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1. 8Renewal of spermatogonia in man. World rally championship 2.
2. 6 Efficient mans organizer pro v1. EUGT 11 polypeptides also can transfer a glucose moiety to the C-2′ of the O-glucose of the acceptor molecule, rubusoside, to produce a ,2-diglycosylated rubusoside.http://softik.org/animation-maker-2-0-4-software-serial-key-bb/ http://softik.org/animated-wallpaper-maker-v2-5-1-serial-setup/Male development of chromosomally female mice transgenic for Sry. The steviol glycoside can be stevioside, wherein the second sugar moiety is glucose, and Rebaudioside E is produced upon transfer of the second glucose moiety.
3. 3 A regulatory region can, however, be positioned as much as about 5, nucleotides upstream of the translation initiation site, or about 2, nucleotides upstream of the transcription start site. http://softik.org/blazedvd-professional-6-1-1-3-with-crack-free-by-totalfreesofts/However, farnesol synthesis can be regulated:
4. 4 Sucrose synthases can be used to generate UDP-glucose and remove UDP, facilitating efficient glycosylation of compounds in various systems. Windows 8 start menu button by rajs.Dpp1 5 29996ru mp 31 10 2011Extreme movie manager 7.
5. 2 Chemical structures for several of the compounds found in Stevia extracts are shown in FIG. How to harmonize any tune on the piano.
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7. 5 The order in which each glucosylation reaction occurs can vary.
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Country of ref document: Kind code of ref document: Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases UGTs.
Such microorganisms, plants, or plant cells can produce steviol glycosides, e. This application claims priority to U. This disclosure relates to the recombinant production of steviol glycosides.
In particular, this disclosure relates to the production of steviol glycosides such as rebaudioside D by recombinant hosts such as recombinant microorganisms, plants, or plant cells.
This disclosure also provides compositions containing steviol glycosides. The disclosure also relates to tools and methods for producing terpenoids by modulating the biosynthesis of terpenoid precursors of the squalene pathway.
Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries. The sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking.
Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine and sucralose. Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevia rebaudiana.
Stevia is commonly grown in South America and Asia for commercial production of stevia extract. Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener.
Extracts of the Stevia plant contain rebaudiosides and other steviol glycosides that contribute to the sweet flavor, although the amount of each glycoside often varies among different production batches.
Existing commercial products are predominantly rebaudioside A with lesser amounts of other glycosides such as rebaudioside C, D, and F. Stevia extracts may also contain contaminants such as plant-derived compounds that contribute to off-flavors.
These off-flavors can be more or less problematic depending on the food system or application of choice. Provided herein is a recombinant host, such as a microorganism, plant, or plant cell, comprising one or more biosynthesis genes whose expression results in production of steviol glycosides such as rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, or dulcoside A.
As described herein, EUGT11 has a strong 1,O-glucose glycosylation activity, which is an important step for rebaudioside D production. Typically, stevioside and rebaudioside A are the primary compounds in commercially-produced stevia extracts.
Stevioside is reported to have a more bitter and less sweet taste than rebaudioside A. The composition of stevia extract can vary from lot to lot depending on the soil and climate in which the plants are grown.
Other steviol glycosides are present in varying amounts in stevia extracts. The amount of the minor steviol glycosides affects the flavor profile of a Stevia extract.
In addition, Rebaudioside D and other higher glycosylated steviol glycosides are thought to be higher quality sweeteners than Rebaudioside A. As such, the recombinant hosts and methods described herein are particularly useful for producing steviol glycoside compositions having an increased amount of Rebaudioside D for use, for example, as a non-caloric sweetener with functional and sensory properties superior to those of many high-potency sweeteners.
This document also features a recombinant host that includes a recombinant gene encoding a polypeptide having the ability to transfer a second sugar moiety to the C-2′ of a O-glucose of rubusoside.
This document also features a recombinant host that includes a recombinant gene encoding a polypeptide having the ability to transfer a second sugar moiety to the C-2′ of a O-glucose of stevioside.
In another aspect, this document features a recombinant host that includes a recombinant gene encoding a polypeptide having the ability to transfer a second sugar moiety to the C-2′ of the O-glucose of rubusoside and to the C-2′ of the O-glucose of rubusoside.
This document also features a recombinant host that includes a recombinant gene encoding a polypeptide having the ability to transfer a second sugar moiety to the C-2′ of a O-glucose of rebaudioside A to produce rebaudioside D, wherein the catalysis rate of the polypeptide is at least 20 times faster e.
The UGT85C polypeptide can include one or more amino acid substitutions at residues 9, 10, 13, 15, 21, 27, 60, 65, 71, 87, 91,,,,,and of SEQ ID NO: The UGT76G polypeptide can have one or more amino acid substitutions at residues 29, 74, 87, 91,,,,,,,and of SEQ ID NO: Any of the hosts described herein further can include a gene e.
Any of the hosts described herein further can include one or more of i a gene encoding a geranylgeranyl diphosphate synthase; ii a gene encoding a bifunctional copalyl 2011 synthase and kaurene synthase, or a gene encoding a copalyl diphosphate synthase and a gene encoding a kaurene synthase; iii a gene encoding a kaurene oxidase; and iv a gene encoding a steviol synthetase.
Each of the genes of iiiiiiand iv can be a recombinant gene. Any of the hosts described herein further can include one or more of v a gene encoding a truncated HMG-CoA; vi a gene encoding a CPR; vii a gene encoding a rhamnose synthetase; viii a gene encoding a UDP-glucose dehydrogenase; and ix a gene encoding a UDP-glucuronic acid decarboxylase.
At least one of the genes of iiiiiiivv29996ruviiviiior ix can be a recombinant gene. Any of the recombinant hosts can produce at least one steviol glycoside when cultured under conditions in which each of the genes is expressed.
The steviol glycoside can be selected from the group consisting of rubusoside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, stevioside, steviol O -Glucoside, steviol O -glucoside, steviol-1,2-bioside, steviol-1,3-bioside, 1,3-stevioside, as well as other rhamnosylated or xylosylated intermediates.
The steviol glycoside e. This document also features a method of producing a steviol glycoside. The method includes growing any of the hosts described herein in a culture medium, under conditions in which the genes 2011 expressed; and recovering the steviol glycoside produced by the host.
The growing step can include inducing expression of one or more of the genes. For example, the steviol glycoside can be rebaudioside D or rebaudioside E.
Other examples of steviol glycosides can include rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside F, and dulcoside A. This document also features a recombinant host. The host includes i a gene encoding a UGT74G1; ii a gene encoding a UGT85C2; iii a gene encoding a UGT76G1; iv a gene encoding a glycosyltransferase having the ability to transfer a second sugar moiety to the C-2′ of a O-glucose of rubusoside or stevioside; and v optionally a gene encoding a UGT91D2e, wherein at least one of the genes is a recombinant gene.
In some embodiments, each of the genes is a recombinant gene. The host can produce at least one steviol glycoside e. The host further can include a a gene encoding a bifunctional copalyl diphosphate synthase and kaurene synthase, or a gene encoding a copalyl diphosphate synthase and a gene encoding a kaurene synthase; b a gene encoding a kaurene oxidase; c a gene encoding a steviol synthetase; d a gene encoding a geranylgeranyl diphosphate synthase.
This document also features a steviol glycoside composition produced by any of the hosts described herein. The composition has reduced levels of dpp1 plant-derived contaminants relative to a stevia extract.
In another aspect, this document features a steviol glycoside composition produced by any of the hosts described herein. The composition has a steviol glycoside composition enriched for rebaudioside D relative to the steviol glycoside composition of a wild-type Stevia plant.
In yet another aspect, this document features a method of producing a steviol glycoside composition. The method includes growing a host described herein in a culture medium, under conditions in which each of the genes is expressed; and recovering the steviol glycoside composition produced by the host e.
The composition is enriched for rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F or dulcoside A relative to the steviol glycoside composition of a wild-type Stevia plant.
The steviol glycoside composition produced by the host e. This document also features a method for transferring a second sugar moiety to the C-2′ of a O -glucose or the C-2′ of a O -glucose in a steviol glycoside.
The steviol glycoside can be rubusoside, wherein the second sugar moiety is glucose, and stevioside is produced upon transfer of the second glucose moiety. The steviol glycoside can be stevioside, wherein the second sugar moiety is glucose, and Rebaudioside E is produced upon transfer of the second glucose moiety.
The steviol glycoside can be Rebaudioside A, and Rebaudioside D is produced upon transfer of the second glucose moiety. In another embodiment of an improved downstream steviol glycoside pathway as disclosed herein, materials and methods are provided for the recombinant production of sucrose synthase, and to materials and methods for increasing production of UDP-glucose in a host, specifically for increasing the availability of UDP-glucose in vivo, with the purpose of promoting glycosylation reactions in the cells, and methods for reducing UDP concentrations in the cells are provided.
The document also provides a recombinant host comprising one or more exogenous nucleic acids encoding a sucrose transporter and a sucrose synthase, wherein expression of the one or more exogenous nucleic acids with a glucosyltransferase results in increased levels of UDP-glucose in the host.
Optionally, the one or more exogenous nucleic acids comprise a SUS1 sequence. Optionally, the SUS1 sequence is from Coffea arabic a, or encodes a functional homolog of the sucrose synthase encoded by the Coffea arabica SUS1 sequence, but equally an Arabidopsis thaliana or Stevia rebaudiana SUS may be used as described herein.
In the recombinant host of the invention, the one or more exogenous nucleic acids may comprise a sequence encoding a polypeptide having the sequence set forth in Dpp1 ID NO: In the recombinant host, the one or more exogenous nucleic acids may comprise a sequence encoding a polypeptide having the sequence set forth in SEQ ID NO: The recombinant host has reduced ability to degrade external sucrose, as 2011 to a corresponding host that lacks the one or more exogenous nucleic acids.
The recombinant host may be a microorganism, such as a Saccharomycete, for example Saccharomyces cerevisiae. Alternatively, the microorganism is Dpp1 coli. In an alternative embodiment, the recominbant host is a plant or plant cell.
The invention also provides a method for increasing the level of UDP-glucose and reducing the level of UDP in a cell, the method comprising expressing in the cell a recombinant sucrose synthase sequence and a recombinant sucrose transporter sequence, in a medium comprising sucrose, wherein the cell is deficient in sucrose degradation.
The invention additionally provides a method for promoting a glycosylation reaction in a cell, comprising expressing in the cell a recombinant sucrose synthase sequence and a recombinant sucrose transporter 29996ru, in a medium comprising sucrose, wherein the expressing results in a decreased level of UDP in the cell and an increased level of UDP-glucose in the cell, such that glycosylation in the cell is increased.
In either method for increasing the level of UDP-glucose or promoting glycosylation, the cell may produce vanillin glucoside, resulting in increased production of vanillin glucoside by the cell, or may produce steviol glucoside, resulting in increased production of steviol glucoside by the cell.
Optionally, the SUS1 sequence is a A. The recombinant sucrose synthase sequence optionally comprises a nucleic acid encoding a polypeptide having the sequence set forth in SEQ ID NO: In either method, the host is a microorganism, for example a Saccharomycete, optionally such as Saccharomyces cerevisiae.
Or the host may be Escherichia coli. Or the host may be a plant cell. Also provided herein is a recombinant host, such as a microorganism, comprising one or more biosynthesis genes whose expression results in production of diterpenoids.
At least one of the genes is a recombinant gene. The 2011 can also be a plant cell. Expression of these gene s in a Stevia plant can result in increased steviol glycoside levels in the plant.
In some embodiments the recombinant host further comprises a plurality of copies of a recombinant gene encoding a CDPS polypeptide EC 5. The host can further comprise a plurality of copies of a recombinant gene encoding a KAH polypeptide, e.
The host can further comprise one or more dpp1 The host can further comprise one or more of iv a gene encoding a truncated HMG-CoA; v a gene encoding a CPR; vi a gene encoding a rhamnose synthetase; vii a gene encoding a UDP-glucose dehydrogenase; and viii a gene encoding a UDP-glucuronic acid decarboxylase.
Two or more exogenous CPRs can be present, for example. The expression of one or 29996ru of such genes can be inducible. At least one of genes iiiiiiivvviviior viii can be a recombinant gene, and in some cases each of the genes of iiiiiiivvviviiand viii is a recombinant gene.
In one aspect, this document features an isolated nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: For example, the polypeptide can include a methionine at position and an alanine at position The polypeptide can have the amino acid sequence set forth in FIG.
This document also features a recombinant host that includes a recombinant gene e. The host can be a microorganism such 29996ru a saccharomycete e. The host can be a plant or plant cell e.
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Visual studio pro with msdn library download. RNA editing is a process which results in sequence variation in the RNA molecule, and is catalyzed by enzymes. For in vitro reactions, one with skill in the art will recognize that addition of different levels of UGT enzymes in combination or under conditions which impact the relative activities of the different UGTS in combination will direct synthesis towards a desired proportion of each steviol glycosides. Joboshare 3gp video converter 3. Schmahl J, Capel B. Dance instructor deluxe edition.
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