Method for producing patchoulol and 7-epi-alpha-selinene

A method of producing patchoulol and 7-epi-α-selinene by contacting at least one polypeptide with farnesyl phyrophosphate (fpp). The method may be carried out in vitro or in vivo to produce patchoulol and 7-epi-α-selinene, compounds which can be useful in the field of perfumery.

MICROBIAL ENGINEERING FOR THE PRODUCTION OF CHEMICAL AND PHARMACEUTICAL PRODUCTS FROM THE ISOPRENOID PATHWAY

The invention relates to recombinant expression of a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in cells and the production of terpenoids. 1. A method comprising:recombinantly expressing a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in a cell that overexpresses one or more components of the non-mevalonate (MEP) pathway, optionally wherein the cell is a bacterial cell, a yeast cell or an algal cell.210-. (canceled)11TaxusTaxus brevifoliaTaxusTaxus canadenis. The method of claim 1 , wherein the taxadiene synthase enzyme is a enzyme claim 1 , optionally a enzyme; and/or wherein the GGPPS enzyme is a enzyme claim 1 , optionally enzyme.1216-. (canceled)17. The method of claim 1 , wherein the one or more components of the non-mevalonate (MEP) pathway is selected from the group consisting of dxs claim 1 , ispC claim 1 , ispD claim 1 , ispE claim 1 , ispF claim 1 , ispG claim 1 , ispH claim 1 , idi claim 1 , ispA and ispB claim 1 , optionally wherein dxs claim 1 , idi claim 1 , ispD and ispF are overexpressed.1820-. (canceled)21. The method of claim 1 , wherein the cell further expresses a taxadiene 5α-hydroxylase (T5αOH) or a catalytically active portion thereof.22. The method of claim 21 , wherein the T5αOH enzyme or a catalytically active portion thereof is fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof claim 21 , optionally wherein the T5αOH enzyme is At24T5αOH-tTCPR.23. (canceled)24. The method of claim 1 , wherein the expression of the taxadiene synthase enzyme claim 1 , the GGPPS enzyme and the one or more components of the MEP pathway are balanced to maximize production of the taxadiene.25. The method of claim 1 , further comprising culturing the cell.26. The method of claim 1 , wherein the cell produces taxadiene or taxadiene-5α-ol.2730-. (canceled)31. The method of claim 26 , wherein the percentage of taxadiene conversion to taxadiene-5α-ol and the ...

Microbial Conversion of Glucose to Styrene and Its Derivatives

A method for the in vivo production of styrene from renewable substrates using a recombinant microorganism is disclosed. Additionally, a method for the in vivo production of styrene oxide from renewable substrates using a recombinant microorganism is also disclosed. In both cases, the host cell expresses at least one gene encoding a polypeptide that possesses phenylalanine ammonia lyase activity in addition to at least one gene encoding a polypeptide that possesses trans-cinnamic acid decarboxylase activity. In the case of styrene oxide, the host cell must additionally express at least one gene encoding a polypeptide that possesses styrene monooxygenase activity. 1. A method for the production of styrene comprising: a) at least one gene encoding a polypeptide having phenylalanine ammonia lyase activity', 'b) at least one gene encoding a polypeptide having trans-cinnamic acid decarboxylase activity, '(i) contacting a recombinant host cell with a fermentable carbon substrate, said recombinant host comprising(ii) growing said recombinant cell for a time sufficient to produce styrene; and(iii) optionally recovering said styrene.2. A method for the production of styrene oxide comprising: a) at least one gene encoding a polypeptide having phenylalanine ammonia lyase activity', 'b) at least one gene encoding a polypeptide having trans-cinnamic acid decarboxylase activity', 'c) at least one gene encoding a polypeptide having styrene oxygenase activity, '(i) contacting a recombinant host cell with a fermentable carbon substrate, said recombinant host comprising(ii) growing said recombinant cell for a time sufficient to produce styrene oxide.3. A method according to wherein the sequence of gene encoding a polypeptide having phenylalanine ammonia lyase activity is as set forth in SEQ ID NO:1 claim 1 , 2 claim 1 , 4 claim 1 , or 5.4. A method according to wherein the sequence of gene encoding a polypeptide having trans-cinnamic acid decarboxylase activity is as set forth in SEQ ...

Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway

The invention relates to recombinant expression of a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in cells and the production of terpenoids.

BIOSYNTHETIC PRODUCTION OF STEVIOL GLYCOSIDE REBAUDIOSIDE I VIA VARIANT ENZYMES

The present invention relates, at least in part, to the production of steviol glycoside rebaudioside I through the use of variant UGT enzymes having activity to transfer a glucosyl group from UDP-glucose to rebaudioside A to produce rebaudioside I. 1. A method for synthesizing rebaudioside I , the method comprising preparing a reaction mixture comprising:(a) a steviol glycoside composition comprising rebaudioside A;(b) a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and(c) a UDP-glycosyltransferase enzyme comprising the amino acid sequence of SEQ ID NO: 1; andincubating the reaction mixture for a sufficient time to produce rebaudioside I.2stevia. The method of claim 1 , wherein the steviol glycoside composition is extract.3. The method of claim 1 , further comprising adding a sucrose synthase to the reaction mixture.4Arabidopsis thaliana. The method of claim 3 , wherein the sucrose synthase is an sucrose synthase 1 (AtSUS1) comprising the amino acid sequence of SEQ ID NO: 11.5. The method of claim 1 , wherein the reaction mixture is in vitro.6. The method of . wherein the reaction mixture is a cell-based reaction mixture.7. The method of claim 6 , wherein the UDP-glycosyltransferase enzyme is expressed in a host cell.8. The method of claim 7 , wherein the host cell is selected from the group consisting of a yeast claim 7 , a non-steviol glycoside producing plant claim 7 , an alga claim 7 , a fungus claim 7 , and a bacterium.9. The method of claim 7 , wherein the host cell is a bacterial cell.10E. coli. The method of claim 9 , wherein the bacterial cell is an cell.11. The method of claim 7 , wherein the host cell is a yeast cell.12. The method of claim 1 , wherein the subject is UDP-glucose.13. The method of claim 1 , wherein the rebaudioside A has a concentration of 15 to 50 g/L in the reaction mixture.14. The method to claim 1 , wherein the reaction mixture ...

TERPENE AND TERPENOID PRODUCTION IN PROKARYOTES AND EUKARYOTES

Terpene synthases are enzymes that directly convert IPP & DMAPP to terpenes, such as fusicoccadiene. Described herein are methods and compositions for the production of terpenes and terpenoids for use as fuel molecules or other useful components. Genetically engineered enzymes capable of producing terpenes and terpenoids are also described. 1. A non-vascular photosynthetic organism comprising a nucleic acid encoding a protein comprising (a) an amino acid sequence of SEQ ID NO: 2 , SEQ ID NO: 10 , SEQ ID NO: 16 , SEQ ID NO: 22 , SEQ ID NO: 27 , SEQ ID NO: 33 , SEQ ID NO: 38 , SEQ ID NO: 45 , SEQ ID NO: 50 , or SEQ ID NO: 55; or (b) an amino acid sequence of at least 90% identity to SEQ ID NO: 2 , SEQ ID NO: 10 , SEQ ID NO: 16 , SEQ ID NO: 22 , SEQ ID NO: 27 , SEQ ID NO: 33 , SEQ ID NO: 38 , SEQ ID NO: 45 , SEQ ID NO: 50 , or SEQ ID NO: 55.2. The organism of claim 1 , wherein said nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID No. 2 or an amino acid sequence of at least 90% identity to SEQ ID NO. 2.3. The organism of wherein said nucleic acid comprises SEQ ID NO. 1 claim 2 , SEQ ID NO. 4 claim 2 , SEQ ID NO claim 2 , 5 claim 2 , SEQ ID NO. 7 or SEQ ID NO claim 2 , 8.4. The organism of claim 1 , wherein said nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID No. 38 or an amino acid sequence of at least 90% identity to SEQ ID NO. 38.5. The organism of wherein said nucleic acid comprises SEQ ID NO. 37 claim 4 , SEQ ID NO. 39 or SEQ ID NO. 40.6. The organism of claim 1 , wherein said nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID No. 10 or an amino acid sequence of at least 90% identity to SEQ ID NO. 10.7. The organism of wherein said nucleic acid comprises SEQ ID NO. 9 claim 6 , SEQ ID NO. 11 or SEQ ID NO. 13.8. The organism of claim 1 , wherein said nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID No. 16 or an amino acid sequence of at least 90% identity to SEQ ...

MICROBIAL ENGINEERING FOR THE PRODUCTION OF CHEMICAL AND PHARMACEUTICAL PRODUCTS FROM THE ISOPRENOID PATHWAY

The invention relates to the production of one or more terpenoids through microbial engineering, and relates to the manufacture of products comprising terpenoids. 1207.-. (canceled)208. A method for making a product containing limonene , or derivative thereof , the method comprising:{'i': Escherichia coli', 'E. coli, 'providing an () that produces isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through an upstream methylerythritol (MEP) pathway and converts the IPP and DMAPP to limonene, or derivative thereof, through a recombinantly expressed downstream terpenoid synthesis pathway comprising geranyl pyrophosphate synthase and limonene synthase;'}{'i': 'E. coli', 'culturing the to produce the limonene, or derivative thereof, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase production of limonene, or derivative thereof; and'}incorporating the limonene, or derivative thereof, into a product.209. The method of claim 208 , wherein the product is a food product claim 208 , food additive claim 208 , beverage claim 208 , chewing gum claim 208 , candy claim 208 , or oral care product.210. The method of claim 208 , wherein the product is a fragrance product claim 208 , a cosmetic claim 208 , a cleaning product claim 208 , or a soap.211. The method of claim 208 , wherein the product is an insecticide claim 208 , pesticide claim 208 , or pest control agent.212. The method of claim 208 , wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream terpenoid synthesis pathway.213. The method of claim 208 , further comprising measuring the amount or concentration of indole continuously or intermittently.214. The method of claim 208 , wherein accumulation of indole in the culture is maintained to below 50 mg/L.215. The method of claim 208 , wherein accumulation of indole in the culture is maintained to below 10 mg/L.216. The method of claim 208 , ...

PRODUCTION OF STEVIOL GLYCOSIDE IN RECOMBINANT HOSTS

The invention relates to recombinant microorganisms and methods for producing steviol glycosides, glycosylated ent-kaurenol, and glycosylated ent-kaurenoic acid. 1. An in vitro method for producing a steviol glycoside , a glycosylated ent-kaurenol compound , and/or a glycosylated ent-kaurenoic acid compound , comprising: a first polypeptide capable of catalyzing beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside and having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:11;', '(ii) a second polypeptide capable of catalyzing beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose and having 70% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or 18;', '(iii) a polypeptide capable of capable of catalyzing glycosylation of steviol or the steviol glycoside at its C-13 hydroxyl group and having 55% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or', '(iv) a polypeptide capable of capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside having 50% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:9;, '(a) adding one or more of 'wherein at least one of the polypeptides is a recombinant polypeptide; and', 'and plant-derived- or synthetic steviol or steviol glycosides to a reaction mixture;'}(b) synthesizing steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound in the reaction mixture.2. The method of claim 1 , further comprising:(c) recovering the steviol glycoside, glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound alone or a composition comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent- ...

Epoxidation Using Peroxygenase

The invention relates to enzymatic methods for epoxidation of a non-cyclicaliphatic alken, or a terpen. 1. A method for producing an epoxide , comprising contacting a non-cyclic aliphatic alkene or a terpene with hydrogen peroxide and a peroxygenase; wherein the peroxygenase comprises an amino acid sequence which has at least 60% identity to SEQ ID NO: 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , or 20.2. The method of claim 1 , wherein the amino acid sequence comprises the motif: E-H-D-[G claim 1 ,A]-S-[L claim 1 ,I]-S-R (SEQ ID NO: 21).3. The method of claim 1 , wherein the peroxygenase comprises or consists of an amino acid sequence having at least 65% identity to the amino acid sequence of SEQ ID NO: 1 claim 1 , 2 claim 1 , 3 claim 1 , 4 claim 1 , 5 claim 1 , 6 claim 1 , 7 claim 1 , 8 claim 1 , 9 claim 1 , 10 claim 1 , 11 claim 1 , 12 claim 1 , 13 claim 1 , 14 claim 1 , 15 claim 1 , 16 claim 1 , 17 claim 1 , 18 claim 1 , 19 claim 1 , or 20.4. The method of claim 1 , wherein the peroxygenase comprises or consists of an amino acid sequence having at least 65% identity to the amino acid sequence of SEQ ID NO: 1.5. The method of claim 1 , wherein the peroxygenase comprises or consists of the amino acid sequence of SEQ ID NO: 1 claim 1 , 2 claim 1 , 3 claim 1 , 4 claim 1 , 5 claim 1 , 6 claim 1 , 7 claim 1 , 8 claim 1 , 9 claim 1 , 10 claim 1 , 11 claim 1 , 12 claim 1 , 13 claim 1 , 14 claim 1 , 15 claim 1 , 16 claim 1 , 17 claim 1 , 18 claim 1 , 19 claim 1 , or 20; or a fragment thereof having peroxygenase activity.6. The method of claim 1 , wherein the peroxygenase comprises or consists of the amino acid sequence of SEQ ID NO: 1; or a fragment thereof having peroxygenase activity.7. The method of claim 1 , wherein the aliphatic alkene has one or more substituents selected from the group consisting of halogen claim 1 , hydroxyl claim 1 , carboxyl claim 1 , amino claim 1 , nitro claim 1 , cyano claim 1 , thiol claim 1 , ...

BIOLOGICAL PRODUCTION OF ß-LACTONES

Methods of using β-lactone biosynthetic enzyme genes and host cells expressing one or more of those genes, e.g., heterologous expression, are provided. 1. A method to prepare β-lactones in vitro , comprising:combining one or more acyl CoA substrates, one or more activated acyl substrates, one or more carboxylic acid substrates, or one or more fatty acid substrates with OleA or a homolog thereof, OleC or a homolog thereof, and OleD or a homolog thereof but not OleB, under conditions that yield one or more oxetan-2-ones.2. The method of wherein one or more 3-hydroxy acid substrates are combined with GleC or a homolog thereof claim 1 , but not OleD or a homolog thereof or OleB or a homolog thereof that is enzymatically active in the decarboxylation of oxetan-2-ones.3. The method of wherein one or more acyl CoA substrates claim 1 , one or more carboxylic acid substrates claim 1 , or one or more fatty acid substrates are combined with OleA or a homolog thereof claim 1 , OleC or a homolog thereof and OleD or a homolog thereof but not OleB or a homolog thereof that is enzymatically active in the decarboxylation of oxetan-2-ones.4. The method of wherein the one or more acyl CoA substrates are prepared by combining one or more carboxylic acids claim 1 , CoA and a ligase.5. The method of wherein the OleA or homolog thereof claim 1 , the OleD or homolog claim 1 , thereof or the OleC or the homolog thereof or any combination thereof claim 1 , are expressed in a heterologous cell.6. The method of wherein the heterologous cell is a bacterial cell claim 5 , a fungal cell claim 5 , or a yeast cell.7. The method of wherein the OleC or homolog thereof is isolated OleC or the homolog thereof claim 1 , the OleA or homolog thereof is isolated OleA or the homolog thereof claim 1 , or the OleD or the homolog thereof is isolated OleD or the homolog thereof.8. The method of wherein the combining yields a plurality of distinct oxetan-2-ones claim 1 , an oxetan-2-one or a plurality of ...

Method for Rapidly Characterizing Content Variations of Triterpenoids in Liquid Fermentation Process of Antrodia Camphorata

The present invention discloses a method for rapidly characterizing content variations of triterpenoids in a liquid fermentation process of , belonging to the field of microbial fermentation. According to the method of the present invention, in the liquid fermentation of , an analysis method of rapidly judging the content variations of triterpenoids by rapid on-line or off-line detection and analysis of the content of a volatile aromatic substance α-terpineol is utilized to implement automatic control of the fermentation process. Predictive analysis of triterpenoids in the fermentation process based on variations in on-line real-time parameters increases the controllability and production predictability of the fermentation process. This is of great significance for the development and utilization of products having various bioactivities and application thereof in industrial production. 1Antrodia camphorata. A method of rapid characterizing a liquid fermentation process of , comprising establishing a quantitative relationship between a certain volatile substance and triterpenoids so that the certain volatile substance becomes a characterizing parameter of a fermentation process , wherein the fermentation process comprising a fermentation process of triterpenoids.2. The method of claim 1 , wherein the certain volatile substance is α-terpineol.3. The method of claim 2 , wherein a variation trend of content of the α-terpineol is consistent with a variation trend of content of triterpenoids.4. The method of claim 2 , wherein the content of α-terpineol is determined by an on-line detection or an off-line detection.5. The method of claim 4 , wherein the on-line detection comprises mounting a process mass spectrometer claim 4 , an electronic nose and other on-line detection instruments claim 4 , and a parameter acquisition system in an exhaust gas analysis part of a fermentor and performing an analysis on the α-terpineol in the fermentation process.6. The method of claim 5 ...

MICROBIAL ENGINEERING FOR THE PRODUCTION OF CHEMICAL AND PHARMACEUTICAL PRODUCTS FROM THE ISOPRENOID PATHWAY

The invention relates to recombinant expression of a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in cells and the production of terpenoids. 199.-. (canceled)100. A method for increasing terpenoid production in a cell that produces one or more terpenoids , comprisingproviding a bacterial cell that produces isopentenylpyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through an upstream methylerythritol phosphate pathway (MEP) and converts the IPP and DMAPP to a terpenoid through a recombinantly expressed downstream terpenoid synthesis pathway; andculturing the bacterial cell to produce the terpenoid.101. The method of claim 100 , wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream terpenoid synthesis pathway.102. The method of claim 101 , wherein the step of culturing comprises removing the accumulated indole.103. The method of claim 100 , wherein the one or more terpenoids is a monoterpenoid claim 100 , a sesquiterpenoid claim 100 , a diterpenoid claim 100 , a triterpenoid or a tetraterpenoid.104. The method of claim 103 , wherein the one or more terpenoids is taxadiene or a taxol precursor.105. The method of claim 101 , further comprising measuring the amount or concentration of indole in the culture.106. The method of claim 105 , wherein the method comprises measuring the amount or concentration of indole two or more times.107. The method of claim 105 , wherein the measured amount or concentration of indole is used to guide a process of producing one or more terpenoids.108. The method of claim 105 , wherein the measured amount or concentration of indole is used to guide strain construction.109. The method of claim 100 , wherein the method amplifies metabolic flux through IPP and DMAPP claim 100 , and at least one intermediate selected from geranyl diphosphate (GPP) claim 100 , geranylgeranyl diphosphate (GGPP) claim 100 , farnesyl diphosphate (FPP) ...

Production of steviol glycosides in recombinant hosts

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.

BIOSYNTHETIC PRODUCTION OF STEVIOL GLYCOSIDES REBAUDIOSIDE J AND REBAUDIOSIDE N

The present disclosure relates to the production of steviol glycosides rebaudioside J and rebaudioside N through the use of rebaudioside A as a substrate and a biosynthetic pathway involving various 1,2 RhaT-rhamnosyltransferases. 1. A biosynthetic method of preparing rebaudioside N , the method comprising:reacting a steviol glycoside composition with a rhamnose donor moiety in the presence of a first recombinant polypeptide having 1,2-rhamnosytransferase activity; wherein said first recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3.2. The method of claim 1 , wherein said first recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3.34.-. (canceled)5. The method of claim 1 , comprising expressing said first recombinant polypeptide in a transformed cellular system.6. The method of claim 5 , wherein the transformed cellular system is selected from the group consisting of a yeast claim 5 , a non-steviol glycoside producing plant claim 5 , an alga claim 5 , a fungus claim 5 , and a bacterium.7. (canceled)8. The method of claim 5 , wherein the reacting step is performed in the transformed cellular system.9. The method of claim 5 , comprising isolating said first recombinant polypeptide from the transformed cellular system and the reacting step is performed in vitro.10. The method of claim 1 , wherein the rhamnose donor moiety is UDP-L-rhamnose.11. The method of claim 1 , wherein the steviol glycoside composition comprises rebaudioside A and the reacting step leads to the production of rebaudioside J.12. The method of claim 11 , comprising reacting said rebaudioside J with a glucose donor moiety in the presence of a second recombinant polypeptide having glucosytransferase activity.13. The method of claim 12 , wherein the glucose donor moiety is generated in situ.14. The method of claim 12 , wherein said second recombinant polypeptide has both ...

METHOD FOR PRODUCING PATCHOULOL AND 7-EPI-ALPHA-SELINENE

A method of producing patchoulol and 7-epi-α-selinene by contacting at least one polypeptide with farnesyl phyrophosphate (fpp). The method may be carried out in vitro or in vivo to produce patchoulol and 7-epi-α-selinene, compounds which can be useful in the field of perfumery. 1. A method for producing patchoulol and 7-epi-α-selinene comprisinga) contacting FPP with at least one polypeptide having a patchoulol and 7-epi-α-selinene synthase activity and comprising an amino acid sequence at least 50% identical to SEQ ID NO:1;b) optionally, isolating the patchoulol and 7-epi-α-selinene produced in step a).2. The method of claim 1 , wherein step a) comprises cultivating a non-human host organism or cell capable of producing FPP and transformed to express at least one polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 and having a patchoulol and 7-epi-α-selinene synthase activity claim 1 , under conditions conducive to the production of patchoulol and 7-epi-α-selinene.3. The method of claim 2 , which further comprises claim 2 , prior to step a) claim 2 , transforming a non human host organism or cell capable of producing FPP with at least one nucleic acid encoding a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 and having a patchoulol and 7-epi-α-selinene synthase activity claim 2 , so that said organism expresses said polypeptide.4. The method of claim 3 , wherein the nucleic acid comprises a nucleotide sequence at least 50% claim 3 , preferably at least 70% claim 3 , preferably at least 90% identical to SEQ ID NO:2 or the complement thereof claim 3 , or the nucleic acid comprises or consists of the nucleotide sequence SEQ ID NO:2 or the complement thereof.5. The method of claim 2 , wherein the non-human host organism is a plant claim 2 , a prokaryote or a fungus claim 2 , a microorganism such as a bacteria or yeast claim 2 , or a plant cell or a fungal cell.6E. coliSaccharomyces cerevisiae.. The ...

NON-CALORIC SWEETENERS AND METHODS FOR SYNTHESIZING

Disclosed are steviol glycosides referred to as rebaudioside V and rebaudioside W. Also disclosed are methods for producing rebaudioside M (Reb M), rebausoside G (Reb G), rebaudioside KA (Reb KA), rebaudioside V (Reb V) and rebaudioside (Reb W). 1. A method for synthesizing a mixture stevioside and rebaudioside KA from rubusoside , the method comprising:preparing a reaction mixture comprising rubusoside, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase fusion enzyme; andincubating the reaction mixture for a sufficient time to produce a mixture of stevioside and rebaudioside KA, wherein a glucose is covalently coupled to C2′-19-O-glucose of rubusoside to produce rebaudioside KA, and a glucose is convalently coupled to C2′-13-O-glucose of rubusoside to produce stevioside.2. The method of further comprising adding a sucrose synthase to the reaction mixture.3ArabidopsisArabidopsisVigna radiate. The method of claim 2 , wherein the sucrose synthase is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a sucrose synthase.4Arabidopsis thaliana. The method of claim 3 , wherein the sucrose synthase is an sucrose synthase 1.5. The method of claim 1 , wherein the UDP-glycosyltransferase fusion enzyme is selected from the group consisting of an EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain.6ArabidopsisArabidopsisVigna radiate. The method of claim 5 , wherein the sucrose synthase domain is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a sucrose synthase.7Arabidopsis thaliana. The method of claim 6 , wherein the sucrose synthase domain is an sucrose synthase 1.8. A method for synthesizing rebaudioside KA from rubusoside claim 6 , the method comprising:preparing a reaction mixture ...

NON-CALORIC SWEETENERS AND METHODS FOR SYNTHESIZING

Disclosed are steviol glycosides referred to as rebaudioside V and rebaudioside W. Also disclosed are methods for producing rebaudioside M (Reb M), rebausoside G (Reb G), rebaudioside KA (Reb KA), rebaudioside V (Reb V) and rebaudioside (Reb W). 3. A method for synthesizing rebaudioside V from rebaudioside G , the method comprising:preparing a reaction mixture comprising rebaudioside G, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a HV1 UDP-glycosyltransferase; andincubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to the rebaudioside G to produce rebaudioside V.4. The method of further comprising adding a sucrose synthase to the reaction mixture.5ArabidopsisArabidopsisVigna radiate. The method of claim 3 , wherein the sucrose synthase is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a sucrose synthase.6Arabidopsis thaliana. The method of claim 5 , wherein the sucrose synthase is an sucrose synthase 1.7. A method for synthesizing rebaudioside V from rebaudioside G claim 5 , the method comprising:preparing a reaction mixture comprising rebaudioside G, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and a uridine dipospho glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of an EUGT11 and a UDP-glycosyltransferase fusion enzyme; andincubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to the rebaudioside G to produce rebaudioside V.8. The method of further comprising adding a sucrose synthase to the reaction mixture.9ArabidopsisArabidopsisVigna radiate. The method of claim 8 , wherein the sucrose synthase is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a ...

NON-CALORIC SWEETENERS AND METHODS FOR SYNTHESIZING

Disclosed are steviol glycosides referred to as rebaudioside V and rebaudioside W. Also disclosed are methods for producing rebaudioside M (Reb M), rebaudioside G (Reb G), rebaudioside KA (Reb KA), rebaudioside V (Reb V) and rebaudioside (Reb W). 1. A method for synthesizing rebaudioside E from rebaudioside KA , the method comprising:preparing a reaction mixture comprising rebaudioside KA, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and a UDP-glycosyltransferase selected from the group consisting of HV1, EUGT11 and a UDP-glycosyltransferase fusion enzyme; andincubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to the C2′ 13-O-glucose of rebaudioside KA to produce rebaudioside E.2. The method of further comprising adding a sucrose synthase to the reaction mixture.3ArabidopsisArabidopsisVigna radiate. The method of claim 2 , wherein the sucrose synthase is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a sucrose synthase.4Arabidopsis thaliana. The method of claim 3 , wherein the sucrose synthase is an sucrose synthase 1.5. The method of claim 1 , wherein the UDP-glycosyltransferase fusion enzyme is selected from the group consisting of an EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain.6ArabidopsisArabidopsisVigna radiate. The method of claim 5 , wherein the sucrose synthase domain is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a sucrose synthase.7Arabidopsis thaliana. The method of claim 6 , wherein the sucrose synthase domain is an sucrose synthase 1.814.-. (canceled)15. A method for synthesizing rebaudioside D2 from rubusoside claim 6 , the method comprising:preparing a reaction mixture comprising rubusoside, substrates selected from the group consisting of sucrose, uridine diphosphate ( ...

PRODUCTION OF TERPENES AND TERPENOIDS

The present invention relates to a nucleic acid construct comprising a nucleic acid molecule encoding a protein involved in the biosynthesis of a terpenoid or a precursor thereof, wherein said nucleic acid molecule is operably linked to a derepressible promoter. 1. A nucleic acid construct comprising a nucleic acid molecule encoding a protein involved in the biosynthesis of a terpenoid or a precursor thereof , wherein said nucleic acid molecule is operably linked to a derepressible promoter.2. The nucleic acid construct according to claim 1 , wherein the protein involved in the biosynthesis of a terpenoid or a precursor thereof is selected from the group consisting of geranylgeranyl diphosphate synthases or taxadiene synthases.3. The nucleic acid construct according to claim 1 , wherein the derpressible promoter is selected from the group consisting of CAT1 promoter claim 1 , FDH1 promoter claim 1 , FLD1 promoter claim 1 , PEX5 promoter claim 1 , DAK1 promoter claim 1 , FGH1 promoter claim 1 , GTH1 promoter claim 1 , G1 promoter claim 1 , G2 promoter claim 1 , G3 promoter claim 1 , G4 promoter claim 1 , G5 promoter claim 1 , G6 promoter claim 1 , FMD promoter and a functional variant thereof.4. The nucleic acid construct according to claim 1 , wherein the promoter is an orthologous promoter.5. The nucleic acid construct according to claim 1 , wherein the derepressible promoter is linked to a second promoter forming a bidirectional promoter or a bidirectional derepressible promoter.6. The nucleic acid construct according to claim 5 , wherein the second promoter is a constitutive claim 5 , derepressible or inducible promoter.7. The nucleic acid construct according to claim 6 , wherein the constitutive promoter is selected from the group consisting of a GAP promoter claim 6 , PGCW14 promoter claim 6 , TEF1 promoter claim 6 , TPI promoter claim 6 , PGK1 promoter or a histone promoter.8. The nucleic acid construct according to claim 6 , wherein the inducible promoter is ...

CYTOCHROME P450 AND CYTOCHROME P450 REDUCTASE POLYPEPTIDES, ENCODING NUCLEIC ACID MOLECULES AND USES THEREOF

Provided are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and/or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells. 1173-. (canceled)174. A host cell , comprising a nucleic acid molecule encoding a cytochrome P450 reductase or a catalytically active portion thereof , wherein:(a) the encoded cytochrome P450 reductase or catalytically active portion thereof exhibits at least 80% sequence identity to a cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 12 or 13 or a corresponding catalytically active portion thereof;(b) the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof catalyzes the transfer of two electrons from NADPH to an electron acceptor; and(c) the nucleic acid molecule is heterologous to the host cell.175. The host cell of claim 174 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active fragment thereof exhibits at least 85% claim 174 , 86% claim 174 , 87% claim 174 , 88% claim 174 , 89% claim 174 , 90% claim 174 , 91% claim 174 , 92% claim 174 , 93% claim 174 , 94% claim 174 , 95% claim 174 , 96% claim 174 , 97% claim 174 , 98% or 99% amino acid sequence identity to a sequence of amino acids set forth in SEQ ID NO: 12 or 13.176Santalum. The host cell of claim 174 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof is a P450 reductase polypeptide.177Santalum album. The host cell of claim 174 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof is a P450 reductase polypeptide.178. The host cell of claim 174 , wherein the encoded cytochrome P450 reductase ...

METHOD FOR PRODUCING STEVIOL GLYCOSIDE

The invention provides a method for producing steviol glycosides. The invention provides a transformant having introduced therein the steviol glucosyltransferase and a method for producing steviol glycosides using the transformant. 2. The method according to claim 1 , wherein said sugar in the UDP-sugar is a hexose.3. The method according to claim 1 , wherein said sugar in the UDP-sugar is one selected from the group consisting of glucose claim 1 , mannose and galactose.4. The method according to claim 1 , wherein said sugar added to the carboxyl at position 19 is a hexose.5. The method according to claim 1 , wherein said sugar added to the carboxyl at position 19 is one selected from the group consisting of glucose claim 1 , mannose and galactose.6. The method according to claim 1 , wherein said Ris H or the sugar residue which is a glucose monomer claim 1 , a glucose dimer or a glucose trimer.7. The method according to claim 1 , wherein said compound is steviolmonoside claim 1 , steviolbioside or steviol.8. The method according to claim 1 , wherein said steviol glycoside is steviolbioside claim 1 , stevioside claim 1 , rebaudioside A claim 1 , rebaudioside B claim 1 , rebaudioside C claim 1 , rebaudioside D claim 1 , rebaudioside E claim 1 , rebaudioside F claim 1 , or a combination thereof.10. The transformant according to claim 9 , wherein said sugar molecule is a hexose.11. The transformant according to claim 9 , wherein said sugar molecule is one selected from the group consisting of glucose claim 9 , mannose and galactose.12. The transformant according to claim 9 , wherein said Ris H or the sugar residue which is a glucose monomer claim 9 , a glucose dimer or a glucose trimer.13. The transformant according to claim 9 , wherein said compound is steviolmonoside claim 9 , steviolbioside or steviol.14. The transformant according to claim 9 , wherein said polynucleotide is inserted into an expression vector.15. The transformant according to claim 9 , which is a ...

KAURENOIC ACID 13-HYDROXYLASE (KAH) VARIANTS AND USES THEREOF

Provided herein are genetically modified host cells, compositions, and methods for improved production of steviol glycosides. The host cells are genetically modified to contain a heterologous nucleic acid that expresses novel and optimized variants of Ro.KAH. The host cell further contains one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing one or more steviol glycosides in the host cell. The host cells, compositions, and methods described herein provide an efficient route for the heterologous production of steviol glycosides including rebaudioside A, rebaudioside D, and rebaudioside M. 1. A variant kaurenoic acid hydroxylase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , wherein the sequence further comprises one or more amino acid substitutions.2. The variant polypeptide of claim 1 , wherein the one or more amino acid substitutions are selected from K69R claim 1 , V343G claim 1 , T403V claim 1 , H491 P claim 1 , P72D claim 1 , L64D claim 1 , Q84C claim 1 , L64G claim 1 , E206D claim 1 , Y238C claim 1 , A210G claim 1 , L64N claim 1 , I237C claim 1 , L11V claim 1 , N207F claim 1 , M73G claim 1 , W8G claim 1 , E6OR claim 1 , Y55S claim 1 , N475G claim 1 , D292P claim 1 , P161C claim 1 , K267D claim 1 , L485F claim 1 , A396F claim 1 , R507A claim 1 , P72T claim 1 , I132G claim 1 , N61P claim 1 , K119V claim 1 , T220E claim 1 , P72G claim 1 , Q513R claim 1 , S133G claim 1 , Y506V claim 1 , K69P claim 1 , E60G claim 1 , K224C claim 1 , M73H claim 1 , H379G claim 1 , P72C claim 1 , K314P claim 1 , W202A claim 1 , G466F claim 1 , N49A claim 1 , S339G claim 1 , N160D claim 1 , T216G claim 1 , D102Y claim 1 , F246G claim 1 , M58P claim 1 , T220R claim 1 , R458D claim 1 , M58G claim 1 , A681 claim 1 , 570P claim 1 , F88V claim 1 , T240D claim 1 , L2051 claim 1 , K167G claim 1 , L232M claim 1 , S62R claim 1 , G56D claim 1 , Q244G claim 1 , A242D claim 1 , N49R claim 1 , Q513G claim 1 , W29T claim 1 , L303D ...

NON-CALORIC SWEETENERS AND METHODS FOR SYNTHESIZING

Disclosed are steviol glycosides referred to as rebaudioside V and rebaudioside W. Also disclosed are methods for producing rebaudioside M (Reb M), rebausoside G (Reb G), rebaudioside KA (Reb KA), rebaudioside V (Reb V) and rebaudioside (Reb W). 17.-. (canceled)8. A method for synthesizing rebaudioside E from rubusoside , the method comprising:preparing a reaction mixture comprising rubusoside, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and a UDP-glycosyltransferase selected from the group consisting of HV1, EUGT11 and a UDP-glycosyltransferase fusion enzyme (EUS); and optionally, a sucrose synthase; andincubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA or stcviosidc, and a glucose is covalently coupled to rebaudioside KA to produce rebaudioside E.9. The method of further comprising adding a sucrose synthase to the reaction mixture.10ArabidopsisArabidopsisVigna radiate. The method of claim 9 , wherein the sucrose synthase is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a sucrose synthase.11Arabidopsis thaliana. The method of claim 10 , wherein the sucrose synthase is an sucrose synthase 1.12. The method of claim 8 , wherein the UDP-glycosyltransferase fusion enzyme is selected from the group consisting of an EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain.13ArabidopsisArabidopsisVigna radiate. The method of claim 12 , wherein the sucrose synthase domain is selected from the group consisting of an sucrose synthase 1; an sucrose synthase 3; and a sucrose synthase.14Arabidopsis thaliana. The method of claim 13 , wherein the sucrose synthase domain is an sucrose synthase 1.1528.-. (canceled) This application claims priority to U.S. Provisional Patent Application No. 62/059,498, filed Oct. 3, ...

FERMENTATION METHODS FOR PRODUCING STEVIOL GLYCOSIDES WITH MULTI-PHASE FEEDING

Disclosed are methods for producing steviol glycosides, such as rebaudioside D and rebaudioside M, using engineered yeast. The methods include at acetyl-CoA least two phases: first and second phases where a glucose-containing feed composition is provided to the medium in different modes of feeding in each phase, such as variable feeding and then constant feeding. The two phase feeding can result in a acetoacetyl-CoA growth rate that is slower in the second phase than in the first phase, and consequently increased steviol glycoside production rates, reduced fermentation times, and reduced biomass concentrations. 1. A method for producing steviol glycoside(s) , the method comprising steps of:(a) growing engineered yeast capable of producing one or more steviol glycoside(s) in a medium, wherein the engineered yeast is grown at one or more growth rate(s) (dilution rate(s)) within a first range; and wherein a composition comprising glucose is added to the medium according to a first mode; and(b) fermenting the medium with the engineered yeast to produce the one or more steviol glycoside(s), wherein during fermenting, a composition comprising glucose is added to the medium according to a second mode that is different than the first mode, and during fermenting the yeast grown at one or more growth rate(s) (dilution rate(s)) within a second range, wherein the second range is less than the first range.2. The method of where in step (a) the growth rate (dilution rate) is 0.06 hor greater.3. The method of where in step (a) the first range is 0.06 hto 0.17 h.4. The method of where in step (a) the first range is 0.09 hto 0.15 h.5. The method of where in step (b) the growth rate (dilution rate) is 0.09 hor less.6. The method of where in step (b) the second range is 0.015 hto 0.09 h.7. The method of where in step (b) the second range is 0.015 hto 0.06 h.8. The method of wherein the growth rate (dilution rate) in step (b) is in the range of 50-90% of a maximum growth rate (dilution ...

TERPENE SYNTHASES FROM SANTALUM

An isolated nucleic acid molecule that encodes a terpene synthase and is selected from among: a) a nucleic acid molecule comprising the sequence of nucleotides set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; b) a nucleic acid molecule that is a fragment of (a); c) a nucleic acid molecule comprising a sequence of nucleotides that is complementary to (a) or (b); and d) a nucleic acid molecule that encodes a terpene synthase having at least or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to any one of (a)-(c); wherein the nucleic acid molecule encodes a terpene synthase. 1Santalum. A host cell , comprising a nucleic acid molecule that encodes a species terpene synthase that comprises:(i) the sequence of amino acid resides set forth in SEQ ID NO:2, 4 or 6 or a catalytically active fragment thereof; or(ii) a sequence of amino acid residues that has at least 95% or greater sequence identity to the sequence of amino acid residues set forth in SEQ ID NO: 2, or a catalytically active fragment thereof, wherein:the nucleic acid molecule is heterologous to the cell; andthe encoded synthase catalyzes the production of an α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene concurrently.2. The host cell of claim 1 , wherein the encoded synthase comprises a serine in place of the proline at residue 143 of SEQ ID NO: 2.3Santalum. A vector claim 1 , comprising a nucleic acid molecule that encodes a species terpene synthase that comprises:(i) the sequence of amino acid resides set forth in SEQ ID NO:2, 4 or 6 or a catalytically active fragment thereof; or(ii) a sequence of amino acid residues that has at least 95% or greater sequence identity to the sequence of amino acid residues set forth in SEQ ID NO: 2, or a catalytically active fragment thereof,wherein the encoded synthase catalyzes the production of an α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene concurrently.4. The vector of claim 3 , wherein the ...

BIOSYNTHETIC PRODUCTION OF STEVIOL GLYCOSIDES REBAUDIOSIDE J AND REBAUDIOSIDE N

The present disclosure relates to the production of steviol glycosides rebaudioside J and rebaudioside N through the use of rebaudioside A as a substrate and a biosynthetic pathway involving various 1,2 RhaT-rhamnosyltransferases. 138.-. (canceled)39. A biosynthetic method of preparing rebaudioside N , the method comprising:reacting a steviol glycoside composition comprising rebaudioside A with a glucose donor moiety in the presence of a first recombinant polypeptide having glucosyltransferase activity to produce rebaudioside I, wherein said first recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; andreacting said rebaudioside I with a rhamnose donor moiety in the presence of a second recombinant polypeptide having 1,2-rhamnosytransferase activity to produce rebaudioside N; wherein said second recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3.40. The method of claim 39 , wherein said second recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 3.41. The method of claim 39 , wherein said first recombinant polypeptide comprises an amino acid sequence having at least 90% identity of SEQ ID NO: 7 claim 39 , SEQ ID NO: 11 claim 39 , SEQ ID NO: 13 claim 39 , or SEQ ID NO: 15.42. The method of claim 41 , wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 7 claim 41 ,43. The method of claim 41 , wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 11.44. The method of claim 41 , wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 13.45. The method of claim 41 , wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 15.46. The method of claim 39 , wherein the rhamnose donor moiety is UDP-L-rhamnose.47. The method of claim 39 , wherein the ...

ENZYMATIC METHOD FOR PREPARING REBAUDIOSIDE N

Provided is a method for preparing rebaudioside N using an enzymatic method, comprising using rebaudioside A or rebaudioside J as a substrate, and making the substrate, in the presence of a glycosyl donor, react under the catalysis of a UDP-glycosyl-transferase and/or a UDP-glycosyltransferase-containing recombinant cell to generate rebaudioside N. 1. A method for preparing rebaudioside N using an enzymatic method , comprising: using rebaudioside A or rebaudioside J as a substrate , in the presence of a glycosyl donor , and reacting under catalysis of a UDP-glycosyltransferase containing recombinant cell and/or a prepared UDP-glycosyltransferase to generate the rebaudioside N.2. (canceled)3. The method according to claim 1 , wherein the glycosyl donor comprises one or two of a glucose-based donor and a rhamnosyl donor claim 1 , wherein the glucose-based donor is UDP-glucose or a UDP-glucose regeneration system consisting of sucrose claim 1 , sucrose synthase and UDP claim 1 , and the rhamnosyl donor is UDP-rhamnose.4Stevia rebaudianaOryza sativa.. The method according to claim 1 , wherein the UDP-glycosyltransferase comprises one or two of UGT-A from and UGT-B from5Stevia rebaudianaOryza sativa. The method according to claim 1 , wherein the UDP-glycosyltransferase comprises UGT-A from and UGT-B from ; the UDP-glycosyltransferase is added into the reaction system in two steps; wherein the UGT-B is added in the first step and the UGT-A is added in the second step.6. The method according to claim 5 , wherein an amino acid sequence of the UGT-A has at least 60% identity to sequence 2 listed in a sequence listing; and/or an amino acid sequence of the UGT-B has at least 60% identity to sequence 4 listed in the sequence listing.7. The method according to claim 6 , wherein the amino acid sequence of the UGT-A has at least 70% identity to sequence 2 listed in the sequence listing; and/or the amino acid sequence of the UGT-B has at least 70% identity to sequence 4 listed in ...

NON-CALORIC SWEETENER

Disclosed is a steviol glycoside referred to as rebaudioside D2. Rebaudioside D2 has five β-D-glucosyl units connected to the aglycone steviol. Also disclosed are methods for producing rebaudioside D2, a UDP-glycosyltransferase fusion enzyme, and methods for producing rebaudioside D and rebaudioside E. 1103.-. (canceled)104. A method for synthesizing rebaudioside D2 , the method comprising:preparing a reaction mixture comprising stevioside; a substrate selected from the group consisting of sucrose, uridine diphosphate and uridine diphosphate-glucose; and a UDP-glycosyltransferase selected from the group consisting of a uridine diphospho glycosyltransferase and a UDP-glycosyltransferase fusion enzyme comprising a uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain; and incubating the reaction mixture for a sufficient time to produce rebaudioside D2, wherein a glucose is covalently coupled to the stevioside to produce a rebaudioside E intermediate, and wherein a glucose is covalently coupled to the rebaudioside E intermediate to produce rebaudioside D2.105Oryza sativa. The method of wherein the UDP-glycosyltransferase is a uridine diphospho glycosyltransferase EUGT11.106. The method of further comprising adding a sucrose synthase to the reaction mixture.107ArabidopsisCoffeaStevia. The method of wherein the sucrose synthase is selected from the group consisting of an sucrose synthase 1 claim 106 , a sucrose synthase 1 and a sucrose synthase 1.108Oryza sativa. The method of wherein the uridine diphospho glycosyltransferase domain is an uridine diphospho glycosyltransferase EUGT11.109ArabidopsisCoffeaStevia. The method of wherein the sucrose synthase domain is selected from the group consisting of an sucrose synthase 1 claim 104 , a sucrose synthase 1 and a sucrose synthase 1.110. The method of wherein the UDP-glycosyltransferase fusion enzyme comprises an amino acid sequence having about 90% sequence identity to SEQ ID NO:5. A paper copy of ...

METHOD FOR PREPARING REBAUDIOSIDE M BY USING ENZYME METHOD

Provided is a method for preparing Rebaudioside M by using an enzyme method. In the method, Rebaudioside A or Rebaudioside D is used as a substrate, and in the presence of sucrose and UDP, Rebaudioside M is generated by means of reaction of the substrate under the catalysis of a mixture of UDP-glucosyl transferase and sucrose synthetase, or recombinant cells containing the UDP-glucosyl transferase and sucrose synthetase. The reaction is carried out in an aqueous-phase system at a temperature of 20 to 60° C. and pH 5.0 to 9.0. The reaction system further contains dimethyl sulfoxide at a concentration of 3% to 5% according to the ratio by volume for facilitating solubilization of the substrate. 114-. (canceled)15. A method for preparing rebaudioside M , the method comprising reacting rebaudioside D in a reaction solution with a glucosyl donor in the presence of recombinant cells comprising:a) a UDP-glucosyl transferase having an amino acid sequence with at least 90% identity to SEQ ID NO: 2; andb) a sucrose synthetase.16. The method according to claim 15 , wherein the glucosyl donor is UDP-glucose generated in situ from UDP and sucrose in the presence of the sucrose synthetase.17Escherichia coli, Saccharomyces cerevisiae,Pichia pastoris.. The method according to claim 15 , wherein the recombinant cells are microbial cells selected from the group consisting of: and18. The method according to claim 15 , wherein the rebaudioside D has a concentration of 15 to 50 g/L in the reaction solution.19. The method according to claim 15 , wherein the reaction solution is an aqueous phase system comprising phosphate buffer at a temperature from 35° C. to 45° C. and at a pH ranging from 6.5 to 8.5.20. The method according to claim 19 , wherein the aqueous phase system further comprises a cellular permeating agent.21. The method according to claim 20 , wherein the cellular permeating agent is toluene at a concentration of 1% to 3% by volume.22. The method according to claim 15 , ...

STEVIOL GLYCOSIDE TRANSPORT

The disclosure provides a recombinant cell capable of producing a steviol glycoside, wherein the cell comprises a nucleic acid coding for a variant of a parent polypeptide, wherein the variant has steviol glycoside transport mediating activity, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence of the parent polypeptide, comprises at least one modification of the amino acid residue corresponding to any of the amino acids in the amino acid sequence of the parent polypeptide, wherein the variant has an improved ability to produce rebaudioside M and optionally other steviol glycosides extracellularly if compared with the parent polytpeptide when measured under the same conditions. 1. A variant of a parent polypeptide wherein the variant has steviol glycoside transport mediating activity , wherein the variant comprises an amino acid sequence which , when aligned with the amino acid sequence of the parent polypeptide , comprises at least one modification of an amino acid residue corresponding to any of the amino acids in the amino acid sequence of the parent polypeptide , wherein , when measured under the same conditions:a) the ratio between the molar concentration of rebaudioside M produced by a recombinant cell expressing the variant and the molar concentration of rebaudioside M produced by a reference cell is at least 0.1; and/orb) the ratio between the molar concentration of rebaudioside M and the molar concentration of rebaudioside A produced by a recombinant cell expressing the variant is higher than the ratio between the molar concentration of rebaudioside M and the molar concentration of rebaudioside A produced by a reference cell; and/orc) the ratio between the molar concentration of rebaudioside M and the molar concentration of total steviol glycosides produced by a recombinant cell expressing the variant is higher than the ratio between the molar concentration of rebaudioside M and the molar concentration of ...

METHOD FOR PRODUCING STEVIOL AND STEVIOL GLYCOSIDE BY USING AOBGL11 HOMOLOG

There has been a demand for a new method for producing a steviol glycoside and steviol. The present invention provides a method for producing a steviol glycoside and/or steviol, the method comprising a step of breaking bonds of steviol glycoside: a glucosidic bond at position 13; a glucosyl ester bond at position 19; and/or a glycosidic bond in a side chain (excluding a rhamnoside bond). 1. A method comprising reacting a protein selected from the group consisting of proteins (a) to (c) shown below with a first steviol glycoside:(a) a protein consisting of the amino acid sequence of SEQ ID NO: 2;(b) a protein consisting of an amino acid sequence wherein 1 to 83 amino acids have been deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 2, and having an activity to hydrolyze at least one of glucoside bond at the 13 position, glucosyl ester bond at the 19 position, and glycoside bond (except for rhamnoside bond) within a side chain of the first steviol glycoside; and(c) a protein having an amino acid sequence having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 2, and having an activity to hydrolyze at least one of glucoside bond at the 13 position, glucosyl ester bond at the 19 position, and glycoside bond (except for rhamnoside bond) within a side chain of the first steviol glycoside.2. The method according to claim 1 , wherein the first steviol glycoside is selected from the group consisting of steviolmonoside claim 1 , steviol monoglucosyl ester claim 1 , rubusoside claim 1 , steviolbioside claim 1 , stevioside claim 1 , rebaudioside B claim 1 , rebaudioside A claim 1 , rebaudioside C claim 1 , rebaudioside D claim 1 , rebaudioside E claim 1 , rebaudioside F claim 1 , rebaudioside M claim 1 , dulcoside A claim 1 , steviol glycoside B claim 1 , steviol glycoside C claim 1 , and steviol glycoside D.3. The method according to claim 19 , wherein the second steviol glycoside is selected from the group ...

HETEROLOGOUS PRODUCTION OF PATCHOULOL, BETA-SANTALENE, AND SCLAREOL IN MOSS CELLS

The present invention relates to methods for preparing patchoulol, β-santalene, and sclareol in transgenic moss cells that include heterologous nucleic acid molecules encoding a polypeptide or synthase capable of using FPP or GGPP as a substrate. Methods for producing the transgenic moss cell, as well as the transgenic moss cell itself are also disclosed. 1. A method for producing a transgenic moss cell comprising introducing into a moss cell capable of producing farnesyl-pyrophosphate (FPP) or geranylgeranyl-pyrophosphate (GGPP) a heterologous nucleic acid molecule encoding a polypeptide , the polypeptide having a patchoulol synthase activity or β-santalene synthase activity to catalyze the conversion of FPP to patchoulol or β-santalene , or a sclareol synthase activity to catalyse the conversion of GGPP to sclareol , wherein the heterologous nucleic acid molecule is at least 70% identical to SEQ ID NO 1 , SEQ ID NO 2 , or SEQ ID NO 3.2. A method for preparing patchoulol , β-santalene , or sclareol in a transgenic moss cell comprising;a) introducing into a moss cell capable of producing farnesyl-pyrophosphate (FPP) or geranylgeranyl-pyrophosphate (GGPP) a heterologous nucleic acid molecule encoding a polypeptide, the polypeptide having a patchoulol synthase activity or β-santalene synthase activity to catalyze the conversion of FPP to patchoulol or β-santalene, or a sclareol synthase activity to catalyse the conversion of GGPP to sclareol, wherein the heterologous nucleic acid molecule is at least 70% identical to SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3,b) culturing the transgenic moss cell to express or overexpress the polypeptide encoded by the heterologous nucleic acid molecule, andc) isolating patchoulol, β-santalene, and/or sclareol produced in step b).3. A method according to claim 2 , wherein step c) is performed by steam distillation and/or vacuum distillation.4. A method according to claim 1 , wherein the moss cell is selected from the group comprising of ...

High-Throughput Enzymatic Preparation of Glucosylated Steviol Glycosides under Programming Temperatures

The present invention discloses a method for preparing glucosyl steviol glycosides through enzymatic catalysis under programming temperatures in high throughput, belonging to the technical field of biosynthesis of sweeteners. By using cyclodextrin glucosyltransferase from sp. as a catalyst, steviol glycosides as the glycosyl receptor and dextrin or oligosaccharide as the glycosyl donor, taking a calcium/barium ion salt bridge as the main stabilizer and combining with glycerol to adjust the conformation and binding domain openness of the enzyme, and utilizing transglucosylation and hydrolytic activities of amylase at variable temperatures in different stages, thereby preparing the glucosyl steviol glycosides through enzymatic catalysis under programming temperatures in high throughput. The technology of the present invention can improve the utilization rate of the enzyme, and obtain glucosyl steviol glycosides with good sweetness and good taste. 1. A method for preparing glucosyl steviol glycosides through enzymatic catalysis under programming temperatures , comprising using cyclodextrin glucosyltransferase as a catalyst , steviol glycosides as a glycosyl receptor , dextrin or oligosaccharide as a glycosyl donor , and salt and polyol as an enzyme composite stabilizer to prepare glucosyl steviol glycosides in an aqueous phase via enzymatic catalysis under programming temperatures in high throughput.2. The method for preparing glucosyl steviol glycosides through enzymatic catalysis under programming temperatures according to claim 1 , comprising dissolving the steviol glycosides and the glycosyl donor in water or a buffer solution under a certain pH environment at 80-85° C. claim 1 , cooling to 60-65° C. and following by addition of 10-100 U/g steviol glycoside of cyclodextrin glucosyltransferase and an enzyme stabilizer claim 1 , wherein temperature is kept for a reaction to start for 0.5-5 h claim 1 , and the temperature is raised to 70-76° C. for 8-24 h and finally ...

FERMENTATION METHODS FOR PRODUCING STEVIOL GLYCOSIDES

Disclosed are methods for producing steviol glycosides, such as rebaudioside D and rebaudioside M, using engineered yeast. The methods include growing yeast on non-fermentative carbon sources. Other methods include growing yeast on one or more polysaccharides in which saccharification and fermentation of the polysaccharides occurs simultaneously. 1. A method for producing steviol glycoside(s) , comprising growing engineered yeast capable of producing one or more steviol glycoside(s) in a glucose-limited medium that contains carbohydrates fermentable by the engineered yeast , whereina. less than 50% by weight (wt %), preferably less than 20 wt %, more preferably less than 10 wt % or less than 5 wt %, of said fermentable carbohydrates in the glucose-limited medium is glucose, fructose, or glucose and fructose; andb. at least 50 wt %, preferably at least 60 wt %, at least 70 wt %, at least 80 wt %, at least 90 wt %, or at least 95 wt %, of said fermentable carbohydrates in the glucose-limited medium is an ethanol-limiting substrate selected from the group consisting of raffinose, mannose, trehalose, galactose, maltose, glycerol, and combinations thereof.2. The method of wherein the ethanol-limiting substrate is selected from the group consisting of raffinose claim 1 , mannose claim 1 , trehalose claim 1 , galactose claim 1 , and combinations thereof.3. The method of wherein the ethanol-limiting substrate is selected from the group consisting of raffinose claim 1 , mannose claim 1 , trehalose claim 1 , and combinations thereof.4. The method of claim 1 , wherein the steviol glycoside(s) comprise rebaudioside M claim 1 , rebaudioside D claim 1 , or both rebaudioside M and rebaudioside D.5Candida, KloeckeraHanseniasporaKluyveromyces, Lipomyces, PichiaHansenulaRhodotorula, Saccharomycete, Saccharomyces, Schizosaccharomyces, Torulopsis, Torulaspora, YarrowiaZygosaccharomyces.. The method of claim 1 , wherein the engineered yeast comprise () claim 1 , () claim 1 , claim 1 , ...

EPOXIDATION USING PEROXYGENASE

The invention relates to enzymatic methods for epoxidation of a non-cyclic aliphatic alkene, or a terpene. 115-. (canceled)16. A method for producing an epoxide , comprising contacting a non-cyclic aliphatic alkene or a terpene with hydrogen peroxide and a peroxygenase (EC 1.11.2.1) , wherein the peroxygenase has at least 90% sequence identity to SEQ ID NO: 1.17. The method of claim 16 , wherein the peroxygenase has at least 95% sequence identity to SEQ ID NO: 1.18. The method of claim 16 , wherein the peroxygenase has at least 97% sequence identity to SEQ ID NO: 1.19. The method of claim 16 , wherein the peroxygenase has at least 98% sequence identity to SEQ ID NO: 1.20. The method of claim 16 , wherein the peroxygenase has at least 99% sequence identity to SEQ ID NO: 1.21. The method of claim 16 , wherein the peroxygenase has the amino acid sequence of SEQ ID NO: 1.22. The method of claim 16 , wherein the amino acid sequence comprises the motif E-H-D-[G claim 16 ,A]-S-[L claim 16 ,I]-S-R (SEQ ID NO: 21).23. The method of claim 16 , wherein the aliphatic alkene has one or more substituents selected from the group consisting of halogen claim 16 , hydroxyl claim 16 , carboxyl claim 16 , amino claim 16 , nitro claim 16 , cyano claim 16 , thiol claim 16 , sulphonyl claim 16 , formyl claim 16 , acetyl claim 16 , methoxy claim 16 , ethoxy claim 16 , carbamoyl and sulfamoyl.24. The method of claim 23 , wherein the substituent(s) are selected from the group consisting of chloro claim 23 , hydroxyl claim 23 , carboxyl and sulphonyl.25. The method of claim 16 , wherein the aliphatic alkene consists of at least three carbons claim 16 , and has a carbon-carbon double bond at one end.26. The method of claim 16 , wherein the aliphatic alkene is propene claim 16 , butene claim 16 , pentene claim 16 , hexene claim 16 , heptene claim 16 , octene claim 16 , nonene claim 16 , decene claim 16 , undecene claim 16 , dodecene claim 16 , tridecene claim 16 , tetradecene claim 16 , ...

Recombinant Production of Steviol Glycosides

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.g., Rebaudioside A and/or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements. 1. A recombinant host cell comprising a recombinant gene encoding a polypeptide having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:152; (a) a recombinant gene encoding a UGT91d2e or a UGT91d2m polypeptide, wherein the UGT91d2e polypeptide comprises a UGT91d2e polypeptide having a substitution at residues 211 and 286 of SEQ ID NO:5 or a UGT91D2e-b polypeptide having a methionine at residue 211 and an alanine at residue 286 of SEQ ID NO:5;', '(b) a recombinant gene encoding a steviol synthase polypeptide (KAH) having at least 80% identity to the amino acid sequence set forth in (SEQ ID NO:164);', '(d) one or more exogenous nucleic acids encoding a sucrose transporter (SUC1) and a sucrose synthase (SUS1) wherein expression of the one or more exogenous nucleic acids encoding a sucrose transporter and expression of a glucosyltransferase results in increased levels of uridine diphosphate glucose (UDP-glucose) in the host;', '(c) a recombinant gene encoding an ent-copalyl diphosphate synthaseCDPS) polypeptide lacking a chloroplast transit peptide;'}, {'i': 'S. rebaudiana', '(e) a recombinant gene encoding a cytochrome P450 reductase (CPR) polypeptide having at least 80% sequence identity to the CPR amino acid sequence set forth in (SEQ ID NO: 170;'}, {'br': None, '-X1-X2-X3-X4-X5-\u2003\u2003(I)'}, '(f) a nucleic acid sequence, said nucleic acid comprising a heterologous insert sequence operably linked to an open reading frame, wherein the heterologous insert sequence has a formula (I)], 'said host optionally comprising one or more of the following [{'i': S. rebaudiana', 'A. thaliana, '( ...

RECOMBINANT PRODUCTION OF STEVIOL GLYCOSIDES

Recombinant polypeptides having UDP-glycosyltransferase activities, including a 1,2-19-O-glucose glycosylation activity and a 1,2-13-O-glucose glycosylation activity for synthesizing of steviol glucosides, are provided. A method of producing a steviol glycoside composition using such recombinant polypeptide is also provided. Also disclosed are steviol glycosides referred to as rebaudioside Z1 and rebaudioside Z2. 1. A recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:6.2. The recombinant polypeptide of claim 1 , wherein the amino acid sequence has at least 90% identity to SEQ ID NO:6.3. The recombinant polypeptide of claim 1 , wherein the amino acid sequence is at least 95% identical to SEQ ID NO:6.4. The recombinant polypeptide of claim 1 , wherein the amino acid sequence consists of SEQ ID NO:6.5. An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant polypeptide of .6. The isolated nucleic acid of claim 5 , wherein the nucleotide sequence has at least 80% identity to SEQ ID NO:7.7. A vector comprising the isolated nucleic acid of .8. A host cell comprising the vector of .9. The host cell of claim 8 , wherein the host cell is selected from the group consisting of a bacteria claim 8 , a yeast claim 8 , a filamentous fungi claim 8 , a cyanobacteria algae and a plant cell.10Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotrys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Escherichia; Klebsiella; Pantoea; Salmonella Corynebacterium; ClostridiumClostridium acetobutylicum.. The host cell of claim 9 , wherein the host cell is selected from the group consisting of ; and11Arabidopsis thalianaOryza sativaHordeum vulgarePanicum ...

HOST CELLS AND METHODS FOR PRODUCING TRICYCLIC SESQUITERPENES, AVIATION AND MISSILE FUEL PRECURSORS

The present invention provides for a fuel compositions are provided comprising a hydrogenation product of a tricyclic sesquiterpene (epi-isozizaene, pentalenene, or α-isocomene) and a fuel additive. Methods of making and using the fuel compositions are also disclosed. 1. A genetically modified host cell capable of producing one or more tricyclic sesquiterpenes , said genetically modified host cell comprising one or more tricyclic sesquiterpenes synthase.2. The genetically modified host cell of claim 1 , wherein (a) the tricyclic sesquiterpene is epi-isozizaene claim 1 , and the tricyclic sesquiterpene synthase is epi-isozizaene synthase (EIZS);(b) the tricyclic sesquiterpene is pentalenene, and the tricyclic sesquiterpene synthase is pentalenene synthase (PentS); or (c) the tricyclic sesquiterpene is α-isocomene, and the tricyclic sesquiterpene synthase is α-isocomene synthase (MrTPS2); wherein the EIZS comprises (i) an amino acid sequence having at least 70% identity with SEQ ID NO:1, and (ii) the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5; the PentS comprises (i) an amino acid sequence having at least 70% identity with SEQ ID NO:2, and (ii) the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5; and the MrTPS2 comprises (i) an amino acid sequence having at least 70% identity with SEQ ID NO:3, and (ii) the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.3. The genetically modified host cell of claim 2 , wherein the tricyclic sesquiterpene is epi-isozizaene claim 2 , and the tricyclic sesquiterpene synthase is epi-isozizaene synthase (EIZS).4. The genetically modified host cell of claim 2 , wherein the tricyclic sesquiterpene is pentalenene claim 2 , and the tricyclic sesquiterpene synthase is pentalenene synthase (PentS).5. The genetically modified host cell of claim 2 , wherein the tricyclic sesquiterpene is α-isocomene claim 2 , and the tricyclic sesquiterpene synthase is α-isocomene synthase (MrTPS2).6. The genetically modified host cell of claim 1 , ...

TRITERPENE OXIDASE DERIVED FROM PLANT BELONGING TO GENUS GLYCYRRHIZA, GENE ENCODING THE SAME, AND METHOD OF USING THE SAME

Identification of a protein having an activity of oxidizing oleanane-type triterpene, and a gene encoding the protein, the protein and the gene, and use thereof are provided. For example, a protein having an activity of oxidizing oleanane-type triterpene obtained from a plant in the family Fabaceae, a gene encoding the protein and use thereof are provided. The protein is shown in, for example, SEQ ID NO: 4, 14 or 18, and the gene encoding the protein is shown in, for example, SEQ ID NO: 3, 13 or 17. A transformant into which the gene is introduced can be produced, and thereby a triterpene oxidase can be obtained. 1. A polypeptide having an activity of oxidizing carbon at a position 30 of oleanane-type triterpene , wherein the isolated polypeptide comprises:an amino acid sequence of SEQ ID NO:4, SEQ ID NO: 14 or SEQ ID NO:18; oran amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NO:4, SEQ ID NO:14 or SEQ ID NO:18.2. The polypeptide according to claim 1 , wherein the oleanane-type triterpene is β-amyrin claim 1 , 11-oxo-β-amyrin claim 1 , or 30-hydroxy-11-oxo-β-amyrin.3. The polypeptide according to claim 1 , being derived from a plant in the family Fabaceae.4. The polypeptide according to claim 3 , wherein the plant in the family Fabaceae is a plant in the subfamily Faboideae claim 3 ,5GlycyrrhizaMedicago.. The polypeptide according to claim 4 , wherein the plant in the subfamily Faboideae is a plant belonging to the genus or the genus6GlycyrrhizaG. uralensisG. glabra.. The polypeptide according to claim 5 , wherein the plant belonging to the genus is or7MedicagoM. truncatula.. The polypeptide according to claim 5 , wherein the plant belonging to the genus is8. The polypeptide according to claim 1 , being a protein belonging to cytochrome P450.9. An isolated cDNA polynucleotide encoding a polypeptide having an activity of oxidizing carbon at a position 30 of an oleanane-type triterpene claim 1 , wherein the isolated cDNA ...

CHEMOENZYMATIC SYNTHESIS OF PEPTIDE BETA-LACTONES AND BETA-HYDROXY ACIDS

Methods of producing peptide beta-lactones and beta-hydroxy acids are disclosed that include contacting a beta-hydroxy-alpha-amino acid, an aryl carrier protein (ObiD), and ATP with a non-ribosomal protein synthetase. A continuous flow reactor is disclosed that includes an elongate conduit with at least one region that includes a first region with a non-ribosomal protein synthetase immobilized to a substrate. The non-ribosomal protein synthetase of the continuous flow reactor is configured to contact a flow of a reaction mixture that includes a beta-hydroxy-alpha-amino acid and an aryl carrier protein. The non-ribosomal protein synthetase is further configured to release a peptide beta-lactone into the flow of the reaction mixture. 1. A method of producing a peptide beta-lactone , the method comprising contacting a beta-hydroxy-alpha-amino acid , a benzoic acid derivative , an aryl carrier protein , and ATP with a non-ribosomal protein synthetase , wherein:{'sub': '2', 'the beta-hydroxy-alpha-amino acid is selected from the group consisting of beta-OH-p-NO-homoPhe and beta-OH-homoPhe;'}the aryl carrier protein is selected from the group consisting of ObiD, a homolog of ObiD, recombinant ObiD, and any variation thereof comprising the amino acid sequence of SEQ ID NO: 1 or fragment thereof;the non-ribosomal protein synthetase is selected from the group consisting of ObiF, a homolog of ObiF, recombinant ObiF, and any variation thereof comprising the amino acid sequence of SEQ ID NO:2 or fragment thereof; andthe benzoic acid derivative is 2,3-dihydroxoybenzoic acid.3. The method of claim 1 , further comprising forming the beta-hydroxy-alpha-amino acid by contacting an aliphatic or aryl aldehyde or derivative thereof claim 1 , an amino acid claim 1 , and a pyridoxyl phosphate (PLP) cofactor with a serine hydroxymethyltransferase/threonine aldoloase claim 1 , wherein:the aliphatic or aryl aldehyde or derivative thereof is selected from the group consisting of aliphatic ...

BIOSYNTHESIS OF PACLITAXEL INTERMEDIATE

The invention relates to methods of making compounds useful for production of paclitaxel and analogs or derivatives thereof. 1. A method of making paclitaxel or an analog thereof , comprising preparing an aminopropanoyl-CoA in a reaction catalyzed by a Tyrocidine synthetase A (TycA) to thereby make paclitaxel or an analog or derivative thereof.5. (canceled)6. (canceled)9. The method of claim 1 , wherein the Tyrocidine synthetase Aa. has a bacterial amino acid sequence;{'i': Bacillus brevis', 'Brevibacillus parabrevis, 'b. is a or Tyrocidine synthetase A;'}c. has an amino acid sequence of a Tyrocidine synthetase A from bacteria deposited with the American Type Culture Collection under deposit number ATCC 8185;d. has a serine that is replaced with a substitute amino acid that does not have a hydroxy in its side chain;e. has a serine that is replaced with a substitute amino acid that does not have a hydroxy in its side chain and the substitute amino acid is an alanine, valine, isovaline, leucine, isoleucine, proline, glycine, arginine, lysine, histidine, tryptophan, phenylalanine, methionine or cysteine;f. has an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and an amino acid sequence with 85% sequence identity to any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6;g. is encoded by a nucleic acid sequence comprising a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7, and an nucleic acid sequence with 85% sequence identity to any of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:7; orh. a combination thereof.10. (canceled)11. (canceled)12. (canceled)13. (canceled)14. (canceled)15. (canceled)16. The method of claim 1 , wherein the reaction comprises a mixture of the Tyrocidine synthetase A claim 1 , the substrate claim 1 , ATP and a divalent cation.17. The method of claim 1 , which is performed in vitro claim 1 , in a cell-free reaction claim 1 , in a ...

RECOMBINANT CELLS AND METHODS FOR BIOSYNTHESIS OF ENT-ATISERENOIC ACID

This disclosure describes recombinant cells and methods for microbial biosynthesis of ent-atiserenoic acid. Thus, in one aspect, this disclosure describes a recombinant cell genetically modified to exhibit increased biosynthesis of ent-atiserenoic acid compared to a comparable control cell. In some cases, the recombinant cell can include a host cell modified to include at least one heterologous polynucleotide encoding at least one enzyme in a biosynthetic pathway that produces ent-atiserenoic acid. In some cases, the recombinant cell can include a host cell and at least one heterologous enzyme in a biosynthetic pathway that produces ent-atiserenoic acid. 1. A recombinant cell genetically modified to exhibit increased biosynthesis of ent-atiserenoic acid compared to a comparable control cell.2. A recombinant cell comprising:a host cell; andat least one heterologous polynucleotide encoding at least one enzyme in a biosynthetic pathway that produces ent-atiserenoic acid.3. A recombinant cell comprising:a host cell; andat least one heterologous enzyme in a biosynthetic pathway that produces ent-atiserenoic acid.4Streptomyces. The recombinant cell of wherein the recombinant cell comprises a spp. host cell.5. The recombinant cell of wherein the comparable control cell comprises a wild-type cell.6. A method of biosynthesizing ent-atiserenoic acid claim 1 , the method comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'culturing the recombinant cell of under conditions effective for the recombinant cell to biosynthesize ent-atiserenoic acid.'}7. The method of further comprising isolating at least a portion of the ent-atiserenoic acid.8. The method of further comprising converting the ent-atiserenoic acid to serofendic acid. This application claims priority to U.S. Provisional Patent Application No. 62/311,596, filed Mar. 22, 2016, which is incorporated herein by reference.This application contains a Sequence Listing electronically submitted via EFS-Web to the ...

METHOD FOR PRODUCING PATCHOULOL AND 7-EPI-ALPHA-SELINENE

The present invention provides a method of producing patchoulol and 7-epi-α-selinene, said method comprising contacting at least one polypeptide with farnesyl phyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce patchoulol and 7-epi-α-selinene, compounds which can be useful in the field of perfumery. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing patchoulol and 7-epi-α-selinene is also an object of the present invention. 1. A recombinant polypeptide having sesquiterpene synthase activity and comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 , wherein the polypeptide when expressed produces a mixture of terpenes comprising one or more of patchoulol and 7-epi-α-selinene.2. The polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 95% or 98% sequence identity to SEQ ID NO: 1.3. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.4. An isolated recombinant nucleic acid molecule comprising{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'a) a nucleotide sequence encoding the polypeptide of ;'}b) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 or the complement thereof.5. The nucleic acid molecule of claim 4 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2 or the complement thereof.6. An expression vector comprising the nucleic acid molecule of or a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2 or the complement thereof.7. The expression vector of claim 6 , wherein the vector is in the form of a viral ...

CYTOCHROME P450 AND CYTOCHROME P450 REDUCTASE POLYPEPTIDES, ENCODING NUCLEIC ACID MOLECULES AND USES THEREOF

Provided are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and/or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells. 1. A method for producing a santalol , a bergamotol , and/or a mixture thereof , comprising:(a) contacting an acyclic pyrophosphate terpene precursor with a santalene synthase or catalytically active portion thereof under conditions suitable for the formation of a santalene, a bergamotene, and/or a mixture thereof;(b) contacting the santalene, the bergamotene, and/or the mixture thereof with a cytochrome P450 oxidase polypeptide; and(c) optionally isolating the santalol, the bergamotol, and/or the mixture thereof; wherein steps (a) and (b) are effected simultaneously or sequentially; andstep (b) occurs in the presence of a cytochrome P450 reductase polypeptide or catalytically active portion thereof; and whereinthe cytochrome P450 reductase polypeptide or catalytically active portion thereof is encoded by a nucleic acid molecule, wherein:the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 95% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically active portion thereof; andthe encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof catalyzes the transfer of two electrons from NADPH to an electron acceptor.2. The method of claim 1 , wherein the encoded cytochrome P450 reductase polypeptide or catalytically active portion thereof exhibits at least 96% sequence identity to the cytochrome P450 reductase polypeptide set forth in SEQ ID NO: 13 or a corresponding catalytically ...

Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway

The invention relates to the production of one or more terpenoids through microbial engineering, and relates to the manufacture of products comprising terpenoids.

TERPENOID DERIVATIVES

It is intended to provide a novel terpenoid derivative that exhibits anti-inflammatory action and a cytoprotective action by activating the Keap1/Nrf2/ARE signaling pathway. The present invention provides terpenoid derivative A represented by the following formula (I): 2. A terpenoid derivative having the following physicochemical properties:1) article description: a colorless powdery substance{'sub': 32', '43', '6, '2) molecular formula: CHNO'}3) molecular weight: 537 (measured by ESI mass spectrometry){'sup': '+', '4) the accurate mass, [M+H], measured by high-resolution LC-ESI mass spectrometry is as given belowfound: 538.31549calculated: 538.31631{'sup': 1', '1, '5) H-nuclear magnetic resonance spectra: the H-nuclear magnetic resonance spectra (500 MHz) measured with the signal of TMS defined as 0.00 ppm in deuterated chloroform are as given belowσ: 0.92 (3H, s), 1.08 (3H, s), 1.12 (3H, s), 1.14-1.17 (1H, m), 1.19 (3H, s), 1.22-1.24 (1H, m), 1.18-1.25 (1H, m), 1.28 (3H, s), 1.28 (3H, s), 1.46-1.48 (1H, m), 1.48-1.52 (1H, m), 1.50-1.54 (1H, m), 1.59-1.67 (2H, m), 1.62-1.66 (1H, m), 1.64-1.67 (1H, m), 1.75 (1H, d, J=13.5 Hz), 1.80-1.83 (1H, m), 1.85-1.87 (2H, m), 1.98 (1H, dd, J=9.0 Hz, 4.5 Hz), 2.95 (1H, s), 2.98 (1H, brs), 3.45 (1H, d, J=11.5 Hz), 3.70 (3H, s), 3.73 (1H, d, J=11.5 Hz), 4.34 (1H, s), 6.11 (1H, s) ppm{'sup': 13', '13, '6) C-nuclear magnetic resonance spectra: the C-nuclear magnetic resonance spectra (125 MHz) measured with the signal of TMS defined as 0.00 ppm in deuterated chloroform are as given belowσ: 18.5 (t), 21.1 (q), 21.6 (q), 23.0 (q), 24.0 (q), 24.1 (t), 27.3 (q), 28.2 (q), 28.6 (t), 29.6 (t), 31.0 (d), 31.5 (t), 31.6 (t), 32.4 (t), 35.7 (s), 41.2 (s), 42.3 (s), 42.8 (d), 45.3 (s), 45.8 (s), 47.1 (s), 49.6 (d), 52.3 (q), 53.5 (s), 67.6 (t), 69.3 (d), 113.7 (s), 124.8 (d), 169.8 (s), 178.0 (s), 199.6 (s), 202.5 (s) ppm7) high-performance liquid chromatography: '(4.6 mm in diameter×75 mm in length; manufactured by Imtakt Corp.)', 'column: ...

GERANYLGERANYL PYROPHOSPHATE SYNTHASES

The present invention relates a variant polypeptide having geranylgeranyl pyrophosphate synthase activity, which variant polypeptide comprises an amino acid sequence which, when aligned with a geranylgeranyl pyrophosphate synthase comprising the sequence set out in SEQ ID NO: 1, comprises at least one substitution of an amino acid residue corresponding to any of amino acids at positions 92, 100 or 235 said positions being defined with reference to SEQ ID NO: 1 and wherein the variant has one or more modified properties as compared with a reference polypeptide having geranylgeranyl pyrophosphate synthase activity. A variant polypeptide of the invention may be used in a recombinant host for the production of steviol or a steviol glycoside. 1. A variant polypeptide having geranylgeranyl pyrophosphate synthase activity , which variant polypeptide comprises an amino acid sequence which , when aligned with a geranylgeranyl pyrophosphate synthase comprising the sequence set out in SEQ ID NO: 1 , comprises at least one substitution of an amino acid residue corresponding to any of amino acids at positions92, 100 or 235said positions being defined with reference to SEQ ID NO: 1 and wherein the variant has one or more modified properties as compared with a reference polypeptide having geranylgeranyl pyrophosphate synthase activity.2. A variant polypeptide according to claim 1 , wherein the modified property is modified geranylgeranyl pyrophosphate synthase activity.3. A variant polypeptide according to claim 1 , wherein the reference polypeptide comprises the geranylgeranyl pyrophosphate synthase of SEQ ID NO: 1 or SEQ ID NO: 17.4. A variant polypeptide according to claim 1 , wherein the variant polypeptide is a non-naturally occurring polypeptide.5. A variant polypeptide according to claim 1 , which comprises additional substitutions other than 92 claim 1 , 100 or 235.6. A variant polypeptide according to claim 1 , having at least 70% claim 1 , at least 75% claim 1 , at least ...

MICROBIAL ENGINEERING FOR THE PRODUCTION OF CHEMICAL AND PHARMACEUTICAL PRODUCTS FROM THE ISOPRENOID PATHWAY

The invention relates to the production of one or more terpenoids through microbial engineering, and relates to the manufacture of products comprising terpenoids. 1207. -. (canceled)208. A method for making a pharmaceutical product , the method comprising:{'i': Escherichia coli', 'E. coli, 'providing an () that produces isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through an upstream methylerythritol pathway (MEP) and converts the IPP and DMAPP to amorphadiene or derivative thereof through a recombinantly expressed downstream synthesis pathway comprising farnesyl diphosphate synthase and amorphadiene synthase;'}{'i': 'E. coli', 'culturing the to produce the amorphadiene or derivative thereof, wherein the accumulation of indole in the culture is controlled to below 100 mg/L to thereby increase production of amorphadiene or derivative thereof; and'}incorporating the amorphadiene or derivative thereof, into a pharmaceutical product.209. The method of claim 208 , wherein accumulation of indole in the culture is controlled by balancing the upstream MEP pathway with the downstream terpenoid synthesis pathway.210. The method of claim 208 , further comprising measuring the amount or concentration of indole continuously or intermittently.211. The method of claim 208 , wherein accumulation of indole in the culture is maintained to below 50 mg/L.212. The method of claim 208 , wherein accumulation of indole in the culture is maintained to below 10 mg/L.213. The method of claim 208 , wherein the amorphadiene or derivative thereof is produced at 10 mg/L or more.214. The method of claim 208 , wherein the amorphadiene or derivative thereof is produced at 100 mg/L or more.215E. coli. The method of claim 208 , wherein the has additional copies of one or more of the dxs claim 208 , idi claim 208 , ispD claim 208 , and ispF genes of the MEP pathway.216E. coli. The method of claim 215 , wherein the has a heterologous dxs-idi-ispDF operon.217. The method of ...

HYDROLYSIS OF STEVIOL GLYCOSIDES BY BETA-GLUCOSIDASE

The present disclosure relates to the use of beta-glucosidase to enhance the production efficiency of desired steviol glycosides, such as rebaudioside M (reb M).

MELLEOLIDE-BIOSYNTHESIS GENE CLUSTER AND ITS APPLICATIONS

The present invention relates to the production of hydroxylated protoilludenes and/or sesquiterpenoid protoilludene-type aryl esters using newly identified genes that can be employed. The present invention accordingly relates to a host microorganism that has been transformed with the newly identified nucleotide sequences and to methods employing the transformed microorganism. 1. An isolated polynucleotide encoding a polypeptide with cytochrome-P450 monooxygenase activity , comprising or consisting of:a) one of SEQ ID NOs: 5 to 8;b) a nucleic acid sequence complementary to one of SEQ ID NOs: 5 to 8;c) a nucleic acid sequence which hybridize under stringent conditions to the nucleic acid sequences defined in a) and b) or their complementary strands.2. A vector , comprising a nucleic acid sequence encoding a polypeptide with cytochrome-P450 monooxygenase activity , the nucleic acid sequence being operably linked to control sequences recognized by a host microorganism transformed with the vector.3. The vector of claim 2 , wherein the vector is an expression vector.4. An isolated host microorganism with a transformed with the vector of .5. The isolated host microorganism of claim 4 , wherein the host microorganism is selected from the group consisting of fungi claim 4 , including yeast claim 4 , and bacteria.6. The isolated host microorganism of claim 4 , wherein that the polynucleotide encoding the polypeptide with cytochrome P450-monooxygenase activity is adapted to the codon usage of the host microorganism.7SaccharomycesSaccharomyces cerevisiaeEscherichia coliAspergillus nidulansArmillariaArmillaria gallicaArmillaria. The host microorganism of claim 4 , wherein the host microorganism is a spp. in particular ; an ; an ; a homobasidiomycetes claim 4 , in particular a member of the genus claim 4 , such as or mellea.8ArmillariaArmillaria gallica.. The host microorganism of claim 4 , wherein the host microorganism is further transformed to comprise a protoilludene synthase ...

METHOD FOR PREPARING REBAUDIOSIDE M BY USING SACCHAROMYCES CEREVISIAE ENZYMATIC PROCESS

Provided in the present invention is a method for preparing rebaudioside M by using an enzymatic process, which catalyzes the formation of rebaudioside M using a recombinant containing a UDP-glucosyltransferase or a UDP-glucosyltransferase prepared therefrom. The recombinant is obtained by transforming an expression vector containing the UDP-glucosyltransferase gene under the control of a strong promoter. 113-. (canceled)14Saccharomyces cerevisiae. A method for preparing rebaudioside M , the method comprising reacting rebaudioside D in a reaction solution with a glucosyl donor in the presence of a first UDP-glucosyltransferase or a first recombinant comprising the first UDP-glucosyltransferase ,wherein the first UDP-glucosyltransferase has an amino acid sequence according to SEQ ID NO: 1; and{'i': 'Saccharomyces cerevisiae.', 'wherein the first UDP-glucosyltransferase is expressed from a first pYES2 expression vector comprising a first strong promoter in the first recombinant'}15Saccharomyces cerevisiae. The method of claim 14 , wherein the rebaudioside D is prepared by reacting rebaudioside A in the reaction solution with the glucosyl donor in the presence of a second UDP-glucosyltransferase or a second recombinant comprising the second UDP-glucosyltransferase claim 14 ,wherein the second UDP-glucosyltransferase has an amino acid sequence according to SEQ ID NO: 3; and{'i': 'Saccharomyces cerevisiae.', 'wherein the second UDP-glucosyltransferase is expressed from a second pYES2 expression vector comprising a second strong promoter in the second recombinant'}16Saccharomyces cerevisiaeSaccharomyces cerevisiae. The method of claim 14 , wherein the first recombinant is BY4742.17Saccharomyces cerevisiaeSaccharomyces cerevisiae. The method of claim 15 , wherein the second recombinant is BY4742.18. The method of claim 14 , wherein the first strong promoter and the second strong promoter are each independently ADH2 or TEF1.19. The method of claim 15 , wherein the first ...

Steviol Glycoside Compositions Sensory Properties

Materials and methods for producing particular steviol glycosides, and high-purity compositions of particular steviol glycosides with improved sensory profilesare provided herein. The steviol glycosides can be, for example, rebaudioside D, rebaudioside A, and rebaudioside B, and can be produced by recombinant microorganisms. 1. A composition comprising at least 90% w/w rebaudioside A , said composition having one or more of the following:{'i': 'Stevia', 'a) a statistically significant decrease in a sweetness build score relative to a -derived rebaudioside A composition;'}{'i': 'Stevia', 'b) a statistically significant decrease in an artificial sweetness score relative to a -derived rebaudioside A composition;'}{'i': 'Stevia', 'c) a statistically significant decrease in a bitterness score relative to a -derived rebaudioside A composition; and'}{'i': 'Stevia', 'd) a statistically significant decrease in two-minute acid score relative to a -derived rebaudioside A composition, said scores determined in a standardized sensory panel evaluation.'}2. The composition of claim 1 , wherein said rebaudioside A is produced in a recombinant microorganism.3. The composition of claim 2 , wherein said recombinant microorganism is a yeast.4Saccharomyces cerevisiae.. The composition of claim 3 , wherein said yeast is5. A food product comprising the composition of .6. A method for producing a steviol glycoside product claim 1 , comprising:a) fermenting a recombinant microorganism capable of producing at least 1 g/L of said steviol glycoside in a culture medium or carrying out biocatalysis in a reaction mixture with one or more of the enzymes listed in Sections I-A, I-B, I-C, or I-D of the specification, to produce the steviol glycoside; andb) purifying said steviol glycoside from said culture medium or from said reaction mixture, using one or more purification steps selected from the group consisting of:(i) fractionation on an adsorbent resin;(ii) fractionation on a reversed phase ...

METHOD FOR PREPARING STALLIMYCIN

Provided is a method for preparing stallimycin. The method comprises: fermenting in a fermentation medium comprising a carbon source, a nitrogen source and 3-hydroxy-4-aminobutyric acid, and adding vegetable oil into the fermentation medium during the fermentation. 1streptomyces. A method for preparing stallimycin comprising the step of fermenting that can produce stallimycin in a fermentation medium comprising an available carbon source , an available nitrogen source and 3-hydroxy-4-aminobutyric acid , and the step of adding vegetable oil into the fermentation medium during the fermentation.2streptomycesStreptomyces distallicusStreptomyces distallicusStreptomyces distallicus. The method according to claim 1 , wherein the is selected from the group consisting of NRRL NO. 2886 claim 1 , D32 or DZ206.3. The method according to claim 1 , wherein the weight concentration of the 3-hydroxy-4-aminobutyric acid in the fermentation medium is 0.005%-0.05%.4. The method according to claim 1 , wherein during the fermentation means at the time when the fermentation has been conducted for 48-120 hours.5. The method according to claim 1 , wherein the step of adding vegetable oil comprising adding vegetable oil in more than one addition claim 1 , the interval between the additions of vegetable oil is once every 24 hours.6. The method according to claim 5 , wherein the weight ratio of the amount of vegetable oil added for each addition to the fermentation medium is 0.3-1.0%; the weight ratio of the total amount of the added vegetable oil to the fermentation medium is 1-4%.7. The method according to claim 1 , wherein the vegetable oil is selected from the group consisting of rapeseed oil claim 1 , sunflower oil claim 1 , peanut oil claim 1 , soybean oil claim 1 , olive oil or the mixtures thereof.8. The method according to claim 1 , wherein the weight concentration of the available carbon source in the fermentation medium is 4-10%; the weight concentration of the available nitrogen ...

STREPSESQUITRIOL, PREPARATION METHOD THEREOF, AND APPLICATION THEREOF

The invention discloses a new strepsesquitriol A, a preparation method thereof and an application thereof. Strepsesquitriol A, a structure as shown in Formula (I), is a compound having a new skeleton and strongly inhibiting the formation of LPS-induced TNFα but showing no cytotoxic activity, so it can be used for the preparation of anti-inflammatory drug or act as a precursor of anti-inflammatory drugs, for the treatment of multiple inflammations. Therefore, the invention provides a new candidate compound for the development of anti-inflammatory drugs, and is of great significance for developing Chinese marine drug resources. 2Streptomyces. A method for preparing the strepsesquitriol of claim 1 , wherein said strepsesquitriol is separated from a fermentation culture of sp. SCSIO 10355.3. The preparation method of claim 2 , wherein the strepsesquitriol is specifically prepared by the following steps:{'i': 'Streptomyces', '(a) preparing a fermentation culture of sp. SCSIO 10355;'}(b) separating a fermentation liquid fraction of the fermentation culture from a mycelia-containing fraction thereof, extracting the fermentation liquid with ethyl acetate, thereby forming a first ethyl acetate extract; performing concentration of the first ethyl acetate extract to obtain an extract A; leaching the mycelia from the mycelia-containing fraction of the fermentation liquid with ethanol to form a mycelia leaching solution, and recovering the ethanol from the mycelia leaching solution wherein an aqueous solution remains after said recovering of ethanol; extracting the aqueous solution with ethyl acetate, thereby forming as second ethyl acetate extract; performing concentration of the second ethyl acetate extract to obtain an extract B; mixing the extract A and the extract B to obtain a crude extract;(c) subjecting the crude extract to medium-pressure liquid chromatography over octadecylsilyl silica gel and performing a gradient elution with water:methanol as an eluent in a volume ...

CYTOCHROME P450 AND CYTOCHROME P450 REDUCTASE POLYPEPTIDES, ENCODING NUCLEIC ACID MOLECULES AND USES THEREOF

Provided are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and/or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells. 1. A host cell , comprising a nucleic acid molecule encoding a cytochrome P450 oxidase polypeptide or a catalytically active portion thereof , wherein:(a) the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof exhibits at least 85% sequence identity to a P450 oxidase polypeptide set forth in SEQ ID NO:6, 7, 8, 9, 50, 74, 75, 76, or 77, or a corresponding catalytically active portion thereof;(b) the encoded cytochrome P450 oxidase or catalytically active fragment thereof catalyzes hydroxylation or monooxygenation of santalene and/or bergamotene; and(c) the nucleic acid molecule is heterologous to the host cell.2. The host cell of claim 1 , wherein the encoded cytochrome P450 oxidase polypeptide exhibits at least 95% sequence identity to the cytochrome P450 oxidase polypeptide set forth in SEQ ID NO:6 claim 1 , 7 claim 1 , 8 claim 1 , 9 claim 1 , 50 claim 1 , 74 claim 1 , 75 claim 1 , 76 claim 1 , or 77.3Santalum album. The host cell of claim 1 , wherein the encoded cytochrome P450 oxidase polypeptide or catalytically active portion thereof is a P450 oxidase polypeptide.4. The host cell of claim 1 , wherein the cytochrome P450 oxidase polypeptide or catalytically active fragment catalyzes formation of a santalol from a santalene claim 1 , or a bergamotol from a bergamotene.5. The host cell of claim 1 , wherein the nucleic acid molecule comprises a sequence of nucleotides selected from among:(a) a sequence of nucleic acids set forth in any of SEQ ID NO:2, 3, 4, 5, 67, 68, 69, 70, or 71;(b) a ...

Production of steviol glycoside in recombinant hosts

The invention relates to recombinant microorganisms and methods for producing steviol glycosides, glycosylated ent-kaurenol, and glycosylated ent-kaurenoic acid.

ENZYMATIC METHOD FOR PREPARING REBAUDIOSIDE C

Provided is a method for preparing Rebaudioside C using an enzymatic method, comprising using rubusoside or dulcoside A as a substrate, and making the substrate, in the presence of a glycosyl donor, react under the catalysis of UDP-glycosyltransferase-containing recombinant cell and/or UDP-glycosyltransferase prepared therefrom to generate Rebaudioside C. 1. A method for preparing Rebaudioside C using an enzymatic method , wherein in the method , dulcoside A or rubusoside is used as a substrate; and in the presence of a glycosyl donor , Rebaudioside C is produced by means of a reaction under the catalysis of recombinant cells containing UDP-glycosyltransferase and/or UDP-glycosyltransferase prepared therefrom.2. (canceled)3. The method according to claim 1 , wherein the glycosyl donor comprises one or two of glucosyl donor and rhamnosyl donor; the glucosyl donor is UDP-glucose or a UDP-glucose regeneration system consisting of sucrose claim 1 , sucrose synthase claim 1 , and UDP; and the rhamnosyl donor is UDP-rhamnose.4Stevia rebaudianaOryza sativa.. The method according to claim 1 , wherein the UDP-glucosyltransferase comprises one or two of UGT-A from and UGT-B from5Stevia rebaudiana. The method according to claim 1 , wherein the UDP-glucosyltransferase is a UGT-A from claim 1 , and an amino acid sequence of the UGT-A is at least 60% consistent with Sequence 2 as shown in the Sequence Listing.6. The method according to claim 5 , wherein the amino acid sequence of the UGT-A is at least 70% consistent with Sequence 2 as shown in the Sequence Listing.7. The method according to claim 6 , wherein the amino acid sequence of the UGT-A is at least 80% consistent with Sequence 2 as shown in the Sequence Listing.8. The method according to claim 7 , wherein the amino acid sequence of the UGT-A is at least 90% consistent with Sequence 2 as shown in the Sequence Listing.9Stevia rebaudianaOryza sativa. The method according to claim 1 , wherein the UDP-glucosyltransferase ...

METHOD OF MANUFACTURING TRANSFRUCTOSYLATION STEVIOL GLYCOSIDES USING THE LACTOBACILLUS MALI

The present disclosure relates to a method for preparing a transglucosylated steviol glycoside using a crude enzyme liquid of a strain. 1Lactobacillus mali. A method for preparing a transglucosylated steviol glycoside using a microorganism or a culture thereof.2Lactobacillus mali. The method according to claim 1 , wherein the method comprises contacting a steviol glycoside with sugar in presence of the microorganism or culture thereof.3. The method according to claim 2 , wherein the steviol glycoside is one or more selected from the group consisting of Stevioside claim 2 , Rubusoside claim 2 , Dulcoside A claim 2 , Rebaudioside A claim 2 , Rebaudioside C claim 2 , Rebaudioside D claim 2 , Rebaudioside E claim 2 , Rebaudioside F claim 2 , and Rebaudioside M.4. The method according to claim 2 , wherein contacting the steviol glycoside with the sugar is carried out at a pH of 3 to 8 at a temperature of 10° C. to 60° C.5. The method according to claim 1 , wherein the transglucosylated steviol glycoside is a steviol glycoside in which glucose is added by linking via a glucose linked to a 19-OH site of the steviol glycoside via an α-(1 claim 1 ,6) bond.6. The method according to claim 5 , wherein the transglucosylated steviol glycoside comprises 1 to 4 glucose molecules.7. A transglucosylated steviol glycoside prepared via the method according to .8. The transglucosylated steviol glycoside according to claim 7 , wherein the transglucosylated steviol glycoside is one or more selected from the group consisting of transglucosylated Stevioside claim 7 , transglucosylated Rubusoside claim 7 , transglucosylated Dulcoside A claim 7 , transglucosylated Rebaudioside A claim 7 , transglucosylated Rebaudioside C claim 7 , transglucosylated Rebaudioside D claim 7 , transglucosylated Rebaudioside E claim 7 , transglucosylated Rebaudioside F claim 7 , and transglucosylated Rebaudioside M.9Lactobacillus mali. A composition for producing the transglucosylated steviol glycoside claim 7 , ...

BIOSYNTHETIC PRODUCTION OF STEVIOL GLYCOSIDE REBAUDIOSIDE I VIA VARIANT ENZYMES

The present invention relates, at least in part, to the production of steviol glycoside rebaudioside I through the use of variant UGT enzymes having activity to transfer a glucosyl group from UDP-glucose to rebaudioside A to produce rebaudioside I. 2. The method of claim 1 , wherein the steviol glycoside composition is stevia extract.3. The method of claim 1 , further comprising adding a sucrose synthase to the reaction mixture.4Arabidopsis thaliana. The method of claim 3 , wherein the sucrose synthase is an sucrose synthase 1 (AtSUS1) comprising the amino acid sequence of SEQ ID NO: 11.5. The method of claim 1 , wherein the reaction mixture is in vitro.6. The method of . wherein the reaction mixture is a cell-based reaction mixture.7. The method of claim 6 , wherein the UDP-glycosyltransferase enzyme is expressed in a host cell.8. The method of claim 7 , wherein the host cell is selected from the group consisting of a yeast claim 7 , a non-steviol glycoside producing plant claim 7 , an alga claim 7 , a fungus claim 7 , and a bacterium.9. The method of claim 7 , wherein the host cell is a bacterial cell.10E. coli. The method of claim 9 , wherein the bacterial cell is an cell.11. The method of claim 7 , wherein the host cell is a yeast cell.12. The method of claim 1 , wherein the subject is UDP-glucose.13. The method of claim 1 , wherein the rebaudioside A has a concentration of 15 to 50 g/L in the reaction mixture.14. The method to claim 1 , wherein the reaction mixture has a range of 6.5 to 9.5 at a temperature of 35° C. to 45° C.15. The method of claim 1 , further comprising isolating crude rebaudioside I.16. The method of claim 15 , further comprising crystallizing the crude rebaudioside I to obtain rebaudioside I with a purity of greater than 98%.17. A UGT76G1 mutant comprising a L200A mutation relative to SEQ ID NO: 9.18. The UGT75G1 mutant of claim 18 , comprising the amino acid sequence of SEQ ID NO: 1. This application is a continuation-in-part of ...

Способ получения таксола и таксанов из эмбриональных культур растений taxus

В изобретении предложены способы получения таксола и его производных (таксанов) путем экстракции тканей различных видов тисса (Taxus) или полученных из них клеток. Способ получения таксола или его производных путем экстракции тканей и/или культурной среды характеризуется тем, что указанные ткани различных видов тисса представляют собой зиготные эмбрионы. Клеточные культуры зиготных эмбрионов различных видов тисса используют для индукции соматических эмбрионов или эмбриогенного каллуса.

manufacture of certain cyclic ester oligomers

Enzymes which are capable of catalyzing esterifications and/or transesterifications such as selected lipases and esterases can, under specified conditions, convert certain lower linear oligomers of polyesters to their cyclic ester oligomers in quantities greater than would be predicted by thermodynamic calculation or prior art methods. The cyclic ester oligomers are useful for the production of higher molecular weight linear polyesters.

一种酶法制备环氧烷烃的工艺

本发明涉及环氧烷烃制备领域，公开了一种酶法制备环氧烷烃的工艺，解决了传统制备方法中使用大量的有机溶剂，生产过程中存在较大安全风险，对环境污染较大等问题，其技术方案要点是在烯烃中加入氧化剂、催化剂以及助剂，在35℃~65℃下混合搅拌反应，再进行过滤分层，将分层得到的有机层进行减压蒸馏后得到1,2‑环氧烷烃，助剂包括长链脂肪酸，达到不采用有机溶剂，便可以高效的制备出环氧烷烃的效果。

Method for producing alpha-santalene

Heterologous production of patchoulol, beta-santalene, and sclareol in moss cells

The present invention relates to methods for preparing patchoulol, β-santalene, and sclareol in transgenic moss cells comprising heterologous nucleic acid molecules encoding a polypeptide or synthasecapable of using FPP or GGPP as substrate, methods for producing the transgenic moss cell, as well as the transgenic moss cell itself.

Method for producing alpha- santalene

The present invention provides a method of producing α-santalene, said method comprising contacting at least one polypeptide with farnesyl phyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce α-santalene, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing α-santalene is also an object of the present invention.

Method for producing taxol and its derivatives from embryo cultures of taxus species

The taxol production method using somatic cell culture consists of (1) culturing seeds of Taxus cuspidata to form callus, (2) isolating somatic cells from callus in hormone free medium, (3) culturing somatic cells in nicotinic acid 1-2 ppm containing medium, (4) adding phenylalanine, gibberelline, pestalotiopsis sp. which is contaminated on Taxus tree to produce taxol in 4.1-6.3 mg/l.

Steviol glycoside transport

The disclosure provides a recombinant cell capable of producing a steviol glycoside, wherein the cell comprises a nucleic acid coding for a variant of a parent polypeptide, wherein the variant has steviol glycoside transport mediating activity, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence of the parent polypeptide, comprises at least one modification of the amino acid residue corresponding to any of the amino acids in the amino acid sequence of the parent polypeptide, wherein the variant has an improved ability to produce rebaudioside M and optionally other steviol glycosides extracellularly if compared with the parent polytpeptide when measured under the same conditions.

Mass production of taxol by changing temperature during the plant cell culture

PURPOSE: Provided is a method for mass producing taxol by changing incubation temperature during the incubation of plant cells. Therefore, taxol can be simply mass produced in higher yield. CONSTITUTION: The method for mass producing taxol comprises the steps of: incubating Taxus sp. plant cells in a growth medium at 20 to 25 deg. C; and then incubating Taxus sp. plant cells at 26 to 32 deg. C during log growth phase of Taxus sp. plant cells. Wherein, the plant cells reach the log growth phase about 10 to 20 days after incubation, the carbon source is added into the fermenting medium of Taxus sp. plant cells in an amount of 10 to 100g/l more than one time, 5 to 30 days after incubation, the Taxus sp. plant cells are inoculated in an amount of 4 to 10 dried weight g/l, and the growth medium contains sugar in an amount of 40 to 100 g/l.

Mass production of paclitaxel by changing the medium temperature during cultivation of plant cells

Methods and compositions

The present invention relates to a biosynthetic route to intermediates of the QS-21 molecule, as well as routes to make the QS-21 molecule, enzymes involved, the products produced and uses of the product.

Method of producing taxol and taxones from embryo plant culture of taxus genus

FIELD: biotechnology. SUBSTANCE: method involves using zygote embryos from yew seeds of species Taxus cuspidata as explants. Callus tissue is cultured up to formation of somatic embryos and then to embryogenic callus preparing. The latter is passaged in liquid nutrient medium and cultured on Hamburg medium mB 5 containing 2-4 ppm 2,4-D (2,4-dichlorophenoxyacetic acid) for cellular mass increase. Then culturing is continued on Murasige-Skoog Ms or Hamburg mB 5 medium containing 1-2 ppm NAA (α--naphthylacetic acid) that stimulates the yield of the end product. The end product is isolated by extraction from somatic embryos at stage of callus culturing or from embryogenic callus at stage of disinfected embryos culturing. The end product can be isolated from cellular suspension mass and cultural fluid. EFFECT: increased yield of taxol and taxones. 7 cl, 7 dwg, 3 tbl, 11 ex ОтДОСКсС ПЧ ГЭ РОССИЙСКОЕ АГЕНТСТВО ПО ПАТЕНТАМ И ТОВАРНЫМ ЗНАКАМ (19) ВИ "” 2120 740 ' 13) СЛ ОМК АОлН 4/00, С 12 М 5/00 12) ОПИСАНИЕ ИЗОБРЕТЕНИЯ К ПАТЕНТУ РОССИЙСКОЙ ФЕДЕРАЦИИ (21), (22) Заявка: 95109640/13, 06.07.1994 (30) Приоритет: 06.07.1993 КК 1993-12641 20.06.1994 КВ 1994-13914 (46) Дата публикации: 27.10.1998 (56) Ссылки: МО 92/13961 АЛ, 20.08.92. МО 93/19585 АЛ, 14.10.93. (71) Заявитель: Форест Генетик Рисеч Инститют Форести, Администрейшн (КК) (72) Изобретатель: Ли, Бо-Сик (КК), Сан Санг-Хо (КК) (73) Патентообладатель: Форест Генетик Рисеч Инститют Форести, Администрейшн (КК) (54) СПОСОБ ПОЛУЧЕНИЯ ТАКСОЛА И ТАКСАНОВ ИЗ ЭМБРИОНАЛЬНЫХ КУЛЬТУР РАСТЕНИЙ РОДА ТАХО$ (57) Реферат: Изобретение относится к биотехнологии и предназначено для получения таксола в промышленном масштабе. Способ предусматривает использовать в качестве эксплантов вычлененные зиготные эмбрионы из семян Тисса вида Тахиз сизраав. Культивируют каллусную ткань до образования соматических эмбриоидов, затем до получения эмбриогенного каллуса. Последний пассируют в жидкую питательную среду и культивируют на среде Гамборга т В 5, ...

Method for preparing 2,3-epoxy pinane through enzymatic reaction

本发明涉及一种酶促反应制备2,3-环氧蒎烷的方法，包括以下步骤：取降冰片烯、双氧水和氯过氧化物酶溶液加入磷酸缓冲溶液，混合形成反应体系进行反应，在反应体系中，降冰片烯、H 2 O 2 、氯过氧化物酶和磷酸缓冲溶液的摩尔比为(0.005～0.015)umol：(10～20)umol：(12～30)×10 -6 umol：(2～3)mL。本发明通过CPO作为催化剂，双氧水作为氧化剂，催化高级烯烃——降冰片烯进行环氧化反应合成2,3-环氧蒎烷，操作简单、快速高效、酶及氧化剂用量极少、反应温度较低、产率较高、选择性高，本发明采用酶促反应制备2,3-环氧蒎烷，克服了现有方法在有机溶剂中进行的问题，绿色环保。

Process for producing optically active 3-phenylglycidate compounds

Semi-continuous culture method of Taxus plant cells

본 발명은 택솔의 대량생산을 위한 택서스속 식물세포의 반연속식 배양방법에 관한 것이다. 본 발명의 반연속적 배양방법은 택서스속 식물세포의 배양도중 당을 첨가하여 거듭 배양한 다음, 배양액의 일부를 새로운 세포 배양배지로 재희석하고 다시 배양하여 택솔 생산의 새로운 사이클이 시작되도록 하고, 나머지 배양액은 다른 발효조로 옮겨 다시 당을 첨가하고 배양함으로써, 택솔 생산 식물세포를 배양하는 방법인다. 본 발명의 반연속식 배양법을 이용하여 택서스속 식물세포를 배양하면, 미생물에 의한 오염을 방지하면서 택속을 생산하는 식물세포를 고수율로 배양할 수 있다.

VARIABLE DOMAINS OF LIGHT AND HEAVY CHAIN OF THE MOUSE MONOCLONAL ANTIBODY AGAINST INTERFERON ALPHA (IFN-α) HUMAN ANTIGEN BINDING FRAGMENTS (FAb) AGAINST HUMAN IFN-α COMPRISING SAID DOMAINS

Изобретение относится к области биохимии, в частности к вариабельным доменам тяжелой и легкой цепей мышиного антитела против интерферона альфа (IFN-α) человека. Также раскрыт антигенсвязывающий фрагмент (Fab), включающий указанные вариабельные домены. Изобретение позволяет эффективно связываться с IFN-α человека. 3 н.п. ф-лы, 7 ил., 7 табл., 6 пр. РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) (13) 2 694 672 C1 (51) МПК C07K 16/24 (2006.01) C12N 15/00 (2006.01) C12P 21/08 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ОПИСАНИЕ ИЗОБРЕТЕНИЯ К ПАТЕНТУ (52) СПК C07K 16/24 (2018.08); C12P 15/00 (2018.08) (21)(22) Заявка: 2018116027, 27.04.2018 (24) Дата начала отсчета срока действия патента: Дата регистрации: 16.07.2019 (45) Опубликовано: 16.07.2019 Бюл. № 20 Адрес для переписки: 196158, Санкт-Петербург, Московское ш., 10, кв. 184, Цветковой Елене Олеговне 2 6 9 4 6 7 2 C 1 (56) Список документов, цитированных в отчете о поиске: RU 2431638 C2 (БАНШЕРО Ж. и др. [US]), 20.10.2011. RU 2499000 C1, 20.11.2013. HUANG T-H and et.al., Targeting IFN-α to B Cell Lymphoma by a Tumor-Specific Antibody Elicits Potent Antitumor Activities, The Journal of Immunology, 2007, n.179, pp.6881-6888. US 20170202962 A1, 20.07.2017. WO 2017120501 A1, 13.07.2017. (54) Вариабельные домены лёгкой и тяжёлой цепи мышиного моноклонального антитела против интерферона альфа (IFN-α) человека, антигенсвязывающий фрагмент (Fab) против IFN-α человека, содержащий указанные домены (57) Реферат: Изобретение относится к области биохимии, раскрыт антигенсвязывающий фрагмент (Fab), в частности к вариабельным доменам тяжелой и включающий указанные вариабельные домены. легкой цепей мышиного антитела против Изобретение позволяет эффективно связываться интерферона альфа (IFN-α) человека. Также с IFN-α человека. 3 н.п. ф-лы, 7 ил., 7 табл., 6 пр. Стр.: 1 C 1 R U (73) Патентообладатель(и): Открытое акционерное общество "Всероссийский научный Центр молекулярной диагностики и лечения" (ОАО ВНЦМДЛ) (RU) 2 6 9 4 6 ...

Enhanced production of taxanes by cell cultures of taxus species

본 발명은 모든 공지의 탁수스 종, 예를 들면, 브레비폴리아(brevifolia), 카나덴시스(canadensis), 쿠스피다타(cuspidata), 바카타(baccata), 글로보사(globosa), 플로리다나(floridana), 왈리치아나(wallichiana), 메디아(media) 및 키넨시스(chinensis)로부터 매우 고수율로 탁솔, 바카틴 III 및 기타 탁솔 유사 화합물을 생산할 수 있는 방법을 제공한다. 배양 조건(즉, 배지 조성 및 조작 방식)의 특정의 변형은 모든 탁수스종의 세포 배양으로부터 다양한 탁산의 수율을 증가시키는 것으로 발견되었다. 특히 바람직한 증강제로는 은 이온 또는 은 착물, 자스몬산(특히 그 메틸 에스테르), 옥신 관련 성장 조절제 및 페닐프로파노이드 경로의 억제제(예; 3,4-메틸렌디옥시-6-니트로신남산)가 있다. 이들 증강제는 단독으로 사용하거나 서로 또는 다른 수율 증가 조건을 조합하여 사용할 수 있다. 탁수스 키넨시스의 식물 세포 배양으로부터의 탁산의 수율이 이들 조건의 하나 이상을 사용하여 특히 증가되는 반면에, 탁수스종에 대한 탁산의 수율은 이들 조건의 사용으로부터 이점을 얻을 수 있는 것으로 확인되었다.

Method for mass production of taxol from Taxus plants

(57)【要約】 本発明は、イチイ属植物の細胞培養物からタキソールを高い純度と回収率で大量生産するための迅速で簡単な方法に関する。本発明によるイチイ属植物からのタキソールの大量生産方法は、（i）イチイ属植物からのバイオマスを有機溶媒で抽出して粗製抽出物を得て、（ii）この粗製抽出物を合成吸着剤で処理し、濾過して濾液を得て、（iii）ヘキサンをこの濾液に加えて粗製タキソールを沈殿させ、（iv）この粗製タキソールをアルコールと水の混合物中で分別沈殿し、この沈殿物を真空乾燥してタキソール粉末を得て、そして（v）このタキソール粉末を高速液体クロマトグラフィーにかける上記の各ステップを含む。本発明の方法によれば、９９％を超える純度のタキソールがイチイ属植物から９０％を超える高い回収率で簡単に得ることができる。

(-) (cis-1,2-epoxypropyl) phosphonic acid

Process for introducing oxygen into 1-amidoadamantanes

New colchicines and their process for obtaining

NEW HYDROXYL PATCHOULOLS

L'invention concerne de nouveaux dérivés de patchoulol hydroxylé. Les composés de l'invention répondent à la formule : The invention relates to novel hydroxylated patchoulol derivatives. The compounds of the invention correspond to the formula:

(-)cis-1,2-epoxypropyl phosphonic acid anti- - biotic prodn by aerobic culture of strep

The cpd. is prepd. by aerobic culture of a microorganism which produces it, pref. a Streptomyces, in aqs. medium until the medium shows substantial antibiotic activity; a phosphorus cpd. is added to the medium to stimulate the prodn. of the antibiotic. The P content of the medium is pref. adjusted to exceed 0.04 g./l.; optimum concn. is 0.10-1.0 g./l. medium. Strains of S. fradiae, S. wedmorensis or S. viridochromogenes are suitable fementation organisms. The products are active against bacteria which are resistant to other antibiotics.

A method of producing a taxane-type diterpene and method of obtaining cultured cells which produce the taxane-type diterpene at a high rate

This invention relates to a method of producing a taxane-type diterpene(s) wherein tissues or cells of a plant which produces taxane-type diterpene(s) is cultured in the presence of at least one selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents, a method of producing a taxane-type diterpene wherein the tissues or the cells of the plant are cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture, a method of producing a taxane-type diterpene wherein the tissue or the cell of the plant is cultured in a culture vessel, while oxygenic gas containing 0.03 - 10 % of carbon dioxide is used as aeration gas to be introduced to the vessel, and a method of obtaining highly productive cultured cells for the taxane-type diterpene wherein cultured cells of the plant which produces the taxane-type diterpene are separated into a plurality of layers according to the difference in their specific gravities, and the cells contained in at least one layer are cultured, then highly productive cultured cells for the taxane-type diterpene are selected from among those cultured cells. The present invention allows industrial production of a taxane-type diterpene such as taxol which is useful as a therapeutic agent for cancer.

Process for the preparation of c-4 deacetyltaxanes

A process useful for the preparation of intermediates in synthesis or semi- synthesis of paclitaxel analogs wherein a starting taxane such as 10- deacetylbaccatin III is deacetylated at the C-4 position using a microorgani sm or an enzyme derived therefrom to provide 4-deacetyltaxanes, such as 4,10- dideacetylbaccatin III.

Mass production of paclitaxel by changing the temperature of the medium during the plant cell culture

The present invention relate to a method to increase the production level of paclitaxel by changing the temperature during the plant cell culture. According to the present invention, the production of the paclitaxel compris es the following procedure: (i) cultivating the Taxus genus plant cells in a medium at ca. 20 to 25 ~C; and (ii) when growth of the plant cells has progressed sufficiently, changing the cultivation temperature to ca. 26 to 3 2 ~C to continue the culture. The present invention comprises also the method of increasing the paclitaxel production by inoculating the cells at a high initial concentration and by increasign the saccharide concentration in the medium. According to the present invention, paclitaxel can be mass-produced conveniently and therefore has an industrial application.

Process for preparing an alpha chromomycin

Mass production of paclitaxel by changing temp. of medium during plant cell culture

本发明涉及一种生产紫杉醇的方法，其中在植物细胞培养期间通过改变温度增加紫杉醇的产量。依据本发明，紫杉醇的生产包括以下步骤：(i)在培养基中于约20至25℃下培养红豆杉属植物细胞；和(ii)当植物细胞充分生长后，将培养温度变为约26至32℃并继续培养。本发明还包括一种增加紫杉醇产量的方法，其中以高初始浓度接种细胞并增加培养基中糖浓度。依据本发明，可方便地大量生产紫杉醇，并因而具有工业应用价值。

Alpha-santalene synthetase, gene and application

本发明公开了一种α‑檀香烯合成酶、基因及应用。所述α‑檀香烯合成酶来源于温郁金(Curcuma wenyujin Y.H.Chen et C.Ling)，氨基酸序列为SEQ ID NO.2所示。本发明对温郁金来源的α‑檀香烯合成酶进行功能验证并在大肠杆菌中进行α‑檀香烯合成方法的发明与应用。发现温郁金来源的α‑檀香烯合成酶可在原核表达体系中催化FPP生成大量的α‑檀香烯、以及少量的β‑檀香烯和α‑法尼烯，根据峰面积计算α‑檀香烯的含量达到了90％以上，从而在大肠杆菌中快速，大量地制备所述的催化剂，并应用于α‑檀香烯的合成，进一步可应用于α‑檀香醇的合成，具有重要的意义。

Method for producing α-santalene

The present invention provides a method of producing α-santalene by contacting at least one polypeptide with farnesyl phyrophosphate (fpp). In particular, the method may be carried out in vitro or in vivo to produce α-santalene, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing the nucleic acid represent part of the present invention. A non-human host organism and a cell transformed to be used in the method of producing α-santalene are also part of the present invention.

Cytochrome P450 and cytochrome P450 reductase polypeptides, encoding nucleic acid molecules and uses thereof

Provided are cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and/or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells.

PROCESS FOR THE PREPARATION OF PATCHOULOL DERIVATIVES

L'invention concerne la synthèse de dérivés de patchoulol. Il s'agit d'un procédé pour l'hydroxylation de patchoulol, caractérisé en ce que le patchoulol est hydroxyle dans des conditions aérobies aqueuses en présence d'un micro-organisme oxydant. Ce procédé ouvre la voie à un procédé synthétique partiel de synthese du norpatchoulénol à partir du patchoulol relativement abondant. The invention relates to the synthesis of patchoulol derivatives. This is a process for the hydroxylation of patchoulol, characterized in that the patchoulol is hydroxylated under aqueous aerobic conditions in the presence of an oxidizing microorganism. This process opens the way to a partial synthetic process for the synthesis of norpatchoulenol from relatively abundant patchoulol.

Antibiotics

An antibiotic substance designated danomycin is produced by cultivating a danomycin-producing strain of Streptomyces albaduncus (A.T.C.C. 14698) in a liquid medium containing a glyceride oil or carbohydrate, and a nitrogenous nutrient, under submerged aerobic conditions until substantial activity versus Gram-positive bacteria is imported to the solution. The danomycin may be removed from the solution by methods such as adsorption on active carbon, precipitation by addition of acetone or extraction by organic solvents. Danomycin is soluble in water and insoluble in acetone, is reddish orange in colour, exhibits negative ninhydrin, Tollens and Fehling reactions, melts with decomposition at 135-138 DEG C., exhibits in water an ultraviolet absorption spectrum having maxima at 270mm (E1%1 cm.=48), 325 mm (E1%1 cm=12.6) and 430 mm (E11 cm.=15.6), is found by analysis to contain C, 48.82%; H 7.05%; N, 7.81%; Fe, 3.13% and O (by difference) 33.19%; and when pelleted in potassium bromide exhibits characteristic absorption bands in the infra-red region of the spectrum at 3200-3280, 2920, 1720-1725, 1630-1640, 1570-1580, 1495-1500, 1455-1470, 1220-1230, 1160-1165, 1010-1040, 865-880, 810, 755 and 680-690 cm-1. Pharmaceutical compositions comprise danomycin and a suitable pharmaceutical carrier. Pharmaceutical compositions comprise danomycin and a suitable pharmaceutical carrier.

Method for producing rebaudioside D by one-pot method

本发明公开了一锅法生产莱鲍迪苷D的方法，包括如下步骤：构建能分泌表达糖基转移酶UGT1的重组菌1，所述糖基转移酶UGT1的氨基酸序列如SEQ ID NO.1所示；在pH3.5‑7.8的培养基中培养重组菌1,得到培养液；在培养液中加入底物莱鲍迪苷A，加入尿苷二磷酸葡萄糖，硫酸镁或氯化镁，甲醇，反应，得到莱鲍迪苷D。本发明的一锅法生产莱鲍迪苷D在重组菌1发酵生成糖基转移酶UGT1的同时可催化莱鲍迪苷A生成莱鲍迪苷D，无需纯化UGT1和离心分离就能获得莱鲍迪苷D的。本发明的方法简单、高效、可以降低分离糖基转移酶UGT1的生产成本，可以商业化获得甜度高、口感好、高价值的莱鲍迪苷D。

Method for obtaining high-cultured taxane-type diterpene-producing cells

Process for preparing doxorubicin

The present invention is directed to a process for improving daunorubicin and doxorubicin production by means of a recombinant microorganism in which a gene of daunorubicin metabolism is mutated. The mutated gene is preferably dnrU and/or dnrX.

Patent BE634041A

Process for the preparation of (-)-(2R,3S)-2,3-epoxy-3-(4-methoxyphenyl)methylpropionate

Enhanced production of taxanes by cell cultures of taxus species

This invention provides methods whereby taxol, baccatin III, and other taxol-like compounds, or taxanes, can be produced in very high yield from all known Taxus species, e.g. brevifolia, canadensis, cuspidata, baccata, globosa, floridana, wallichiana, media and chinensis . Particular modifications of culture conditions (i.e. media composition and operating modes) have been discovered to enhance the yield of various taxanes from cell culture of all species of Taxus. Particularly preferred enhancement agents include silver ion or complex, jasmonic acid (especially the methyl ester), auxin-related growth regulators, and inhibitors of the phenylpropanoid pathway, such as 3,4-methylenedioxy-6-nitrocinnamic acid. These enhancement agents may be used alone or in combination with one another or other yield-enhancing conditions. While the yield of taxanes from plant cell culture of T. chinensis is particularly enhanced by use of one or more of these conditions, yield of taxanes for all Taxus species has been found to benefit from use of these conditions.

Production of steviol glycosides in recombinant hosts

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.

The method that Antrodia camphorata liquid fermentation process quickly characterizes triterpenoid changes of contents

本发明公开了牛樟芝液体发酵过程快速表征三萜类化合物含量变化的方法，属于微生物发酵领域。本发明方法为牛樟芝液体发酵中，通过快速在线或者离线检测分析一种挥发性芳香物质α‑松油醇含量，进而能够快速预测判定三萜类化合物含量变化的分析方法，实现了发酵过程的自动化控制。根据在线实时参数的变化进行发酵过程的三萜类化合物预测分析增加了发酵过程的可控性和生产可预测性。这对于开发利用兼具多种生物活性的牛樟芝产品以及对其在工业生产上的应用具有十分重要的意义。