process for preparing rebaudioside m

专利摘要:
PRODUCTION OF DITERPENE. The present invention relates to a recombinant microorganism comprising nucleotide sequence (s) which encodes: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having enthalurene synthase activity; a polypeptide having enthalurene oxidase activity; and a polypeptide having kurenoic acid 13-hydroxylase activity, a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviol, a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviolmonoside or at the position C-19 of rebaudioside A, a polypeptide capable of catalyzing the addition of a glucose at the C-19 position of steviolbioside; and a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of stevioside or at the C-19 position of rebaudioside D, where the expression of the nucleotide sequence (s) gives the microorganism the ability to produce at least eebaudioside M. Such a recombinant microorganism can be used in a method of producing rebaudioside M.
公开号:BR112016000745B1
申请号:R112016000745-0
申请日:2014-07-15
公开日:2021-01-05
发明作者:Viktor Marius Boer；Igor Galaev；Robertus Antonius Mijndert Van Der Hoeven
申请人:Dsm Ip Assets B.V.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[01] The present invention relates to a recombinant microorganism capable of producing a glycosylated diterpene and / or diterpene and to a process for the production of a glycosylated diterpene and / or diterpene using such a cell. The invention also relates to a fermentation broth comprising a glycosylated diterpene and / or diterpene obtainable by such a process. The invention also relates to a method for converting a first glycosylated diterpene into a second glycosylated diterpene BACKGROUND OF THE INVENTION
[02] The world demand for high potency sweeteners is increasing and, with the mixture of different artificial sweeteners, becoming a standard practice. However, the demand for alternatives is expected to increase. The leaves of the perennial herb, Stevia rebaudiana Bert., Accumulate amounts of intensely sweet compounds known as steviol glycosides. Although the biological function of these compounds is unclear, they have commercial significance as high-potency alternative sweeteners, with the added advantage that Stevia sweeteners are natural plant products.
[03] These sweet steviol glycosides have functional and sensory properties that appear to be superior to many high potency sweeteners. In addition, studies suggest that stevioside may lower blood glucose levels in Type II diabetics and may lower blood pressure in mildly hypertensive patients.
[04] Steviol glycosides accumulate in Stevia leaves where they can comprise 10 to 20% of the dry weight of the leaf. Stevioside and rebaudioside A are both heat and pH stable and suitable for use in carbonated drinks 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 that accumulates in Stevia leaves. It can be about 200 times sweeter than sucrose. Rebaudioside M is also a high potency diterpene glycoside sweetener present in trace amounts in certain Stevia leaf varieties, but it has been suggested to have a better flavor profile.
[05] Currently, steviol glycosides are extracted from the Stevia plant. In Stevia, (-) - kaurenoic acid, an intermediate in the biosynthesis of gibberellic acid (GA), is converted to tetracyclic dipterepene steviol, which then proceeds through a several-step glycosylation pathway to form the various steviol glycosides. However, yields can be variable and affected by agriculture and environmental conditions. In addition, growing Stevia requires substantial land area, long before harvest, labor intensive and additional costs for the extraction and purification of glycosides.
[06] New, more standardized sources, with a unique clean composition, with no after-taste, of glycosides, such as rebaudioside M, are necessary to satisfy the growing commercial demand for natural high-power sweeteners. SUMMARY OF THE INVENTION
[07] Development of fermentation technologies for the production of high-value steviol glycosides that can be low-cost, more abundant, and currently perceived as trace by-products, is desired.
[08] There are more than 30 different steviol glycosides found within the stevia leaf, including Reb A, and next generation sweeteners, such as Reb D and Reb M, which have superior flavor profiles, but which are found in very large quantities lower within the stevia leaf. Because the most desirable steviol glycosides are rare within the stevia leaf, fermentation processes offer attractive commercial advantages for large-scale production. This invention illustrates a method for quickly and reliably producing a variety of next-generation stevia sweeteners on an “on-demand” basis that could still be adapted to evolving consumer requirements for stevia.
[09] In Stevia, steviol is synthesized from GGPP, which is formed by the deoxyxylulose 5-phosphate pathway. The activity of two diterpene (-) - copalyl diphosphate synthase (CPS) and (-) - kaurene synthase (KS) cyclases, results in the formation of (-) - kaurene which is then oxidized in a three-step reaction by (-) - kaurene oxidase (KO) to form (-) - kaurenoic acid.
[010] In stevia leaves, (-) - kaurenoic acid is then hydroxylated by ent-kaurenoic acid 13-hydroxylase (KAH) to form steviol. Steviol is then glycosylated by a series of UDP-glycosyltransferases (UGTs).
[011] This invention relates to a microorganism capable of producing a diterpene, such as steviol, or a glycosylated diterpene (i.e., a diterpene glycoside), such as steviolmonoside, steviolbioside, stevioside, rebaudioside A, reaudioside B, rebaudioside B C, rebaudioside D, rebaudioside E, rebaudioside F, rubusoside or dulcoside A.
[012] According to the invention, there is thus provided a recombinant microorganism comprising one or more nucleotide sequences which codes for: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having enthalurene synthase activity; a polypeptide having enthalurene oxidase activity; and a polypeptide having kurenoic acid 13-hydroxylase activity, a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviol, a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviolmonoside or at the position C-19 of rebaudioside A, a polypeptide capable of catalyzing the addition of a glucose at the C-19 position of steviolbioside; and a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of stevioside or at the C-19 position of rebaudioside D, in which the expression of the nucleotide sequence (s) gives the microorganism the ability to produce at least rebaudioside M.
[013] Normally, a recombinant microorganism of the present invention will comprise all of the above-mentioned nucleotide sequences.
[014] The invention also provides: - a process for the preparation of rebaudioside M which comprises fermenting a recombinant microorganism of the invention in a suitable fermentation medium and, optionally, recovering the glycosylated diterpene or diterpene; - a fermentation broth comprising M rebaudioside obtainable by the process of the invention; - rebaudioside M obtained by a process according to the invention or obtainable from a fermentation broth according to the invention; - a food product, food or drink comprising rebaudioside M according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS
[015] Figure 1 shows a schematic representation of the plasmid pUG7-EcoRV.
[016] Figure 2 presents a schematic representation of the method by which the ERG20, tHMG1 and BTS1 overexpression cassettes are designed (A) and integrated (B) in the yeast genome. (C) shows the final situation after removal of the KANMX marker by Cre-recombinase.
[017] Figure 3 presents a schematic representation of the ERG9 knockdown construct. This consists of a 3 '500 bp long part of ERG9, 98 bp of the TRP1 promoter, the TRP1 open reading frame and terminator, followed by a 400 bp long sequence downstream of the ERG9. Due to the introduction of an Xbal site at the end of the ERG9 open reading frame the last amino acid changes to Ser and the stop codon to Arg. A new stop codon is located on the TPR1 promoter, resulting in an extension of 18 amino acids.
[018] Figure 4 presents a schematic representation of how UGT2 is integrated into the genome. A. different fragments used in the transformation; B. situation after integration; C. situation after expression of Cre recombinase.
[019] Figure 5 presents a schematic representation of how the GGPP pathway to Reba is integrated into the genome. A. different fragments used in the transformation; B. situation after integration.
[020] Figure 6 presents a schematic representation of how deletions of specific genes are constructed. A. in the genome of the parental strain; B. situation after integration; C. situation after “out” recombination.
[021] Figure 7 establishes a schematic diagram of the potential pathways that lead to biosynthesis of steviol glycosides.
[022] Figure 8 shows analytical HPLC chromatograms generated in the preparation of RebM. From the top: concentrated extraction; centrifuged food (diluted concentrate); eluate after 1st chromatographic run; eluate after 2nd chromatographic run (pH 8.5); and Reba standard.
[023] Figure 9 establishes a schematic representation of plasmid MB6969.
[024] Figure 10 establishes a schematic representation of plasmid MB6856.
[025] Figure 11 establishes a schematic representation of plasmid MB6857.
[026] The schematic Figure 12 of the plasmid establishes MB6948. a representation
[027] Schematic figure 13 of the plasmid establishes an MB6958. representation
[028] The schematic Figure 14 of the plasmid establishes MB7015. a representation
[029] The schematic Figure 15 of the plasmid establishes MB6986. a representation
[030] The schematic Figure 16 of the plasmid establishes MB7059. a representation
[031] The schematic Figure 17 of the plasmid establishes MB7100. a representation
[032] The schematic Figure 18 of the plasmid establishes MB6988. a representation
[033] The schematic Figure 19 of the plasmid establishes MB7044. a representation
[034] The schematic Figure 20 of the plasmid establishes MB7094. a representation
[035] Schematic Figure 21 of the plasmid DESCRIPTION OF THE LISTING OF pRS417 Con5-3. SEQUENCES a representation
[036] A description of the sequences is set out in Table 1. The sequences described here can be defined with reference to the sequence listing, or with reference to the database access numbers also set out in Table 1. DETAILED DESCRIPTION OF THE INVENTION
[037] Throughout this specification and in the appended claims, the words "comprises", "includes" and "having" and variations such as "comprise", "comprising", "include" and "including" are to be interpreted inclusive . That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.
[038] The articles "one" and "one" are used here to refer to one or more than one (that is, one or at least one) of the grammatical object of the article. For example, "an element" can mean an element or more than one element.
[039] The invention relates to a recombinant microorganism that is capable of producing M rebaudioside (RebM). The structure of RebM is described in the Figure and in J. Appl. Glycosci. 57, 199-209, 2010.
[040] RebM is a glycosylated diterpene. For the purposes of the present invention, a diterpene usually means an organic compound made up of four isoprene units. Such a compound can be derived from geranylgeranyl pyrophosphate. A glycosylated diterpene or diterpene glycoside is a diterpene in which a sugar is typically attached to a different portion of carbohydrate.
[041] Typically, in a diterpene a glycoside, the sugar group can be linked through its anomeric carbon to another group via a glycosidic bond.
[042] According to the invention, a recombinant microorganism is provided. The recombinant microorganism comprises one or more nucleotide sequences that code for: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having enthalurene synthase activity; a polypeptide having enthalurene oxidase activity; and a polypeptide having kurenoic acid 13-hydroxylase activity.
[043] For the purposes of the present invention, a polypeptide having ent-copalyl pyrophosphate synthase (EC 5.5.1.13) is capable of catalyzing the chemical reaction:

[044] This enzyme has a substrate, geranylgeranyl pyrophosphate, and a product, ent-copalyl pyrophosphate. This enzyme participates in the biosynthesis of gibberellin. This enzyme belongs to the isomerase family, specifically from the class of intramolecular lyases. The systematic name of this class of enzymes is ent-copalyl-diphosphate lyase (deciclizing). Other names in common use include having ent-copalyl pyrophosphate synthase, ent-kaurene synthase A and ent-kaurene synthase A.
[045] For the purposes of the present invention, a polypeptide with 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
[046] Thus, this enzyme has a substrate, ent-copalyl diphosphate, and two products, ent-kaurene and diphosphate.
[047] This enzyme belongs to the lyase family, specifically those carbon-oxygen lyases that act on phosphates. The systematic name of this class of enzymes is ent-copalyl diphosphate diphosphate lyase (cyclizing, ent-kurene-forming). Other commonly used names include ent-kurene synthase B, ent-kurene synthase B, ent-copalyl diphosphate diphosphate lyase and (cyclizing). This enzyme participates in diterpenoid biosynthesis.
[048] The ent-copalyl diphosphate synthas may also have a distinct ent-kaurene synthase activity that is associated with the same protein molecule. The reaction catalyzed by ent-kaurene synthase is the next step in the biosynthetic pathway of gibberellins. The two types of enzyme activity are distinct and site-directed mutagenesis to suppress protein ent-kuren synthase activity leads to the accumulation of ent-copalyl pyrophosphate.
[049] Thus, a single nucleotide sequence used in the present invention can code for a polypeptide with ent-copalyl pyrophosphate synthase activity and ent-kurene synthase activity. Alternatively, the two activities can be coded for two separate separate nucleotide sequences.
[050] For the purposes of the present invention, a polypeptide with ent-kaurene oxidase activity (EC 1.14.13.78) is a polypeptide that is capable of catalyzing three successive oxidations of the 4-methyl group of ent-kaurene to give kaurenoic acid. This activity typically requires the presence of a cytochrome P450.
[051] For the purposes of the present invention, a polypeptide having kurenoic acid 13-hydroxylase activity (EC 1.14.13) is one that is capable of catalyzing the formation of steviol (ent-caur-16-en-13-ol acid -19-oico) using NADPH and O2. Such an activity can also be referred to as ent-Ka 13-hydroxylase activity.
[052] A recombinant microorganism of the present invention also comprises nucleotide sequences encoding polypeptides with UDP-glycosyltransferase (UGT) activity, in which the expression of the nucleotide sequences gives the microorganism the possibility to produce at least M. rebaudioside.
[053] For the purposes of the present invention, a polypeptide having UGT activity is one that has glycosyltransferase activity (EC 2.4), that is, it can act as a catalyst for the transfer of a monosaccharide unit from a sugar from activated nucleotide (also known as the "glycosyl donor") to a glycosyl acceptor molecule, usually an alcohol. The glycosyl donor for a UGT is typically the nucleotide sugar uridine diphosphate glucose (uracil diphosphate glucose, UDP-glucose).
[054] The UGTs used are selected to produce a desired diterpene glycoside, such as a steviol glycoside. Schematic diagrams of steviol glycoside formation are established in Humphrey et al., Plant Molecular Biology (2006) 61: 47-62 and Mohamed et al., J. Plant Physiology 168 (2011) 1136-1141. In addition, Figure 7 shows a schematic diagram of steviol glycoside formation.
[055] Rebaudioside A biosynthesis involves glycosylation of aglycone steviol. Specifically, rebaudioside A can be formed by glycosylation of the steviol 13-OH which forms the 13-O-steviolmonoside, C-2 'glycosylation of the steviolmonoside 13-O-glucose which forms steviol-1,2-bioside, glycosylation of the C-19 carboxyl of 1,2-steviol-bioside forming stevioside, and glycosylation of C-3 'of stevioside C-13-glucose. The order in which each glycosylation reaction occurs can vary - see Figure 7. A UGT may be able to catalyze more than one conversion, as set out in the present scheme.
[056] It has been shown that the conversion of steviol to M rebaudioside can be performed in a recombinant host through the expression of gene (s) encoding (m) for the following functional UGTs: UGT74G1, UGT85C2, UGT76G1 and UGT2.
[057] Thus, a recombinant microorganism that expresses these four UGTs can make rebaudioside M, if it produces steviol or when fed steviol in the medium. Typically, one or more of these genes are recombinant genes that have been transformed into a microorganism that does not naturally possess them.
[058] Examples of all these enzymes are shown in Table 1. A microorganism of the invention can comprise any combination of a UGT74G1, UGT85C2, UGT76G1 and UGT2. In Table 1, UGT64G1 sequences are indicated as UGT1 sequences, UGT74G1 sequences are indicated as UGT3 sequences and UGT76G1 sequences are indicated as UGT4 sequences. UGT2 sequences are indicated as UGT2 sequences in Table 1.
[059] A recombinant microorganism of the invention comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-13-glucose to steviol. That is to say, a microorganism of the invention can comprise a UGT that is capable of catalyzing a reaction in which steviol is converted to steviolmonoside.
[060] Such a microorganism of the invention may comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT85C2, in which the nucleotide sequence, upon transformation of the microorganism, gives the cell the ability to convert steviol to steviolmonoside.
[061] UGT85C2 Activity is transferred from a glucose unit to steviol 13-OH. Thus, a suitable UGT85C2 can function as a 5'-diphospho glycosyl uridine: steviol 13-OH-transferase and a 5'-diphospho glycosyl uridine: steviol-19-O-glycoside 13-OH transferase. A functional UGT85C2 polypeptide can also catalyze glycosyl transferase reactions that use steviol glycoside substrates other than steviol and steviol-19-O-glycoside. Such sequences are indicated as UGT1 sequences in Table 1.
[062] A recombinant microorganism of the invention also comprises a nucleotide sequence that encodes a polypeptide having UGT activity that can comprise a nucleotide sequence that encodes a polypeptide capable of catalyzing the addition of a 13-C-glucose to steviol or steviolmonoside. That is to say, a microorganism of the invention can comprise a UGT that is capable of catalyzing a reaction in which steviolmonoside is converted to steviolbioside. Therefore, such a microorganism may be able to convert steviolmonoside to steviolbioside. Expression of such a nucleotide sequence can give the microorganism the ability to produce at least steviolbioside.
[063] A microorganism of the invention can thus comprise a nucleotide sequence encoding a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT2, in which the nucleotide sequence upon transformation of the microorganism gives the cell the capacity to convert steviolmonoside to steviolbioside.
[064] A suitable UGT2 polypeptide functions as a 5'-diphospho glycosyl uridine: steviol-13-O-glycoside transferase (also referred to as a steviol-13-monoglycoside 1,2-glycosylase), transferring a glucose portion to the C- 2 'of the 13-O-glucose of the acceptor molecule, steviol-13-O-glycoside. Typically, a suitable UGT2 polypeptide also functions as a 5'-diphospho glycosyl: rubusoside transferase transferring a glucose portion to the C-2 'of the 13-O-glucose of the acceptor molecule, rubusoside.
[065] Functional UGT2 polypeptides can also catalyze reactions that use steviol glycoside substrates other than steviol-13-O-glycoside and rubusoside, for example, functional UGT2 polypeptides can use stevioside as a substrate, transferring a glucose portion to the C- 2 'of the 19-O-glucose residue to produce Rebaudioside E. A functional UGT2 polypeptide can also use Rebaudioside A as a substrate, transferring a glucose portion to the C-2' of the 19-O-glucose residue to produce Rebaudioside D However, a functional UGT2 polypeptide does not normally transfer a glucose portion to steviol compounds having a 1,3-linked glucose at the C-13 position, that is, the transfer of a glucose portion to 1,3-bioside and steviol 1,3-stevioside does not occur. Functional UGT2 polypeptides can also transfer sugar portions from donors other than uridine diphosphate glucose. For example, a functional UGT2 polypeptide can act as a 5'-diphospho D-xylosyl uridine: steviol-13-O-glycoside transferase, transferring a portion of C-2 'xylose from the 13-O-glucose receptor molecule, steviol -13-O-glycoside. As another example, a functional UGT2 polypeptide can act as a 5'-diphospho L-ramnosyl: steviol-13-O-glycoside transferase uridine, transferring a portion of rhamnose to the C-2 'of the 13-O-glucose from the acceptor , steviol-13-O-glycoside. Such sequences are indicated as UGT2 sequences in Table 1.
[066] A recombinant microorganism of the invention also comprises a nucleotide sequence that encodes a polypeptide having UGT activity that can comprise a nucleotide sequence that encodes a polypeptide capable of catalyzing the addition of a C-19-glucose to steviolbioside. That is to say, a microorganism of the invention can comprise a UGT that is capable of catalyzing a reaction in which steviolbioside is converted to stevioside. Therefore, such a microorganism may be able to convert steviolbioside to stevioside. Expression of such a nucleotide sequence can give the microorganism the ability to produce at least stevioside.
[067] A microorganism of the invention can thus also comprise a nucleotide sequence that encodes a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT74G1, in which the nucleotide sequence, upon transformation of the microorganism, confers to the cell the ability to convert steviolbioside to stevioside.
[068] Suitable UGT74G1 polypeptides may be able to transfer a glucose unit to 13-OH or 19-COOH, respectively, of steviol. A suitable UGT74G1 polypeptide can function as a 5'-diphospho glycosyl uridine: steviol 19-COOH transferase and a 5'-diphospho glycosyl uridine: steviol-13-O-glycoside 19-COOH transferase. Functional UGT74G1 polypeptides can also catalyze glycosyl transferase reactions that use steviol glycoside substrates other than steviol and steviol-13-O-glycoside, or that transfer portions of sugar from different uridine diphosphate glucose donors. Such sequences are indicated as UGT1 sequences in Table 3.
[069] A recombinant microorganism of the invention also comprises a nucleotide sequence encoding a polypeptide capable of catalyzing the glycosylation of C-3 'of glucose at the C-13 position of stevioside. That is to say, a microorganism of the invention can comprise a UGT that is capable of catalyzing a reaction, in which stevioside to Rebaudioside A. Thus, such a microorganism may be able to convert stevioside to Rebaudioside A. The expression of such a nucleotide sequence can give the microorganism the ability to produce at least rebaudioside A.
[070] A microorganism of the invention can thus also comprise a nucleotide sequence that codes for a polypeptide having the activity shown by UDP-glycosyltransferase (UGT) UGT76G1, in which the nucleotide sequence, upon transformation of the microorganism, gives the cell the ability to convert stevioside to rebaudioside A.
[071] A suitable UGT76G1 adds a glucose unit with the C-3 'end of the C-13-O-glucose of the acceptor molecule, a steviol glycoside 1,2. Thus, the UGT76G1 functions, for example, as a 5'-diphospho glycosyl uridine: 13-O-1,2-glycoside steviol C-3 'glycosyl transferase, and a 5'-diphospho glycosyl uridine: steviol-19-O -glucose, 13 -O-1,2 C-3 'bioside gliosyl ransferase. Functional UGT76G1 polypeptides can also catalyze glucosyl transferase reactions that use steviol glycoside substrates that contain sugars other than glucose, for example, steviol ramnosides and steviol xylosides. Such sequences are indicated as UGT4 sequences in Table 1.
[072] A microorganism of the invention comprises nucleotide sequences that encode polypeptides that possess all four UGT activities described above. A nucleic acid can encode a given polypeptide having one or more of the above activities. For example, a nucleic acid encodes a polypeptide having two, three or four of the activities mentioned above. Preferably, a recombinant microorganism of the invention comprises UGT1, UGT2 and UGT3 and UGT4 activity.
[073] A microorganism of the invention comprises a nucleotide sequence that encodes a polypeptide having UGT activity capable of catalyzing the glycosylation of stevioside or rebaudioside A. That is, a microorganism of the invention can comprise a UGT that is capable of catalyzing a reaction in which stevioside or rebaudioside A is converted to Rebaudioside D. Therefore, such a microorganism may be able to convert stevioside or rebaudioside A to Rebaudioside D. Expression of such a nucleotide sequence may give the microorganism the ability to produce at least Rebaudioside D.
[074] It has been shown that the expression of a microorganism from a combination of UGT85C2, UGT2, UGT74G1 and UGT76G1 polypeptides is capable of producing rebaudioside M.
[075] A microorganism of the invention comprising a nucleotide sequence encoding a polypeptide having UGT activity may comprise a nucleotide sequence encoding a polypeptide capable of catalyzing stevioside glycosylation. That is to say, a microorganism of the invention can comprise a UGT that is capable of catalyzing a reaction in which stevioside is converted to Rebaudioside E. Therefore, such a microorganism may be able to convert stevioside to Rebaudioside E. The expression of such a sequence of nucleotides can give the microorganism the ability to produce at least Rebaudioside E.
[076] A microorganism of the invention comprising a nucleotide sequence encoding a polypeptide having UGT activity can comprise a nucleotide sequence encoding a polypeptide capable of catalyzing the glycosylation of rebaudioside E. That is, a microorganism of the invention can understand a UGT that is capable of catalyzing a reaction in which rebaudioside E is converted to Rebaudioside D. Therefore, such a microorganism may be able to convert stevioside or rebaudioside A to Rebaudioside D. The expression of such a nucleotide sequence can confer to the microorganism the ability to produce at least rebaudioside D.
[077] A recombinant microorganism of the present invention may be able to express a nucleotide sequence that encodes a polypeptide having NADPH-cytochrome P450 reductase activity. This means that a recombinant microorganism of the present invention can comprise a sequence encoding a polypeptide having NADPH-cytochrome P450 reductase activity.
[078] For the purposes of the present invention, a polypeptide having NADPH-cytochrome P450 reductase activity (CE 1.6.2.4; also known as NADPH: ferrihemoprotein oxidoreductase, NADPH: hemoprotein oxidoreductase, NADPH: P450 oxidoreductase, P450 reductase, POR, RCP , CYPOR) is typically one that is a membrane-bound enzyme, allowing electron transfer to cytochrome P450 in the eukaryotic cell microsome from an NADPH enzyme: cytochrome P450 reductase containing Fad and FMN (POR; CE 1.6.2.4) .
[079] Preferably, a recombinant microorganism according to any one of the preceding claims, which is capable of expressing one or more of the following: a. a nucleotide sequence encoding a polypeptide having NADPH-cytochrome P450 reductase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having NADPH-cytochrome P450 reductase activity, said polypeptide comprising an amino acid sequence that is at least about 20%, preferably at least 25, 30, 40, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 54, 56, 58 or 78; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 53, 55, 57 or 77; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a sequence of nucleotides that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code,
[080] Preferably, a recombinant microorganism of the present invention is one that is capable of expressing one or more of the following: a. a nucleotide sequence encoding a polypeptide having ent-copalyl pyrophosphate synthase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having ent-copalyl pyrophosphate synthase activity, said polypeptide comprising an amino acid sequence that is at least about 20%, preferably at least 25, 30, 40, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 18, 20 , 60 or 62; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 17, 19, 59 or 61, 141, 142, 151, 152, 153, 154, 159, 160 , 182 or 184; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code, b. a nucleotide sequence encoding a polypeptide having enthalurene synthase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having enthalurene synthase activity, said polypeptide comprising an amino acid sequence that is at least about 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 64 or 66; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, 157, 158, 159, 160 , 183 or 184; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code, c. a nucleotide sequence encoding a polypeptide with ent-kaurene oxidase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide with ent-kaurene oxidase activity, said polypeptide comprising an amino acid sequence that is at least about 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 22, 24, 26, 68 or 86; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 21, 23, 25, 67, 85, 145, 161, 162, 163, 180 or 186; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code; or d. a nucleotide sequence encoding a polypeptide having kurenoic acid 13-hydroxylase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having kurenoic acid 13-hydroxylase activity, said polypeptide comprising an amino acid sequence which is at least about 20%, preferably at least 25, 30, 40, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 28, 30, 32, 34, 70, 90 , 92, 94, 96 or 98; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 27, 29, 31, 33, 69, 89, 91, 93, 95, 97, 146, 164, 165, 166, 167 or 185 ; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[081] In a recombinant microorganism of the present invention, which is capable of expressing a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-13-glucose to steviol (addition of glucose to the C-13 position of steviol ), said nucleotide may comprise: i. a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-13-glucose to steviol, said polypeptide comprising an amino acid sequence that is at least about 20%, preferably at least 25, 30 , 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 36, 38 or 72 ; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 35, 37, 71, 147, 168, 169 or 189; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[082] In a recombinant microorganism of the present invention, which is capable of expressing a nucleotide sequence that codes for a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviolmonoside (this usually indicates glycosylation of C- 2 'of C-13-glucose / steviolmonoside 13-O-glucose), said nucleotide sequence may comprise: i. a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a C-13-glucose to steviol or steviolmonoside, said polypeptide comprising an amino acid sequence that is at least about 20%, preferably at least 25 , 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 88, 100 , 102, 104, 106, 108, 110 or 112; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 87, 99, 101, 103, 105, 107, 109, 111, 181 or 192; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[083] In a recombinant microorganism of the present invention, which is capable of expressing a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-19 position of steviolbioside, said nucleotide sequence may comprise: i. a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-19 position of steviolbioside, said polypeptide comprising an amino acid sequence that has at least about 20% sequence identity with the sequence amino acids of SEQ ID NOs: 40, 42, 44, 46, 48 or 74; ii. a nucleotide sequence that has at least about 15% sequence identity with the nucleotide sequence of SEQ ID NOs: 39, 41, 43, 45, 47, 73, 148, 170, 171, 172, 173, 174 or 190; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[084] In a recombinant microorganism of the present invention, which expresses a nucleotide sequence encoding a polypeptide capable of catalyzing glucose C-3 'glycosylation at the stevioside C-13 position, said nucleotide sequence may comprise: i . a nucleotide sequence encoding a polypeptide capable of catalyzing the glycosylation of glucose C-3 'at the C-13 position of stevioside, said polypeptide comprising an amino acid sequence which is at least about 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 50, 52 or 76; ii. a nucleotide sequence that is at least about 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 49, 51, 75, 149, 175, 176 or 191; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[085] In a recombinant microorganism of the present invention, which expresses a nucleotide sequence that codes for a polypeptide capable of catalyzing one or more of: the glycosylation of stevioside or rebaudioside A to rebaudioside D; glycosylation of stevioside to rebaudioside E; glycosylation of rebaudioside E to rebaudioside D; or the glycosylation of rebaudioside D to Rebaudioside M, said nucleotide sequence may comprise: i. a nucleotide sequence encoding a polypeptide capable of catalyzing one or more of: the glycosylation of stevioside or rebaudioside A to rebaudioside D; glycosylation of stevioside to rebaudioside E; glycosylation of rebaudioside E to rebaudioside D; or the glycosylation of rebaudioside D to Rebaudioside M, said polypeptide comprising an amino acid sequence that has at least about 20% sequence identity with the amino acid sequence of SEQ ID NOs: 88, 100, 102, 104, 106, 108, 110, 112; ii. a nucleotide sequence that has at least about 15% sequence identity to the nucleotide sequence of SEQ ID NOs: 87, 99, 101, 103, 105, 107, 109, 111, 181 or 192; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[086] A microorganism according to the invention, may be one in which the ability of the microorganism to produce geranylgeranyl pyrophosphate (GGPP) is positively regulated.
[087] Positively regulated, in the context of the present invention, implies that the microorganism produces more GGPP than an equivalent untransformed strain.
[088] Therefore, a microorganism of the invention can comprise one or more nucleotide sequences encoding hydroxymethylglutaryl-CoA reductase, farnesyl pyrophosphate synthase and geranylgeranyl synthase diphosphate, wherein the nucleotide sequence (s), upon transformation of the microorganism, gives (m) the microorganism the ability to produce high levels of GGPP.
[089] Preferably, a microorganism according to the invention is one that is capable of expressing one or more of the following: a. a nucleotide sequence encoding a polypeptide having hydroxymethylglutaryl-CoA reductase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having hydroxymethylglutaryl-CoA reductase activity, said polypeptide comprising an amino acid sequence that has at least about 20% sequence identity with the amino acid sequence of SEQ ID NO: 80 ; ii. a nucleotide sequence that has at least about 15% sequence identity with the nucleotide sequence of SEQ ID NO: 79; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code, b. a nucleotide sequence encoding a polypeptide having farnesyl pyrophosphate synthase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having farnesyl pyrophosphate synthase activity, said polypeptide comprising an amino acid sequence that has at least about 20% sequence identity with the amino acid sequence of SEQ ID NO: 82; ii. a nucleotide sequence that has at least about 15% sequence identity to the nucleotide sequence of SEQ ID NO: 81; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code; or c. a nucleotide sequence encoding a polypeptide having geranylgeranyl synthase diphosphate activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having geranylgeranyl synthase diphosphate activity, said polypeptide comprising an amino acid sequence that has at least about 20% sequence identity with the amino acid sequence of SEQ ID NO: 84 ; ii. a nucleotide sequence that has at least about 15% sequence identity with the nucleotide sequence of SEQ ID NO: 83; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[090] The invention relates to a recombinant microorganism. A microorganism or microbe, for the purposes of the present invention, is typically an organism that is not visible to the human eye (i.e. microscopic). A microorganism can be from bacteria, fungi, protists or archaea. Typically a microorganism will be a single cell or unicellular organism.
[091] As used herein, a recombinant microorganism is defined as a microorganism that is genetically modified or transformed / transfected with one or more of the nucleotide sequences, as defined herein. The presence of one or more of such nucleotide sequences alters the microorganism's ability to produce a diterpene diterpene or glycoside, namely steviol or steviol glycoside. A microorganism that is not transformed / transfected, or genetically modified, is not a recombinant microorganism and normally does not comprise one or more of the nucleotide sequences that allow the cell to produce a diterpene or diterpene glycoside. Thus, an untransformed / non-transfected microorganism is typically a microorganism that does not naturally produce a diterpene, although a microorganism that naturally produces a diterpene diterpene or glycoside and that has been modified according to the invention (and therefore therefore , has an altered ability to produce a diterpene diterpene / glycoside) to be considered a recombinant microorganism according to the invention.
[092] Sequence identity is defined herein as a relationship between two or more amino acid sequences (polypeptide or protein) or two or more nucleic acid sequences (polynucleotides), as determined by comparing the sequences. Typically, sequence identities or similarities are compared over the entire length of the compared sequences. In the art, "identity" also means the degree of sequence relationship between amino acid or nucleic acid sequences, as the case may be, as determined by the correspondence between strands of such sequences. "Identity" and "similarity" can be easily calculated by several methods, known to those skilled in the art. The preferred methods for determining identity are designed to match the tested strings as closely as possible. Typically, then, identities and similarities are calculated over the entire length of the strings being compared. Methods for determining identity and similarity are encoded in publicly available computer programs. Preferred computer program methods to determine the identity and similarity between two sequences include, for example, BestFit, BLASTP, BLASTN and FASTA (Altschul, SF et al., J. Mol. Biol. 215: 403-410 (1990), publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894). Preferred parameters for comparing amino acid sequences using BLASTP are open gap 10.0, extension of gap 0.5, Blosum 62 matrix. Preferred parameters for comparing nucleic acid sequences using BLASTP are open gap 10.0, extension of gap 0.5, complete DNA matrix (DNA identity matrix).
[093] The nucleotide sequences encoding the enzymes expressed in the cell of the invention can also be defined by their ability to hybridize to the nucleotide sequences of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 or 84 or any other sequence mentioned here, respectively, under moderate, or, preferably, under stringent hybridization conditions. Stringent hybridization conditions are defined herein as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50, 75 or 100 nucleotides and more preferably about 200 or more nucleotides, to hybridize at a temperature of about 65 ° C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65 ° C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is carried out overnight, that is, for at least 10 hours and, preferably, the washing is carried out for at least one hour with at least two changes of the washing solution. These conditions will generally allow for specific hybridization of sequences having about 90% or more of sequence identity.
[094] Moderate conditions are defined herein as conditions that allow nucleic acid sequences of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at a temperature of about 45 ° C in a solution comprising about 1 M of salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M of salt, preferably 6 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is carried out overnight, that is, for at least 10 hours, and, preferably, the washing is carried out for at least one hour with at least two changes of the washing solution. These conditions will generally allow for specific hybridization of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridization conditions in order to specifically identify sequences ranging in identity between 50% and 90%.
[095] The nucleotide sequences that encode an ent-copalyl pyrophosphate synthase; ent-kaurene synthase; ent-kaurene oxidase; kaurenoic acid 13-hydroxylase; UGT; hydroxymethylglutaryl-CoA reductase, farnesyl pyrophosphate synthetase; geranylgeranyl synthase diphosphate; NADPH- cytochrome P450 reductase, can be of prokaryotic or eukaryotic origin.
[096] A nucleotide sequence encoding an ENT-copalyl pyrophosphate synthase can, for example, 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.
[097] A nucleotide sequence encoding an ent-kaurene synthase may, for example, 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.
[098] A nucleotide sequence encoding an ent-kaurene oxidase can, for example, comprise a sequence as set out in SEQ ID. NO: 21, 23, 25, 67, 85, 145, 161, 162, 163, 180 or 186. A preferred KO is the polypeptide encoded by the nucleic acid set forth in SEQ ID NO: 85.
[099] The nucleotide sequence encoding a 13-hydroxylase kurenoic acid can, for example, 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 preferred KAH sequence is the nucleic acid encoded polypeptide set out in SEQ ID NO : 33.
[0100] Another preferred recombinant microorganism of the invention can express a combination of the polypeptides encoded by SEQ ID NO: 85 and SEQ ID NO: 33 or a variant of any of them as described herein. A preferred recombinant microorganism of the invention can express the combination of sequences indicated in Table 8 (in combination with any UGT2, but in particular that encoded by SEQ ID NO: 87).
[0101] A nucleotide sequence encoding a UGT can, for example, comprise a sequence as set out in SEQ ID. NO: 35, 37, 39, 41, 43, 45, 47, 49, 51, 71, 73, 75, 168, 169, 170, 171, 172, 173, 174, 175, 176, 147, 148, 149, 87, 181, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 189, 190, 191 or 192.
[0102] The nucleotide sequence encoding a hydroxymethylglutaryl-CoA reductase can, for example, comprise a sequence as set out in SEQ ID. NO: 79.
[0103] A nucleotide sequence encoding a farnesyl pyrophosphate synthetase can, for example, comprise a sequence as set out in SEQ ID. NO: 81.
[0104] A nucleotide sequence encoding a May geranylgeranyl diphosphate synthase, for example, comprising a sequence as set out in SEQ ID. NO: 83.
[0105] A nucleotide sequence encoding a NADPH-cytochrome P450 reductase can, for example, comprise a sequence as set out in SEQ ID. NO: 53, 55, 57 or 77.
[0106] In the case of UGT sequences, combinations of at least one of each of: (i) SEQ ID NOs: 35, 37, 168, 169, 71, 147 or 189; (ii) SEQ ID NOs: 87, 99, 101, 103, 105, 107, 109, 111, 181 or 192; (iii) SEQ ID NOs: 39, 41, 43, 45, 47, 170, 171, 172, 173, 174, 73, 148 or 190; and (iv) SEQ ID NOs: 49, 51, 175, 176, 75, 149 or 191 may be preferred. Typically, at least one UGT from group (i) can be used. If at least one UGT from group (iii) is used, in general, at least one UGT from group (i) is also used. If at least one UGT from group (iv) is used, generally at least one UGT from group (i) and at least one UGT from group (iii) is used. Typically, at least one UGT from group (ii) is used.
[0107] A sequence that is at least about 10%, about 15%, about 20%, preferably at least about 25%, about 30%, about 40%, about 50 %, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96% , about 97%, about 98%, or about 99% sequence identity with a sequence, as mentioned, can be used in the invention.
[0108] To increase the probability that the introduced enzymes will be expressed in an active form in a eukaryotic cell of the invention, the corresponding coding nucleotide sequence can be adapted to optimize its codon usage to that of the chosen eukaryotic host cell. The adaptability of the nucleotide sequences that encode the enzymes for the use of codons of the chosen host cell can be expressed as the codon adaptation index (CAI). The codon adaptation index is defined here as a measure of the relative adaptability of the use of codons of a gene to the use of codons of highly expressed genes. The relative adaptability (W) of each codon is the relationship between the use of each codon, for the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptability values. Non-synonymous codons and termination codons (dependent on the genetic code) are excluded. CAI values vary between 0 and 1, with higher values indicating a greater proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; see also: Jansen et al., 2003, Nucleic Acids Res. 31 (8): 2242-51). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.
[0109] In a preferred embodiment, the eukaryotic cell, according to the present invention, is genetically modified with a nucleotide sequence (s) that is (are) adapted for the use of eukaryotic cell codons using codon pair optimization technology, as disclosed in PCT / EP2007 / 05.594. Codon pair optimization is a method for the production of a polypeptide in a host cell, in which the nucleotide sequences encoding the polypeptide have been modified with respect to their codon of use, in particular the codon pairs that are used , to obtain better expression of the nucleotide sequence encoding the polypeptide and / or improved production of the polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.
[0110] Furthermore, the improvement of enzyme activity in vivo in a eukaryotic host cell of the invention, can be achieved by well-known methods such as error-prone PCR or directed evolution. A preferred method of directed evolution is described in WO03010183 and WO03010311.
[0111] The microorganism according to the present invention can be any suitable host cell of microbial origin. Preferably, the host cell is a yeast or a filamentous fungus. More preferably, the host cell belongs to one of the genera Saccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces, Yarrowia, Candida, Hansenula, Humicola, Torulaspora, Trichosporon, Brettanomyces, Pachysolen or Yamadazyma or Zygosaccharomyces.
[0112] A most preferred microorganism belongs to the species Aspergillus niger, Penicillium chrysogenum, Pichia stipidis, Kluyveromyces marxianus, K. lactis, K. thermotolerans, Yarrowia lipolytica, Candida sonorensis, C. glabrata, Hansenula polymorpha, Torulaspora brizyellis, bailii, Saccharomyces uvarum, Saccharomyces Bayanus or Saccharomyces cerevisiae. Preferably, the eukaryotic cell is a Saccharomyces cerevisiae.
[0113] A recombinant yeast cell, according to the invention, can be modified so that the ERG9 gene is down-regulated and / or the ERG5 / ERG6 genes are excluded. The corresponding genes can be modified in this way in other microorganisms.
[0114] Such a microorganism can be transformed as defined here, wherein the nucleotide sequence (s) with which the microorganism is transformed gives the cell the ability to produce RebM.
[0115] A preferred microorganism according to the invention is a yeast, such as a cell of Saccharomyces cerevisiae or Yarrowia lipolytica. A recombinant microorganism of the present invention, such as a recombinant Saccharomyces cerevisiae cell or Yarrowia lipolytica cell can comprise one or more nucleotide sequences from each of the following groups; (i) SEQ. ID NO: 1, 3, 5, 7, 17, 19, 59, 61, 141, 142, 152, 153, 154, 159, 160, 182 or 184. (ii) SEQ ID. NO: 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, 157, 158, 159, 160, 183 or 184. (iii) SEQ ID. NO: 21, 23, 25, 67 85, 145, 161, 162, 163, 180 or 186. (iv) SEQ ID. NO: 27, 29, 31, 33, 69, 89, 91, 93, 95, 97, 146, 164, 165, 166, 167 or 185.
[0116] Such a microorganism will typically also comprise one or more nucleotide sequences, as defined in SEQ ID. NO: 53, 55, 57 or 77.
[0117] Such a microorganism may also comprise one or more nucleotide sequences as set out in 35, 37, 39, 41, 43, 45, 47, 49, 51, 71, 73, 75, 168, 169, 170, 171, 172, 173, 174, 175, 176, 147, 148, 149, 87, 181, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 189, 190, 191 or 192. In the case of these sequences, combinations of at least one of each of (i) SEQ ID NOs: 35, 37, 168, 169, 71, 147 or 189; (ii) SEQ ID NOs: 87, 99, 101, 103, 105, 107, 109, 111, 181 or 192; (iii) SEQ ID NOs: 39, 41, 43, 45, 47, 170, 171, 172, 173, 174, 73, 148 or 190; and (iv) SEQ ID NOs: 49, 51, 175, 176, 75, 149 or 191 may be preferred. Typically, at least one UGT of group (i) can be used. If at least one UGT from group (iii) is used, in general, at least one UGT from group (i) is also used. If at least one UGT from group (iv) is used, generally at least one UGT from group (i) and at least one UGT from group (iii) is used. Typically, at least one UGT from group (ii) is used.
[0118] Such a microorganism may also comprise the following nucleotide sequences: SEQ ID. NO: 79; SEQ ID. NO: 81; and SEQ ID. NO: 83.
[0119] For each sequence described above (or any sequence mentioned here), a variant having at least about 15%, preferably at least about 20, about 25, about 30, about 40, about about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 98 or about 99% sequence identity to the indicated sequence can be used.
[0120] Nucleotide sequences encoding ent-copalyl pyrophosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, kurenoic acid 13-hydroxylase, UGTs, hydroxymethylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, and geranium diphosphate - cytochrome P450 reductase can be linked to one or more nucleic acid constructs to facilitate the transformation of the microorganism, according to the present invention.
[0121] A nucleic acid construct can be a plasmid carrying the genes encoding enzymes in the RebM pathway as described above, or a nucleic acid construct can comprise two or three plasmids each carrying three or two genes, respectively, which code for the enzymes of the distributed diterpene pathway in any appropriate manner.
[0122] Any suitable plasmid can be used, for example, a few copy plasmid or a multiple copy plasmid.
[0123] It may be possible that the enzymes selected from the group consisting of ent-copalyl pyrophosphate synthase, ent-kaurene synthase, ent-kaurene oxidase and kurenoic acid 13-hydroxylase, UGTs, hydroxymethylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, geranylgeranyl synthase diphosphate and NADPH-cytochrome P450 reductase are native to the host microorganism and that transformation with one or more of the nucleotide sequences encoding for these enzymes may not be necessary to give the host cell the ability to produce a diterpene or glycoside of diterpene. A further improvement in the production of diterpene / diterpene glycoside by the host microorganism can be obtained by classical strain improvement.
[0124] The nucleic acid construct can be maintained episomically and thus comprise an autonomous replication sequence, such as an autosomal replication sequence. If the host cell is of fungal origin, a suitable episomal nucleic acid construct may be, for example, based on yeast 2μ plasmids or PKD1 (Gleer et al., 1991, Biotechnology 9: 968-975), or plasmids from AMA (Fierro et al., 1995, Curr Genet 29: 482- 489).
[0125] Alternatively, each nucleic acid construct can be integrated into one or more copies in the host cell genome. Integration into the host cell genome can occur at random by non-homologous recombination, but preferably the nucleic acid construct can be integrated into the host cell genome by homologous recombination, as is well known in the art (see for example, WO90 / 14423 , EP-A-0481008, EP-A-0635 574 and US 6,265,186).
[0126] Optionally, a selectable marker can be present in the nucleic acid construct. As used herein, the term "marker" refers to a gene that codes for a trait or phenotype, which allows for the selection of, or screening for, a microorganism containing the marker. The marker gene can be an antibiotic resistance gene in which the appropriate antibiotic can be used to select transformed cells from cells that are not transformed. Alternatively, or also, non-antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1, LEU2). Host cells transformed with the nucleic acid constructs can be free of the marker gene. Methods for building microbial host cells free of recombinant marker genes are described in EP-A-0 635 574 and are based on the use of bidirectional markers. Alternatively, a detectable marker such as a Green Fluorescent Protein, lacZ, luciferase, chloramphenicol-acetyltransferase, beta-glucuronidase, can be incorporated into the nucleic acid constructs of the invention, allowing for the screening of transformed cells. A preferred marker-free method for introducing heterologous polynucleotides is described in WO0540186.
[0127] In a preferred embodiment, the nucleotide sequences encoding ent-copalyl pyrophosphate synthase, ent-kaurene synthase, ent-kaurene oxidase and kurenoic acid 13-hydroxylase, UGTs, hydroxymethylglutaryl-CoA reductase, farnesyl pyrophosphate synthetase geranylgeranyl synthase diphosphate and NADPH cytochrome P450 reductase, are each operatively linked to a promoter that causes sufficient expression of the corresponding nucleotide sequences in the eukaryotic cell, in accordance with the present invention, to give the cell the ability to produce RebM.
[0128] As used herein, the term "operably linked" refers to a bonding of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
[0129] As used herein, the term "promoter" refers to a fragment of nucleic acid that functions to control the transcription of one or more genes, located upstream from the transcription direction of the transcription initiation site of the gene and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and other DNA sequences, including, but not limited to, transcription factor binding sites, binding sites repressor and activator protein, and any other nucleotide sequences known to one skilled in the art to act, directly or indirectly, to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or development regulations.
[0130] The promoter that can be used to achieve expression of the nucleotide sequences that encode an enzyme as defined herein above, may be non-native to the nucleotide sequence that encodes the enzyme to be expressed, that is, a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operationally linked. Preferably, the promoter is homologous, that is, endogenous to the host cell.
[0131] Suitable promoters in microorganisms of the invention can be GAL7, GAL10 or GAL 1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI and AOX1. Other suitable promoters include PDC, GPD1, PGK1, TEF1 and TDH. In addition, suitable promoters are established in the Examples.
[0132] Any terminator, which is functional in the cell, can be used in the present invention. Preferred terminators are obtained from natural host cell genes. Suitable termination sequences are well known in the art. Preferably, such terminators are combined with mutations that prevent nonsense-mediated mRNA degradation in the host cell of the invention (see, for example: Shirley et al., 2002, Genetics 161: 1465-1482).
[0133] The nucleotide sequences used in the present invention may include sequences that target them for intended compartments of the microorganism. For example, in a preferred microorganism of the invention, all nucleotide sequences, except for ent-kaurene oxidase, kaurenoic acid 13-hydroxylase and sequences encoding NADPH-cytochrome P450 reductase, can be targeted to the cytosol. This approach can be used in a yeast cell.
[0134] The term "homologous", when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that, in nature, the nucleic acid molecule or polypeptide is produced by a host cell or organisms of the same species, preferably of the same strain or variety.
[0135] The term "heterologous", when used with respect to a nucleic acid (DNA or RNA) or protein, refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA sequence or RNA in which it is present, or which is found in a cell or at a location or locations in the RNA or DNA genome or sequence that differs from that in which it is found in nature. Nucleic acids or heterologous proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or produced synthetically or recombinantly.
[0136] Typically, the recombinant microorganism of the invention will comprise heterologous nucleotide sequences. Alternatively, a recombinant microorganism of the invention can comprise entirely homologous sequence that has been modified as set forth herein, so that the microorganism produces increased amounts of RebM compared to an unmodified version of the same microorganism.
[0137] One or more enzymes from the diterpene pathway, as described herein, can be overexpressed to achieve sufficient production of diterpene by the cell.
[0138] There are several means available in the art for the overexpression of enzymes in the host cells of the invention. In particular, an enzyme can be overexpressed by increasing the number of copies of the gene encoding the enzyme in the host cell, for example, by integrating additional copies of the gene into the host cell's genome.
[0139] A preferred host cell, according to the present invention, can be a recombinant cell that is naturally capable of producing GGPP.
[0140] A recombinant microorganism, according to the present invention, may be able to grow on any suitable carbon source known in the art and convert it to RebM. The recombinant microorganism may be able to directly convert plant biomass, celluloses, hemicelluloses, pectins, rhamnose, galactose, fucose, maltose, maltodextrins, ribose, ribulose or starch, derivatives of starch, sucrose, lactose and glycerol. Thus, a preferred host organism expresses enzymes, such as cellulases (endocellulases and exocellulases) and hemicellulases (for example, endo- and exo-xylanases, arabinases), necessary for the conversion of cellulose into glucose and hemicellulose monomers in xylose and monomers of arabinose, pectinases capable of converting pectins into glucuronic acid and galacturonic acid or amylases to convert starch into glucose monomers. Preferably, the host cell is capable of converting a carbon source selected from the group consisting of glucose, xylose, arabinose, sucrose, lactose and glycerol. The host cell can, for example, be a eukaryotic host cell, as described in WO03 / 062430, WO06 / 009434, EP1499708B1, WO2006096130 or WO04 / 099381.
[0141] In another aspect, the present invention relates to a process for the production of RebM comprising fermenting a eukaryotic cell transformed in accordance with the present invention into a suitable fermentation medium, and optionally recovering the RebM .
[0142] The fermentation medium used in the process for the production of RebM can be any suitable fermentation medium that allows the growth of a particular eukaryotic host cell. The essential elements of the fermentation medium are known to the person skilled in the art and can be adapted to the selected host cell.
[0143] Preferably, the fermentation medium includes a carbon source selected from the group consisting of plant biomass, celluloses, hemicelluloses, pectins, rhamnose, galactose, fucose, fructose, maltose, maltodextrins, 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 sulfate, ammonium chloride, ammonium nitrate or ammonium phosphate.
[0144] The fermentation process, according to the present invention, can be carried out in batch, semi-continuous or continuous mode. A separate hydrolysis and fermentation process (SSF) or a simultaneous saccharification and fermentation process (SHF) can also be applied. A combination of these fermentation process modes may also be possible for optimum productivity. An SSF process can be particularly attractive if starch, cellulose, pectin or hemicellulose is used as a carbon source in the fermentation process, in which it may be necessary to add hydrolytic enzymes, such as cellulases, hemicellulases or pectinases to hydrolyze the substrate.
[0145] The recombinant microorganism used in the process for the preparation of RebM can be any suitable microorganism, as defined here above. It may be advantageous to use a recombinant eukaryotic microorganism, according to the invention, in the process for producing RebM, because most eukaryotic cells do not require sterile conditions for propagation and are insensitive to bacteriophage infections. In addition, eukaryotic host cells can be grown at low pH to prevent bacterial contamination.
[0146] The recombinant microorganism according to the present invention can be an optional anaerobic microorganism. An optional anaerobic microorganism can be propagated under aerobic conditions at a high cell concentration. This anaerobic phase can then be performed at high cell density, which reduces the volume of fermentation required substantially and can minimize the risk of contamination with aerobic microorganisms.
[0147] The fermentation process for the production of a diterpene, according to the present invention, can be one of an anaerobic or aerobic fermentation process.
[0148] An anaerobic fermentation process can be defined here 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 in which organic molecules serve as both electron donors and electron acceptors. The fermentation process according to the present invention can also be carried out first under aerobic conditions and subsequently under anaerobic conditions.
[0149] The fermentation process can also be performed under limited oxygen or micro aerobic conditions. Alternatively, the fermentation process can be performed, first, under aerobic conditions and, subsequently, under limited oxygen conditions. An oxygen-limited fermentation process is a process in which oxygen consumption is limited by the transfer of oxygen from gas to liquid. The degree of oxygen limitation is determined by the amount and composition of the inlet gas flow, as well as the actual mixture / mass transfer properties of the fermentation equipment used.
[0150] The production of a diterpene, in the process according to the present invention, can occur during the growth phase of the host cell, during the stationary phase (steady state) or during both phases. It may be possible to carry out the fermentation process at different temperatures.
[0151] The process for producing RebM can be performed at a temperature that is optimal for the eukaryotic cell. The optimal growth temperature may be different for each transformed eukaryotic cell and is known to the person skilled in the art. The optimum temperature can be higher than the ideal for wild type organisms, to grow the organism efficiently in non-sterile conditions under minimal infection sensitivity and lower cooling cost. Alternatively, the process can be carried out at a temperature that is not ideal for the growth of the recombinant microorganism.
[0152] The process for the production of RebM, according to the present invention, can be carried out at any suitable pH value. If the recombinant microorganism is yeast, the pH of the fermentation medium is preferably 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 a pH below 2.5, preferably a pH greater than 2. An advantage of carrying out fermentation at these low pH values is that growth contaminating bacteria in the fermentation medium can be avoided.
[0153] Such a process can be carried out on an industrial scale.
[0154] The product of such a process is rebaudioside M.
[0155] The recovery of RebM from the fermentation medium can be carried out by methods known in the art, for example, by distillation, vacuum extraction, solvent extraction or evaporation.
[0156] In the process for the production of RebM, according to the invention, it may be possible to reach a fermentation broth concentration above 5 mg / L, preferably above 10 mg / L, preferably above 20 mg / L, preferably above 30 mg / l of fermentation broth, preferably above 40 mg / l, more preferably above 50 mg / l, preferably above 60 mg / l, preferably above 70, preferably above 80 mg / l, preferably above 100 mg / L, preferably above 1 g / L, preferably above 5 g / L, preferably above 10 g / L, but usually below 70 g / L.
[0157] The present invention also relates to a fermentation broth that comprises RebM obtainable by the process according to the present invention.
[0158] In the case where RebM is expressed within the microorganism, such cells may need to be treated in order to release RebM. Preferably, RebM is produced extracellularly.
[0159] The invention also relates to a method for converting a first glycosylated diterpene into a second glycosylated diterpene, which method comprises: contacting said first glycosylated diterpene with a microorganism, as described herein, a cell-free extract derived from such a microorganism or an enzyme preparation derived from any of them, to thereby convert the first glycosylated diterpene into the second glycosylated diterpene.
[0160] the second glycosylated diterpene can be rebaudioside A, rebaudioside D or rebuadioside M. In particular, the method can be carried out in a format such that the first glycosylated diterpene is steviol, rebaudioside A or rebaudioside D and, preferably, the second glycosylated diterpene is rebaudioside M.
[0161] That is, the present invention relates to a method of bioconversion or biotransformation.
[0162] RebM produced by the fermentation process according to the present invention can be used in any application known for these compounds. In particular, they can, for example, be used as a sweetener, for example, in a food or drink. For example RebM can be formulated into soft drinks, such as a table sweetener, chewing gum, dairy products such as yogurt (eg plain yogurt), cake, cereal or cereal-based foods, nutraceutical, pharmaceutical, edible gel , confectionery, cosmetics, toothpaste or other oral cavity composition, etc. In addition, RebM can be used as a sweetener not only for food products, beverages and other products intended for human consumption, but also in animal feed and fodder with improved characteristics.
[0163] Accordingly, the invention provides, inter alia, a food product, food or drink, which comprises a diterpene or glycosylate, prepared according to a process of the invention.
[0164] During the manufacture of food products, beverages, pharmaceuticals, cosmetics, table top products, chewing gums, conventional methods such as mixing, kneading, dissolving, stripping, permeation, percolation, spraying, atomization, infusion and others methods can be used.
[0165] The RebM obtained in the present invention can be used in dry or liquid forms. It can be added before or after heat treatment of food products. The amount of sweetener depends on the purpose of use. It can be added alone or in combination with other compounds.
[0166] The compounds produced according to the method of the invention can be mixed with one or more non-caloric or caloric sweeteners. Such a mixture can be used to improve flavor or time profile or stability. A wide range of both non-caloric and caloric sweeteners may be suitable for mixing with RebM. For example, non-caloric sweeteners such as mogroside, Monatin, aspartame, acesulfame salts, cyclamate, sucralose, saccharine salts or erythritol. Caloric sweeteners suitable for mixing with RebM include sugar alcohols and carbohydrates such as sucrose, glucose, fructose and HFCS (high fructose corn syrup). Sweet-flavored amino acids, such as glycine, alanine or serine, can also be used.
[0167] RebM can be used in combination with a sweetener suppressor, such as a natural sweetener suppressant. It can be combined with an umami flavor enhancer, such as an amino acid or a salt thereof.
[0168] RebM can be combined with a polyol or alcohol sugar, a carbohydrate, a physiologically active substance or functional ingredient (for example, a carotenoid, dietary fiber, fatty acids, saponin, antioxidant, nutraceutical, flavonoids, isothiocyanate, phenol, vegetable sterol or stanol (phytosterols and phytostanols), a polyol, a prebiotic, probiotic, a phytoestrogen, soy protein, sulfides / thiols, amino acids, a protein, a vitamin, a mineral and / or a substance classified based on a benefit for health, such as cardiovascular, cholesterol-lowering or anti-inflammatory.
[0169] A composition with RebM can include a flavoring agent, a flavoring 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 thereof.
[0170] The RebM of the invention can be applied as a high intensity sweetener to produce zero calorie, reduced calorie or diabetic drinks and food products with better flavor characteristics. It can also be used in drinks, food products, pharmaceuticals and other products in which sugar cannot be used.
[0171] Furthermore, RebM of the invention can be used as a sweetener not only for food products, beverages and other products intended for human consumption, but also in animal feed and forages with improved characteristics.
[0172] Examples of products where the RebM of the invention can be used as a sweetening compound can be as alcoholic beverages, such as vodka, wine, beer, liquors, sakes, etc .; natural juices, refreshing drinks, soft drinks, diet drinks, zero calorie drinks, low calorie drinks and foods, yogurt drinks, instant juices, instant coffee, types of instant beverage powder, canned products, syrups, fermented soy paste , soy sauce, vinegar, sauces, mayonnaise, ketchup, curry, soup, instant broth, powdered soy sauce, powdered vinegar, types of cookies, rice cracker, cookies, breads, chocolates, caramel, sweets, chewing gum, jam, pudding, pickled fruit and vegetables, fresh cream, jam, marmalade, flower paste, powdered milk, ice cream, sorbet, fruits and vegetables packed in bottles, canned and cooked beans, meat and food cooked in sauce sweetened, agricultural vegetable food products, seafood, ham, sausage, fish ham, fish sausage, fish paste, fried fish products, dry seafood products, frozen food products, seaweed grass, canned meat, tobacco, medicinal products and many others. In principle, it can have unlimited applications.
[0173] The sweetened composition comprises a drink, non-limiting examples of which include non-carbonated and carbonated drinks, such as soft drinks, ginger beers, root beers, cider, fruit-flavored soft drinks (for example, flavored soft drinks) citrus fruit such as lime or orange), powdered soft drinks, and the like; fruit juices from fruits or vegetables, fruit juices including juices or the like, fruit juices containing fruit particles, fruit drinks, fruit juices, drinks containing fruit juices, fruit flavored drinks, vegetable juices, juices containing vegetables and mixed juices containing fruits and vegetables; sports drinks, energy drinks, drinks close to water and similar drinks (for example, water with natural or synthetic flavorings); tea-type or favorite-type drinks, such as coffee, cocoa, black tea, green tea, oolong tea and the like; beverages containing dairy components, such as dairy beverages, coffee containing milk components, café au lait (with milk), milk tea, milk fruit drinks, drinkable yogurt, lactic acid bacteria drinks or the like; and dairy products.
[0174] 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.
[0175] The RebM of the invention, 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 sweetener depends on the purpose of use. It can be added alone or in combination with other compounds.
[0176] During the manufacture of food products, beverages, pharmaceuticals, cosmetics, table top products, chewing gums, conventional methods such as mixing, kneading, dissolving, stripping, permeation, percolation, spraying, atomization, infusion and others methods can be used.
[0177] Thus, the compositions of the present invention can be made by any method known to those skilled in the art that provide uniform or homogeneous mixtures of ingredients. These methods include dry mixing, spray drying, agglomeration, wet granulation, compaction, co-crystallization and the like.
[0178] In solid form, the RebM of the present invention of the present invention can be supplied to consumers in any form suitable for delivery in the edible product to be sweetened, including sachets, packages, bags, bulk boxes or cubes, lozenges, mists or dissolvable strips. The composition can be delivered as a unit dose or in a bulky form.
[0179] For liquid sweetener systems and compositions, convenient variations of fluid, semi-fluid forms, pastes and creams, suitable packaging with appropriate packaging material, in any shape or form must be invented, that is easy to load or distribute or store or carry any combination containing any of the above sweetener products or product combination produced above.
[0180] The composition can include various bulking agents, functional ingredients, dyes, flavorings.
[0181] The reference here to a patent document or other matter that is given as prior art should not be taken as an admission that that document or matter was known or that the information contained therein was part of common general knowledge, as in priority date of any of the claims.
[0182] The disclosure of each reference established herein is hereby incorporated by reference in its entirety.
[0183] The present invention is further illustrated by the following examples: EXAMPLES General
[0184] Conventional genetic techniques, such as overexpression of enzymes in host cells, as well as for additional genetic modification of host cells, are methods known in the art, such as those described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual) "(3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Publisher, or F. Ausubel et al., Eds.," Current protocols in molecular biology. molecular biology) ", Publication Green and Wiley Interscience, New York (1987). Methods of transformation and genetic modification of fungal host cells are known for example from EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671. A description of the sequences is defined in Table 1. The sequences described here can be defined with reference to the sequence listing or with reference to the database access numbers also set out in Table 1. Example 1. Overexpression of ERG20, BTS1 and tHMG in S. cerevisiae
[0185] For the overexpression of ERG20, BTS1 tHMG1, expression cassettes were designed to be integrated into a locus using the technology described in co-pending patent application no. PCT / EP2013 / 056623. To amplify the 5 'and 3' integration flanks for the integration locus, suitable primers and genomic DNA from a yeast strain CEN.PK (van Dijken et al., Enzyme Microbial and Tecnology 26 (2000) 706-714 ) were used. The different genes were ordered as cassettes (containing homologous sequence, promoter, gene, terminator, homologous sequence) in DNA2.0. The genes in these cassettes were flanked by constitutive promoters and terminators. See Table 2. Plasmid DNA2.0 DNA containing the ERG20, tHMG1 and BTS1 cassettes were dissolved at a concentration of 100 ng / L. In a 20 ng model of 50 μL PCR mix it was used in conjunction with 20 pmol of primers. The material was dissolved at a concentration of 0.5 g / L. TABLE 2: COMPOSITION OF OVEREXPRESSION CONSTRUCTIONS

[0186] For the amplification of the selection marker, the construct pUG7-EcoRV (Figure 1) and the appropriate primers were used. The KanMX fragment was purified from the gel using the Gel Zymoclean DNA recovery kit (ZymoResearch). The yeast strain Cen.PK113-3C was transformed with the fragments listed in Table 3. TABLE 3: DNA FRAGMENTS USED FOR THE TRANSFORMATION OF ERG20, THMG1 AND BTS1

[0187] After transformation and recovery for 2.5 hours in YEPhD (yeast extract glucose phytone peptone; BBL phytone peptone from BD) at 30 ° C, the cells were plated on YEPhD agar with 200 g / ml G418 ( Sigma). The plates were incubated at 30 ° C for 4 days. Correct integration was established with PCR diagnosis and sequencing. The excess of expression was confirmed by CL / EM in the proteins. The scheme of the ERG20, tHMG1 and BTS1 set is illustrated in Figure 2. This strain is called STV002.
[0188] The expression of Cre-recombinase in this strain led to the "out" recombination of the KanMX marker. The correct out-recombination and presence of ERG20, tHMG and BTS1 was established with diagnostic PCR. Example 2. Kngdown of Erg9
[0189] To reduce Erg9 expression, a knockdown Erg9 construct was designed and used that contains a modified 3 'end, which continues on the TRP1 promoter directing TRP1 expression.
[0190] The construct containing the Erg9-KD fragment was transformed into E. coli TOP10 cells. The transformants were grown in 2PY (2 times Fitone 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 into S. cerevisiae, and the colonies were seeded on agar plates of mineral medium (Verduyn et al., 1992. Yeast 8: 501-517) without tryptophan. The correct integration of the Erg9-KD construct was confirmed with a diagnosis of PCR and sequencing. The transformation scheme carried out for the Erg9-KD construct is illustrated in Figure 3. The strain was named STV003. Example 3. UGT2 1a overexpression
[0191] For the overexpression of UGT2_1a, technology was used as described in the pending patent applications PCT / EP2013 / 056623 and PCT / EP2013 / 055047. UGT2_1a was ordered as a cassette (containing homologous sequence, promoter, gene, terminator, homologous sequence) in DNA2.0. For more details, see Table 4. To obtain fragments containing the label and Cre-recombinase, technology was used as described in the co-pending patent application in PCT / EP2013 / 055047. The NAT marker, which confers resistance to nourseotrichin, was used for selection. TABLE 4: COMPOSITION OF THE OVEREXPRESSION CONSTRUCTION

[0192] Primers suitable for amplification were used. To amplify the 5 'and 3' flanks of the integration locus for integration, appropriate primers and genomic DNA from a yeast strain CEN.PK.
[0193] Yeast strain STV003 from S. cerevisiae was transformed with the fragments listed in Table 5, and the transformation mixture was plated onto YEPhD agar plates containing 50 g / ml nourseotricin (Lexy NTC from Jena Bioscience). TABLE 5: DNA FRAGMENTS USED FOR UGT2 TRANSFORMATION 1A

[0194] Cre-recombinase expression is activated by the presence of galactose. To induce the expression of Cre-recombinase, the transformants were seeded in YEPh galactose medium. This resulted in the out-recombination of the marker (s) located between the lox sites. The correct integration of UGT2a and out-recombination of the NAT marker was confirmed by diagnostic PCR. The resulting strain was called STV004. The schematic of the transformation carried out for the construct UGT2_1a is illustrated in Figure 4. Example 4. Overexpression of the Reba production pathway: CPS, KS, KO, KAH, RCP, UGT1, UGT3 and UGT4.
[0195] All pathway genes leading to the production of RebA were designed to be integrated into a locus using the technology described in the pending patent application in PCT / EP2013 / 056623. To amplify the 5 'and 3' flanks of the locus of integration for integration, appropriate primers and genomic DNA from a yeast strain CEN.PK. The different genes were ordered as cassettes (containing homologous sequence, promoter, gene, terminator, homologous sequence) in DNA2.0 (see Table 5 for an overview). DNA2.0 DNA was dissolved at 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, the DNA was purified with the NucleoSpin 96 PCR Cleaning Kit (Macherey-Nagel) or, alternatively, concentrated using ethanol precipitation. TABLE 6. SEQUENCES USED FOR THE REBA PRODUCTION ROUTE


[0196] All fragments for the pathway for RebA, the marker and the flanks (see overview in Table 7) were transformed into S. cerevisiae STV004 yeast. After overnight recovery in YEPhD at 20 ° C the transformation mixtures were seeded on YEPhD agar plates containing 200 μg / ml of G418. These were incubated 3 days at 25 ° C and one night at RT. TABLE 7. DNA FRAGMENTS USED FOR THE TRANSFORMATION OF CPS, KS, KO, KANMX, KAH, CPR, UGT1, UGT3 AND UGT4.


[0197] Correct integration was confirmed by diagnostic PCR and sequence analysis (3500 Genetic Analyzer, Applied Biosystems). Sequence reactions were performed with the BigDye Terminator v3.1 cycle sequencing kit (Life Technologies). Each reaction (10 μL) contained 50 ng of model and 3.2 pmol of the initiator. The products were purified by precipitation with ethanol / EDTA, dissolved in 10 μL of Hidi formamide and applied over the apparatus. The strain was named STV016. The schematic of how the GGPP pathway for Reba is integrated into the genome is illustrated in Figure 5. Example 5: Construct of the STV027 strain
[0198] To remove the KanMX marker from the STV016 chromosome, this strain was transformed with the plasmid pSH65, which expresses Cre-recombinase (Guldender, 2002). Subsequently, plasmid pSH65 was cured from the strain by growth in a non-selective medium (YEP 2% glucose). The resulting strains free of KanMX and free of pSH65, as determined by plating onto plates containing 200 mL of G418 ug / mL or 20 μg of phleomycin / mL, where no growth should occur, were named STV027. The absence of the KanMX marker was further confirmed by diagnostic PCR. Example 6. RebM production by Saccharomyces cerevisiae
[0199] Although strain STV027 was initially designed for the production of RebA, in this example, the strain is shown to produce the steviol glycoside, RebM. 6.1 - Construction of recombinant host for the production of glycosylated diterpenes or diterpenes
[0200] The construction of the recombinant host for the production of RebM, STV027, is described above in Example 5. 6.2 - Detection of rebaudioside M and by CL / EM
[0201] The purified fraction (see 6.4 below) was analyzed with an Orbitrap LTQ (Thermo), equipped with a CL Acella and a Waters Acquity UPLC BEH 1.7 μm 2.1 * 150 mm amide column. The eluents used for the separation were A: 10 mM ammonium acetate in MilliQ water, B: acetonitrile, and the gradient started at 65% A and remained so for 1.5 minutes, then increased to 95% B at 0, 5 minutes and maintained this way for 0.5 minutes before regeneration for 1.5 min at 65% A. The flow rate was 0.6 ml / min and the column temperature was maintained at 50 ° C. Mass spectral analysis was carried out in negative electrospray ionization mode, scanning from 100 to 1800 m / z at a resolution of 7500. 6.3 - Fermentation of diterpene or glycosylated diterpene
[0202] The yeast strain STV027, constructed as described above, was grown in a shaken flask (500 ml with 50 ml of medium) for 2 days at 30 ° C and 280 rpm. The medium was based on Verduyn et al., (Verduyn C., Postma E., Scheffers WA, Van Dijken JP Yeast, July 1992; 8 (7): 501-517), with modifications to the carbon and nitrogen sources , as described in Table 8. TABLE 8. COMPOSITION OF THE PRECULTURE MEDIA

the trace element solution
b Vitamin Solution


[0203] Subsequently, 6 mL of the contents of the stirring flask were transferred to a fermenter (starting volume 0.3 L), which contained the medium, as set out in Table 9. TABLE 9. FERMENTATION MEDIA COMPOSITION

[0204] The pH was controlled to 5.0 by the addition of ammonia (12.5% by weight). The temperature was controlled at 27 ° C. pO2 was controlled at 40% by adjusting the agitator speed. The glucose concentration was kept limited by controlled feeding to the fermenter. TABLE 10. COMPOSITION OF FERMENTATION FEED
6.4 - Production of rebaudioside M in the fermentation broth during semi-continuous fermentation
[0205] The fermentation broth was shocked with heat at 70-90 ° C to kill the yeast cells and make them open. The thermal shock broth was spray dried. The dry biomass was extracted twice with 90% ethanol at 50-60 ° C. The extracts were combined and evaporated to about 1/10 - 1/30 of their initial volume. The evaporated extract was diluted with water until it reached an ethanol concentration of 20%. The HPLC analytical chromatogram of the extract is the upper curve in Figure 8. A large precipitate formed by diluting the extract with water was removed by centrifugation. The analytical HPLC chromatogram of the centrifuge is the next upper curve in Figure 8. The 20% ethanol feed with a pH of about 4.5 was applied to a column filled with Diaion® HP20 and eluted with a 20- 80% of 14CV. The analytical HPLC chromatogram of the first eluate is the third from the upper curve in Figure 8. The pH in the pooled fraction was adjusted to 8.5 and it was again applied to a column packed with Diaion® HP20 and eluted by steps with 4 80% ethanol CV. The analytical HPLC chromatogram of the second eluate is the fourth curve from the upper curve in Figure 8. The peak in analytical chromatograms eluting at 8.7 min is eluting at 8.7 min is RebM.
[0206] The presence of RebM was confirmed by CL / EM and performed on the first eluate (as described in the previous paragraph), using the conditions set out in 6.2 above. Reb M elutes at tr = 0.72 min, immediately after Reb D at tr = 0.63. Reb M is characterized by a deprotonated molecule of m / z 1289.5286. The elemental composition can be estimated using precise mass analysis. Example 7. Description of the STV2019 construct
[0207] Two strains of Yarrowia lipolytica of mating types MATA and MATB were designed for the production of steviol glycoside. These strains were mated, the diploid sporulated and spores with steviol glycoside production were selected. One of these spores was further developed for the production of steviol glycosides, including the production of rebaudioside M. Example 7.1. Description of the ML14094 steviol glycoside production strain (strain MAT-A)
[0208] Step 1. Strain ML10371 (MAT-A, lys1-, ura3-, leu2-) was transformed with 5 defined DNA fragments. All transformations were carried out using a lithium acetate / PEG fungal transformation protocol method and the transformants were selected in minimal medium, from YPD + 100 μg / mL of nourseotricin or YPD + 100 μg / mL of hygromycin, as appropriate . 1) a 7.0 kb DNA fragment isolated by gel purification followed by HindIII / Notl digestion of plasmid MB6969 (Figure 9). This construct encodes a synthetic construct for the overexpression of UGT2 (SEQ ID NO: 242) linked to the pPGM promoter (SEQ ID NO: 258) and XPRT terminator (SEQ ID NO: 269) and the hygromycin resistance gene HPH ( SEQ ID NO: 245), flanked by the lox sites (SEQ ID NOs: 232 and 233), and a synthetic material to build the overexpression of the open reading frame of hydroxymethylglutaryl-coenzyme Y. lipolytica optimized codon reductase without the membrane anchor sequence '5 (tHMGopt: SEQ ID NO: 234) linked to the pHSP promoter (SEQ ID NO: 253) and CWPT terminator (SEQ ID NO: 265). 2) a 2.7 kb DNA fragment isolated by gel purification followed by MB6856 HindIII / SspI digestion (Figure 10). This construct encodes tHMGopt linked to the pHYPO promoter (SEQ ID NO: 254) and gpdT terminator (SEQ ID NO: SEQ ID NO: 266). 3) a 2.5 kb DNA fragment isolated by gel purification followed by digestion with MB6857 SspI (Figure 11). This construct encodes tHMGopt linked to the pHSP promoter and CWPT terminator. 4) a 2.0 kb DNA fragment isolated by gel purification followed by digestion with MB6948 SspI (Figure 12). This construct encodes a synthetic construct for the overexpression of Y. lipolytica-optimized codon geranylgeranyl synthase (GGSopt: SEQ ID NO: 235) linked to the pHSP promoter and CWPT terminator. 5) a 2.2 kb DNA fragment isolated by gel purification followed by MB6958 HindIII / SspI digestion (Figure 13).
[0209] This construct codes for GGSopt linked to the pHYPO promoter and gpdT terminator. The resulting strain was designated ML13462.
[0210] Step 2. The ML13462 strain was transformed with a 9.7 kb fragment isolated by gel purification followed by Sfil digestion of plasmid MB7015 (Figure 14). This construct encodes a synthetic construct for the overexpression of UGT1 (SEQ ID NO: 241) linked to the pENO promoter (SEQ ID NO: 255) and gpdT terminator (SEQ ID NO: 272), UGT3 (SEQ ID NO: 243) ) linked to the pHSP promoter and pgmT terminator (SEQ ID NO: 267), UGT4 (SEQ ID NO: 244) linked to the pCWP promoter (SEQ ID NO: 257) and pgkT terminator (SEQ ID NO: 268), and the resistance to lox-flanked nourseothricin (NAT: SEQ ID NO: 246). Note that the placement of lox sites allows for the subsequent removal of nourseotrichin resistance via CRE-recombinase-mediated recombination. A nourseothricin-resistant isolate was designated ML13500.
[0211] Step 3. The ML13500 strain was transformed with a 9.1 kb fragment isolated by gel purification followed by Pvul / SapI digestion of plasmid MB6986 (Figure 15). This construct encodes tHMGopt linked to the pHSP promoter and cWPT terminator, the lox-flanked URA3blaster prototrophic marker (SEQ ID NO: 252), and GGSopt linked to the pHYPO promoter and gpdT terminator (SEQ ID NO: 272). The transformants were selected in a minimal medium without uracil. A selected prototrophic uracil was designated ML13723.
[0212] Step 4. The ML13723 strain was transformed with an 18.1 kb fragment isolated by gel purification followed by digestion of the plasmid with MB7059 Sfil (Figure 16). MB7059 codes for tCPS_SR (SEQ ID NO: 236) linked to the pCWP promoter and cWPT terminator, tKS_SR (SEQ ID NO: 237) linked to the pHYPO promoter and gpdT terminator, KAH_4 (SEQ ID NO: 239), linked to the promoter pHSP and pgmT terminator (SEQ ID NO: 273), KO_Gib (SEQ ID NO: 238) linked to the pTPI promoter (SEQ ID NO: 256) and pgkT terminator (SEQ ID NO: 274), CPR_3 (SEQ ID NO: 240) linked to the pENO promoter and xprT terminator and the LEU2 locus of native Y. lipolytica (SEQ ID NO: 250). A selected rebaudioside A producing transformant was designated ML14032.
[0213] Step 5. The ML14032 strain was targeted for YPD and grown overnight and then targeted for 5-FOA plates to allow for loss-mediated recombination of the URA3 marker introduced in Step 3. A 5-resistant transformant -FOA selected was designated ML14093.
[0214] Step 6. Strain ML14093 was transformed with a 19.0 kb fragment isolated by gel purification followed by Sfil digestion of plasmid MB7100 (Figure 17). MB7100 codes for tCPS_SR linked to the pHYPO promoter and cwpT terminator, tKS_SR (SEQ ID NO: 237) linked to the PCP promoter and gpdT terminator, KAH_4 linked to the pHSP promoter and pgmT terminator, KO_Gib linked to the pENT promoter and pgk terminator , CPR_3 linked to the pTPI promoter and xprT terminator and URA3 Blaster prototrophic marker. The transformants were selected in a minimal medium without uracil. A selected rebaudioside A-producing uracil prototropher was designated ML14094. Example 7.2. Description of the ML14087 steviol glycoside production strain (MAT-B line)
[0215] Step 1. Strain ML13206 (MAT-B, ade1-, ure2-, leu2-) was transformed with 5 defined DNA fragments. All transformations were performed using a lithium acetate / PEG fungal transformation protocol method and the transformants were selected in a minimum medium of YPD + 100 ug / mL of nourseothricin or YPD + 100 ug / mL of hygromycin, as appropriate . 1) a 7.0 kb DNA fragment isolated by gel purification followed by HindIII / Notl digestion of plasmid MB6969 (Figure 9). This construct encodes a synthetic construct for the overexpression of the UGT2 ORF optimized codon pair (CpO) linked to the pPGM promoter and xprT terminator and the HPH hygromycin resistance gene, together flanked by lox sites and a synthetic construct for the over -expression of the hydroxymethylglutaryl-coenzyme open reading frame The Y. lipolytica optimized codon reductase without the 5 'membrane anchor sequence (tHMGopt) linked to the pHSP promoter and cwpT terminator. 2) a 2.7 kb DNA fragment isolated by gel purification followed by MB6856 HindIII / SspI digestion (Figure 10). This construct encodes tHMGopt linked to the pHYPO promoter and gpdT terminator. 3) a 2.5 kb DNA fragment isolated by gel purification followed by MB6857 SspI digestion (Figure 11). This construct encodes tHMGopt linked to the pHSP promoter and cwpT terminator. 4) a 2.0 kb DNA fragment isolated by gel purification followed by MB6948 SspI digestion (Figure 12). This construct encodes a synthetic construct for the overexpression of geranyl-geranyl pyrophosphate synthetase from Y. codon optimized lipolytica (GGSopt) linked to the pHSP promoter and cwpT terminator. 5) a 2.2 kb DNA fragment isolated by gel purification followed by MB6958 HindIII / SspI digestion (Figure 13). This construct encodes GGSopt linked to the pHYPO promoter and gpdT terminator.
[0216] The resulting strain was designated ML13465.
[0217] Step 2. Strain ML13465 was transformed with 2 defined DNA fragments: 1) a 9.7 kb fragment isolated by gel purification followed by Sfil digestion of plasmid MB7015 (Figure 14). This construct encodes a synthetic construct for the overexpression of UGT1 linked to the pENO promoter and gpdT terminator, UGT3 linked to the pHSP promoter and pgmT terminator, UGT4 (SEQ ID NO: 244) linked to the pCWP promoter and pgkT terminator and the resistance marker nourseotricina felanquado in lox (NAT). Note that the placement of lox sites allows for the subsequent removal of nourseotrichin resistance via CRE-recombinase-mediated recombination. 2) a 9.1 kb fragment isolated by gel purification followed by Pvul / SapI digestion of plasmid MB6988 (Figure 18). This construct encodes tHMGopt linked to the pHSP promoter and cwpT terminator, the prototrophic URA2blaster marker flanked in lox (SEQ ID NO: 251), and GGSopt linked to the pHYPO promoter and gpdT terminator. The strains were selected in YPD + 100 ug / mL of nourseotricin and plated in replication for minimal medium without uracil. A prototrophic nourseotricin-resistant uracil isolate was designated ML13490.
[0218] Step 3. The ML13490 strain was targeted for YPD and grown overnight and then targeted for 5-FOA plates to allow for loss-mediated recombination of the URA2 marker introduced in step 3 above. A selected 5-FOA resistant transformant was streaked ML13501.
[0219] Step 4. The ML13501 strain was transformed with a 9.1 kb fragment isolated by gel purification followed by Pvul / SapI digestion of plasmid MB6988 (Figure 18). The transformants were selected in a minimal medium without uracil. A selected prototrophic uracil was designated ML13724.
[0220] Step 5. Strain ML13724 was transformed with an 18.1 kb fragment isolated by gel purification followed by Sfil digestion of plasmid MB7044 (Figure 19). MB7044 codes for the tCPS_SR linked to the pHYPO promoter and cwpT terminator, the tKS_SR linked to the pcwP promoter and gpdT terminator, the KAH_4 linked to the pHSP promoter and pgmT terminator, the KO_Gib linked to the pENO promoter and pgkT promoter and the pRTP3 promoter linked to the pRTP3 promoter terminator xprT and the LEU2 locus. A selected rebaudioside A producing transformant was designated ML14044.
[0221] Step 6. Strain ML14044 was targeted for YPD and grown overnight and then targeted for 5-FOA plates to allow for loss-mediated recombination of the URA2 marker introduced in Step 4 above. A selected 5'-FOA resistant transformant was denoted ML14076.
[0222] Step 7. The ML14076 strain was transformed with a 19.0 kb fragment isolated by gel purification followed by Sfil digestion of plasmid MB7094 (Figure 20). MB7094 codes for the tCPS_SR linked to the pHYPO promoter and cwpT terminator, the tKS_SR linked to the pCWP promoter and gpdT terminator, the KAH_4 linked to the pHSP promoter and pgmT terminator, the KO_Gib linked to the pENO promoter and the pgkT promoter and the pgTP terminator and the pRTP3 promoter xprT terminator and URA2blaster prototrophic marker. The transformants were selected in a minimal medium without uracil. A selected rebaudioside A producing uracil prototropher was designated ML14087. Example 7.3. MATA and MATB mating line and selection of steviol glycoside producing progeny
[0223] Strains of opposite mating types (ML14094 and ML14087) with complementary nutritional deficiencies (ADE1 + lys1- and ade1- LYS1 +) were allowed to mate and then sown in selective medium that would allow only diploids to grow (minimum medium without both) adenine and lysine). Diploid cells (ML14143) were then induced to undergo meiosis and sporulation by starvation, and the resulting haploid progenies were plated in replication to identify prototrophic isolates with resistance to hygromycin and nourseotricin. One strain of rebaudioside A production selected was designated STV2003. Example 7.4. Increased production of steviol glycoside by overexpression of CPS, KAH4, UGT2 and UGT4.
[0224] Additional copies of CPS, KAH, UGT2 and UGT4 have been transformed for STV2003 and integrated into the GSY1 locus (YALI0F18502). The GSY1 locus is thus interrupted. To amplify the 5 'and 3' integration flanks for the GSY1 integration locus, the appropriate primers and genomic DNA from the Yarrowia strain ML326 were used (SEQ ID NOs: 224-227). These flanks contain connector sequences (5 and A and F and 3) for proper mounting on the vector pRS417_3_5 (Figure 21), together with the cassettes (described below). The heterologous genes were ordered as cassettes (containing connector sequence, promoter, gene, terminator, connector sequence) in DNA2.0, or assembled at home. After amplification, the DNA was purified with the NucleoSpin 96 PCR cleaning kit (Macherey-Nagel). TABLE 11. CASSETTES USED FOR TRANSFORMATION TO STV2003

[0225] All cassettes, flanks and linearized pRS417 5_3 vector (SnaBI / PmeI, Figure M) were transformed into S. cerevisiae strain CEN.PK114-7D. Transformation mixtures were plated on YEPhD agar plates containing 200 μg / ml of G418, and incubated 4 days at 30 ° C. A selected number of correct transformants were grown in a YEPhD liquid medium. From these cultures, plasmid DNA was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, 27106) according to the supplier's instructions.
[0226] The isolated plasmid was transformed into chemically competent E. coli 10-Beta cells (NEB, C3019H) according to the suppliers' instructions. The transformed cells were allowed to recover for 1 hour in 1 ml of SOC at 37 ° C, 250 rpm. An aliquot was plated in 2xPY + Amp and incubated overnight at 37 ° C. Isolated colonies were selected from the transformation plate and used to inoculate 2xPY + Amp and incubated at 30 ° C with shaking at 250 rpm.
[0227] Plasmid DNA was isolated from plasmid E. coli clones using the NucleoSpin kit (Machery Nagel, 740,588,250 REF) according to the suppliers' instructions. PCR diagnosis was performed to confirm the appropriate plasmid. The isolated plasmid DNA was used as a template for the amplification of the whole set. Two fragments were amplified, with a protrusion on the KanMX marker with primers DBC-05,793 (SEQ ID NO: 28) and DBC-10726 (SEQ ID NO: 229) for amplification of CPS, KAH and part of the KanMX gene, and primers DBC-10727 (SEQ ID NO: 230) and DBC-05.816 (SEQ ID NO: 231) for the amplification of part of the KanMX and UGT4 and UGT2 gene.
[0228] After amplification, both fragments were purified using NucleoSpin Gel and PCR cleaning (Machery Nagel, REF740609.250) according to the suppliers' instructions. The DNA was then transformed into Y. lipolytica yeast strain STV2003. The transformation mixtures were seeded on YEPhD agar plates containing 200 μg / ml of G418 and incubated for 3 days at 30 ° C. Transformants were checked with diagnostic PCR for correct integration. A correct transformant was named STV2019. Example 7.5. Production of RebM with STV2019 strain
[0229] Strains of Y. lipolytica STV2019 constructed as described above, were grown in a shaken flask (0.5 L with 50 ml of medium) for 2 days at 30 ° C and 280 rpm. The medium was based on Verduyn et al. (Verduyn C., Postma E., Scheffers WA, Van Dijken JP Yeast, July 1992; 8 (7): 501-517), with modifications in carbon and nitrogen sources, as described in Table 12. TABLE 12. COMPOSITION OF THE PRE-CULTURE MEDIA
the trace element solution
b Vitamin solution

[0230] Subsequently, 40 ml of the contents of the stirring flask were transferred to a fermenter (starting volume 0.4 L), which contained the medium, as set out in Table 13. TABLE 13. COMPOSITION OF FERMENTATION MEDIA

[0231] The pH was controlled to 5.0 by adding ammonia (10% by weight). The temperature was controlled at 30 ° C. pO2 was controlled at 20% by adjusting the agitator speed. The glucose concentration was kept limited by a controlled 60% glucose feed to the fermenter. RebM is as determined above in Example 6. The amount of RebM measured in the entire broth is as defined in Table 14. TABLE 14. REBM IN FULL BRUSH
Example 8. Enzymatic conversion of glycosylated diterpenes to rebM Cepa
[0232] Saccharomyces cerevisiae strain STV027 and STV2019 (see Examples 6 and 7 above) are grown in suitable media that allow active transcription and translation of the introduced genes. The cells obtained are pelleted and stored at -20 ° C until analysis. Preparation of cell-free extract
[0233] For 8 g cell pellet, 40 mL of 100 mM Tris buffer pH 7.18 is added and homogenized. Thereafter, 8 g glass beads (50-200um) are added. The samples were cooled on ice for 15 minutes. The cells are disrupted for 4 cycles by vortexing every 2 min at maximum speed, followed by 5 min of cooling on ice. After lysis, the extract is centrifuged at 3000g for 60 min at 4 degrees C. The obtained supernatant is used directly for activity tests. Preparation of Permeabilized Cells
[0234] 8 g of fresh sediment is homogenized with 40 ml of 40% DMSO in Tris buffer (100 mM pH 7.18) and frozen at -20 degrees C. Before analysis, cells are thawed and 5 ml are transferred to a new tube and washed three times with 100 mM Tris buffer pH 0.1 containing 7.18% glucose. The cells were centrifuged each time by centrifuging at 3000g. Finally, the cells are resuspended in 5 ml of 100 mM Tris, pH 7.18. UDP-glucosyltransferase enzyme assay: - 2.5 - 500 μL enzyme sample (CFE, permeabilized cells or isolated enzyme) is added to the reaction mixture containing: - 0.05% glucose (w / v; 5.55 mM ) - UDP-Glc, 5 mM - MnCl2, 3 mM - DMSO, 2.5% (v / v) - Steviol, Rebaudioside A or Rebaudioside D, 0.3 mM - H2O
[0235] The total reaction volume is 1000 μL and the assay is performed on microtiter plates (MTP) heated to 30 degrees C in an Eppendorf MTP incubator. Incubations are performed, shaking at 250 rpm for up to 120 hours. MTPs are sealed to prevent contamination and evaporation. Analyze
[0236] 100 μL samples were taken in time to follow the conversion reaction. The reaction mixtures are centrifuged at 4 degrees C for 20 minutes to stop the enzymatic reaction and collect the samples. 50 μL of acetonitrile is added to 100 μL of sample in order to completely stop the reaction and extract all formed molecules. To this end, the MTPs are sealed and shaken vigorously. Subsequently, the samples are centrifuged for 60 minutes at 4 degrees C and 100 μL are transferred to a new MTP for analysis by CL / EM (as described in Example 6, section 6.2). RESULTS
[0237] Rebaudioside M is easily formed after 24 hours (> 1% of converted substrates) and progressively increases in concentration during the reaction over 120 hours (> 10% of converted substrates).








Gray markings are truncated and thus a mentioned UniProt identification fragment.

权利要求:
Claims (12)
[0001]
1. Process for the preparation of rebaudioside M CHARACTERIZED by understanding to ferment a microorganism belonging to the genus Yarrowia in a fermentation medium suitable to a pH below 6, in which the microorganism is a recombinant microorganism comprising: nucleotide sequences encoding a polypeptide having activity of ent-copalyl pyrophosphate synthase, said polypeptide comprising an amino acid sequence selected from SEQ. ID Nos: 2, 4, 6, 8, 18, 20, 60 or 62; and nucleotide sequences encoding a polypeptide having enthalurene synthase activity, said polypeptide comprising an amino acid sequence selected from SEQ. ID Nos: 10, 12, 14, 16, 18, 20, 64 or 66; and nucleotide sequences encoding a polypeptide having enthalurene oxidase activity, said polypeptide comprising an amino acid sequence selected from SEQ. ID Nos: 22, 24, 26, 68 or 86; and nucleotide sequences encoding a polypeptide having kurenoic acid 13-hydroxylase activity, said polypeptide comprising an amino acid sequence selected from SEQ. ID Nos: 28, 30, 32, 34, 70, 90, 92, 94, 96 or 98; and nucleotide sequences encoding a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of the steviol to obtain 13-O-steviolmonoside, said polypeptide comprising an amino acid sequence selected from SEQ. ID Nos: 36, 38 or 72; and nucleotide sequences encoding a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of the 13-O-steviolmonoside to obtain steviolbioside or at the C-19 position of rebaudioside A to obtain rebaudioside D, said polypeptide comprising a sequence of amino acids selected from SEQ. ID Nos: 88, 100, 102, 104, 106, 108, 110, 112; and nucleotide sequences encoding a polypeptide capable of catalyzing the addition of a glucose at the C-19 position of the steviolbioside to obtain stevioside, said polypeptide comprising an amino acid sequence selected from SEQ. ID Nos: 40, 42, 44, 46, 48 or 74; and nucleotide sequences encoding a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of the stevioside to obtain rebaudioside A or at the C-19 position of the rebaudioside D to obtain M rebaudioside, said polypeptide comprising an amino acid sequence selected from from SEQ. ID Nos: 50, 52 or 76, in which the expression of the nucleotide sequence (s) gives the microorganism the ability to produce Rebaudioside M.
[0002]
2. Process, according to claim 1, CHARACTERIZED by also comprising recovering the rebaudioside M.
[0003]
3. Process, according to claim 1 or 2, CHARACTERIZED by the fact that the nucleotide sequence (s) also gives the microorganism the ability to produce steviolmonoside, steviolbioside, stevioside, rebaudioside A, rebaudioside B , rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rubusoside or dulcoside A.
[0004]
Process according to claim 1, CHARACTERIZED in that the microorganism is capable of expressing a nucleotide sequence encoding a polypeptide having NADPH-cytochrome P450 reductase activity, wherein the nucleotide sequence comprises i. a nucleotide sequence encoding a polypeptide with NADPH-cytochrome p450 reductase activity, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 54, 56, 58 or 78; ii. a nucleotide sequence of SEQ ID NOs: 53, 55, 57 or 77; iii. a strand complementary to the nucleotide sequence which hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a sequence of nucleotides that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneration of the genetic code.
[0005]
Process according to any one of claims 1 to 4, CHARACTERIZED in that the microorganism is capable of expressing one or more of: a. a nucleotide sequence encoding a polypeptide having ent-copalyl pyrophosphate synthase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having ent-copalyl pyrophosphate synthase activity, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 18, 20, 60 or 62; ii. a nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 17, 19, 59, 61, 141, 142, 151, 152, 153, 154, 159, 160, 182 or 184; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code, b. a nucleotide sequence encoding a polypeptide having enthalurene synthase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having enthalurene synthase activity, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 64 or 66; ii. a nucleotide sequence of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, 157, 158, 159, 160, 183 or 184; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code, c. a nucleotide sequence that encodes a polypeptide having ent-kaurene oxidase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having ent-kaurene oxidase activity, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 22, 24, 26, 68 or 86; ii. a nucleotide sequence of SEQ ID NOs: 21, 23, 25, 67, 85, 145, 161, 162, 163, 180 or 186; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code; or d. a nucleotide sequence encoding a polypeptide having kurenoic acid 13-hydroxylase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having kurenoic acid 13-hydroxylase activity, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 28, 30, 32, 34, 70, 90, 92, 94, 96 or 98; ii. a nucleotide sequence of SEQ ID NOs: 27, 29, 31, 33, 69, 89, 91, 93, 95, 97, 146, 164, 165, 166, 167 or 185; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[0006]
Process according to any one of claims 1 to 5, CHARACTERIZED in that the microorganism is capable of expressing a nucleotide sequence that encodes a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviol, where said nucleotide comprises: i. a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviol, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 36, 38 or 72; ii. a nucleotide sequence of SEQ ID NOs: 35, 37, 71, 147, 168, 169, 189; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[0007]
Process according to any one of claims 1 to 6, CHARACTERIZED in that the microorganism is capable of expressing a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviolmonoside or in the position Rebaudioside A C-19, wherein said nucleotide comprises: i. a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of steviolmonoside, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 88, 100, 102, 104, 106, 108, 110 , 112; ii. a nucleotide sequence of SEQ ID NOs: 87, 99, 101, 103, 105, 107, 109, 111, 181 or 192; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[0008]
Process according to any one of claims 1 to 7, CHARACTERIZED in that the microorganism is capable of expressing a nucleotide sequence that encodes a polypeptide capable of catalyzing the addition of a glucose at the C-19 position of steviolbioside, wherein the said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide capable of catalyzing the addition of a glucose at the C-19 position of steviolbioside, said polypeptide comprising an amino acid sequence of SEQ ID Nos: 40, 42, 44, 46, 48 or 74; ii. a nucleotide sequence of SEQ ID NOs: 39, 41, 43, 45, 47, 73, 148, 170, 171, 172, 173, 174 or 190; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[0009]
Process according to any one of claims 1 to 8, CHARACTERIZED that the microorganism expresses a nucleotide sequence that encodes a polypeptide capable of catalyzing the addition of a glucose at the C-13 position of the stevioside or at the C-19 position of rebaudioside D, wherein said nucleotide sequence comprises: i. a nucleotide sequence that encodes a polypeptide capable of catalyzing glycosylation of glucose C-3 'at the C-13 position of stevioside, said polypeptide comprising an amino acid sequence of SEQ ID NOs: 50, 52 or 76; ii. a nucleotide sequence of SEQ ID NOs: 49, 51 or 75, 149, 175, 176 or 191; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.
[0010]
Process according to any one of claims 1 to 9, CHARACTERIZED in that the microorganism is a Yarrowia lipolytic cell.
[0011]
Process according to any one of claims 1 to 10, CHARACTERIZED in that the ability of the microorganism to produce geranylgeranyl diphosphate (GGPP) is positively regulated.
[0012]
12. Process according to claim 11, CHARACTERIZED in that the microorganism is capable of expressing one or more of the following: a. a nucleotide sequence encoding a polypeptide having hydroxymethylglutaryl-CoA reductase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence that encodes a polypeptide having hydroxymethylglutaryl-CoA reductase activity, said polypeptide comprising an amino acid sequence of SEQ ID NO: 80; ii. a nucleotide sequence of SEQ ID NO: 79; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code, b. a nucleotide sequence encoding a polypeptide having farnesyl pyrophosphate synthase activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having farnesyl pyrophosphate synthase activity, said polypeptide comprising an amino acid sequence of SEQ ID NO: 82; ii. a nucleotide sequence of SEQ ID NO: 81; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code; or c. a nucleotide sequence encoding a polypeptide having geranylgeranyl synthase diphosphate activity, wherein said nucleotide sequence comprises: i. a nucleotide sequence encoding a polypeptide having geranylgeranyl synthase diphosphate activity, said polypeptide comprising an amino acid sequence of SEQ ID NO: 84; ii. a nucleotide sequence of SEQ ID NO: 83; iii. a complementary strand nucleotide sequence that hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); or iv. a nucleotide sequence that differs from the sequence of a nucleic acid molecule of (i), (ii) or (iii) due to the degeneracy of the genetic code.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

WO1990014423A1|1989-05-18|1990-11-29|The Infergene Company|Microorganism transformation|

---

DE69031710T2|1989-07-07|1998-03-05|Unilever Nv|METHOD FOR PRODUCING A PROTEIN BY MEANS OF A MUSHROOM TRANSFORMED BY MULTIPLE COPY INTEGRATION OF AN EXPRESSION VECTOR|

---

DE69432543T2|1993-07-23|2003-12-24|Dsm Nv|Selection marker gene-free recombinant strains: process for their preparation and the use of these strains|

---

PL336345A1|1997-04-11|2000-06-19|Dsm Nv|Genic conversion as a tool for constructing recombined filiform fungi|

---

US6265186B1|1997-04-11|2001-07-24|Dsm N.V.|Yeast cells comprising at least two copies of a desired gene integrated into the chromosomal genome at more than one non-ribosomal RNA encoding domain, particularly with Kluyveromyces|

---

ID27054A|1998-05-19|2001-02-22|Dsm Nv|IMPROVING THE PRODUCTION OF SEPALOSPORIN IN LIVING ORGANISMS|

---

WO2000037671A2|1998-12-22|2000-06-29|Dsm N.V.|Improved in vivo production of cephalosporins|

---

AU2002333269A1|2001-07-23|2003-02-17|Dsm Ip Assets B.V.|Process for preparing variant polynucleotides|

---

AT321850T|2001-07-23|2006-04-15|Dsm Ip Assets Bv|METHOD FOR PRODUCING POLYNUCLEOTIVARIANTS|

---

CA2474033C|2002-01-23|2013-06-04|Royal Nedalco B.V.|Fermentation of pentose sugars|

---

SE0202090D0|2002-05-08|2002-07-04|Forskarpatent I Syd Ab|A modified yeast consuming L-arabinose|

---

AU2004236724B2|2003-05-02|2008-07-10|Cargill Inc.|Genetically modified yeast species and fermentation processes using genetically modified yeast|

---

US20070298455A1|2003-10-14|2007-12-27|Van Den Berg Marco A|Method For Preparing A Modified Host Cell|

---

DK1781772T3|2004-07-16|2017-01-09|Dsm Ip Assets Bv|Metabolic MODIFICATION of xylose-fermenting eukaryotic cells|

---

EP1863901A1|2005-03-11|2007-12-12|Forskarpatent i Syd AB|Arabinose- and xylose-fermenting saccharomyces cerevisiae strains|

---

US9284570B2|2010-11-30|2016-03-15|Massachusetts Institute Of Technology|Microbial production of natural sweeteners, diterpenoid steviol glycosides|

---

NZ604915A|2010-06-02|2014-10-31|Evolva Nutrition Inc|Recombinant production of steviol glycosides|

---

SG10201606563PA|2011-08-08|2016-10-28|Evolva Sa|Recombinant production of steviol glycosides|

---

RS56884B1|2011-12-19|2018-04-30|Coca Cola Co|Beverage comprising steviol glycosides|

---

WO2013110673A1|2012-01-23|2013-08-01|Dsm Ip Assets B.V.|Diterpene production|

---

CN103974628B|2012-05-22|2019-04-05|谱赛科有限责任公司|The steviol glycoside of high-purity|

---

AU2014214004B2|2013-02-06|2018-05-24|Evolva Sa|Methods for improved production of Rebaudioside D and Rebaudioside M|

---

SG11201506127WA|2013-02-11|2015-09-29|Evolva Sa|Efficient production of steviol glycosides in recombinant hosts|

---

US10689681B2|2013-05-31|2020-06-23|Dsm Ip Assets B.V.|Microorganisms for diterpene production|

---

WO2015011209A1|2013-07-23|2015-01-29|Dsm Ip Assets B.V.|Diterpene production in yarrowia|NZ604915A|2010-06-02|2014-10-31|Evolva Nutrition Inc|Recombinant production of steviol glycosides|

---

SG11201506127WA|2013-02-11|2015-09-29|Evolva Sa|Efficient production of steviol glycosides in recombinant hosts|

---

WO2015011209A1|2013-07-23|2015-01-29|Dsm Ip Assets B.V.|Diterpene production in yarrowia|

---

JP2017528134A|2014-09-09|2017-09-28|エヴォルヴァ エスアー．Ｅｖｏｌｖａ Ｓａ．|Production of steviol glycosides in recombinant hosts|

---

AU2015327940B2|2014-10-03|2020-04-16|Conagen Inc.|Non-caloric sweeteners and methods for synthesizing|

---

US10472661B2|2014-10-08|2019-11-12|Dsm Ip Assets B.V.|Method for increasing glycosylation of a composition comprising steviol glycosides|

---

CA2973674A1|2015-01-30|2016-08-04|Evolva Sa|Production of steviol glycosides in recombinant hosts|

---

CA2979931A1|2015-03-16|2016-09-22|Dsm Ip Assets B.V.|Udp-glycosyltransferases|

---

WO2016170045A1|2015-04-21|2016-10-27|Dsm Ip Assets B.V.|Geranylgeranyl pyrophosphate synthase|

---

WO2016196368A1|2015-05-29|2016-12-08|Cargill, Incorporated|Fermentation methods for producing steviol glycosides with multi-phase feeding|

---

US10612065B2|2015-05-29|2020-04-07|Cargill, Incorporated|Heat treatment to produce glycosides|

---

CN107949632B|2015-05-29|2021-06-01|嘉吉公司|Fermentation process for the production of steviol glycosides using high pH and compositions obtained thereby|

---

US11066688B2|2015-07-10|2021-07-20|Dsm Ip Assets B.V.|Method for preparing a steviol glycoside composition|

---

EP3331373A4|2015-08-06|2019-07-10|Cargill, Incorporated|Fermentation methods for producing steviol glycosides|

---

BR112018002564A2|2015-08-07|2018-09-25|Evolva Sa|production of steviol glycosides in recombinant hosts|

---

EP3334819A1|2015-08-13|2018-06-20|DSM IP Assets B.V.|Steviol glycoside transport|

---

EP3359654B1|2015-10-05|2022-01-05|DSM IP Assets B.V.|Kaurenoic acid hydroxylases|

---

US10760085B2|2015-10-05|2020-09-01|Dsm Ip Assets B.V.|Kaurenoic acid hydroxylases|

---

EP3364766A1|2015-10-23|2018-08-29|DSM IP Assets B.V.|Low sugar flavoured yogurt|

---

CN108779451A|2016-01-08|2018-11-09|帝斯曼知识产权资产管理有限公司|The mating type of Yarrowia lipolytica is converted|

---

CA3020671A1|2016-04-13|2017-10-19|Evolva Sa|Production of steviol glycosides in recombinant hosts|

---

EP3458599A1|2016-05-16|2019-03-27|Evolva SA|Production of steviol glycosides in recombinant hosts|

---

BR112019002432A2|2016-08-09|2019-06-04|Dsm Ip Assets Bv|crystallization of steviol glycosides|

---

CN109563118A|2016-08-09|2019-04-02|帝斯曼知识产权资产管理有限公司|The crystallization of steviol glycoside|

---

RU2019104496A3|2016-08-12|2021-04-09|

---

WO2018078014A1|2016-10-27|2018-05-03|Dsm Ip Assets B.V.|Geranylgeranyl pyrophosphate synthases|

---

CA3045722A1|2016-12-08|2018-06-14|Dsm Ip Assets B.V.|Kaurenoic acid hydroxylases|

---

WO2018114995A1|2016-12-22|2018-06-28|Dsm Ip Assets B.V.|Fermentation process for producing steviol glycosides|

---

US20210147816A1|2017-06-27|2021-05-20|Dsm Ip Assets B.V.|Udp-glycosyltransferases|

---

WO2018224698A1|2017-08-23|2018-12-13|Dsm Ip Assets B.V.|Yoghurt comprising rebaudioside m|

---

EP3788059A1|2018-04-30|2021-03-10|DSM IP Assets B.V.|Steviol glycoside transport|

---

BR112021001097A2|2018-07-24|2021-04-20|Dsm Ip Assets B.V.|steviol glycoside aggregates with specific particle size distribution|

---

CN110818750B|2019-11-22|2020-11-06|江西师范大学|Synthesis method of stevioside R|

法律状态:
2019-10-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2020-05-05| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

---

2020-09-08| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: A23L 1/00 , C12P 21/00 , C12N 15/00 Ipc: A23L 27/30 (2016.01), C12P 21/00 (2006.01), C12N 1 |

---

2020-10-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-01-05| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/07/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201313942491A| true| 2013-07-15|2013-07-15|

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US13/942,491|2013-07-15|

---

PCT/EP2014/065179|WO2015007748A1|2013-07-15|2014-07-15|Diterpene production|

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