Steviol glycosides

Information

  • Patent Grant
  • 11540544
  • Patent Number
    11,540,544
  • Date Filed
    Friday, April 22, 2022
    2 years ago
  • Date Issued
    Tuesday, January 3, 2023
    a year ago
Abstract
The present invention relates to a steviol glycoside having the formula of (I)
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII
TEXT FILE (.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-compliant text file (entitled “2919208-319002_Sequence_Listing_ST25.txt” created on 20 Apr. 2022, and 1,060,381 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.


FIELD OF THE INVENTION

The present invention relates to steviol glycosides, to methods for producing them, to sweetener compositions, flavour compositions, foodstuffs, feeds and beverages comprising the steviol glycosides and to use of the steviol glycosides in sweetener compositions, flavour compositions, foodstuffs, feeds and beverages.


BACKGROUND TO THE INVENTION

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


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


Steviol glycosides accumulate in Stevia leaves where they may comprise from 10 to 20% of the leaf dry weight. Stevioside and rebaudioside A are both heat and pH stable and suitable for use in carbonated beverages and many other foods. Stevioside is between 110 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320 times sweeter than sucrose. In addition, rebaudioside D is also a high-potency diterpene glycoside sweetener which accumulates in Stevia leaves. It may be about 200 times sweeter than sucrose. Rebaudioside M is a further high-potency diterpene glycoside sweetener. It is present in trace amounts in certain stevia variety leaves, but has been suggested to have a superior taste profile.


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


There is though a need for additional steviol glycosides having alternative and/or improved taste profiles since different steviol glycosides may be better suited to different applications.


SUMMARY OF THE INVENTION

The present invention is based on the identification of new steviol glycosides in fermentation broths obtained from microorganisms which have been modified so as to produce steviol glycosides, including rebA. The new steviol glycosides will have different sensory properties as compared with known steviol glycosides. They may be used alone or in combination with other steviol glycosides, in particular as sweeteners or in sweetener compositions.


Accordingly, the invention relates to:

  • a steviol glycoside having the formula of (I)




embedded image




    • wherein at least 3 sugar moieties are present at position R1 and at least three sugar moieties are present at position R2 and wherein the steviol glycoside comprises at least seven sugar moieties all of which are linked, directly or indirectly, to the steviol aglycon by β-linkages;



  • a steviol glycoside having the formula of (I)





embedded image




    • wherein at least 4 sugar moieties are present at positions R1 and at least three sugar moieties are present at position R2;



  • a steviol glycoside having the formula of (I)





embedded image




    • wherein at least 3 sugar moieties are present at position R1 and at least three sugar moieties are present at position R2, wherein the steviol glycoside comprises at least seven sugar moieties and wherein at least one of the sugars present at position R1 is linked to the steviol aglycon or to a sugar molecule by a α-linkage;



  • a steviol glycoside having the formula of (I)





embedded image




    • wherein at least 3 sugar moieties are present at position R1 and at least four sugar moieties are present at position R2, wherein at least four of the sugar moieties present at position R2 are glucose moieties;



  • a steviol glycoside having the formula (II)





embedded image


  • a steviol glycoside having the formula (III)





embedded image


  • a steviol glycoside having the formula (IV)





embedded image


  • a fermentatively produced steviol glycoside having the formula of (I)





embedded image




    • wherein at least 3 sugar moieties are present at position R1 and at least three sugar moieties are present at position R2 and wherein the steviol glycoside comprises at least seven sugar moieties;



  • a method for the production of a steviol glycoside according to any one of the preceding claims, which method comprises:
    • providing a recombinant yeast cell comprising recombinant nucleic acid sequences encoding polypeptides comprising the amino acid sequences encoded by: SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 71, SEQ ID NO: 87, SEQ ID NO: 73 and SEQ ID NO: 75;
    • fermenting the recombinant yeast cell in a suitable fermentation medium; and, optionally,
    • recovering a steviol glycoside according to any one of the preceding claims.

  • a composition comprising a steviol glycoside of the invention and one or more different steviol glycosides (which different steviol glycosides may or may not be a steviol glycoside of the invention);

  • a sweetener composition, flavor composition, foodstuff, feed or beverage which comprises a steviol glycoside or a composition of the invention;

  • use of a steviol glycoside or a composition of the invention in a sweetener composition or flavor composition; and

  • use of a steviol glycoside or a composition of the invention in a foodstuff, feed or beverage.






BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 sets out a schematic representation of the plasmid pUG7-EcoRV.



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



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



FIG. 4 sets out a schematic representation of how UGT2 is integrated into the genome. A. different fragments used in transformation; B. situation after integration; C. situation after expression of Cre recombinase).



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



FIG. 6a shows an extracted ion chromatogram of m/z 1451.5820 of the mixture of the steviol glycosides containing 7 glucoses (7.1,7.2 and 7.3) in the ethanol extract (starting material for purification), using High Resolution Mass Spectrometry; and FIG. 6b: extracted ion chromatogram of m/z 1451.5 of the purified steviol glycosides containing 7 glucoses (7.1,7.2 and 7.3), using LC-MS.



FIG. 7 shows the structure of Rebaudioside 7.1.



FIG. 8 shows the structure of Rebaudioside 7.2.



FIG. 9 shows the structure of Rebaudioside 7.3.



FIG. 10 shows the structure of Rebaudioside M.



FIG. 11A shows atom numbering of steviol and FIG. 11B shows atom numbering of glucose.



FIG. 12 shows the selected region of the 1H NMR spectrum of a) Reb M (cdcl3/pyr 1:1, 2 drops cdood at 300K), b) Reb 7.1 (cdcl3/pyr 1:3, 2 drops cdood at 320K) c) Reb 7.2 (cdcl3/pyr 1:1, 2 drops cdood at 300K) and d) Reb 7.3 (cdc13/pyr 1:2, 3 drops cdood at 300K).





DESCRIPTION OF THE SEQUENCE LISTING

A description of the sequences is set out in Table 15. Sequences described herein may be defined with reference to the sequence listing or with reference to the database accession numbers also set out in Table 15.


DETAILED DESCRIPTION OF THE INVENTION

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


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


This invention relates to steviol glycosides. For the purposes of this invention, a steviol glycosides is a glycoside of steviol, specifically a steviol molecule with its carboxyl hydrogen atom replaced by a glucose molecule to form an ester, and an hydroxyl hydrogen with glucose to form an acetal.


A steviol glycoside of the invention may be provided in isolated form. An “isolated steviol glycoside” is a substance removed from other material, such as other steviol glycosides, with which it may be naturally associated. Thus, an isolated steviol glycoside may contain at most 10%, at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, even more preferably at most 1% and most preferably at most 0.5% by weight of other material, for example other steviol glycosides, with which it is naturally associated. The isolated steviol glycosides may be free of any other impurities. The isolated steviol glycoside of the invention may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 90% pure, or at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% pure by weight.


The invention provides a steviol glycoside having the formula of (I)




embedded image




    • wherein at least 3 sugar moieties are present at position R1 and at least three sugar moieties are present at position R2 and wherein the steviol glycoside comprises at least seven sugar moieties all of which are linked, directly or indirectly, to the steviol aglycon by β-linkages, or

    • wherein at least 4 sugar moieties are present at positions

    • R1 and at least three sugar moieties are present at position R2, or

    • wherein at least 3 sugar moieties are present at position R1 and at least three sugar moieties are present at position R2, wherein the steviol glycoside comprises at least seven sugar moieties and wherein at least one of the sugars present at position R1 is linked to the steviol aglycon or to a sugar molecule by a α-linkage, or

    • wherein at least 3 sugar moieties are present at position R1 and at least four sugar moieties are present at position R2, wherein at least four of the sugar moieties present at position R2 are glucose moieties.





The invention also provides steviol glycosides having the formula (II), (III) or (IV):




embedded image


embedded image


A steviol glycoside of the invention may be obtained from plant material, but more typically will be obtained by fermentative production, for example via fermentation of a recombinant host cell, such as a yeast cell.


Thus, the invention provides a fermentatively produced steviol glycoside having the formula of (I)




embedded image




    • wherein at least 3 sugar moieties are present at position R1 and at least three sugar moieties are present at position R2 and wherein the steviol glycoside comprises at least seven sugar moieties.





One may distinguish between α- and β-glycosidic bonds based on the relative stereochemistry (R or S) of the anomeric position and the stereocentre furthest from C1 in a saccharide. Typically, an α-glycosidic bond is formed when both carbons have the same stereochemistry, whereas a β-glycosidic bond occurs when the two carbons have different stereochemistry.


Such a fermentatively-produced steviol glycoside may have a structure of any of the steviol glycosides described herein.


The invention further relates to a method for the production of a steviol glycoside. In such a method, a suitable recombinant host cell, such as a yeast cell, is fermented in a suitable fermentation medium such that the steviol glycoside is produced. Optionally, the steviol glycoside may be recovered.


For example, a method for the production of a steviol glycoside as described herein may comprise:

    • providing a recombinant yeast cell comprising recombinant nucleic acid sequences encoding polypeptides comprising the amino acid sequences encoded by: SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 71, SEQ ID NO: 87, SEQ ID NO: 73 and SEQ ID NO: 75;
    • fermenting the recombinant yeast cell in a suitable fermentation medium; and, optionally,
    • recovering a steviol glycoside as described herein.


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


A recombinant yeast cell used in a process of the invention may be any suitable yeast cell. Preferred recombinant yeast cells may be selected from the genera: Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), Brettanomyces, Kluyveromyces, Candida (e.g., C. krusei, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis), Issatchenkia (e.g. I. orientalis) Pichia (e.g., P. pastoris), Schizosaccharomyces, Hansenula, K/oeckera, Pachyso/en, Schwanniomyces, Trichosporon, Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)) or Yamadazyma. Preferably, the recombinant yeast cell is a Saccharomyces cerevisiae, Yarrowia lipolitica or Issatchenkia orientalis cell.


A recombinant yeast cell for use in a method according to the invention may comprise one or more recombinant nucleotide sequence(s) encoding one of more of:

    • a polypeptide having ent-copalyl pyrophosphate synthase activity;
    • a polypeptide having ent-Kaurene synthase activity;
    • a polypeptide having ent-Kaurene oxidase activity; and
    • a polypeptide having kaurenoic acid 13-hydroxylase activity.


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




embedded image


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


Suitable nucleic acid sequences encoding an ent-copalyl pyrophosphate synthase may for instance comprise a sequence as set out in SEQ ID. NO: 1, 3, 5, 7, 17, 19, 59, 61, 141, 142, 151, 152, 153, 154, 159, 160, 182 or 184.


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

ent-copalyl diphosphate ⇄ent-kaurene+diphosphate


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


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


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


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


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


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


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


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


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


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


Suitable nucleic acid sequences encoding a NADPH-cytochrome p450 reductase may for instance comprise a sequence as set out in SEQ ID. NO: 53, 55, 57 or 77.


A recombinant yeast cell suitable for use in a method of the invention may also comprise one or more recombinant nucleic acid sequences encoding one or more of:

    • (i) a polypeptide having UGT74G1 activity;
    • (ii) a polypeptide having UGT2 activity;
    • (iii) a polypeptide having UGT85C2 activity; and
    • (iv) a polypeptide having UGT76G1 activity.


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


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


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


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


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


A polypeptide having UGT2 activity may also catalyze reactions that utilize steviol glycoside substrates other than steviol-13-O-glucoside and rubusoside, e.g., functional UGT2 polypeptides may utilize stevioside as a substrate, transferring a glucose moiety to the C-2′ of the 19-O-glucose residue to produce rebaudioside E. A functional UGT2 polypeptides may also utilize rebaudioside A as a substrate, transferring a glucose moiety to the C-2′ of the 19-O-glucose residue to produce rebaudioside D. However, a functional UGT2 polypeptide typically does not transfer a glucose moiety to steviol compounds having a 1,3-bound glucose at the C-13 position, i.e., transfer of a glucose moiety to steviol 1,3-bioside and 1,3-stevioside typically does not occur.


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


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


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


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


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


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


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


A recombinant yeast suitable for use in a method of the invention typically comprises nucleotide sequences encoding at least one polypeptide having UGT1 activity, at least one polypeptide having UGT2 activity, least one polypeptide having UGT3 activity and at least one polypeptide having UGT4 activity. One or more of these nucleic acid sequences may be recombinant. A given nucleic acid may encode a polypeptide having one or more of the above activities. For example, a nucleic acid encode for a polypeptide which has two, three or four of the activities set out above. Preferably, a recombinant yeast for use in the method of the invention comprises UGT1, UGT2 and UGT3 and UGT4 activity. Suitable UGT1, UGT2, UGT3 and UGT4 sequences are described in in Table 15 herein. A preferred combination of sequences encoding UGT1, 2, 3 and 4 activities is SEQ ID NOs: 71, 87, 73 and 75.


In the method of the invention, a recombinant host, such as a yeast, may be able to grow on any suitable carbon source known in the art and convert it to one or more steviol glycosides. The recombinant host may be able to convert directly plant biomass, celluloses, hemicelluloses, pectins, rhamnose, galactose, fucose, maltose, maltodextrines, ribose, ribulose, or starch, starch derivatives, sucrose, lactose and glycerol. Hence, a preferred host expresses enzymes such as cellulases (endocellulases and exocellulases) and hemicellulases (e.g. endo- and exo-xylanases, arabinases) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, pectinases able to convert pectins into glucuronic acid and galacturonic acid or amylases to convert starch into glucose monomers. Preferably, the host is able to convert a carbon source selected from the group consisting of glucose, xylose, arabinose, sucrose, lactose and glycerol. The host cell may for instance be a eukaryotic host cell as described in WO03/062430, WO06/009434, EP149970861, WO2006096130 or WO04/099381.


The fermentation medium used in the process for the production of a steviol glycoside of the invention may be any suitable fermentation medium which allows growth of a particular eukaryotic host cell. The essential elements of the fermentation medium are known to the person skilled in the art and may be adapted to the host cell selected. Preferably, the fermentation medium comprises a carbon source selected from the group consisting of plant biomass, celluloses, hemicelluloses, pectins, rhamnose, galactose, fucose, fructose, maltose, maltodextrines, ribose, ribulose, or starch, starch derivatives, sucrose, lactose, fatty acids, triglycerides and glycerol. Preferably, the fermentation medium also comprises a nitrogen source such as ureum, or an ammonium salt such as ammonium sulphate, ammonium chloride, ammonium nitrate or ammonium phosphate.


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


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


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


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


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


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


The process for the production of a steviol glycoside according to the present invention may be carried out at any suitable pH value. If the recombinant host is a yeast, the pH in the fermentation medium preferably has a value of below 6, preferably below 5,5, preferably below 5, preferably below 4,5, preferably below 4, preferably below pH 3,5 or below pH 3,0, or below pH 2,5, preferably above pH 2. An advantage of carrying out the fermentation at these low pH values is that growth of contaminant bacteria in the fermentation medium may be prevented.


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


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


In the process for the production of a steviol glycoside according to the invention, it may be possible to achieve a concentration of above 0.5 mg/l, preferably above about 1 mg/l.


In the event that one or more steviol glycosides of the invention is expressed within a recombinant host, such cells may need to be treated so as to release them.


The invention also provides a composition comprising a steviol glycoside of the invention in combination with one or more different steviol glycosides. One or more of the one or more different steviol glycosides may be a steviol glycoside of the invention. One or more of the one or more different steviol glycosides may be a glycosylated diterpene (i.e. a diterpene glycoside), such as steviolmonoside, steviolbioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rubusoside, dulcoside A, steviol-13-monoside, steviol-19-monoside or 13-[(β-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester.


A composition of the invention may comprise a relatively low amount of a steviol glycoside of the invention in combination with a greater amount of a different steviol glycoside.


For example, a composition of the invention may comprise at least about 80%, at least about 90%, at least about 95% rebaudioside A in combination with a steviol glycoside of the invention. A composition of the invention may comprise at least about 80%, at least about 90%, at least about 95% rebaudioside D in combination with a steviol glycoside of the invention. A composition of the invention may comprise at least about 80%, at least about 90%, at least about 95% rebaudioside M in combination with a steviol glycoside of the invention. A composition of the invention may comprise at least about 80%, at least about 90%, at least about 95% rebaudioside A in combination with a steviol glycoside of the invention and rebaudioside D. A composition of the invention may comprise at least about 80%, at least about 90%, at least about 95% rebaudioside A in combination with a steviol glycoside of the invention and rebaudioside M. Percentages referred to are on a dry weight basis.


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


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


A composition of the invention may comprise one or more non-naturally occurring components.


Furthermore, the invention provides:

    • use of a steviol glycoside or a composition of the invention in a sweetener composition or flavor composition; and
    • use of a steviol glycoside or a composition of the invention in a foodstuff, feed or beverage.


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


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


Compounds produced according to the method of the invention may be blended with one or more further non-calorific or calorific sweeteners. Such blending may be used to improve flavour or temporal profile or stability. The steviol glycoside of the invention may be used to improve the flavour or temporal profile or stability of a second steviol glycoside, such as rebaudiose A, D or M.


A wide range of both non-calorific and calorific sweeteners may be suitable for blending with a steviol glycoside of the invention, including one or more other steviol glycosides according to the invention or one or more other known steviol glycosides such as steviolmonoside, steviolbioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rubusoside, dulcoside A, steviol-13-monoside, steviol-19-monoside or 13-[(β-D-Glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β-D-glucopyranosyl-β-D-glucopyranosyl ester. Alternatively, or in addition, non-calorific sweeteners such as mogroside, monatin, aspartame, acesulfame salts, cyclamate, sucralose, saccharin salts or erythritol. Calorific sweeteners suitable for blending with steviol glycosides include sugar alcohols and carbohydrates such as sucrose, glucose, fructose and HFCS. Sweet tasting amino acids such as glycine, alanine or serine may also be used.


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


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


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


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


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


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


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


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


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


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


Thus, compositions of the present invention can be made by any method known to those skilled in the art that provide homogenous even or homogeneous mixtures of the ingredients. These methods include dry blending, spray drying, agglomeration, wet granulation, compaction, co-crystallization and the like.


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


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


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


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


EMBODIMENTS OF THE INVENTION

1. A steviol glycoside having the formula of (I)




embedded image




    • wherein at least 3 sugar moieties are present at positions R1 and at least three sugar moieties are present at position R2 and wherein the steviol glycoside comprises at least seven sugar moieties all of which are linked, directly or indirectly, to the steviol aglycon by β-linkages.





2. A steviol glycoside having the formula of (I)




embedded image




    • wherein at least 4 sugar moieties are present at positions R1 and at least three sugar moieties are present at position R2.





3. A steviol glycoside having the formula of (I)




embedded image




    • wherein at least 3 sugar moieties are present at positions R1 and at least three sugar moieties are present at position R2, wherein the steviol glycoside comprises at least seven sugar moieties and wherein at least one of the sugars present at position R1 is linked to the steviol aglycon or to a sugar molecule by a α-linkage.





4. A steviol glycoside having the formula of (I)




embedded image




    • wherein at least 3 sugar moieties are present at positions R1 and at least four sugar moieties are present at position R2, wherein at least four of the sugar moieties present at position R2 are glucose moieties.





5. A steviol glycoside having the formula (II)




embedded image


6. A steviol glycoside having the formula (III)




embedded image


7. A steviol glycoside having the formula (IV)




embedded image


8. A steviol glycoside according to any one of the preceding embodiments which is fermentatively produced.


9. A fermentatively produced steviol glycoside having the formula of (I)




embedded image




    • wherein at least 3 sugar moieties are present at positions R1 and at least three sugar moieties are present at position R2 and wherein the steviol glycoside comprises at least seven sugar moieties.





10. A steviol glycoside according to embodiment 9 having a structure according to any one of embodiments 1 to 7.


11. A method for the production of a steviol glycoside according to any one of the preceding embodiments, which method comprises:

    • providing a recombinant yeast cell comprising recombinant nucleic acid sequences encoding polypeptides comprising the amino acid sequences encoded by: SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 59, SEQ ID NO: 71, SEQ ID NO: 87, SEQ ID NO: 73 and SEQ ID NO: 75;
    • fermenting the recombinant yeast cell in a suitable fermentation medium; and, optionally,
    • recovering a steviol glycoside according to any one of the preceding embodiments.


12. A composition comprising a steviol glycoside according to any one of embodiments 1 to 11 and one or more different steviol glycosides.


13. A foodstuff, feed or beverage which comprises a steviol glycoside according to any one of embodiments 1 to 10 or a composition according to embodiment 12.


14. Use of a steviol glycoside according to any one of embodiments 1 to 10 or a composition according to embodiment 12 in a sweetener composition or flavor composition.


15. Use of a steviol glycoside according to any one of embodiment 1 to 10 or a composition according to embodiment 12 in a foodstuff, feed or beverage.


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


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


The present invention is further illustrated by the following Examples:


EXAMPLES

Example 1: Construction of STV016



S. cerevisiae Strain STV016 was constructed for the fermentative production of steviol glycosides.


1.1 Over-Expression of ERG20, BTS1 and tHMG in S. cerevisiae


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









TABLE 1







Composition of the over-expression constructs











Promoter
ORF
Terminator






Eno2 (SEQ ID
Erg20 (SEQ ID
Adh1 (SEQ ID



NO: 201)
NO: 81)
NO: 212)



Fba1 (SEQ ID
tHMG1 (SEQ ID
Adh2 (SEQ ID



NO: 202)
NO: 79)
NO: 213)



Tef1 (SEQ ID
Bts1 (SEQ ID
Gmp1 (SEQ ID



NO: 203)
NO: 83)
NO: 214)









For amplification of the selection marker, the pUG7-EcoRV construct (FIG. 1) and suitable primers were used. The KanMX fragment was purified from gel using the Zymoclean Gel DNA Recovery kit (ZymoResearch). Yeast strain Cen.PK113-3C was transformed with the fragments listed in Table 2.









TABLE 2





DNA fragments used for transformation of ERG20, tHMG1 and BTS1


Fragment

















5′YPRcTau3



ERG20 cassette



tHMG1 cassette



KanMX cassatte



BTS1 cassette



3′YPRcTau3









After transformation and recovery for 2.5 hours in YEPhD (yeast extract phytone peptone glucose; BBL Phytone Peptone from BD) at 30° C. the cells were plated on YEPhD agar with 200 μg/ml G418 (Sigma). The plates were incubated at 30° C. for 4 days. Correct integration was established with diagnostic PCR and sequencing. Over-expression was confirmed with LC/MS on the proteins. The schematic of the assembly of ERG20, tHMG1 and BTS1 is illustrated in FIG. 2. This strain was named STV002.


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


1.2 Knock-Down of Erg9


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


The construct containing the Erg9-KD fragment was transformed to E. coli TOP10 cells. Transformants were grown in 2PY(2 times Phytone peptone Yeast extract), sAMP medium. Plasmid DNA was isolated with the QIAprep Spin Miniprep kit (Qiagen) and digested with Sall-HF (New England Biolabs). To concentrate, the DNA was precipitated with ethanol. The fragment was transformed to S. cerevisiae, and colonies were plated on mineral medium (Verduyn et al, 1992. Yeast 8:501-517) agar plates without tryptophan. Correct integration of the Erg9-KD construct was confirmed with diagnostic PCR and sequencing. The schematic of performed transformation of the Erg9-KD construct is illustrated in FIG. 3. The strain was named STV003.


1.3 Over-Expression of UGT2_1a


For over-expression of UGT2_1a, technology was used as described in co-pending patent application nos. WO2013/076280 and WO2013/144257. The UGT2a was ordered as a cassette (containing homologous sequence, promoter, gene, terminator, homologous sequence) at DNA2.0. For details, see Table 3. To obtain the fragments containing the marker and Cre-recombinase, technology was used as described in co-pending patent application no. WO2013/135728. The NAT marker, conferring resistance to nourseothricin was used for selection.









TABLE 3







Composition of the over-expression construct











Promoter
ORF
Terminator






Pgk1 (SEQ ID
UGT2_1a (SEQ ID
Adh2 (SEQ ID



NO: 204)
NO: 87)
NO: 213)









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



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









TABLE 4





DNA fragments used for transformation of UGT2a


Fragment

















5′Chr09.01



UGT2a cassette



NAT-CR



RE



3′Chr09.01









Expression of the CRE recombinase is activated by the presence of galactose. To induce the expression of the CRE recombinase, transformants were restreaked on YEPh Galactose medium. This resulted in out-recombination of the marker(s) located between lox sites. Correct integration of the UGT2a and out-recombination of the NAT marker was confirmed with diagnostic PCR. The resulting strain was named STV004. The schematic of the performed transformation of the UGT2a construct is illustrated in FIG. 4.


1.4 Over-Expression Of Production Pathway to RebA: CPS, KS, KO, KAH, CPR, UGT1, UGT3 and UGT4


All pathway genes leading to the production of RebA were designed to be integrated in one locus in the STV004 strain background. To amplify the 5′ and 3′ integration flanks for the integration locus (site 3), suitable primers and genomic DNA from a CEN.PK yeast strain was used. The different genes were ordered as cassettes (containing homologous sequence, promoter, gene, terminator, homologous sequence) at DNA2.0 (see Table 5 for overview). The DNA from DNA2.0 was dissolved to 100 ng/μl. This stock solution was further diluted to 5 ng/μl, of which 1 μl was used in a 50 μl-PCR mixture. The reaction contained 25 pmol of each primer. After amplification, DNA was purified with the NucleoSpin 96 PCR Clean-up kit (Macherey-Nagel) or alternatively concentrated using ethanol precipitation.









TABLE 5







Composition of the over-expression constructs


for CPS, KS, KO, KAH, CPR, UGT1, UGT3 and UGT4









Promoter
ORF
Terminator





Kl prom 12.pro (SEQ ID NO: 205)
CPS (SEQ ID NO: 61)
Sc Adh2.ter (SEQ ID NO: 213)


Sc Pgk1.pro (SEQ ID NO: 204)
KS (SEQ ID NO: 65)
Sc Tal1.ter (SEQ ID NO: 215)


Sc Eno2.pro (SEQ ID NO: 201)
KO (SEQ ID NO: 23)
Sc Tpi1.ter (SEQ ID NO: 216)


Ag lox_Tef1.pro (SEQ ID NO: 206)
KANMX (SEQ ID NO: 211)
Ag Tef1_lox.ter (SEQ ID NO: 217)


Sc Tef1.pro (SEQ ID NO: 203)
KAH (SEQ ID NO: 33)
Sc Gpm1.ter (SEQ ID NO: 214)


Kl prom 6.pro (SEQ ID NO: 207)
CPR (SEQ ID NO: 77)
Sc Pdc1.ter (SEQ ID NO: 218)


Sc Pma1.pro (SEQ ID NO: 208)
UGT1 (SEQ ID NO: 71)
Sc Tdh1.ter (SEQ ID NO: 219)


Sc Vps68.pro (SEQ ID NO: 209)
UGT3 (SEQ ID NO: 73)
Sc Adh1.ter (SEQ ID NO: 212)


Sc Oye2.pro (SEQ ID NO: 210)
UGT4 (SEQ ID NO: 75)
Sc Eno1.ter (SEQ ID NO: 220)









All fragments for the pathway to RebA, the marker and the flanks (see overview in Table 6) were transformed to a S. cerevisiae yeast strain STV004. After overnight recovery in YEPhD at 20° C. the transformation mixes were plated on YEPhD agar containing 200 μg/ml G418. These were incubated 3 days at 30° C.









TABLE 6





DNA fragments used for transformation of CPS, KS, KO,


KanMX, KAH, CPR, UGT1, UGT3 and UGT4


Fragment

















5′ INT1



CPS cassette



KS cassette



KO cassette



KanMX cassette



KAH cassette



CPR cassette



UGT1 cassette



UGT3 cassette



UGT4 cassette



3′INT1









Correct integration was confirmed with diagnostic PCR and sequence analysis (3500 Genetic Analyzer, Applied Biosystems). The sequence reactions were done with the BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies). Each reaction (10 μl) contained 50 ng template and 3.2 pmol primer. The products were purified by ethanol/EDTA precipitation, dissolved in 10 μl HiDi formamide and applied onto the apparatus. The strain was named STV016. The schematic of how the pathway from GGPP to RebA is integrated into the genome is illustrated in FIG. 5. Table 7 sets out the strains used in this Example 1.









TABLE 7







Table of strains









Strain
Background
Genotype





Cen.PK113-3C

MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2


STV002
Cen.PK113-3C
MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2




YPRcTau3::ERG20, tHMG1, KanMX, BTS1


STV003
STV002
MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2




YPRcTau3::ERG20, tHMG1, KanMX, BTS1




ERG9::ERG9-KD TRP1


STV004
STV003
MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2




YPRcTau3::ERG20, tHMG1, BTS1 ERG9::ERG9-KD




TRP1 Chr09.01::UGT91D2


STV016
STV004
MATa URA3 HIS3 LEU2 trp1-289 MAL2-8C SUC2




YPRcTau3::ERG20, tHMG1, BTS1 ERG9::ERG9-KD




TRP1 Chr09.01::UGT2_1a INT1::CPS, KS,




KO, KanMX, KAH, CPR, UGT1, UGT3, UGT4










1.5 Fermentation of STV016


The S. cerevisiae strain STV016 constructed as described above, were cultivated in shake-flasks (2 l with 200 ml medium) for 32 hours at 30° C. and 220 rpm. The medium was based on Verduyn et al. (Verduyn C, Postma E, Scheffers W A, Van Dijken J P. Yeast, 1992 Jul; 8(7):501-517), with modifications in the carbon and nitrogen sources, as described in Table 8.









TABLE 8





Preculture medium composition



















Concentration


Raw material
Formula
(g/kg)





Galactose
C6H12O6
20.0


Urea
(NH2)2CO
2.3


Potassium dihydrogen phosphate
KH2PO4
3.0


Magnesium sulphate
MgSO4•7H2O
0.5


Trace element solution

1


Vitamin solution

1














Concentration


Component
Formula
(g/kg)











aTrace elements solution










EDTA
C10H14N2Na2O8•2H2O
15.00


Zincsulphate•7H2O
ZnSO4•7H2O
4.50


Manganesechloride•2H2O
MnCl2•2H2O
0.84


Cobalt (II) chloride•6H2O
CoCl2•6H2O
0.30


Cupper (II) sulphate•5H2O
CuSO4•5H2O
0.30


Sodium molybdenum•2H2O
Na2MoO4•2H2O
0.40


Calciumchloride•2H2O
CaCl2•2H2O
4.50


Ironsulphate•7H2O
FeSO4•7H2O
3.00


Boric acid
H3BO3
1.00


Potassium iodide
KI
0.10








bVitamin solution










Biotin (D−)
C10H16N2O3S
0.05


Ca D(+) panthothenate
C18H32CaN2O10
1.00


Nicotinic acid
C6H5NO2
1.00


Myo-inositol
C6H12O6
25.00


Thiamine chloride
C12H18Cl2N4OS•xH2O
1.00


hydrochloride




Pyridoxol hydrochloride
C8H12ClNO3
1.00


p-aminobenzoic acid
C7H7NO2
0.20









Subsequently, 200 ml of the content of the shake-flask was transferred into a fermenter (starting volume 5 L), which contained the medium as set out in Table 9.









TABLE 9







Composition fermentation medium











Final




Concentration


Raw material

(g/kg)












Glucose•1aq
C6H12O6•1H2O
4.4


Ammonium sulphate
(NH4)2SO4
1


Potassium dihydrogen phosphate
KH2PO4
10


Magnesium sulphate
MgSO4•7H2O
5


Trace element solution

8


Vitamin solution

8









The pH was controlled at 5.0 by addition of ammonia (25 wt %). Temperature was controlled at 27° C. pO2 was controlled at 40% by adjusting the stirrer speed. Glucose concentration was kept limited by controlled feed to the fermenter as set out in Table 10.









TABLE 10







Composition of the fermentation feed medium













Final





Concentration



Raw material
Formula
(g/kg)














Glucose•1aq
C6H12O6•1H2O
550



Potassium dihydrogen
KH2PO4
15.1



phosphate





Magnesium sulphate
MgSO4•7H2O
7.5



heptahydrate





Verduyn trace elements

12



solution





Verduyn vitamin solution

12









Example 2: Observation of 7.1, 7.2 and 7.3 Using LC-MS

Steviol glycosides containing 7 glucose molecules (further referred to as 7.1, 7.2 and 7.3) were observed with the LC-MS system described below in the mother liquid after crystallization of rebaudioside A in a water/ethanol mixture (strain STV016). Prior to purification the sample was concentrated by evaporation.


7.1, 7.2 and 7.3 were analyzed on an Acquity UPLC (Waters) coupled to a XEVO-TQ Mass Spectrometer (Waters) equipped with an electrospray ionization source operated in the negative-ion mode in MRM mode at the deprotonated molecules for all steviol glycosides studied, among these m/z 1451.5, representing the deprotonated molecule of a steviol glycoside containing 7 glucose molecules.


The chromatographic separation was achieved with a 2.1×100 mm 1.8 μm particle size, Acquity UPLC® HSS T3 column, using a gradient elution with (A) 50 mM ammonium acetate in LC-MS grade water, and B) LC-MS grade acetonitrile as mobile phases. The 4 min gradient started from 30% B linearly increasing to 35% B in 0.5 minutes and kept at 35% B for 0.8 minutes, then linearly increased to 95% B in 0.7 minutes and kept there for 0.5 minutes, then re-equilibrating with 30% B for 1.5 min. The flow rate was kept at 0.6 ml/min, using an injection volume of 5 μl and the column temperature was set to 50° C. The individual compounds, 7.1, 7.2 and 7.3, observed for m/z 1451.5 elute at retention times 0.59, 0.71 and 0.74 minutes.


For the analysis of elemental composition of 7.1, 7.2 and 7.3 HRMS (High Resolution Mass Spectrometry) analysis was performed with an LTQ-Orbitrap Fourier Transform Mass Spectrometer (Thermo Electron) equipped with an electrospray ionization source operated in the negative-ion mode, scanning from m/z 300-2000. The chromatographic separation was achieved with an Acella LC system (Thermo Fisher) with the same column and gradient system as described above.


Using this chromatographic system the individual compounds elute at retention times 0.84, 1.20 and 1.30 minutes, respectively as shown in FIGS. 6a, and 7.1,7.2 and 7.3 were characterized at respectively m/z 1451.5786, 1451.5793, and 1451.5793, which is in good agreement with the theoretical m/z value of 1451.5820 (respectively −1.8 and −2.3 ppm). The corresponding chemical formula of these components is C62H100O38 for the uncharged species.


Example 3: Purification of 7.1, 7.2. and 7.3 Using Preparative LC-UV

Purification of 7.1, 7.2 and 7.3 was performed from the ethanol extract of Saccharomyces broth (strain STV016) containing minimal amount of the compounds of interest. Preparative separation was performed using Reversed Phase chromatography (Waters Atlantis T3, 30*150 mm, 5 um), gradient elution with LC-MS grade water and acetonitrile as eluent. A flow-rate of 40 ml/min and an injection volume of 300 ul was used.


Approximately 100 injections were performed and the compounds of interest were triggered by UV detection at 210 nm. All fractions of 7.1, 7.2 and 7.3 were pooled and freeze dried, before LC-MS and NMR analysis.


LC-MS of 71, 72 and 7.3 for Mass Confirmation and Purity Determination After Preparative Purification, Using LC-MS


The purity of 7.1, 7.2 and 7.3 was analyzed on an Acquity UPLC (Waters) coupled to a XEVO-TQ Mass Spectrometer (Waters) equipped with an electrospray ionization source operated in the negative-ion mode in MRM mode at the deprotonated molecules for all steviol glycosides studied, among these m/z 1451.5, representing the deprotonated molecule of a steviol glycoside containing 7 glucose molecules.


7.1 eluting at retention time 0.59 minutes could be estimated to be over 80% pure, whereas 7.2 and 7.3, eluting at retention times 0.71 and 0.74 minutes, could be estimated to be over 90% pure and 7.3 still contains about 5% of 7.2, shown in FIG. 6b.


Using HRMS (High Resolution Mass Spectrometry) analysis was performed with an LTQ-Orbitrap Fourier Transform Mass Spectrometer (Thermo Electron) equipped with an electrospray ionization source operated in the negative-ion mode the elemental composition of the individual compound was checked and found to be in agreement with the theoretical mass corresponding to the chemical formula of C62H100O38 for the uncharged species.


Example 4: Analysis of Rebaudioside 7.1

1.1 mg of fraction 7.1 obtained as described in Example 3 was dissolved in 1.3 mL of CDCl3/pyridine-d5 ⅓ (w/w) and 2 drops of DCOOD.


A series of COSY and TOCSY 2D NMR spectra with small increments of the mixing time afforded the assignment of almost all protons of each spin system (of the seven sugar units) for all three Rebaudiosides as well as the ent-kaurane diterpenoid core. The HSQC experiment allowed for the assignment of corresponding C—H couples.


The anomeric H of glcI and glcII were identified based on their long range correlation in HMBC to the protons of the ent-kaurane diterpenoid core.


The long range correlation of H2I-H1V and H3I-H1VI and H2II-H1III and H3II-H1III observed in corresponding ROESY spectra allowed the assignment of the substitution sites of glcI and glcII. The assignment was also corroborated by the long range correlation in HMBC experiment of the anomeric protons of glcIII up to glcVI with the 13C atoms of glcI and glcII, namely H1III-C2II, H1III-C3II, H1V-C2I and H1VI-C3I. The position of sugars glcIII, glcIV, glcV and glcVI is identical as in the structure of Rebaudioside M (FIG. 10).


The down field shift of the anomeric H1VII (5.86 ppm vs. 4.5-4.6 ppm) and small coupling constant (3.8 Hz vs. 7.8 Hz) indicates that the seventh sugar residue has the a configuration.


The position of the 7th sugar in Rebaudioside 7.1 could be identified from the long range HMBC coupling of H1VII and C3III long range proton coupling of H1VII-H3III in ROESY experiment and the low field shift of the C3III (83.8 ppm as compared to unsubstituted C3 atoms around 78-79 ppm). The structure of rebaudioside 7.1 is depicted in FIG. 7. All 1H and 13C NMR chemical shifts for Rebaudioside 7.1 are listed in Table 11. For the sake of comparison also the data of Rebaudioside M are included.









TABLE 11








1H and 13C NMR chemical shifts of Rebaudioside 7.1 in CDCl3/pyridine



1/3 and 3 drops of DCOOD recorded at 320K and Rebaudioside M in CDCl3/pyridine


1/1 and 3 drops of DCOOD recorded at 300K, δTMS = 0












Rebaudioside M

Rebaudioside 7.1



Position

1H


13C


1H


13C















 1
0.77 (dt, 13.5 & 4 Hz) & 1.76 (m)
39
0.74 (dt, 13.6 & 4.2 Hz) & 1.72 (m)
41.6


 2
1.35 & 2.12 (m)
19.1
1.69 & 2.06 (m)
17.4


 3
1.0 (dt, 13.2 & 4.7) & 2.14 (m)
38.2
0.99 (m) & 2.33 (d, 13 Hz)
38.2


 4

43.8

43.8


 5
1.02 (t, 13 Hz)
57
1.01 (d, 14.1 Hz)
58.6


 6
2.03 (m) & 2.17 (m)
23.2
1.93 & 2.11 (m)
23.9


 7
1.37 & 1.66 (m)
42.2
1.38 & 1.53 (m)
43.5


 8

40.6

42.5


 9
0.89 (d, 8.1 Hz)
54.1
0.88 (d, 7.7 Hz)
55.3


10

40

40.9


11
1.57 & 1.68(m)
19.8
1.56 & 1.64 (m)
21.3


12
1.66 & 2.43 (m)
37.9
1.66 & 2.28 (m)
39.5


13

87.3

88.6


14
1.76 (m) & 2.49 (d, 10.9 Hz)
42.7
1.72 & 2.40 (m)
44.8


15
1.83 & 1.99 (d, 17.3 Hz)
45.9
1.89 & 1.99 (d, 17 Hz)
48.1


16

152.2

154


17
4.78 & 5.42 (s)
104.6
4.85 & 5.45 (s)
105.9


18
1.21 (s)
27
1.22 (s)
29.8


19

176.4

176.1


20
1.15 (s)
16.1
1.08 (s)
15.9


 1I
5.98 (d, 8.3 Hz)
94.6
5.99 (d, 8.2 Hz)
94.9


 2I
4.22 (t, 8.6 Hz)
76.1
4.28 (m)
77.5


 3I
4.88 (t, 8.7 Hz)
87.9
4.52 (t, 9.2 Hz)
89.9


 4I
3.88 (m)
69.6
4.00 (m)
71.1


 5I
3.86 (m)
77.5
4.11 (m)
78.8


 6I
4.06 & 3.92 (m)
61.4
5.06 & 3.98 (m)
62.7


 1II
5.15 (d, 7.7 Hz)
95.3
5.07 (d, 7.8 Hz)
97.3


 2II
3.81 (m)
80.8
3.87 (m)
82.4


 3II
4.67 (t, 9 Hz)
87
4.58 (t, 9.1 Hz)
88


 4II
3.68 (m)
69.9
3.76 (m)
71


 5II
3.74 (m)
77.1
3.63 (m)
78.4


 6II
4.06 & 3.92 (m)
62.3
4.09 & 3.95 (m)
63.6


 1III
5.13 (d, 7.6 Hz)
104
5.22 (d, 8.2 Hz)
105.24


 2III
3.81 (m)
74.9
3.72 (m)
75.1


 3III
3.81 (m)
77.8
4.04 (m)
83.8


 4III
3.60 (m)
72.8
3.87 (m)
74.1


 5III
3.45 (m)
76.3
3.42 (m)
77.9


 6III
4.2 & 3.91 (m)
63.6
4.12 & 3.92 (m)
64.4


 1IV
5.147 (d, 8.1 Hz)
103.1
5.34 (d, 7.8 Hz)
104.6


 2IV
3.69 (m)
74.6
3.75 (m)
76.2


 3IV
4.20 (m)
76.9
4.21 (m)
78.9


 4IV
3.74 (m)
69.9
3.87 (m)
72.5


 5IV
3.81 (m)
77.2
3.88 (m)
78.9


 6IV
4.07 & 3.85 (m)
61.5
4.18 & 3.95 (m)
63.2


 1V
5.47 (d, 7.8 Hz)
103.5
5.42 (d, 7.8 Hz)
104.9


 2V
3.88 (m)
74.7
3.83 (m)
75.8


 3V
3.76 (m)
77.1
3.95 (m)
78.9


 4V
3.79 (m)
73.2
3.79 (m)
74.5


 5V
3.57 (m)
76.2
3.69 (m)
78.5


 6V
4.28 (dd, 11.1 & 4.1 Hz) & 4.01 (m)
63.6
4.32 & 4.08 (m)
64.8


 1VI
5.05 (d, 7.8 Hz)
103.4
5.3 (d, 7.8 Hz)
105.6


 2VI
3.68 (m)
76.9
3.76 (m)
76.2


 3VI
4.06 (m)
77.1
3.98 (m)
78.9


 4VI
3.77 (m)
70.5
3.77 (m)
72.4


 5VI
3.59 m)
77.2
4.07 (m)
78.8


 6VI
4.08 & 3.83 (m)
61.6
4.28 & 3.92 (m)
63.5


 1VII


5.86 (d, 3.6 Hz)
100.7


 2VII


3.87 (m)
74.2


 3VII


4.93 (t, 9.5 Hz)
75.7


 4VII


3.68 (m)
74.3


 5VII


4.65 (m)
74.2


 6VII


4.31 & 4.02 (m)
64.8









Example 5: Analysis of Rebaudioside 7.2

2.5 mg of sample was dissolved in 1 mL of CDCl3/pyridine-d5 1/1 (w/w) and 2 drops of DCOOD.


A series of COSY and TOCSY 2D NMR spectra with small increments of the mixing time afforded the assignment of almost all protons of each spin system (of the seven sugar units) for all three Rebaudiosides as well as the ent-kaurane diterpenoid core. The HSQC experiment allowed for the assignment of corresponding C—H couples.


The anomeric H of glcI and glcII were identified based on their long range correlation in HMBC to the protons of the ent-kaurane diterpenoid core.


The position of sugars glcIII, glcIV, glcV and glcVI is identical as in the structure of Rebaudioside M and the assignment is described in more detail in section dedicated to assignment of structure of Rebaudioside 7.1.


The position of the 7th sugar in Rebaudioside 7.2 could be identified from the long range HMBC coupling of H6IV and C1VII, long range proton coupling of H1VII-H6IV in ROESY experiment and the low field shift of the C6IV (69.4 ppm as compared to remaining C6 atoms 62-64 ppm). The 7th sugar is attached via β-glycosidic bond to GlcIV.The structure of rebaudioside 7.2 is depicted in FIG. 8. All 1H and 13C NMR chemical shifts of Rebaudioside 7.2 are listed in Table 12. For the sake of comparison also the data of Rebaudioside M are included.









TABLE 12








1H and 13C NMR chemical shifts of Rebaudioside 7.2 in CDCl3/pyridine



1/1 and 2 drops of DCOOD and Rebaudioside M in CDCl3/pyridine 1/1 and


3 drops of DCOOD recorded at 300K, δTMS = 0












Rebaudioside M

Rebaudioside 7.2



Position

1H


13C


1H


13C















 1
0.77 (dt, 13.5 & 4 Hz) & 1.76 (m)
39
0.77 (dt, 13.3 & 4 Hz) & 1.76 (m)
39.9


 2
1.35 & 2.12 (m)
19.1
1.35 & (2.13 (m)
18.6


 3
1.0 (dt, 13.2 & 4.7) & 2.14 (m)
38.2
0.99 (dt, 14 & 4.6) & 2.12 (m)
38.3


 4

43.8

43.1


 5
1.02 (t, 13 Hz)
57
1.02 (13.1 Hz)
57.1


 6
2.03 (m) & 2.17 (m)
23.2
2.02 & 2.15 (m)
22.7


 7
1.37 & 1.66 (m)
42.2
1.37 (m) & 1.66 (m)
42.3


 8

40.6

40


 9
0.89 (d, 8.1 Hz)
54.1
0.89 (d, 7.6 Hz)
54.1


10

40

39.1


11
1.57 & 1.68(m)
19.8
1.57 & 1.69 (m)
19.3


12
1.66 & 2.43 (m)
37.9
1.64 & 2.42 (m)
37.9


13

87.3

86.8


14
1.76 (m) & 2.49(d, 10.9 Hz)
42.7
1.75 (m) & 2.46 (d, 10.9 Hz)
42.8


15
1.83 & 1.99 (d, 17.3 Hz)
45.9
1.84 & 1.99 (d, 17 Hz)
45.9


16

152.2

151.6


17
4.78 & 5.42 (s)
104.6
4.77 & 5.39 (s)
104.5


18
1.21 (s)
27
1.22 (s)
27.5


19

176.4

176.1


20
1.15 (s)
16.1
1.14 (s)
15.7


 1I
5.98 (d, 8.3 Hz)
94.6
5.97 (d, 8.4 Hz)
94.7


 2I
4.22 (t, 8.6 Hz)
76.1
4.21 (t, 8.8 Hz)
76.4


 3I
4.88 (t, 8.7 Hz)
87.9
4.92 (t, 8 Hz)
87.1


 4I
3.88 (m)
69.6
3.84 (m)
70.7


 5I
3.86 (m)
77.5
3.84 (m)
76.9


 6I
4.06 & 3.92 (m)
61.4
4.04 & 3.91 (m)
61.5


 1II
5.15 (d, 7.7 Hz)
95.3
5.13 (d, 7.4 Hz)
95.2


 2II
3.81 (m)
80.8
3.78 (m)
80.5


 3II
4.67 (t, 9 Hz)
87
4.62 (t, 9.1 Hz)
88


 4II
3.68 (m)
69.9
3.63(m)
70.5


 5II
3.74 (m)
77.1
3.65 (m)
76.6


 6II
4.06 & 3.92 (m)
62.3
4.1 & 3.99 (m)
61.9


 1III
5.13 (d, 7.6 Hz)
104
5.04 (d, 8.1 Hz)
104.3


 2III
3.81 (m)
74.9
3.81 (m)
74.7


 3III
3.81(m)
77.8
3.76 (m)
77.6


 4III
3.60 (m)
72.8
3.57 (m)
73


 5III
3.45 (m)
76.3
3.42 (m)
76.2


 6III
4.2 & 3.91 (m)
63.6
4.17 & 3.91 (m)
63.8


 1IV
5.147 (d, 8.1 Hz)
103.1
4.94 (d, 8 Hz)
103.5


 2IV
3.69 (m)
74.6
3.65 (m)
74.4


 3IV
4.20 (m)
76.9
4.06 (m)
76.6


 4IV
3.74 (m)
69.9
3.83 (m)
69.7


 5IV
3.81 (m)
77.2
3.58 (m)
77.3


 6IV
4.07 & 3.85 (m)
61.5
4.43 (d, 9.6 Hz) & 3.63 (m)
69.4


 1V
5.47 (d, 7.8 Hz)
103.5
5.51 (d, 7.4 Hz)
103.4


 2V
3.88 (m)
74.7
3.88 (m)
74.8


 3V
3.76 (m)
77.1
3.89(m)
77.6


 4V
3.79 (m)
73.2
3.77 (m)
73.3


 5V
3.57 (m)
76.2
3.55 (m)
76.1


 6V
4.28 (dd, 11.1 & 4.1 Hz) & 4.01 (m)
63.6
4.25 (dd, 11.1 & 3.8 Hz) & 3.98 (m)
63.7


 1VI
5.05 (d, 7.8 Hz)
103.4
5.18 (d, 8.1 Hz)
103.14


 2VI
3.68 (m)
76.9
3.69 (m)
74.7


 3VI
4.06 (m)
77.1
4.12 (t, 9.1 Hz)
77.2


 4VI
3.77 (m)
70.5
3.82 (m)
70.6


 5VI
3.59 m)
77.2
3.71 (m)
77.2


 6VI
4.08 & 3.83 (m)
61.6
4.03 & 3.82(m)
61.4


 1VII


4.47 (d, 7.8 Hz)
103.6


 2VII


3.62 (m)
74.5


 3VII


3.84 (m)
77.7


 4VII


3.57 (m)
75.6


 5VII


3.82 (m)
77.3


 6VII


4.2 & 4.06 (m)
62









Example 6: Analysis of Rebaudioside 7.3

2.3 mg of sample was dissolved in 1 mL of CDCl3/pyridine-d5 ½ (w/w) and 3 drops of DCOOD.


A series of COSY and TOCSY 2D NMR spectra with small increments of the mixing time afforded the assignment of almost all protons of each spin system (of the seven sugar units) for all three Rebaudiosides as well as the ent-kaurane diterpenoid core. The HSQC experiment allowed for the assignment of corresponding C—H couples.


The anomeric H of glcI and glcII were identified based on their long range correlation in HMBC to the protons of the ent-kaurane diterpenoid core.


The position of sugars glcIII, glcIV, glcV and glcVI is identical as in the structure of Rebaudioside M and the assignment is described in more detail in section dedicated to assignment of structure of Rebaudioside 7.1.


The position of the 7th sugar in Rebaudioside 7.3 could be identified from the long range HMBC coupling of H1VII and C6VI, long range proton coupling of H1VII-H6VI in ROESY experiment and the low field shift of the C6VI (69.5 ppm as compared to remaining C6 atoms 61-63 ppm). The 7th sugar is attached via β-glycosidic bond to GlcVI. The structure of rebaudioside 7.3 is depicted in FIG. 9. All 1H and 13C NMR chemical shifts of Rebaudioside 7.3 are listed in Table 13. For the sake of comparison also the data of Rebaudioside M are included.









TABLE 13








1H and 13C NMR chemical shifts of Rebaudioside 7.3 in CDCl3/pyridine



1/2 and 3 drops of DCOOD and Rebaudioside M in CDCl3/pyridine 1/1 and


3 drops of DCOOD recorded at 300K, δTMS = 0












Rebaudioside M

Rebaudioside 7.3



Position

1H


13C


1H NMR


13C















 1
0.77 (dt, 13.5 & 4 Hz) & 1.76 (m)
39
0.79 (dt, 14 & 4.3 Hz) & 1.77 (m)
39.9


 2
1.35 & 2.12 (m)
19.1
1.38 & 2.18 (m)
19.1


 3
1.0 (dt, 13.2 & 4.7) & 2.14 (m)
38.2
1.03 (m) & 2.17 (m)
38.1


 4

43.8

43.7


 5
1.02 (t, 13 Hz)
57
1.04 (t, 12.8 Hz)
57


 6
2.03 (m) & 2.17 (m)
23.2
2.09 & 2.21 (m)
23.3


 7
1.37 & 1.66 (m)
42.2
1.41 & 1.69 (m)
42.1


 8

40.6

40.5


 9
0.89 (d, 8.1 Hz)
54.1
0.91 (d, 8 Hz)
53.9


10

40

39.4


11
1.57 & 1.68(m)
19.8
1.59 & 1.72 (m)
19.9


12
1.66 & 2.43 (m)
37.9
1.68 & 2.48 (m)
38


13

87.3

87.4


14
1.76 (m) & 2.49 (d, 10.9 Hz)
42.7
1.81 (d, 10.4 Hz) & 2.53 (d, 10.4 Hz)
42.1


15
1.83 & 1.99 (d, 17.3 Hz)
45.9
1.87 (d, 17.9 Hz) & 2.01 (d, 17.9 Hz)
46


16

152.2

152.3


17
4.78 & 5.42 (s)
104.6
4.84 & 5.47 (s)
104.6


18
1.21 (s)
27
1.27 (s)
27.9


19

176.4

176.3


20
1.15 (s)
16.1
1.2 (s)
16.4


 1I
5.98 (d, 8.3 Hz)
94.6
6.06 (d, 8.6 Hz)
94.5


 2I
4.22 (t, 8.6 Hz)
76.1
4.29 (t, 8.7 Hz)
76.1


 3I
4.88 (t, 8.7 Hz)
87.9
4.87 (t, 8.7 Hz)
88.6


 4I
3.88 (m)
69.6
3.99 (m)
69.3


 5I
3.86 (m)
77.5
3.89 (m)
77.3


 6I
4.06 & 3.92 (m)
61.4
4.04 & 4.10 (m)
61


 1II
5.15 (d, 7.7 Hz)
95.3
5.19 (d, 7.1 Hz)
95.4


 2II
3.81 (m)
80.8
3.87 (m)
80.8


 3II
4.67 (t, 9 Hz)
87
4.72 (t, 9.5 Hz)
86.9


 4II
3.68 (m)
69.9
3.81 (m)
69.6


 5II
3.74 (m)
77.1
3.70 (m)
77


 6II
4.06 & 3.92 (m)
62.3
4.10 & 3.99 (m)
61.9


 1III
5.13 (d, 7.6 Hz)
104
5.22 (d, 7.4 Hz)
104.1


 2III
3.81 (m)
74.9
3.90 (m)
74.9


 3III
3.81(m)
77.8
3.92 (m)
77.6


 4III
3.60 (m)
72.8
3.65 (m)
72.7


 5III
3.45 (m)
76.3
3.55 (m)
76.8


 6III
4.2 & 3.91 (m)
63.6
4.27 & 3.99 (m)
63.4


 1IV
5.147 (d, 8.1 Hz)
103.1
5.32 (d, 8.0 Hz)
103.2


 2IV
3.69 (m)
74.6
3.77 (m)
74.8


 3IV
4.20 (m)
76.9
4.23 (m)
77.3


 4IV
3.74 (m)
69.9
3.89 (m)
70.7


 5IV
3.81 (m)
77.2
3.90 (m)
77.5


 6IV
4.07 & 3.85 (m)
61.5
4.15 (d, 11.3 Hz) & 3.91 (m)
61.5


 1V
5.47 (d, 7.8 Hz)
103.5
5.53 (d, 7.8 Hz)
103.5


 2V
3.88 (m)
74.7
3.91 (m)
74.9


 3V
3.76 (m)
77.1
3.93 (m)
77.6


 4V
3.79 (m)
73.2
3.84 (m)
73


 5V
3.57 (m)
76.2
3.66 (m)
77.3


 6V
4.28 (dd, 11.1 & 4.1 Hz) & 4.01 (m)
63.6
4.37 (dd, 12 & 4.3 Hz) & 4.08 (m)
63.4


 1VI
5.05 (d, 7.8 Hz)
103.4
5.08 (d, 8.0 Hz)
103.6


 2VI
3.68 (m)
76.9
3.74 (m)
74.4


 3VI
4.06 (m)
77.1
4.09 (m)
77.1


 4VI
3.77 (m)
70.5
3.70 (m)
70.6


 5VI
3.59 m)
77.2
3.71 (m)
75.9


 6VI
4.08 & 3.83 (m)
61.6
4.52 (d, 8.7 Hz) & 3.70 (m)
69.5


 1VII


4.59 (d, 7.8 Hz)
103.6


 2VII


3.68 (m)
74.6


 3VII


3.93 (m)
77


 4VII


3.91 (m)
70.7


 5VII


3.67 (m)
76.7


 6VII


4.21 (m) & 4.1 (m)
61.8









In summary, three new rebaudiosides were determined as set out in Table 14.









TABLE 14







Summary of new Rebaudiosides









Steviol




glycosides compound
R1
R2





7.1


embedded image




embedded image







7.2


embedded image




embedded image







7.3


embedded image




embedded image











General Materials and Methods (NMR Analysis)

The solvent mixture was optimized for each of the Rebaudioside samples to obtain the best possible resolution of the signals of the anomeric protons. The amount of samples and the amount of solvent is critical for the resolution of the peaks as the shift of the peaks, especially the anomeric ones, are concentration and pH dependent (FIG. 12).


The spectra of Rebaudiosides 7.2 and 7.3 were recorded at 300K while in case of Rebaudioside 7.1 higher temperature had to be used. At 300K the resonances in the spectrum of Rebaudioside 7.1 were rather broad, indicating either bad solubility or slow conformational processes. Therefore, the final assignment of all signals was achieved at a sample temperature of 320K.


For each example, various 2D NMR experiments were conducted: COSY, TOCSY (with 40, 50, 60, 70, 80, 90 and 100 ms mixing time), HSQC, HMBC and ROESY (225, 400 ms mixing time) spectra were recorded at 320 K on a Bruker Avance III 600 and 700 MHz spectrometer. The detailed assignment for each example is specified in the example section.


In Examples 4, 5 and 6, the atom numbering of steviol and glucose is as set out in FIGS. 11A and 11B, respectively.









TABLE 15







Description of the sequence listing












Nucleic
Nucleic






acid (CpO
acid (CpO






for S.
for Y.
Amino






cerevisiae)


lipolytica)

acid
Id*
UniProt{circumflex over ( )}
Organism





SEQ ID NO:
SEQ ID NO:
SEQ ID
CPS_1
Q9FXV9

Lactuca sativa



1
151
NO: 2


(Garden Lettuce)


SEQ ID NO:
SEQ ID NO:
SEQ ID
tCPS_l
Q9FXV9

Lactuca sativa



3
152
NO: 4


(Garden Lettuce)


SEQ ID NO:
SEQ ID NO:
SEQ ID
CPS_2
D2X8G0

Picea glauca



5
153
NO: 6





SEQ ID NO:
SEQ ID NO:
SEQ ID
CPS_3
Q45221

Bradyrhizobium



7
154
NO: 8



japonicum



SEQ ID NO:
SEQ ID NO:
SEQ ID
KS_1
Q9FXV8

Lactuca sativa



9
155
NO: 10


(Garden Lettuce)


SEQ ID NO:
SEQ ID NO:
SEQ ID
tKS_l
Q9FXV8

Lactuca sativa



11
156
NO: 12


(Garden Lettuce)


SEQ ID NO:
SEQ ID NO:
SEQ ID
KS_2
D2X8G1

Picea glauca



13
157
NO: 14





SEQ ID NO:
SEQ ID NO:
SEQ ID
KS_3
Q45222

Bradyrhizobium



15
158
NO: 16



japonicum



SEQ ID NO:
SEQ ID NO:
SEQ ID
CPSKS_1
O13284

Phaeosphaeria sp



17
159
NO: 18





SEQ ID NO:
SEQ ID NO:
SEQ ID
CPSKS_2
Q9UVY5

Gibberella fujikuroi



19
160
NO: 20





SEQ ID NO:
SEQ ID NO:
SEQ ID
KO_1
B5MEX5

Lactuca sativa



21
161
NO: 22


(Garden Lettuce)


SEQ ID NO:
SEQ ID NO:
SEQ ID
KO_2
B5MEX6

Lactuca sativa



23
162
NO: 24


(Garden Lettuce)


SEQ ID NO:
SEQ ID NO:
SEQ ID
KO_3
B5DBY4

Sphaceloma manihoticola



25
163
NO: 26





SEQ ID NO:
SEQ ID NO:
SEQ ID
KAH_1
Q2HYU7

Artemisia annua



27
164
NO: 28


(Sweet wormwood).


SEQ ID NO:
SEQ ID NO:
SEQ ID
KAH_2
B9SBP0

Ricinus communis



29
165
NO: 30


(Castor bean).


SEQ ID NO:
SEQ ID NO:
SEQ ID
KAH_3
Q0NZP1

Stevia rebaudiana



31
166
NO: 32





SEQ ID NO:
SEQ ID NO:
SEQ ID
KAH_4
JP2009065886

Arabidopsis thaliana



33
167
NO: 34


(Mouse-ear cress)


SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT1_1
A9X3L6

Ixeris dentata var.



35
168
NO: 36



albiflora.



SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT1_2
B9SIN2

Ricinus communis



37
169
NO: 38


(Castor bean).


SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT3_1
A9X3L7

Ixeris dentata var.



39
170
NO: 40



Albiflora



SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT3_2
B9IEM5

Populus trichocarpa



41
171
NO: 42


(Western balsam poplar)


SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT3_3
Q9M6E7

Nicotiana tabacum



43
172
NO: 44





SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT3_4
A3E7Y9

Vaccaria hispanica



45
173
NO: 46





SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT3_5
P10249

Streptococcus mutans



47
174
NO: 48





SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT4_1
A4F1T4

Lobelia erinus



49
175
NO: 50


(Edging lobelia)


SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT4_2
Q9M052

Arabidopsis thaliana



51
176
NO: 52


(Mouse-ear cress)


SEQ ID NO:
SEQ ID NO:
SEQ ID
CPR_1
Q7Z8R1

Gibberella fujikuroi



53
177
NO: 54





SEQ ID NO:
SEQ ID NO:
SEQ ID
CPR_2
Q9SB48

Arabidopsis thaliana



55
178
NO: 56


(Mouse-ear cress)


SEQ ID NO:
SEQ ID NO:
SEQ ID
CPR_3
Q9SUM3

Arabidopsis thaliana



57
179
NO: 58


(Mouse-ear cress)


SEQ ID NO:
SEQ ID NO:
SEQ ID
CPS_SR
O22667

Stevia rebaudiana



59
141
NO: 60





SEQ ID NO:
SEQ ID NO:
SEQ ID
tCPS_SR
O22667

Stevia rebaudiana



61
142
NO: 62





SEQ ID NO:
SEQ ID NO:
SEQ ID
KS_SR
Q9XEI0

Stevia rebaudiana



63
143
NO: 64





SEQ ID NO:
SEQ ID NO:
SEQ ID
tKS_SR
Q9XEI0

Stevia rebaudiana



65
144
NO: 66





SEQ ID NO:
SEQ ID NO:
SEQ ID
KO_SR
Q4VCL5

Stevia rebaudiana



67
145
NO: 68





SEQ ID NO:
SEQ ID NO:
SEQ ID
KAH_SR
US7927851

Stevia rebaudiana



69
146
NO: 70





SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT1_SR
Q6VAB0

Stevia rebaudiana



71
147
NO: 72





SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT3_SR
Q6VAA6

Stevia rebaudiana



73
148
NO: 74





SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT4_SR
Q6VAB4

Stevia rebaudiana



75
149
NO: 76





SEQ ID NO:
SEQ ID NO:
SEQ ID
CPR_SR
Q2I6J8

Stevia rebaudiana



77
150
NO: 78





SEQ ID NO:

SEQ ID
tHMG1
G2WJY0

Saccharomyces cerevisiae



79

NO: 80





SEQ ID NO:

SEQ ID
ERG20
E7LW73

Saccharomyces cerevisiae



81

NO: 82





SEQ ID NO:

SEQ ID
BTS1
E7Q9V5

Saccharomyces cerevisiae



83

NO: 84





SEQ ID NO:
SEQ ID NO:
SEQ ID
KO_Gibfu
O94142

Gibberella fujikuroi



85
180
NO: 86





SEQ ID NO:
SEQ ID NO:
SEQ ID
UGT2_1a
B3VI56/99%

Stevia rebaudiana



87
181
NO: 88





SEQ iD NO:

SEQ ID
KAH_ASR1
Xxx

S. rebaudiana



89

NO: 90





SEQ ID NO:

SEQ ID
KAH_ASR2
Q0NZP1_STERE

S. rebaudiana



91

NO: 92





SEQ ID NO:

SEQ ID
KAH_AAT
Q6NKZ8_ARATH

A. thaliana



93

NO: 94





SEQ ID NO:

SEQ ID
KAH_AVV
F6H1G0_VITVI/98%

Vitis vinifera



95

NO: 96





SEQ ID NO:

SEQ ID
KAH_AMT
Q2MJ20_MEDTR

Medicago truncatula



97

NO: 98





SEQ ID NO:

SEQ ID
UGT2_1b
B3VI56/99%

S. rebaudiana



99

NO: 100





SEQ ID NO:

SEQ ID
UGT2_2
Q53UH5_IPOPU

I. purpurea



101

NO: 102





SEQ ID NO:

SEQ ID
UGT2_3
UGAT_BELPE/99%

Bellis perennis



103

NO: 104





SEQ ID NO:

SEQ ID
UGT2_4
B3VI56

S. rebaudiana



105

NO: 106





SEQ iD NO:

SEQ ID
UGT2_5
Q6VAA8

S. rebaudiana



107

NO: 108





SEQ ID NO:

SEQ ID
UGT2_6
Q8LKG3

S. rebaudiana



109

NO: 110





SEQ ID NO:

SEQ ID
UGT2_7
B9HSH7_POPTR

Populus trichocarpa



111

NO: 112





SEQ ID NO:

SEQ ID
UGT_RD1
Q6VAA3

S. rebaudiana



113

NO: 114





SEQ ID NO:

SEQ ID
UGT_RD2
Q8H6A4

S. rebaudiana



115

NO: 116





SEQ ID NO:

SEQ ID
UGT_RD3
Q6VAA4

S. rebaudiana



117

NO: 118





SEQ ID NO:

SEQ ID
UGT_RD4
Q6VAA5

S. rebaudiana



119

NO: 120





SEQ ID NO:

SEQ ID
UGT_RD5
Q6VAA7

S. rebaudiana



121

NO: 122





SEQ ID NO:

SEQ ID
UGT_RD6
Q6VAA8

S. rebaudiana



123

NO: 124





SEQ ID NO:

SEQ ID
UGT_RD7
Q6VAA9

S. rebaudiana



125

NO: 126





SEQ ID NO:

SEQ ID
UGT_RD8
Q6VAB1

S. rebaudiana



127

NO: 128





SEQ ID NO:

SEQ ID
UGT_RD9
Q6VAB2

S. rebaudiana



129

NO: 130





SEQ ID NO:

SEQ ID
UGT_RD10
Q6VAB3

S. rebaudiana



131

NO: 132





SEQ ID NO:

SEQ ID
UGT_RD11
B9VVB1

S. rebaudiana



133

NO: 134





SEQ ID NO:

SEQ ID
UGT_RD12
C7EA09

S. rebaudiana



135

NO: 136





SEQ ID NO:

SEQ ID
UGT_RD13
Q8LKG3

S. rebaudiana



137

NO: 138





SEQ ID NO:

SEQ ID
UGT_RD14
B3VI56

S. rebaudiana



139

NO: 140






SEQ ID NO:

tCPS





182







SEQ ID NO:

tKS





183







SEQ ID NO:

CPSKS





184







SEQ ID NO:

KAH4





185







SEQ ID NO:

KO_Gibfu





186







SEQ ID NO:

CPR1





187







SEQ ID NO:

CPR3





188







SEQ ID NO:

UGT1





189







SEQ ID NO:

UGT3





190







SEQ ID NO:

UGT4





191







SEQ ID NO:

UGT2_1a





192







SEQ ID NO:

pTPI





193







SEQ ID NO:

gpdT-pGPD





194







SEQ ID NO:

pgmT-pTEF





195







SEQ ID NO:

pgkT-pPGM





196







SEQ ID NO:

LEU2 and





197

flanking







sequences





SEQ ID NO:

vector sequences





198







SEQ ID NO:

pENO





199







SEQ ID NO:

HPH





200






SEQ ID NO:


Sc Eno2.pro




201







SEQ ID NO:


Sc Fba1.pro




202







SEQ ID NO:


Sc Tef1.pro




203







SEQ ID NO:


Sc Pgk1.pro




204







SEQ ID NO:


Kl prom 12.pro




205







SEQ ID NO:


Ag lox_TEF1.pro




206







SEQ ID NO:


Kl prom 6.pro




207







SEQ ID NO:


Sc Pma1.pro




208







SEQ ID NO:


Sc Vps68.pro




209







SEQ ID NO:


Sc Oye2.pro




210







SEQ ID NO:


KANMX ORF




211







SEQ ID NO:


Adh1.ter




212







SEQ ID NO:


Adh2.ter




213







SEQ ID NO:


Gmp1.ter




214







SEQ ID NO:


Sc Tal1.ter




215







SEQ ID NO:


Sc Tpi1.ter




216







SEQ ID NO:


Ag Tef1_lox.ter




217







SEQ ID NO:


Sc Pdc1.ter




218







SEQ ID NO:


Sc Tdh1.ter




219







SEQ ID NO:


Sc Eno1.ter




220







SEQ ID NO:


Kl prom3.pro




221







SEQ ID NO:


Kl prom2.pro




222







SEQ ID NO:


Sc PRE3. Pro




223








Claims
  • 1. A steviol glycoside having the formula (II)
  • 2. The steviol glycoside according to claim 1, wherein it is fermentatively produced.
  • 3. A method for production of a steviol glycoside according to claim 1, which method comprises: providing a recombinant yeast cell comprising recombinant nucleic acid sequences encoding polypeptides comprising the amino acid sequences encoded by: SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 77, SEQ ID NO: 71, SEQ ID NO: 87, SEQ ID NO: 73 and SEQ ID NO: 75;fermenting the recombinant yeast cell in a suitable fermentation medium.
  • 4. The method of claim 3, further comprising recovering the steviol glycoside from the fermentation medium.
  • 5. A composition comprising a steviol glycoside according to claim 1 and one or more different steviol glycosides.
  • 6. The composition according to claim 5, which is a sweetener composition or flavor composition.
  • 7. A foodstuff, feed and/or beverage which comprises a steviol glycoside according to claim 1.
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/563,475, filed 29 Sep. 2017, which is a National Stage entry of International Application No. PCT/EP2016/057360, filed 4 Apr. 2016, which claims priority to U.S. Provisional Application No. 62/142,631, filed 3 Apr. 2015, the contents of each of which are hereby incorporated by reference in their entirety.

US Referenced Citations (31)
Number Name Date Kind
6180157 Fotos et al. Jan 2001 B1
6365216 Dron et al. Apr 2002 B1
9522929 Mao et al. Dec 2016 B2
9527880 Mao et al. Dec 2016 B2
9567619 Mao et al. Feb 2017 B2
9643990 Mao et al. May 2017 B2
9783566 Mao et al. Oct 2017 B2
9850270 Mao et al. Dec 2017 B2
10689681 Boer et al. Jun 2020 B2
20060083838 Jackson et al. Apr 2006 A1
20090162500 Mui et al. Jun 2009 A1
20110027446 Gelov Feb 2011 A1
20110033525 Liu Feb 2011 A1
20110183056 Morita et al. Jul 2011 A1
20130209658 Spelman et al. Aug 2013 A1
20130251881 Mutilangi et al. Sep 2013 A1
20130309389 Carlson et al. Nov 2013 A1
20140017378 Purkayastha et al. Jan 2014 A1
20140171519 Prakash et al. Jun 2014 A1
20140227421 Markosyan Aug 2014 A1
20140296499 Chen et al. Oct 2014 A1
20140329281 Houghton-Larsen et al. Nov 2014 A1
20140342043 Bell et al. Nov 2014 A1
20140343262 Prakash et al. Nov 2014 A1
20150257424 Catani et al. Sep 2015 A1
20160097070 Mao et al. Apr 2016 A1
20170190727 Krammer et al. Jul 2017 A1
20170240942 Robertson et al. Aug 2017 A1
20170362268 Carlson et al. Dec 2017 A1
20170369922 Olsson et al. Dec 2017 A1
20200283815 Boer et al. Sep 2020 A1
Foreign Referenced Citations (44)
Number Date Country
3055288 Sep 2018 CA
102216313 Oct 2011 CN
103404833 Nov 2013 CN
104684414 Jun 2015 CN
105051195 Nov 2015 CN
3383201 Oct 2018 EP
2001048727 Feb 2001 JP
2012504552 Feb 2012 JP
0160842 Aug 2001 WO
2010038911 Apr 2010 WO
2010151653 Dec 2010 WO
201153378 May 2011 WO
2013022989 Feb 2013 WO
2013066490 May 2013 WO
2013096420 Jun 2013 WO
2013148177 Oct 2013 WO
2014052457 Apr 2014 WO
2014086890 Jun 2014 WO
2014122227 Aug 2014 WO
2014122328 Aug 2014 WO
2014146135 Sep 2014 WO
2014172055 Oct 2014 WO
2014191580 Dec 2014 WO
2014191581 Dec 2014 WO
2014193888 Dec 2014 WO
2014193889 Dec 2014 WO
2014193934 Dec 2014 WO
2015006764 Jan 2015 WO
2015023928 Feb 2015 WO
2015051454 Apr 2015 WO
2015065650 May 2015 WO
2015171555 Nov 2015 WO
2016023844 Feb 2016 WO
2016028899 Feb 2016 WO
2016038095 Mar 2016 WO
2016043926 Mar 2016 WO
2016054534 Apr 2016 WO
2016054548 Apr 2016 WO
2016086233 Jun 2016 WO
2016100689 Jun 2016 WO
2016120486 Aug 2016 WO
2016156616 Oct 2016 WO
2017095932 Jun 2017 WO
2018164747 Sep 2018 WO
Non-Patent Literature Citations (35)
Entry
Chaturvedula, Venkata Sai Prakash et al. “Additional Minor Diterpene Glycosides from Stevia rebaudiana”, Natural Product Communications, 2011, pp. 1059-1062, vol. 6, No. 8.
Chaturvedula, Venkata Sai Prakash et al., “Minor diterpenoid glycosides from the leaves of Stevia rebaudiana,” Phytochemistry Letters, 2011, pp. 209-212, vol. 4.
Chaturvedula, Venkata Sai Prakash et al., “Structure of the novel [alpha]-glucosyl linked diterpene glycosides from Stevia rebaudiana,” Carbohydrate Research, 2011, pp. 2034-2038, vol. 346.
Chaturvedula, Venkata Sai Prakash et al., “Structures of the novel diterpene glycosides from Stevia rebaudiana,” Carbohydrate Research, 2011, pp. 1057-1060, vol. 346.
Chaturvedula, Venkata Sai Prakash et al., “Utilization of RP-HPLC fingerprinting analysis for identification of diterpene glycosides from stevia rebaudinana,” International Journal of Research in Phytochemistry & Pharmacology, 2011, pp. 88-92, vol. 1, No. 2.
Chaturvedula, Venkata Sai Prakash et al., “A new diterpene glycoside from Stevia rebaudiana,” Molecules, 2011, pp. 2937-2943, vol. 16.
Chaturvedula, Venkata Sai Prakash et al., “Diterpene Glycosides from Stevia rebaudiana,” Molecules, 2011, pp. 3552-3562, vol. 16.
Chaturvedula, Venkata Sai Prakash et al., “IR Spectral Analysis of Diterpene Glycosides Isolated from Stevia rebaudiana,” Food and Nutrition Sciences, 2012, pp. 1467-1471, vol. 3.
Chaturvedula, Venkata Sai Prakash et al., “NMR Spectral Analysis and Hydrolysis Studies of Rebaudioside N, a Minor Steviol Glycoside of Stevia rebaudiana Bertoni,” Food and Nutrition Sciences, 2013, pp. 1004-1008, vol. 4.
Chaturvedula, Venkata Sai Prakash, “Isolation and Structural Characterization of a New Minor Penta β-D-Glucopyranosyl Diterpene from Stevia rebaudiana Bertoni,” American Journal of Plant Sciences, 2014, pp. 3519-3525, vol. 5.
EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), “Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive,” EFSA Journal 8(4):1537 (2010).
EFSA Panel on Food Additives and Nutrient Sources added to Food, “Scientific opinion on the safety of the proposed amendment of the specifications for steviol glycosides (E 960) as a food additive,” EFSA Journal 13(12):4316.
EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), “Scientific opinion on the safety of the extension of use of steviol glycosides (E 960) as a food additive,” EFSA Journal 13(6):4146 (2015).
FDA GRAS Notice (GRN) No. 626; http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInvtentory/default.htm. (68 pages)(2016).
Hsu, Che-Hsiung et al., “Toward Automated Oligosaccharide Synthesis,” Angewandte Chemie Int. Ed., 2011, pp. 11872-11923, vol. 50.
Ibrahim, Mohamed A. et al., “Minor Diterpene Glycosides from the Leaves of Stevia rebaudiana,” Journal of Natural Products, 2014, 1231-1235, vol. 77.
Kusama, Satoru et al., “Transglucosylation into stevioside by the enzyme system from Streptomyces sp.,” Agricultural and Biological Chemistry, Oct. 1986, pp. 2445-2451, vol. 50, No. 10.
Prakash, Indra et al., “Catalytic hydrogenation of the sweet principles of Stevia rebaudiana, Rebaudioside B, Rebaudioside C, and Rebaudioside D and sensory evaluation of their reduced derivatives,” International Journal of Molecular Sciences, 2012, pp. 15126-15136, vol. 13, No. 11.
Prakash, Indra et al., “Stability of rabaudioside A under acidic conditions and its degradation products,” Food Research International, 2012, pp. 65-75, vol. 48.
Prakash, Indra et al., “Isolation, characterization and sensory evaluation of a Hexa β-D-glucopyranosyl diterpene from Stevia rebaudiana,” Natural Product Communications, 2013, pp. 1523-1526, vol. 8, No. 11.
Prakash, Indra et al., “Additional Minor Diterpene Glycosides from Stevia rebaudiana Bertoni,” Molecules, 2013, pp. 13510-13519, vol. 18.
Chaturvedula, Venkata Sai Prakash and Prakash, Indra et al., “Structural Characterization and Hydrolysis Studies of Rebaudioside E, A Minor Sweet Component of Stevia Rebaudiana,” European Chemical Bulletin, 2013, pp. 298-302, vol. 2, No. 5.
Prakash, Indra et al., “Development of Next Generation Stevia Sweetener Rebaudioside M,” Foods, 2014, pp. 162-175, vol. 3.
Prakash, Indra et al., “Bioconversion of Rebaudioside I from Rebaudioside A,” Molecules, 2014, pp. 17345-17355, vol. 19.
Prakash, Indra et al, “Isolation and Characterization of a Novel Rebaudioside M Isomer from a Bioconversion Reaction of Rebaudioside A and NMR Comparison Studies of Rebaudioside M Isolated from Stevia rebaudiana Bertoni and Stevia rebaudiana Morita,” Biomolecules, 2014, pp. 374-389, vol. 4, No. 2.
Prakash, Indra et al., “Structural Characterization of the Degradation Products of a Minor Natural Sweet Diterpene Glycoside Rebaudioside M under Acidic Conditions,” International Journal of Molecular Sciences, 2014, pp. 1014-1025, vol. 15.
Prakash, Indra et al., “A New Diterpene Glycoside: 15α-Hydroxy-Rebaudioside M Isolated from Stevia rebaudiana,” Natural Product Communications, 2015, pp. 1159-1161, vol. 10, No. 7.
Starratt, Alvin N. et al., “Rebaudioside F, a diterpene glycoside from Stevia rebaudiana”, Phytochemistry, 2002, pp. 367-370, vol. 59.
Verduyn, Cornelis et al., “Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation,” Yeast, 1992, pp. 501-517, vol. 8.
Praksh, I. et al., “Stability of rebaudioside A under acidic conditions and its degradation products”, Food Research International, 2012, XP-002717610.
“Chemistry Glossary”, Epimer @ Chemistry Dictionary & Glossary, Jan. 21, 2021, pp. 1-2, [retrieved online at http://glossary.periodni.com/glossary.php?en=epimer].
Bautista, Vanesa et al., “Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei”, Extremophiles, 2012, pp. 147-159, vol. 16.
Le, Anh S. and Mulderrig, Kathleen Bowe, “Sorbitol and Mannitol”, Alternative Sweeteners: Third Edition, Revised and Expanded, Chapter 18, 2001, pp. 317-334.
Ohta, Masaya, “Characterization of Novel Steviol Glycosides from Leaves of Stevia rebaudiana Morita”, Journal of Applied Glycoscience, Aug. 17, 2010, pp. 199-209, vol. 57.
International Search Report of International Patent Application No. PCT/EP2016/057360 dated Jun. 16, 2016.
Related Publications (1)
Number Date Country
20220256903 A1 Aug 2022 US
Provisional Applications (1)
Number Date Country
62142631 Apr 2015 US
Continuations (1)
Number Date Country
Parent 15563475 US
Child 17727130 US