US 20160213039A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0213039 A1 KUMAR et al. (43) Pub. Date: Jul. 28, 2016

(54) STEVIOL GLYCOSIDES A23G 9/34 (2006.01) A618/24 (2006.01) (71) Applicant: DSM IPASSETS B.V., Heerlen (NL) A68/73 (2006.01) A618/34 (2006.01) (72) Inventors: Manoj KUMAR, Echt (NL); Marieke A618/86 (2006.01) NIJMEIJER, Echt (NL) A61K 8/44 (2006.01) A68/60 (2006.01) (73) Assignee: DSM IPASSETS B.V., Heerlen (NL) A2.3L 2/60 (2006.01) A61O 11/00 (2006.01) (21) Appl. No.: 14/908,146 (52) U.S. Cl. 22) PCT Fled:1-1. Jul. 31, 2014 CPC ...... A23L I/2363 (2013.01);s A23L 2/60 (22) 1. l. 3, (2013.01); A23L 2/02 (2013.01); A2ID 13/062 (86). PCT No.: PCT/EP2014/066SSS (2013.01); A2ID 2/36 (2013.01); A23G 1/40 (2013.01); A23G 9/34 (2013.01); A23L I/238 S371 (c)(1), (2013.01); A61O II/00 (2013.01); A61K 8/24 (2) Date: Jan. 28, 2016 (2013.01); A61K 8/73 (2013.01); A61K 8/731 O O (2013.01); A61K 8/345 (2013.01); A61K 8/738 Related U.S. Application Data (2013.01); A61K 8/86 (2013.01); A61K 8/442 (63) Continuation of application No. 13/956,226, filed on (2013.01); A61K 8/602 (2013.01); A23V Jul. 31, 2013, now abandoned. 2002/00 (2013.01); A61 K 2800/59 (2013.01) Publication Classification (57) ABSTRACT (51) Int. Cl. The present invention relates to a product which is a foodstuff, A2.3L I/236 (2006.01) beverage, pharmaceutical composition, tobacco, nutraceuti A2.3L 2/02 (2006.01) cal, oral hygiene composition or cosmetic comprising a A2ID I3/06 (2006.01) Sweetener composition, wherein the Sweetener composition A2ID 2/36 (2006.01) comprises one or more fermentatively-produced Steviol gly A23G L/40 (2006.01) cosides. Patent Application Publication Jul. 28, 2016 Sheet 1 of 11 US 2016/0213039 A1

Not Hind Pst Sai

Nde ECOR

pUG7-EcoRV kanMX 3982 bps ind

Fig. 1 Patent Application Publication Jul. 28, 2016 Sheet 2 of 11 US 2016/0213039 A1

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PCR:

Transformation

-5-YPRcTau3r- ERG20,or- orthmG1 > s:------g. KAN is Bisis 3'-YPRcTau3

x Cre recombinase

r-r-r-r

Fig. 2 Patent Application Publication Jul. 28, 2016 Sheet 3 of 11 US 2016/0213039 A1

A g SO bp i -- - ERG9 . < CTF8

recy TRP1 > rap - a CTF8 ----- 98 bp

Transformation

B - ERG9 > pe TRP repH < CTF8 -

Fig. 3 Patent Application Publication Jul. 28, 2016 Sheet 4 of 11 US 2016/0213039 A1

RA28 UGT2.1a

s

s-chro5.0 3 NAT-T cra 3'-ch:89.01.

- s-chroea usta ass NATs cRE - is 3-chro90

x Cre reconnairase

w 5'-Chr09,81 ug2.1a :- 2 3'-C39,

Fig. 4 Patent Application Publication Jul. 28, 2016 Sheet 5 of 11 US 2016/0213039 A1

GGPP - RebA A NA2.0: KOs. UGTGT1 .

momen B

Fig. 5 Patent Application Publication Jul. 28, 2016 Sheet 6 of 11 US 2016/0213039 A1

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Fig. 6 Patent Application Publication Jul. 28, 2016 Sheet 7 of 11 US 2016/0213039 A1

Fig. 7 Patent Application Publication Jul. 28, 2016 Sheet 8 of 11 US 2016/0213039 A1

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Fig. 8 Patent Application Publication Jul. 28, 2016 Sheet 9 of 11 US 2016/0213039 A1

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Fig. 9 Patent Application Publication Jul. 28, 2016 Sheet 10 of 11 US 2016/0213039 A1

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Fig. 10 Patent Application Publication Jul. 28, 2016 Sheet 11 of 11 US 2016/0213039 A1

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Fig. 11 US 2016/0213039 A1 Jul. 28, 2016

STEVOL, GLYCOSDES may be further processed to remove spent fermentation medium components by down-stream purification processes. FIELD OF THE INVENTION 0010. In contrast to plant-extracted Steviol glycosides, fer 0001. The present invention relates to products compris mentatively-produced rebA may readily be produced in a ing a Sweetener composition and to a method for the prepa highly purified grade and in a form which has less residual ration of such products. The invention further relates to the bitterness and aftertaste. use of Sweetener compositions in the preparation of the prod 0011. Accordingly, the invention relates to a process of ucts and to a Steviol-glycoside containing composition. fermentative production of high purity rebA. The process is useful for producing high purity rebA with a product speci BACKGROUND TO THE INVENTION fication of at least about 95% rebA (dry weight basis). 0002. The use of artificial sweeteners such as dulcin, 0012. This product specification of fermentatively-pro Sodium cyclamate, aspartame, acylsulfame and saccharin has duced rebA is useful as a non-caloric Sweetener in various been seeing lower consumer demand for Some time. How food and beverage compositions as well as being useful in ever, non-caloric high potency Sweeteners with natural origin combination with other caloric and non-caloric Sweeteners. Such as Steviol glycosides have been receiving increasing 0013 This fermentatively-produced reb-A is useful as a demand. In addition, the Sweet Steviol glycosides have func non-caloric Sweetener in edible and chewable compositions tional and sensory properties Superior to those of many high Such as any beverages, confectioneries, bakeries, cookies, potency Sweeteners. chewing gums, and alike. 0003 Steviol glycosides (Masaya Ohta et al., J. Appl. Gly 0014. An object of the present invention is thus to provide cosci, V 57, (2010), pages 199-209) are a group of different a commercially valuable fermentative production process for Sweet diterpene glycosides, which have a single base—ste producing a highly purified Sweetener with known product viol diterpene and differ by the presence of carbohydrate specifications from a microbial production system and its use residues at positions C13 and C19. in various food products and beverages, overcoming the dis 0004. The physical and sensory properties of Rebaudio advantages of known plant extracted Steviol glycosides. side A and a number of other steviol glycosides have been 0015. In particular, fermentatively-produced rebA may be studied (Caroline Hellfritschet. al. J. Agric Food Chem, v60, (2012) pages 6782-6793). They have been tested for stability used to enhance citrus or sour attributes, total aroma impact, in carbonated beverages and found to be both heat and pH sweet aromatic complex, ethyl maltol (strawberry flavor) or stable (Chang and Cook, 1983). The Sweetness potency of brown fruit (flavor/aroma). Reb-A is between 200-400 times higher than sucrose, 0016. Herein is described a fermentation process where 0005. However, apart from its high level of sweetness, Steviol glycosides are produced via microbial fermentation. they have also intrinsic properties of post-bitter taste and Also described is a down-stream purification and recovery metallic and liquorish aftertaste. Some undesirable taste char process for recovery of fermentatively-produced steviol gly acteristics of Steviol glycosides as a result of contamination of cosides along with a highly-purified rebA of a given product other Substances such as polyphenolics and other flavor com specification. pound present in Stevia plant extracts. There are thus limita 0017. The fermentatively produced Steviol glycoside (of tions associated with the use of plant-derived Steviol glyco specific product specification) may be applied in various side extracts. foods and beverages as a Sweetener. That is to say, the present invention is based on fermentatively-produced Steviol glyco SUMMARY OF THE INVENTION sides Suitable for various food and beverage applications. 0006. One of the main ways to improve the taste quality is 0018. According to the invention, there is thus provided a the fermentative production of steviol glycosides. Another fermentatively derived product which is a foodstuff, bever way to produce highly purified individual glycosides with age, pharmaceutical composition, tobacco, nutraceutical, desired clean taste characteristics and minimal content of oral hygiene composition or cosmetic comprising a Sweet accompanying compounds is to tailor make specific Steviol ener composition, wherein the Sweetener composition com glycosides via designed biosynthesis in a fermentation pro prises one or more fermentatively-produced Steviol glyco duction host and fermentation thereof. sides. 0007 Accordingly, the invention is related to processes 0019. The invention also provides a method for the prepa for microbial production of steviol glycosides by fermenta ration of a product which is a foodstuff, beverage, pharma tion and to fermentatively-produced Steviol glycosides, such ceutical composition, tobacco, nutraceutical, oral hygiene as rebaudioside A (rebA), of certain product specification and composition or cosmetic comprising a Sweetener composi use thereof. That is to say, the present invention relates to a tion, which method comprises preparing a said product and microbial fermentation process for producing a highly puri incorporating a Sweetener composition comprising one or fied rebA and to its use thereof in various food products and more fermentatively-produced Steviol glycosides. Such a beverages. method may also comprise fermenting a microorganism as 0008. A process for the fermentative production and herein described and recovering rebaudioside A from the recovery of diterpene glycosides, from microbial fermenta microorganism and/or extracellular medium. tion broth leading to a product specification is disclosed. In 0020. Further provided by the invention is use of a sweet particular, a method for the recovery of rebA from microbial ener composition comprising one or more fermentatively fermentation broth is described. Individual sweet glycosides produced Steviol glycosides in the preparation of a foodstuff. can be obtained from a microbial fermentation process. beverage, pharmaceutical composition, tobacco, nutraceuti 0009. A mixture of sweet steviol glycosides can also be cal, oral hygiene composition or cosmetic comprising a obtained from a designed microbial fermentation process and Sweetener composition. US 2016/0213039 A1 Jul. 28, 2016

0021. Also provided by the invention is a composition description and specific examples, while indicating preferred which comprises, on a dry solids basis, at least about 95% embodiments of the invention, are given by way of illustration fermentatively-produced Rebaudioside A. only, since various changes and modifications within the spirit and scope of the invention will become apparent to BRIEF DESCRIPTION OF THE DRAWINGS those skilled in the art from this detailed description. 0035. Throughout the present specification and the 0022 FIG. 1 sets out a schematic representation of the accompanying claims, the words "comprise'.99 &“include’ and plasmid puG7-EcoRV. "having and variations such as "comprises', 'comprising. 0023 FIG. 2 sets out a schematic representation of the “includes and “including are to be interpreted inclusively. method by which the ERG20, thMG1 and BTS1 over-expres That is, these words are intended to convey the possible sion cassettes are designed (A) and integrated (B) into the inclusion of other elements or integers not specifically yeast genome. (C) shows the final situation after removal of recited, where the context allows. the KANMX marker by the Cre recombinase. 0036. The invention relates to products comprising sweet 0024 FIG. 3 sets out a schematic representation of the ener compositions. The Sweetener compositions comprise ERG9 knock down construct. This consists of a 500 bp long one or more Steviol glycosides, one or more of which is 3' part of ERG9,98bp of the TRP1 promoter, the TRP1 open prepared fermentatively. reading frame and terminator, followed by a 400 bp long 0037. The invention thus provides a solution comprising downstream sequence of ERG9. Due to introduction of a one or more Steviol glycosides. Such a solution may comprise Xbal site at the end of the ERG9 open reading frame the last rebaudioside A. amino acid changes into Ser and the stop codon into Arg. A 0038. Such a solution may comprise, on a dry solids basis, new stop codon is located in the TPR1 promoter, resulting in at least about 60%, at least about 70%, at least about 80%, at an extension of 18 amino acids. least about 90%, at least about 95%, at least about 99% weight 0025 FIG. 4 sets out a schematic representation of how of Rebaudioside A. UGT2 is integrated into the genome. A. different fragments 0039. Accordingly, the invention provides a composition used in transformation; B. situation after integration; C. situ which may comprise, on a dry solids basis, at least about 60%, ation after expression of Cre recombinase. at least about 70%, at least about 80%, at least about 90%, at 0026 FIG. 5 sets out a schematic representation of how least about 95%, at least about 99% weight offermentatively the pathway from GGPP to RebA is integrated into the produced Rebaudioside A. genome. A. different fragments used in transformation; B. 0040. Such a composition may be a granulate or powder. situation after integration. Such a solid composition may comprise at least about 60%, at 0027 FIG. 6 sets out a schematic diagram of the potential least about 70%, at least about 80%, at least about 90%, at pathways leading to biosynthesis of Steviol glycosides. least about 95%, at least about 99% by weight of fermenta 0028 FIG. 7 sets out a process for recovery of steviol tively-produced Rebaudioside A. glycosides from a fermentation broth. 0041. Such solutions and compositions may be prepared 0029 FIG. 8 shows sweetness intensity score per applica by fermentation of a recombinant microorganism that is tion (acidified water, near water, juice and cola), fermentative capable of producing a Steviol glycoside. Suitable recombi Reb A versus Plant based Reb A. nant microorganisms are described herein below. Such a 0030 FIG. 9 shows the intensity scores for fermentative recombinant microorganism may comprise one or more Reb A versus Plant based Reb Ain acidified water nucleotide sequence(s) encoding: 0031 FIG. 10 shows the intensity scores fermentative Reb 0042 a polypeptide having ent-copalyl pyrophosphate A versus Plant based RebA in near water. synthase activity; 0032 FIG. 11 shows the intensity scores fermentative Reb 0.043 a polypeptide havingent-Kaurene synthase activ A versus Plant based Reb A in juice. ity; 0044 a polypeptide having ent-Kaurene oxidase activ DESCRIPTION OF THE SEQUENCE LISTING ity; 0.045 a polypeptide having kaurenoic acid 13-hydroxy 0033. A description of the sequences is set out in Table 1. lase activity; and Sequences described herein may be defined with reference to 0046 one or more polypeptides having UDP-glucosyl the sequence listing or with reference to the database acces transferase (UGT) activity, sion numbers also set out in Table 1. 0047 whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least DETAILED DESCRIPTION OF THE INVENTION one Steviol glycoside. 0034 Advantages of the present invention will become 0048 For the purposes of this invention, a polypeptide more apparent from the detailed description given hereinaf having ent-copalyl pyrophosphate synthase (EC 5.5.1.13) is ter. However, it should be understood that the detailed capable of catalyzing the chemical reaction:

O O O O

S. S S. Sn | O-P-O-P-O O O O O US 2016/0213039 A1 Jul. 28, 2016

0049. This enzyme has one substrate, geranylgeranyl (also known as the glycosyl donor) to a glycosyl acceptor pyrophosphate, and one product, ent-copalyl pyrophosphate. molecule, usually an alcohol. The glycosyl donor for a UGT This enzyme participates in gibberellin biosynthesis. This is typically the nucleotide Sugar uridine diphosphate glucose enzyme belongs to the family of isomerase, specifically the (uracil-diphosphate glucose, UDP-glucose). class of intramolecular lyases. The systematic name of this 0060. The UGTs used may be selected so as to produce a enzyme class is ent-copalyl-diphosphate lyase (decyclizing). desired diterpene glycoside, Such as a Steviol glycoside. Sche Other names in common use include havingent-copalyl pyro matic diagrams of Steviol glycoside formation are set out in phosphate synthase, ent-kaurene synthase A, and ent-kaurene Humphrey et al., Plant Molecular Biology (2006) 61: 47-62 synthetase A. and Mohamed et al., J. Plant Physiology 168 (2011) 1136 0050 For the purposes of this invention, a polypeptide 1141. In addition, FIG. 6 sets out a schematic diagram of having ent-kaurene synthase activity (EC 4.2.3.19) is a Steviol glycoside formation. polypeptide that is capable of catalyzing the chemical reac 0061 The biosynthesis of rebaudioside A involves gluco tion: sylation of the aglycone steviol. Specifically, rebaudioside A 0051 ent-copalyl diphosphateve ent-kaurene+diphos can beformed by glucosylation of the 13-OH of steviol which phate forms the 13-O-Steviolmonoside, glucosylation of the C-2" of 0052 Hence, this enzyme has one substrate, ent-copalyl the 13-O-glucose of Steviolmonoside which forms steviol-1, diphosphate, and two products, ent-kaurene and diphosphate. 2-bioside, glucosylation of the C-19 carboxyl of steviol-1,2- 0053. This enzyme belongs to the family of lyases, spe bioside which forms stevioside, and glucosylation of the C-3' cifically those carbon-oxygen lyases acting on phosphates. of the C-13-O-glucose of stevioside. The order in which each The systematic name of this enzyme class is ent-copalyl glucosylation reaction occurs can vary—see FIG. 6. One diphosphate diphosphate-lyase (cyclizing, ent-kaurene UGT may be capable of catalyzing more than one conversion forming). Other names in common use include ent-kaurene as set out in this scheme. synthase B, ent-kaurene synthetase B, ent-copalyl-diphos phate diphosphate-lyase, and (cyclizing). This enzyme par 0062 Conversion of steviol to rebaudioside A or rebau ticipates in diterpenoid biosynthesis. dioside D may be accomplished in a recombinant host by the 0054 ent-copalyl diphosphate synthases may also have a expression of gene(s) encoding the following functional distinct ent-kaurene synthase activity associated with the UGTs: UGT74G1, UGT85C2, UGT76G1 and UGT2. Thus, a same protein molecule. The reaction catalyzed by ent-kau recombinant microorganism expressing these four UGTs can rene synthase is the next step in the biosynthetic pathway to make rebaudioside A if it produces steviol or when fed steviol gibberellins. The two types of enzymic activity are distinct, in the medium. Typically, one or more of these genes are and site-directed mutagenesis to Suppress the ent-kaurene recombinant genes that have been transformed into a micro synthase activity of the protein leads to build up of ent organism that does not naturally possess them. Examples of copalyl pyrophosphate. all of these enzmyes are set out in Table 1. A recombinant 0055 Accordingly, a single nucleotide sequence may microorganism may comprise any combination of a encode a polypeptide having ent-copalyl pyrophosphate Syn UGT74G1, UGT85C2, UGT76G1 and UGT2. In Table 1 thase activity and ent-kaurene synthase activity. Alterna UGT64G1 sequences are indicated as UGT1 sequences, tively, the two activities may be encoded two distinct, sepa UGT74G1 sequences are indicated as UGT3 sequences and rate nucleotide sequences. UGT76G1 sequences are indicated as UGT4 sequences. 0056. For the purposes of this invention, a polypeptide UGT2 sequences are indicated as UGT2 sequences in Table 1. having ent-kaurene oxidase activity (EC 1.14.13.78) is a 0063 A recombinant microorganism which comprises a polypeptide which is capable of catalysing three Successive nucleotide sequence encoding a polypeptide having UGT oxidations of the 4-methyl group of ent-kaurene to give kau activity may comprise a nucleotide sequence encoding a renoic acid. Such activity typically requires the presence of a polypeptide capable of catalyzing the addition of a C-13 cytochrome P450. glucose to Steviol. That is to say, a recombinant microorgan 0057 For the purposes of the invention, a polypeptide ism may comprise a UGT which is capable of catalyzing a having kaurenoic acid 13-hydroxylase activity (EC 1.14.13) reaction in which steviol is converted to steviolmonoside. is one which is capable of catalyzing the formation of Steviol Accordingly, expression of Such a nucleotide sequence may (ent-kaur-16-en-13-ol-19-oic acid) using NADPH and O. confer on the microorganism the ability to produce at least Such activity may also be referred to as ent-ka 13-hydroxy Steviolmonoside. lase activity. 0064. Such a microorganism may comprise a nucleotide 0058. A recombinant microorganism which may be fer sequence encoding a polypeptide having the activity shown mented to produce a fermentation broth for use in the process by UDP-glycosyltransferase (UGT) UGT85C2, whereby the of the invention comprises one or more nucleotide sequences nucleotide sequence upon transformation of the microorgan encoding a polypeptide having UDP-glucosyltransferase ism confers on the cell the ability to convert Steviol to stevio (UGT) activity, whereby expression of the nucleotide lmonoside. sequence(s) confer(s) on the microorganism the ability to 0065. UGT85C2activity is transfer of a glucose unit to the produce at least one of Steviolmonoside, Steviolbioside, 13-OH of steviol. Thus, a suitable UGT85C2 may function as Stevioside or rebaudioside A, rebaudioside B, rebaudioside C, a uridine 5'-diphospho glucosyl: Steviol 13-OH transferase, rebaudioside D, rebaudioside E, rebaudioside F, rubusoside, and a uridine 5'-diphosphoglucosyl: Steviol-19-0-glucoside dulcoside A or rebaudioside M. 13-OH transferase. A functional UGT85C2 polypeptides 0059 For the purposes of this invention, a polypeptide may also catalyze glucosyl transferase reactions that utilize having UGT activity is one which has glycosyltransferase Steviol glycoside substrates other than steviol and steviol-19 activity (EC 2.4), i.e. that can act as a catalyst for the transfer O-glucoside. Such sequences are indicated as UGT1 of a monosaccharide unit from an activated nucleotide Sugar sequences in Table 1. US 2016/0213039 A1 Jul. 28, 2016

0066. A recombinant microorganism which comprises a a Suitable recombinant microorganism may comprise a UGT nucleotide sequence encoding a polypeptide having UGT which is capable of catalyzing a reaction in which Steviolbio activity may comprise a nucleotide sequence encoding a side is converted to Stevioside. Accordingly, Such a microor polypeptide capable of catalyzing the addition of a C-13 ganism may be capable of converting Steviolbioside to Stevio glucose to Steviol or Steviolmonoside. That is to say, a recom side. Expression of such a nucleotide sequence may confer on binant microorganism may comprise a UGT which is capable the microorganism the ability to produce at least Stevioside. of catalyzing a reaction in which Steviolmonoside is con 0073. A suitable recombinant microorganism may also Verted to Steviolbioside. Accordingly, such a microorganism comprise a nucleotide sequence encoding a polypeptide hav may be capable of converting Steviolmonoside to Steviolbio ing the activity shown by UDP-glycosyltransferase (UGT) side. Expression of such a nucleotide sequence may confer on UGT74G1, whereby the nucleotide sequence upon transfor the microorganism the ability to produce at least Steviolbio mation of the microorganism confers on the cell the ability to side. convert Steviolbioside to stevioside. 0067. A suitable recombinant microorganism may also (0074) Suitable UGT74G1 polypeptides may be capable of comprise a nucleotide sequence encoding a polypeptide hav transferring a glucose unit to the 13-OH or the 19-000H. ing the activity shown by UDP-glycosyltransferase (UGT) respectively, of steviol. A suitable UGT74G1 polypeptide UGT74G1, whereby the nucleotide sequence upon transfor may function as a uridine 5'-diphospho glucosyl: Steviol mation of the microorganism confers on the cell the ability to 19-000H transferase and a uridine 5'-diphospho glucosyl: convert Steviolmonoside to steviolbioside. Steviol-13-O-glucoside 19-000H transferase. Functional 0068 A suitable recombinant microorganism may also UGT74G1 polypeptides also may catalyze glycosyl trans comprise a nucleotide sequence encoding a polypeptide hav ferase reactions that utilize Steviol glycoside substrates other ing the activity shown by UDP-glycosyltransferase (UGT) than Stevioland Steviol-13-O-glucoside, or that transfer Sugar UGT2, whereby the nucleotide sequence upon transforma moieties from donors other than uridine diphosphate glucose. tion of the microorganism confers on the cell the ability to Such sequences are indicated as UGT1 sequences in Table 3. convert Steviolmonoside to steviolbioside. 0075 A recombinant microorganism which comprises a 0069. A suitable UGT2 polypeptide functions as a uridine nucleotide sequence encoding a polypeptide having UGT 5'-diphospho glucosyl: Steviol-13-O-glucoside transferase activity may comprise a nucleotide sequence encoding a (also referred to as a Steviol-13-monoglucoside 1.2-glucosy polypeptide capable of catalyzing glucosylation of the C-3' of lase), transferring a glucose moiety to the C-2 of the 13-0- the glucose at the C-13 position of stevioside. That is to say, glucose of the acceptor molecule, Steviol-13-O-glucoside. a recombinant microorganism may comprise a UGT which is Typically, a suitable UGT2 polypeptide also functions as a capable of catalyzing a reaction in which Stevioside to rebau uridine 5'-diphosphoglucosyl: rubusoside transferase trans dioside A. Accordingly, Such a microorganism may be ferring a glucose moiety to the C-2 of the 13-O-glucose of the capable of converting Stevioside to rebaudioside A. Expres acceptor molecule, rubusoside. sion of such a nucleotide sequence may confer on the micro 0070 Functional UGT2 polypeptides may also catalyze organism the ability to produce at least rebaudioside A. reactions that utilize Steviol glycoside Substrates other than Steviol-13-O-glucoside and rubusoside, e.g., functional 0076 A suitable recombinant microorganism may also UGT2 polypeptides may utilize stevioside as a substrate, comprise a nucleotide sequence encoding a polypeptide hav transferring a glucose moiety to the C-2 of the 19-O-glucose ing the activity shown by UDP-glycosyltransferase (UGT) residue to produce Rebaudioside E. A functional UGT2 UGT76G1, whereby the nucleotide sequence upon transfor polypeptides may also utilize Rebaudioside A as a Substrate, mation of the microorganism confers on the cell the ability to transferring a glucose moiety to the C-2 of the 19-O-glucose convert Stevioside to rebaudioside A. residue to produce Rebaudioside D. However, a functional (0077. A suitable UGT76G1 adds a glucose moiety to the UGT2 polypeptide typically does not transfer a glucose moi C-3' of the C-13-O-glucose of the acceptor molecule, a steviol ety to Steviol compounds having a 1.3-bound glucose at the 1.2 glycoside. Thus, UGT76G1 functions, for example, as a C-13 position, i.e., transfer of a glucose moiety to Steviol uridine 5'-diphospho glucosyl: Steviol 13-O-1.2 glucoside 1,3-bioside and 1.3-stevioside does not occur. C-3 glucosyltransferase and a uridine 5'-diphosphogluco 0071 Functional UGT2 polypeptides may also transfer syl: Steviol-19-O-glucose, 13-O-1.2 bioside C-3' glucosyl Sugar moieties from donors other than uridine diphosphate transferase. Functional UGT76G1 polypeptides may also glucose. For example, a functional UGT2 polypeptide may catalyze glucosyltransferase reactions that utilize Steviol gly act as a uridine 5'-diphospho D-xylosyl: Steviol-13-O-gluco coside Substrates that contain Sugars other than glucose, e.g., side transferase, transferring a xylose moiety to the C-2 of the Steviol rhamnosides and Steviol Xylosides. Such sequences 13-O-glucose of the acceptor molecule, steviol-13-O-gluco are indicated as UGT4 sequences in Table 1. side. As another example, a functional UGT2 polypeptide can 0078. A recombinant microorganism may comprise act as a uridine 5'-diphospho L-rhamnosyl: Steviol-13-O-glu nucleotide sequences encoding polypeptides having one or coside transferase, transferring a rhamnose moiety to the C-2 more of the four UGT activities described above. Preferably, of the 13-O-glucose of the acceptor molecule, Steviol-13-O- a recombinant microorganism may comprise nucleotide glucoside. Such sequences are indicated as UGT2 sequences sequences encoding polypeptides having all four of the UGT in Table 1. activities described above. A given nucleic acid may encode a 0072 A recombinant microorganism which may be fer polypeptide having one or more of the above activities. For mented to produce a fermentation broth for use in a process of example, a nucleic acid encode for a polypeptide which has the invention which comprises a nucleotide sequence encod two, three or four of the activities set out above. Preferably, a ing a polypeptide having UGT activity may comprise a nucle recombinant microorganism comprises UGT1, UGT2 and otide sequence encoding a polypeptide capable of catalyzing UGT3 activity. More preferably, such a recombinant micro the addition of a C-19-glucose to steviolbioside. That is to say, organism will also comprise UGT4 activity. US 2016/0213039 A1 Jul. 28, 2016

0079 A recombinant microorganism which comprises a I0087 (b) a nucleotide sequence that has at least about nucleotide sequence encoding a polypeptide having UGT 15%, preferably at least 20, 25, 30, 40, 50,55, 60, 65, activity may comprise a nucleotide sequence encoding a 70, 75,80, 85,90, 95, 96, 97,98, or 99%, sequence polypeptide capable of catalyzing the glucosylation of stevio identity with the nucleotide sequence of SEQID NOs: side or rebaudioside A. That is to say, a recombinant micro 53, 55, 57 or 77; organism may comprise a UGT which is capable of cata I0088 (c) a nucleotide sequence the complementary lyzing a reaction in which Stevioside or rebaudioside A is strand of which hybridizes to a nucleic acid molecule converted to rebaudioside D. Accordingly, Such a microor ganism may be capable of converting Stevioside or rebaudio of sequence of (i) or (ii); or side A to rebaudioside D. Expression of such a nucleotide I0089 (d) a nucleotide sequence which differs from sequence may confer on the microorganism the ability to the sequence of a nucleic acid molecule of (i), (ii) or produce at least rebaudioside D. We have shown that a micro (iii) due to the degeneracy of the genetic code, organism expression a combination of UGT85C2, UGT2, 0090 Preferably, a recombinant microorganism is one UGT74G1 and UGT76G1 polypeptides may be capable of which is capable of expressing one or more of: rebaudioside D production. 0091 a. a nucleotide sequence encoding a polypeptide 0080 A microorganism which comprises a nucleotide having ent-copalyl pyrophosphate synthase activity, sequence encoding a polypeptide having UGT activity may wherein said nucleotide sequence comprises: comprise a nucleotide sequence encoding a polypeptide 0092 1. a nucleotide sequence encoding a polypep capable of catalyzing the glucosylation of Stevioside. That is tide having ent-copalyl pyrophosphate synthase to say, a microorganism may comprise a UGT which is activity, said polypeptide comprising an amino acid capable of catalyzing a reaction in which Stevioside is con sequence that has at least about 20%, preferably at Verted to rebaudioside E. Accordingly, such a microorganism least 25, 30, 40, 50, 55, 60, 65,70, 75, 80, 85,90, 95, may be capable of converting stevioside to rebaudioside E. 96.97, 98, or 99%, sequence identity with the amino Expression of such a nucleotide sequence may confer on the acid sequence of SEQID NOs: 2, 4, 6, 8, 18, 20, 60 or microorganism the ability to produce at least rebaudioside E. 62: 0081. A microorganism which comprises a nucleotide 0093. 2. a nucleotide sequence that has at least about sequence encoding a polypeptide having UGT activity may 15%, preferably at least 20, 25, 30, 40, 50,55, 60, 65, comprise a nucleotide sequence encoding a polypeptide 70, 75,80, 85,90, 95, 96, 97,98, or 99%, sequence capable of catalyzing the glucosylation of rebaudioside E. identity with the nucleotide sequence of SEQID NOs: That is to say, a microorganism may comprise a UGT which 1, 3, 5, 7, 17, 19, 59 or 61, 141, 142, 151, 152, 153, is capable of catalyzing a reaction in which rebaudioside E is 154, 159, 160, 182 or 184: converted to rebaudioside D. Accordingly, Such a microor ganism may be capable of converting Stevioside or rebaudio 0094) 3. a nucleotide sequence the complementary side A to rebaudioside D. Expression of such a nucleotide strand of which hybridizes to a nucleic acid molecule sequence may confer on the microorganism the ability to of sequence of (i) or (ii); or produce at least rebaudioside D. 0.095 4. a nucleotide sequence which differs from the 0082. A recombinant microorganism may be capable of sequence of a nucleic acid molecule of (i), (ii) or (iii) expressing a nucleotide sequence encoding a polypeptide due to the degeneracy of the genetic code, having NADPH-cytochrome p450 reductase activity. That is 0.096 b. a nucleotide sequence encoding a polypeptide to say, a recombinant microorganism may comprise sequence having ent-Kaurene synthase activity, wherein said encoding a polypeptide having NADPH-cytochrome p450 nucleotide sequence comprises: 0097. 1. a nucleotide sequence encoding a polypep reductase activity. tide having ent-Kaurene synthase activity, said I0083. A polypeptide having NADPH-Cytochrome P450 polypeptide comprising an amino acid sequence that reductase activity (EC 1.6.2.4; also known as NADPH:ferri has at least about 20%, preferably at least 25, 30, 40, hemoprotein oxidoreductase, NADPH:hemoprotein oxi 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 96, 97,98, or doreductase, NADPH:P450 oxidoreductase, P450 reductase, 99%, sequence identity with the amino acid sequence POR, CPR, CYPOR) is typically one which is a membrane of SEQID NOs: 10, 12, 14, 16, 18, 20, 64 or 66: bound enzyme allowing electron transfer to cytochrome P450 0.098 2. a nucleotide sequence that has at least about in the microsome of the eukaryotic cell from a FAD- and 15%, preferably at least 20, 25, 30, 40, 50,55, 60, 65, FMN-containing enzyme NADPH:cytochrome P450 reduc 70, 75,80, 85,90, 95, 96, 97,98, or 99%, sequence tase (POR; EC 1.6.2.4). identity with the nucleotide sequence of SEQID NOs: 0084 Preferably, a recombinant microorganism, capable 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, 157, of being fermented to prepare a fermentation broth suitable 158, 159, 160, 183 or 184: for use in the process of the invention, is capable of expressing 0099 3. a nucleotide sequence the complementary one or more of: strand of which hybridizes to a nucleic acid molecule I0085 a. a nucleotide sequence encoding a polypeptide of sequence of (i) or (ii); or having NADPH-cytochrome p450 reductase activity, 0100. 4. a nucleotide sequence which differs from the wherein said nucleotide sequence comprises: sequence of a nucleic acid molecule of (i), (ii) or (iii) 0086 (a) a nucleotide sequence encoding a polypep due to the degeneracy of the genetic code, tide having NADPH-cytochrome p450 reductase 0101 c. a nucleotide sequence encoding a polypeptide activity, said polypeptide comprising an amino acid having ent-Kaurene oxidase activity, wherein said sequence that has at least about 20%, preferably at nucleotide sequence comprises: least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 0102 1. a nucleotide sequence encoding a polypep 96.97, 98, or 99%, sequence identity with the amino tide having ent-Kaurene oxidase activity, said acid sequence of SEQID NOs: 54, 56, 58 or 78: polypeptide comprising an amino acid sequence that US 2016/0213039 A1 Jul. 28, 2016

has at least about 20%, preferably at least 25, 30, 40, lation of the C-2" of the C-13-glucose/13-O-glucose of ste 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 96, 97,98, or violmonoside), said nucleotide sequence may comprise: 99%, Sequence identity with the amino acid sequence 0117 1. a nucleotide sequence encoding a polypeptide of SEQID NOs: 22, 24, 26, 68 or 86; capable of catalyzing the addition of a C-13-glucose to 0103 2. a nucleotide sequence that has at least about Steviol or Steviolmonoside, said polypeptide comprising 15%, preferably at least 20, 25, 30, 40, 50,55, 60, 65, an amino acid sequence that has at least about 20%, 70, 75,80, 85,90, 95, 96, 97,98, or 99%, sequence preferably at least 25, 30, 40, 50, 55, 60, 65,70, 75, 80, identity with the nucleotide sequence of SEQID NOs: 85,90, 95, 96.97,98, or 99%, sequence identity with the 21, 23, 25, 67, 85, 145, 161, 162, 163, 180 or 186: amino acid sequence of SEQID NOs: 88, 100, 102,104, 0104 3. a nucleotide sequence the complementary 106, 108, 110 or 112: strand of which hybridizes to a nucleic acid molecule 0118 2. a nucleotide sequence that has at least about of sequence of (i) or (ii); or 15%, preferably at least 20, 25, 30, 40, 50,55, 60, 65,70, 0105. 4. a nucleotide sequence which differs from the 75, 80, 85,90, 95, 96, 97,98, or 99%, sequence identity sequence of a nucleic acid molecule of (i), (ii) or (iii) with the nucleotide sequence of SEQID NOs: 87, 99, due to the degeneracy of the genetic code; or 101, 103, 105,107, 109, 111, 181 or 192: 0106 d. a nucleotide sequence encoding a polypeptide 0119. 3. a nucleotide sequence the complementary having kaurenoic acid 13-hydroxylase activity, wherein strand of which hybridizes to a nucleic acid molecule of said nucleotide sequence comprises: sequence of (i) or (ii); or 0107 1 a nucleotide sequence encoding a polypep 0120 4. a nucleotide sequence which differs from the tide having kaurenoic acid 13-hydroxylase activity, sequence of a nucleic acid molecule of (i), (ii) or (iii) due said polypeptide comprising an amino acid sequence to the degeneracy of the genetic code. that has at least about 20%, preferably at least 25, 30, 0121. In a recombinant microorganism which is capable 40, 50,55, 60, 65,70, 75, 80, 85,90, 95, 96, 97,98, or of expressing a nucleotide sequence encoding a polypeptide 99%, Sequence identity with the amino acid sequence capable of catalyzing the addition of a glucose at the C-19 of SEQID NOs: 28, 30, 32, 34, 70,90, 92,94, 96 or position of Steviolbioside, said nucleotide sequence may 98: comprise: 0108) 2 a nucleotide sequence that has at least about 0.122 1. a nucleotide sequence encoding a polypeptide 15%, preferably at least 20, 25, 30, 40, 50,55, 60, 65, capable of catalyzing the addition of a glucose at the 70, 75, 80, 85,90, 95, 96, 97,98, or 99%, sequence C-19 position of Steviolbioside, said polypeptide com identity with the nucleotide sequence of SEQID NOs: prising an amino acid sequence that has at least about 27, 29, 31, 33, 69, 89,91, 93, 95, 97, 146, 164, 165, 20% sequence identity with the amino acid sequence of 166, 167 or 185: 0109) 3 a nucleotide sequence the complementary SEQ ID NOs: 40, 42, 44, 46, 48 or 74: strand of which hybridizes to a nucleic acid molecule 0123 2. a nucleotide sequence that has at least about of sequence of (i) or (ii); or 15% sequence identity with the nucleotide sequence of 0110 4 a nucleotide sequence which differs from the SEQID NOs: 39, 41, 43,45, 47,73, 148, 170, 171, 172, sequence of a nucleic acid molecule of (i), (ii) or (iii) 173, 174 or 190; due to the degeneracy of the genetic code. 0.124 3. a nucleotide sequence the complementary 0111. In a recombinant microorganism which is capable strand of which hybridizes to a nucleic acid molecule of of expressing a nucleotide sequence encoding a polypeptide sequence of (i) or (ii); or capable of catalyzing the addition of a C-13-glucose to Ste 0.125 4. a nucleotide sequence which differs from the viol, said nucleotide may comprise: sequence of a nucleic acid molecule of (i), (ii) or (iii) due 0112 a nucleotide sequence encoding a polypeptide to the degeneracy of the genetic code. capable of catalyzing the addition of a C-13-glucose to 0.126 In a recombinant microorganism which expresses a Steviol, said polypeptide comprising an amino acid nucleotide sequence encoding a polypeptide capable of cata sequence that has at least about 20%, preferably at least lyzing glucosylation of the C-3' of the glucose at the C-13 25, 30, 40, 50,55, 60, 65,70, 75, 80, 85,90, 95, 96.97, position of Stevioside, said nucleotide sequence may com 98, or 99%, sequence identity with the amino acid prise: sequence of SEQID NOs: 36, 38 or 72: 0127. 1. a nucleotide sequence encoding a polypeptide 0113 a nucleotide sequence that has at least about 15%, capable of catalyzing glucosylation of the C-3' of the preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, glucose at the C-13 position of Stevioside, said polypep 80, 85,90,95, 96, 97.98, or 99%, sequence identity with tide comprising an amino acid sequence that has at least the nucleotide sequence of SEQID NOS:35, 37, 71,147, about 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 168, 169 or 189: 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%, sequence 0114 a nucleotide sequence the complementary Strand identity with the amino acid sequence of SEQID NOs: of which hybridizes to a nucleic acid molecule of 50, 52 or 76; sequence of (i) or (ii); or 0.128 2. a nucleotide sequence that has at least about 0115 a nucleotide sequence which differs from the 15%, preferably at least 20, 25, 30, 40, 50,55, 60, 65,70, sequence of a nucleic acid molecule of (i), (ii) or (iii) due 75, 80, 85,90, 95, 96, 97,98, or 99%, sequence identity to the degeneracy of the genetic code. with the nucleotide sequence of SEQID NOs:49, 51,75, 0116. In a recombinant microorganism which is capable 149, 175, 176 or 191; of expressing a nucleotide sequence encoding a polypeptide 0129. 3. a nucleotide sequence the complementary capable of catalyzing the addition of a glucose at the C-13 strand of which hybridizes to a nucleic acid molecule of position of Steviolmonoside (this typically indicates glucosy sequence of (i) or (ii); or US 2016/0213039 A1 Jul. 28, 2016

0.130. 4. a nucleotide sequence which differs from the ity, said polypeptide comprising an amino acid sequence of a nucleic acid molecule of (i), (ii) or (iii) due sequence that has at least about 20% sequence iden to the degeneracy of the genetic code. tity with the amino acid sequence of SEQID NO:82; 0131. In a recombinant microorganism which expresses a 0146 2. a nucleotide sequence that has at least about nucleotide sequence encoding a polypeptide capable of catal 15% sequence identity with the nucleotide sequence ysing one or more of the glucosylation of Stevioside or rebau of SEQID NOs: 81; dioside A to rebaudioside D; the glucosylation of stevioside to 0147 3. a nucleotide sequence the complementary rebaudioside E; or the glucosylation of rebaudioside E to strand of which hybridizes to a nucleic acid molecule rebaudioside D, said nucleotide sequence may comprise: of sequence of (i) or (ii); or 0132) i. a nucleotide sequence encoding a polypeptide 0148 4. a nucleotide sequence which differs from the capable of catalysing one or more of the glucosylation sequence of a nucleic acid molecule of (iii) due to the of Stevioside or rebaudioside A to rebaudioside D; the degeneracy of the genetic code; or glucosylation of stevioside to rebaudioside E; or the 0149 c. a nucleotide sequence encoding a polypeptide glucosylation of rebaudioside E to rebaudioside D, said having geranylgeranyl diphosphate synthase activity, polypeptide comprising an amino acid sequence that has wherein said nucleotide sequence comprises: at least about 20% sequence identity with the amino acid 0150 a nucleotide sequence encoding a polypeptide sequence of SEQID NOs: 88, 100, 102, 104, 106, 108, having geranylgeranyl diphosphate synthase activity, 110, 112: said polypeptide comprising an amino acid sequence 0.133 ii. a nucleotide sequence that has at least about that has at least about 20% sequence identity with the 15% sequence identity with the nucleotide sequence of amino acid sequence of SEQID NO: 84; SEQID NOs: 87,99, 101, 103,105,107,109, 111, 181 0151 a nucleotide sequence that has at least about or 192: 15% sequence identity with the nucleotide sequence I0134) iii. a nucleotide sequence the complementary of SEQID NOs: 83: strand of which hybridizes to a nucleic acid molecule of 0152 a nucleotide sequence the complementary sequence of (i) or (ii); or strand of which hybridizes to a nucleic acid molecule 0.135 iv. a nucleotide sequence which differs from the of sequence of (i) or (ii); or sequence of a nucleic acid molecule of (i), (ii) or (iii) due 0153 a nucleotide sequence which differs from the to the degeneracy of the genetic code. sequence of a nucleic acid molecule of (i), (ii) or (iii) 0136. A suitable microorganism may be one in which the due to the degeneracy of the genetic code. ability of the microorganism to produce geranylgeranyl pyro 0154) A microorganism or microbe, for the purposes of phosphate (GGPP) is upregulated. Upregulated in the context this invention, is typically an organism that is not visible to the of this invention implies that the microorganism produces human eye (i.e. microscopic). A microorganism may be from more GGPP than an equivalent non-transformed strain. bacteria, fungi, archaea or protists. Typically a microorgan 0.137 Accordingly, a suitable recombinant microorgan ism will be a single-celled or unicellular organism. ism may comprise one or more nucleotide sequence(s) encod 0.155. As used herein a recombinant microorganism is ing hydroxymethylglutaryl-CoA reductase, farnesyl-pyro defined as a microorganism which is genetically modified or phosphate synthetase and geranylgeranyl diphosphate transformed/transfected with one or more of the nucleotide synthase, whereby the nucleotide sequence(s) upon transfor sequences as defined herein. The presence of the one or more mation of the microorganism confer(s) on the microorganism Such nucleotide sequences alters the ability of the microor the ability to produce elevated levels of GGPP. ganism to produce a diterpene or diterpene glycoside, in 0138 Preferably, a suitable recombinant microorganism particular Steviol or Steviol glycoside. A microorganism that is one which is capable of expressing one or more of: is not transformed/transfected or genetically modified, is not 0.139 a. a nucleotide sequence encoding a polypeptide a recombinant microorganism and does typically not com having hydroxymethylglutaryl-CoA reductase activity, prise one or more of the nucleotide sequences enabling the wherein said nucleotide sequence comprises: cell to produce a diterpene or diterpene glycoside. Hence, a 0140) 1. a nucleotide sequence encoding a polypep non-transformed/non-transfected microorganism is typically tide having hydroxymethylglutaryl-CoA reductase a microorganism that does not naturally produce a diterpene, activity, said polypeptide comprising an amino acid although a microorganism which naturally produces a diter sequence that has at least about 20% sequence iden pene or diterpene glycoside and which has been modified, as tity with the amino acid sequence of SEQID NO: 80: described herein for example (and which thus has an altered 0141 2. a nucleotide sequence that has at least about ability to produce a diterpenefditerpenegylcoside), is consid 15% sequence identity with the nucleotide sequence ered a recombinant microorganism. of SEQID NO: 79; 0156 Sequence identity is herein defined as a relationship 0142. 3. a nucleotide sequence the complementary between two or more amino acid (polypeptide or protein) strand of which hybridizes to a nucleic acid molecule sequences or two or more nucleic acid (polynucleotide) of sequence of (i) or (ii); or sequences, as determined by comparing the sequences. Usu 0.143 4. a nucleotide sequence which differs from the ally, sequence identities or similarities are compared over the sequence of a nucleic acid molecule of (i), (ii) or (iii) whole length of the sequences compared. In the art, “identity” due to the degeneracy of the genetic code, also means the degree of sequence relatedness between 014.4 b. a nucleotide sequence encoding a polypeptide amino acid or nucleic acid sequences, as the case may be, as having farnesyl-pyrophosphate synthetase activity, determined by the match between strings of Such sequences. wherein said nucleotide sequence comprises: “Identity” and “similarity' can be readily calculated by vari 0145 1. a nucleotide sequence encoding a polypep ous methods, known to those skilled in the art. Preferred tide having farnesyl-pyrophosphate synthetase activ methods to determine identity are designed to give the largest US 2016/0213039 A1 Jul. 28, 2016 match between the sequences tested. Typically then, identi 0.161. A nucleotide sequence encoding an ent-Kaurene ties and similarities are calculated over the entire length of the synthase may for instance comprise a sequence as set out in sequences being compared. Methods to determine identity SEQID. NO: 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, and similarity are codified in publicly available computer 157, 158, 159, 160, 183 or 184. programs. Preferred computer program methods to determine 0162. A nucleotide sequence encoding an ent-Kaurene identity and similarity between two sequences include e.g. oxidase may for instance comprise a sequence as set out in the BestFit, BLASTP. BLASTN, and FASTA (Altschul, S. F. SEQ ID. NO: 21, 23, 25, 67, 85, 145, 161, 162, 163, 180 or et al., J. Mol. Biol. 215:403-410 (1990), publicly available 186. A preferred KO is the polypeptide encoded by the from NCBI and other sources (BLAST Manual. Altschul, S., nucleic acid set out in SEQID NO: 85. et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred 0163 A nucleotide sequence encoding a kaurenoic acid parameters for amino acid sequences comparison using 13-hydroxylase may for instance comprise a sequence as set BLASTP are gap open 10.0, gap extend 0.5, Blosum 62 out in SEQID. NO: 27, 29, 31, 33, 69,89, 91.93, 95,97, 146, matrix. Preferred parameters for nucleic acid sequences com 164, 165, 166, 167 or 185. A preferred KAH sequence is the parison using BLASTP are gap open 10.0, gap extend 0.5, polypeptide encoded by the nucleic acid set out in SEQ ID DNA full matrix (DNA identity matrix). NO: 33. 0157 Nucleotide sequences encoding the enzymes 0164. A Suitable recombinant microorganism may express expressed in the cells described herein may also be defined by a combination of the polypeptides encoded by SEQID NO: their capability to hybridize with the nucleotide sequences of 85 and SEQID NO:33 or a variant of either thereofas herein SEQID NO.'s 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, described. A preferred recombinant microorganism may 29, 31,33,35, 37,39, 41,43, 45, 47,49,51,53,55, 57, 59, 61, express the combination of sequences set out in Table 8 (in 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 or 84 it any other combination with any UGT2, but in particular that encoded sequence mentioned herein respectively, under moderate, or by SEQID NO: 87). preferably under Stringent hybridisation conditions. Stringent 0.165. A nucleotide sequence encoding a UGT may for hybridisation conditions are herein defined as conditions that instance comprise a sequence as set out in SEQID. NO:35, allow a nucleic acid sequence of at least about 25, preferably 37, 39, 41, 43, 45, 47,49, 51,71, 73, 75, 168, 169, 170, 171, about 50 nucleotides, 75 or 100 and most preferably of about 172,173, 174, 175, 176, 147,148, 149, 87, 181,99, 100, 101, 200 or more nucleotides, to hybridise at a temperature of 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, about 65° C. in a solution comprising about 1 M salt, prefer 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, ably 6xSSC or any other solution having a comparable ionic 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, strength, and washing at 65°C. in a solution comprising about 138, 139, 140, 189, 190, 191 or 192. 0.1 M salt, or less, preferably 0.2xSSC or any other solution 0166 A nucleotide sequence encoding a hydroxymethyl having a comparable ionic strength. Preferably, the hybridi glutaryl-CoA reductase may for instance comprise a sation is performed overnight, i.e. at least for 10 hours and sequence as set out in SEQID. NO: 79. preferably washing is performed for at least one hour with at 0.167 A nucleotide sequence encoding a farnesyl-pyro least two changes of the washing solution. These conditions phosphate synthetase may for instance comprise a sequence will usually allow the specific hybridisation of sequences as set out in SEQID. NO: 81. having about 90% or more sequence identity. 0168 A nucleotide sequence encoding a geranylgeranyl 0158 Moderate conditions are herein defined as condi diphosphate synthase may for instance comprise a sequence tions that allow a nucleic acid sequences of at least 50 nucle as set out in SEQID. NO:83. otides, preferably of about 200 or more nucleotides, to hybri 0169. A nucleotide sequence encoding a NADPH-cyto dise at a temperature of about 45°C. in a solution comprising chrome p450 reductase may for instance comprise a sequence about 1 M salt, preferably 6xSSC or any other solution having as set out in SEQID, NO. 53, 55, 57 or 77. a comparable ionic strength, and washing at room tempera 0170 In the case of the UGT sequences, combinations of ture in a solution comprising about 1 M salt, preferably at least one from each of: (i) SEQID NOS: 35, 37, 168, 169, 6xSSC or any other solution having a comparable ionic 71, 147 or 189; (ii) SEQID NOs: 87,99, 101, 103, 105, 107, strength. Preferably, the hybridisation is performed over 109, 111, 181 or 192; (iii) SEQ ID NOs: 39, 41, 43, 45, 47, night, i.e. at least for 10 hours, and preferably washing is 170, 171, 172,173, 174,73, 148 or 190; and (iv) SEQID NOs: performed for at least one hour with at least two changes of 49, 51, 175, 176, 75, 149 or 191 may be preferred. Typically, the washing solution. These conditions will usually allow the at least one UGT from group (i) may be used. If at least one specific hybridisation of sequences having up to 50% UGT from group (iii) is used, generally at least one UGT from sequence identity. The person skilled in the art will be able to group (i) is also used. If at least one UGT from group (iv) is modify these hybridisation conditions in order to specifically used, generally at least one UGT from group (i) and at least identify sequences varying in identity between 50% and 90%. one UGT from group (iii) is used. Typically, at least one UGT 0159. The nucleotide sequences encoding an ent-copalyl form group (ii) is used. pyrophosphate synthase; ent-Kaurene synthase; ent-Kaurene 0171 A sequence which has at least about 10%, about oxidase; kaurenoic acid 13-hydroxylase; UGT: hydroxym 15%, about 20%, preferably at least about 25%, about 30%, ethylglutaryl-CoA reductase, farnesyl-pyrophosphate Syn about 40%, about 50%, about 55%, about 60%, about 65%, thetase; geranylgeranyl diphosphate synthase, NADPH-cyto about 70%, about 75%, about 80%, about 85%, about 90%, chrome p450 reductase, may be from prokaryotic or about 95%, about 96%, about 97%, about 98%, or about 99% eukaryotic origin. sequence identity with a sequence as mentioned may be used 0160 A nucleotide sequence encoding an ent-copalyl in the invention. pyrophosphate synthase may for instance comprise a 0172 To increase the likelihood that the introduced sequence as set out in SEQID. NO: 1, 3, 5, 7, 17, 19, 59, 61, enzymes are expressed in active formina recombinant micro 141, 142, 151, 152, 153, 154, 159, 160, 182 or 184. organism, the corresponding encoding nucleotide sequence US 2016/0213039 A1 Jul. 28, 2016 may be adapted to optimise its codon usage to that of the recombinant Saccharomyces cerevisiae cell or Yarrowia chosen eukaryote host cell. The adaptiveness of the nucle lipolytica cell may comprise one or more nucleotide sequence otide sequences encoding the enzymes to the codon usage of (s) from each of the following groups; the chosen host cell may be expressed as codon adaptation 0180 (i) SEQID. NO: 1, 3, 5, 7, 17, 19, 59, 61,141, 142, index (CAI). The codon adaptation index is herein defined as 152, 153, 154, 159, 160, 182 or 184. a measurement of the relative adaptiveness of the codon usage 0181 (ii) SEQID. NO: 9, 11, 13, 15, 17, 19, 63, 65, 143, of a gene towards the codon usage of highly expressed genes. 144, 155, 156, 157, 158, 159, 160, 183 or 184. The relative adaptiveness (w) of each codon is the ratio of the 0182 (iii) SEQID. NO: 21, 23, 25, 6785, 145, 161, 162, usage of each codon, to that of the most abundant codon for 163, 180 or 186. the same amino acid. The CAI index is defined as the geo 0183 (iv) SEQID. NO: 27, 29, 31,33, 69, 89,91, 93, 95, metric mean of these relative adaptiveness values. Non-Syn 97, 146, 164, 165, 166, 167 or 185. onymous codons and termination codons (dependent on 0.184 Such a microorganism will typically also comprise genetic code) are excluded. CAI values range from 0 to 1, one or more nucleotide sequence(s)as set out in SEQID. NO: with higher values indicating a higher proportion of the most 53, 55, 57 or 77. abundant codons (see Sharp and Li, 1987, Nucleic Acids 0185. Such a microorganism may also comprise one or Research 15: 1281-1295; also see: Jansen et al., 2003, more nucleotide sequences as set out in 35, 37, 39, 41, 43, 45, Nucleic Acids Res. 31 (8):2242-51). An adapted nucleotide 47,49, 51,71, 73, 75, 168, 169,170, 171, 172,173, 174, 175, sequence preferably has a CAI of at least 0.2,0.3, 0.4,0.5,0.6 176, 147,148, 149, 87, 181,99, 100, 101, 102, 103,104,105, or 0.7. 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 0173. In a preferred embodiment the recombinant is 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, genetically modified with (a) nucleotide sequence(s) which is 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 189, (are) adapted to the codon usage of the eukaryotic cell using 190, 191 or 192. In the case of these sequences, combinations codon pair optimisation technology as disclosed in PCT/ of at least one from each of (i) SEQID NOS:35, 37,168, 169, EP2007/05594. Codon-pair optimisation is a method for pro 71, 147 or 189; (ii) SEQID NOs: 87,99, 101, 103, 105, 107, ducing a polypeptide in a host cell, wherein the nucleotide 109, 111, 181 or 192; (iii) SEQ ID NOs: 39, 41, 43, 45, 47, sequences encoding the polypeptide have been modified with 170, 171, 172,173, 174,73, 148 or 190; and (iv) SEQID NOs: respect to their codon-usage, in particular the codon-pairs that 49, 51, 175, 176, 75, 149 or 191 may be preferred. Typically, are used, to obtain improved expression of the nucleotide at least one UGT from group (i) may be used. If at least one sequence encoding the polypeptide and/or improved produc UGT from group (iii) is used, generally at least one UGT from tion of the polypeptide. Codon pairs are defined as a set of two group (i) is also used. If at least one UGT from group (iv) is Subsequent triplets (codons) in a coding sequence. used, generally at least one UGT from group (i) and at least 0.174 Further improvement of the activity of the enzymes one UGT from group (iii) is used. Typically, at least one UGT in vivo in a recombinant microorganism, can be obtained by form group (ii) is used. well-known methods like error prone PCR or directed evolu 0186 Such a microorganism may also comprise the fol tion. A preferred method of directed evolution is described in lowing nucleotide sequences: SEQID. NO: 79; SEQID. NO: WOO3O10183 and WOO3O10311. 81; and SEQID. NO: 83. 0.175. A suitable recombinant microorganism may be any 0187. For each sequence set out above (or any sequence suitable host cell from microbial origin. Preferably, the host mentioned herein), a variant having at least about 15%, pref cell is a yeast or a filamentous fungus. More preferably, the erably at least about 20, about 25, about 30, about 40, about host cell belongs to one of the genera Saccharomyces, 50, about 55, about 60, about 65, about 70, about 75, about 80, Aspergillus, Penicillium, Pichia, Kluyveromyces, Yarrowia, about 85, about 90, about 95, about 96, about 97, about 98, or Candida, Hansenula, Humicola, Torulaspora, TrichospOron, about 99%, sequence identity with the stated sequence may Brettanomyces, Pachysolen or Yamadazyma or Zygosaccha be used. romyces. 0188 The nucleotide sequences encoding the ent-copalyl 0176 A more preferred microorganism belongs to the spe pyrophosphate synthase, ent-Kaurene synthase, ent-Kaurene cies Aspergillus niger, Penicillium chrysogenium, Pichia Sti oxidase, kaurenoic acid 13-hydroxylase, UGTs, hydroxym pidis, Kluyveromyces marxianus, K. lactis, K. thermotoler ethylglutaryl-CoA reductase, farnesyl-pyrophosphate Syn ans, Yarrowia lipolytica, Candida Sonorensis, C. glabrata, thetase, geranylgeranyl diphosphate synthase and NADPH Hansenula polymorpha, Torulaspora delbruecki, Brettano cytochrome p450 reductase may be ligated into one or more myces bruxellensis, Zygosaccharomyces bailii, Saccharomy nucleic acid constructs to facilitate the transformation of the ces uvarum, Saccharomyces bayanus or Saccharomyces cer microorganism. evisiae species. Preferably, the eukaryotic cell is a 0189 A nucleic acid construct may be a plasmid carrying Saccharomyces cerevisiae. the genes encoding enzymes of the diterpene, e.g. Steviol/ 0177. A recombinant yeast cell may be modified so that Steviol glycoside, pathway as described above, or a nucleic the ERG9 gene is down-regulated and or the ERG5/ERG6 acid construct may comprise two or three plasmids carrying genes are deleted. Corresponding genes may be modified in each three or two genes, respectively, encoding the enzymes this way in other microorganisms. of the diterpene pathway distributed in any appropriate way. 0178. Such a microorganism may be transformed, 0190. Any suitable plasmid may be used, for instance a whereby the nucleotide sequence(s) with which the microor low copy plasmid or a high copy plasmid. ganism is transformed confer(s) on the cell the ability to 0191 It may be possible that the enzymes selected from produce a diterpene or glycoside thereof. the group consisting of ent-copalyl pyrophosphate synthase, 0179 A preferred suitable recombinant microorganism is ent-Kaurene synthase, ent-Kaurene oxidase, and kaurenoic a yeast, Such as a Saccharomyces cerevisiae or Yarrowia acid 13-hydroxylase, UGTs, hydroxymethylglutaryl-CoA lipolytica cell. A recombinant microorganism, Such as a reductase, farnesyl-pyrophosphate synthetase, geranylgera US 2016/0213039 A1 Jul. 28, 2016 nyl diphosphate synthase and NADPH-cytochrome p450 tion of one or more genes, located upstream with respect to reductase are native to the host microorganism and that trans the direction of transcription of the transcription initiation site formation with one or more of the nucleotide sequences of the gene, and is structurally identified by the presence of a encoding these enzymes may not be required to confer the binding site for DNA-dependent RNA polymerase, transcrip host cell the ability to produce a diterpene or diterpene gly tion initiation sites and any other DNA sequences, including, cosidase. Further improvement of diterpenefditerpene gly but not limited to transcription factor binding sites, repressor cosidase production by the host microorganism may be and activator proteinbinding sites, and any other sequences of obtained by classical strain improvement. nucleotides known to one of skilled in the art to act directly or 0.192 The nucleic acid construct may be maintained epi indirectly to regulate the amount of transcription from the Somally and thus comprise a sequence for autonomous repli promoter. A “constitutive' promoter is a promoter that is cation, such as an autosomal replication sequence sequence. active under most environmental and developmental condi If the host cell is of fungal origin, a suitable episomal nucleic tions. An “inducible' promoter is a promoter that is active acid construct may e.g. be based on the yeast 2L or pKD1 under environmental or developmental regulation. plasmids (Gleer et al., 1991, Biotechnology 9:968-975), or 0198 The promoter that could be used to achieve the the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482 expression of the nucleotide sequences coding for an enzyme 489). as defined herein above, may be not native to the nucleotide 0193 Alternatively, each nucleic acid construct may be sequence coding for the enzyme to be expressed, i.e. a pro integrated in one or more copies into the genome of the host moter that is heterologous to the nucleotide sequence (coding cell. Integration into the host cells genome may occur at sequence) to which it is operably linked. Preferably, the pro random by non-homologous recombination but preferably moter is homologous, i.e. endogenous to the host cell the nucleic acid construct may be integrated into the host 0199 Suitable promoters for use in recombinant microor cells genome by homologous recombination as is well ganisms may be GAL7, GAL10, or GAL 1, CYC1, HIS3, known in the art (see e.g. WO90/14423, EP-A-0481008, EP ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, A-0635 574 and U.S. Pat. No. 6,265,186). ENO, TPI, and AOX1. Other suitable promoters include 0194 Optionally, a selectable marker may be present in PDC, GPD1, PGK1, TEF1, and TDH. the nucleic acid construct. As used herein, the term “marker” 0200 Any terminator, which is functional in the cell, may refers to a gene encoding a trait or a phenotype which permits be used. Preferred terminators are obtained from natural the selection of, or the screening for, a microorganism con genes of the host cell. Suitable terminator sequences are well taining the marker. The marker gene may be an antibiotic known in the art. Preferably, such terminators are combined resistance gene whereby the appropriate antibiotic can be with mutations that prevent nonsense mediated mRNA decay used to select for transformed cells from among cells that are in the host cell (see for example: Shirley et al., 2002, Genetics not transformed. Alternatively or also, non-antibiotic resis 161:1465-1482). tance markers are used. Such as auxotrophic markers (URA3, TRP1, LEU2). The host cells transformed with the nucleic 0201 Nucleotide sequences used may include sequences acid constructs may be marker gene free. Methods for con which target them to desired compartments of the microor structing recombinant marker gene free microbial host cells ganism. For example, in a preferred recombinant microorgan are disclosed in EP-A-0 635 574 and are based on the use of ism, all nucleotide sequences, except for ent-Kaurene oxi bidirectional markers. Alternatively, a screenable marker dase, kaurenoic acid 13-hydroxylase and NADPH such as Green Fluorescent Protein, lac7, luciferase, chloram cytochrome p450 reductase encoding sequences may be phenicol acetyltransferase, beta-glucuronidase may be incor targeted to the cytosol. This approach may be used in a yeast porated into the nucleic acid constructs allowing for screen cell. ing for transformed cells. A preferred marker-free method for 0202 The term “homologous' when used to indicate the the introduction of heterologous polynucleotides is described relation between a given (recombinant) nucleic acid or in WOO540186. polypeptide molecule and a given host organism or host cell, 0.195. In a preferred embodiment, the nucleotide is understood to mean that in nature the nucleic acid or sequences encoding ent-copalyl pyrophosphate synthase, polypeptide molecule is produced by a host cell or organisms ent-Kaurene synthase, ent-Kaurene oxidase, and kaurenoic of the same species, preferably of the same variety or strain. acid 13-hydroxylase, UGTs, hydroxymethylglutaryl-CoA 0203 The term "heterologous' when used with respect to reductase, farnesyl-pyrophosphate synthetase, geranylgera a nucleic acid (DNA or RNA) or protein refers to a nucleic nyl diphosphate synthase and NADPH-cytochrome p450 acid or protein that does not occur naturally as part of the reductase, are each operably linked to a promoter that causes organism, cell, genome or DNA or RNA sequence in which it Sufficient expression of the corresponding nucleotide is present, or that is found in a cell or location or locations in sequences in the recombinant microorganism to confer to the the genome or DNA or RNA sequence that differ from that in cell the ability to produce a diterpene or diterpene glycoside. which it is found in nature. Heterologous nucleic acids or 0196. As used herein, the term “operably linked’ refers to proteins are not endogenous to the cell into which it is intro a linkage of polynucleotide elements (or coding sequences or duced, but have been obtained from another cell or syntheti nucleic acid sequence) in a functional relationship. A nucleic cally or recombinantly produced. acid sequence is “operably linked when it is placed into a 0204 Typically, a suitable recombinant microorganism functional relationship with another nucleic acid sequence. will comprise heterologous nucleotide sequences. Alterna For instance, a promoter or enhancer is operably linked to a tively, a recombinant microorganism may comprise entirely coding sequence if it affects the transcription of the coding homologous sequence which has been modified as set out Sequence. herein so that the microorganism produces increased amounts (0197). As used herein, the term “promoter” refers to a of a diterpene and/or diterpene glycoside in comparison to a nucleic acid fragment that functions to control the transcrip non-modified version of the same microorganism. US 2016/0213039 A1 Jul. 28, 2016

0205 One or more enzymes of the diterpene pathway as 0213. The recombinant microorganism used in the process described herein may be overexpressed to achieve a sufficient for the preparation of a Steviol glycoside may be any Suitable diterpene production by the cell. microorganism as defined herein above. It may be advanta 0206. There are various means available in the art for geous to use a recombinant eukaryotic microorganism as overexpression of enzymes in the host cell. In particular, an described herein in the process for the production of a diter enzyme may be overexpressed by increasing the copy number pene or diterpene glycoside, because most eukaryotic cells do of the gene coding for the enzyme in the host cell, e.g. by not require sterile conditions for propagation and are insen integrating additional copies of the gene in the host cells sitive tobacteriophage infections. In addition, eukaryotic host genome. cells may be grown at low pH to prevent bacterial contami 0207. A preferred recombinant microorganism may be a nation. recombinant microorganism which is naturally capable of 0214. The recombinant microorganism may be a faculta producing GGPP. tive anaerobic microorganism. A facultative anaerobic micro 0208. A suitable recombinant microorganism may be able organism can be propagated aerobically to a high cell con to grow on any Suitable carbon source known in the art and centration. This anaerobic phase can then be carried out at convert it to one or more Steviol glycosides. The recombinant high cell density which reduces the fermentation volume microorganism may be able to convert directly plant biomass, required substantially, and may minimize the risk of contami celluloses, hemicelluloses, pectines, rhamnose, galactose, nation with aerobic microorganisms. fucose, maltose, maltodextrines, ribose, ribulose, or starch, 0215. The fermentation process for the production of a starch derivatives, Sucrose, lactose and glycerol. Hence, a Steviol glycoside may be an aerobic or an anaerobic fermen preferred host organism expresses enzymes such as cellulases tation process. (endocellulases and exocellulases) and hemicellulases (e.g. 0216. An anaerobic fermentation process may be herein endo- and exo-Xylanases, arabinases) necessary for the con defined as a fermentation process run in the absence of oxy version of cellulose into glucose monomers and hemicellu gen or in which Substantially no oxygen is consumed, pref lose into Xylose and arabinose monomers, pectinases able to erably less than 5, 2.5 or 1 mmol/L/h, and wherein organic convert pectines into glucuronic acid and galacturonic acid or molecules serve as both electron donor and electron accep amylases to convert starch into glucose monomers. Prefer tors. The fermentation process may also first be run under ably, the host cell is able to convert a carbon source selected aerobic conditions and Subsequently under anaerobic condi from the group consisting of glucose, Xylose, arabinose, tions. Sucrose, lactose and glycerol. The host cell may for instance 0217. The fermentation process may also be run under be a eukaryotic host cell as described in WO03/062430, oxygen-limited, or micro-aerobical, conditions. Alterna WO06/009.434, EP1499708B1, WO2006096130 or WOO4/ tively, the fermentation process may first be run under aerobic O99381. conditions and Subsequently under oxygen-limited condi 0209. A recombinant microorganism as described above tions. An oxygen-limited fermentation process is a process in may be used in a process for the production of a Steviol which the oxygen consumption is limited by the oxygen glycoside, which method comprises fermenting a trans transfer from the gas to the liquid. The degree of oxygen formed a suitable recombinant microorganism (as described limitation is determined by the amount and composition of herein) in a suitable fermentation medium, and optionally the ingoing gas flow as well as the actual mixing/mass trans recovering the diterpene and/or diterpene glycoside. fer properties of the fermentation equipment used. 0210. The fermentation medium used in the process for 0218. The production of a steviol glycoside in the fermen the production of a diterpene or diterpene glycoside may be tation process may occur during the growth phase of the host any suitable fermentation medium which allows growth of a cell, during the stationary (steady state) phase or during both particular eukaryotic host cell. The essential elements of the phases. It may be possible to run the fermentation process at fermentation medium are known to the person skilled in the different temperatures. art and may be adapted to the host cell selected. 0219. The process for the production of a steviol glycoside 0211 Preferably, the fermentation medium comprises a may be run at a temperature which is optimal for the recom carbon Source selected from the group consisting of plant binant microorganism. The optimum growth temperature biomass, celluloses, hemicelluloses, pectines, rhamnose, may differ for each transformed cell and is known to the galactose, fucose, fructose, maltose, maltodextrines, ribose, person skilled in the art. The optimum temperature might be ribulose, or starch, starch derivatives, sucrose, lactose, fatty higher than optimal for wild type organisms to grow the acids, triglycerides and glycerol. Preferably, the fermentation organism efficiently under non-sterile conditions under mini medium also comprises a nitrogen source Such as ureum, or mal infection sensitivity and lowest cooling cost. Alterna an ammonium salt such as ammonium Sulphate, ammonium tively, the process may be carried out at a temperature which chloride, ammonium nitrate or ammonium phosphate. is not optimal for growth of the recombinant microorganism. 0212. A suitable fermentation process may be carried out 0220. The temperature for growth of the recombinant in batch, fed-batch or continuous mode. A separate hydrolysis microorganism in a process for production of a diterpene or and fermentation (SHF) process or a simultaneous sacchari diterpene glycoside may be above 20° C., 22°C., 25°C., 28° fication and fermentation (SSF) process may also be applied. C., or above 30° C., 35° C., or above 37° C., 40° C., 42°C., A combination of these fermentation process modes may also and preferably below 45° C. During the production phase of be possible for optimal productivity. A SSF process may be a diterpene or diterpene glycoside however, the optimum particularly attractive if starch, cellulose, hemicellulose or temperature might be lower than average in order to optimize pectin is used as a carbon Source in the fermentation process, biomass stability. The temperature during this phase may be where it may be necessary to add hydrolytic enzymes, such as below 45° C., for instance below 42°C., 40°C., 37° C., for cellulases, hemicellulases or pectinases to hydrolyse the Sub instance below 35°C., 30°C., or below 28°C., 25°C., 22°C. Strate. or below 20° C. preferably above 15° C. US 2016/0213039 A1 Jul. 28, 2016

0221) The process for the production of a steviol glycoside 0231. 3. Stevioside (wt/wt %): 2% (on dry basis) maxi may be carried out at any suitable pH value. If the recombi U nant microorganism is yeast, the pH in the fermentation 0232 4. Steviol (wit/wt %): Less than 0.005% (on dry medium preferably has a value of below 6, preferably below basis) 5.5, preferably below 5, preferably below 4.5, preferably 0233 5. Moisture Content (%) by loss on drying: 6% below 4, preferably below pH 3.5 or below pH 3.0, or below maximum pH 2.5, preferably above pH 2. An advantage of carrying out 0234 6. Optical Rotation: -29 to 37 Degrees the fermentation at these low pH values is that growth of 0235 7. pH: 4.5-7.0 (1 g in 100 ml water) contaminant bacteria in the fermentation medium may be 0236 8. Arsenic (as As): 1 mg/kg maximum prevented. 0237 9. Lead (as Pb): 1 mg/kg maximum 0222 Such a process may be carried out on an industrial 0238 10. Mercusry (Hb): 1 mg/kg maximum scale. 0239 11. Cadmium (Cd): 1 mg/kg maximum 0223) The product of such a process may be one or more of 0240 12. Total Aerobic Plate Count: 1000 CFU/g maxi Steviolmonoside, Steviolbioside, Stevioside or rebaudioside mum (CFU=colony forming unit) A, rebaudioside B, rebaudioside C, rebaudioside D, rebau 0241 13. Total Aerobic Mold count: 1000 CFU/g maxi dioside E, rebaudioside F, rubusoside, dulcoside A. Prefer U ably, rebaudioside A or rebaudioside D is produced. 0242) 14. Total Aerobic Yeast count: 1000 CFU/g maxi 0224 Recovery of the diterpene or diterpene glycoside U from the resulting broth may be carried out by known meth 0243 15. Coliform: Less than 10 CFU/g ods in the art, for instance by filtration, crystallization, distil 0244 16. E. coli: less than 3 MPN/g (MPN=most probable lation, vacuum extraction, Solvent extraction, or evaporation. number) In the event that Reb-A is expressed within the microorgan 0245 17. Residue on Ignition: 1.0% maximum (synonym ism, such cells may need to be treated so as to release Reb-A. for Ash) FIG. 7 sets out a scheme for recovery of steviol glycosides 0246) 18. Residual solvents: MeOH <200 ppm: EtOH from a fermentation broth <5000 ppm 0225. In the process for the fermentative production of a 0247 The sweetener composition (i.e. a composition Steviol glycoside, it may be possible to achieve a concentra comprises a Steviol glycoside Such as rebA) and product tion of above 5 mg/l fermentation broth, preferably above 10 specifications described herein may be applied for use in any mg/l, preferably above 20 mg/l, preferably above 30 mg/1 suitable product such as Zero calorie, reduced calorie or dia fermentation broth, preferably above 40 mg/l, more prefer betic beverages and food products with improved taste char ably above 50 mg/l, preferably above 60 mg/l, preferably acteristics. Also it can be used in drinks, foodstuffs, pharma above 70, preferably above 80 mg/l, preferably above 100 ceuticals, and other products in which Sugar cannot be used. mg/l, preferably above 1 g/l, preferably above 5 g/l, prefer 0248. In addition, the sweetener composition can be used ably above 10 g/l, but usually below 70 g/l in the broth. as a sweetener not only for drinks, foodstuffs, and other 0226. As described above, in the event that a diterpene or products dedicated for human consumption, but also in ani diterpene glycoside is expressed within the microorganism, mal feed and fodder with improved characteristics. such cells may need to be treated so as to release the steviol 0249. The examples of products where the Sweetener glycoside. composition can be used as Sweetening compound can be as alcoholic beverages such as Vodka, wine, beer, liquor, sake, 0227 Steviol glycosides produced as described herein etc., natural juices, refreshing drinks, carbonated Soft drinks, may be blended with one or more further non-calorific or diet drinks, Zero calorie drinks, reduced calorie drinks and calorific sweeteners. Such blending may be used to improve foods, yogurt drinks, instantjuices, instant coffee, powdered flavor or temporal profile or stability. A wide range of both types of instant beverages, canned products, syrups, fer non-calorific and calorific sweeteners may be suitable for mented Soybean paste, soy sauce, vinegar, dressings, mayon blending with Reb-A. For example, non-calorific sweeteners naise, ketchups, curry, Soup, instant bouillon, powdered soy Such as mogroside, monatin, aspartame, acesulfame salts, sauce, powdered vinegar, types of biscuits, rice biscuit, crack cyclamate. Sucralose, Saccharin salts or erythritol. Calorific ers, bread, chocolates, caramel, candy, chewing gum, jelly, sweeteners suitable for blending with Reb-A include sugar pudding, preserved fruits and vegetables, fresh cream, jam, alcohols and carbohydrates such as Sucrose, glucose, fructose marmalade, flower paste, powdered milk, ice cream, Sorbet, and HFCS. Sweet tastingamino acids such as glycine, alanine Vegetables and fruits packed in bottles, canned and boiled or serine may also be used. beans, meat and foods boiled in Sweetened sauce, agricultural 0228 Steviol glycosides (containing Rebaudioside-A Vegetable food products, seafood, ham, sausage, fish ham, 95%) may be a white granular material that has a Sweet taste. fish sausage, fish paste, deep fried fish products, dried seafood A microbial fermentation process is used for production of products, frozen food products, preserved seaweed, pre this material. Reb-A or steviol glycosides is soluble in water served meat, tobacco, medicinal products, and many others. at a level greater than 3000 ppm (>0.3%). This material con In principal it can have unlimited applications. tains no detectable microbial residues. This material is a Food 0250. The sweetened composition comprises a beverage, Additive and Kosher Pareve. The ingredients in this material non-limiting examples of which include non-carbonated and are derived from recombinant microbial sources. Following carbonated beverages such as colas, ginger ales, root beers, are detailed product specifications: ciders, fruit-flavored soft drinks (e.g., citrus-flavored soft 0229. 1. Assay (wit/wt %): Greater than or equal to 95% drinks such as lemon-lime or orange), powdered soft drinks, Reb-A (on dry basis) and the like, fruit juices originating in fruits or vegetables, 0230 2. Total Steviol Glycosides (2 t?wt %): Greater than fruit juices including Squeezed juices or the like, fruit juices 95% (on dry basis) containing fruit particles, fruit beverages, fruit juice bever US 2016/0213039 A1 Jul. 28, 2016 ages, beverages containing fruit juices, beverages with fruit 1-L-phenylalanine 1-methyl ester, N—N-3-(3-methoxy-4- flavorings, vegetablejuices, juices containing vegetables, and hydroxyphenyl)propyl-L-a-aspartyl-L-phenylalanine mixed juices containing fruits and vegetables; sport drinks, 1-methyl ester, salts thereof, and the like, and combinations energy drinks, near water and the like drinks (e.g., water with thereof. natural or synthetic flavorants); tea type or favorite type bev 0259. In one embodiment rebA can be used in the combi erages such as coffee, cocoa, black tea, green tea, oolong tea nation with natural Sweetener Suppressors such as gymnemic and the like; beverages containing milk components such as acid, hodulcin, Ziziphin, lactisole, and the like. milk beverages, coffee containing milk components, cafe au 0260 The sweetener composition can be combined with lait, milk tea, fruit milk beverages, drinkable yogurt, lactic various umami taste enhancers. acid bacteria beverages or the like; and dairy products. 0261 The Sweetener composition can be formulated with 0251. The Sweetener composition described herein may amino acids including, but not limited to, aspartic acid, argi be incorporated as a high intensity natural Sweetener in food nine, glycine, glutamic acid, proline, threonine, theanine, stuffs, beverages, pharmaceutical compositions, cosmetics, cysteine, cystine, alanine, Valine, tyrosine, leucine, isoleu chewing gums, table top products, cereals, dairy products, cine, asparagine, serine, lysine, histidine, ornithine, methion toothpastes and other oral cavity compositions, etc. ine, carnitine, aminobutyric acid (alpha-, beta-, or gamma 0252. In addition, the Sweetener composition can be used isomers), glutamine, hydroxyproline, taurine, norvaline, as a sweetener not only for drinks, foodstuffs, and other sarcosine, and their salt forms such as Sodium or potassium products dedicated for human consumption, but also in ani salts or acid salts. The amino acid additives also may be in the mal feed and fodder with improved characteristics D- or L-configuration and in the mono-, di-, or tri-form of the 0253 During the manufacturing of foodstuffs, drinks, same or different amino acids. Additionally, the amino acids pharmaceuticals, cosmetics, table top products, chewing gum may be alpha-, beta-, y-, delta-, and -isomers if appro the conventional methods such as mixing, kneading, dissolu priate. Combinations of the foregoing amino acids and their tion, pickling, permeation, percolation, sprinkling, atomiz corresponding salts (e.g., Sodium, potassium, calcium, mag ing, infusing and other methods can be used. nesium salts or other alkali or alkaline earth metal salts 0254 The sweetener composition can be used in dry or thereof, or acid salts) also are suitable additives. The amino liquid forms. It can be added before or after heat treatment of acids may be natural or synthetic. The amino acids also may food products. The amount of the sweetener depends on the be modified. Modified amino acids refers to any amino acid purpose of usage. It can be added alone or in the combination wherein at least one atom has been added, removed, Substi with other compounds. tuted, or combinations thereof (e.g., N-alkyl amino acid, 0255. The Sweetener composition may be employed as the N-acyl amino acid, or N-methyl amino acid). Non-limiting sole sweetener, or it may be used together with other naturally examples of modified amino acids includeamino acid deriva occurring high intensity Sweeteners. tives such as trimethylglycine, N-methyl-glycine, and N-me 0256 The phrase “natural high intensity Sweeteners’, as thyl-alanine. As used herein, amino acids encompass both used herein, refers to any compositions which are found in modified and unmodified amino acids. As used herein, modi nature and which have Sweetness potency higher than fied amino acid also may encompass peptides and polypep Sucrose, fructose, or glucose. tides (e.g., dipeptides, tripeptides, tetrapeptides, and pen 0257 Non-limiting examples of natural high intensity tapeptides) such as glutathione and L-alanyl-L-glutamine. sweeteners include Stevioside, Rebaudioside A, Rebaudio 0262 The Sweetener composition may beformulated with side B, Rebaudioside C, Rebaudioside E. Rebaudioside F, polyamino acid additives include poly-L-aspartic acid, poly Rebaudioside M, Rebaudioside X, Steviolbioside, Dulcoside L-lysine (e.g., poly-L-a-lysine or poly-L--lysine), poly-L- A. Rubusoside, mogrosides, braZZein, glycyrrhizic acid and ornithine (e.g., poly-L- -ornithine or poly-L-f-ornithine), its salts, thaumatin, perillartine, pernandulcin, mukurozio poly-L-arginine, other polymeric forms of amino acids, and sides, baiyunoside, phlomisoside-I, dimethyl-hexahydrof salt forms thereof (e.g., magnesium, calcium, potassium, or luorene-dicarboxylic acid, abrusosides, periandrin, carnosi Sodium salts such as L-glutamic acid mono sodium salt). The flosides, cyclocarioside, pterocaryosides, polypodoside A, polyamino acid additives also may be in the D- or L-configu brazilin, hernandulcin, philodulcin, glycyphyllin, phlorizin, ration. Additionally, the polyamino acids may be a -, beta-, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, gamma-, delta-, and epsilon-isomers if appropriate. neoastilibin, ira"5-cinnamaldehyde, monatin and its salts, Combinations of the foregoing polyamino acids and their selligueain A, hematoxylin, monellin, osladin, pterocaryo corresponding salts (e.g., Sodium, potassium, calcium, mag side A, pterocaryoside B, mabinlin, pentadin, miraculin, cur nesium salts or other alkali or alkaline earth metal salts culin, neoculin, chlorogenic acid, cynarin, Luo Han Guo thereof or acid salts) also are suitable Sweet taste improving Sweetener, siamenoside and alike, and combinations thereof. additives in embodiments of this invention. The polyamino 0258. The sweetener composition may be used together acids described herein also may comprise co-polymers of with synthetic or artificial high intensity sweeteners. The differentamino acids. The polyamino acids may be natural or phrase “synthetic' or “artificial high intensity sweeteners', as synthetic. The polyamino acids also may be modified, such used herein, refers to any compositions which are not found in that at least one atom has been added, removed, Substituted, or nature and which have—Sweetness potency higher than combinations thereof (e.g., N-alkyl polyamino acid or N-acyl Sucrose, fructose, or glucose. Non-limiting examples of syn polyamino acid). As used herein, polyamino acids encompass thetic or artificial high intensity Sweeteners include Sucralose, both modified and unmodified polyamino acids. In accor potassium acesulfame, aspartame, alitame, Saccharin, neo dance with particular embodiments, modified polyamino hesperidin dihydrochalcone, cyclamate, neotame, dulcin, acids include, but are not limited to polyamino acids of vari Suosan, N N-3-(3-hydroxy-4-methoxyphenyl)propyl-L- ous molecular weights (MW), such as poly-L-a-lysine with a a-aspartyl-L-phenylalanine 1-methyl ester, N—N-3-(3- MW of 1,500, MW of 6,000, MW of 25,200, MW of 63,000, hydroxy-4-methoxyphenyl)-3-methylbutyl-L- -asparty MW of 83,000, or MW of 300,000. US 2016/0213039 A1 Jul. 28, 2016

0263. The Sweetener composition can be combined with cellobiose, amylopectin, glucosamine, mannosamine, polyols or sugar alcohols. The term “polyol refers to a mol fucose, glucuronic acid, gluconic acid, glucono-lactone, abe ecule that contains more than one hydroxyl group. A polyol quose, galactosamine, beet oligosaccharides, isomalto-oli may be a diol, triol, or a tetraol which contain 2, 3, and 4 gosaccharides (isomaltose, isomaltotriose, panose and the hydroxyl groups, respectively. A polyol also may contain like), Xylo-oligosaccharides (Xylotriose, Xylobiose and the more than four hydroxyl groups, such as a pentaol, hexaol, like), Xylo-terminated oligosaccharides, gentio-oligosaccha heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, rides (gentiobiose, gentiotriose, gentiotetraose and the like), respectively. Additionally, a polyol also may be a Sugar alco Sorbose, nigero-oligosaccharides, palatinose oligosaccha hol, polyhydric alcohol, or polyalcohol which is a reduced rides, fructooligosaccharides (kestose, nystose and the like), form of carbohydrate, wherein the carbonyl group (aldehyde maltotetraol, maltotriol, malto-oligosaccharides (maltotri or ketone, reducing Sugar) has been reduced to a primary or ose, maltotetraose, maltopentaose, maltohexaose, maltohep secondary hydroxyl group. taose and the like), starch, inulin, inulo-oligosaccharides, 0264. Non-limiting examples of polyols include erythri lactulose, melibiose, raffinose, ribose, isomerized liquid Sug tol, maltitol, mannitol, Sorbitol, lactitol. Xylitol, inositol, iso ars such as high fructose corn Syrups, coupling Sugars, and malt, propylene glycol, glycerol, threitol, galactitol, hydro Soybean oligosaccharides. Additionally, the carbohydrates as genated isomalitulose, reduced isomalto-oligosaccharides, used herein may be in either the D- or L-configuration. In the reduced Xylo-oligosaccharides, reduced gentio-oligosaccha formulations any combinations of the compounds can be rides, reduced maltose syrup, reduced glucose syrup, hydro used. genated Starch hydrolyzates, polyglycitols and Sugar alcohols 0268. In a particular embodiment rebA may beformulated or any other carbohydrates capable of being reduced which do with Sugar acids which is include, but are not limited to, not adversely affect the taste of the sweetener composition, aldonic, uronic, aldaric, alginic, gluconic, glucuronic, glu and combinations thereof. caric, galactaric, galacturonic, and their salts (e.g., Sodium, 0265. In one particular embodiment rebA can be com potassium, calcium, magnesium salts or other physiologi bined with reduced calorie Sweeteners such as D-tagatose, cally acceptable salts), and combinations thereof. L-Sugars, L-Sorbose, L-arabinose, and others and combina 0269. The Sweetener composition can be used in the com tions thereof. bination with various physiologically active Substances or 0266 The Sweetener composition can be combined with functional ingredients. Functional ingredients generally are various carbohydrates. The term “carbohydrate generally classified into categories such as carotenoids, dietary fiber, refers to aldehyde or ketone compounds substituted with fatty acids, Saponins, antioxidants, nutraceuticals, flavonoids, multiple hydroxyl groups, of the general formula (CH2O)m isothiocyanates, phenols, plant sterols and stanols (phytoster wherein “n” is 3-30, as well as their oligomers and polymers. ols and phytostanols); polyols; prebiotics, probiotics; phy The carbohydrates of the present invention can, in addition, toestrogens; soy protein, Sulfides/thiols; amino acids; pro be substituted or deoxygenated at one or more positions. teins; vitamins; and minerals. Functional ingredients also Carbohydrates, as used herein, encompass unmodified carbo may be classified based on their health benefits, such as hydrates, carbohydrate derivatives, substituted carbohy cardiovascular, cholesterol-reducing, and anti-inflammatory. drates, and modified carbohydrates. As used herein, the 0270. The Sweetener composition may include a flavoring phrases “carbohydrate derivatives”, “substituted carbohy agent which may be natural or artificial origin. As used herein, drate', and “modified carbohydrates' are synonymous. unless otherwise indicated, the term “flavor” means any food Modified carbohydrate means any carbohydrate wherein at grade material that may be added to the present compositions least one atom has been added, removed, Substituted, or com to provide a desired flavor to a foodstuff. The flavors useful in binations thereof. Thus, carbohydrate derivatives or substi the present invention include, for example, an essential oil, tuted carbohydrates include substituted and unsubstituted Such as an oil derived from a plant or a fruit, peppermint oil, monosaccharides, disaccharides, oligosaccharides, and spearmint oil, other mint oils, clove oil, cinnamon oil, oil of polysaccharides. The carbohydrate derivatives or substituted wintergreen, bay, thyme, cedar leaf, nutmeg, allspice, sage, carbohydrates optionally can be deoxygenated at any corre mace, and almonds. The flavoring agent may be a plant sponding C-position, and/or substituted with one or more extract or a fruit essence Such as apple, banana, watermelon, moieties such as hydrogen, halogen, haloalkyl, carboxyl, pear, peach, grape, Strawberry, raspberry, cherry, plum, pine acyl, acyloxy, amino, amido, carboxyl derivatives, alky apple, apricot, and mixtures thereof. The flavoring agent may lamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, be a citrus flavor, such as an extract, essence, or oil of lemon, cyano, Sulfo, mercapto, imino, Sulfonyl, Sulfenyl, Sulfinyl, lime, orange, tangerine, grapefruit, citron, or kumquat. Fla Sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphi Vors useful in the present invention also can include cream, nyl, phosphoryl, phosphino, thioester, thioether, oXimino, hazelnut, Vanilla, chocolate, cinnamon, pecan, lemon, lime, hydrazino, carbamyl, phospho, phosphonato, or any other raspberry, peach, mango, Vanillin, butter, butterscotch, tea, viable functional group provided the carbohydrate derivative orange, tangerine, caramel, Strawberry, banana, grape, plum, or substituted carbohydrate functions to improve the sweet cherry, blueberry, pineapple, elderberry, watermelon, bubble taste of the Sweetener composition. gum, cantaloupe, guava, kiwi, papaya, coconut, mint, spear 0267. Non-limiting examples of carbohydrates in embodi mint, derivatives, and combinations thereof. ments of this invention include tagatose, trehalose, galactose, 0271 The sweetener composition may include an aroma rhamnose, various cyclodextrins, cyclic oligosaccharides, component. As used herein, unless otherwise indicated, the various types of maltodextrins, dextran, Sucrose, glucose, term 'aroma component’ means any food-grade Volatile Sub ribulose, fructose, threose, arabinose, Xylose, lyxose, allose, stance that may be employed to produce a desired scent, for altrose, mannose, idose, lactose, maltose, invert Sugar, isotre example, when mixed with a foodstuff. Aromas useful in the halose, neotrehalose, isomalitulose, erythrose, deoxyribose, present invention include, for example, essential oils (citrus gulose, idose, talose, erythrulose, Xylulose, psicose, turanose, oil), expressed oils (orange oil), distilled oils (rose oil), US 2016/0213039 A1 Jul. 28, 2016

extracts (fruits), anethole (liquorice, anise seed, ouZo, fen selected from the group consisting of hydrogen, alkyl, alk nel), anisole (anise seed), benzaldehyde (marzipan, almond), enyl, alkynyl, halo, haloalkyl, carboxyl, acyl, acyloxy, amino, benzyl alcohol (marzipan, almond), camphor (cinnamomum amido, carboxyl derivatives, alkylamino, dialkylamino, ary camphora), cinnamaldehyde (cinnamon), citral (citronella lamino, alkoxy, aryloxy, nitro, cyano, Sulfo, thiol, imine, Sul oil, lemon oil), d-limonene (orange) ethylbutanoate (pine fonyl, Sulfenyl, Sulfinyl, Sulfamyl, carboxalkoxy, carboxa apple), eugenol (clove oil), furaneol (Strawberry), furfural mido, phosphonyl, phosphinyl, phosphoryl, phosphino, (caramel), linalool (coriander, rose wood), menthol (pepper thioester, thioether, anhydride, oximino, hydrazino, car mint), methylbutanoate (apple, pineapple), methyl salicylate bamyl, phospho, phosphonato, and any other viable func (oil of wintergreen), neral (orange flowers), nerolin (orange tional group, provided the Substituted organic acid salt addi flowers), pentylbutanoate (pear, apricot), pentyl pentanoate tive functions to improve the sweet taste of the Sweetener (apple, pineapple), Sotolon (maple syrup, curry, fennugreek), composition. Strawberry ketone (strawberry), Substituted pyrazines, e.g., 0275. The compositions with rebA can comprise an inor 2-ethoxy-3-isopropylpyrazine, 2-methoxy-3-sec-butylpyra ganic acid additive for use in embodiments of this invention. Zine; and 2-methoxy-3-methylpyrazine (toasted seeds of They include, but are not limited to, phosphoric acid, phos fenugreek, cumin, and coriander), thujone (juniper, common phorous acid, polyphosphoric acid, hydrochloric acid, Sulfu sage, Nootka cypress, and wormwood), thymol (camphor ric acid, carbonic acid, Sodium dihydrogen phosphate, and like), trimethylamine (fish), Vanillin (vanilla), and combina their corresponding alkali or alkaline earth metal salts thereof tions thereof. Preferred aroma components according to the (e.g., inositol hexaphosphate Mg,Ca). present invention include, essential oils (citrus oil), expressed 0276. The sweetener composition can comprise a bitter oils (orange oil), distilled oils (rose oil), extracts (fruits), compound additive for use in embodiments of this invention, benzaldehyde, d-limonene, furfural, menthol, methyl but are not limited to, caffeine, quinine, urea, bitter orange oil, butanoate, pentylbutanoate, salts, derivatives, and combina naringin, quassia, and salts thereof. tions thereof. 0277. The sweetener composition can comprise an artifi 0272. The sweetener composition can comprise a nucle cial or natural Sweetness enhancers and combinations otide additive for use in embodiments of this invention. They thereof. include, but are not limited to, inosine monophosphate, gua 0278. The sweetener composition may include a polymer nosine monophosphate, adenosine monophosphate, cytosine additives for use in embodiments of this invention, but are not monophosphate, uracil monophosphate, inosine diphos limited to, chitosan, pectin, pectic, pectinic, polyuronic, phate, guanosine diphosphate, adenosine diphosphate, polygalacturonic acid, starch, food hydrocolloid or crude cytosine diphosphate, uracil diphosphate, inosine triphos extracts thereof (e.g., gum acacia Senegal (FibergumTM), gum phate, guanosine triphosphate, adenosine triphosphate, acacia Seyal, carageenan), poly-L-lysine (e.g., poly-L-a- cytosine triphosphate, uracil triphosphate, and their alkali or lysine or poly-L-f-lysine), poly-L-ornithine (e.g., poly-L-a- alkaline earth metal salts, and combinations thereof. The ornithine or poly-L-epsilon-ornithine), polyarginine, nucleotides described herein also may comprise nucleotide polypropylene glycol, polyethylene glycol, poly(ethylene related additives such as nucleosides or nucleic acid bases glycol methyl ether), polyaspartic acid, polyglutamic acid, (e.g., guanine, cytosine, adenine, thymine, uracil). polyethyleneimine, alginic acid, sodium alginate, propylene 0273. The sweetener composition can comprise an glycol alginate, Sodium hexametaphosphate (SHMP) and its organic acid additive. Organic acids are compounds which salts, and sodium polyethyleneglycolalginate and other cat comprises a —COOH moiety. Suitable organic acid additives ionic and anionic polymers. for use in embodiments of this invention include, but are not 0279. The Sweetener compositions may include a protein limited to, C2-C30 carboxylic acids, substituted hydroxy or protein hydrolyzates additives for use in embodiments of 1C1-C30 carboxylic acids, benzoic acid, substituted benzoic this invention, but are not limited to, bovine serum albumin, acids (e.g. 2,4-dihydroxybenzoic acid), Substituted cinnamic whey protein (including fractions or concentrates thereof acids, hydroxyacids, Substituted hydroxybenzoic acids, Sub such as 90% instant whey protein isolate, 34% whey protein, stituted cyclohexyl carboxylic acids, tannic acid, lactic acid, 50% hydrolyzed whey protein, and 80% whey protein con tartaric acid, citric acid, gluconic acid, glucoheptonic acids, centrate), soluble rice protein, soy protein, protein isolates, adipic acid, hydroxycitric acid, malic acid, fruitaric acid (a protein hydrolyzates, reaction products of protein hydrolyZ blend of malic, fumaric, and tartaric acids), fumaric acid, ates, glycoproteins, and/or proteoglycans containing amino maleic acid. Succinic acid, chlorogenic acid, Salicylic acid, acids (e.g., glycine, alanine, Senrne, threonine, asparagine, creatine, glucosamine hydrochloride, glucono delta lactone, glutamine, arginine, Valine, isoleucine, leucine, norvaline, caffeic acid, bile acids, acetic acid, ascorbic acid, alginic acid, methionine, proline, tyrosine, hydroxyproline, and the like), crythorbic acid, polyglutamic acid, and their alkali or alkaline collagen (e.g., gelatin), partially hydrolyzed collagen (e.g., earth metal salt derivatives thereof. In addition, the organic hydrolyzed fish collagen), and collagen hydrolyzates (e.g., acid additives also may be in either the D- or L-configuration. porcine collagen hydrolyzates). 0274 The sweetener composition can comprise an 0280. The Sweetener composition may include a surfac organic acid salt additive. They include, but are not limited to, tant additives for use in embodiments of this invention, but are Sodium, calcium, potassium, and magnesium salts of all not limited to, polysorbates (e.g., polyoxyethylene Sorbitan organic acids, such as salts of citric acid, malic acid, tartaric monooleate (polysorbate 80), polysorbate 20, polysorbate acid, flunaric acid, lactic acid (e.g., sodium lactate), alginic 60), sodium dodecylbenzenesulfonate, dioctylsulfosuccinate acid (e.g., Sodium alginate), ascorbic acid (e.g., Sodium ascor or dioctyl Sulfo Succinate sodium, sodium dodecyl sulfate, bate), benzoic acid (e.g., sodium benzoate or potassium ben cetylpyridinium chloride (hexadecylpyridinium chloride), Zoate), and adipic acid. The examples of the Sweet taste hexadecyltrimethylammonium bromide, Sodium cholate, improving organic acid salt additives described optionally carbamoyl, choline chloride, Sodium glycocholate, sodium may be substituted with one or more of the following moiety taurodeoxycholate, lauric arginate, sodium Stearoyl lactylate, US 2016/0213039 A1 Jul. 28, 2016

Sodium taurocholate, lecithins, sucrose oleate esters, Sucrose green beans, broccoli, cauliflower, carrots, potatoes, celery. Stearate esters, sucrose palmitate esters, Sucrose laurate Plant foods such as bran, nuts, and seeds (such as flaxseeds) esters, and other emulsifiers, and the like. are also sources of dietary fiber. Parts of plants providing 0281. A rebA formulation may include a flavonoid addi dietary fiber include, but are not limited to, the stems, roots, tives for use in embodiments of this invention generally are leaves, seeds, pulp, and skin. classified as flavonols, flavones, flavanones, flavan-3-ols, 0288 Although dietary fiber generally is derived from isoflavones, oranthocyanidins. Non-limiting examples offla plant sources, indigestible animal products such as chitins are vonoid additives include catechins (e.g., green tea extracts), also classified as dietary fiber. Chitin is a polysaccharide polyphenols, rutins, neohesperidin, naringin, neohesperidin composed of units of acetylglucosamine joined by 5(I-4) dihydrochalcone, and the like. linkages, similar to the linkages of cellulose. 0282. The formulation may include an alcohol additives 0289. The Sweetener composition may comprise an anti for use in embodiments of this invention include, but are not oxidant. Examples of suitable antioxidants for embodiments limited to, ethanol. 00192 The formulation may include an of this invention include, but are not limited to, vitamins, astringent compound additives include, but are not limited to, Vitamin cofactors, minerals, hormones, carotenoids, caro tannic acid, europium chloride (EUC3), gadolinium chloride tenoid terpenoids, non-carotenoid terpenoids, flavonoids, fla (GdC), terbium chloride (TbCb), alum, tannic acid, and vonoid polyphenolics (e.g., bioflavonoids), fiavonols, fla polyphenols (e.g., tea polyphenols). Vones, phenols, polyphenols, esters of phenols, esters of 0283. The sweetener composition may include a vitamin. polyphenols, nonflavonoid phenolics, isothiocyanates, and Vitamins are organic compounds that the human body needs combinations thereof. In some embodiments, the antioxidant in Small quantities for normal functioning. The body uses may include vitamin A, vitamin C, Vitamin E., ubiquinone, Vitamins withoutbreaking them down, unlike other nutrients mineral selenium, manganese, melatonin, a-carotene, Such as carbohydrates and proteins. The vitamins for use in f-carotene, lycopene, lutein, Zeanthin, crypoxanthin, reser embodiment include, but not limited to, vitamin A (retinol, Vatol, eugenol, quercetin, catechin, gossypol, hesperetin, cur retinaldehyde, retinoic acid, retinoids, retinal, retinoic acid), cumin, ferulic acid, thymol, hydroxytyrosol, tumeric, thyme, vitamin D (vitamins D1–D5; cholecalciferol, lumisterol, olive oil, lipoic acid, glutathinone, gulamine, oxalic acid, ergocalciferol, dihydrotachysterol, 7-dehydrocholesterol), tocopherol-derived compounds, butylated hydroxyanisole, Vitamin E (eocopherol, tocotrienol), vitamin (phylloquinone, butylated hydroxyoluene, ethylenediaminetetraacetic acid, naphthoquinone), vitamin BI (thiamin), vitamin B2 (ribofla tert-butylhydroquinone, acetic acid, pectin, tocotrienol, toco vin, Vitamin G), vitamin B3 (niacin, nicotinic acid, vitamin pherol, coenzyme Q10, zeaxanthin, astaxanthin, canthaxan PP), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, tin, Saponins, limonoids, kaempfedrol, myricetin, isorhamne pyridoxal, pyridoxamine), vitamin B7 (biotin, vitamin H), tin, proanthocyanidins, quercetin, rutin, luteolin, apigenin, vitamin B9 (folic acid, folate, folacin, vitamin M, pteroyl-L- tangeritin, hesperetin, naringenin, erodictyol, flavan-3-ols glutamic acid), Vitamin B12 (cobalamin, cyanocobalamin), (e.g., anthocyanidins), gallocatechins, epicatechin and its and vitamin C (ascorbic acid). gallate forms, epigallocatechin and its gallate forms theafla 0284 Various other compounds have been classified as vin and its gallate forms, thearubigins, isotlavone phytoestro Vitamins by some authorities. These compounds may be gens, genistein, daidZein, glycitein, anythocyanins, cyanid termed pseudo-Vitamins and include, but are not limited to, ing, delphinidin, malvidin, pelargonidin, peonidin, petunidin, compounds Such as ubiquinone (coenzyme Q10), pangamic ellagic acid, gallic acid, salicylic acid, rosmarinic acid, cin acid, dimethylglycine, taestrile, amygdaline, flavanoids, namic acid and its derivatives (e.g., ferulic acid), chlorogenic para-aminobenzoic acid, adenine, adenylic acid, and S-meth acid, chicoric acid, gallotannins, ellagitannins, anthoxan ylmethionine. As used herein, the term vitamin includes thins, betacyanins and other plant pigments, silymarin, citric pseudo-Vitamins. acid, lignan, antinutrients, bilirubin, uric acid, R-.alpha.-li 0285. The formulation with rebA may include a dietary poic acid, N-acetylcysteine, emblicanin, apple extract, apple fiber. Dietary fiber, also known as bulk or roughage, is the skin extract (applephenon), rooibos extract red, rooibos portion of food resistant to hydrolysis by human digestive extract, green hawthornberry extract, red raspberry extract, enzymes and generally comprises the indigestible portion of green coffee antioxidant, aronia extract 20% grape seed plant materials that moves through the digestive system and extract, cocoa extract, hops extract, mangosteen extract, man stimulates the intestine to peristalsis. gosteen hull extract, cranberry extract, pomegranate extract, 0286 Numerous polymeric carbohydrates having signifi pomegranate hull extract, pomegranate seed extract, haw cantly different structures in both composition and linkages thornberry extract, pomella pomegranate extract, cinnamon fall within the definition of dietary fiber. Such compounds are hark extract, grape skin extract, bilberry extract, pine bark well known to those skilled in the art, non-limiting examples extract, pycnogenol, elderberry extract, mulberry root of which include non-starch polysaccharides, lignin, cellu extract, wolfberry (gogi) extract, blackberry extract, blue lose, methylcellulose, the hemicelluloses, 2-glucans, pectins, berry extract, blueberry leaf extract, raspberry extract, tur gums, mucilage, waxes, inulin, oligosaccharides, fructooli meric extract, citrus bioflavonoids, black currant, ginger, acai gosaccharides, cyclodextrins, chitins, and combinations powder, green coffee bean extract, green tea extract, and thereof. phytic acid, or combinations thereof. In alternate embodi 0287 Food sources of dietary fiber include, but are not ments, the antioxidant may comprise a synthetic antioxidant limited to, grains, legumes, fruits, and vegetables. Grains such as butylated hydroxytolune or butylated hydroxyani providing dietary fiber include, but are not limited to, oats, sole, for example. Other sources of suitable antioxidants for rye, barley, wheat. Legumes providing fiber include, but are embodiments of this invention include, but are not limited to, not limited to, peas and beans such as soybeans. Fruits and fruits, vegetables, tea, cocoa, chocolate, spices, herbs, rice, Vegetables providing a source of fiber include, but are not organ meats from livestock, yeast, whole grains, or cereal limited to, apples, oranges, pears, bananas, berries, tomatoes, grains. US 2016/0213039 A1 Jul. 28, 2016

0290. Some antioxidants belong to the class of phytonu omega-9 fatty acid precursors), and esterified fatty acids. As trients called polyphenols (also known as "polyphenolics’), used herein, “long chain polyunsaturated fatty acid refers to which are a group of chemical Substances found in plants, any polyunsaturated carboxylic acid or organic acid with a characterized by the presence of more than one phenol group longaliphatic tail. As used herein, "omega-3 fatty acid refers per molecule. A variety of health benefits may derived from to any polyunsaturated fatty acid having a first double bond as polyphenols, including prevention of cancer, heart disease, the third carbon-carbon bond from the terminal methylend of and chronic inflammatory disease and improved mental its carbon chain. In particular embodiments, the omega-3 strength and physical strength, for example. Suitable fatty acid may comprise a long chain omega-3 fatty acid. As polyphenols for embodiments of this invention, include cat used herein, "omega-6 fatty acid any polyunsaturated fatty echins, proanthocyanidins, procyanidins, anthocyanins, acid having a first double bond as the sixth carbon-carbon quercerin, rutin, reservatrol, isoflavones, curcumin, punicala bond from the terminal methyl end of its carbon chain. gin, ellagitannin, hesperidin, naringin, citrus flavonoids, 0293. The Sweetener composition may include a salt. The chlorogenic acid, other similar materials, and combinations term "salt” also refers to complexes that retain the desired thereof. chemical activity of the Sweet taste improving compositions 0291 Suitable sources of catechins for embodiments of of the present invention and are safe for human or animal this invention include, but are not limited to, green tea, white consumption in a generally acceptable range. Alkali metal tea, black tea, oolong tea, chocolate, cocoa, red wine, grape (for example, sodium or potassium) or alkaline earth metal seed, red grape skin, purple grape skin, red grape juice, purple (for example, calcium or magnesium) salts also can be made. grape juice, berries, pycnogenol, and red apple peel. Suitable Salts also may include combinations of alkali and alkaline Sources of Such antioxidants as proanthocyanidins and pro earth metals. Non-limiting examples of Such salts are (a) acid cyanidins for embodiments of this invention include, but are addition salts formed with inorganic acids and salts formed not limited to, red grapes, purple grapes, cocoa, chocolate, with organic acids; (b) base addition salts formed with metal grape seeds, red wine, cacao beans, cranberry, apple peel, cations such as calcium, bismuth, barium, magnesium, alu plum, blueberry, black currants, chokeberry, green tea, Sor minum, copper, cobalt, nickel, cadmium, sodium, potassium, ghum, cinnamon, barley, red kidney bean, pinto bean, hops, and the like, or with a cation formed from ammonia, N.Nr almonds, hazelnuts, pecans, pistachio, pycnogenol, and col dibenzylethylenediamine, D-glucosamine, tetraethylammo orful berries. Suitable sources of anthocyanins for embodi nium, or ethylenediamine; or (c) combinations of (a) and (b). ments of this invention include, but are not limited to, red Thus, any salt forms which may be derived from the Sweet berries, blueberries, bilberry, cranberry, raspberry, cherry, taste improving compositions may be used with the embodi pomegranate, Strawberry, elderberry, chokeberry, red grape ments of the present invention as long as the salts of the Sweet skin, purple grape skin, grape seed, red wine, black currant, taste improving additives do not adversely affect the taste of red currant, cocoa, plum, apple peel, peach, red pear, red the Sweetener compositions comprising the at least one natu cabbage, red onion, red orange, and blackberries. Suitable ral and/or synthetic high-potency Sweetener. The salt forms of Sources of quercetin and rutin for embodiments of this inven the additives can be added to the natural and/or synthetic tion include, but are not limited to, red apples, onions, kale, Sweetener composition in the same amounts as their acid or bog whortleberry, lingonberrys, chokeberry, cranberry, base forms. blackberry, blueberry, strawberry, raspberry, black currant, green tea, black tea, plum, apricot, parsley, leek, broccoli, 0294 Suitable inorganic salts may include, but are not chili pepper, berry wine, and ginkgo. Suitable sources of limited to, sodium chloride, potassium chloride, Sodium Sul resveratrol for embodiments of this invention include, but are fate, potassium citrate, europium chloride (EuC), gadolinium not limited to, red grapes, peanuts, cranberry, blueberry, bil chloride (GdCb), terbium chloride (TbCb), magnesium sul berry, mulberry, Japanese Itadori tea, and red wine. Suitable fate, alum, magnesium chloride, mono-di-, tri-basic sodium sources of isoflavones for embodiments of this invention or potassium salts of phosphoric acid (e.g., inorganic phos include, but are not limited to, soy beans, soy products, phates), salts of hydrochloridic acid (e.g., inorganic chlo legumes, alfalfa spouts, chickpeas, peanuts, and red clover. rides), sodium carbonate, Sodium bisulfate, and Sodium bicar Suitable sources of curcumin for embodiments of this inven bonate. Furthermore, in particular embodiments, suitable tion include, but are not limited to, turmeric and mustard. organic salts useful as Sweet taste improving additives Suitable sources of punicalagin and elagitannin for embodi include, but are not limited to, choline chloride, alginic acid ments of this invention include, but are not limited to, pome Sodium salt (sodium alginate), glucoheptonic acid sodium granate, raspberry, Strawberry, walnut, and oak-aged red salt, gluconic acid sodium salt (sodium gluconate), gluconic wine. Suitable sources of citrus flavonids, such as hesperidin acid potassium salt (potassium gluconate), guanidine HC1. ornaringin, for embodiments of this invention include, but are glucosamine HC1, amriloride HC1, monosodium glutamate, not limited to, oranges, grapefruits, and citrus juices. Suitable adenosine monophosphate salt, magnesium gluconate, potas Sources of chlorogenic acid for embodiments of this invention sium tartrate (monohydrate), and Sodium tartrate (dihydrate). include, but are not limited to, green coffee, yerba mate, red 0295 The Sweetener composition can be applied as high wine, grape seed, red grape skin, purple grape skin, red grape intensity Sweetener to produce Zero calorie, reduced calorie juice, purple grape juice, applejuice, cranberry, pomegranate, or diabetic beverages and food products with improved taste blueberry, Strawberry, Sunflower, Echinacea, pycnogenol, characteristics. Also it can be used in drinks, foodstuffs, phar and apple peel. maceuticals, and other products in which Sugar cannot be 0292. The sweetener composition may include fatty acids. used. As used herein, “fatty acid refers to any straight chain mono 0296. The Sweetener composition can be used as a sweet carboxylic acid and includes saturated fatty acids, unsatur ener not only for drinks, foodstuffs, and other products dedi ated fatty acids, long chain fatty acids, medium chain fatty cated for human consumption, but also in animal feed and acids, short chain fatty acids, fatty acid precursors (including fodder with improved characteristics. US 2016/0213039 A1 Jul. 28, 2016 18

0297. The sweetener composition can be used as sweet including Sachets, packets, bulk bags or boxes, cubes, tablets, ening compound can be as alcoholic beverages such as Vodka, mists, or dissolvable strips. The composition can be delivered wine, beer, liquor, sake, etc.; natural juices, refreshing drinks, as a unit dose or in bulk form. carbonated Soft drinks, diet drinks, Zero calorie drinks, 0305 For liquid sweetener systems and compositions con reduced calorie drinks and foods, yogurt drinks, instant Venient ranges of fluid, semi-fluid, paste and cream forms, juices, instant coffee, powdered types of instant beverages, appropriate packing using appropriate packing material in canned products, syrups, fermented soybean paste, soy sauce, any shape or form shall be invented which is convenient to Vinegar, dressings, mayonnaise, ketchups, curry, Soup, instant carry or dispense or store or transport any combination con bouillon, powdered soy sauce, powdered vinegar, types of taining any of the above Sweetener products or combination biscuits, rice biscuit, crackers, bread, chocolates, caramel, of product produced above. candy, chewing gum, jelly, pudding, preserved fruits and 0306 The sweetener composition may include various Vegetables, fresh cream, jam, marmalade, flower paste, pow bulking agents, functional ingredients, colorants, flavors. A dered milk, ice cream, Sorbet, vegetables and fruits packed in reference hereinto a patent document or other matter which is bottles, canned and boiled beans, meat and foods boiled in given as prior art is not to be taken as an admission that that Sweetened sauce, agricultural vegetable food products, sea document or matter was known or that the information it food, ham, sausage, fish ham, fish sausage, fish paste, deep contains was part of the common general knowledge as at the fried fish products, dried seafood products, frozen food prod priority date of any of the claims. ucts, preserved seaweed, preserved meat, tobacco, medicinal 0307. The disclosure of each reference set forth herein is products, and many others. In principal it can have unlimited incorporated herein by reference in its entirety. applications. 0308 The following Examples illustrate preferred 0298. The Sweetened composition comprises a beverage, embodiments of the invention for the fermentatively pro non-limiting examples of which include non-carbonated and duced and purified Rebaudioside A as per product specifica carbonated beverages such as colas, ginger ales, root beers, tions and related compounds and the use thereof in foodstuffs ciders, fruit-flavored soft drinks (e.g., citrus-flavored soft and pharmaceuticals. Accordingly, the present invention is drinks such as lemon-lime or orange), powdered soft drinks, further illustrated by the following Examples: and the like, fruit juices originating in fruits or vegetables, fruit juices including Squeezed juices or the like, fruit juices EXAMPLES containing fruit particles, fruit beverages, fruit juice bever ages, beverages containing fruit juices, beverages with fruit General flavorings, vegetablejuices, juices containing vegetables, and 0309 Standard genetic techniques, such as overexpres mixed juices containing fruits and vegetables; sport drinks, sion of enzymes in the host cells, as well as for additional energy drinks, near water and the like drinks (e.g., water with genetic modification of host cells, are known methods in the natural or synthetic flavorants); tea type or favorite type bev art, such as described in Sambrook and Russel (2001) erages such as coffee, cocoa, black tea, green tea, oolong tea “Molecular Cloning: A Laboratory Manual (3" edition), and the like; Cold Spring Harbor Laboratory, Cold Spring Harbor Labo 0299 beverages containing milk components such as milk ratory Press, or F. Ausubel et al., eds. “Current protocols in beverages, coffee containing milk components, cafe au lait, molecular biology'. Green Publishing and Wiley Inter milk tea, fruit milk beverages, drinkable yogurt, lactic acid science, New York (1987). Methods for transformation and bacteria beverages or the like; and dairy products. genetic modification of fungal host cells are known from e.g. 0300 Generally, the amount of sweetener composition EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO present in a Sweetened composition varies widely depending OO/37671. on the particular type of Sweetened composition and its 0310. A description of the sequences is set out in Table 1. desired sweetness. Those of ordinary skill in the art can Sequences described herein may be defined with reference to readily discern the appropriate amount of Sweetener to put in the sequence listing or with reference to the database acces the Sweetened composition. sion numbers also set out in Table 1. 0301 The sweetener composition can be used in dry or liquid forms. It can be added before or after heat treatment of Example 1 food products. The amount of the sweetener depends on the purpose of usage. It can be added alone or in the combination Over-Expression of ERG20, BTS1 and thMG in S. with other compounds. cerevisiae 0302. During the manufacturing of foodstuffs, drinks, 0311. For over-expression of ERG20, BTS1 thMG1, pharmaceuticals, cosmetics, table top products, chewing gum expression cassettes were designed to be integrated in one the conventional methods such as mixing, kneading, dissolu locus using technology described in co-pending patent appli tion, pickling, permeation, percolation, sprinkling, atomiz cation no. PCT/EP2013/056623. To amplify the 5' and 3' ing, infusing and other methods can be used. integration flanks for the integration locus, Suitable primers 0303 Thus, products of the present invention can be made and genomic DNA from a CEN.PKyeaststrain (van Dijken et by any method known to those skilled in the art that provide al. Enzyme and Microbial Technology 26 (2000) 706-714) homogenous even or homogeneous mixtures of the ingredi was used. The different genes were ordered as cassettes (con ents. These methods include dry blending, spray drying, taining homologous sequence, promoter, gene, terminator, agglomeration, wet granulation, compaction, co-crystalliza homologous sequence) at DNA2.0. The genes in these cas tion and the like. settes were flanked by constitutive promoters and termina 0304. In solid form the Sweetening composition of the tors. SeeTable 2. Plasmid DNA from DNA2.0 containing the present invention can be provided to consumers in any form ERG20, thMG 1 and BTS1 cassettes were dissolved to a suitable for delivery into the comestible to be sweetened, concentration of 100 ng/ul. In a 50 ul PCR mix 20 ng template US 2016/0213039 A1 Jul. 28, 2016 was used together with 20 pmol of the primers. The material diagnostic PCR and sequencing. The schematic of performed was dissolved to a concentration of 0.5 g/ul. transformation of the Erg9-KD construct is illustrated in FIG. 3. The Strain was named STV003. TABLE 2 Example 3 Composition of the over-expression constructs.

Promoter ORF Terminator Over-Expression of UGT2 1 a Eno.2 Erg20 Adh1 0317 For over-expression of UGT2 1a, technology was (SEQID NO: 201) (SEQID NO: 81) (SEQID NO: 212) used as described in co-pending patent application nos. PCT/ Fba.1 tEHMG1 Adh2 EP2013/056623 and PCT/EP2013/055047. The UGT2 1a (SEQID NO: 202) (SEQID NO: 79) (SEQID NO: 213) was ordered as a cassette (containing homologous sequence, Tef1 BtS1 Gmp1 promoter, gene, terminator, homologous sequence) at DNA2. (SEQID NO: 203) (SEQID NO: 83) (SEQID NO: 214) 0. For details, see Table 4. To obtain the fragments containing the marker and Cre-recombinase, technology was used as 0312 For amplification of the selection marker, the described in co-pending patent application no. PCT/EP2013/ pUG7-EcoRV construct (FIG. 1) and suitable primers were 055047. The NAT marker, conferring resistance to nourseo used. The KanMX fragment was purified from gel using the thricin was used for selection. Zymoclean Gel DNA Recovery kit (ZymoResearch). Yeast strain Cen.PK113-3C was transformed with the fragments TABLE 4 listed in Table 3. Composition of the over-expression construct TABLE 3 Promoter ORF Terminator DNA fragments used for transformation Pgk1 UGT2 1 a Adh2 of ERG20, tEHMG1 and BTS1 (SEQID NO:204) (SEQID NO: 87) (SEQID NO: 213) Fragment

SYPRcTal3 0318 Suitable primers were used for amplification. To ERG20 cassette amplify the 5' and 3' integration flanks for the integration tHMG1 cassette locus, suitable primers and genomic DNA from a CEN.PK KanMX cassatte yeast Strain was used. BTS1 cassette 0319 S. cerevisiae yeast strain STV003 was transformed 3YPRcTal3 with the fragments listed in Table 5, and the transformation mix was plated on YEPhD agar plates containing 50 g/ml 0313. After transformation and recovery for 2.5 hours in nourseothricin (Lexy NTC from Jena Bioscience). YEPhD (yeast extract phytone peptone glucose: BBL Phy tone Peptone from BD) at 30° C. the cells were plated on TABLE 5 YEPhD agar with 200 g/ml G418 (Sigma). The plates were incubated at 30°C. for 4 days. Correct integration was estab DNA fragments used for transformation of UGT2 la lished with diagnostic PCR and sequencing. Over-expression Fragment was confirmed with LC/MS on the proteins. The schematic of S'Chr09.01 the assembly of ERG20, thMG1 and BTS1 is illustrated in UGT2 1a cassette FIG. 2. This Strain is named STV002. NATCR 0314 Expression of the CRE-recombinase in this strain RE led to out-recombination of the KanMX marker. Correct out 3'Chr09.01 recombination, and presence of ERG20, thMG and BTS1 was established with diagnostic PCR. 0320 Expression of the CRE recombinase is activated by the presence of galactose. To induce the expression of the Example 2 CRE recombinase, transformants were restreaked on YEPh Galactose medium. This resulted in out-recombination of the Knock Down of Erg9 marker(s) located between lox sites. Correct integration of the 0315 For reducing the expression of Erg9, an Erg9 knock UGT2a and out-recombination of the NAT marker was con down construct was designed and used that contains a modi firmed with diagnostic PCR. The resulting strain was named fied 3' end, that continues into the TRP1 promoter driving STV004. The schematic of the performed transformation of TRP1 expression. the UGT2 la construct is illustrated in FIG. 4. 0316 The construct containing the Erg9-KD fragment was transformed to E. coli TOP10 cells. Transformants were Example 4 grown in 2PY (2 times Phytone peptone Yeast extract), SAMP medium. Plasmid DNA was isolated with the QIAprep Spin Over-Expression of Production Pathway to RebA: Miniprep kit (Qiagen) and digested with Sall-HF (New CPS, KS, KO, KAH, CPR, UGT1, UGT3 and UGT4 England Biolabs). To concentrate, the DNA was precipitated 0321 All pathway genes leading to the production of with ethanol. The fragment was transformed to S. cerevisiae, RebA were designed to be integrated in one locus using and colonies were plated on mineral medium (Verduyn et al. technology described in co-pending patent application no. 1992. Yeast 8:501-517) agar plates without tryptophan. Cor PCT/EP2013/056623. To amplify the 5' and 3' integration rect integration of the Erg9-KD construct was confirmed with flanks for the integration locus, Suitable primers and genomic US 2016/0213039 A1 Jul. 28, 2016 20

DNA from a CEN.PK yeast strain was used. The different Example 5 genes were ordered as cassettes (containing homologous sequence, promoter, gene, terminator, homologous sequence) Construction of Strain STVO27 at DNA2.0 (see Table 5 for overview). The DNA from DNA2.0 was dissolved to 100 ng/ul. This stock solution was 0324. To remove the KanMX marker from the chromo further diluted to 5 ng/ul, of which 1 ul was used in a 50 some of strain STV016, this strain was transformed with ul-PCR mixture. The reaction contained 25 pmol of each plasmid pSH65, expressing Cre-recombinase (Gildender, primer. After amplification, DNA was purified with the 2002). Subsequently plasmid pSH65 was cured from the NucleoSpin 96 PCR Clean-up kit (Macherey-Nagel) or alter strain by growing on non-selective medium (YEP 2% glu natively concentrated using ethanol precipitation. cose). The resulting, KanMX-free and pSH65-free strains, as determined by plating on plates containing 200 ug G418/ml TABLE 6 or 20 ug phleomycin/ml, where no growth should occur, was named STV027. Absence of the KanMX marker was further Sequences used for production pathway to RebA more confirmed with diagnostic PCR. SEQ Promoter ORF ID Terminator Example 6 KI prom 12-pro ErCPS SR 61 ScADH2ter Preparation of Fermentative Steviol Glycoside (SEQID NO: 205) (SEQID NO:) Sc PGK1.pro ErKS SR 65 ScTAL1ter Compositions (SEQID NO:204) (SEQID NO:215) Sc ENO2.pro KO 2 23 SCTPI1.ter 0325 The microbial production strain STV027 con (SEQID NO: 201) (SEQID NO: 216) structed as described above is used for production offermen Aglox TEF1.pro KANMIX 211 Ag TEF1 lox.ter tative RebaudiosideA. The pH is controlled at 5.0 by addition (SEQID NO: 206) (SEQID NO:217) Sc TEF1 pro KAH 4 33 SC GPM1.ter of ammonia (12.5 wt %). Temperature is controlled at 27°C. (SEQID NO: 203) (SEQID NO: 214) pO2 is controlled at 40% by adjusting the stirrer speed. Glu KI prom 6.pro CPR SR S9 SC PDC1 ter cose concentration is kept limited by controlled feed to the (SEQID NO: 207) (SEQID NO: 218) fermenter. Subsequently, 6 ml of the content of the shake KI prom 3-pro UGT1 SR 71 ScTDH1.ter (SEQID NO: 221) (SEQID NO: 219) flask is transferred into a fermenter (starting volume 0.3 L), KI prom 2-pro UGT3 SR 73 ScADH1ter which contained the medium as set out in the Examples of (SEQID NO: 222) (SEQID NO. 212) PCT/EP2013/051262. The pH is controlled between pH 4.0 Sc FBA1, pro UGT4 SR 7S SCENO1ter and pH 8.0 by addition of ammonia (12.5 wt %). Temperature (SEQID NO: 202) (SEQID NO: 220) is controlled between 20 and 45° C. p02 is controlled between 05-40% by adjusting the stirrer speed. Glucose concentration 0322 All fragments for the pathway to RebA, the marker is kept limited by controlled feed to the fermenter. After the and the flanks (see overview in Table 7) were transformed to completion of fermentation, microbial production host cells S. cerevisiae yeaststrainSTV004. After overnight recovery in are removed and the fermentation broth was processed as per YEPhD at 20° C. the transformation mixes were plated on the unit operations illustrated in FIG. 7 and/or modification YEPhD agar containing 200 ug/ml G418. These were incu thereof. In the case of Example 7, microbial production host bated 3 days at 25°C. and one night at RT. cells were removed and the fermentation broth was processed as per Example 7 of U.S. Ser. No. 13/956,144. TABLE 7 0326 Fermentatively produced Reb-A was analytically characterized meeting purity and product specification using DNA fragments used for transformation of CPS, KS KO, KanMX, KAH, CPR, UGT1, UGT3 and UGT4. methods known in the art. Fragment Example 7 5INT1 CPS cassette Comparison of Fermentatively-Produced KS cassette Rebaudioside A with Plant Derived Rebaudioside A KO cassette KanMX cassette in Four Applications KAH cassette CPR cassette 0327 Fermentatively-produced rebA was used as UGT1 cassette described in Examples 1 to 6. The plant derived product used UGT3 cassette was Reb A 97% from Pure Circle/Prinova. UGT4 cassette 3INT1 7.1 Products Tested 0323 Correct integration was confirmed with diagnostic Acidified Water PCR and sequence analysis (3500 Genetic Analyzer, Applied Biosystems). The sequence reactions were done with the 0328 BigDye Terminator v3.1 Cycle Sequencing kit (Life Tech nologies). Each reaction (10 ul) contained 50 ng template and 3.2 pmol primer. The products were purified by ethanol/ Acidified water gll EDTA precipitation, dissolved in 10 ul Hili formamide and Citric Acid O6 applied onto the apparatus. The strain was named STV016. Reb A O.24 The schematic of how the pathway from GGPP to RebA is integrated into the genome is illustrated in FIG. 5. US 2016/0213039 A1 Jul. 28, 2016

Procedure for Preparation of the Beverage: Procedure for Preparation of Beverage: 0329. One liter of water was weighed out and citric acid 0341 water was weighed out and then stevia, Stevia and rebaudioside A dissolved while stirring with a stan masking flavor, pomegranate flavor, citric acid and malic dard mixer. No further processing was needed. acid was added 0342 apple juice concentrate, pomegranate juice con Near Water centrate and grape juice concentrate was then added while using a standard mixer 0330 0343 the resulting composition was mixed well, pas teurized at 95°C. for 30 seconds and then hot filled into Mixed Berry Sobe Knockoff gll bottles Filtered Water 984.5 Cola Erythritol 13 Citric Acid 1 Mixed Berry 1 0344 Canthaxanthin 10% CWSS 1.5 Stock Solution Xanthan Gum 0.4 Reduced Calorie Cola gll Reb A O.3 Stevia Masker Flavor 1 Filtered Water 944.31 Car Color DS400 O.8 Sugar 78.75 Phosphoric Acid (85%) 1.1 Reb A97% O.13S Procedure for Preparation of the Beverage: Nat. Anhydrous Caffeine O.09S (57 mg per 20 oz) 0331 60% of the required water was weighed out. High Tri-Sodium Citrate O46 shear xanthan gum was then added into the water (30 Potassium Sorbate O.25 seconds at 600 rpm) Cola Flavor 2.5 0332 While mixing with a standard mixer. eythritol, Stevia Masker Flavor 1 citric acid, flavor, Canthaxanthin Stock Solution (see for preparation below), Stevia and the Stevia masking flavor were added Procedure for Preparation of Beverage: 0333 the remaining water was then added and the resulting composition was mixed well 0345 the water was weighed out and then potassium 0334 the resulting composition was pasteurized at 95° sorbate was dissolved in the water while stirring. Next C. for 30 seconds and hot filled into bottles. Stevia, tri-sodium citrate and caffeine were dissolved 0346 Sugar, caramel color, Stevia masking flavor, cola Procedure for Preparation of Stock Solution: flavor and phosphoric acid were then added 0347 the resulting composition was then mixed well, 0335 Weighout specified product form (Canthaxanthin carbonated to 3.6 Carbonation Units and then filled into 10%-CWS/S) plastic bottles. 0336. In a beaker, measure out 60 ml of warm deionized water 45° C. to 55° C. 7.2 Sensory Evaluation Method 0337 Slowly add the powder to the warm water while stirring. Minimize incorporation of air while stirring 0348 Six experienced and trained panelists evaluated all samples with the Sensory Spectrum(R) method which is used 0338 Stir for 10-15 minutes to ensure complete disper for detailed flavor analysis. Per application type, each sample sion of the powder was evaluated twice by each panelist in two different sessions. 0339 Fill to 100 ml total volume with room tempera Scoring was done on a 0 (very low intensity) to 15 (very high ture water and then stir. intensity) Sensory Spectrum Scale with discussions to reach consensus on the scores. The products were presented in a Juice balanced order. 0340 0349 Sweetness was rated after the products was held in the mouth for 3 seconds after which the product was expec torated. The Sweetness score, also on a scale ranging from 0 to 50 call bev Pomegranate (45% Juice) gll 15, is the mean intensity score of individual data. To evaluate the significance of the difference an ANOVA was done. Filtered Water 922.02 Applejuice concentrate 34.29 Pomegranate Juice Concentrate 32.1 7.3 Results GrapeJuice Concentrate 32.1 Pomegranate Flavor 1 Overall Sweetness Malic Acid O.S Citric Acid O.S Reb A O.2 0350. In FIG. 8, the Sweetness per sample is shown. No Stevia Masker Flavor 1 significant differences on Sweetness (p<0.05) were found between the fermentative and plant based RebA in each of the tested applications. US 2016/0213039 A1 Jul. 28, 2016 22

Results Per Application TABLE 10

Raw data for acidified water Acidified Water Juice Fermentative Reb A Plant based Reb A 0351. In FIG.9, the consensus data for the acidified water Total impact aroma 7 6.3 application is shown. Fermentative Reb A showed an impact Sweet aromatic complex 2 2 on citrus and Sour attributes, indicating an enhancement Sugarbag 1 1.3 Floral 1 1 effect of fermentative Reb Abased on the used ingredients. Red fruit O 1.5 Brown fruit 4 2 Sweet 10.8 11 Near Water Sour 3.5 3 Bitter O.S O.8 0352. In FIG. 10, the consensus data for the near water Bitter after taste 1.5 1.8 application is shown. Fermentative Reb A showed an impact Astringent 4 4.8 on total aroma impact, Sweet aromatic complex and ethyl Mouthdrying 1.5 1 maltol (also known as Strawberry flavor), indicating an enhancement effect of fermentative Reb Abased on the used ingredients. Example 8 Low-Calorie Orange Juice Drink Juice 0355 60 g of concentrated orange juice is mixed with 1.1 g of citric acid, 0.24 g of vitamin C, 1.0 g of orange essence, 0353. In FIG. 11, the consensus data for the juice applica 0.76 g offermentatively-produced Rebaudioside A and water, tion is shown. Fermentative Reb A showed an impact on total to create a homogeneously dissolved mixture of 1000 mL aroma impact and brown fruit, indicating an enhancement total amount. Then, the mixture is pasteurized for a period of effect of fermentative Reb Abased on the used ingredients. 20 seconds at about 95.degree centigrade in order to prepare 0354. The raw data is shown in Tables 8, 9 and 10 below an orange juice similar to one made by conventional method. The product is Subjected to sensory evaluation in terms of TABLE 8 flavour, aftertaste and mouthfeel. The data shows that excel lent taste and mouth-feel results were obtained for fermenta Raw data for acidified water tively produced Rebaudioside A. 0356) Juices from other fruits, such as apple, lemon, apri Acidified water Fermentative Reb A Plant based Reb A cot, cherry, pineapple, etc. can be prepared using the same Total impact aroma 4.5 4.5 approach. Sweet aromatic complex 2.3 2.5 Sugarbag 1.5 2 Example 8 Citrus 2.5 1.5 Plastic/vinyl O.S O.8 Ice-Cream Sweet 9.1 8.3 Sour 3.5 2.5 Bitter 1.5 1.8 0357 1.50 kg of whole milk is heated to 45° C., and 300 Bitter after taste 2 2 grams of milk cream, 100 grams of tagatose, 90 grams of Astringent 4 3.3 Sorbitol. 6 grams of carrageenan as a stabilizer, 3 grams of Cooling O O.S polysorbate-80 as an emulsifier and 1.0 gram of fermenta Stabilizer O.8 O.S tively-produced Rebaudioside A are added into the milk and Mouthdrying 1 O.S stirred until the ingredients completely dissolved. 0358. The mixture is then pasteurized at a temperature of 80° C. for 25 seconds. After homogenization the samples are kept at a temperature of 4°C. for 24 hours to complete the TABLE 9 aging process. Vanilla flavor (1.0% of the mixture weight) and Raw data for acidified water coloring (0.025% of the mixture weight) are added into the mixture after aging. The mixture is then transferred to an Near water Fermentative Reb A Plant based Reb A icecream maker to produce icecream automatically. Samples Total impact aroma 6 5.3 of produced ice creams are transferred to sealed containers Sweet aromatic complex 3 2.3 and are kept in the freezer at a temperature of -18°C. Sugarbag 1.5 1.5 Ethyl Maltol 1.5 O 0359 The physicochemical properties of the ice cream, as Citrus 1 O.8 well as the overall attributes of color, smoothness, surface Stone Fruit 2.3 2.5 texture, air cell, Vanilla aroma intensity, Vanilla taste, chalki Sweet 9.3 9.2 ness, iciness and melting rate are assessed. Sour 3 3.3 Bitter 1.4 1.5 Bitter after taste 2 2 Example 9 Astringent 3 2.5 Cooling 0.4 1 Yoghurt Mouthdrying O.S O 0360. In 1 kg of defatted milk, 0.8 grams of fermenta tively-produced Rebaudioside A, prepared according to the US 2016/0213039 A1 Jul. 28, 2016

invention is dissolved. After pasteurization at 82° C. for 20 Example 14 minutes, the milk was cooled to 40°C. A starter in an amount of 30 grams is added and the mixture is incubated at 37°C. for Chocolate 6 hours. Then, the fermented mass is maintained at 10-15°C. 0368. A composition containing 30 kg of cacao liquor, for 12 hours. 11.5 kg of cacao butter, 14 kg of milk powder, 44 kg of 0361. The product is a low-calorie and low-cariogenic sorbitol, 0.1 kg of salt, and 0.1 kg offermentatively produced yoghurt and is assessed for taste and odor. Rebaudioside-A is kneaded sufficiently and the mixture is then placed in a refiner to reduce its particle size for 24 hours. Example 10 Thereafter, the content is transferred into a conche 300 grams of lecithin is added, and the composition is kneaded at 50° C. Ice Lemon Tea for 48 hours. Then, the content is placed in a shaping appa ratus and solidified. 0362. The formula for the beverage is as below: 0369. The products are low-cariogenic and low-calorie 95% high purity fermentatively-produced Rebaudioside A, chocolate and are assessed for texture and the presence of any 0.08 Sodium benzoate, 0.02 Citric acid, 0.27 Ascorbic acid, after-taste. 0.01 Tea extract, 0.03 Lemon flavor 0.10 Water to 100 0363 All ingredients are blended and dissolved in the Example 15 water and pasteurized. The product is assessed for taste and flavour. Sensory and physicochemical characteristics are Tooth Paste compared to that of caloric lemon-flavored iced tea. 0370. A toothpaste is prepared by kneading a composition comprising of calcium phosphate, 45.0%; carboxymethylcel Example 11 lulose, 1.5%: carrageenan, 0.5%; glycerol. 18.0%; polyoxy ethylene sorbitan mono-ester, 2.0%; beta-cyclodextrin, 1.5%: Bread sodium laurylsarcosinate, 0.2%; flavoring, 1.0%; preserva tive, 0.1%; fermentatively-produced Rebaudioside A, 0364) 1 kg of flour, 37.38 grams of fructooligosaccharide obtained according to this invention, 0.2%; and water to syrup, 80 grams of margarine, 20 grams of salt, 20 grams of 100%, by usual way. yeasts, and 0.25 grams of 95% high purity fermentatively 0371. The product is assessed for foaming and cleaning produced Rebaudioside A, obtained as described above are abilities along with appropriate Sweetness. placed into a blender and mixed well. 600 ml of water is poured into the mixture and kneaded sufficiently. At the Example 16 completion of the kneading process, the dough is shaped and raised for 30 to 45 minutes. The ready dough is placed in an Low-Calorie Carbonated Drink oven and baked for 45 minutes. Bread samples are assessed 0372. The formula for the beverage was as below: for color and texture. Example 12 Ingredients Quantity,% Cola flavor O.340 Diet to Mid Caloric Cookie Phosphoric acid (85%) O.100 Sodium citrate O.310 0365 Flour, 50.0%; margarine, 30.0%; fructose, 10.0%; Sodium benzoate O.O18 maltitol, 8.0%; whole milk, 1.0%; salt, 0.2%; baking powder, Citric acid O.O18 0.15%; vanillin, 0.1%; fermentatively-produced Rebaudio Sweetener O.O3O side A, 0.55% obtained according to this invention are Carbonated water to 100 kneaded well in a dough-mixing machine. After moulding of the dough, the cookies are baked at 200°C. for 15 minutes. 0373 The beverages are prepared with different sweeten 0366. The product is a low-calorie diet cookie and is ers (plant-extracted Rebaudioside A (95%) and fermenta assessed for taste and appropriate Sweetness. tively-produced Rebaudioside A (95%)) and given to an 8 judge panel for comparison. The beverages are assessed for Example 13 bitter taste, astringent taste, after-taste, quality of the Sweet taste and the overall evaluation, Soy Sauce 0374. The above assessments set out in Examples 7 to 17 are to demonstrate that the various products prepared using 0367 0.8g offermentatively-produced Rebaudioside A is fermentatively-produced Rebaudioside A possess improved added to 1000 mL of soy sauce and mixed homogenously. organoleptic characteristics as compared with similar prod The product is assessed for taste and texture. ucts made with plant-extracted similar grade Rebaudioside A. TABLE 1. Description of the sequence listing Nucleic acid Nucleic (CpO for S. acid (CpO for Amino cerevisiae) Y. lipolytica) acid Id: UniProt Organism SEQ ID NO: SEQID NO: SEQID CPS 1 Q9FXV9 La Citica Saiiva 1 151 NO: 2 (Garden Lettuce)

US 2016/0213039 A1 Jul. 28, 2016 27

TABLE 1-continued Description of the sequence listing

Nucleic acid Nucleic (CpO for S. acid (CpO for Amino - cerevisiae) Y. lipolytica) acid Id: UniProt Organism SEQID NO: Sc Oye2.pro 210 SEQID NO: KANMXORF 211 SEQID NO: Adh1.ter 212 SEQID NO: Adh2.ter 213 SEQID NO: Gmp1..ter 214 SEQID NO: Sc Tal1ter 215 SEQID NO: Sc Tpi1.ter 216 SEQID NO: Ag Tefl Iox.ter 217 SEQID NO: Sc Pdc1ter 218 SEQID NO: Sc Tch1ter 219 SEQID NO: Sc Eno1.ter 220 SEQID NO: KI prom3.pro 221 SEQID NO: KI prom2.pro 222 SEQID NO: SC PRE3. Pro 223 greyed out ids are truncated and thus a fragment of mentioned UniProtid

SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing section. A copy of the “Sequence Listing is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160213039A1). An electronic copy of the “Sequence Listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. A product which is a foodstuff, beverage, pharmaceuti mented Soybean paste, a soy sauce, a Vinegar, a dressing, a cal composition, tobacco, nutraceutical, oral hygiene compo mayonnaise, a ketchup, a curry, a Soup, an instant bouillon, a sition or cosmetic comprising a Sweetener composition, powdered soy sauce, a powdered vinegar, a biscuit, a rice wherein the Sweetener composition comprises one or more biscuit, a cracker, a bread, a chocolate, a caramel, a candy, a fermentatively-produced Steviol glycoside. chewing gum, a jelly, a pudding, a preserved fruit or Veg 2. A product according to claim 1, wherein the at least one etable, a fresh cream, a jam, a marmalade, a flower paste, a of the one or more fermentatively-produced Steviol glyco powdered milk, an ice cream, a sorbet, a vegetable or fruits sides is rebaudioside A. packed in a bottle, a canned or boiled bean, a meat or food 3. A product according to claim 1, wherein the Sweetener boiled in Sweetened sauce, an agricultural vegetable food composition comprises at least about 95% by dry weight of product, a seafood, a ham, a sausage, a fish ham, a fish fermentatively-produced rebaudioside A. sausage, a fish paste, a deep fried fish product, a dried seafood 4. A product according to claim 1, which is a Zero calorie, product, a frozen food product, a preserved seaweed, a pre reduced calorie or diabetic product. served meat, a tobacco or a medicinal product. 5. A product according to claim 1, which is dedicated for 7. A product according to claim 1, which is a non-carbon human consumption or is an animal feed or fodder. ated or carbonated beverages optionally comprising a cola, a 6. A product according to claim 1, which is an alcoholic fruit juices originating in fruits or vegetables, a fruit juices, a beverages, a natural juices, a carbonated Soft drinks, a diet fruit juice containing fruit particles, a fruit beverage, a fruit drink, a Zero calorie drink, a reduced calorie drink or food, a juice beverage, a beverage containing fruit juice, a beverage yogurt drink, an instant juice, an instant coffee, a powdered with fruit flavoring, a vegetable juice, a juice containing veg type of instant beverages, a canned product, a syrup, a fer etables, a mixed juice containing fruit and/or vegetables, a US 2016/0213039 A1 Jul. 28, 2016 28 sport drink, an energy drinks, near water or the like drinks, a paring a said product and incorporating a Sweetener compo tea type or favorite type beverage, a beverage containing milk sition comprising one or more fermentatively-produced components or a dairy products. Steviol glycosides. 8. A product according to claim 1, wherein the Sweetener 10. A method according to claim 9, wherein the product is composition further comprises a natural high intensity Sweet a product according to claim 1. ener, a synthetic or artificial high intensity Sweetener, a natu 11. A Sweetener composition comprising one or more fer ral Sweetness Suppresser, a umami taste enhancer, an amino mentatively-produced Steviol glycosides used in preparation acid, a polyamino acid additive, a polyol or Sugar alcohol, a of a foodstuff, beverage, pharmaceutical composition, reduced calorie Sweetener, a carbohydrate, a Sugar acid, a tobacco, nutraceutical, oral hygiene composition or cosmetic. flavoring agent, an aroma component, a nucleotide additive, 12. Composition comprising a product according to claim an organic acid additive, an organic acid salt additive, an 1. inorganic acid salt additive, a bitter component additive, an 13. Composition according to claim 12 capable of being artificial or natural Sweetness enhancer, a polymer additive, a protein or protein hydrolyzate additive, a Surfactant additive, used to enhance citrus or sour attributes, total aroma impact, a flavonoid additive, an alcohol additive, an astringent com sweet aromatic complex, ethyl maltol (strawberry flavor) or pound additive, a vitamin, a dietary fiber, an antioxidant, a brown fruit. fatty acid, or a salt. 14. A composition which comprises, on a dry solids basis, 9. A method for the preparation of a product which is a at least about 60%, at least about 70%, at least about 80%, at foodstuff, beverage, pharmaceutical composition, tobacco, least about 90%, at least about 95%, at least about 99% weight nutraceutical, oral hygiene composition or cosmetic compris of fermentatively-produced Rebaudioside A. ing a Sweetener composition, which method comprises pre k k k k k