STEVIOL GLYCOSIDE TRANSPORT

Abstract

A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

Description

FIELD OF THE INVENTION

[0001] 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. The invention also relates to a fermentation broth comprising a steviol glycoside, a steviol glycoside and to a composition comprising two or more steviol glycosides. The invention further relates to a foodstuff, feed or beverage which comprises a steviol glycoside or a composition comprising two or more steviol glycosides.

BACKGROUND TO THE INVENTION

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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 D and rebaudioside M.

[0007] 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

[0008] The present invention is based on the identification of proteins which are capable of mediating steviol glycoside transport.

[0009] Accordingly, one or more such proteins may be overexpressed in a recombinant host (such as a microbial cell) in order to increase steviol glycoside transport out of the host. Alternatively, a host (such as a microbial cell) may be modified so as to express less of one or more such proteins than a corresponding non-modified version of the host. In this case, more steviol glycoside may be retained within the host which is then glycosylated to a steviol glycoside comprising a higher number of sugar moieties.

[0010] Thus, the invention relates to a recombinant host, for example a cell such as a microbial cell, which produces steviol glycoside outside the host to a greater degree than a corresponding host not overexpressing the protein. This may facilitate easier recovery of steviol glycosides. The invention also relates to a recombinant host capable of producing a steviol glycoside which overexpresses a heterologous polypeptide which mediates steviol glycoside transport.

[0011] Accordingly, the invention relates to a recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

[0012] The invention also relates to a recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 of SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

[0013] The invention also relates to a recombinant host which comprises steviol glycosides (inside and/or outside the host) having a higher or lower average glycosylation number than a corresponding host not modified according to the invention.

[0014] The invention also relates to:

[0015] a process for the preparation of a steviol glycoside which comprises fermenting a recombinant host according to any one of the preceding claims in a suitable fermentation medium and, optionally, recovering the steviol glycoside;

[0016] a fermentation broth comprising a steviol glycoside obtainable by a process of the invention;

[0017] a steviol glycoside obtained by a process or a fermentation broth of the invention;

[0018] a composition comprising two or more steviol glycosides of the invention or obtainable by a process of the invention;

[0019] a foodstuff, feed or beverage which comprises a steviol glycoside or a composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 sets out a schematic representation of the plasmid pUG7-KanMX.

[0021] FIG. 2 sets out a schematic representation of the method by which the ERG20, tHMG1 and BTS1 over-expression cassettes are designed (A) and integrated (B) into the yeast genome. (C) shows the final situation after removal of the KANMX marker by the Cre recombinase.

[0022] FIG. 3 sets out a schematic representation of the ERG9 knock down construct. This consists of a 500 bp long 3' part of ERG9, 98 bp of the TRP1 promoter, the TRP1 open reading frame and terminator, followed by a 400 bp long downstream sequence of ERG9. Due to introduction of a XbaI site at the end of the ERG9 open reading frame the last amino acid changes into Ser and the stop codon into Arg. A new stop codon is located in the TPR1 promoter, resulting in an extension of 18 amino acids.

[0023] FIG. 4 sets out a schematic representation of how UGT2_1a is integrated into the genome. A. different fragments used in transformation; B. situation after integration; C. situation after expression of Cre recombinase.

[0024] FIG. 5 sets out a schematic representation of how the pathway from GGPP to RebA is integrated into the genome. A. different fragments used in transformation; B. situation after integration.

[0025] FIG. 6 sets out a schematic representation of how KAH and CPR are integrated in the genome. A. different fragments used in transformation; B. situation after integration.

[0026] FIG. 7 sets out a schematic representation of the plasmid pUG7-NAT.

[0027] FIG. 8 sets out a schematic representation of how CPS is integrated in the genome. A. different fragments used in transformation; B. situation after integration.

[0028] FIG. 9 sets out a schematic representation of plasmid Sc_2_5-2_a.bbn

[0029] FIG. 10 sets out a schematic representation of the plasmid pUG7-HYG

[0030] FIG. 11 sets out a schematic representation of how the transporter genes ALNQ_007_38000 and ALNQ_214_12000 are integrated into the genome. A. different fragments used in transformation; B. situation after integration.

[0031] FIG. 12 sets out the production of Rebaudioside A in the supernatant in strains with over-expressed transporters ALNQ_007_38000 and ALNQ_214_12000.

[0032] FIG. 13 sets out the production of Rebaudioside B in the supernatant in strains with over-expressed transporters ALNQ_007_38000 and ALNQ_214_12000.

[0033] FIG. 14 sets out a schematic diagram of the potential pathways leading to biosynthesis of steviol glycosides. The compound shown with an asterisk is 13-[(.beta.-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl ester.

DESCRIPTION OF THE SEQUENCE LISTING

[0034] A description of the sequences is set out in Table 15. 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 15.

DETAILED DESCRIPTION OF THE INVENTION

[0035] 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.

[0036] 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.

[0037] The invention relates to the identification of polypeptides which are capable of mediating steviol glycoside transport. Such a polypeptide may directly mediate steviol glycoside transport, i.e. may be a transporter protein, or may indirectly mediate steviol glycoside transport. Such a polypeptide may be capable of mediating transport of one or more steviol glycoside.

[0038] The invention relates to a recombinant host either overexpressing or having reduced expression of such a polypeptide. The terms recombinant host or recombinant cell may, depending on the context, be used interchangeably.

[0039] Such a polypeptide as described herein may be overexpressed in a recombinant host, such as a recombinant host cell, capable of producing one or more steviol glycosides. Such a cell may be capable of producing more of one or more steviol glycosides external to the cell than a corresponding cell which does not overexpress the polypeptide. That is to say, a recombinant cell according to the invention may have increased or decreased steviol glycoside transport in a comparison with a corresponding non-recombinant cell.

[0040] Accordingly, the invention provides a recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide, the polypeptide being one which is capable of mediating steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

[0041] The expression of such a polypeptide may also be modified in a host, such as a recombinant host cell, such that it is reduced compared to a corresponding cell which has not been similarly modified. In this way, the amount of one or more steviol glycosides outside the cell may be reduced in comparison with a corresponding cell which has not been similarly modified. This may allow for increased glycosylation of one or more steviol glycosides within the cell compared with a corresponding cell which has not been similarly modified. Such a host may thus comprise steviol glycosides having a higher average glycosylation number compared with a corresponding cell which has not been similarly modified.

[0042] Accordingly, the invention provides a recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide, the polypeptide being one which is capable of mediating steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

[0043] A host cell of the invention is a recombinant host cell. "Recombinant" in this sense means that the host cell is a non-naturally occurring host cell, for example modified by introduction of one or more nucleic acids using recombinant techniques. A nucleic acid used to modify a host cell to arrive at a recombinant host cell of the invention may be a naturally-occurring nucleic acid or a non-naturally occurring nucleic acid.

[0044] Thus, when used in reference to a host of the invention, "recombinant" indicates that a cell has been modified by the introduction of one or more 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. The term "heterologous" as used herein refers to nucleic acid or amino acid sequences not naturally occurring in a host cell. In other words, the nucleic acid or amino acid sequence is not identical to that naturally found in the host cell.

[0045] The invention relates to a recombinant host capable of producing a steviol glycoside which overexpresses a heterologous polypeptide which mediates steviol glycoside transport. Such a heterologous polypeptide may be obtained from or derived from a genus or species other than that of the host. Accordingly, if the recombinant host is a yeast, the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from a different genus or species of yeast.

[0046] For example, if the host cell is a Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from a Candida (e.g., C. krusei, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis), an Issatchenkia (eg. I. orientalis) or a Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)).

[0047] For example, if the host cell is a Candida (e.g., C. krusei, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis), the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from a Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), an Issatchenkia (eg. I. orientalis) or a Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)).

[0048] For example, if the host cell is an Issatchenkia (eg. I. orientalis), the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from a Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), a Candida (e.g., C. krusei, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis) or a Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)).

[0049] For example, if the host cell is a Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)), the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from a Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis). a Candida (e.g., C. krusei, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis) or an Issatchenkia (eg. I. orientalis).

[0050] If the host cell is Saccharomyces cerevisiae, the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from Yarrowia lipolytica (formerly classified as Candida lipolytica)), Candida krusei or Issatchenkia orientalis.

[0051] If the host cell is Yarrowia lipolytica, the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from Saccharomyces cerevisiae, Yarrowia lipolytica (formerly classified as Candida lipolytica)) or Candida krusei or Issatchenkia orientalis.

[0052] If the host cell is Candida krusei or Issatchenkia orientalis, the heterologous polypeptide which mediates steviol glycoside transport may be obtained from or derived from Saccharomyces cerevisiae or Yarrowia lipolytica.

[0053] 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 may indicate that the substance is native to that microorganism or is a substance native to that microorganism, but may also indicate a substance that has been altered from a native version.

[0054] Thus, for example, a recombinant cell may express a polypeptide as defined herein not found within the native (non-recombinant) form of the cell. Alternatively, a recombinant cell may be modified so as to express a native gene encoding a polypeptide as defined herein to a greater degree than takes place within the native "non-recombinant" form of the cell.

[0055] Alternatively, a recombinant cell may be modified so as to express a native gene encoding a polypeptide as defined herein to a lesser degree than takes place within the native "non-recombinant" form of the cell.

[0056] In a cell of the invention, a polypeptide as defined herein may be overexpressed. Herein, "overexpressed", "overexpression" or the like implies that the recombinant host cell expresses more of the polypeptide than a corresponding cell which does not overexpress the polypeptide or, alternatively, that the polypeptide is expressed in a cell which would not typically express that protein. Alternatively, overexpression may be achieved by expressing a variant polypeptide having a higher specific activity.

[0057] A recombinant cell of the invention cell may be modified, preferably in its genome, to result in a deficiency in the production of a polypeptide as defined herein.

[0058] Such a cell may be from a parent host cell and be modified, preferably in its genome, if compared to the parent host cell to obtain a different genotype and/or a different phenotype if compared to the parent host cell from which it is derived.

[0059] Such a cell which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide as defined herein, is a mutant host cell which has been modified, preferably in its genome, to result in a phenotypic feature wherein the cell: a) produces less of the product or produces substantially no product and/or b) produces a product having a decreased activity or decreased specific activity or a product having no activity or no specific activity and combinations of one or more of these possibilities as compared to the parent microbial host cell that has not been modified, when analyzed under the same conditions.

[0060] Such a recombinant host may be a full or partial knock-out of a nucleic acid sequence encoding a polypeptide as described herein.

[0061] The term "recombinant" is synonymous with "genetically modified".

[0062] The invention thus concerns recombinant hosts overexpressing or deficient in a polypeptide identified as having steviol glycoside transport mediating 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).

[0063] For the purpose of this invention, a polypeptide having steviol glycoside transport mediating activity (i.e. a polypeptide which mediates steviol glycoside transport) is one which has an effect on transport of one or more steviol glycosides across a cell membrane. The effect may be direct, i.e. the polypeptide may be a transporter protein or comprise a functional transporter region. Alternatively, the effect may be indirect, i.e. the polypeptide is not a transporter protein, but its activity nevertheless has an effect on steviol glycoside transport.

[0064] Typically, the effect will be such that increasing the level of expression of the polypeptide increases the amount of transport of one or more steviol glycosides across the membrane of a cell (in comparison with a corresponding cell having a lower level of expression of the polypeptide). Conversely, decreasing the level of expression of the polypeptide may decrease the amount of transport of one or more steviol glycosides across the membrane of a cell (in comparison with a corresponding cell having a higher level of expression of the polypeptide).

[0065] Typically, a recombinant host of the invention is capable of producing a steviol glycoside. For example, a recombinant host of the invention may be capable of producing one or more of, for example but not limited to, steviol-13-monoside, steviol-19-monoside, 13-[(.beta.-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl ester, rubusoside, stevioside, steviol-19-diside, steviolbioside, rebA, rebB, rebC, rebD, rebE or rebM. A recombinant host of the invention may be capable of producing one or more of the steviol glycosides set out in Ceunen and Geuns, Journal of Natural Products 76(6), 1201-1228, 2013.

[0066] Thus, a cell of the invention may be one in which the amount of total amount of steviol glycosides outside the cell as compared with inside the cell is greater or less than compared with a corresponding cell which either does not overexpress or does not have a reduced level of expression of a cell of the invention.

[0067] Alternatively, a cell of the invention may have the same total amount of steviol glycosides outside the cell as compared with inside the cell compared with a corresponding cell which either does not overexpress or does not have a reduced level of expression of a cell of the invention, but may have an altered distribution of steviol glycosides inside and outside the cell.

[0068] Thus, a recombinant host of the invention is capable of producing a steviol glycoside. For example, a recombinant host of the invention may be capable of producing one or more of, for example, steviol-.beta.-monoside, steviol-19-monoside, 13-[(.beta.-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl ester, rubusoside, stevioside, steviol-19-diside, steviolbioside, rebA, rebB, rebC, rebD, rebE or rebM.

[0069] Thus, a recombinant host of the invention may be one in which at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% of the rebA produced by the cell is outside the cell.

[0070] Thus, a recombinant host of the invention may be one in which at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% of the rebD produced by the cell is outside the cell.

[0071] Thus, a recombinant host of the invention may be one in which at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% of the rebM produced by the cell is outside the cell.

[0072] A recombinant cell of the invention may be one in which no more than about 50%, no more than about 40%, no more than about 30%, no more than about 20%, no more than about 10% of the rebA produced by the cell is outside the cell.

[0073] A recombinant cell of the invention may be one in which no more than about 50%, no more than about 40%, no more than about 30%, no more than about 20%, no more than about 10% of the rebD produced by the cell is outside the cell.

[0074] A recombinant cell of the invention may be one in which no more than about 50%, no more than about 40%, no more than about 30%, no more than about 20%, no more than about 10% of the rebM produced by the cell is outside the cell.

[0075] A recombinant cell of the invention may be one where the average glycosylation number of the steviol glycosides is at least 3, at least 4, at least 5, at least 6 or more. The average glycosylation number may be increased or decreased in comparison with a corresponding cell not modified according to the invention. For example, average glycosylation may decrease when a polypeptide as described herein is overexpressed. For example, average glycosylation may increase (in particular in a cell itself) when expression of a polypeptide of the invention is reduced.

[0076] The average glycosylation may refer to that in the supernatant of a recombinant cell of the invention or to the average glycosylation in the broth (pellet+supernatant).

[0077] The invention thus provides a recombinant cell capable of producing a steviol glycoside either overexpressing or deficient in the expression of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto. Such an amino acid sequence has an effect of steviol glycoside transport, i.e. is a mediator of steviol glycoside transport.

[0078] The polypeptide may also be defined as one comprising the following amino acid sequence (or an amino acid sequence having at least about 45% sequence identity thereto):

TABLE-US-00001 (SEQ ID NO: 35) MSALNTDALESQPDFKFQRQKRLMSPFMSKKVPPIPTKEERKPYGEYHTN ILFRIMFWWLNPILNVGYKRTLTEQDLFYLDNSQTMDTLYETFKSHLKTT IEKSMKKYLQEKYSKEGKTYDPSSIPTAEDLKDFQIPIYAIPLCLFKTLY WQYSLGNLYKVLSDCTSATTPLLQKKLINFVQMKSFTALGSTGKGVGYAI GVCLMIFFQAITVNHAFHNLQICGAKSKAILTRMLLDKSMSVDARGNHFF PASKVQSMISTDLNRVDLAIGFFPFALTCVFPIAICIGLLIWNVGVSALV GIAIFVANVGLLAVSIPRLMRFRIKAMVFTDKRVTLMKELLKNFKMIKFY SWENSYARRIQDARFKEMKLILSLQSLRNIVMSVSFAMPTLASMATFCTA FDITSGKNAASLFSSLSLFQVLSMQFMLAPVALNTAADMMVSMKKFNQFL AHADLDPEQYRIEEFHDDKLAVKVDNATFEWDTFDDDKVEDPALEFEKQD NDSLEKVSSHNTVDYDSTEKIRNDTSSIDSTKILEKTAFPGLRNINLEIK KGEFVVVTGSIGAGKSSLLQAISGLMKRVSGKVYVDGDLLLCGYPWVQNA TIRDNILFGLPFDQEKYDQVVYACSLQSDFNQFQGGDMTEVGERGITLSG GQKARINLARSVYADKDIILLDDVLSAVDAKVGRHIVDTCLLGLLKDKTR IMATHQLSLIDSADRMIFLNGDGSIDCGTISELKDRNEKLNELLSHQKDK ANDSDEELELQEEIESKEQHLKEDLSEVKHEIKEEQKKMEISGDVGEEFE HADEHKEIVRIIGDEERAVNALKADVYINYAKLAFGKLGLFSLMLFVTVA ALQTYCMFTNTWLSFWIEEKFHGRSKSFYMGIYIMFAFLYTFFLAAFFYS MCYFCNRASKYLNYKASEKILHVPMSFMDISPIGRVLNRFTKDTDVLDNE ILDQFRQFLSPFCNAIGTIVLCIIYIPWFAIAVPLIVTFYVLVANYYQAS AREIKRLEAVKRSLVFGHFNEALSGKETIKAYRAIDRVKQRLNKLIDGQN EAYFLTIVNQRWLGANLSILSFCMVFIISFLCVFRVFNISAASTGLLLTY VINLTNTITMMMRAMTQVENEFNSVERLNHYAFDLVQEAPYEIPENDPPQ DWPKYGEIIFKDVSMRYRPELPFVLKNINLSIGKGEKIGFCGRTGAGKST FMTCLYRISEFEGTIVIDDVDISKLGLHKLRSKLTIIPQDPVLFVGSIRE NLDPFGEYSDEELWEALTISGLINKEDLNEVKKQNENDDNLNKFHLIRMV EDDGVNFSIGERQLIALARALVRKTKILILDEATSSVDYATDSRIQKTIA TEFDDCMILCIAHRLNTILNYDKIVVMDKGEIVEFDKPRSLFMREEGVFR SMCEQANITIEDFP; or (SEQ ID NO: 38) MKSDNIAMEDLPDSKYLKQRRLLTPLMSKKVPPIPSEDERKAYGEYYTNP VSRMMFWWLNPILKVGYRRTLTENDLFYLEDRQRTETLYEIFRGYLDEEI ARAWKKSQESSDDPREFKLPIYIIPLCLFKTMKWEYSRGILQKILGDCAS ATTPLLQKKLINFVQVKTFSNVGNTGQGVGYAIGVCLMIFFQVLMLTHAF HNFQISGAKAKAVLTRLLLDKSLTVDARGNHYFPASKIQSMISTDLNRID LAVGFAPVGFVTIFPIIICIALLIWNVGVSALVGIGVFIANIFVLGLFVS SLMLYREKAMVFTDKRVNLVKELLKNFKMIKFYSWENSYQDRIENARNNE MKYILRLQLLRNFVFSLAFAMPVLASMATFCTAFKITDGKSAASVFSSLS LFEVLSLQFILAPFSLNSTVDMMVSVKKINQFLQHKDTNPNEFSVEKFSD STLAIKVDNASFEWDTFEDEEKDYEEEAKTKDNIEDEDHNCATETIKGKI TVDYKSDSDSISSTLTKGVKTAFPGLNNINLEIAKGEFIVVTGAIGSGKS SLLQAISGLMKRTSGEVYVDGDLLLCGYPWVQNSTIRENILFGLPFNKER YDQVVYSCSLQSDFDQFQGGDMTEVGERGITLSGGQKARINLARSVYADK DIILLDDVLSAVDAKVGKHIVNTCILGLLGGKTRIMATHQLSLIDSADRM VFLNGDGTIDFGTIPELRKRNQKLIELLQHQRDPGQDKEDLSNDLDIQGS TDEGQQIEHADEHKEIVKIIGDEEKAVNALSFQVYYNYCKLAFGKLGYIS MLVFIIVSSLETFTQIFTNTWLSFWIEDKFVSRSKNFYMGIYIMFAFLYA IMLCFFLFLLGYFCVKAAERLNIKASRKILHVPMSFMDISPIGRVLNRFT KDTDVLDNELLEQLIQFLSPLFNCFGIIILCIVYIPWFAIGVPIILGFYF IIASYYQASAREIKRLEAVKRSFVFGHFHEVLTGKDTIKAYNAIDRMKLK LNKLIDEQNEAYYLTIANQRWLGANLAIVSFSMVFVISFLCIFRVFNISA ASTGLLLTYVIALTDSITMIMRAMTQVENEFNSVERVNHYAFDLIQEAPY EIPENDPAEDWPQHGKIEFKDVSMRYRPELPFVLKNINLSVREQEKIGFC GRTGAGKSTFMTCLYRITEYEGLISIDGVDISRLGLHRLRSKLTIIPQDP VLFVGTIRENLDPFTEHSDDELWEALAISGLIEREDLEVVKGQEKIGGND SGKLHKFHLVRMVEDDGINFSLGERQLIALARALVRKSKILILDEATSSV DYATDSKIQRTIASEFRDCTILCIAHRLNTILGYDKIVVMDNGEIVEFEN PKLLFMRENSVFRSMCEQANITINDFE

[0079] A polypeptide, typically having steviol glycoside transport mediating activity, may comprise an amino acid sequence having 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: 35.

[0080] A polypeptide, typically having steviol glycoside transport mediating activity, may comprise an amino acid sequence having 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: 38.

[0081] A polypeptide, typically having steviol glycoside transport mediating 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.

[0082] That is to say, the invention also a recombinant host overexpressing a biologically active fragment of a polypeptide having steviol glycoside transport mediating activity as described herein.

[0083] Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of SEQ ID NO: 29 which include fewer amino acids than the full-length polypeptide as given in SEQ ID NO: 29, but which exhibit at least one biological activity of the corresponding full-length polypeptide.

[0084] Typically, biologically active fragments comprise a domain or motif with at least one activity of the polypeptide of the invention. A biologically active fragment of a polypeptide of the invention can be a polypeptide which is, for example, about 10, about 25, about 50, about 100 or more amino acids in length or at least about 100 amino acids, at least 150, 200, 250, 300, 350, 400, 600, 1000 amino acids in length, or of a length up to the total number of amino acids of the polypeptide of the invention. Moreover, other biologically active portions, in which other regions of the polypeptide are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention. The invention also features nucleic acid fragments which encode the above biologically active fragments of the polypeptide of the invention.

[0085] A recombinant host of the invention may overexpress or be deficient in such a polypeptide.

[0086] 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 or different.

[0087] A recombinant cell of the invention may be modified so as to reduce the expression level of more than one such polypeptide, for example two, three, four or more such polypeptides.

[0088] An overexpressed 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 Pichia, for example one which is obtainable from or derived from or found in a Pichia kudriavzeii.

[0089] 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.

[0090] 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 of the invention comprises a signal peptide and/or a propeptide, sequence identity may be calculated over the mature polypeptide sequence.

[0091] 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 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide described herein may be generated according to methods well known to those skilled in the art. A sequence encoding a polypeptide as described herein may be modified such that less or no expression of the polypeptide takes place. A sequence encoding a polypeptide as described herein may be partially or entirely deleted, for example.

[0098] A recombinant host of the invention may comprise any polypeptide as described herein. A recombinant host of the invention may overexpress or be deficient in any polypeptide described herein. Typically, a recombinant host of the invention is capable of producing a steviol glycoside. For example, a recombinant host of the invention may be capable of producing one or more of, for example, steviol-13-monoside, steviol-19-monoside, 13-[(.beta.-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl ester, rubusoside, stevioside, steviol-19-diside, steviolbioside, rebA, rebE, rebD or rebM.

[0099] 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.

[0100] 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).

[0101] 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, FIG. 14 sets out a schematic diagram of steviol glycoside formation.

[0102] A recombinant host of the invention may thus comprise one or more recombinant nucleic acid sequences encoding one or more of:

[0103] (i) a polypeptide having UGT74G1 activity;

[0104] (ii) a polypeptide having UGT2 activity;

[0105] (iii) a polypeptide having UGT85C2 activity; and

[0106] (iv) a polypeptide having UGT76G1 activity.

[0107] 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.

[0108] 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.

[0109] 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-O-glucoside. Such sequences may be referred to as UGT1 sequences herein.

[0110] A recombinant yeast suitable for use in the invention may comprise a nucleotide sequence encoding a polypeptide which has UGT2 activity.

[0111] 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.

[0112] A polypeptide having UGT2 activity may also catalyze reactions that utilize steviol glycoside substrates other than steviol-13-0-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. However, a functional UGT2 polypeptide may be one which 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.

[0113] 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: steviol-13-0-glucoside transferase, transferring a rhamnose moiety to the C-2' of the 13-O-glucose of the acceptor molecule, steviol.

[0114] 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.

[0115] 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.

[0116] Suitable UGT74G1 polypeptides may be capable of transferring a glucose unit to the 13-OH and/or the 19-COOH of steviol. A suitable UGT74G1 polypeptide may function as a uridine 5'-diphospho glucosyl: steviol 19-COOH transferase and/or 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] A recombinant host according to the invention may comprise one or more recombinant nucleotide sequence(s) encoding one of more of:

[0123] a polypeptide having ent-copalyl pyrophosphate synthase activity;

[0124] a polypeptide having ent-Kaurene synthase activity;

[0125] a polypeptide having ent-Kaurene oxidase activity; and

[0126] a polypeptide having kaurenoic acid 13-hydroxylase activity.

[0127] 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:

##STR00001##

[0128] 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.

[0129] 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.

[0130] 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:

[0131] ent-copalyl diphosphateent-kaurene+diphosphate

[0132] Hence, this enzyme has one substrate, ent-copalyl diphosphate, and two products, ent-kaurene and diphosphate.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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 O.sub.2. Such activity may also be referred to as ent-ka 13-hydroxylase activity.

[0140] 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.

[0141] 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).

[0142] 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.

[0143] 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.

[0144] 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.

[0145] Accordingly, a recombinant host of the invention may comprise nucleic acid sequences encoding one or more of:

[0146] a polypeptide having hydroxymethylglutaryl-CoA reductase activity;

[0147] a polypeptide having farnesyl-pyrophosphate synthetase activity; and

[0148] 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.

[0149] 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, an insect host cell.

[0150] 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.

[0151] "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.

[0152] 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 (eg. I. orientalis) Pichia (e.g., P. pastoris and P. kudriavzevii), Schizosaccharomyces, Hansenula, Kloeckera, Pachysolen, Schwanniomyces, Trichosporon, Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)), Yamadazyma.

[0153] 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)).

[0154] 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).

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] The fermentation process for the production of a steviol glycoside according to the present invention may be an aerobic or an anaerobic fermentation process.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] Such a process may be carried out on an industrial scale. The product of such a process is one or more steviol glycosides.

[0169] Recovery of steivol 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.

[0170] 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/l fermentation broth, preferably above 10 mg/l, preferably above 20 mg/l, preferably above 30 mg/l fermentation broth, preferably above 40 mg/l, more preferably above 50 mg/l, preferably above 60 mg/l, preferably above 70, preferably above 80 mg/l, preferably above 100 mg/l, preferably above 1 g/I, preferably above 5 g/I, preferably above 10 g/I, for example at least about 15 g/L, such as at least about 20 g/I. The invention further provides a fermentation broth comprising a steviol glycoside obtainable by the process of the invention for the preparation of a steivol glycoside.

[0171] 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

[0172] 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.

[0173] Also provided is a composition obtainable by a process of the invention (which typically comprises one or more steviol glycosides). 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. These are all compositions of the invention.

[0174] A composition of the invention may be used in any application known for such compounds. In particular, such a composition 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 composition of the invention.

[0175] For example a composition of the invention 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, 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.

[0176] Accordingly, the invention provides, inter alia, a foodstuff, feed or beverage which comprises a composition of the invention.

[0177] 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.

[0178] A composition of 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.

[0179] A composition of the invention may be blended with one or more further non-caloric or caloric sweeteners. Such blending may be used to improve flavour or temporal profile or stability. A wide range of both non-caloric and caloric sweeteners may be suitable for blending with a composition of the invention. For example, non-caloric sweeteners such as mogroside, monatin, aspartame, acesulfame salts, cyclamate, sucralose, saccharin salts or erythritol. Caloric sweeteners suitable for blending with a steviol glycoside or a composition of the invention 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.

[0180] A composition of the invention 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.

[0181] A composition of the invention 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.

[0182] A composition of the invention 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.

[0183] A composition of the invention 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.

[0184] In addition, a composition of the invention 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.

[0185] The examples of products where a composition of the invention 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 principal it can have unlimited applications.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] Thus, compositions which incorporate a composition 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.

[0190] In solid form a composition of the invention 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.

[0191] 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.

[0192] A composition of the invention may include various bulking agents, functional ingredients, colorants, flavors.

[0193] 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 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.

[0194] 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) pp 276-277, 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.

[0195] 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".

[0196] 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/.

[0197] 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.sup.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

[0198] 1. A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

[0199] 2. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

[0200] 3. A recombinant host according to embodiment 1, which comprises a recombinant nucleic acid encoding a polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

[0201] 4. A recombinant host according to any one of the preceding embodiments which comprises one or more recombinant nucleotide sequence(s) encoding:

[0202] a polypeptide having ent-copalyl pyrophosphate synthase activity;

[0203] a polypeptide having ent-Kaurene synthase activity;

[0204] a polypeptide having ent-Kaurene oxidase activity; and

[0205] a polypeptide having kaurenoic acid 13-hydroxylase activity.

[0206] 5. 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.

[0207] 6. A recombinant host according to any one of the preceding embodiments which comprises a recombinant nucleic acid sequence encoding one or more of:

[0208] (i) a polypeptide having UGT74G1 activity;

[0209] (ii) a polypeptide having UGT2 activity;

[0210] (iii) a polypeptide having UGT85C2 activity; and

[0211] (iv) a polypeptide having UGT76G1 activity.

[0212] 7. 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.

[0213] 8. A recombinant host according to embodiment 7, wherein the recombinant host is a Saccharomyces cerevisiae cell, a Yarrowia lipolitica cell, a Candida krusei cell, an Issatchenkia orientalis cell or an Escherichia coli cell.

[0214] 9. A recombinant host according to any one of the preceding embodiments, wherein the ability of the host to produce geranylgeranyl diphosphate (GGPP) is upregulated.

[0215] 10. A recombinant host according to any one of the preceding embodiments which comprises a nucleic acid sequence encoding one or more of:

[0216] a polypeptide having hydroxymethylglutaryl-CoA reductase activity; or

[0217] a polypeptide having farnesyl-pyrophosphate synthetase activity.

[0218] 11. A recombinant host capable of producing a steviol glycoside which overexpresses a heterologous polypeptide which mediates steviol glycoside transport.

[0219] 12. 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.

[0220] 13. A process according to embodiment 12 for the preparation of a steviol glycoside, optionally wherein the process is carried out on an industrial scale.

[0221] 14. A fermentation broth comprising a steviol glycoside obtainable by the process according to embodiment 12 or 13.

[0222] 15. A steviol glycoside obtained by a process according to embodiment 12 or 13 or obtained from a fermentation broth according to embodiment 14.

[0223] 16. A composition obtainable by a process according to embodiment 12 or 13, a composition comprising two or more steviol glycosides obtained by a process according to embodiment 12 or 13 or a composition obtained from a fermentation broth according to embodiment 14.

[0224] 17. A foodstuff, feed or beverage which comprises a steviol glycoside according to claim 15 or a composition according to claim 16.

[0225] 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.

[0226] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

[0227] The present invention is further illustrated by the following Examples:

EXAMPLES

General

[0228] 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.sup.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.

Example 1: Over-Expression of ERG20, BTS1 and tHMG in S. cerevisiae

[0229] For over-expression of ERG20, BTS1 tHMG1, expression cassettes were designed to be integrated in one locus using technology described in WO2013/076280. To amplify the 5' and 3' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN.PK yeast strain (van Dijken et al. Enzyme and Microbial Technology 26 (2000) 706-714) was used. The different genes were ordered as cassettes (containing homologous sequence, promoter, gene, terminator, homologous sequence) at DNA2.0. The genes in these cassettes were flanked by constitutive promoters and terminators. See Table 1. Plasmid DNA from DNA2.0 containing the ERG20, tHMG1 and BTS1 cassettes were dissolved to a concentration of 100 ng/.mu.l. In a 50 .mu.l PCR mix 20 ng template was used together with 20 pmol of the primers. The material was dissolved to a concentration of 0.5 .mu.g/.mu.l.

TABLE-US-00002 TABLE 1 Composition of the over-expression constructs Promoter ORF Terminator Eno2 (SEQ ID NO: 1) ERG20 (SEQ ID NO: 2) Adh1 (SEQ ID NO: 3) Fba1 (SEQ ID NO: 4) tHMG1 (SEQ ID NO: 5) Adh2 (SEQ ID NO: 6) Tef1 (SEQ ID NO: 7) BTS1 (SEQ ID NO: 8) Gmp1 (SEQ ID NO: 9)

[0230] For amplification of the selection marker, the pUG7-EcoRV construct (FIG. 1) 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 2.

TABLE-US-00003 TABLE 2 DNA fragments used for transformation of ERG20, tHMG1 and BTS1 Fragment 5'YPRcTau3 ERG20 cassette tHMG1 cassette KanMX cassette BTS1 cassette 3'YPRcTau3

[0231] After transformation and recovery for 2.5 hours in YEPhD (yeast extract phytone peptone glucose; BBL Phytone Peptone from BD) at 30.degree. C. the cells were plated on YEPhD agar with 200 .mu.g/ml G418 (Sigma). The plates were incubated at 30.degree. C. for 4 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. 2. This strain is named STV002.

[0232] Expression of 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.

Example 2. Knock Down of Erg9

[0233] For reducing the expression of Erg9, an Erg9 knock down construct was designed and used that contains a modified 3' end, that continues into the TRP1 promoter driving TRP1 expression.

[0234] The construct containing the Erg9-KD fragment was transformed to E. coli TOP10 cells. Transformants were grown in 2PY (2 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, 1992. Yeast 8:501-517) 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. 3. The strain was named STV003.

Example 3. Over-Expression of UGT2_1a

[0235] For over-expression of UGT2_1a, technology was used as described in patent application nos. WO2013/076280 and WO2013/144257. The UGT2_1a was ordered as a cassette (containing homologous sequence, promoter, gene, terminator, homologous sequence) at DNA2.0. For details, see Table 3. To obtain the fragments containing the marker and Cre-recombinase, technology was used as described in patent application no. WO2013/135728. The NAT marker, conferring resistance to nourseothricin was used for selection.

TABLE-US-00004 TABLE 3 Composition of the over-expression construct Promoter ORF Terminator Pgk1 (SEQ ID NO: 10) UGT2_1a Adh2 (SEQ ID NO: 6) (SEQ ID NO: 11)

[0236] Suitable primers were used for amplification. To amplify the 5' and 3' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN.PK yeast strain was used.

[0237] S. cerevisiae yeast strain STV003 was transformed with the fragments listed in Table 4, and the transformation mix was plated on YEPhD agar plates containing 50 .mu.g/ml nourseothricin (Lexy NTC from Jena Bioscience).

TABLE-US-00005 TABLE 4 DNA fragments used for transformation of UGT2 1a Fragment 5'Chr09.01 UGT2_1a cassette NAT-CR RE 3'Chr09.01

[0238] 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 UGT2_1a 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. 4.

Example 4. Over-Expression of Production Pathway to RebA: CPS, KS, KO, KAH, CPR, UGT1, UGT3 and UGT4

[0239] All pathway genes leading to the production of RebA were designed to be integrated in one locus using technology described in patent application nos. WO2013/076280 and WO2013/144257. To amplify the 5' and 3' 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 DNA2.0 (see Table 5 for overview). The DNA from DNA2.0 was dissolved to 100 ng/.mu.l. This stock solution was further diluted to 5 ng/.mu.l, of which 1 .mu.l was used in a 50 .mu.l-PCR mixture. The reaction contained 25 pmol of each primer. After amplification, DNA was purified with the NucleoSpin 96 PCR Clean-up kit (Macherey-Nagel) or alternatively concentrated using ethanol precipitation.

TABLE-US-00006 TABLE 5 Sequences used for production pathway to RebA Promoter ORF SEQ ID Terminator KI prom 12.pro (SEQ ID NO: 12) trCPS_SR 13 Sc ADH2.ter(SEQ ID NO: 9) Sc PGK1.pro (SEQ ID NO: 10) trKS_SR 14 Sc TAL1.ter (SEQ ID NO: 15) Sc ENO2.pro (SEQ ID NO: 1) KO_2 16 Sc TPl1.ter (SEQ ID NO: 17) Ag lox_TEF1.pro (SEQ ID NO: 18) KANMX 19 Ag TEF1_lox.ter (SEQ ID NO: 20) Sc TEF1.pro (SEQ ID NO: 7) KAH_4 21 Sc GPM1.ter (SEQ ID NO: 9) KI prom 6.pro (SEQ ID NO: 22) CPR_3 23 Sc PDC1.ter (SEQ ID NO: 24) KI prom 3.pro (SEQ ID NO: 25) UGT1_SR 26 Sc TDH1.ter (SEQ ID NO: 27) KI prom 2.pro (SEQ ID NO: 28) UGT3_SR 29 Sc ADH1.ter (SEQ ID NO: 3) Sc FBA1.pro (SEQ ID NO: 4) UGT4_SR 30 Sc ENO1.ter (SEQ ID NO: 31)

[0240] All fragments for the pathway to RebA, the marker and the flanks (see overview in Table 6) were transformed to S. cerevisiae yeast strain STV004. After overnight recovery in YEPhD at 20.degree. C. the transformation mixes were plated on YEPhD agar containing 200 .mu.g/ml G418. These were incubated 3 days at 25.degree. C. and one night at RT.

TABLE-US-00007 TABLE 6 DNA fragments used for transformation of CPS, KS, KOKanMX, KAH, CPR, UGT1, UGT3 and UGT4 Fragment 5'INT1 CPS cassette KS cassette KO cassette KanMX cassette KAH cassette CPR cassette UGT1 cassette UGT3 cassette UGT4 cassette 3'INT1

[0241] Correct integration was confirmed with diagnostic PCR and sequence analysis (3500 Genetic Analyzer, Applied Biosystems). The sequence reactions were done with the BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies). Each reaction (10 .mu.l) contained 50 ng template and 3.2 pmol primer. The products were purified by ethanol/EDTA precipitation, dissolved in 10 .mu.l 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. 5. Table 7 sets out the strains used in Examples 1 to 5.

Example 5: Construction of Strain STV027

[0242] To remove the KanMX marker from the chromosome of strain STV016, this strain was transformed with plasmid pSH65, expressing Cre-recombinase (Guldender, 2002). Subsequently plasmid pSH65 was cured from the strain by growing on non-selective medium (YEP 2% glucose). The resulting, KanMX-free and pSH65-free strains, as determined by plating on plates containing 200 .mu.g G418/ml or 20 .mu.g phleomycin/ml, where no growth should occur, was named STV027. Absence of the KanMX marker was furthermore confirmed with diagnostic PCR. The resulting strain was named STV027.

Example 6: Construction of Strain STV035

[0243] To introduce additional copies of KAH and CPR available PCR fragments were used (see Table 6 and Table 7). The KanMX selection marker fragment was amplified from pUG7-EcoRV (FIG. 1) with the appropriate primers. To amplify the 5' and 3' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN.PK yeast strain was used.

[0244] STV027 was transformed with these fragments (Table 7) according the Gietz method. After 2 h recovery in YEPhD at 30.degree. C. the transformation mixes were plated on YEPhD agar containing 200 .mu.g/ml G418. These were incubated for 4 days at 30.degree. C.

TABLE-US-00008 TABLE 7 DNA fragments used for transformation of KanMX, KAH and CPR to STV027. Fragment 5'Chr11.04 KanMX cassette KAH cassette CPR cassette 3' Chr11.04

[0245] The schematic of how KAH and CPR are integrated into the genome is illustrated in FIG. 6. Correct integration was confirmed with diagnostic PCR. The resulting strain was named STV035.

Example 7: Construction of Strain STV058

[0246] For the integration of a second copy of CPS this gene was amplified together with a TDH3 promoter and ADH2 terminator.

TABLE-US-00009 TABLE 8 Sequences in CPS cassette (2) Promoter ORF SEQ ID Terminator Sc TDH3.pro trCPS_SR 12 Sc ADH2.ter (SEQ ID NO: 32) (SEQ ID NO: 9)

[0247] Due to presence of a KanMX marker in STV035 a NAT marker was amplified from pUG7-NAT (FIG. 7) with the appropriate primers. To amplify the 5' and 3' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN.PK yeast strain was used.

TABLE-US-00010 TABLE 9 DNA fragments used for transformation of CPS and NAT to STV035. Fragment 5'Chr2.06 CPS cassette(2) NAT 3' Chr2.06

[0248] The different fragments for integration of the second copy of CPS (Table 9) were combined and transformed to STV035. After recovery the transformation mix was plated on YEPhD agar plates containing 50 .mu.g/ml nourseothricin. These were incubated for 3 days at 30.degree. C. Correct integration was confirmed with diagnostic PCR. The new strain was named STV058. The schematic of how the CPS is integrated into the genome is illustrated in FIG. 8.

TABLE-US-00011 TABLE 10 Table of strains Strain Background Genotype Cen.PK113-3C -- MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 STV002 Cen.PK113- MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 3C YPRcTau3::ERG20, tHMG1, KanMX, BTS1 STV003 STV002 MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 YPRcTau3::ERG20, tHMG1, KanMX, BTS1 ERG9::ERG9- KD TRP1 STV004 STV003 MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 YPRcTau3::ERG20, tHMG1, BTS1 ERG9::ERG9-KD TRP1 Chr09.01::UGT2_1a STV016 STV004 MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 YPRcTau3::ERG20, tHMG1, BTS1 ERG9::ERG9-KD TRP1 Chr09.01::UGT2_1a INT1::CPS, KS, KO, KanMX, KAH, CPR, UGT1, UGT3, UGT4 STV027 STV016 MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 YPRcTau3::ERG20, tHMG1, BTS1 ERG9::ERG9-KD TRP1 Chr09.01::UGT2_1a INT1::CPS, KS, KO, KAH, CPR, UGT1, UGT13, UGT4 STV035 STV027 MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 YPRcTau3::ERG20, tHMG1, BTS1 ERG9::ERG9-KD TRP1 Chr09.01::UGT2_1a INT1::CPS, KS, KO, KAH, CPR, UGT1, UGT3, UGT4 5'Chr11.04::KanMX, KAH, CPR STV058 STV035 MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2 YPRcTau3::ERG20, tHMG1, BTS1 ERG9::ERG9-KD TRP1 Chr09.01::UGT2_1a INT1::CPS, KS, KO, KAH, CPR, UGT1, UGT3, UGT4 5'Chr11.04::KanMX, KAH, CPR 5'Chr2.06::CPS, NAT

Example 8. Expression of I. orientalis ALNQ 007_38000 and ALNQ 214_12000 in S. cerevisiae Strain STV058

[0249] For expression of ALNQ_007_38000 (SEQ ID NO: 33) and ALNQ_214_12000 (SEQ ID NO: 36), expression cassettes were designed to be integrated in the S. cerevisiae STV058 Chr01.05 locus, using technology described in patent application nos. WO2013/076280 and WO2013/144257. To amplify the 5' and 3' integration flanks for the integration locus, suitable primers and genomic DNA from a CEN.PK yeast strain were used.

[0250] The two transporter genes were amplified from I. orientalis CBS 5147 genomic DNA using suitable primers. The PCR amplicons were sub-cloned in a in a Zero Blunt TOPO vector (Life Technologies). The genes were cloned into the Sc_2_5-2_a.bbn vector using BspMI or BsaI in which they were flanked by the constitutive promoters KI_ENO1 or Sc_GPM1 and Sc_TAL1 terminator resulting in two expression cassettes for each transporter gene. The expression cassettes were PCR-amplified in six times 50 .mu.l PCR mix. The PCR product was purified and concentrated using NucleoSpin Gel and PCR Clean-up Kit (Machery Nagel).

[0251] For amplification of the selection marker, the pUG7-HygB construct (FIG. 10) and suitable primers were used. The PCR product was purified and concentrated using NucleoSpin Gel and PCR Clean-up Kit (Machery Nagel). Yeast strain S. cerevisiae STV058 was transformed with the fragments listed in Table 11. The in-vivo assembly is illustrated in FIG. 11.

TABLE-US-00012 TABLE 11 Fragments transformed to S. cerevisiae STV058 Over expression strain ENO1_ALNQ_007_38000 GPM1_ALNQ_007_38000 ENO1_ALNQ_214_12000 GPM1_ALNQ_214_12000 5' Chr01.05 205 ng 205 ng 205 ng 205 ng Transporter 477 ng 258 ng 529 ng 246 ng ORF cassette HygB 204 ng 204 ng 204 ng 204 ng cassette 3' Chr01.05 201 ng 201 ng 201 ng 201 ng

[0252] After transformation and recovery for 2 hours in YEPhD at 30.degree. C. the cells were plated on YEPhD agar with 200 .mu.g/ml HygB (Invitrogen). The plates were incubated at 30.degree. C. for 2 days. Transformants were purified by re-streaking them on YEPhD agar with 200 .mu.g/ml HygB. Correct integration and assembly was established with diagnostic PCR.

Example 9. Fermentation of STV058 and ALNQ 007_38000 and ALNQ 214_12000 Transporter Overexpression Strains

[0253] A pre-culture was inoculated with colony material from YEPh-D agar. The pre-culture was grown in 96-Half Deep Well Plate in 200 .mu.l mineral medium with glucose as carbon source. The pre-culture was incubated 72 hours in an Infors incubator at 27.degree. C., 750 rpm and 80% humidity.

[0254] 40 .mu.l of pre-culture was used to inoculate 2.5 ml mineral medium with glucose as carbon source in a 24-Deep Well Plate. These production cultures were incubated 120 hours in an Infors incubator at 27.degree. C., 550 rpm, 80% humidity. The production cultures were well homogenized and 0.5 ml of culture was transferred to a 96-well plate. This sample was used as whole broth sample. The remainder of the production cultures were pelleted by centrifugation at 3000.times.g for 10 minutes. After centrifugation 0.5 supernatant was transferred to a 96-well plate. This sample was used as supernatant sample. Both the whole broth 96-well plates and supernatant 96-well plates were incubated for 10 minutes at 90.degree. C. in a water bath and cooled down to room temperature. To each well 0.25 ml of acetonitrile was added and homogenized. The plates were then centrifuged at 3000.times.g for 10 minutes to pellet cell material and debris. The whole broth and supernatant samples were diluted 200 times in 33% acetonitrile. Samples were analyzed for RebA and other steviolglycosides using LC/MS. We found that the strains that had the particular transporter gene over-expressions as described, produced higher titers of Rebaudioside A or other steviolglycosides such as Rebaudioside B in the supernatant fraction compared to the parent strain. For an overview of the results, see Tables 12, 13.

TABLE-US-00013 TABLE 12 Rebaudioside A concentrations in supernatant and broth Reb A Strain supernatant (mg/L) Reb A broth (mg/L) STV058 29 137 ENO1p_ALNQ_007_38000 51 95 GPM1p_ALNQ_007_38000 53 79 ENO1p_ALNQ_214_12000 171 178 GPM1p_ALNQ_214_12000 155 151

[0255] Strains with over-expression of the ALNQ_007_38000 transporter or the ALNQ_214_12000 transporter have increased levels of Rebaudioside A in the supernatant. With the over-expression of the ALNQ_214_12000 transporter, the amount of RebA in the supernatant was increased with 5 to 6 fold compared to the reference strain. See also FIG. 12.

TABLE-US-00014 TABLE 13 Rebaudioside B concentrations in supernatant and broth Reb B Strain supernatant (mg/L) Reb B broth (mg/L) STV058 7 41 ENO1p_ALNQ_007_38000 124 139 GPM1p_ALNQ_007_38000 165 170 ENO1p_ALNQ_214_12000 15 19 GPM1p_ALNQ_214_12000 15 17

[0256] Strains with over-expression of the ALNQ_007_38000 transporter or the ALNQ_214_12000 transporter have increased levels of Rebaudioside B in the supernatant. In the case of the ALNQ_007_38000 transporter this also results in a higher RebB concentration in the broth. See also FIG. 13. The observation that such high amounts of RebB are exported into the supernatant offer an explanation for the reduced Rebaudioside A production in the broth (Table 12), as Rebaudioside B in the supernatant is no longer available as substrate for Rebaudioside A production, which occurs inside the cell. For the extracellular production of Rebaudioside A or products downstream of Rebaudioside A, therefore the ALNQ_007_38000 (or equivalent) transporter may be a target for deletion in a host that contains such a stransporter, such as I. orientalis, in combination with over-expression of a transporter that more specifically transports Rebaudioside A, such as the ALNQ_214_12000 transporter.

[0257] Strains with over-expression of the ALNQ_007_38000 transporter or the ALNQ_214_12000 transporter have decreased levels of Rebaudioside M in the broth. Because both transporters are efficient in exporting steviol glycosides such as Rebaudioside A and Rebaudioside B, lower amounts of these intermediates are available inside the cell for the conversion towards Rebaudioside M. Therefore, these transporters (or equivalent) may be target for deletion in a host that would have such a transporter, such as I. orientalis, to increase Rebaudioside M production.

TABLE-US-00015 TABLE 14 Rebaudioside M concentrations in supernatant and broth Strain Reb M broth (mg/L) STV058 60 ENO1p_ALNQ_007_38000 25 GPM1p_ALNQ_007_38000 13 ENO1p_ALNQ_214_12000 7 GPM1p_ALNQ_214_12000 2

TABLE-US-00016 TABLE 15 Description of the sequence listing SEQ ID NO Description SEQ ID NO: 1 Eno2 promoter from S. cerevisiae SEQ ID NO: 2 ERG20 nucleic acid from S. cerevisiae SEQ ID NO: 3 Adh1 terminator from S. cerevisiae SEQ ID NO: 4 Fba1 promoter from S. cerevisiae SEQ ID NO: 5 tHMG nucleic acid from S. cerevisiae SEQ ID NO: 6 Adh2 terminator from S. cerevisiae SEQ ID NO: 7 Tef1 promoter from S. cerevisiae SEQ ID NO: 8 BTS1 nucleic acid from S. cerevisiae SEQ ID NO: 9 Gmp1 terminator from S. cerevisiae SEQ ID NO: Pgk1 10 promoter from S. cerevisiae SEQ ID NO: UGT2_1a 11 CpO for S. cerevisiae SEQ ID NO: KI prom 12 12 promoter SEQ ID NO: trCPS from S. rebaudiana 13 CpO for S. cerevisiae SEQ ID NO: trKS from S. rebaudiana 14 CpO for S. cerevisiae SEQ ID NO: TAL1 15 terminator from S. cerevisiae SEQ ID NO: KO_2_Lactuca_sativa 16 CpO for S. cerevisiae SEQ ID NO: Tpi1 17 terminator from S. cerevisiae SEQ ID NO: Ag 18 lox_TEF1.pro nucleic acid construct SEQ ID NO: KANMX ORF 19 CpO for S. cerevisiae SEQ ID NO: Ag 20 Tef1_lox.ter nucleic acid construct SEQ ID NO: KAH_4 from 21 Arabidopsis thaliana CpO for S. cerevisiae SEQ ID NO: KI prom 6.pro 22 promoter SEQ ID NO: CPR_3 from 23 Arabidopsis thaliana CpO for S. cerevisiae SEQ ID NO: Pdc1 24 terminator from S. cerevisiae SEQ ID NO: KI prom3 25 promoter SEQ ID NO: UGT1 from S. rebaudiana 26 CpO for S. cerevisiae SEQ ID NO: Tdh1 27 terminator from S. cerevisiae SEQ ID NO: KI prom 2 28 promoter SEQ ID NO: UGT3 from S. rebaudiana 29 CpO for S. cerevisiae SEQ ID NO: UGT4 from S. rebaudiana 30 CpO for S. cerevisiae SEQ ID NO: Eno1 31 terminator from S. cerevisiae SEQ ID NO: TDH3 promoter 32 from S. cerevisiae SEQ ID NO: ALNQ_007_38000 33 CpO for S. cerevisiae SEQ ID NO: ALNQ_007_38000 34 VVT CDS from I. orientalis SEQ ID NO: ALNQ_007_38000 35 VVT from I. orientalis SEQ ID NO: ALNQ_214_12000 36 CpO for S. cerevisiae SEQ ID NO: ALNQ_214_12000 37 VVT CDS from I. orientalis SEQ ID NO: ALNQ_214_12000 38 VVT from I. orientalis

Sequence CWU 1

1

381600DNAArtificial sequenceScEno2 promoter 1gtgtcgacgc tgcgggtata gaaagggttc tttactctat agtacctcct cgctcagcat 60ctgcttcttc ccaaagatga acgcggcgtt atgtcactaa cgacgtgcac caacttgcgg 120aaagtggaat cccgttccaa aactggcatc cactaattga tacatctaca caccgcacgc 180cttttttctg aagcccactt tcgtggactt tgccatatgc aaaattcatg aagtgtgata 240ccaagtcagc atacacctca ctagggtagt ttctttggtt gtattgatca tttggttcat 300cgtggttcat taattttttt tctccattgc tttctggctt tgatcttact atcatttgga 360tttttgtcga aggttgtaga attgtatgtg acaagtggca ccaagcatat ataaaaaaaa 420aaagcattat cttcctacca gagttgattg ttaaaaacgt atttatagca aacgcaattg 480taattaattc ttattttgta tcttttcttc ccttgtctca atcttttatt tttattttat 540ttttcttttc ttagtttctt tcataacacc aagcaactaa tactataaca tacaataata 60021056DNASaccharomyces cerevisiae 2atggcttctg aaaaggaaat cagaagagaa cgtttcttga atgttttccc aaaattggtt 60gaagaattga acgcttctct attagcttac ggtatgccaa aggaagcttg tgactggtac 120gctcactctt tgaactacaa caccccaggt ggtaagttga acagaggtct atccgttgtt 180gacacctacg ccattttgtc caacaagacc gtcgaacaat taggtcaaga agaatacgaa 240aaggttgcca tcttaggttg gtgtatcgaa ttgttgcaag cttacttctt ggttgctgat 300gacatgatgg acaaatctat caccagaaga ggtcaaccat gttggtacaa ggttccagaa 360gtcggtgaaa ttgccatcaa cgatgctttc atgttggaag ctgccatcta caagttgttg 420aagtctcact tcagaaacga aaagtactac attgacatca ctgaattatt ccacgaagtt 480actttccaaa ccgaattggg tcaattgatg gacttgatta ccgctccaga agataaggtc 540gatttgtcca aattttcctt gaagaaacac tctttcattg tcactttcaa gactgcttac 600tactcctttt acttgcctgt tgctttggcc atgtatgtcg ctggtatcac cgatgaaaag 660gacttgaagc aagctcgtga tgtcttgatt ccattaggtg aatacttcca aatccaagat 720gactacttgg actgtttcgg tactccagaa caaatcggta agattggtac tgatatccaa 780gacaacaagt gttcctgggt tatcaacaag gctttggaat tggcttctgc tgaacaaaga 840aagactttgg acgaaaacta cggtaagaag gactctgttg ctgaagctaa gtgtaagaag 900atcttcaacg atttgaaaat tgaacaatta taccatgaat acgaagaatc tattgccaag 960gacttgaaag ccaagatctc tcaagtcgac gaatccagag gtttcaaggc tgatgtcttg 1020actgctttct tgaacaaggt ctacaagaga tcaaaa 10563301DNAArtificial sequenceAdh1 terminator 3agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt 60atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc 120tttcctgtag gtcaggttgc tttctcaggt atagcatgag gtcgctctta ttgaccacac 180ctctaccggc atgccgagca aatgcctgca aatcgctccc catttcaccc aattgtagat 240atgctaactc cagcaatgag ttgatgaatc tcggtgtgta ttttatgtcc tcagaggaca 300a 3014600DNAArtificial sequenceSc Fba1 promoter 4ctacttggct tcacatacgt tgcatacgtc gatatagata ataatgataa tgacagcagg 60attatcgtaa tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat 120gataggaatg ggattcttct atttttcctt tttccattct agcagccgtc gggaaaacgt 180ggcatcctct ctttcgggct caattggagt cacgctgccg tgagcatcct ctctttccat 240atctaacaac tgagcacgta accaatggaa aagcatgagc ttagcgttgc tccaaaaaag 300tattggatgg ttaataccat ttgtctgttc tcttctgact ttgactcctc aaaaaaaaaa 360aatctacaat caacagatcg cttcaattac gccctcacaa aaactttttt ccttcttctt 420cgcccacgtt aaattttatc cctcatgttg tctaacggat ttctgcactt gatttattat 480aaaaagacaa agacataata cttctctatc aatttcagtt attgttcttc cttgcgttat 540tcttctgttc ttctttttct tttgtcatat ataaccataa ccaagtaata catattcaaa 60051575DNASaccharomyces cerevisiae 5atggaccaat tggtcaagac tgaagtcacc aagaaatctt tcactgctcc agtccaaaag 60gcttccactc cagttttgac caacaagacc gtcatctccg gttccaaggt taaatctttg 120tcctctgctc aatcttcctc ctctggtcca tcttcttctt ctgaagaaga tgattccaga 180gatatcgaat ctttggacaa gaaaatcaga ccattggaag aattggaagc tctattgtcc 240tctggtaaca ctaagcaatt aaagaacaag gaagttgctg ctttggttat ccacggtaaa 300ttgccattgt acgctttgga aaagaaatta ggtgacacca ccagagctgt tgctgtcaga 360agaaaggctt tgtccatttt ggctgaagct ccagtcttgg cttccgacag attaccatac 420aagaactacg actacgaccg tgtctttggt gcttgttgtg aaaatgtcat tggttacatg 480ccattaccag ttggtgtcat tggtccattg gttatcgacg gtacttctta ccacatccca 540atggctacca ctgaaggttg tttggttgct tctgccatga gaggttgtaa ggccatcaac 600gctggtggtg gtgctaccac cgttttgact aaggatggta tgaccagagg tcctgttgtc 660agattcccaa ctttgaagag atctggtgct tgtaagatct ggttggattc tgaagaaggt 720caaaacgcca tcaagaaggc tttcaactcc acttccagat tcgctagatt gcaacacatt 780caaacttgtt tagctggtga cttgttgttc atgagattca gaaccaccac tggtgacgct 840atgggtatga acatgatctc caagggtgtt gaatactctt tgaagcaaat ggttgaagaa 900tacggttggg aagatatgga agttgtctct gtttctggta actactgtac cgacaagaag 960ccagctgcca tcaactggat cgaaggtcgt ggtaagtccg ttgttgctga agctaccatt 1020ccaggtgacg ttgtcagaaa ggttttgaaa tctgatgttt ctgctttagt cgaattgaac 1080attgccaaga acttggtcgg ttctgccatg gctggttccg tcggtggttt caacgctcat 1140gccgctaact tggtcactgc tgttttcttg gctttaggtc aagatccagc tcaaaatgtc 1200gaatcctcta actgtatcac tttgatgaag gaagttgacg gtgatttgag aatttctgtt 1260tccatgccat ccattgaagt cggtactatc ggtggtggta ctgtcttgga accacaaggt 1320gccatgttgg acttgttggg tgttcgtggt ccacacgcta ccgctccagg tactaacgcc 1380agacaattgg ccagaattgt tgcctgtgcc gtcttggctg gtgaattgtc tctatgtgcc 1440gctttggctg ctggtcactt ggttcaatct cacatgaccc acaacagaaa gcctgctgaa 1500ccaaccaaac caaacaactt ggatgctact gacattaaca gattaaagga cggttctgtc 1560acctgtatca agtct 15756301DNAArtificial sequenceAdh2 terminator 6agcggatctc ttatgtcttt acgatttata gttttcatta tcaagtatgc ctatattagt 60atatagcatc tttagatgac agtgttcgaa gtttcacgaa taaaagataa tattctactt 120tttgctccca ccgcgtttgc tagcacgagt gaacaccatc cctcgcctgt gagttgtacc 180cattcctcta aactgtagac atggtagctt cagcagtgtt cgttatgtac ggcatcctcc 240aacaaacagt cggttatagt ttgtcctgct cctctgaatc gtgtccctcg atatttctca 300t 3017600DNAArtificial sequenceSc Tef1 promoter 7ttggctgata atagcgtata aacaatgcat actttgtacg ttcaaaatac aatgcagtag 60atatatttat gcatattaca tataatacat atcacatagg aagcaacagg cgcgttggac 120ttttaatttt cgaggaccgc gaatccttac atcacaccca atcccccaca agtgatcccc 180cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 240tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 300tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagacacc 360gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 420ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 480aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 540ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 60081005DNASaccharomyces cerevisiae 8atggaagcta agattgacga attgatcaac aacgaccctg tctggtcctc tcaaaacgaa 60tctttgatct ccaagccata caaccacatc ttgttgaagc caggtaagaa cttcagatta 120aacttgattg ttcaaatcaa cagagttatg aacttgccaa aggaccaatt ggccattgtt 180tcccaaattg tcgaattgtt gcacaactcc tctctattga tcgatgacat tgaagataat 240gctccattaa gaagaggtca aaccacttct catttgattt tcggtgtccc atccaccatc 300aacactgcta actacatgta cttcagagcc atgcaattgg tttctcaatt gaccaccaag 360gaaccattat accacaactt gatcactatc tttaacgaag aattgattaa cttgcaccgt 420ggtcaaggtt tggacatcta ctggagagat ttcttgccag aaattattcc aactcaagaa 480atgtacttga acatggtcat gaacaagact ggtggtttat tcagattgac tttacgtttg 540atggaagctt tgtctccatc ttcccaccac ggtcactctt tggttccatt catcaatcta 600ttaggtatca tctaccaaat cagagatgat tacttgaact tgaaggactt ccaaatgtcc 660tctgaaaagg gtttcgctga agatatcact gaaggtaaat tgtctttccc aattgtccac 720gccttgaact ttaccaagac caagggtcaa actgaacaac acaacgaaat tttgagaatc 780ttattgttga gaacttctga caaggacatc aagttgaaat tgatccaaat cttggaattc 840gataccaact ctttggctta caccaagaac ttcatcaacc aattggttaa catgatcaag 900aatgacaacg aaaacaaata cttgccagac ttggcttccc actccgatac cgctaccaac 960ttgcacgacg aattgttgta cattattgac catttgtctg agtta 10059301DNAArtificial sequenceSc Gmp1 terminator 9agtctgaaga atgaatgatt tgatgatttc tttttccctc catttttctt actgaatata 60tcaatgatat agacttgtat agtttattat ttcaaattaa gtagctatat atagtcaaga 120taacgtttgt ttgacacgat tacattattc gtcgacatct tttttcagcc tgtcgtggta 180gcaatttgag gagtattatt aattgaatag gttcattttg cgctcgcata aacagttttc 240gtcagggaca gtatgttgga atgagtggta attaatggtg acatgacatg ttatagcaat 300a 30110600DNAArtificial sequenceSc Pgk1 promoter 10gggccagaaa aaggaagtgt ttccctcctt cttgaattga tgttaccctc ataaagcacg 60tggcctctta tcgagaaaga aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa 120ctgaaaaaac ccagacacgc tcgacttcct gtcttcctat tgattgcagc ttccaatttc 180gtcacacaac aaggtcctag cgacggctca caggttttgt aacaagcaat cgaaggttct 240ggaatggcgg gaaagggttt agtaccacat gctatgatgc ccactgtgat ctccagagca 300aagttcgttc gatcgtactg ttactctctc tctttcaaac agaattgtcc gaatcgtgtg 360acaacaacag cctgttctca cacactcttt tcttctaacc aagggggtgg tttagtttag 420tagaacctcg tgaaacttac atttacatat atataaactt gcataaattg gtcaatgcaa 480gaaatacata tttggtcttt tctaattcgt agtttttcaa gttcttagat gctttctttt 540tctctttttt acagatcatc aaggaagtaa ttatctactt tttacaacaa atataaaaca 600111422DNAArtificial sequenceUGT2_1a CpO for S. cerevisiae 11atggctacat ctgattctat tgttgatgac aggaagcagt tgcatgtggc tactttccct 60tggcttgctt tcggtcatat actgccttac ctacaactat caaaactgat agctgaaaaa 120ggacataaag tgtcattcct ttcaacaact agaaacattc aaagattatc ttcccacata 180tcaccattga ttaacgtcgt tcaattgaca cttccaagag tacaggaatt accagaagat 240gctgaagcta caacagatgt gcatcctgaa gatatccctt acttgaaaaa ggcatccgat 300ggattacagc ctgaggtcac tagattcctt gagcaacaca gtccagattg gatcatatac 360gactacactc actattggtt gccttcaatt gcagcatcac taggcatttc tagggcacat 420ttcagtgtaa ccacaccttg ggccattgct tacatgggtc catccgctga tgctatgatt 480aacggcagtg atggtagaac taccgttgaa gatttgacaa ccccaccaaa gtggtttcca 540tttccaacta aagtctgttg gagaaaacac gacttagcaa gactggttcc atacaaggca 600ccaggaatct cagacggcta tagaatgggt ttagtcctta aagggtctga ctgcctattg 660tctaagtgtt accatgagtt tgggacacaa tggctaccac ttttggaaac attacaccaa 720gttcctgtcg taccagttgg tctattacct ccagaaatcc ctggtgatga gaaggacgag 780acttgggttt caatcaaaaa gtggttagac gggaagcaaa aaggctcagt ggtatatgtg 840gcactgggtt ccgaagtttt agtatctcaa acagaagttg tggaacttgc cttaggtttg 900gaactatctg gattgccatt tgtctgggcc tacagaaaac caaaaggccc tgcaaagtcc 960gattcagttg aattgccaga cggctttgtc gagagaacta gagatagagg gttggtatgg 1020acttcatggg ctccacaatt gagaatcctg agtcacgaat ctgtgtgcgg tttcctaaca 1080cattgtggtt ctggttctat agttgaagga ctgatgtttg gtcatccact tatcatgttg 1140ccaatctttg gtgaccagcc tttgaatgca cgtctgttag aagataaaca agttggaatt 1200gaaatcccac gtaatgagga agatggatgt ttaaccaagg agtctgtggc cagatcatta 1260cgttccgttg tcgttgaaaa ggaaggcgaa atctacaagg ccaatgcccg tgaactttca 1320aagatctaca atgacacaaa agtagagaag gaatatgttt ctcaatttgt agattaccta 1380gagaaaaacg ctagagccgt agctattgat catgaatcct aa 1422121001DNAArtificial sequenceKl prom 12 promoter 12cgtaaaaact aaaacgagcc cccaccaaag aacaaaaaag aaggtgctgg gcccccactt 60tcttcccttg cacgtgatag gaagatggct acagaaacaa gaagatggaa atcgaaggaa 120agagggagac tggaagctgt aaaaactgaa atgaaaaaaa aaaaaaaaaa aaaaaaacaa 180gaagctgaaa atggaagact gaaatttgaa aaatggtaaa aaaaaaaaag aaacacgaag 240ctaaaaacct ggattccatt ttgagaagaa gcaagaaagg taagtatggt aacgaccgta 300caggcaagcg cgaaggcaaa tggaaaagct ggagtccgga agataatcat ttcatcttct 360tttgttagaa cagaacagtg gatgtccctc atctcggtaa cgtattgtcc atgccctaga 420actctctgtc cctaaaaaga ggacaaaaac ccaatggttt ccccagcttc cagtggagcc 480accgatccca ctggaaacca ctggacagga agagaaaatc acggacttcc tctattgaag 540gataattcaa cactttcacc agatcccaaa tgtcccgccc ctattcccgt gttccatcac 600gtaccataac ttaccatttc atcacgttct ctatggcaca ctggtactgc ttcgactgct 660ttgcttcatc ttctctatgg gccaatgagc taatgagcac aatgtgctgc gaaataaagg 720gatatctaat ttatattatt acattataat atgtactagt gtggttattg gtaattgtac 780ttaattttga tatataaagg gtggatcttt ttcattttga atcagaattg gaattgcaac 840ttgtctcttg tcactattac ttaatagtaa ttatatttct tattaacctt ttttttaagt 900caaaacacca aggacaagaa ctactcttca aaggtatttc aagttatcat acgtgtcaca 960cacgcttcac agtttcaagt aaaaaaaaag aatattacac a 1001132229DNAStevia rebaudiana 13atgtgtaaag ctgtttccaa ggaatactct gacttgttgc aaaaggatga agcctccttc 60accaaatggg atgatgacaa agttaaggac catttagaca ctaacaagaa cttgtaccca 120aacgatgaaa tcaaggaatt cgtcgaatct gtcaaagcta tgttcggttc catgaatgat 180ggtgaaatca acgtttccgc ttacgacacc gcttgggttg ctttggttca agacgttgat 240ggttccggtt ctccacaatt cccatcttct ttggaatgga ttgccaacaa ccaattgtct 300gatggttctt ggggtgacca tttgttattc tctgctcacg acagaattat taacacttta 360gcttgtgtca ttgctttgac ttcctggaat gtccatccat ccaagtgtga aaagggtttg 420aacttcttga gagaaaacat ctgtaagttg gaagatgaaa atgctgaaca catgccaatt 480ggtttcgaag ttaccttccc atctttgatt gatatcgcca agaagttgaa catcgaagtc 540ccagaagaca ccccagcttt gaaggaaatc tacgccagaa gagatatcaa gttgaccaaa 600atcccaatgg aagttttgca caaggttcca accaccttgt tgcactcttt ggaaggtatg 660ccagacttgg aatgggaaaa gttgttaaag ttgcaatgta aggacggttc tttcttgttc 720tctccatctt ctaccgcctt tgctttgatg caaactaagg acgaaaagtg tctacaatac 780ttaactaata tcgttaccaa attcaacggt ggtgtcccaa acgtttaccc tgttgacttg 840tttgaacaca tctgggttgt tgacagattg caacgtttgg gtattgctcg ttatttcaag 900tctgaaatca aggactgtgt tgaatacatc aacaagtact ggactaagaa cggtatctgt 960tgggctcgta acacccacgt tcaagatatc gacgacactg ctatgggttt cagagtcttg 1020agagctcatg gttacgatgt caccccagat gtcttcagac aattcgaaaa ggatggtaag 1080ttcgtttgtt ttgccggtca atccactcaa gccgtcactg gtatgttcaa cgtctacaga 1140gcttctcaaa tgttgttccc aggtgaaaga atcctagaag acgctaagaa gttctcctac 1200aactacttga aagaaaagca atctactaac gaattgttgg acaaatggat cattgccaaa 1260gacttaccag gtgaagtcgg ttacgctttg gatattccat ggtacgcttc tctaccaaga 1320ttagaaacca gatactactt ggaacaatac ggtggtgaag acgatgtctg gatcggtaag 1380accttgtaca gaatgggtta cgtttccaac aacacttact tggaaatggc caaattggac 1440tacaacaact acgtcgccgt cttacaattg gaatggtaca ccattcaaca atggtacgtt 1500gacattggta ttgaaaagtt tgaatccgac aacatcaagt ccgtcttggt ttcctactac 1560ttggctgctg cttccatctt tgaaccagaa agatccaagg aaagaattgc ttgggctaag 1620accaccatct tggttgacaa gatcacttct attttcgact cttcccaatc ttccaaggaa 1680gatatcaccg ctttcattga caaattcaga aacaagtctt cttccaagaa gcactccatt 1740aacggtgaac catggcacga agttatggtt gctttgaaga agactttgca cggttttgct 1800ttggatgctt tgatgactca ctctcaagat attcaccctc aattacacca agcttgggaa 1860atgtggttaa ccaagttgca agatggtgtc gatgtcactg ctgaattgat ggttcaaatg 1920atcaacatga ctgccggtag atgggtttct aaggaattgt tgactcaccc tcaataccaa 1980cgtttgtcca ccgtcaccaa ctctgtctgt cacgacatca ctaagttgca caacttcaaa 2040gaaaactcca ctactgtcga ttctaaggtt caagaattgg ttcaattagt tttctctgac 2100accccagatg acttggacca agacatgaag caaactttct tgactgtcat gaagaccttc 2160tactacaagg cttggtgtga cccaaacacc atcaacgacc atatttctaa ggtcttcgaa 2220attgttatc 2229142271DNAStevia rebaudiana 14atgacttctc acggtggtca aaccaaccca accaacttga ttattgacac caccaaggaa 60agaatccaaa agcaattcaa gaatgttgaa atctccgttt cctcctacga cactgcttgg 120gttgccatgg ttccatctcc aaactcccca aagtctccat gtttcccaga atgtttgaac 180tggttaatca acaaccaatt gaacgatggt tcctggggtt tagtcaatca cacccacaac 240cacaatcacc cattgttgaa ggactctcta tcctccactt tggcttgtat cgttgctttg 300aagagatgga acgttggtga agaccaaatc aacaagggtt tgtcctttat tgaatccaac 360ttggcttctg ctactgaaaa gtcccaacca tctcctatcg gttttgacat cattttccca 420ggtttattgg aatacgctaa gaacttggac atcaacttat tatctaagca aaccgatttc 480tccttgatgt tgcacaagag agaattggaa caaaagagat gtcactccaa cgaaatggac 540ggttacttgg cttacatttc tgaaggtttg ggtaacttgt acgactggaa catggtcaag 600aaataccaaa tgaagaacgg ttccgttttc aactctccat ctgctaccgc tgctgctttc 660atcaaccatc aaaacccagg ttgtttgaac tacttgaact ctttgttgga caaattcggt 720aacgctgttc caactgtcta cccacacgat ttgtttatca gattatccat ggttgacacc 780attgaacgtt tgggtatttc tcatcacttc agagtcgaaa tcaagaacgt tttggatgaa 840acttacagat gttgggttga aagagatgaa caaatcttca tggatgtcgt cacttgtgcc 900ttggccttca gattattgag aattaacggt tacgaagttt ctccagaccc attggctgaa 960atcactaacg aattggcttt gaaggacgaa tacgccgctt tggaaactta ccatgcctct 1020cacatcttat accaagaaga cttgtcctct ggtaagcaaa tcttgaagtc tgctgacttc 1080ttgaaggaaa ttatctctac tgattctaac agattgtcca agttgattca caaggaagtt 1140gaaaacgcct tgaaattccc aatcaacact ggtttggaaa gaattaacac cagaagaaac 1200atccaattat acaacgttga caacactaga atcttgaaga ctacttatca ctcttccaac 1260atctccaaca ctgactactt gagattggct gtcgaagatt tctacacctg tcaatctatt 1320tacagagaag aattgaaggg tttggaaaga tgggttgtcg aaaacaaatt ggaccaattg 1380aaatttgcta gacaaaagac cgcctactgt tacttctccg ttgctgccac tttgtcctct 1440ccagaattat ctgacgccag aatctcctgg gctaagaatg gtatcttgac caccgttgtc 1500gatgacttct tcgatattgg tggtaccatt gacgaattga ccaacttgat tcaatgtgtt 1560gaaaagtgga acgtcgatgt cgataaggac tgttgttctg aacacgtcag aatcttattc 1620ttggctttga aagatgctat ctgttggatc ggtgacgaag ctttcaaatg gcaagctcgt 1680gacgttacct ctcacgtcat ccaaacctgg ttggaattga tgaactctat gttgagagaa 1740gccatctgga cccgtgatgc ttacgtccca actttgaacg aatacatgga aaatgcttac 1800gtttctttcg ctttgggtcc aattgtcaag cctgctattt acttcgttgg tccaaagttg 1860tccgaagaaa ttgttgaatc ttctgaatac cacaacttgt tcaaattgat gtctactcaa 1920ggtcgtttgt tgaacgatat ccactctttc aagcgtgaat tcaaggaagg taagttgaat 1980gctgttgctt tgcatttgtc taacggtgaa tctggtaagg tcgaagaaga agttgtcgaa 2040gaaatgatga tgatgatcaa gaacaagaga aaggaattga tgaagttgat ctttgaagaa 2100aacggttcta ttgtcccaag agcttgtaag gatgctttct ggaacatgtg tcacgtcttg 2160aacttcttct acgctaacga tgacggtttc actggtaaca ccatcttaga caccgtcaag 2220gacatcattt acaacccatt agtcttggtt aacgaaaacg aagaacaaag a 227115301DNAArtificial sequenceSc Tal1 terminator 15aggaagtatc tcggaaatat taatttaggc catgtcctta tgcacgtttc ttttgatact 60tacgggtaca tgtacacaag tatatctata tatataaatt aatgaaaatc ccctatttat 120atatatgact ttaacgagac agaacagttt tttatttttt atcctatttg atgaatgata 180cagtttctta ttcacgtgtt atacccacac caaatccaat agcaataccg gccatcacaa 240tcactgtttc

ggcagcccct aagatcagac aaaacatccg gaaccacctt aaatcaacgt 300c 301161539DNAArtificial sequenceKO_2_ Lactuca_sativa_Sc_CpO 16atggatggtg tcattgacat gcaaaccatt ccattgagaa ccgccattgc cattggtggt 60actgctgttg ctttggttgt tgctctatac ttctggttct tgagatctta cgcttctcca 120tctcaccact ctaaccattt gccacctgtt ccagaagttc caggtgtccc agtcttgggt 180aacttgttgc aattgaaaga aaagaagcca tacatgactt tcaccaaatg ggctgaaatg 240tacggtccaa tctactctat cagaactggt gctacctcca tggttgttgt ttcctctaac 300gaaattgcca aggaagttgt tgtcactaga ttcccatcca tctccaccag aaagttgtct 360tacgctttga aggtcttgac tgaagataag tccatggttg ctatgtctga ttaccatgac 420taccacaaga ccgtcaaaag acacattttg actgctgtct taggtccaaa cgcccaaaag 480aagttccgtg ctcacagaga caccatgatg gaaaacgttt ccaatgaatt gcatgccttc 540tttgaaaaga acccaaacca agaagtcaac ttgagaaaga tcttccaatc tcaattgttc 600ggtttggcca tgaagcaagc tttgggtaag gatgtcgaat ctatctacgt caaggacttg 660gaaactacca tgaagagaga agaaatcttt gaagtcttgg ttgttgaccc aatgatgggt 720gccattgaag tcgattggag agacttcttc ccatacttga aatgggttcc aaacaaatct 780ttcgaaaaca tcattcacag aatgtacacc cgtcgtgaag ctgtcatgaa ggctttgatc 840caagaacaca agaagagaat tgcttctggt gaaaacttaa actcctacat tgactacttg 900ttgtctgaag ctcaaacttt gactgacaag caattgttga tgtccctatg ggaaccaatc 960attgaatctt ccgacaccac catggtcacc actgaatggg ctatgtacga attggctaag 1020aatccaaaca tgcaagacag attgtacgaa gaaatccaat ctgtttgtgg ttccgaaaag 1080atcactgaag aaaacttgtc tcaattacca tacttgtacg ctgttttcca agaaactttg 1140agaaagcact gtccagttcc aatcatgcca ttgagatacg tccacgaaaa caccgttttg 1200ggtggttacc acgttccagc tggtactgaa gttgctatca acatctatgg ttgtaacatg 1260gacaagaagg tctgggaaaa cccagaagaa tggaacccag aaagattctt atccgaaaag 1320gaatccatgg acttgtacaa gaccatggcc ttcggtggtg gtaagagagt ttgtgctggt 1380tctttgcaag ctatggtcat ctcttgtatc ggtattggta gattagtcca agattttgaa 1440tggaaattga aagatgacgc tgaagaagat gtcaacactt taggtttaac cactcaaaag 1500ttgcacccat tattggcttt gatcaaccct cgaaagtaa 153917301DNAArtificial sequenceSc Tpi1 terminator 17agattaatat aattatataa aaatattatc ttcttttctt tatatctagt gttatgtaaa 60ataaattgat gactacggaa agctttttta tattgtttct ttttcattct gagccactta 120aatttcgtga atgttcttgt aagggacggt agatttacaa gtgatacaac aaaaagcaag 180gcgctttttc taataaaaag aagaaaagca tttaacaatt gaacacctct atatcaacga 240agaatattac tttgtctcta aatccttgta aaatgtgtac gatctctata tgggttactc 300a 30118403DNAArtificial sequenceAg lox_TEF1 promoter 18taccgttcgt ataatgtatg ctatacgaag ttatgtcccc gccgggtcac ccggccagcg 60acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat 120gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac 180attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag acctgcgagc 240agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa 300atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct 360tgctaggata cagttctcac atcacatccg aacataaaca aca 40319810DNAArtificial sequenceKANMX 19atgggtaagg aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat 60ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 120ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 180aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 240accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 300ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 360gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 420agcgatcgcg tatttcgttt ggctcaggcg caatcacgaa tgaataacgg tttggttgat 480gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 540cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 600aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 660gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 720ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 780tttcatttga tgctcgatga gtttttctaa 81020285DNAArtificial sequenceAg Tef1_lox terminator 20atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt 60tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc 120gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt 180atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca tccagtgtcg 240aaaacgagct cataacttcg tataatgtat gctatacgaa cggta 285211575DNAArabidopsis thaliana 21atggaatctt tagtcgttca caccgtcaat gccatctggt gtattgtcat tgttggtatt 60ttctctgttg gttaccacgt ttacggtcgt gccgttgttg aacaatggag aatgagaaga 120tctttgaaat tgcaaggtgt caagggtcca ccaccatcca ttttcaacgg taatgtctct 180gaaatgcaaa gaatccaatc tgaagctaag cactgttccg gtgacaacat catttctcac 240gattactcct cctctttgtt ccctcacttt gaccactgga gaaagcaata cggtagaatc 300tacacctact ccactggttt gaaacaacat ttgtacatca accatccaga aatggtcaag 360gaattatctc aaaccaacac tttgaactta ggtcgtatca ctcacatcac caagagattg 420aacccaatct taggtaacgg tatcatcact tccaacggtc cacactgggc tcatcaaaga 480agaattattg cttacgaatt cacccacgac aaaatcaagg gtatggtcgg tttgatggtc 540gaatctgcca tgccaatgtt gaacaaatgg gaagaaatgg ttaagagagg tggtgaaatg 600ggttgtgaca tccgtgttga cgaagatttg aaggatgttt ctgctgatgt cattgctaag 660gcttgtttcg gttcctcttt ctccaagggt aaggctatct tctccatgat cagagacttg 720ttgactgcca tcactaagag atctgttttg ttcagattca acggtttcac cgacatggtt 780ttcggttcca agaagcatgg tgatgtcgat atcgatgctt tggaaatgga attggaatct 840tctatctggg aaaccgttaa ggaaagagaa attgaatgta aggacactca caagaaggat 900ttgatgcaat taatcttgga aggtgccatg agatcttgtg acggtaactt gtgggacaag 960tctgcttaca gaagatttgt tgtcgacaac tgtaaatcca tctactttgc cggtcacgac 1020tctactgctg tctccgtttc ctggtgtttg atgttgctag ctttgaaccc atcctggcaa 1080gtcaagatca gagatgaaat cttatcttct tgtaagaacg gtattccaga tgctgaatcc 1140attccaaact tgaagaccgt taccatggtc attcaagaaa ctatgagatt gtacccacca 1200gctccaattg tcggtagaga agcttccaag gacatcagat taggtgactt ggttgttcca 1260aagggtgttt gtatctggac tttgattcca gctttgcacc gtgacccaga aatctggggt 1320ccagatgcta acgacttcaa gccagaaaga ttctctgaag gtatttccaa ggcttgtaaa 1380tacccacaat cttacatccc attcggtttg ggtccaagaa cctgtgtcgg taagaacttc 1440ggtatgatgg aagtcaaagt tttggtttct ttgattgttt ccaagttctc tttcaccttg 1500tctccaactt accaacactc tccatctcac aagttgttgg ttgaacctca acacggtgtt 1560gtcattagag tcgtt 1575221000DNAArtificial sequenceKl prom 6 promoter 22caaagggggg gcagggacag ggatacgaca agggctgggg aaaaaaaaaa agatagatac 60gattggccgg gtaagcctgg ggaaatgtag caagtgcggg taagttaaaa ggtaaccacg 120tgactccgga agagtcacgt ggttacggac ttttttctct agatctcagc tttttatcgg 180tcttaccctg ccctcctgcc ccctgcccct tccctttgcc ccaaaaagaa aggaaatctg 240ttggatttcg ctcaggccat ccctttcgtt aatatcggtt atcgctttac acactgcaca 300tccttctgtc caaaaggaat ccagaagttt agcttttcct tcctttccca cagacattag 360cctaggccct ctctcatcat ttgcatgcct cagccaatgt accaagaata acgcaacgag 420gttgggaaat tttaacccaa caatcgatgc agatgtgaca agagattaga cacgttccag 480ataccagatt acacagcttg tgctagcaga gtgacatatg gtggtgttgt gtctcgttta 540gtacctgtaa tcgagagtgt tcaaatcagt cgatttgaac acccttactg ccactgaata 600ttgattgaat accgtttatt gaaggtttta tgagtgatct tctttcggtc caggacaatt 660tgttgagctt tttctatgta gagttccgtc cctttttttt ttttttttgc tttctcgcac 720ttactagcac tatttttttt tcacacacta aaacacttta ttttaatcta tatatatata 780tatatatata tgtaggaatg gaatcacaga catttgatac tcatcctcat ccttattaat 840tcttgtttta atttgtttga cttagccaaa ccaccaatct caacccatcg tatttcaggt 900attgtgtgtc tagtgtgtct ctggtatacg gaaataagtg ccagaagtaa ggaagaaaca 960aagaacaagt gtctgaatac tactagcctc tcttttcata 1000232133DNAArabidopsis thaliana 23atgtcctctt cttcttcttc ttctacttcc atgattgatt tgatggctgc catcatcaag 60ggtgaaccag tcattgtctc tgacccagcc aacgcttctg cttacgaatc cgttgctgct 120gaattgtcct ccatgttgat tgaaaacaga caattcgcta tgattgtcac tacttccatt 180gctgtcttga ttggttgtat cgtcatgttg gtctggagaa gatccggttc cggtaactcc 240aagagagttg aaccattgaa gccattagtc atcaagccaa gagaagaaga aattgatgac 300ggtagaaaga aggtcaccat cttctttggt actcaaaccg gtactgctga aggttttgct 360aaggctttgg gtgaagaagc caaagctaga tacgaaaaga ccagattcaa gatcgttgac 420ttggacgact acgctgctga tgacgacgaa tacgaagaaa agttgaagaa ggaagatgtt 480gccttcttct tcttggctac ttacggtgat ggtgaaccaa ctgacaatgc tgccagattc 540tacaaatggt tcaccgaagg taacgacaga ggtgaatggt taaagaactt gaaatacggt 600gttttcggtc taggtaacag acaatacgaa cacttcaaca aggttgccaa ggttgtcgat 660gacatcttgg ttgaacaagg tgctcaaaga ttagtccaag tcggtttggg tgatgatgac 720caatgtatcg aagatgactt cactgcttgg agagaagctt tgtggccaga attggacacc 780atcttaagag aagaaggtga taccgctgtt gccaccccat acactgctgc tgttttggaa 840tacagagttt ctatccacga ctctgaagat gccaagttca acgacatcaa catggctaac 900ggtaacggtt acactgtttt cgacgctcaa cacccataca aggccaatgt tgctgtcaag 960agagaattgc acactccaga atctgatcgt tcttgtatcc acttggaatt tgacattgct 1020ggttctggtt tgacctacga aaccggtgac cacgtcggtg tcttatgtga caacttgtct 1080gaaactgtcg atgaagcttt gagattattg gacatgtctc cagacactta tttctccttg 1140catgctgaaa aggaagatgg tactccaatt tcttcttcct tgcctcctcc attcccacca 1200tgtaacttga gaaccgcttt aaccagatac gcttgtttgc tatcctctcc aaagaagtcc 1260gctttggttg ctttggctgc tcacgcttct gacccaactg aagctgaaag attgaaacat 1320ttggcttccc cagctggtaa ggatgaatac tccaaatggg ttgttgaatc tcaaagatct 1380ttgttggaag tcatggctga attcccatct gccaagccac cattgggtgt tttcttcgcc 1440ggtgttgctc caagattgca accaagattt tactccatct cttcttctcc aaagattgct 1500gaaaccagaa ttcacgttac ctgtgccttg gtctacgaaa agatgccaac cggtagaatt 1560cacaagggtg tttgttccac ctggatgaag aacgctgttc catacgaaaa gtctgaaaac 1620tgttcttctg ctccaatctt cgtccgtcaa tccaacttca agttgccatc tgactccaag 1680gtcccaatca tcatgatcgg tccaggtact ggtttagctc cattcagagg tttcttgcaa 1740gaaagattgg ccttagttga atctggtgtc gaattgggtc cttctgtttt gttcttcggt 1800tgtagaaacc gtcgtatgga cttcatctac gaagaagaat tgcaaagatt tgtcgaatct 1860ggtgctttgg ctgaattgtc cgttgctttc tctcgtgaag gtccaaccaa agaatacgtt 1920caacacaaga tgatggacaa agcctccgac atctggaaca tgatctccca aggtgcttac 1980ttgtacgttt gtggtgatgc taaaggtatg gccagagatg tccacagatc tttacatacc 2040attgcccaag aacaaggttc catggactcc accaaggctg aaggtttcgt taagaacttg 2100caaacttctg gtcgttactt gagagatgtt tgg 213324301DNAArtificial sequenceSc Pdc1 terminator 24agcgatttaa tctctaatta ttagttaaag ttttataagc atttttatgt aacgaaaaat 60aaattggttc atattattac tgcactgtca cttaccatgg aaagaccaga caagaagttg 120ccgacagtct gttgaattgg cctggttagg cttaagtctg ggtccgcttc tttacaaatt 180tggagaattt ctcttaaacg atatgtatat tcttttcgtt ggaaaagatg tcttccaaaa 240aaaaaaccga tgaattagtg gaaccaagga aaaaaaaaga ggtatccttg attaaggaac 300a 301251000DNAArtificial sequenceKl prom 3 promoter 25gagcctgtcc aagcaaatgc cttctcataa atggtgccaa agacccgcaa gcccaaagca 60attacccccc aaaaagaaat gatatagtgc aagatacgta tatgaccatg acttgactag 120gtgaaacagt gcagaaacag ccgcacaaaa gcagccctaa ccctcagagt cgattttact 180ctttcaggta ataaagcctc gacatcaatt ttagacagaa gccaggctgg cctcgagatt 240atagccatag gcaagcaaga ggagagaagg ggaggccccc catggggggc ctcccccccg 300ctgtcaaggt ttggcagaac ctagcttcat taggccacta gcccagccta aaacgtcaac 360gggcaggagg aacactccca caagacggcg tagtattctc gattcataac cattttctca 420atcgaattac acagaacaca ccgtacaaac ctctctatca taactactta atagtcacac 480acgtactcgt ctaaatacac atcatcgtcc tacaagttca tcaaagtgtt ggacagacaa 540ctataccagc atggatctct tgtatcggtt cttttctccc gctctctcgc aataacaatg 600aacactgggt caatcatagc ctacacaggt gaacagagta gcgtttatac agggtttata 660cggtgattcc tacggcaaaa atttttcatt tctaaaaaaa aaaagaaaaa tttttctttc 720caacgctaga aggaaaagaa aaatctaatt aaattgattt ggtgattttc tgagagttcc 780ctttttcata tatcgaattt tgaatataaa aggagatcga aaaaattttt ctattcaatc 840tgttttctgg ttttatttga tagttttttt gtgtattatt attatggatt agtactggtt 900tatatgggtt tttctgtata acttcttttt attttagttt gtttaatctt attttgagtt 960acattatagt tccctaactg caagagaagt aacattaaaa 1000261443DNAStevia rebaudiana 26atggacgcta tggccaccac tgaaaagaag cctcacgtta tctttattcc attcccagct 60caatctcata tcaaggctat gttgaaattg gctcaattat tgcaccacaa gggtttgcaa 120atcacttttg tcaacaccga cttcattcac aaccaattct tggaatcttc tggtcctcac 180tgtttggacg gtgctccagg tttcagattc gaaaccattc cagatggtgt ttcccactct 240ccagaagcct ccatcccaat cagagaatcc ttgttgagat ctattgaaac caacttcttg 300gaccgtttca tcgatttggt taccaaattg ccagacccac caacctgtat catttctgac 360ggtttcttgt ccgttttcac catcgatgct gccaagaaat tgggtattcc agtcatgatg 420tactggactt tggctgcttg tggtttcatg ggtttctacc atattcactc tttgattgaa 480aagggtttcg ctccattaaa ggatgcttct tacttgacca acggttactt ggacaccgtc 540attgactggg ttccaggtat ggaaggtatc agattgaaag atttcccatt ggactggtct 600actgacttga atgacaaggt cttgatgttc actactgaag ctccacaaag atctcataag 660gtttctcacc acatcttcca cactttcgat gaattagaac catctatcat caagactcta 720tccttgagat acaaccatat ctacaccatt ggtccattac aattgttgtt ggaccaaatc 780ccagaagaaa agaagcaaac cggtatcact tctttgcacg gttactcttt agtcaaggaa 840gaaccagaat gtttccaatg gttacaatcc aaggaaccaa actctgttgt ctacgttaac 900tttggttcca ccactgttat gtccttggaa gatatgactg aatttggttg gggtttggct 960aactctaacc actacttctt atggatcatc agatctaact tggtcattgg tgaaaacgcc 1020gttttgcctc cagaattgga agaacacatc aagaagagag gtttcattgc ttcctggtgt 1080tctcaagaaa aggtcttgaa gcacccatct gttggtggtt tcttgaccca ctgtggttgg 1140ggttccacca ttgaatccct atctgctggt gttccaatga tctgttggcc atactcctgg 1200gaccaattga ctaactgtcg ttacatctgt aaggaatggg aagttggttt ggaaatgggt 1260actaaggtca agagagatga agtcaagaga ttagtccaag aattgatggg tgaaggtggt 1320cacaagatga gaaacaaagc caaggactgg aaggaaaagg ccagaattgc tattgctcca 1380aacggttctt cctccttgaa catcgataaa atggttaagg aaatcactgt cttggctcga 1440aac 144327301DNAArtificial sequenceSc TDH1 terminator 27aataaagcaa tcttgatgag gataatgatt tttttttgaa tatacataaa tactaccgtt 60tttctgctag attttgtgaa gacgtaaata agtacatatt actttttaag ccaagacaag 120attaagcatt aactttaccc ttttctcttc taagtttcaa tactagttat cactgtttaa 180aagttatggc gagaacgtcg gcggttaaaa tatattaccc tgaacgtggt gaattgaagt 240tctaggatgg tttaaagatt tttccttttt gggaaataag taaacaatat attgctgcct 300t 301281000DNAArtificial sequenceKl prom 2 promoter 28gagcctgtcc aagcaaatgc cttctcataa atggtgccaa agacccgcaa gcccaaagca 60attacccccc aaaaagaaat gatatagtgc aagatacgta tatgaccatg acttgactag 120gtgaaacagt gcagaaacag ccgcacaaaa gcagccctaa ccctcagagt cgattttact 180ctttcaggta ataaagcctc gacatcaatt ttagacagaa gccaggctgg cctcgagatt 240atagccatag gcaagcaaga ggagagaagg ggaggccccc catggggggc ctcccccccg 300ctgtcaaggt ttggcagaac ctagcttcat taggccacta gcccagccta aaacgtcaac 360gggcaggagg aacactccca caagacggcg tagtattctc gattcataac cattttctca 420atcgaattac acagaacaca ccgtacaaac ctctctatca taactactta atagtcacac 480acgtactcgt ctaaatacac atcatcgtcc tacaagttca tcaaagtgtt ggacagacaa 540ctataccagc atggatctct tgtatcggtt cttttctccc gctctctcgc aataacaatg 600aacactgggt caatcatagc ctacacaggt gaacagagta gcgtttatac agggtttata 660cggtgattcc tacggcaaaa atttttcatt tctaaaaaaa aaaagaaaaa tttttctttc 720caacgctaga aggaaaagaa aaatctaatt aaattgattt ggtgattttc tgagagttcc 780ctttttcata tatcgaattt tgaatataaa aggagatcga aaaaattttt ctattcaatc 840tgttttctgg ttttatttga tagttttttt gtgtattatt attatggatt agtactggtt 900tatatgggtt tttctgtata acttcttttt attttagttt gtttaatctt attttgagtt 960acattatagt tccctaactg caagagaagt aacattaaaa 1000291380DNAStevia rebaudiana 29atggctgaac aacaaaagat caagaaatct ccacacgtct tgttgattcc attcccattg 60caaggtcaca tcaacccatt catccaattc ggtaagagat tgatttccaa gggtgtcaag 120accactttag tcaccactat tcacacttta aactccactt taaaccactc taacactact 180accacctcta ttgaaatcca agccatttct gacggttgtg acgaaggtgg tttcatgtct 240gctggtgaat cttacttgga aactttcaag caagtcggtt ccaagtcttt ggctgatttg 300atcaagaaat tgcaatccga aggtactacc atcgatgcta tcatctacga ctccatgact 360gaatgggttt tggatgttgc cattgaattt ggtattgacg gtggttcttt cttcacccaa 420gcctgtgttg ttaactcttt gtactaccac gtccacaagg gtttgatctc tctaccatta 480ggtgaaaccg tttccgtccc aggtttccca gtcttgcaaa gatgggaaac tccattgatc 540ttacaaaacc atgaacaaat ccaatctcca tggtcccaaa tgttgtttgg tcaattcgct 600aacattgacc aagctagatg ggttttcacc aactctttct acaagttgga agaagaagtc 660attgaatgga ccagaaagat ctggaacttg aaggttatcg gtccaactct accatccatg 720tacttggaca agagattgga tgacgacaag gacaacggtt tcaacttgta caaggctaac 780catcacgaat gtatgaactg gttggatgac aagccaaagg aatctgttgt ttacgttgct 840ttcggttctt tggtcaagca tggtccagaa caagttgaag aaatcaccag agctttgatt 900gactccgatg ttaacttctt atgggttatc aagcacaagg aagaaggtaa attgccagaa 960aacttgtctg aagttatcaa gaccggtaag ggtttgattg ttgcttggtg taagcaattg 1020gatgttttgg ctcacgaatc cgtcggttgt ttcgtcactc actgtggttt caactctact 1080ttggaagcta tctccttggg tgttccagtt gttgccatgc ctcaattctc tgaccaaacc 1140accaacgcca aattgttgga tgaaatcttg ggtgtcggtg tccgtgtcaa ggctgatgaa 1200aacggtattg ttagaagagg taacttagct tcctgtatca agatgatcat ggaagaagaa 1260cgtggtgtca ttatcagaaa gaatgctgtc aaatggaagg acttggctaa ggttgctgtc 1320cacgaaggtg gttcctctga caatgacatt gttgaatttg tctctgaatt gatcaaagcg 1380301374DNAStevia rebaudiana 30atggaaaaca agactgaaac cactgttaga agaagaagaa gaatcatctt attcccagtt 60ccattccaag gtcacattaa cccaatcttg caattggcta acgtcttata ctccaagggt 120ttctccatca ccatcttcca caccaacttc aacaaaccta aaacttccaa ctacccacac 180ttcaccttca gatttatctt ggacaacgac ccacaagatg aaagaatttc taacttgcca 240acccatggtc cattggccgg tatgagaatt ccaatcatca acgaacacgg tgctgacgaa 300ttgagaagag aattggaatt gttgatgttg gcttctgaag aagatgaaga agtctcttgt 360ttgatcactg atgctttatg gtactttgct caatctgttg ctgactcttt gaacttgaga

420agattagtct tgatgacctc ttctttgttc aacttccacg ctcacgtttc tctaccacaa 480tttgatgaat tgggttactt ggacccagat gacaagacca gattggaaga acaagcctcc 540ggtttcccaa tgttgaaggt caaggatatc aagtctgcct actccaactg gcaaatcttg 600aaggaaattt tgggtaagat gatcaagcaa accaaggctt cttctggtgt catctggaac 660tccttcaagg aattggaaga atctgaattg gaaaccgtca tcagagaaat tccagctcca 720tctttcttga ttccattacc aaagcatttg actgcttcct cctcttctct attggaccac 780gacagaactg ttttccaatg gttggaccaa caaccaccat cttccgtctt atacgtttcc 840tttggttcca cttctgaagt tgacgaaaag gacttcttgg aaattgctcg tggtttggtt 900gactccaagc aatctttctt atgggttgtc agaccaggtt tcgtcaaggg ttccacctgg 960gttgaacctt tgccagacgg tttcttgggt gaaagaggta gaattgtcaa atgggttcca 1020caacaagaag ttttggctca cggtgccatt ggtgctttct ggactcactc tggttggaac 1080tctactttgg aatccgtttg tgaaggtgtt ccaatgattt tctctgactt cggtttggac 1140caaccattga atgctcgtta catgtccgat gttttgaagg ttggtgtcta cttggaaaac 1200ggttgggaac gtggtgaaat tgctaacgcc atcagaagag tcatggtcga tgaagaaggt 1260gaatacatca gacaaaatgc tcgtgtcttg aaacaaaagg ctgatgtttc tttgatgaag 1320ggtggttctt cttacgaatc tttggaatct ttggtttcct acatctccag tctc 137431602DNAArtificial sequenceSc Eno1 terminator 31aagcttttga ttaagccttc tagtccaaaa aacacgtttt tttgtcattt atttcatttt 60cttagaatag tttagtttat tcattttata gtcacgaatg ttttatgatt ctatataggg 120ttgcaaacaa gcatttttca ttttatgtta aaacaatttc aggtttacct tttattctgc 180ttgtggtgac gcgtgtatcc gcccgctctt ttggtcaccc atgtatttaa ttgcataaat 240aattcttaaa agtggagcta gtctatttct atttacatac ctctcatttc tcatttcctc 300caagcttttg attaagcctt ctagtccaaa aaacacgttt ttttgtcatt tatttcattt 360tcttagaata gtttagttta ttcattttat agtcacgaat gttttatgat tctatatagg 420gttgcaaaca agcatttttc attttatgtt aaaacaattt caggtttacc ttttattctg 480cttgtggtga cgcgtgtatc cgcccgctct tttggtcacc catgtattta attgcataaa 540taattcttaa aagtggagct agtctatttc tatttacata cctctcattt ctcatttcct 600cc 60232600DNAArtificial sequenceSc_TDH3.pro 32ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa aatagggggc 60gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt cctggcatcc 120actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag aatcccagca 180ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt agcgcaacta 240cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc 300tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat ctcattttct 360tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa aaaggttgaa 420accagttccc tgaaattatt cccctacttg actaataagt atataaagac ggtaggtatt 480gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta tagttagtct 540tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata aacaaacaaa 600334248DNAArtificial sequenceALNQ_007_38000 CpO for S. cerevisiae 33atgtctgctt tgaacaccga tgctttggaa tctcaaccag acttcaagtt ccaaagacaa 60aagagattga tgtctccatt tatgtctaag aaggttccac caatcccaac caaagaagaa 120agaaagccat acggtgaata ccacaccaac atcttattca gaattatgtt ctggtggttg 180aaccctatct tgaacgtcgg ttacaagaga actttgactg aacaagactt gttctatttg 240gacaactccc aaaccatgga cactttatac gaaactttca aatcccactt gaagaccact 300atcgaaaagt ccatgaagaa gtacttgcaa gaaaaatact ctaaggaagg taagacctac 360gacccatctt ccattccaac tgctgaagat ttgaaggact tccaaattcc aatctacgct 420atcccattgt gtttattcaa gactttgtac tggcaatact ctttgggtaa tttgtacaag 480gtcttgtctg actgtacttc tgctactacc ccattgttgc aaaagaagtt aatcaacttc 540gttcaaatga agtctttcac cgctttgggt tctaccggta agggcgttgg ttacgccatc 600ggtgtgtgtt tgatgatctt cttccaagcc attaccgtca accatgcttt ccacaacttg 660caaatttgtg gtgccaaatc caaggctatt ttgactagaa tgttgttgga caagtctatg 720tctgttgatg ctagaggtaa ccacttcttc ccagcctcca aggtccaatc tatgatctct 780accgatttga acagagtcga tttggccatc ggtttcttcc cattcgcttt gacttgtgtt 840tttccaattg ctatctgtat cggtttgtta atctggaacg ttggtgtctc cgccttggtt 900ggtattgcca tcttcgttgc taacgttggt ttgttggctg tttccatccc aagattgatg 960agattcagaa tcaaagctat ggtttttacc gacaagcgtg tcactttgat gaaggaattg 1020ttgaagaact tcaagatgat caagttctac tcttgggaaa actcttacgc tagaagaatc 1080caagatgctc gtttcaagga aatgaagttg atcttgtcat tacaatcttt aagaaacatt 1140gtcatgtccg tttctttcgc catgccaact ttggcttcta tggctacttt ctgtaccgct 1200ttcgatatca cttctggtaa gaacgctgct tccttgttct cctctttgtc tttattccaa 1260gttttatcca tgcaattcat gttggctcca gttgccttaa acaccgctgc tgacatgatg 1320gtttctatga agaaattcaa ccagttcttg gctcacgctg atttggatcc tgaacaatac 1380agaatcgaag aattccacga tgataagttg gccgttaagg ttgacaacgc caccttcgaa 1440tgggacacct tcgatgatga caaggtcgaa gacccagctt tagaatttga aaaacaagat 1500aatgactcct tggaaaaagt ttcctcccac aacaccgttg actacgactc tactgaaaag 1560atcagaaacg acacttcttc tatcgattcc accaagattt tggaaaagac tgctttccct 1620ggtttgagaa acatcaactt ggaaatcaaa aagggtgaat tcgttgttgt taccggttcc 1680attggtgctg gtaaatcctc tttgctacaa gctatctctg gtttgatgaa aagagtctcc 1740ggtaaggttt acgtcgatgg tgacttgttg ttgtgtggtt acccttgggt tcaaaacgct 1800actatcagag acaacatctt gttcggttta ccattcgacc aagaaaagta cgaccaagtt 1860gtttacgctt gttctttgca atctgacttc aaccaattcc aaggtggtga catgactgaa 1920gttggtgaaa gaggtattac cttgtctggt ggtcaaaagg ctagaattaa cttggccaga 1980tccgtctacg ctgacaagga cattattttg ttggatgacg ttttgtctgc tgtcgatgct 2040aaggttggta gacatattgt cgatacctgt ttgttgggtt tattgaagga caagacccgt 2100atcatggcta cccaccaatt gtctttaatt gactctgctg acagaatgat tttcttgaac 2160ggtgatggtt ctattgactg tggtactatt tccgaattaa aggaccgtaa cgaaaaattg 2220aacgaattgt tgtctcacca aaaggacaag gccaacgact ctgatgaaga attggaattg 2280caagaagaaa tcgaatctaa ggaacaacac ttgaaggaag atttgtctga agttaagcac 2340gaaatcaagg aagaacaaaa gaagatggaa atctccggtg atgtcggtga agaattcgaa 2400cacgctgacg aacacaagga aattgttaga attattggtg atgaggaaag agctgtcaac 2460gctttgaagg ccgatgtcta catcaactac gctaaattgg ccttcggtaa gttgggtcta 2520ttctccttga tgttgttcgt caccgttgct gctttgcaaa cttactgtaa catgttcact 2580aacacctggt tatccttctg gattgaagaa aagttccatg gtagatccaa gtccttctac 2640atgggtattt acatcatgtt cgctttcttg tacactttct tcctagctgc ctttttctac 2700tctatgtgtt acttctgtaa cagagcttcc aagtatttga actacaaggc ctccgaaaag 2760attttgcacg ttccaatgtc tttcatggac atttctccaa tcggtcgtgt tttgaataga 2820ttcaccaagg acaccgatgt cttggacaac gaaattttgg accaattcag acaattcttg 2880tccccattct gtaacgccat tggtactatc gtcttgtgca tcatctacat tccatggttc 2940gctattgccg tcccattgat tgtcactttc tacgtcttgg tcgccaacta ctaccaagct 3000tctgctcgtg aaatcaagag attggaagct gtcaagcgtt ctttggtctt tggtcacttc 3060aacgaagctc tatctggtaa ggaaactatc aaggcttaca gagctatcga cagagtcaag 3120caaagattga acaaattgat cgatgggcaa aacgaagctt actttttgac cattgttaac 3180caaagatggt tgggtgccaa tttgtctatc ttgtctttct gtatggtttt catcatctct 3240ttcttgtgtg tcttcagagt cttcaacatt tctgctgctt ccactggttt attattgact 3300tacgttatca acttgaccaa taccatcact atgatgatga gagctatgac ccaagtcgaa 3360aacgaattta actccgttga gagattgaac cactacgctt tcgacttagt ccaagaagct 3420ccatacgaaa tcccagaaaa cgatccacca caagactggc caaagtacgg tgaaatcatt 3480ttcaaggatg tttccatgag atacagacca gaattgccat tcgttttgaa gaacatcaac 3540ctatccatcg gtaagggtga aaagattggt ttctgtggta gaaccggtgc cggtaagtct 3600actttcatga cttgtttgta cagaatttct gagttcgaag gtaccatcgt tatcgatgac 3660gtcgatatct ccaagttggg tttgcacaaa ttgcgttcta agttgactat tatcccacaa 3720gacccagtct tattcgttgg ttccatcaga gaaaacttag acccatttgg tgaatactct 3780gacgaagaat tatgggaagc tttgaccatc tccggtttga tcaacaaaga agacttgaac 3840gaggttaaga agcaaaatga aaatgacgac aacttgaaca agttccactt gattagaatg 3900gtcgaagatg atggtgttaa cttctccatc ggtgaacgtc aattgattgc tctagccaga 3960gctttggtca gaaagactaa gattttgatc ttggacgaag ctacttcttc tgtcgattac 4020gctactgact ccagaattca aaagaccatt gctactgaat tcgacgactg tatgatcttg 4080tgtatcgctc acagattaaa caccatcttg aactacgaca agatcgtcgt catggacaaa 4140ggtgaaatcg ttgaattcga taagccaaga tctttgttca tgagagaaga aggtgttttc 4200agatccatgt gtgaacaagc taacatcacc attgaagatt ttccataa 4248344248DNAIssatchenkia orientalis 34atgtcggcgc ttaatacgga tgccttagag agtcagcctg acttcaagtt ccagagacaa 60aagaggttga tgtctccatt tatgtcgaaa aaagtgcctc ctatacctac aaaggaagag 120aggaaaccct atggggagta ccatacaaac atactatttc gtattatgtt ttggtggttg 180aatcctattt tgaatgtcgg gtacaaaagg acattgactg aacaggattt attctatctt 240gataatagcc agactatgga caccctctat gagaccttca aaagccatct aaagacgaca 300attgagaaat caatgaaaaa ataccttcag gagaaataca gtaaagaggg gaagacatat 360gacccaagta gtatacctac agctgaagac ttaaaagatt tccaaatacc catttatgca 420atcccattgt gcttgttcaa aactctatac tggcagtata gtcttggtaa tctctacaaa 480gtattatccg actgtacatc tgctacaaca cctttattgc agaagaaact cattaatttt 540gttcagatga aatcctttac cgcattagga agtacgggga aaggtgtagg gtacgccatc 600ggtgtatgct tgatgatctt cttccaagca ataactgtca atcatgcctt tcataatttg 660caaatctgtg gtgccaaatc taaggcgatt cttacgagaa tgttattgga taaatcaatg 720tcagtcgatg ccagaggtaa tcattttttt cctgctagta aagttcagag catgatatcc 780acagatttga atagggtaga tttggctatt ggctttttcc catttgcact tacgtgtgta 840tttccaattg ctatttgtat aggcttgctt atttggaacg ttggtgtgtc agcattagtg 900ggaattgcca ttttcgttgc caatgtcggt ctcttagccg tttctatacc tagacttatg 960cggttcagga taaaggccat ggtattcaca gacaagagag ttactttaat gaaagagctg 1020ttaaaaaatt ttaaaatgat caagttctat agctgggaaa attcatacgc cagaaggatc 1080caggatgcca gattcaagga gatgaaattg attttgtctt tacagtcttt aagaaatatt 1140gtgatgtcag tttcatttgc tatgccgaca ttggcttcaa tggcaacatt ttgtactgcc 1200tttgatatca caagtggtaa aaatgctgcc tctttattct cctctctttc tctcttccaa 1260gttttatcaa tgcaattcat gttagcccca gttgccctta atactgctgc tgatatgatg 1320gtgtcaatga aaaagttcaa tcaattttta gctcatgctg acttggatcc agagcagtat 1380cgtatagaag aatttcacga cgataaattg gcggtgaaag ttgataatgc cacttttgaa 1440tgggatacgt ttgacgatga taaagttgaa gatcctgctc ttgaatttga aaaacaggac 1500aatgacagtc ttgaaaaagt ttccagtcat aatacggtcg attatgacag taccgaaaag 1560attagaaatg atacaagctc gatagattct accaaaatct tggagaaaac agcatttcct 1620ggcctaagaa acataaatct agagattaag aaaggcgaat ttgttgttgt tacaggtagt 1680atcggtgctg gtaaatcatc ccttctccag gccatctcgg gattgatgaa aagagtatct 1740ggtaaggttt atgttgatgg tgacttattg ctatgcggtt atccatgggt tcaaaatgct 1800acgattcggg acaacatttt gtttggtttg ccatttgacc aagaaaagta cgaccaagtt 1860gtttatgctt gttcattgca gagcgacttc aatcagttcc aaggtggtga catgaccgag 1920gttggtgaac gaggtattac attatctggt ggtcaaaagg ctagaatcaa cttggcaaga 1980tctgtatatg ctgacaagga tataatttta cttgatgatg ttttaagtgc tgttgatgcg 2040aaggtgggtc gacatatcgt tgatacttgc cttcttggtt tgttaaagga taaaactaga 2100attatggcaa ctcaccaact aagtttgatt gactctgcag atcgaatgat tttcctcaat 2160ggggatggaa gtattgattg cggtaccatc tcagaattga aagacagaaa tgaaaaactg 2220aacgaacttt tgtctcatca aaaggataag gcaaatgact ccgacgaaga gttagaattg 2280caagaagaaa tcgagtcgaa agaacaacat ctcaaagaag atttatctga ggtgaaacat 2340gaaatcaaag aagaacagaa gaagatggaa ataagcggtg atgtaggaga agagtttgaa 2400catgcagatg aacataaaga gattgttagg attattggtg atgaagaaag agccgtgaat 2460gccctgaagg cggatgttta tatcaattat gctaaacttg catttggtaa actcggattg 2520ttttcactga tgttgtttgt cacagttgct gctttacaga cttactgcaa tatgtttacc 2580aacacatggt tatctttttg gatagaagaa aaattccacg gcagatccaa aagtttttac 2640atggggatct acattatgtt tgccttctta tacacatttt tccttgctgc atttttctat 2700tcaatgtgct atttctgcaa tagggcttcc aagtatctta attataaagc ttcagaaaaa 2760atcttgcatg ttccaatgtc cttcatggat atttctccaa ttggtcgagt tttgaataga 2820tttacaaaag atactgatgt gttagataac gagatactag atcaatttag acagtttttg 2880agtcccttct gcaatgctat cggtaccatt gttctatgta ttatttacat tccatggttt 2940gcaattgctg ttcccttaat tgttacattt tatgttttgg ttgccaatta ctaccaagcc 3000agcgctagag agatcaaaag gttagaagca gttaaaaggt cgttggtctt tggccatttc 3060aatgaagcat tatccggaaa ggagacaatc aaagcttata gggcaatcga cagagtcaag 3120caaaggttga acaaattgat tgatgggcag aacgaggctt attttctaac cattgttaac 3180cagagatggt taggtgccaa tttatcgatc ttatcatttt gtatggtctt cattatctcg 3240ttcttgtgtg ttttcagagt tttcaatatc agtgcagcgt cgactggttt acttttaacc 3300tatgtcataa atttgacaaa taccattact atgatgatga gagctatgac gcaagttgaa 3360aatgagttca attcagttga aagattaaac cattatgcct ttgatcttgt ccaggaggcc 3420ccttatgaga ttcctgagaa tgatccaccc caggactggc ctaagtatgg tgaaattatt 3480ttcaaagatg ttagtatgag atatagacca gaattaccat ttgttttgaa gaatatcaac 3540ttaagtattg ggaaaggtga gaaaattgga ttttgtggaa gaaccggtgc tggtaagtct 3600acgttcatga cttgcttgta caggatttcc gaatttgaag gaactattgt tattgatgat 3660gttgatatca gtaaattggg tttacataaa ctaagatcca aattaactat tattccacaa 3720gatccagtgt tatttgtcgg ttcgatccga gagaatctgg atccatttgg cgagtattct 3780gatgaagaat tgtgggaggc acttacaatt tccggtttga tcaacaagga agatctaaac 3840gaagtaaaaa aacagaacga aaatgatgat aacttaaaca aattccacct tatcagaatg 3900gtggaggatg atggtgtgaa tttctctatt ggagagagac agttgattgc acttgccaga 3960gctttggtta ggaaaaccaa aattctaatc ttagatgagg caacatcaag tgttgactat 4020gccaccgatt caagaatcca aaagaccatt gccaccgaat tcgacgactg tatgatactg 4080tgtattgctc acagattgaa tacgattcta aactacgata agattgtcgt tatggataag 4140ggtgagattg ttgagtttga taaaccaaga tcattgttta tgagggagga aggggtcttt 4200aggtctatgt gtgagcaggc aaatatcaca atcgaagatt tcccttaa 4248351415PRTIssatchenkia orientalis 35Met Ser Ala Leu Asn Thr Asp Ala Leu Glu Ser Gln Pro Asp Phe Lys 1 5 10 15 Phe Gln Arg Gln Lys Arg Leu Met Ser Pro Phe Met Ser Lys Lys Val 20 25 30 Pro Pro Ile Pro Thr Lys Glu Glu Arg Lys Pro Tyr Gly Glu Tyr His 35 40 45 Thr Asn Ile Leu Phe Arg Ile Met Phe Trp Trp Leu Asn Pro Ile Leu 50 55 60 Asn Val Gly Tyr Lys Arg Thr Leu Thr Glu Gln Asp Leu Phe Tyr Leu 65 70 75 80 Asp Asn Ser Gln Thr Met Asp Thr Leu Tyr Glu Thr Phe Lys Ser His 85 90 95 Leu Lys Thr Thr Ile Glu Lys Ser Met Lys Lys Tyr Leu Gln Glu Lys 100 105 110 Tyr Ser Lys Glu Gly Lys Thr Tyr Asp Pro Ser Ser Ile Pro Thr Ala 115 120 125 Glu Asp Leu Lys Asp Phe Gln Ile Pro Ile Tyr Ala Ile Pro Leu Cys 130 135 140 Leu Phe Lys Thr Leu Tyr Trp Gln Tyr Ser Leu Gly Asn Leu Tyr Lys 145 150 155 160 Val Leu Ser Asp Cys Thr Ser Ala Thr Thr Pro Leu Leu Gln Lys Lys 165 170 175 Leu Ile Asn Phe Val Gln Met Lys Ser Phe Thr Ala Leu Gly Ser Thr 180 185 190 Gly Lys Gly Val Gly Tyr Ala Ile Gly Val Cys Leu Met Ile Phe Phe 195 200 205 Gln Ala Ile Thr Val Asn His Ala Phe His Asn Leu Gln Ile Cys Gly 210 215 220 Ala Lys Ser Lys Ala Ile Leu Thr Arg Met Leu Leu Asp Lys Ser Met 225 230 235 240 Ser Val Asp Ala Arg Gly Asn His Phe Phe Pro Ala Ser Lys Val Gln 245 250 255 Ser Met Ile Ser Thr Asp Leu Asn Arg Val Asp Leu Ala Ile Gly Phe 260 265 270 Phe Pro Phe Ala Leu Thr Cys Val Phe Pro Ile Ala Ile Cys Ile Gly 275 280 285 Leu Leu Ile Trp Asn Val Gly Val Ser Ala Leu Val Gly Ile Ala Ile 290 295 300 Phe Val Ala Asn Val Gly Leu Leu Ala Val Ser Ile Pro Arg Leu Met 305 310 315 320 Arg Phe Arg Ile Lys Ala Met Val Phe Thr Asp Lys Arg Val Thr Leu 325 330 335 Met Lys Glu Leu Leu Lys Asn Phe Lys Met Ile Lys Phe Tyr Ser Trp 340 345 350 Glu Asn Ser Tyr Ala Arg Arg Ile Gln Asp Ala Arg Phe Lys Glu Met 355 360 365 Lys Leu Ile Leu Ser Leu Gln Ser Leu Arg Asn Ile Val Met Ser Val 370 375 380 Ser Phe Ala Met Pro Thr Leu Ala Ser Met Ala Thr Phe Cys Thr Ala 385 390 395 400 Phe Asp Ile Thr Ser Gly Lys Asn Ala Ala Ser Leu Phe Ser Ser Leu 405 410 415 Ser Leu Phe Gln Val Leu Ser Met Gln Phe Met Leu Ala Pro Val Ala 420 425 430 Leu Asn Thr Ala Ala Asp Met Met Val Ser Met Lys Lys Phe Asn Gln 435 440 445 Phe Leu Ala His Ala Asp Leu Asp Pro Glu Gln Tyr Arg Ile Glu Glu 450 455 460 Phe His Asp Asp Lys Leu Ala Val Lys Val Asp Asn Ala Thr Phe Glu 465 470 475 480 Trp Asp Thr Phe Asp Asp Asp Lys Val Glu Asp Pro Ala Leu Glu Phe 485 490 495 Glu Lys Gln Asp Asn Asp Ser Leu Glu Lys Val Ser Ser His Asn Thr 500 505 510 Val Asp Tyr Asp Ser Thr Glu Lys Ile Arg Asn Asp Thr Ser Ser Ile 515 520 525 Asp Ser Thr Lys Ile Leu Glu Lys Thr Ala Phe Pro Gly Leu Arg Asn 530 535 540 Ile Asn Leu Glu Ile Lys Lys Gly Glu Phe Val Val Val Thr Gly Ser 545 550 555 560 Ile Gly Ala Gly Lys Ser Ser Leu Leu Gln Ala Ile Ser Gly Leu Met 565 570 575 Lys Arg Val Ser Gly Lys Val Tyr Val Asp Gly Asp Leu Leu Leu Cys 580 585 590 Gly Tyr Pro Trp Val Gln Asn Ala Thr Ile Arg Asp Asn Ile Leu Phe 595 600 605 Gly Leu Pro Phe Asp Gln Glu Lys Tyr Asp Gln Val Val Tyr Ala Cys 610 615 620 Ser Leu Gln Ser Asp Phe Asn Gln Phe Gln Gly

Gly Asp Met Thr Glu 625 630 635 640 Val Gly Glu Arg Gly Ile Thr Leu Ser Gly Gly Gln Lys Ala Arg Ile 645 650 655 Asn Leu Ala Arg Ser Val Tyr Ala Asp Lys Asp Ile Ile Leu Leu Asp 660 665 670 Asp Val Leu Ser Ala Val Asp Ala Lys Val Gly Arg His Ile Val Asp 675 680 685 Thr Cys Leu Leu Gly Leu Leu Lys Asp Lys Thr Arg Ile Met Ala Thr 690 695 700 His Gln Leu Ser Leu Ile Asp Ser Ala Asp Arg Met Ile Phe Leu Asn 705 710 715 720 Gly Asp Gly Ser Ile Asp Cys Gly Thr Ile Ser Glu Leu Lys Asp Arg 725 730 735 Asn Glu Lys Leu Asn Glu Leu Leu Ser His Gln Lys Asp Lys Ala Asn 740 745 750 Asp Ser Asp Glu Glu Leu Glu Leu Gln Glu Glu Ile Glu Ser Lys Glu 755 760 765 Gln His Leu Lys Glu Asp Leu Ser Glu Val Lys His Glu Ile Lys Glu 770 775 780 Glu Gln Lys Lys Met Glu Ile Ser Gly Asp Val Gly Glu Glu Phe Glu 785 790 795 800 His Ala Asp Glu His Lys Glu Ile Val Arg Ile Ile Gly Asp Glu Glu 805 810 815 Arg Ala Val Asn Ala Leu Lys Ala Asp Val Tyr Ile Asn Tyr Ala Lys 820 825 830 Leu Ala Phe Gly Lys Leu Gly Leu Phe Ser Leu Met Leu Phe Val Thr 835 840 845 Val Ala Ala Leu Gln Thr Tyr Cys Asn Met Phe Thr Asn Thr Trp Leu 850 855 860 Ser Phe Trp Ile Glu Glu Lys Phe His Gly Arg Ser Lys Ser Phe Tyr 865 870 875 880 Met Gly Ile Tyr Ile Met Phe Ala Phe Leu Tyr Thr Phe Phe Leu Ala 885 890 895 Ala Phe Phe Tyr Ser Met Cys Tyr Phe Cys Asn Arg Ala Ser Lys Tyr 900 905 910 Leu Asn Tyr Lys Ala Ser Glu Lys Ile Leu His Val Pro Met Ser Phe 915 920 925 Met Asp Ile Ser Pro Ile Gly Arg Val Leu Asn Arg Phe Thr Lys Asp 930 935 940 Thr Asp Val Leu Asp Asn Glu Ile Leu Asp Gln Phe Arg Gln Phe Leu 945 950 955 960 Ser Pro Phe Cys Asn Ala Ile Gly Thr Ile Val Leu Cys Ile Ile Tyr 965 970 975 Ile Pro Trp Phe Ala Ile Ala Val Pro Leu Ile Val Thr Phe Tyr Val 980 985 990 Leu Val Ala Asn Tyr Tyr Gln Ala Ser Ala Arg Glu Ile Lys Arg Leu 995 1000 1005 Glu Ala Val Lys Arg Ser Leu Val Phe Gly His Phe Asn Glu Ala 1010 1015 1020 Leu Ser Gly Lys Glu Thr Ile Lys Ala Tyr Arg Ala Ile Asp Arg 1025 1030 1035 Val Lys Gln Arg Leu Asn Lys Leu Ile Asp Gly Gln Asn Glu Ala 1040 1045 1050 Tyr Phe Leu Thr Ile Val Asn Gln Arg Trp Leu Gly Ala Asn Leu 1055 1060 1065 Ser Ile Leu Ser Phe Cys Met Val Phe Ile Ile Ser Phe Leu Cys 1070 1075 1080 Val Phe Arg Val Phe Asn Ile Ser Ala Ala Ser Thr Gly Leu Leu 1085 1090 1095 Leu Thr Tyr Val Ile Asn Leu Thr Asn Thr Ile Thr Met Met Met 1100 1105 1110 Arg Ala Met Thr Gln Val Glu Asn Glu Phe Asn Ser Val Glu Arg 1115 1120 1125 Leu Asn His Tyr Ala Phe Asp Leu Val Gln Glu Ala Pro Tyr Glu 1130 1135 1140 Ile Pro Glu Asn Asp Pro Pro Gln Asp Trp Pro Lys Tyr Gly Glu 1145 1150 1155 Ile Ile Phe Lys Asp Val Ser Met Arg Tyr Arg Pro Glu Leu Pro 1160 1165 1170 Phe Val Leu Lys Asn Ile Asn Leu Ser Ile Gly Lys Gly Glu Lys 1175 1180 1185 Ile Gly Phe Cys Gly Arg Thr Gly Ala Gly Lys Ser Thr Phe Met 1190 1195 1200 Thr Cys Leu Tyr Arg Ile Ser Glu Phe Glu Gly Thr Ile Val Ile 1205 1210 1215 Asp Asp Val Asp Ile Ser Lys Leu Gly Leu His Lys Leu Arg Ser 1220 1225 1230 Lys Leu Thr Ile Ile Pro Gln Asp Pro Val Leu Phe Val Gly Ser 1235 1240 1245 Ile Arg Glu Asn Leu Asp Pro Phe Gly Glu Tyr Ser Asp Glu Glu 1250 1255 1260 Leu Trp Glu Ala Leu Thr Ile Ser Gly Leu Ile Asn Lys Glu Asp 1265 1270 1275 Leu Asn Glu Val Lys Lys Gln Asn Glu Asn Asp Asp Asn Leu Asn 1280 1285 1290 Lys Phe His Leu Ile Arg Met Val Glu Asp Asp Gly Val Asn Phe 1295 1300 1305 Ser Ile Gly Glu Arg Gln Leu Ile Ala Leu Ala Arg Ala Leu Val 1310 1315 1320 Arg Lys Thr Lys Ile Leu Ile Leu Asp Glu Ala Thr Ser Ser Val 1325 1330 1335 Asp Tyr Ala Thr Asp Ser Arg Ile Gln Lys Thr Ile Ala Thr Glu 1340 1345 1350 Phe Asp Asp Cys Met Ile Leu Cys Ile Ala His Arg Leu Asn Thr 1355 1360 1365 Ile Leu Asn Tyr Asp Lys Ile Val Val Met Asp Lys Gly Glu Ile 1370 1375 1380 Val Glu Phe Asp Lys Pro Arg Ser Leu Phe Met Arg Glu Glu Gly 1385 1390 1395 Val Phe Arg Ser Met Cys Glu Gln Ala Asn Ile Thr Ile Glu Asp 1400 1405 1410 Phe Pro 1415 364134DNAArtificial sequenceALNQ_214_12000 36atgaagtctg acaacattgc tatggaagac ttgccagact ccaaatactt gaaacaacgt 60agattattga ccccattgat gtctaagaag gtcccaccaa ttccatctga agacgaaaga 120aaggcttacg gtgaatacta caccaaccca gtctctcgta tgatgttctg gtggttgaac 180ccaatcctaa aagtcggtta cagaagaact ttgactgaaa acgacttgtt ctacctagaa 240gatagacaaa gaactgaaac tttgtacgaa attttcagag gttacctaga cgaagaaatc 300gctagagctt ggaaaaagtc tcaagaatcc tctgacgacc caagagaatt caagttgcca 360atttacatta ttccattgtg tttgttcaag accatgaaat gggaatactc cagaggtatc 420ttgcaaaaga ttttgggtga ctgtgcttct gctaccaccc cattattgca gaaaaagctc 480atcaactttg tgcaagttaa gactttctct aacgtcggta acaccggtca aggtgttggt 540tacgctattg gtgtctgttt gatgatcttc ttccaagttc taatgttgac tcacgctttc 600cacaacttcc aaatctccgg tgctaaggct aaggctgttt tgaccagatt gttgttggac 660aagtctttga ctgtcgatgc tagaggtaac cactacttcc cagcctccaa gatccaatct 720atgatttcca ctgacttgaa cagaattgac ttggctgttg gtttcgctcc agtcggtttt 780gtcaccattt tcccaatcat tatctgtatt gctttgttga tttggaacgt cggtgtttct 840gctttggttg gtattggtgt tttcattgct aacattttcg ttttgggttt gttcgtttct 900tccttgatgt tgtacagaga aaaggccatg gttttcactg acaagagagt taacttggtt 960aaggaattgt tgaagaactt caaaatgatc aaattctact cctgggaaaa ctcttaccaa 1020gatcgtatcg aaaacgctag aaacaatgaa atgaagtaca tcttgagatt acaattgttg 1080agaaacttcg tcttctcttt agctttcgcc atgccagttt tggcttccat ggctaccttc 1140tgtactgctt tcaagatcac cgatggtaaa tccgctgcct ctgttttctc ctccttatct 1200ttgttcgaag tcttgtcttt acaattcatc ttggctccat tttccttgaa ctctactgtc 1260gacatgatgg tcagtgttaa gaagattaac caattcttgc aacacaagga cactaaccca 1320aacgaatttt ctgtcgaaaa gttttctgac tccactttgg ctatcaaggt cgataatgct 1380tctttcgaat gggacacctt tgaagatgaa gaaaaggact acgaagaaga agctaagact 1440aaggacaaca tcgaagatga ggaccataac tgtgccactg aaaccattaa gggtaagatc 1500actgtcgact acaagtctga ttctgattcc atctcttcta ccttgaccaa gggtgtcaag 1560accgctttcc caggtttgaa caacattaac ctagaaatcg ccaagggtga attcatcgtt 1620gtcaccggtg ccatcggttc tggtaagtct tctttgttgc aagccatctc tggtttaatg 1680aagagaactt ctggtgaagt ctacgtcgat ggtgacttgt tgttgtgtgg ttatccatgg 1740gttcaaaact ccactatcag agaaaacatc ttgttcggtt tgccattcaa caaggaaaga 1800tacgaccaag ttgtttactc ctgttctttg caatctgatt ttgatcaatt ccaaggtggt 1860gacatgaccg aagtcggtga aagaggtatc actttgtctg gtggtcaaaa agccagaatt 1920aacttagcta gatctgtcta tgctgacaag gatatcatct tattggatga tgtcttgtct 1980gctgttgacg ctaaagtcgg taagcacatc gtcaacacct gtattttggg tctattgggt 2040ggtaagacca gaattatggc tactcaccaa ttgtctttga ttgattccgc tgatagaatg 2100gttttcttga acggtgacgg taccattgac ttcggtacca tcccagaatt acgtaagaga 2160aaccaaaagt tgattgaatt gttacaacac caacgtgacc ctggtcaaga taaagaagat 2220ttgtctaacg acttggacat tcaaggttct actgatgaag gtcaacaaat cgaacacgct 2280gatgaacata aggaaatcgt taaaattatc ggtgatgagg aaaaggccgt taatgctttg 2340tctttccaag tttactacaa ctactgtaag ttggctttcg gtaaattggg ttacatttct 2400atgttggttt tcattatcgt cagctctttg gaaactttca cccaaatctt caccaacact 2460tggttgtctt tctggatcga agataagttc gtttccagat ctaagaactt ctacatgggt 2520atctacatca tgtttgcttt cttatacgct atcatgttgt gtttcttttt gttcttgttg 2580ggttacttct gtgttaaggc tgctgaaaga ttaaacatta aggcttctag aaagatcttg 2640cacgttccaa tgtccttcat ggacatttct ccaatcggtc gtgtcttaaa cagattcacc 2700aaggataccg atgtcttgga caacgaattg ttggaacaat tgatccaatt cttatctcca 2760ttgttcaact gtttcggtat catcatcttg tgtattgttt acatcccatg gttcgctatc 2820ggtgttccaa ttatcttggg tttctacttc atcatcgctt cttactacca agcctctgct 2880agagaaatca agagattgga agctgtcaag agatcctttg ttttcggtca tttccacgaa 2940gtccttactg gtaaggatac catcaaggct tacaacgcca ttgacagaat gaagttgaaa 3000ctaaacaagt tgatcgacga acaaaacgaa gcttactact tgactattgc taaccaaaga 3060tggttgggtg ctaacttggc tatcgtttct ttctctatgg ttttcgttat ttctttctta 3120tgtatcttca gagttttcaa catctccgcc gcttccactg gtttgttgtt aacctacgtt 3180atcgccttga ctgactctat caccatgatt atgagagcta tgacccaagt cgaaaacgaa 3240ttcaactccg tcgaacgtgt caaccactac gcttttgact tgatccaaga agccccatac 3300gaaatcccag aaaacgaccc agctgaagac tggccacaac acggtaagat cgaattcaag 3360gacgtttcca tgagatacag acctgaattg ccattcgttt tgaagaacat taacttgtcc 3420gtcagagaac aagagaaaat tggtttctgt ggtagaactg gtgccggtaa gtccactttc 3480atgacctgtt tgtacagaat cactgaatac gaaggtttga tctccatcga cggtgtcgat 3540atctctagat tgggtttgca cagattgaga tccaagttga ccattattcc tcaagaccca 3600gttctattcg ttggtaccat tagagaaaac ttggacccat tcaccgaaca ctccgatgac 3660gaattgtggg aagctttggc catttccggt ttgatcgaac gtgaagactt ggaagtcgtc 3720aagggtcaag aaaagattgg tggtaacgat tccggtaagt tgcacaagtt ccacttggtt 3780cgtatggttg aagacgatgg tatcaacttc tctttgggtg agagacaatt gattgctttg 3840gctagagcct tggttagaaa gtccaagatc ttaatcttgg acgaagctac ttcttccgtc 3900gactacgcta ctgattccaa gattcaaaga accattgcct ccgaattcag agactgtact 3960attttgtgta tcgcccatag attgaacacc atcttaggtt acgacaagat cgtcgttatg 4020gacaacggtg aaatcgttga atttgaaaac ccaaagttgt tgttcatgcg tgaaaactcc 4080gttttcagat ccatgtgtga acaagctaac attaccatca atgactttga ataa 4134374134DNAIssatchenkia orientalis 37atgaagtcag ataacattgc aatggaggac ctacccgact ccaaatactt gaaacagaga 60cgattattaa ctccgttgat gtcaaaaaaa gttccaccta ttccaagtga agatgagagg 120aaggcatacg gggaatatta tacgaatccg gtatcacgga tgatgttttg gtggttaaat 180cccattttga aggtgggata taggcgaaca ttgacggaga atgatctttt ctaccttgaa 240gacagacaac gtacagagac attatatgaa atatttcgtg gttacttgga tgaggaaatt 300gcacgtgcat ggaaaaaatc tcaagaaagc tcagatgatc caagagagtt caagcttccg 360atctatatta tccctttatg tttatttaag acaatgaaat gggaatacag ccggggaatt 420ctccagaaaa tcttgggtga ttgtgcttct gcaacgaccc cactattaca gaaaaaacta 480atcaactttg tccaggtcaa gactttcagt aatgtcggaa atactggtca gggtgtagga 540tatgctattg gcgtttgctt gatgattttc tttcaagttt taatgttaac tcatgcattt 600cacaatttcc aaatttcggg tgcaaaggca aaagctgttt tgacaagatt gctcttggat 660aaatcgttga ctgttgatgc cagaggcaac cattattttc cagcgagtaa gattcaaagc 720atgatttcta ctgatttgaa tagaatagac cttgctgtcg gatttgcacc agtggggttt 780gttactatat tccctattat tatctgcatt gccttattga tttggaatgt tggagtctcg 840gcattggttg gtattggggt attcattgca aacatttttg tgttgggact ttttgtatca 900agtctaatgt tatacaggga gaaggcgatg gtattcaccg acaaaagagt caatttggtt 960aaggaactat tgaaaaattt taaaatgatt aagttctaca gttgggaaaa ctcttatcag 1020gatagaattg aaaatgcaag gaacaacgaa atgaaatata ttttgaggtt acaattgcta 1080agaaattttg tgttttcgtt agcttttgcg atgcctgttt tggcatcaat ggctacattt 1140tgtacagcct ttaagataac tgacggcaaa agtgccgcat ctgtgttttc atctctctca 1200ttgtttgaag ttttatcgct acaatttatt ttggcccctt tctcactaaa ttctactgtg 1260gatatgatgg tttcagttaa aaagataaac cagtttctgc agcataaaga taccaatcca 1320aatgagttca gcgttgaaaa gttcagtgat agtacattgg caatcaaggt cgataatgca 1380tcatttgaat gggatacgtt tgaggatgaa gagaaagatt atgaagagga agctaaaact 1440aaagacaaca ttgaagatga agatcataat tgtgctacgg aaacaatcaa gggaaaaata 1500acagtggact ataagagcga cagtgactca atttctagta ctttaacgaa aggggttaaa 1560actgcatttc ctggactaaa taatatcaat cttgaaattg caaaagggga attcattgtt 1620gttactggtg caattggttc cggcaaatcc tctctacttc aggccatttc tgggctaatg 1680aagagaacat caggggaagt gtatgttgat ggtgatttgt tgttatgtgg ttacccgtgg 1740gtacaaaact caacaatacg agagaatata ctatttggat taccatttaa taaggagagg 1800tatgaccaag tcgtgtattc atgctcgttg cagagtgatt ttgatcaatt tcaaggaggt 1860gatatgacgg aagttggcga gagagggata accttatcag gaggccaaaa ggccagaatt 1920aatttagctc ggagtgttta tgctgataag gatattattt tattagacga cgttctcagt 1980gcggtcgacg ctaaagttgg caagcatatt gtgaatacat gcatcttagg attattggga 2040ggtaaaacaa gaatcatggc tacccaccaa ctaagtttga tcgactctgc tgatcgtatg 2100gtttttctca atggagatgg aactattgat tttggtacta ttcctgagct aagaaagaga 2160aaccagaagc tgattgagtt actacaacat caaagggatc caggtcaaga taaagaggat 2220ctctcaaatg atttggatat tcaaggaagc acagatgagg gtcagcaaat tgagcatgca 2280gacgagcata aagagatagt caagattatt ggtgacgagg aaaaagcagt taatgcattg 2340agtttccagg tttattataa ctattgtaag cttgcatttg gtaagcttgg atatatttcg 2400atgttggtat tcattattgt ttctagtttg gagaccttca cccaaatatt caccaatact 2460tggttgtcat tttggatcga ggataaattt gttagtagat ccaaaaattt ctatatggga 2520atatacatca tgtttgcatt tttgtatgca ataatgctat gtttttttct ctttttactg 2580ggatattttt gtgtaaaggc agcagagaga ctaaatatta aagcatccag gaagattcta 2640catgttccaa tgtcttttat ggacatatca cctattggta gagtcttgaa tcggtttact 2700aaagatacag atgtattaga caatgaattg ttagagcaat taattcaatt tttaagccca 2760ctattcaact gttttggtat tatcatattg tgcattgttt atataccatg gtttgctatt 2820ggtgtcccta taattcttgg attttatttt ataatagcca gttattatca ggccagtgca 2880agagaaatca agagattaga ggctgtaaag aggtcgtttg tttttggcca ttttcacgaa 2940gttctaacag ggaaagacac catcaaagca tataatgcta ttgatcggat gaaattgaag 3000ctgaacaaat tgattgatga acagaatgaa gcttattatc taacaatcgc caatcagaga 3060tggttaggag caaatttggc gattgtctca ttttcaatgg tttttgttat ttcgtttctg 3120tgtatcttta gggttttcaa cataagtgca gcatccactg gtttgctttt gacctacgtc 3180atagcactga cagattctat taccatgatt atgagagcaa tgactcaagt tgagaatgag 3240ttcaactctg tggaacgggt caaccattat gcatttgatc ttatacaaga agcaccatat 3300gaaataccgg agaatgatcc cgctgaggat tggccacaac atggcaaaat tgaattcaaa 3360gatgtgagta tgagatatag accggaacta ccatttgttt tgaaaaatat caatttgagt 3420gttagagaac aagaaaagat tggattttgt ggtagaacag gtgcaggtaa gtctacattt 3480atgacatgtc tatataggat aacagaatac gaaggcctta tatcaatcga tggtgttgat 3540attagccgat tagggttgca tagactacga tctaaattga ccattatacc gcaagacccg 3600gtattatttg ttggtaccat tagggagaac ctcgatccat ttacagagca ttctgatgat 3660gaattatggg aagcgcttgc gatatctgga ttgattgagc gggaagatct agaagtcgtc 3720aagggacaag agaagatagg tgggaatgat agtgggaagt tacacaagtt ccacctagtt 3780cgaatggttg aggatgatgg catcaatttt tcacttggtg agaggcagtt gattgcctta 3840gccagggcat tagttaggaa aagcaagata ttgatattgg atgaggctac atcgagtgtt 3900gactatgcta cagactctaa aatccagcga acgattgcta gtgagtttag ggattgtact 3960atattatgta ttgctcatcg attgaacacc attttaggtt atgataaaat tgtcgttatg 4020gacaatggtg agattgttga atttgagaat cccaaattgt tgtttatgag ggagaatagt 4080gtatttcgat ccatgtgtga gcaggcaaac atcaccatca atgattttga ataa 4134381377PRTIssatchenkia orientalis 38Met Lys Ser Asp Asn Ile Ala Met Glu Asp Leu Pro Asp Ser Lys Tyr 1 5 10 15 Leu Lys Gln Arg Arg Leu Leu Thr Pro Leu Met Ser Lys Lys Val Pro 20 25 30 Pro Ile Pro Ser Glu Asp Glu Arg Lys Ala Tyr Gly Glu Tyr Tyr Thr 35 40 45 Asn Pro Val Ser Arg Met Met Phe Trp Trp Leu Asn Pro Ile Leu Lys 50 55 60 Val Gly Tyr Arg Arg Thr Leu Thr Glu Asn Asp Leu Phe Tyr Leu Glu 65 70 75 80 Asp Arg Gln Arg Thr Glu Thr Leu Tyr Glu Ile Phe Arg Gly Tyr Leu 85 90 95 Asp Glu Glu Ile Ala Arg Ala Trp Lys Lys Ser Gln Glu Ser Ser Asp 100 105 110 Asp Pro Arg Glu Phe Lys Leu Pro Ile Tyr Ile Ile Pro Leu Cys Leu 115 120 125 Phe Lys Thr Met Lys Trp Glu Tyr Ser Arg Gly Ile Leu Gln Lys Ile 130 135 140 Leu Gly Asp Cys Ala Ser Ala Thr Thr Pro Leu Leu Gln Lys Lys Leu 145 150 155 160 Ile Asn Phe Val Gln Val Lys Thr Phe Ser Asn Val Gly Asn Thr Gly 165 170 175 Gln Gly Val Gly Tyr Ala Ile Gly Val Cys Leu Met Ile Phe Phe Gln 180 185 190 Val Leu Met Leu Thr His Ala Phe His Asn Phe Gln Ile Ser Gly Ala 195 200 205 Lys Ala Lys Ala Val Leu Thr Arg Leu Leu Leu Asp Lys

Ser Leu Thr 210 215 220 Val Asp Ala Arg Gly Asn His Tyr Phe Pro Ala Ser Lys Ile Gln Ser 225 230 235 240 Met Ile Ser Thr Asp Leu Asn Arg Ile Asp Leu Ala Val Gly Phe Ala 245 250 255 Pro Val Gly Phe Val Thr Ile Phe Pro Ile Ile Ile Cys Ile Ala Leu 260 265 270 Leu Ile Trp Asn Val Gly Val Ser Ala Leu Val Gly Ile Gly Val Phe 275 280 285 Ile Ala Asn Ile Phe Val Leu Gly Leu Phe Val Ser Ser Leu Met Leu 290 295 300 Tyr Arg Glu Lys Ala Met Val Phe Thr Asp Lys Arg Val Asn Leu Val 305 310 315 320 Lys Glu Leu Leu Lys Asn Phe Lys Met Ile Lys Phe Tyr Ser Trp Glu 325 330 335 Asn Ser Tyr Gln Asp Arg Ile Glu Asn Ala Arg Asn Asn Glu Met Lys 340 345 350 Tyr Ile Leu Arg Leu Gln Leu Leu Arg Asn Phe Val Phe Ser Leu Ala 355 360 365 Phe Ala Met Pro Val Leu Ala Ser Met Ala Thr Phe Cys Thr Ala Phe 370 375 380 Lys Ile Thr Asp Gly Lys Ser Ala Ala Ser Val Phe Ser Ser Leu Ser 385 390 395 400 Leu Phe Glu Val Leu Ser Leu Gln Phe Ile Leu Ala Pro Phe Ser Leu 405 410 415 Asn Ser Thr Val Asp Met Met Val Ser Val Lys Lys Ile Asn Gln Phe 420 425 430 Leu Gln His Lys Asp Thr Asn Pro Asn Glu Phe Ser Val Glu Lys Phe 435 440 445 Ser Asp Ser Thr Leu Ala Ile Lys Val Asp Asn Ala Ser Phe Glu Trp 450 455 460 Asp Thr Phe Glu Asp Glu Glu Lys Asp Tyr Glu Glu Glu Ala Lys Thr 465 470 475 480 Lys Asp Asn Ile Glu Asp Glu Asp His Asn Cys Ala Thr Glu Thr Ile 485 490 495 Lys Gly Lys Ile Thr Val Asp Tyr Lys Ser Asp Ser Asp Ser Ile Ser 500 505 510 Ser Thr Leu Thr Lys Gly Val Lys Thr Ala Phe Pro Gly Leu Asn Asn 515 520 525 Ile Asn Leu Glu Ile Ala Lys Gly Glu Phe Ile Val Val Thr Gly Ala 530 535 540 Ile Gly Ser Gly Lys Ser Ser Leu Leu Gln Ala Ile Ser Gly Leu Met 545 550 555 560 Lys Arg Thr Ser Gly Glu Val Tyr Val Asp Gly Asp Leu Leu Leu Cys 565 570 575 Gly Tyr Pro Trp Val Gln Asn Ser Thr Ile Arg Glu Asn Ile Leu Phe 580 585 590 Gly Leu Pro Phe Asn Lys Glu Arg Tyr Asp Gln Val Val Tyr Ser Cys 595 600 605 Ser Leu Gln Ser Asp Phe Asp Gln Phe Gln Gly Gly Asp Met Thr Glu 610 615 620 Val Gly Glu Arg Gly Ile Thr Leu Ser Gly Gly Gln Lys Ala Arg Ile 625 630 635 640 Asn Leu Ala Arg Ser Val Tyr Ala Asp Lys Asp Ile Ile Leu Leu Asp 645 650 655 Asp Val Leu Ser Ala Val Asp Ala Lys Val Gly Lys His Ile Val Asn 660 665 670 Thr Cys Ile Leu Gly Leu Leu Gly Gly Lys Thr Arg Ile Met Ala Thr 675 680 685 His Gln Leu Ser Leu Ile Asp Ser Ala Asp Arg Met Val Phe Leu Asn 690 695 700 Gly Asp Gly Thr Ile Asp Phe Gly Thr Ile Pro Glu Leu Arg Lys Arg 705 710 715 720 Asn Gln Lys Leu Ile Glu Leu Leu Gln His Gln Arg Asp Pro Gly Gln 725 730 735 Asp Lys Glu Asp Leu Ser Asn Asp Leu Asp Ile Gln Gly Ser Thr Asp 740 745 750 Glu Gly Gln Gln Ile Glu His Ala Asp Glu His Lys Glu Ile Val Lys 755 760 765 Ile Ile Gly Asp Glu Glu Lys Ala Val Asn Ala Leu Ser Phe Gln Val 770 775 780 Tyr Tyr Asn Tyr Cys Lys Leu Ala Phe Gly Lys Leu Gly Tyr Ile Ser 785 790 795 800 Met Leu Val Phe Ile Ile Val Ser Ser Leu Glu Thr Phe Thr Gln Ile 805 810 815 Phe Thr Asn Thr Trp Leu Ser Phe Trp Ile Glu Asp Lys Phe Val Ser 820 825 830 Arg Ser Lys Asn Phe Tyr Met Gly Ile Tyr Ile Met Phe Ala Phe Leu 835 840 845 Tyr Ala Ile Met Leu Cys Phe Phe Leu Phe Leu Leu Gly Tyr Phe Cys 850 855 860 Val Lys Ala Ala Glu Arg Leu Asn Ile Lys Ala Ser Arg Lys Ile Leu 865 870 875 880 His Val Pro Met Ser Phe Met Asp Ile Ser Pro Ile Gly Arg Val Leu 885 890 895 Asn Arg Phe Thr Lys Asp Thr Asp Val Leu Asp Asn Glu Leu Leu Glu 900 905 910 Gln Leu Ile Gln Phe Leu Ser Pro Leu Phe Asn Cys Phe Gly Ile Ile 915 920 925 Ile Leu Cys Ile Val Tyr Ile Pro Trp Phe Ala Ile Gly Val Pro Ile 930 935 940 Ile Leu Gly Phe Tyr Phe Ile Ile Ala Ser Tyr Tyr Gln Ala Ser Ala 945 950 955 960 Arg Glu Ile Lys Arg Leu Glu Ala Val Lys Arg Ser Phe Val Phe Gly 965 970 975 His Phe His Glu Val Leu Thr Gly Lys Asp Thr Ile Lys Ala Tyr Asn 980 985 990 Ala Ile Asp Arg Met Lys Leu Lys Leu Asn Lys Leu Ile Asp Glu Gln 995 1000 1005 Asn Glu Ala Tyr Tyr Leu Thr Ile Ala Asn Gln Arg Trp Leu Gly 1010 1015 1020 Ala Asn Leu Ala Ile Val Ser Phe Ser Met Val Phe Val Ile Ser 1025 1030 1035 Phe Leu Cys Ile Phe Arg Val Phe Asn Ile Ser Ala Ala Ser Thr 1040 1045 1050 Gly Leu Leu Leu Thr Tyr Val Ile Ala Leu Thr Asp Ser Ile Thr 1055 1060 1065 Met Ile Met Arg Ala Met Thr Gln Val Glu Asn Glu Phe Asn Ser 1070 1075 1080 Val Glu Arg Val Asn His Tyr Ala Phe Asp Leu Ile Gln Glu Ala 1085 1090 1095 Pro Tyr Glu Ile Pro Glu Asn Asp Pro Ala Glu Asp Trp Pro Gln 1100 1105 1110 His Gly Lys Ile Glu Phe Lys Asp Val Ser Met Arg Tyr Arg Pro 1115 1120 1125 Glu Leu Pro Phe Val Leu Lys Asn Ile Asn Leu Ser Val Arg Glu 1130 1135 1140 Gln Glu Lys Ile Gly Phe Cys Gly Arg Thr Gly Ala Gly Lys Ser 1145 1150 1155 Thr Phe Met Thr Cys Leu Tyr Arg Ile Thr Glu Tyr Glu Gly Leu 1160 1165 1170 Ile Ser Ile Asp Gly Val Asp Ile Ser Arg Leu Gly Leu His Arg 1175 1180 1185 Leu Arg Ser Lys Leu Thr Ile Ile Pro Gln Asp Pro Val Leu Phe 1190 1195 1200 Val Gly Thr Ile Arg Glu Asn Leu Asp Pro Phe Thr Glu His Ser 1205 1210 1215 Asp Asp Glu Leu Trp Glu Ala Leu Ala Ile Ser Gly Leu Ile Glu 1220 1225 1230 Arg Glu Asp Leu Glu Val Val Lys Gly Gln Glu Lys Ile Gly Gly 1235 1240 1245 Asn Asp Ser Gly Lys Leu His Lys Phe His Leu Val Arg Met Val 1250 1255 1260 Glu Asp Asp Gly Ile Asn Phe Ser Leu Gly Glu Arg Gln Leu Ile 1265 1270 1275 Ala Leu Ala Arg Ala Leu Val Arg Lys Ser Lys Ile Leu Ile Leu 1280 1285 1290 Asp Glu Ala Thr Ser Ser Val Asp Tyr Ala Thr Asp Ser Lys Ile 1295 1300 1305 Gln Arg Thr Ile Ala Ser Glu Phe Arg Asp Cys Thr Ile Leu Cys 1310 1315 1320 Ile Ala His Arg Leu Asn Thr Ile Leu Gly Tyr Asp Lys Ile Val 1325 1330 1335 Val Met Asp Asn Gly Glu Ile Val Glu Phe Glu Asn Pro Lys Leu 1340 1345 1350 Leu Phe Met Arg Glu Asn Ser Val Phe Arg Ser Met Cys Glu Gln 1355 1360 1365 Ala Asn Ile Thr Ile Asn Asp Phe Glu 1370 1375

Claims

1. A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

2. A recombinant host capable of producing a steviol glycoside which has been modified, optionally in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

3. The recombinant host according to claim 1, which comprises a recombinant nucleic acid encoding a polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

4. The recombinant host according to claim 1, which further 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.

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

6. The recombinant host according to claim 1, which further 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.

7. The 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.

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

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

10. The recombinant host according to claim 1, which further 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.

11. A recombinant host capable of producing a steviol glycoside which overexpresses a heterologous polypeptide which mediates steviol glycoside transport.

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

13. The process according to claim 12 for the preparation of a steviol glycoside, wherein the process is carried out on an industrial scale.

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

15. A steviol glycoside obtained by the process according to claim 12.

16. A composition obtainable by the process according to claim 12.

17. A foodstuff, feed or beverage which comprises the steviol glycoside according to claim 15.