Miniprep kit (Qiagen) and digested with Sall-HF (New England Biolabs). To concentrate, the DNA was precipitated with ethanol. The fragment was transformed to S. cerevisiae, and colonies were plated on mineral medium (Verduyn et al, <NUM>. Yeast <NUM>:<NUM>-<NUM>) agar plates without tryptophan. Correct integration of the Erg9-KD construct was confirmed with diagnostic PCR and sequencing. The schematic of performed transformation of the Erg9-KD construct is illustrated in <FIG>. The strain was named STV003.

For over-expression of UGT2_1a, technology was used as described in co-pending patent application nos. <CIT> and <CIT>. The UGT2_1a was ordered as a cassette (containing homologous sequence, promoter, gene, terminator, homologous sequence) at DNA2. For details, see Table <NUM>. To obtain the fragments containing the marker and Cre-recombinase, technology was used as described in co-pending patent application no. The NAT marker, conferring resistance to nourseothricin was used for selection.

Suitable primers were used for amplification. To amplify the <NUM>' and <NUM>' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN. PK yeast strain was used.

cerevisiae yeast strain STV003 was transformed with the fragments listed in Table <NUM>, and the transformation mix was plated on YEPhD agar plates containing <NUM>µg/ml nourseothricin (Lexy NTC from Jena Bioscience).

Expression of the CRE recombinase is activated by the presence of galactose. To induce the expression of the CRE recombinase, transformants were restreaked on YEPh Galactose medium. This resulted in out-recombination of the marker(s) located between lox sites. Correct integration of the UGT2a and out-recombination of the NAT marker was confirmed with diagnostic PCR. The resulting strain was named STV004. The schematic of the performed transformation of the UGT2_1a construct is illustrated in <FIG>.

All pathway genes leading to the production of RebA were designed to be integrated in one locus using technology described in co-pending patent application no. To amplify the <NUM>' and <NUM>' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN. PK yeast strain was used. The different genes were ordered as cassettes (containing homologous sequence, promoter, gene, terminator, homologous sequence) at DNA2. <NUM> (see Table <NUM> for overview). The DNA from DNA2. <NUM> was dissolved to <NUM> ng/µl. This stock solution was further diluted to <NUM> ng/µl, of which <NUM>µl was used in a 50µl-PCR mixture. The reaction contained <NUM> pmol of each primer. After amplification, DNA was purified with the NucleoSpin <NUM> PCR Clean-up kit (Macherey-Nagel) or alternatively concentrated using ethanol precipitation.

All fragments for the pathway to RebA, the marker and the flanks (see overview in Table <NUM>) were transformed to S. cerevisiae yeast strain STV004. After overnight recovery in YEPhD at <NUM> the transformation mixes were plated on YEPhD agar containing <NUM>µg/ml G418. These were incubated <NUM> days at <NUM> and one night at RT.

Correct integration was confirmed with diagnostic PCR and sequence analysis (<NUM> Genetic Analyzer, Applied Biosystems). The sequence reactions were done with the BigDye Terminator v3. <NUM> Cycle Sequencing kit (Life Technologies). Each reaction (<NUM>µl) contained <NUM> ng template and <NUM> pmol primer. The products were purified by ethanol/EDTA precipitation, dissolved in <NUM>µl HiDi formamide and applied onto the apparatus. The strain was named STV016. The schematic of how the pathway from GGPP to RebA is integrated into the genome is illustrated in <FIG>.

To remove the KanMX marker from the chromosome of strain STV016, this strain was transformed with plasmid pSH65, expressing Cre-recombinase (Güldender, <NUM>). Subsequently plasmid pSH65 was cured from the strain by growing on non-selective medium (YEP <NUM>% glucose). The resulting, KanMX-free and pSH65-free strains, as determined by plating on plates containing <NUM>µg G418/ml or <NUM>µg phleomycin/ml, where no growth should occur, was named STV027. Absence of the KanMX marker was furthermore confirmed with diagnostic PCR.

Although the STV027 strain was initially designed for production of RebA, in this Example the strain is shown to produce the steviol glycoside, RebM.

The construction of the recombinant host for the production of RebM, STV027, is described above in Example <NUM>.

The purified fraction (see <NUM> below) was analyzed with a LTQ orbitrap (Thermo), equipped with a Acella LC and a Waters Acquity UPLC BEH amide <NUM> <NUM>*<NUM> column. Eluentia used for the separation were A: <NUM> Ammonium acetate in MilliQ water, B: Acetonitrile, and the gradient started at <NUM> % A and was kept here for <NUM> minutes, then increased to <NUM> % B in <NUM> minutes and kept here for <NUM> minutes before regeneration for <NUM> at <NUM> % A. The flow-rate was <NUM>/min and the column temperature was kept at <NUM> C. Mass spectral analysis was performed in electrospray negative ionization mode, scanning from m/z <NUM>-<NUM> at a resolution of <NUM>.

The yeast strain STV027 constructed as described above, was cultivated in shake-flask (<NUM> with <NUM> medium) for <NUM> days at <NUM> and <NUM> rpm. The medium was based on Verduyn et al. (<NPL>), with modifications in the carbon and nitrogen sources, as described in Table <NUM>.

Subsequently, <NUM> of the content of the shake-flask was transferred into a fermenter (starting volume <NUM>), which contained the medium as set out in Table <NUM>.

The pH was controlled at <NUM> by addition of ammonia (<NUM> wt%). Temperature was controlled at <NUM>. pOz was controlled at <NUM>% by adjusting the stirrer speed. Glucose concentration was kept limited by controlled feed to the fermenter.

Fermentation broth was heat shocked at <NUM>-<NUM> to kill the yeast cells and make them open. The heat shocked broth was spray dried. Dried biomass was extracted twice with <NUM> % ethanol at <NUM>-<NUM>. The extracts were combined and evaporated to about <NUM>/<NUM> - <NUM>/<NUM> of their original volume. The evaporated extract was diluted with water to reach the ethanol concentration <NUM> %. The analytical HPLC chromatogram of the extract is the top curve in <FIG>. A voluminous precipitate formed on dilution of extract with water was removed by centrifugation. The analytical HPLC chromatogram of the centrifugate is the next top curve in <FIG>. The <NUM> % ethanol feed with pH around <NUM> was applied on a column packed with DIAION® HP20 and eluted with gradient 14CV <NUM>-<NUM>% ethanol. The analytical HPLC chromatogram of the first eluate is the third from the top curve in <FIG>. pH in the pooled fraction was adjusted to <NUM> and it was applied again on a column packed with DIAION® HP20 and eluted stepwise with <NUM> CV of <NUM> % ethanol. The analytical HPLC chromatogram of the second eluate is the fourth curve from the top curve in <FIG>. The peak in analytical chromatograms eluting at <NUM> is eluting at <NUM> is RebM.

The presence of RebM was confirmed by LC and MS carried out on the first eluate (as described in the preceding paragraph) using the conditions set out at <NUM> above. Reb M elutes at tr=<NUM>, just after reb D at tr=<NUM>. Reb M is characterized by a deprotonated molecule of m/z <NUM>. The elemental composition could be estimated using accurate mass analysis.

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 M.

Step <NUM>. Strain ML10371 (MAT-A, lys1-, ura3-, leu2-) was transformed with <NUM> 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 + <NUM> ug/ml nourseothricin or YPD + <NUM> ug/ml hygromycin, as appropriate.

This construct encodes GGSopt linked to the pHYPO promoter and gpdT terminator. The resulting strain was denoted ML13462.

Step <NUM>. Strain ML13462 was transformed with a <NUM> kb fragment isolated by gel purification following Sfil digestion of plasmid MB7015 (<FIG>). This construct encodes a synthetic construct for the overexpression of UGT1 (SEQ ID NO: <NUM>) linked to the pENO promoter (SEQ ID NO: <NUM>) and gpdT terminator (SEQ ID NO: <NUM>), UGT3 (SEQ ID NO: <NUM>) linked to the pHSP promoter and pgmT (SEQ ID NO: <NUM>) terminator, UGT4 (SEQ ID NO: <NUM>) linked to the pCWP promoter (SEQ ID NO: <NUM>) and pgkT terminator (SEQ ID NO: <NUM>), and the lox-flanked nourseothricin resistance marker (NAT: SEQ ID NO: <NUM>). 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 <NUM>. Strain ML13500 was transformed with a <NUM> kb fragment isolated by gel purification following Pvul/Sapl digestion of plasmid MB6986 (<FIG>). This construct encodes tHMGopt linked to the pHSP promoter and cwpT terminator, the lox-flanked URA3blaster prototrophic marker (SEQ ID NO: <NUM>), and GGSopt linked to the pHYPO promoter and gpdT terminator (SEQ ID NO: <NUM>). Transformants were selected on minimal medium lacking uracil. One selected uracil prototroph was denoted ML13723.

Step <NUM>. Strain ML13723 was transformed with an <NUM> kb fragment isolated by gel purification following Sfil digestion of plasmid MB7059 (<FIG>). MB7059 encodes the tCPS_SR (SEQ ID NO: <NUM>) linked to pCWP promoter and cwpT terminator, the tKS_SR (SEQ ID NO: <NUM>) linked to the pHYPO promoter and gpdT terminator, the KAH_4 (SEQ ID NO: <NUM>) linked to the pHSP promoter and pgmT terminator (SEQ ID NO: <NUM>), the KO_Gib (SEQ ID NO: <NUM>) linked to the pTPI promoter (SEQ ID NO: <NUM>) and pgkT terminator (SEQ ID NO: <NUM>), the CPR_3 (SEQ ID NO: <NUM>) linked to the pENO promoter and xprT terminator and the native Y. lipolytica LEU2 locus (SEQ ID NO: <NUM>). One selected rebaudioside A-producing transformant was denoted ML14032.

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

Step <NUM>. Strain ML14093 was transformed with a <NUM> kb fragment isolated by gel purification following Sfil digestion of plasmid MB7100 (<FIG>). MB7100 encodes the tCPS_SR linked to the pHYPO promoter and cwpT terminator, the tKS_SR (SEQ ID NO: <NUM>) linked to the pCWP promoter and gpdT terminator, the KAH_4 linked to the pHSP promoter and pgmT terminator, the KO_Gib linked to the pENO promoter and pgkT terminator, the CPR_3 linked to the pTPI promoter and xprT terminator and URA3blaster prototrophic marker. Transformants were selected on minimal medium lacking uracil. One selected rebaudioside A producing uracil prototroph was denoted ML14094.

Step <NUM>. Strain ML13206 (MAT-B, ade1-, ure2-, leu2-) was transformed with <NUM> 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 + <NUM> ug/ml nourseothricin or YPD + <NUM> ug/ml hygromycin, as appropriate.

The resulting strain was denoted ML13465.

Step <NUM>. Strain ML13465 was transformed with <NUM> defined DNA fragments:.

Step <NUM>. Strain ML13490 was struck to YPD and grown overnight and then struck to <NUM>-FOA plates to allow for recombination mediated loss of the URA2 marker introduced in step <NUM> above. One selected <NUM>-FOA resistant transformant was denoted ML13501.

Step <NUM>. Strain ML13501 was transformed with a <NUM> kb fragment isolated by gel purification following Pvul/Sapl digestion of plasmid MB6988 (<FIG>). Transformants were selected on minimal medium lacking uracil. One selected uracil prototroph was denoted ML13724.

Step <NUM>. Strain ML13724 was transformed with an <NUM> kb fragment isolated by gel purification following Sfil digestion of plasmid MB7044 (<FIG>). MB7044 encodes the tCPS_SR linked to the pHYPO promoter and cwpT terminator, the tKS_SR linked to the pCWP promoter and gpdT terminator, the KAH_4 linked to the pHSP promoter and pgmT terminator, the KO_Gib linked to the pENO promoter and pgkT terminator, the CPR_3 linked to the pTPI promoter and xprT terminator and the LEU2 locus. One selected rebaudioside A-producing transformant was denoted ML14044.

Step <NUM>. Strain ML14044 was struck to YPD and grown overnight and then struck to <NUM>-FOA plates to allow for recombination mediated loss of the URA2 marker introduced in Step <NUM> above. One selected <NUM>'-FOA resistant transformant was denoted ML14076.

Step <NUM>. Strain ML14076 was transformed with a <NUM> kb fragment isolated by gel purification following Sfil digestion of plasmid MB7094 (<FIG>). MB7094 encodes the tCPS_SR linked to the pHYPO promoter and cwpT terminator, the tKS_SR linked to the pCWP promoter and gpdT terminator, the KAH_4 linked to the pHSP promoter and pgmT terminator, the KO_Gib linked to the pENO promoter and pgkT terminator, the CPR_3 linked to the pTPI promoter and xprT terminator and URA2blaster prototrophic marker. Transformants were selected on minimal medium lacking uracil. One selected rebaudioside A producing uracil prototroph was denoted ML14087.

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 STV2003.

Additional copies of CPS, KAH, UGT2 and UGT4 were transformed to STV2003, and integrated in the GSY1 (YALI0F18502) locus. The GSY1 locus is thereby disrupted. To amplify the <NUM>' and <NUM>' integration flanks for the GSY1 integration locus, suitable primers and genomic DNA from Yarrowia strain ML326 was used (SEQ ID NOs: <NUM> to <NUM>). These flanks contain connector sequences (<NUM> and a and f and <NUM>) for proper assembly in the pRS417_3_5 vector (<FIG>) together with the cassettes (described below). The heterologous genes were ordered as cassettes (containing connector sequence, promoter, gene, terminator, connector sequence) at DNA2. <NUM>, or assembled in house. After amplification, DNA was purified with the NucleoSpin <NUM> PCR Clean-up kit (Macherey-Nagel).

All cassettes, flanks and the linearized pRS417 5_3 vector (SnaBl/Pmel, Figure M) were transformed to S. cerevisiae strain CEN. Transformation mixes were plated on YEPhD agar containing <NUM>µg/ml G418, and incubated <NUM> days at <NUM>. A select number of correct transformants were grown in YEPhD liquid medium. From these cultures plasmid DNA was isolated using the the Qiaprep Spin Miniprep Kit (Qiagen, <NUM>) according to suppliers'instruction.

The isolated plasmid was transformed to chemically competent <NUM>-Beta E. coli cells (NEB, C3019H) according to suppliers' instructions. The transformed cells were allowed to recovered for <NUM> hour in <NUM> SOC at <NUM> <NUM> rpm. An aliquot was plated on 2xPY + Amp and incubated overnight at <NUM>. Single colonies were selected from the transformation plate and used to inoculate 2xPY + Amp, and incubated at <NUM> with shaking at <NUM> rpm.

Plasmid DNA was isolated from the E. coli clones using the NucleoSpin Plasmid Kit (Machery Nagel, REF <NUM>) according to suppliers' instructions. Diagnostic PCR was performed to confirm proper plasmid. The plasmid DNA isolated was used as template for amplification of the complete assembly. Two fragments were amplified, with overhang in the KanMX marker with primers DBC-<NUM> (SEQ ID NO:<NUM>) and DBC-<NUM> (SEQ ID NO: <NUM>) for amplification of the CPS, KAH and part of the KanMX gene, and primers DBC-<NUM> (SEQ ID NO: <NUM>) and DBC-<NUM> (SEQ ID NO: <NUM>) for the amplification of part of the KanMX gene and UGT4 and UGT2.

After amplification, both fragments were purified using the Nucleospin Gel and PCR Clean-up (Machery Nagel, REF740609. <NUM>) according to suppliers' instructions. The DNA was then transformed to Y. lipolytica yeast strain STV2003. The transformation mixes were plated on YEPhD agar containing <NUM>µg/ml G418 and incubated <NUM> days at <NUM>. Tranformants were checked with diagnostic PCR for correct integration. One correct transformant was named STV2019.

lipolytica strains STV2019 constructed as described above, were cultivated in shake-flask (<NUM> I with <NUM> medium) for <NUM> days at <NUM> and <NUM> rpm. The medium was based on Verduyn et al. (<NPL>), with modifications in the carbon and nitrogen sources, as described in Table <NUM>.

The pH was controlled at <NUM> by addition of ammonia (<NUM> wt%). Temperature was controlled at <NUM>. pOz was controlled at <NUM>% by adjusting the stirrer speed. Glucose concentration was kept limited by a controlled <NUM>% glucose feed to the fermenter. RebM is determined as se out above in Example <NUM>. The amount of RebM measured in the whole broth is as set out in Table <NUM>.

Saccharomyces cerevisiae strain STV027 and STV2019 (see Examples <NUM> and <NUM> above) are cultivated on suitable media enabling active transcription and translation of the introduced genes. Obtained cells are pelleted and stored at -<NUM> until analysis.

To <NUM> gr cell pellet <NUM> of <NUM> Tris buffer pH <NUM> is added and homogenized. Subsequently <NUM> gr glass beads (<NUM>-<NUM>) are added. The samples were cooled on ice for <NUM> minutes. Cells are disrupted by <NUM> cycles of each <NUM> vortexing on full speed followed by <NUM> cooling on ice. After lysis the extract is centrifuged at <NUM> for <NUM> at <NUM> degrees C. The obtained supernatant is used directly for activity assays.

<NUM> gr of fresh pellet is homogenized with <NUM> of <NUM>% DMSO in Tris buffer (<NUM> pH <NUM>) and frozen at -<NUM> degrees C. Before analysis cells are thawed and <NUM> is transferred to a new tube and washed three times with <NUM> Tris buffer pH <NUM> containing <NUM>% glucose. Cells were spun each time via centrifugation at <NUM>. Finally, the cells are resuspended in <NUM> of of <NUM> Tris pH <NUM>.

The total reaction volume is 1000uL and the assay is performed in microtiterplates (MTP) heated to <NUM> degrees C in a MTP eppendorf incubator. Incubations are done while shaking at <NUM> rpm up to <NUM> hrs. The MTP's are sealed to prevent contamination and evaporation.

100uL samples were taken in time to follow the conversion reaction. Reaction mixtures are centrifuged at <NUM> degrees C for <NUM> minutes to stop the enzyme reaction and collect the samples. 50uL of acetonitrile is added to 100uL of sample in order to completely stop the reaction and extract all molecules formed. To this end the MTP's are sealed and shaken vigorously. Subsequently, the samples are centrifuged for <NUM> minutes at <NUM> degrees C and 100ul is transferred to a new MTP for LC-MS analysis (as described in Example <NUM>, section <NUM>).

Rebaudioside M is readily formed after <NUM> hrs (><NUM>% of substrates converted) and steadily increases in concentration during the reaction for <NUM> hrs (><NUM>% of substrates converted).

Claim 1:
A process for the preparation of rebaudioside M by fermentation which comprises fermenting a microorganism of the genus Yarrowia in a suitable fermentation medium at a pH of below <NUM>, and recovering rebaudioside M,
wherein the microorganisms is a recombinant microorganism comprising and expressing 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 <NUM>-hydroxylase activity,
a polypeptide capable of catalyzing the addition of a glucose at the C-<NUM> position of steviol to yield <NUM>-O-steviolmonoside,
a polypeptide capable of catalyzing the addition of a glucose at the C-<NUM> position of <NUM>-steviolmonoside to yield steviolbioside or at the C-<NUM> position of rebaudioside A to yield rebaudioside D,
a polypeptide capable of catalyzing the addition of a glucose at the C-<NUM> position of steviolbioside to yield stevioside; and
a polypeptide capable of catalyzing addition of a glucose at the C-<NUM> position of stevioside to yield rebaudioside A or at the C-<NUM> position of rebaudioside D to yield rebaudioside M,
whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least rebaudioside M.