METHOD FOR PRODUCING MONOTERPENOID COMPOUNDS

The invention relates to enzymes and methods for producing a monoterpenoid compound. In one aspect, the invention is a method for producing a monoterpenoid compound, comprising the steps of (1) providing a monoterpenoid precursor; (2) providing a NEPS enzyme; and (3) contacting the monoterpenoid precursor with the enzyme under catalytic conditions to produce an monoterpenoid compound.

Recombinant microorganism metabolizing 3,6-anhydride-L-galactose and a use thereof

The present invention relates to a recombinant microorganism metabolizing 3,6-anhydro-L-galactose and a use thereof, and, more particularly, can produce ethanol from a recombinant microorganism expressing an enzyme group involved in a metabolic pathway of 3,6-AHG.

STEREO-SPECIFIC SYNTHESIS OF (13R)-MANOYL OXIDE

The present invention relates to a method for manufacturing enantiomerically pure (13R)-manoyl oxide, said method comprising the steps of contacting geranylgeranyl diphosphate (GGPP) with a class II diterpene synthase to obtain labd-13-en-8,15-diol diphosphate (LPP), and then contacting the LPP with a class I diterpene synthase to obtain (13R)-manoyl oxide. The invention further relates to (13R)-manoyl oxide obtained by the method of the invention.

Method

Recombinant microorganisms metabolizing 3,6-anhydro-L-galactose and use thereof

... 본 발명은 3,6-안하이드로-L-갈락토오스를 대사하는 재조합 미생물 및 이의 용도에 관한 것으로, 보다 상세하게는 3,6-AHG의 대사 경로에 관여하는 효소 군을 발현하는 재조합 미생물로부터 에탄올을 제조할 수 있다.

RECOMBINANT MICROORGANISM METABOLIZING 3,6-ANHYDRIDE- L-GALACTOSE AND A USE THEREOF

The present invention relates to a recombinant microorganism metabolizing 3,6-anhydro-L-galactose and a use thereof, and, more particularly, can produce ethanol from a recombinant microorganism expressing an enzyme group involved in a metabolic pathway of 3,6-AHG. 1. A recombinant vector for producing ethanol , comprising:a gene encoding 3,6-anhydro-L-galactose dehydrogenase;a gene encoding 3,6-anhydrogalactonic acid cycloisomerase;a gene encoding 2-keto-3-deoxy-galactonic acid kinase; anda gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase.2. The recombinant vector of claim 1 , wherein the gene encoding 3 claim 1 ,6-anhydro-L-galactose dehydrogenase is represented by the base sequence set forth in SEQ ID NO: 1.37. The recombinant vector of claim 1 , wherein the gene encoding 3 claim 1 ,6-anhydrogalactonic acid cycloisomerase is represented by any one of the base sequences set forth in SEQ ID NOs: 3 claim 1 , 5 and .4. The recombinant vector of claim 1 , wherein the gene encoding 2-keto-3-deoxy-galactonic acid kinase is represented by the base sequence set forth in SEQ ID NO: 9.5. The recombinant vector of claim 1 , wherein the gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase is represented by the base sequence set forth in SEQ ID NO: 11.6. A recombinant microorganism for producing ethanol claim 1 , which is transformed by:a gene encoding 3,6-anhydro-L-galactose dehydrogenase;a gene encoding 3,6-anhydrogalactonic acid cycloisomerase;a gene encoding 2-keto-3-deoxy-galactonic acid kinase; anda gene encoding 2-keto-3-deoxy-phosphogalactonic acid aldolase.7. The recombinant microorganism of claim 6 , wherein the recombinant microorganism is transformed with:a recombinant vector comprising a gene encoding 3,6-anhydro-L-galactose dehydrogenase;a recombinant vector comprising a gene encoding 3,6-anhydrogalactonic acid cycloisomerase;a recombinant vector comprising a gene encoding 2-keto-3-deoxy-galactonic acid kinase; anda recombinant vector ...

METHOD FOR PRODUCING MONOTERPENOID COMPOUNDS

The invention relates to enzymes and methods for producing a monoterpenoid compound. In one aspect, the invention is a method for producing a monoterpenoid compound, comprising the steps of (1) providing a monoterpenoid precursor; (2) providing a NEPS enzyme; and (3) contacting the monoterpenoid precursor with the enzyme under catalytic conditions to produce an monoterpenoid compound. 2. The method of claim 1 , wherein the enzyme comprises an amino acid sequence comprising SEQ ID NO: 1 and is encoded by a nucleotide sequence comprising SEQ ID NO: 4 claim 1 , and/or the enzyme comprises an amino acid sequence comprising SEQ ID NO: 2 and is encoded by a nucleotide sequence comprising SEQ ID NO: 5 claim 1 , and/or the enzyme comprises an amino acid sequence comprising SEQ ID NO: 3 and is encoded by a nucleotide sequence comprising SEQ ID NO: 6.3. The method of claim 1 , wherein the monoterpenoid compound comprises a monoterpene indole alkaloid (MIA).4. The method of or claim 1 , wherein the monoterpenoid precursor comprises 8-oxocitronellyl enol and/or 8-oxocitronellal and/or derivatives thereof.5. The method of any preceding claim claim 1 , wherein the enzyme further comprises an iridoid synthase (ISY) claim 1 , for example an ISY comprising an amino acid sequence of SEQ ID NO: 7 [AmISY] claim 1 , SEQ ID NO: 8 [NmISY2] or SEQ ID NO: 9 [CrISY] claim 1 , or a functional variant or homologue thereof.6. The method of claim 5 , wherein the ISY comprising an amino acid sequence of SEQ ID NO: 7 is encoded by a nucleotide sequence comprising SEQ ID NO: 10 claim 5 , the ISY comprising an amino acid sequence of SEQ ID NO: 8 is encoded by a nucleotide sequence comprising SEQ ID NO: 11 claim 5 , or the ISY comprising an amino acid sequence of SEQ ID NO: 9 is encoded by a nucleotide sequence comprising SEQ ID NO: 12.7. The method of claim 6 , wherein the monoterpenoid precursor comprises 8-oxogeranial.8. The method of any preceding claim claim 6 , wherein the monoterpenoid ...

método para produzir compostos monoterpenoides

Production of manool

Provided herein are methods of producing (+)-manool comprising: contacting geranylgeranyl diphosphate with an copalyl diphosphate (CPP) synthase to form a (9S, 10S)-copalyl diphosphate wherein the CPP synthase comprises an amino acid sequence having at least 90%, 95%, 98%, 99% and 100% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2; and contacting the CPP with a sclareol synthase enzyme to form (+)-manool.

Efficient Production of Steviol Glycosides in Recombinant Hosts

Recombinant microorganisms are disclosed that produce steviol glycosides and have altered expression of one or more endogenous transporter or transcription factor genes, or that overexpress one or more heterologous transporters, leading to increased excretion of steviol glycosides of interest. 1. A recombinant microorganism capable of producing a steviol glycoside in a cell culture ,wherein the microorganism has a modified expression of at least one endogenous transporter gene and at least one endogenous transcription factor gene that regulates expression of the at least one endogenous transporter gene,wherein the modified expression comprises reduced expression or activity of the at least one endogenous transporter gene, having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:113;wherein the reduced expression is produced in the microorganism by disrupting or deleting the gene locus for the at least one endogenous transporter gene;wherein the microorganism further expresses at least one heterologous transporter gene;wherein the steviol glycoside is Rebaudioside A, Rebaudioside B, Rebaudioside D, Rebaudioside E, Rebaudioside M, or an isomer thereof.2. The recombinant microorganism of claim 1 , wherein the endogenous and/or the heterologous transporter gene encodes an ATP-Binding Cassette (ABC) transporter.3Stevia. The recombinant microorganism of claim 1 , wherein the heterologous transporter gene is a transporter gene.4. The recombinant microorganism of claim 1 , further comprising a gene encoding a polypeptide capable of beta 1 claim 1 ,2 glycosylation of the C2′ of the 13-O-glucose claim 1 , 19-O-glucose claim 1 , or both 13-O-glucose and 19-O-glucose of a steviol glycoside; wherein the gene has a copy number of 2 or more; and wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs:51 claim 1 , 54 claim 1 , 55 claim 1 , 86 claim 1 , ...

PRODUCTION OF MANOOL

Described herein are methods of producing (+)-manool, the methods including: contacting geranylgeranyl diphosphate with a copalyl diphosphate (CPP) synthase to form a (9S, 10S)-copalyl diphosphate and contacting the CPP with a sclareol synthase enzyme to form (+)-manool and derivatives thereof. Also described herein are nucleic acids encoding CPP synthases and sclareol synthases for use in the methods. Further described herein are expression vectors and non-human host organisms and cells including nucleic acids encoding a CPP synthase and a sclareol synthase as described herein. 1. A method of producing (+)-manool , the method comprising: a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or', 'b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or', 'c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and, 'a) contacting geranylgeranyl diphosphate (GGPP) with a copalyl diphosphate (CPP) synthase to form a copalyl diphosphate, wherein the CPP synthase comprises'}b) contacting the CPP with a sclareol synthase to form the (+)-manool; andc) optionally isolating the (+)-manool.2. The method of claim 1 , wherein the CPP synthase comprisesa) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; orb) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; orc) a polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21.3. The method of claim 1 , wherein step a) further comprises culturing a non-human host ...

METHOD FOR THE BIOSYNTHESIS OF DIOSMIN AND/OR HESPERIDIN IN A MICROORGANISM

The present invention relates to a recombinant microorganism which is modified to be capable of producing diosmin and hesperidin and to the use thereof for producing diosmin and/or hesperidin. 132-. (canceled)33. A recombinant microorganism comprising:a heterologous nucleic acid sequence coding for a flavanone 7-O-beta-D-glucosyltransferase (UGT) which is capable of adding a glucose in position 7 of hesperetin and/or diosmetin; anda heterologous nucleic acid sequence coding for a 6″-O-rhamnosyltransferase (RhaT) which is capable of transferring a rhamnose into position 6 of the glucose of hesperetin-7-O-glucoside and/or diosmetin-7-O-glucoside; anda heterologous nucleic acid sequence coding for a UDP-glucose 4,6-dehydratase/UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase/UDP-4-keto-L-rhamnose-reductase (RHM) which is capable of producing UDP-rhamnose.34. The microorganism as claimed in claim 33 , in which the flavanone 7-O-beta-D-glucosyltransferase is selected from the group consisting of SEQ ID NOs: 113 claim 33 , 115 claim 33 , 91 claim 33 , 93 claim 33 , 95 claim 33 , 97 claim 33 , 99 and 101 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having flavanone 7-O-beta-D-glucosyltransferase activity.35. The microorganism as claimed in claim 33 , in which the 6″-O-rhamnosyltransferase is selected from the group consisting of SEQ ID NOs: 103 and 105 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having 6″-O-rhamnosyltransferase activity.36. The microorganism as claimed in claim 33 , in which the UDP-glucose 4 claim 33 ,6-dehydratase/UDP-4-keto-6-deoxy-D-glucose 3 claim 33 ,5-epimerase/UDP-4-keto-L-rhamnose-reductase is selected from the group consisting of SEQ ID NOs: 107 claim 33 , 109 and 111 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having UDP-glucose 4 claim 33 ,6-dehydratase/UDP-4- ...

COMPOSITIONS AND METHODS OF BIOSYNTHESIZING CAROTENOIDS AND THEIR DERIVATIVES

The present invention relates to compositions and methods of producing carotenoids and carotenoid derivatives. 1. A recombinant microorganism comprising at least one nucleic acid construct comprising:a) a nucleic acid sequence encoding a lycopene β-cyclase enzyme; andb) a nucleic acid sequence encoding a carotenoid cleavage dioxygenase enzyme;wherein the nucleic acid sequences are operably linked to one or more expression control sequence, and wherein the microorganism further comprises lycopene.2Mucor circinelloidesPhycomyces blakesleeanusErwinia herbicola.. The microorganism of claim 1 , wherein the lycopene β-cyclase is selected from a lycopene cyclase enzyme of a bifunctional lycopene cyclase/phytoene synthase encoded by carRP of claim 1 , lycopene cyclase/phytoene synthase encoded by carRA of claim 1 , and lycopene cyclase encoded by crtY from3Daucus carota.. The microorganism of claim 1 , wherein the carotenoid cleavage dioxygenase enzyme is encoded by CCD1 from4. The microorganism of claim 1 , wherein the microorganism is selected from a microorganism genetically engineered to inhibit the expression of lycopene α-cyclase and a microorganism naturally not capable of expressing lycopene α-cyclase.5Yarrowia lipolytica, Saccharomyces cerevisiaeE. coli.. The microorganism of claim 1 , wherein the microorganism is selected from claim 1 , and6. The microorganism of claim 1 , comprising β-ionone.7. The recombinant microorganism of claim 1 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a carotenoid cleavage dioxygenase enzyme with at least 80% identity to an amino acid sequence of SEQ ID NO: 88.8. The recombinant microorganism of claim 1 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a lycopene β-cyclase enzyme with at least 80% identity to an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO: 64 and SEQ ID NO: 67.9. A recombinant microorganism ...

PRODUCTION OF STEVIOL GLYCOSIDES IN RECOMBINANT HOSTS

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors. 1. A recombinant host cell capable of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture , comprising a recombinant gene encoding damage resistance protein 1 (DAP1) polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:2;wherein expression of the recombinant gene encoding DAP1 polypeptide results in increased production of the one or more steviol glycosides or the steviol glycoside composition in the cell culture.2. The recombinant host cell of claim 1 , further comprising:(a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);(b) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP;(c) a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate;(d) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;(e) a gene encoding a polypeptide 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 a steviol glycoside;(f) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or(g) a gene encoding a polypeptide capable of 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 a steviol glycoside;and 'wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to one of the amino acid sequences set forth in SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:117, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, or SEQ ID NO:76; and', '(h) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent- ...

Recombinant Production of Steviol Glycosides

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements. 1. A method for producing Rebaudioside D (RebD) , Rebaudioside E (RebE) , or a mixture thereof , comprising contacting a precursor steviol glycoside having a 13-O-glucose , a 19-O-glucose , or both the 13-O-glucose and the 19-O-glucose with a polypeptide capable of beta 1 ,2 glycosylation of the C-2′ of the 13-O-glucose , 19-O-glucose , or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and a UDP-glucose in a reaction mixture under suitable conditions for the transfer of one or more glucose moiety to the C2′ of the 13-O-glucose , 19-O-glucose or both 13-O-glucose and 19-O-glucose in the precursor steviol glycoside; thereby producing RebD , RebE , or a mixture thereof;wherein the polypeptide comprises an amino acid motif AA1-AA2-AA3-AA4-AA5-AA6-AA7, corresponding to residues 20-26 in SEQ ID NO:5; and{'claim-text': [{'sub': '1', '#text': 'AAis Proline'}, {'sub': '2', '#text': 'AAis one aromatic amino acid;'}, 'AA3 is one large hydrophobic amino acid;', 'AA4 is one small amino acid;', 'AA5 is one amino acid;', 'AA6 is one small amino acid; and', 'AA7 is Histidine.'], '#text': 'wherein:'}2. The method of claim 1 , wherein AA2 is:{'sup': '3', 'sub': 'R', '#text': '(a) one amino acid having a van der Waals volume ≥130 Åand a side chain hydrophobicity ≥60 Δt;'}{'sup': '3', 'sub': 'R', '#text': '(b) one amino acid having a van der Waals volume ≥130 Åand a side chain hydrophobicity ≥80 Δt;'}(c) Tryptophan, Phenylalanine, or Tyrosine;(d) Tryptophan or Phenylalanine; or(e) Tryptophan.3. The method of claim 1 , wherein AA3 is:{'sup': '3', 'sub': 'R ...

INOSITOL PREPARATION METHOD

An inositol preparation method by enzymatic catalysis uses starch and cellulose or substrates thereof as substrates. Raw materials are converted to inositol by in vitro multi-enzyme reaction system in one pot. The yield from the substrate to inositol is significantly improved by process optimization and adding new enzymes. The new enzymes can promote the phosphorolysis of starch or cellulose and utilization of glucose, which is the final production after the phosphorolysis of starch and cellulose. The inositol preparation method described herein has great potentials in industrial production of inositol because of high inositol yield, easy scale-up, low production cost, and lower impact to environment 1. A method for preparing inositol , comprising:(1) using starch or starch derivative as substrate, adding α-glucan phosphorylase, phosphoglucomutase, inositol-3-phophate synthase and inositol monophosphatase to establish a multi-enzyme reaction system and perform an enzyme-catalyzed reaction; and(2) separating and purifying reaction product to obtain inositol.2. The preparation method according to claim 1 , wherein the multi-enzyme reaction system further comprises starch debranching enzyme claim 1 , maltose phosphorylase and α-amylase claim 1 , or claim 1 , starch debranching enzyme claim 1 , glucanotransferase and α-amylase; the starch debranching enzyme is either one or both of isoamylase and pullulanase.3. The preparation method according to claim 2 , wherein the multi-enzyme reaction system further comprises polyphosphate glucokinase and polyphosphate; wherein the polyphosphate is preferably sodium polyphosphate.4. The preparation method according to claim 1 , wherein the starch derivative of step (1) includes any one or more of partially hydrolyzed starch claim 1 , starch dextrin claim 1 , maltodextrin claim 1 , malto-oligosaccharide or maltose.5. A method for preparing inositol claim 1 , comprising:(1) using cellulose or cellulose derivative as a substrate, ...

EXTRACELLULAR DITERPENE PRODUCTION

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism in a suitable fermentation medium, wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol, whereby a diterpene or glycosylated diterpene is produced extracellularly in the fermentation medium; and b. recovering the diterpene or glycosylated diterpene from the fermentation medium. 1. A method for the production of a diterpene or a glycosylated diterpene , which method comprises:a. fermenting a recombinant microorganism in a suitable fermentation medium,wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol,whereby a diterpene or glycosylated diterpene is produced extracellularly in the fermentation medium; andb. recovering the diterpene or glycosylated diterpene from the fermentation medium.2. The method according to claim 1 , wherein the recombinant microorganism comprises one or more nucleotide sequences encoding a polypeptide having UDP-glucosyltransferase activity claim 1 ,whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least one of steviolmonoside, steviolbioside, stevioside or ...

Production of Steviol Glycosides in Microorganisms

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements. 1. A recombinant host cell capable of producing steviol , a target steviol glycoside or a target steviol glycoside composition , comprising: 'wherein the polypeptide is capable of transferring a sugar moiety to the C2′ of a glucose in the precursor steviol glycoside;', '(a) a gene encoding a polypeptide capable of 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 a precursor steviol glycoside;'}and one or more of:(b) a gene encoding a polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-13 hydroxyl group; and/or(c) a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of the precursor steviol glycoside; and/or(d) a gene encoding a polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-19 carboxyl group;wherein at least one of the genes is a recombinant gene.2. The recombinant host cell of claim 1 , wherein:(a) the precursor steviol glycoside is rubusoside, wherein the sugar moiety is glucose, and stevioside is produced upon transfer of the glucose moiety;(b) the precursor steviol glycoside is stevioside, the sugar moiety is glucose, and rebaudioside E is produced upon transfer of the glucose moiety;(c) the precursor steviol glycoside is stevioside, the sugar moiety is glucose, the stevioside is contacted with the polypeptide capable of 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 precursor steviol glycoside and a polypeptide ...

COMPOSITIONS AND METHODS OF BIOSYNTHESIZING CAROTENOIDS AND THEIR DERIVATIVES

The present invention relates to compositions and methods of producing carotenoids and carotenoid derivatives. 1106-. (canceled)107. A recombinant microorganism comprising at least one nucleic acid construct comprising:a) a nucleic acid sequence encoding a lycopene ε-cyclase enzyme; andb) a nucleic acid sequence encoding a carotenoid cleavage dioxygenase enzyme;wherein the nucleic acid sequences are operably linked to one or more expression control sequences, and wherein the microorganism further comprises lycopene.108Lactuca sativa.. The microorganism of claim 107 , wherein the lycopene ε-cyclase enzyme is encoded by LCYe from109Daucus carota.. The microorganism of claim 107 , wherein the carotenoid cleavage dioxygenase enzyme is encoded by CCD1 from110. The microorganism of claim 107 , wherein the microorganism is selected from a microorganism genetically engineered to inhibit the expression of lycopene β-cyclase and a microorganism naturally not capable of expressing lycopene β-cyclase.111Yarrowia lipolytica, Saccharomyces cerevisiaeE. coli.. The microorganism of claim 107 , wherein the microorganism is selected from claim 107 , and112. The microorganism of claim 107 , wherein the at least one nucleic acid construct is codon optimized for expression in the microorganism.113. The microorganism of claim 107 , comprising α-ionone.114. The recombinant microorganism of claim 107 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a lycopene ε-cyclase enzyme with at least 80% identity to an amino acid sequence of SEQ ID NO: 87.115. The recombinant microorganism of claim 107 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a carotenoid cleavage dioxygenase enzyme with at least 80% identity to an amino acid sequence of SEQ ID NO: 88.117Mucor circinelloidesPhycomyces blakesleeanusErwinia herbicola.. The microorganism of claim 116 , wherein the lycopene β-cyclase is selected from a lycopene ...

Cellular production of glucaric acid

The invention relates to the production of glucuronic and glucaric acid in cells through recombinant expression of myo-inositol 1-phosphate synthase, myo-inositol oxygenase and uronate dehydrogenase. Cloning and characterization of the gene encoding uronate dehydrogenase is also disclosed.

EXTRACELLULAR DITERPENE PRODUCTION

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism in a suitable fermentation medium, wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol, whereby a diterpene or glycosylated diterpene is produced extracellularly in the fermentation medium; and b. recovering the diterpene or glycosylated diterpene from the fermentation medium. 1. A method for the production of a diterpene or a glycosylated diterpene , which method comprises:a. fermenting a recombinant microorganism in a suitable fermentation medium,wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol,whereby a diterpene or glycosylated diterpene is produced extracellularly in the fermentation medium; andb. recovering the diterpene or glycosylated diterpene from the fermentation medium.2. A method according to claim 1 , wherein the recombinant microorganism comprises one or more nucleotide sequences encoding a polypeptide having UDP-glucosyltransferase activity claim 1 ,whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least one of steviolmonoside, steviolbioside, stevioside or ...

COMPOSITIONS AND METHODS OF BIOSYNTHESIZING XANTHOPHYLLS

The present invention relates to compositions and methods of producing xanthophylls in microorganisms. 1. A recombinant microorganism comprising at least one artificial nucleic acid construct comprising:{'i': 'Marchantia polymorpha;', '(a) a nucleic acid comprising a sequence encoding a lycopene ε-cyclase enzyme from and'}{'i': Chlamydomonas reinhardtii,', 'Chromochloris zofingiensis,, '(b) a nucleic acid comprising a sequence encoding a lycopene β-cyclase enzyme selected from a lycopene β-cyclase enzyme from a lycopene β-cyclase enzyme from and a combination thereof;'}wherein the nucleic acid sequences are operably linked to one or more expression control sequences.2. The recombinant microorganism of claim 1 , wherein the microorganism further comprises:a) a nucleic acid comprising a sequence encoding a β-carotene hydroxylase; andb) a nucleic acid comprising a sequence encoding a P450 carotene ε-ring hydroxylase;wherein the nucleic acid sequences are operably linked to one or more expression control sequences.3Marchantia polymorphaMarchantia polymorpha.. The recombinant microorganism of claim 2 , wherein the β-carotene hydroxylase enzyme comprises the β-carotene hydroxylase enzyme from and the P450 carotene ε-ring hydroxylase enzyme comprises the P450 carotene ε-ring hydroxylase enzyme from4. The recombinant microorganism of claim 3 , wherein the microorganism further comprises:a) a nucleic acid comprising a sequence encoding a phytoene synthase enzyme; andb) a nucleic acid comprising a sequence encoding a phytoene dehydrogenase enzyme;wherein the nucleic acid sequences are operably linked to one or more expression control sequences.5Mucor circinelloides, Phycomyces blakesleeanus,Xanthophyllomyces dendrorhous,Mucor circinelloides,Xanthophyllomyces dendrorhous,Phycomyces blakesleeanus.. The recombinant microorganism of claim 4 , wherein the phytoene synthase enzyme comprises a lycopene cyclase/phytoene synthase enzyme modified to decrease lycopene cyclase activity ...

MICROBIAL POLYCULTURES AND METHODS OF USE THEREOF

Disclosed herein are novel microbial polycultures of two or more cell strains, capable of producing flavanones, flavonoids, and anthocyanidin-3-O-glucosides, and methods of use thereof. Also disclosed is a microbial cell capable of producing phenylpropanoic acids, and methods of use thereof. 1. A method of producing a product compound in a microbial polyculture;wherein, optionally, the microbial polyculture comprises a TAL module cell comprising an exogenous gene encoding for a tyrosine ammonia lyase (TAL);wherein, optionally, the microbial polyculture comprises a C5 module cell comprising an exogenous gene encoding for a 4-coumaroyl-CoA ligase (4CL), an exogenous gene encoding for a chalcone synthase (CHS), an exogenous gene encoding for a chalcone isomerase (CHI), and wherein, optionally, the C5 module cell further comprises an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC);wherein, optionally, the microbial polyculture further comprises a p168 module cell comprising an exogenous gene encoding for a flavanone 3β-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR); andwherein, optionally, the microbial polyculture further comprises an Antho module cell comprising an exogenous gene encoding for an anthocyanidin synthase (ANS) and an exogenous gene encoding for a 3-glucosyl transferase (3GT); providing a substrate to the microbial polyculture;', 'culturing the microbial polyculture under conditions permitting synthesis of the product compound by the microbial polyculture; and', 'isolating the product compound synthesized by the microbial polyculture;, 'the method comprising the microbial polyculture comprises the TAL module cell and the C5 module cell, the substrate is glucose, glycerol, or a combination thereof, and the product compound is a flavanone; or', 'the ...

METHOD OF PRODUCING TERPENES OR TERPENOIDS

The present invention relates to a recombinant bacterium exhibiting enhanced 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DXP) pathway, and its use for producing terpene or terpenoid compounds. 122-. (canceled)23Deinococcus. A method of producing a terpene or terpenoid comprising (i) culturing a recombinant bacterium that is genetically modified to increase 1-deoxyxylulose 5-phosphate synthase (DXS) activity and/or isopentenyl pyrophosphatase isomerase (IPP isomerase) activity , under conditions suitable to produce the terpene or terpenoid and optionally (ii) recovering the terpene or terpenoid.24Deinococcus. The method of claim 23 , wherein the recombinant bacterium overexpresses a native claim 23 , homologous or heterologous idi gene.25Deinococcus. The method of claim 23 , wherein the recombinant bacterium overexpresses a native claim 23 , homologous or heterologous dxs gene.26Deinococcus. The method of claim 23 , wherein the recombinant bacterium expresses an improved DXS enzyme.27. The method of claim 26 , wherein the improved DXS enzyme is a mutant DXS enzyme comprising a cysteine at position corresponding to position 244 of SEQ ID NO: 52.28DeinococcusD. radiopugnansD. yunweiensisD. geothermalis. The method of claim 26 , wherein the recombinant bacterium expresses a gene encoding the R244C mutant of the DXP synthase from (SEQ ID NO: 8) claim 26 , a gene encoding the R238C mutant of the DXP synthase from (SEQ ID NO: 14) or a gene encoding the R241C mutant of the DXP synthase from (SEQ ID NO: 56).29Deinococcus. The method of claim 23 , wherein the recombinant bacterium expresses an improved DXS enzyme and overexpresses a native claim 23 , homologous or heterologous idi gene.30Deinococcus. The method of claim 23 , wherein the recombinant bacterium overexpresses a native claim 23 , homologous or heterologous gene encoding FPP synthase.31. The method of claim 30 , wherein the gene encoding FPP synthase encodes a polypeptide selected from ...

MUTANT HAVING ABILITY TO OVERPRODUCE CAROTENOIDS AND METHOD FOR PRODUCING CAROTENOIDS BY USING SAME

Provided is a mutant having an ability to overproduce carotenoids and a method for producing carotenoids by using the mutant. The mutant, of which mutations are induced by irradiation after being transformed with a recombinant vector according to the subject matter, has an excellent ability to produce carotenoids and can be mass-produced, and thus can be useful in various industrial fields, which use carotenoids, such as cosmetics, food, and feed. 1. A mutant strain having an ability to over-produce a carotenoid , which is mutagenized by irradiation ,wherein the mutant strain includes a CrtE gene encoding geranylgeranyl pyrophosphate synthase; a CrtB gene encoding phytoene synthase; a CrtI gene encoding phytoene desaturase; a CrtY gene encoding lycopene beta cyclase; a CrtX gene encoding glucosyl transferase; and a CrtZ gene encoding beta-carotene hydroxylase, and is transformed with a recombinant vector for production of a carotenoid.2Escherichia coli. The mutant strain of claim 1 , wherein the mutant strain is whose accession number is KCTC 12683BP.3Pantoea agglomerans.. The mutant strain of claim 1 , wherein the CrtE gene claim 1 , the CrtB gene claim 1 , the CrtI gene claim 1 , the CrtY gene claim 1 , the CrtX gene claim 1 , and the CrtZ gene are derived from4. The mutant strain of claim 3 , wherein the CrtE gene consists of a nucleotide sequence as set forth in SEQ ID NO: 1.5. The mutant strain of claim 3 , wherein the CrtB gene consists of a nucleotide sequence as set forth in SEQ ID NO: 2.6. The mutant strain of claim 3 , wherein the CrtI gene consists of a nucleotide sequence as set forth in SEQ ID NO: 3.7. The mutant strain of claim 3 , wherein the CrtY gene consists of a nucleotide sequence as set forth in SEQ ID NO: 4.8. The mutant strain of claim 3 , wherein the CrtX gene consists of a nucleotide sequence as set forth in SEQ ID NO: 5.9. The mutant strain of claim 3 , wherein the CrtZ gene consists of a nucleotide sequence as set forth in SEQ ID NO: 6.10. ...

Production of Steviol Glycosides in Microorganisms

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements. 1. A method for producing Rebaudioside D (RebD) , Rebaudioside E (RebE) , or a mixture thereof , comprising contacting a precursor steviol glycoside having a 13-O-glucose , a 19-O-glucose , or both the 13-O-glucose and the 19-O-glucose with a polypeptide capable of beta 1 ,2 glycosylation of the C-2′ of the 13-O-glucose , 19-O-glucose , or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and a UDP-glucose in a reaction mixture under suitable conditions for the transfer of one or more glucose moiety to the C2′ of the 13-O-glucose , 19-O-glucose or both 13-O-glucose and 19-O-glucose in the precursor steviol glycoside; thereby producing RebD , RebE , or a mixture thereof.2. The method of claim 1 , comprising further contacting the reaction mixture with:(a) a polypeptide capable of glycosylating a precursor steviol glycoside having a C-13 hydroxyl group present in the reaction mixture at its C-13 hydroxyl group; and/or(b) a polypeptide capable of glycosylating a precursor steviol glycoside having a C-19 carboxyl group present in the reaction mixture at its C-19 carboxyl group; and/or(c) a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, of the 19-O-glucose or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose present in the reaction mixture.3. The method of claim 1 , which is an in vitro method comprising supplying the UDP-glucose or a cell-free system for regeneration of the UDP-glucose.4. The method of ...

DITERPENE PRODUCTION IN YARROWIA

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism of the genus in a suitable fermentation medium at a temperature of about 29° C. or higher, wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol; and b. recovering the diterpene or glycosylated diterpene. 1. A method for the production of a diterpene or a glycosylated diterpene , which method comprises:{'i': 'Yarrowia', 'a. fermenting a recombinant microorganism of the genus in a suitable fermentation medium at a temperature of about 29° C. or higher,'}wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol; andb. recovering the diterpene or glycosylated diterpene.2. A process according to claim 1 , wherein the recombinant microorganism comprises one or more nucleotide sequences encoding a polypeptide having UDP-glucosyltransferase activity claim 1 ,whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least one of steviolmonoside, steviolbioside, stevioside or rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rubusoside, dulcoside A.3 ...

CONVERSION OF BIOMASS TO USEFUL INTERMEDIATES

An aspect of the present disclosure is a microbial cell that includes a genetic modification resulting in the expression of a deficient form of an endogenous dioxygenase, and a gene encoding an exogenous dioxygenase and a promoter sequence, where the endogenous dioxygenase includes PcaH and PcaG, the exogenous dioxygenase includes LigA and LigB, the microbial cell is capable of growth utilizing at least one of a cellulose decomposition molecule or a lignin decomposition molecule, and the microbial cell is capable of producing 2-hydroxy-2H-pyran-4,6-dicarboxylic acid. 1PseudomonasPseudomonasPseudomonas. A non-naturally occurring not capable of expressing 2-pyrone-4 ,6-dicarboxylic acid hydrolase , and capable of producing 2-oxo-2H-pyran-4 ,6-dicarboxylic acid , wherein the is capable of growing on at least one of a cellulose decomposition molecule or a lignin decomposition molecule , and wherein the lacks the gene encoding for LigI.2PseudomonasPseudomonasP. putida. The of claim 1 , wherein the is KT2440.3Pseudomonas. The of claim 1 , wherein the cellulose decomposition molecule comprises a sugar molecule.4Pseudomonas. The of claim 3 , wherein the sugar molecule is at least one of D-xylose or D-glucose.5Pseudomonas. The of claim 1 , wherein the lignin decomposition molecule comprises an aromatic molecule.6Pseudomonas. The of claim 5 , wherein the aromatic molecule is at least one of catechol claim 5 , benzoate claim 5 , phenol claim 5 , guaiacol claim 5 , protecatechuate claim 5 , ferulate claim 5 , p-coumarate claim 5 , vanillate claim 5 , or 4-hydroxybenzoate.7Pseudomonas. The of claim 6 , wherein the aromatic molecule is protocatechuate.8Pseudomonas. The of claim 1 , further comprising an exogenous gene encoding a carboxylase.9Pseudomonas. The of claim 8 , wherein the exogenous gene encodes for AroY. This application claims priority under 35 U.S.C. 121 to U.S. Nonprovisional application Ser. No. 15/467,761 filed on Mar. 23, 2017 which claims priority under 35 U.S.C ...

MICROBIAL POLYCULTURES AND METHODS OF USE THEREOF

Disclosed herein are novel microbial polycultures of two or more cell strains, capable of producing flavanones, flavonoids, and anthocyanidin-3-O-glucosides, and methods of use thereof. Also disclosed is a microbial cell capable of producing phenylpropanoic acids, and methods of use thereof. 1. A microbial polyculture comprising a first module cell and at least a second module cell , the first and the at least a second module cell including:a TAL module cell comprising an exogenous gene encoding for a tyrosine ammonia lyase (TAL);a C5 module cell comprising an exogenous gene encoding for a 4-coumaroyl-CoA ligase (4CL), an exogenous gene encoding for a chalcone synthase (CHS), an exogenous gene encoding for a chalcone isomerase (CHI), or, the C5 module cell comprises an exogenous gene encoding for a 4-coumaroyl-CoA ligase (4CL), an exogenous gene encoding for a chalcone synthase (CHS), an exogenous gene encoding for a chalcone isomerase (CHI) and further comprises an exogenous gene encoding for malonyl-CoA synthetase (MatB) and an exogenous gene encoding for putative dicarboxylate carrier protein (MatC);a p168 module cell comprising an exogenous gene encoding for a flavanone 3β-hydroxylase (F3H), an exogenous gene encoding for a dihydroflavonol 4-reductase (DFR), and an exogenous gene encoding for a leucoanthocyanidin reductase (LAR); andan Antho module cell comprising an exogenous gene encoding for an anthocyanidin synthase (ANS) and an exogenous gene encoding for a 3-glucosyl transferase (3GT);{'i': Rhodotorula glutinis', 'Rhodobacter capsulatus', 'Arabidopsis, 'wherein the exogenous gene encoding for TAL encodes a polypeptide having at least 85% amino acid identity with the amino acid sequence of tyrosine ammonia lyase (RgTAL) of SEQ ID NO: 29, TAL of SEQ ID NO: 80, Rice TAL of SEQ ID NO: 81, Parsley TAL of SEQ ID NO: 82, Tomato TAL of SEQ ID NO: 83, TAL of SEQ ID NO: 84, or a combination thereof, having TAL activity,'}{'i': Arabidopsis thaliana', 'Petroselinum ...

Method for Producing Myo-Inositol and Myo-Inositol Derivative

To impart significantly improved myo-inositol producing capability, suitable for use in recombinant DNA techniques and synthetic biology methods, to a host microorganism that does not possess an endogenous myo-inositol biosynthesis pathway, such as . Inositol monophosphatase activity is strengthened in a transformant obtained by introducing a myo-inositol biosynthesis pathway into a host microorganism that does not possess an endogenous myo-inositol biosynthesis pathway. 1. A method of producing myo-inositol or a myo-inositol derivative , comprising:(1) preparing a transformant of a microorganism that does not express endogenous inositol-1-phosphate synthase, the transformant containing at least an inositol-1-phosphate synthase-encoding exogenous gene introduced expressibly into the transformant, and the transformant having a gene recombination or mutation to induce overproduction of functional inositol monophosphatase or activation of inositol monophosphatase within the transformant; and{'claim-ref': {'@idref': 'CLM-00021', 'claim 21'}, '(2) contacting the transformant with a carbon source that is converted to the compound of by the transformant under conditions suited to growth and/or maintenance of the transformant.'}2. (canceled)3. The method of claim 1 , wherein the transformant is a prokaryote.4. The method of claim 1 , wherein the transformant is a microorganism having an endogenous inositol monophosphatase gene.5Escherichia coliBacillusCorynebacteriumZymomonas.. The method of claim 1 , wherein the transformant is a bacterium selected from the group consisting of claim 1 , bacteria belonging to the genus claim 1 , bacteria belonging to the genus claim 1 , and bacteria belonging to the genus6Escherichia coli.. The method according to claim 5 , wherein the bacterium is7. The method of claim 1 , wherein the overproduction of inositol monophosphatase is induced in the transformant by claim 1 ,(a) introducing an exogenous inositol monophosphatase gene,(b) ...

Method for producing terpenes

The present invention concerns a method for producing terpenes in fungi comprising the steps of (a) providing a modified terpene biosynthetic gene cluster inside a host cell, wherein one or more of the naturally occurring genes or promoters of the cluster have been replaced, truncated or removed, (b) providing a transcription factor inside the host cell, the transcription factor activating the terpene biosynthetic gene cluster; (c) cultivating said host in conditions allowing the expression of the transcription factor activating the cluster; and optionally (d) recovering the thus produced terpene product. 1. Method for producing terpenes in fungi comprising the steps of:providing a modified terpene biosynthetic gene cluster inside a host cell, the gene cluster having naturally occurring terpene biosynthetic genes and promoters, wherein one or more of these genes or promoters have been replaced, truncated or removed,providing a transcription factor inside the host cell so that the transcription factor is operably linked to one of the natural or modified promoters, the transcription factor activating the terpene biosynthetic gene cluster having terpene biosynthetic genes and regulatory regions operably linked to said genes; andcultivating said host in conditions allowing the expression of the transcription factor activating the cluster.2. The method of claim 1 , wherein terpene is selected from: terpenoid claim 1 , γ-terpinene claim 1 , limonene claim 1 , cymene claim 1 , cineol claim 1 , α-farnesene claim 1 , amorphadiene claim 1 , cadinene claim 1 , a caryophyllene claim 1 , a bisabolene claim 1 , a taxadiene claim 1 , a kaurene claim 1 , a fusicoccadiene claim 1 , a casbene claim 1 , or an abietadiene.3. (canceled)4. (canceled)5. (canceled)6. The method of claim 1 , wherein the host cell carries the terpene biosynthetic gene cluster having terpene biosynthetic genes.7. The method of claim 1 , wherein the terpene biosynthetic gene cluster having terpene biosynthetic ...

METHODS OF PRODUCTION OF PRODUCTS OF METABOLIC PATHWAYS

A plurality of isolated polynucleotide sequences encoding enzymes of the astaxanthin pathway is disclosed. The polynucleotides include: 1. A plurality of isolated polynucleotide sequences encoding enzymes of the astaxanthin pathway comprising:(i) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and a first transcriptional regulatory sequence;(ii) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and a second transcriptional regulatory sequence;(iii) a polynucleotide which encodes Beta-carotene ketolase (crtW) and a third transcriptional regulatory sequence; andwherein said first, second and third regulatory sequence are selected such that the expression of said lcy-B and said crtW is greater than a level of expression of said crtI.2. The plurality of isolated polynucleotide sequences of claim 1 , further comprising at least one of:(iv) a polynucleotide which encodes Isopentenyl pyrophosphate (idi) and a fourth transcriptional regulatory sequence; or(v) a polynucleotide which encodes Geranylgeranyl pyrophosphate synthase (crtE) and a fifth transcriptional regulatory sequence; or(vi) a polynucleotide which encodes Prephytoene pyrophosphate synthase (crtB) and a sixth transcriptional regulatory sequence; or(vii) a polynucleotide which encodes Beta-carotene hydroxylase (crtZ) and a seventh transcriptional regulatory sequence.3. A plurality of isolated polynucleotide sequences encoding enzymes of the astaxanthin pathway comprising:(i) a polynucleotide which encodes Isopentenyl pyrophosphate (idi) and a first transcriptional regulatory sequence;(ii) a polynucleotide which encodes Geranylgeranyl pyrophosphate synthase (crtE) and a second transcriptional regulatory sequence;(iii) a polynucleotide which encodes Prephytoene pyrophosphate synthase (crtB) and a third transcriptional regulatory sequence;(iv) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and a fourth transcriptional regulatory sequence;(v) a polynucleotide which encodes Beta- ...

INOSITOL PREPARATION METHOD

An inositol preparation method by enzymatic catalysis uses starch and cellulose or substrates thereof as substrates. Raw materials are converted to inositol by in vitro multi-enzyme reaction system in one pot. The yield from the substrate to inositol is significantly improved by process optimization and adding new enzymes. The new enzymes can promote the phosphorolysis of starch or cellulose and utilization of glucose, which is the final production after the phosphorolysis of starch and cellulose. The inositol preparation method described herein has great potentials in industrial production of inositol because of high inositol yield, easy scale-up, low production cost, and lower impact to environment 1. A method for preparing inositol , comprising:(1) using starch or starch derivative as substrate, adding α-glucan phosphorylase, phosphoglucomutase, inositol-3-phophate synthase and inositol monophosphatase to establish a multi-enzyme reaction system and perform an enzyme-catalyzed reaction; and(2) separating and purifying reaction product to obtain inositol.2. The preparation method according to claim 1 , wherein the multi-enzyme reaction system further comprises starch debranching enzyme claim 1 , maltose phosphorylase and α-amylase claim 1 , or claim 1 , starch deb ranching enzyme claim 1 , glucanotransferase and α-amylase claim 1 , the starch debranching enzyme is either one or both of isoamylase and pullulanase.3. The preparation method according to claim 2 , wherein the multi-enzyme reaction system further comprises polyphosphate glucokinase and polyphosphate; wherein the polyphosphate is preferably sodium polyphosphate.4. The preparation method according to claim 1 , wherein the starch derivative of step (1) includes any one or more of partially hydrolyzed starch claim 1 , starch dextrin claim 1 , maltodextrin claim 1 , malto-oligosaccharide or maltose.5. A method for preparing inositol claim 1 , comprising:(1) using cellulose or cellulose derivative as a ...

Production of alpha-(R)-(E)-(+)-ionone in recombinant Saccharomyces cerevisiae

This invention provides improved biological synthesis of the apocarotenoid α-ionone in Saccharomyces cerevisiae. The final native step involved in the natural apocarotenoid pathway depends on an endogenous farnesyl pyrophosphate synthase (FPPs). From there, heterologous geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtI), lycopene ε-cyclase (LycE) and a Carotenoid Cleavage Dioxygenase (CCD1) are required to complete the synthesis of α-ionone. Lycopene ε-cyclase from lettuce (Lactuca sativa) or modified cyclase from Arabidopsis thaliana was used to overproduce lycopene which was then cleaved by the carotenoid cleavage dioxygenase from Petunia hybrida (Ph-CCD1).

Production of Steviol Glycosides in Microorganisms

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements. 1. A recombinant host cell capable of producing steviol , a target steviol glycoside or a target steviol glycoside composition , comprising: 'wherein the polypeptide is capable of transferring a sugar moiety to the C2′ of a glucose in the precursor steviol glycoside;', '(a) a gene encoding a polypeptide capable of 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 a precursor steviol glycoside;'}and one or more of:(b) a gene encoding a polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-13 hydroxyl group; and/or(c) a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of the precursor steviol glycoside; and/or(d) a gene encoding a polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-19 carboxyl group;wherein at least one of the genes is a recombinant gene.2. The recombinant host cell of claim 1 , wherein:(a) the precursor steviol glycoside is rubusoside, wherein the sugar moiety is glucose, and stevioside is produced upon transfer of the glucose moiety;(b) the precursor steviol glycoside is stevioside, the sugar moiety is glucose, and rebaudioside E is produced upon transfer of the glucose moiety;(c) the precursor steviol glycoside is stevioside, the sugar moiety is glucose, the stevioside is contacted with the polypeptide capable of 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 precursor steviol glycoside and a polypeptide ...

METHOD FOR THE BIOTECHNOLOGICAL PRODUCTION OF FLAVONE GLYCOSIDE DIHYDROCHALCONES

The invention relates to a method for producing flavone glycoside dihydrochalcones, having the following steps: (a) providing a transgenic microorganism containing (i) a first nucleic acid portion (A) containing a gene which codes for a bacterial chalcone isomerase and (ii) a second nucleic acid portion (B) containing a gene which codes for a bacterial enoate reductase, (b) adding one or more flavone glycosides to the transgenic microorganism under conditions which allow the simultaneous isomerization and reduction of the flavone glycoside into the flavone glycoside dihydrochalcone, and optionally (d) isolating and purifying the final product, wherein the nucleic acid portion (A) (1) is a nucleotide sequence according to SEQ ID NO:1, in which the nucleic acid portion (A′) according to SEQ ID NO:3 has been cut out, or (2) is an amino acid sequence according to SEQ ID NO:2, in which the amino acid portion (A′) according to SEQ ID NO:4 has been cut out. 1. A method for preparing flavanone glycoside dihydrochalcones comprising: (i) a first nucleotide section (A) containing a gene coding for a bacterial chalcone isomerase, and', '(ii) a second nucleic acid section (B) containing a gene coding for a bacterial enoate reductase, '(a) providing a transgenic microorganism containing'}(b) adding one or more flavanone glycosides to the transgenic microorganism,(c) culturing the transgenic microorganism under conditions which allow the simultaneous isomerisation and reduction of the flavanone glycoside to the flavanone glycoside dihydrochalcone, and, wherein the nucleic acid section (A)', '(1) represents a nucleic acid sequence according to SEQ ID NO: 1, in which the nucleic acid section (A′) according to SEQ ID NO: 3 has been cut out, or', '(2) represents an amino acid sequence according to SEQ ID NO: 2, in which the amino acid section (A′) according to SEQ ID NO: 4 has been cut out., '(d) optionally, isolating and purifying the final product,'}2. The method according to claim ...

NOVEL VIOLAXANTHIN-OVERPRODUCING STRAIN OF CHLORELLA VULGARIS AND THE METHOD FOR PRODUCING VIOLAXANTHIN USING THE SAME

The present invention relates to a novel violaxanthin-overproducing strain of and a method of producing violaxanthin therefrom. The inventors have developed a strain that produces violaxanthin at a significantly higher level than a wild-type strain by inducing a random chemical mutation in a strain to, and then as a result of analysis, confirmed that the strain produces violaxanthin up to 0.41% based on dry weight, which reaches the highest level that is possible to be produced in microalgae. Furthermore, as a method of effectively extracting a carotenoid pigment containing violaxanthin from the strain was established, since the strain and the developed pigment extraction method according to the present invention allow effective production and separation of violaxanthin, the strain is expected to increase commercial applications such as cosmetics, health functional foods and feed. 1Chlorella vulgaris. A violaxanthin-overproducing CvLD-01 strain (Accession No. KCTC 14091BP).2Chlorella vulgaris. The strain of claim 1 , wherein the strain is derived from a UTEX395 strain (Accession No. KCTC 14091BP).3. The strain of claim 1 , wherein the strain has alanine (A) claim 1 , which is amino acid 336 of the lycopene epsilon cyclase (CvLCYE) gene claim 1 , substituted with valine (V) (Accession No. KCTC 14091BP).4Chlorella vulgaris. A method of extracting carotenoid pigment from the strain of using glass bead-added sonication.5. The method of claim 4 , wherein the extraction method uses methanol as a solvent.6. The method of claim 4 , wherein the glass bead has a diameter of 0.4 to 0.7 mm.7Chlorella vulgaris. A method of producing violaxanthin on a large scale claim 1 , which comprises culturing the CvLD-01 strain of .8. The method of claim 7 , wherein the culturing is performed for 72 to 96 hours in a medium containing acetic acid as a carbon source under 100 μmol photon ms.9Chlorella vulgaris. A composition for food or a food additive claim 1 , comprising one or more ...

Diterpene production in yarrowia

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism of the genus Yarrowia in a suitable fermentation medium at a temperature of about 29° C. or higher, wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol; and b. recovering the diterpene or glycosylated diterpene.

Steviol glycoside transport

A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto.

Production of Steviol Glycosides in Recombinant Hosts

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors. 1. A recombinant host comprising one or more of:(a) a gene encoding an ent-kaurene oxidase (KO) polypeptide;(b) a gene encoding a cytochrome P450 reductase (CPR) polypeptide; and/or(c) a gene encoding an ent-kaurenoic acid hydroxylase (KAH) polypeptide;wherein at least one of the genes is a recombinant gene; andwherein the recombinant host is capable of producing a steviol glycoside precursor.2. A recombinant host comprising:(a) a gene encoding a geranylgeranyl diphosphate synthase (GGPPS) polypeptide;(b) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide;(c) a gene encoding an ent-kaurene synthase (KS) polypeptide(d) a gene encoding an ent-kaurene oxidase (KO) polypeptide;(e) a gene encoding a cytochrome P450 reductase (CPR) polypeptide; and(f) a gene encoding an ent-kaurenoic acid hydroxylase (KAH) polypeptide;wherein at least one of the genes is a recombinant gene; andwherein the recombinant host is capable of producing steviol.3. The recombinant host of or , wherein:(a) the KO polypeptide comprises a KO polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:72 or SEQ ID NO:75; at least 65% identity to an amino acid sequence set forth in SEQ ID NO:54; at least 70% identity to an amino acid sequence set forth in SED ID NO: 70, SEQ ID NO:71, or SEQ ID NO:79; at least 40% identity to an amino acid sequence set forth in SEQ ID NO:77; or at least 50% identity to an amino acid sequence set forth in SEQ ID NO:78;(b) the CPR polypeptide comprises a CPR polypeptide having at least 70% identity to an amino acid sequences set forth in SEQ ID NO:69, SEQ ID NO:74, SEQ ID NO:76, or SEQ ID NO:87; at least 80% identity to an amino acid sequence set forth in SEQ ID NO:73; at least 85% identity to an amino acid sequence set forth in SEQ ID NO:22; at least 65% identity to an amino acid sequence set ...

METHOD OF FERMENTATIVE ALPHA-IONONE PRODUCTION

The present invention concerns a method of producing and enantiomerically pure alpha-ionone. Further, the invention concerns a nucleic acid that comprises a sequence that encodes a lycopene-epsilon-cyclase (EC), a lycopene-epsilon-cyclase (EC), plasmids, which encode components of the alpha-ionone biosynthesis and a microorganism that contains heterologous nucleotide sequences which encode the enzymes geranylgeranyl-diphosphate-synthase, isopentenyl-diphosphate-isomerase (IPI), phytoene desaturase-dehydrogenase (crtI), phytoene synthase (crtB), lycopene-epsilon-cyclase (EC) and carotenoid-cleavage-dioxygenase (CCD1). Further, the invention concerns a method of producing highly pure epsilon-carotene. 1. A method of producing enantiomerically pure alpha-ionone comprising culturing a microorganism that comprises heterologous nucleotide sequences that encode the following enzymes:a. geranylgeranyl-diphosphate-synthase,b. isopentenyl-diphosphate-isomerase (ipi),c. phytoene-desaturase/dehydrogenase (crtI),d. phytoene synthase (crtB),e. lycopene-epsilon-cyclase (EC) andf. carotenoid-cleavage-dioxygenase (CCD1).2. The method according to claim 1 , wherein the geranylgeranyl-diphosphate-synthase is the geranylgeranyl-diphosphate-synthase crtE for the geranylgeranyl-diphosphate-synthase idsA.3. The method according to or claim 1 , wherein the lycopene-epsilon-cyclase (EC) has at least 80% sequence identity with a sequence according to SEQ ID NO. 19 and deviates at least at one position of 403 claim 1 , 404 and 445 from the sequence according to SEQ ID NO. 19.4. The method according to one of to claim 1 , wherein the lycopene-epsilon-cyclase (EC) comprises one of the following mutations or mutation combinations: ECmut9 (L404S) claim 1 , ECmut10 (A403S/L404T) claim 1 , ECmut3.3 (A403E/L404A/A445S) and ECmut3.2 (A403C/L404C/A445S).5Arabidopsis thalianaOsmanthus fragans.. The method according to one of to claim 1 , wherein the carotenoid-cleavage-dioxygenase (CCD1) is a ...

METHODS OF PRODUCING ASTAXANTHIN OR PRECURSORS THEREOF

The present invention relates to a recombinant bacterium comprising a heterologous biosynthetic pathway converting lycopene to astaxanthin or a precursor thereof, and its use for producing astaxanthin or a precursor thereof. Shown is the production of carotene, zeaxanthin, canthaxanthin and astaxanthin using recombinant expressing genes encoding for lycopene cylase (EC 5.5.1.19), beta-carotene hydroxylase (EC1.14.13.129) and beta-carotene ketolase, namely carotenoid-4,4-beta-ionone ring oxygenase (EC 1.14.11.B16). 133-. (canceled)34Deinococcus. A recombinant bacterium comprising a heterologous nucleic acid sequence encoding a polypeptide exhibiting lycopene cyclase activity and being able to produce beta-carotene when cultured at a temperature greater than 40° C.35. The recombinant bacterium of claim 34 , further comprising a heterologous nucleic acid sequence encoding a polypeptide exhibiting beta-carotene hydroxylase activity.36. The recombinant bacterium of claim 34 , further comprising a nucleic acid sequence encoding a polypeptide exhibiting beta-carotene ketolase activity.37. The recombinant bacterium of claim 34 , wherein the polypeptide exhibiting lycopene cyclase activity is selected from the group consisting of SEQ ID NOs: 1 claim 34 , 3 claim 34 , 5 claim 34 , 7 claim 34 , 9 claim 34 , 75 claim 34 , 77 claim 34 , 79 claim 34 , 81 claim 34 , 83 claim 34 , 85 and 87 claim 34 , and polypeptides exhibiting lycopene cyclase activity and having at least 80% identity to SEQ ID NO: 1 claim 34 , 3 claim 34 , 5 claim 34 , 7 claim 34 , 9 claim 34 , 75 claim 34 , 77 claim 34 , 79 claim 34 , 81 claim 34 , 83 claim 34 , 85 or 87.38. The recombinant bacterium of claim 35 , wherein the polypeptide exhibiting beta-carotene hydroxylase activity is selected from the group consisting of SEQ ID NOs: 11 claim 35 , 13 claim 35 , 15 claim 35 , 17 claim 35 , 19 claim 35 , 21 claim 35 , 23 claim 35 , 25 claim 35 , 89 claim 35 , 91 and 93 and polypeptides exhibiting beta-carotene ...

CONVERSION OF BIOMASS TO USEFUL INTERMEDIATES

An aspect of the present disclosure is a microbial cell that includes a genetic modification resulting in the expression of a deficient form of an endogenous dioxygenase, and a gene encoding an exogenous dioxygenase and a promoter sequence, where the endogenous dioxygenase includes PcaH and PcaG, the exogenous dioxygenase includes LigA and LigB, the microbial cell is capable of growth utilizing at least one of a cellulose decomposition molecule or a lignin decomposition molecule, and the microbial cell is capable of producing 2-hydroxy-2H-pyran-4,6-dicarboxylic acid. 1. A microbial cell comprising:a genetic modification resulting in the expression of a deficient form of an endogenous dioxygenase; anda gene encoding an exogenous dioxygenase and a promoter sequence, wherein:the endogenous dioxygenase comprises PcaH and PcaG,the exogenous dioxygenase comprises LigA and LigB,the microbial cell is capable of growth utilizing at least one of a cellulose decomposition molecule or a lignin decomposition molecule, andthe microbial cell is capable of producing 2-hydroxy-2H-pyran-4,6-dicarboxylic acid.2. A microbial cell comprising:a genetic modification resulting in the expression of a deficient form of an endogenous dioxygenase; anda gene encoding an exogenous dioxygenase, an exogenous dehydrogenase, and a promoter sequence, wherein:the endogenous dioxygenase comprises PcaH and PcaG,the exogenous dioxygenase comprises LigA and LigB,the exogenous dehydrogenase comprises LigC,the microbial cell is capable of growth utilizing at least one of a cellulose decomposition molecule or a lignin decomposition molecule, andthe microbial cell is capable of producing 2-oxo-2H-pyran-4,6-dicarboxylic acid.3. A microbial cell comprising:a first genetic modification resulting in the expression of a deficient form of an endogenous dioxygenase;a second genetic modification resulting in the expression of deficient forms of an endogenous tautomerase, an endogenous hydratase, and an endogenous ...

BIOLOGICAL DEVICES AND METHODS OF USE THEREOF TO PRODUCE CAROTENOIDS

Described herein are devices and methods for using the same to produce carotenoids. The carotenoids produced by the devices and methods disclosed herein do not require the ultra purification that is common in conventional or commercial methods. The devices and methods disclosed herein also enhance one or more physical properties of plants treated with the devices described herein. 1. A DNA construct comprising the following genetic components: (a) a gene that expresses beta-carotene hydroxylase , (b) a gene that expresses lycopene epsilon cyclase , and (c) a gene that expresses 1-deoxy--xylylose phosphate synthase.2. The DNA construct of claim 1 , further comprising at least one promoter claim 1 , wherein the promoter is positioned before the gene that expresses beta-carotene hydroxylase claim 1 , the gene that expresses lycopene epsilon cyclase claim 1 , the gene that expresses 1-deoxy--xylylose phosphate synthase claim 1 , or any combination thereof.3. The DNA construct of claim 1 , further comprising at least one promoter positioned before the gene that expresses beta-carotene hydroxylase claim 1 , the gene that expresses lycopene epsilon cyclase claim 1 , and the gene that expresses 1-deoxy--xylylose phosphate synthase claim 1 , or any combination thereof.4. The DNA construct of claim 1 , wherein the promoter is a GAL1 promoter.5. The DNA construct of claim 1 , wherein the gene that expresses 1-deoxy--xylulose phosphate synthase is SEQ ID NO. 1 or a sequence having at least 90% homology thereof.6. The DNA construct of claim 1 , wherein the gene that expresses beta-carotene hydroxylase is SEQ ID NO. 2 or a sequence having at least 90% homology thereof.7. The DNA construct of claim 1 , wherein the gene that expresses lycopene epsilon-cyclase is SEQ ID NO. 3 or a sequence having at least 90% homology thereof.8. The DNA construct of claim 1 , further comprising at least one terminator.9. The DNA construct of claim 8 , wherein the terminator is a CYC1 terminator.10. ...

STEVIOL GLYCOSIDE PRODUCTION

The present invention relates to a method for increasing the degree of glycosylation of a composition comprising steviol glycosides, which method comprises: 1. A method for increasing the degree of glycosylation of a composition comprising steviol glycosides , which method comprises:a. contacting said composition comprising steviol glycosides with a recombinant microorganism, a cell free extract derived from such a recombinant microorganism or an enzyme preparation derived from either thereof; and 'wherein the recombinant microorganism comprises one or more nucleotide sequence(s) encoding:', 'b. thereby to increase the degree of glycosylation of the composition comprising steviol glycosides,'}a polypeptide having ent-copalyl pyrophosphate synthase activity;a polypeptide having ent-Kaurene synthase activity;a polypeptide having ent-Kaurene oxidase activity;a polypeptide having kaurenoic acid 13-hydroxylase activity; andone or more polypeptides having UDP-glucosyltransferase activitywhereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least one steviol glycoside.2. A method according to claim 1 , wherein the composition comprising steviol glycosides comprises at least one of steviolmonoside claim 1 , steviolbioside claim 1 , stevioside or rebaudioside A claim 1 , rebaudioside B claim 1 , rebaudioside C claim 1 , rebaudioside D claim 1 , rebaudioside E claim 1 , rebaudioside F claim 1 , rebaudioside M claim 1 , rubusoside or dulcoside A.3. A method according to claim 1 , wherein the increase in the degree of glycosylation of the composition comprising steviol glycosides is an increase in the amount of one or more steviol glycosides present in the composition comprising steviol glycosides.4. A method according to claim 1 , wherein the increase in the degree of glycosylation of the composition comprising steviol glycosides is an increase in the amount of four glucose molecule-containing steviol glycosides present in ...

METHOD FOR ENZYMATICALLY PREPARING HIGHLY CONCENTRATED MYO-INOSITOL

The present disclosure relates to a method for preparing myo-inositol using myo-inositol monophosphate synthase consisting of an amino acid sequence of SEQ ID NO: 1 and/or myo-inositol monophosphate phosphatase consisting of an amino acid sequence of SEQ ID NO: 3. 1. A method for preparing myo-inositol , comprising: converting myo-inositol monophosphate to myo-inositol by reacting the myo-inositol monophosphate with myo-inositol monophosphate phosphatase consisting of an amino acid sequence of SEQ ID NO: 3 , a microorganism expressing the same , or a culture of the microorganism.2. The method according to claim 1 , wherein the method further comprises converting glucose-6-phosphate to myo-inositol monophosphate by reacting the glucose-6-phosphate with myo-inositol monophosphate synthase consisting of an amino acid sequence of SEQ ID NO: 1 claim 1 , a microorganism expressing the same claim 1 , or a culture of the microorganism claim 1 , prior to converting the myo-inositol monophosphate to myo-inositol.3. The method according to claim 2 , wherein the method further comprises converting glucose-1-phosphate to glucose-6-phosphate by reacting the glucose-1-phosphate with phosphoglucomutase claim 2 , a microorganism expressing the same claim 2 , or a culture of the microorganism claim 2 , prior to converting the glucose-6-phosphate to myo-inositol monophosphate.4. The method according to claim 2 , wherein the method further comprises converting glucose to glucose-6-phosphate by reacting the glucose with phosphate and glucokinase claim 2 , a microorganism expressing the same claim 2 , or a culture of the microorganism claim 2 , prior to converting the glucose-6-phosphate to myo-inositol monophosphate.5. The method according to claim 3 , wherein the method further comprises converting starch claim 3 , maltodextrin claim 3 , sucrose claim 3 , or a combination thereof to glucose-1-phosphate by reacting the starch claim 3 , maltodextrin claim 3 , sucrose claim 3 , or ...

Efficient Production of Steviol Glycosides in Recombinant Hosts

Recombinant microorganisms are disclosed that produce steviol glycosides and have altered expression of one or more endogenous transporter or transcription factor genes, or that overexpress one or more heterologous transporters, leading to increased excretion of steviol glycosides of interest.

PRODUCTION OF MANOOL

Described herein are methods of producing (+)-manool, the methods including: contacting geranylgeranyl diphosphate with a copalyl diphosphate (CPP) synthase to form a (9S, 10S)-copalyl diphosphate and contacting the CPP with a sclareol synthase enzyme to form (+)-manool and derivatives thereof. Also described herein are nucleic acids encoding CPP synthases and sclareol synthases for use in the methods. Further described herein are expression vectors and non-human host organisms and cells including nucleic acids encoding a CPP synthase and a sclareol synthase as described herein. 1. A method of producing (+)-manool , the method comprising: a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or', 'b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or', 'c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and, 'a) contacting geranylgeranyl diphosphate (GGPP) with a copalyl diphosphate (CPP) synthase to form a copalyl diphosphate, wherein the CPP synthase comprises'}b) contacting the CPP with a sclareol synthase to form the (+)-manool; andc) optionally isolating the (+)-manool.2. The method of claim 1 , wherein the CPP synthase comprisesa) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; orb) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; orc) a polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21.3. The method of claim 1 , wherein step a) further comprises culturing a non-human host ...

Production of Anthocyanin from Simple Sugars

Methods for producing anthocyanin by expression in a microorganism are disclosed including culturing of the microorganism under anthocyanin producing conditions, wherein the microorganism has an operative metabolic pathway including at least one heterologous enzyme activity, the pathway producing anthocyanin from simple sugars or other simple carbon sources. 1. A microorganism , comprising an operative metabolic pathway capable of producing an anthocyanin from a simple sugar , the operative metabolic pathway comprising:a 4-coumaric acid-CoA ligase (4CL);a chalcone synthase (CHS);a flavanone 3-hydroxylase (F3H);a dihydroflavonol-4-reductase (DFR);an anthocyanidin synthase (ANS);an anthocyanidin 3-O-glycosyltransferase (A3GT);a chalcone isomerase (CHI); and a) a tyrosine ammonia lyase (TAL); or', 'b) a phenylalanine ammonia lyase (PAL) and a trans-cinnamate 4-monooxygenase (C4H),, 'at least one of'}wherein at least one enzyme of the operative metabolic pathway is encoded by a gene heterologous to the microorganism.2. The microorganism of claim 1 , wherein the metabolic pathway further comprises:a tyrosine ammonia lyase (TAL);a phenylalanine ammonia lyase (PAL); anda trans-cinnamate 4-monooxygenase (C4H).3. The microorganism of claim 1 , wherein the metabolic pathway further comprises one or more of:a flavonoid 3′-hydroxylase (F3′H);a flavonoid 3′-5′-hydroxylase (F3′5′H);a leucoanthocyanidin reductase (LAR); ora CYP450 reductase (CPR).4. The microorganism of claim 3 , wherein the anthocyanin is pelargonidin-3-O-glucoside (P3G) claim 3 , cyanidin-3-O-glucoside (C3G) claim 3 , or delphinidin-3-O-glucoside (D3G).5. The microorganism of claim 1 , wherein the microorganism is a yeast or a bacteria.6. (canceled)7. (canceled)8. (canceled)9. (canceled)10. The microorganism of claim 1 , wherein a plurality of enzymes comprising the operative metabolic pathway are encoded by genes that are heterologous to the microorganism.11. (canceled)12. (canceled)13. (canceled)14. The ...

Efficient Production of Steviol Glycosides in Recombinant Hosts

Recombinant microorganisms are disclosed that produce steviol glycosides and have altered expression of one or more endogenous transporter or transcription factor genes or that overexpress one or more heterologous transporters, leading to increased excretions of steviol glucosides of interest. 1. A recombinant microorganism capable of producing a steviol glycoside in a cell culture ,wherein the microorganism has a modified expression of at least one endogenous transporter gene and at least one endogenous transcription factor gene that regulates expression of the at least one endogenous transporter gene,wherein the modified expression comprises increasing or decreasing expression or activity of the at least one endogenous transporter gene, the at least one endogenous transcription factor gene that regulates expression of the at least one endogenous transporter gene, or both by at least 5% above or below the level of expression or activity observed in a microorganism without modified expression of the at least one endogenous transporter gene and the at least one endogenous transcription factor gene that regulates expression of the at least one endogenous transporter gene;wherein the microorganism further expresses at least one heterologous transporter gene;wherein the steviol glycoside is Rebaudioside A, Rebaudioside B, Rebaudioside D, Rebaudioside E, Rebaudioside M, or an isomer thereof.2. The recombinant microorganism of claim 1 , wherein the endogenous and/or heterologous transporter gene encodes an ATP-Binding Cassette (ABC) transporter or a Major Facilitator Superfamily (MFS) transporter.3. The recombinant microorganism of claim 1 , wherein the endogenous transporter gene encodes a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:106-147 claim 1 , and wherein the endogenous transcription factor gene encodes a polypeptide having at least 90% sequence identity to the an amino acid sequence set forth in ...

PRODUCTION OF CAROTENOIDS AND APOCAROTENOIDS

A method for producing a carotenoid or apocarotenoid is disclosed. The method comprises the step of expressing in a host cell an expression module comprising an expression vector having a coding region encoding at least one optimised carotenoid or apocarotenoid generating enzyme, the coding region being operably linked to a promoter. A host cell comprising an expression vector having a coding region encoding at least one optimised carotenoid or apocarotenoid generating enzyme, the coding region being operably linked to a promoter is also provided together with a kit. 153.-. (canceled)54. A method for producing a carotenoid or apocarotenoid comprising the step of expressing in a host cell an expression module comprising an expression vector having a coding region encoding at least one optimized apocarotenoid or carotenoid generating enzyme , the coding region being operably linked to a promoter.55. The method as claimed in claim 54 , wherein the carotenoid is selected from phytoene claim 54 , lycopene claim 54 , α-carotene claim 54 , γ-carotene claim 54 , δ-carotene claim 54 , ε-carotene or β-carotene and wherein the apocarotenoid is selected from α-ionone claim 54 , β-ionone claim 54 , pseudo-ionone (or psi-ionone) claim 54 , hydroxy-ionone claim 54 , β-cyclocitral claim 54 , trans-geranylacetone claim 54 , 6-methyl-5-hepten-2-one (MHO) claim 54 , retinal claim 54 , retinol claim 54 , 8′ claim 54 ,10-diapocarotene-8′ claim 54 ,10-dial (C17) or 10′ claim 54 ,6-diapocarotene-10′ claim 54 ,6-dial (C19).56. The method as claimed in claim 54 , wherein said at least one optimised carotenoid or apocarotenoid generating enzyme is selected from crtY claim 54 , CCD1 claim 54 , CCD2 claim 54 , CCD4 claim 54 , BCDO claim 54 , LcyE claim 54 , blh or ybbO claim 54 , optionally wherein said CCD2 is selected from CaCCD2 or CsCCD2; optionally wherein said CCD4 is selected from the group consisting of AtCCD4 claim 54 , BoCCD4b claim 54 , CmCCD4 claim 54 , CsCCD4a claim 54 , MaCCD4 ...

Production of Steviol Glycosides in Recombinant Hosts

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors. 1. A recombinant host cell capable of producing a steviol glycoside precursor and/or one or more steviol glycosides in a cell culture , comprising:(a) a gene encoding a polypeptide that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);(b) a gene encoding a polypeptide that synthesizes ent-copalyl diphosphate from GGPP; and(c) a gene encoding a polypeptide that synthesizes ent-kaurene from ent-copalyl diphosphate; 'wherein the polypeptide that reduces cytochrome P450 complex comprises a polypeptide having at least 70% identity to an amino acid sequences set forth in any one of SEQ ID NO:69, 74, 76, or 87; at least 80% identity to an amino acid sequence set forth in SEQ ID NO:73; at least 85% identity to an amino acid sequence set forth in SEQ ID NO:22; at least 65% identity to an amino acid sequence set forth in SEQ ID NO:28; or at least 50% identity to an amino acid sequence set forth in SEQ ID NO:98;', '(d) a gene encoding a polypeptide that reduces cytochrome P450 complex;'}and further comprising: wherein the polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:72 or 75; at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:54; at least 70% sequence identity to the amino acid sequence set forth in any one of SED ID NO: 70, 71, or 79; at least 40% sequence identity to the amino acid sequence set forth in SEQ ID NO:77; or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:78;', 'or, '(e) a gene encoding a polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene;'} 'wherein the polypeptide that synthesizes steviol from ent-kaurenoic acid comprises a polypeptide having at least 40% identity ...

肌醇的制备方法

本发明公开了一种肌醇的制备方法，属于肌醇的酶催化制备领域。本发明所公开的肌醇的制备方法以淀粉、纤维素或它们的衍生物为底物，在一个多酶反应体系中，通过体外多酶高效催化将底物转化为肌醇；本发明通过过程优化，添加能够促进淀粉或纤维素水解的酶以及利用副产物葡萄糖的酶，从而建立多酶反应体系，显著提升了原料的转化效率和肌醇的产率。本发明方法原料转化率高，肌醇产率高，步骤简捷，生产成本低，对环境影响小，可实现肌醇的规模化生产。

肌醇的制备方法

本发明公开了一种肌醇的制备方法，属于肌醇的酶催化制备领域。本发明所公开的肌醇的制备方法以淀粉、纤维素或它们的衍生物为底物，在一个多酶反应体系中，通过体外多酶高效催化将底物转化为肌醇；本发明通过过程优化，添加能够促进淀粉或纤维素水解的酶以及利用副产物葡萄糖的酶，从而建立多酶反应体系，显著提升了原料的转化效率和肌醇的产率。本发明方法原料转化率高，肌醇产率高，步骤简捷，生产成本低，对环境影响小，可实现肌醇的规模化生产。

Method of Making Inositol

본 발명은 이노시톨의 제조방법에 관한 것이다. 기질로서 전분 및 셀룰로오스 또는 이들의 기질을 사용하고, 전분 또는 셀룰로오스의 가수분해를 촉진시킬 수 있는 효소를 가하고, 부산물 글루코스의 효소를 사용함으로써, 다중효소 반응 시스템이 정착되고, 기질은 이노시톨로 전환된다. 원료의 전환율 및 이노시톨의 수율이 개선된다. The present invention relates to a process for preparing inositol. By using starch and cellulose or a substrate thereof as a substrate, adding an enzyme capable of promoting hydrolysis of starch or cellulose, and using an enzyme of byproduct glucose, a multienzyme reaction system is settled and the substrate is converted to inositol. . The conversion of raw materials and the yield of inositol are improved.

Cellular production of glucaric acid

본 발명은 미오-이노시톨 1-포스페이트 신타제, 미오-이노시톨 옥시게나제 및 우로네이트 데히드로게나제의 재조합 발현을 통해 세포에서 글루쿠론산 및 글루카르산을 제조하는 것에 관한 것이다. 또한, 우로네이트 데히드로게나제를 코딩하는 유전자의 클로닝 및 특성분석이 개시되어 있다. The present invention relates to the production of glucuronic acid and glucuronic acid in cells through the recombinant expression of myo-inositol 1-phosphate synthase, myo-inositol oxygenase and uronate dehydrogenase. In addition, cloning and characterization of a gene encoding uronate dehydrogenase is disclosed.

Diterpene production

The present invention relates to a recombinant microorganism comprising one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity, whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol. The recombinant microorganism may also be capable of expressing one or more UDP-glucosyltransferases such that the microorganism is capable of producing one or more steviol glycosides.

Seed-preferred regulatory elements and uses thereof

The present invention provides compositions and methods for regulating expression of isolated nucleotide sequences in a plant. The compositions are novel nucleic acid sequences for seed-preferred regulatory sequences. Methods for expressing an isolated nucleotide sequence in a plant using the regulatory sequences are also provided. The methods comprise transforming a plant cell to contain an isolated nucleotide sequence operably linked to the seed-preferred regulatory sequences of the present invention and regenerating a stably transformed plant from the transformed plant cell.

Recombinant microoganisms for producing steviolmonoside and method for steviolmonoside using the same

The present invention relates to a recombinant microorganism for producing steviol monoside transformed with a recombinant vector and a method for producing steviol monoside using the recombinant microorganism. The recombinant microorganism transformed with the recombinant vector according to the present invention has an excellent effect of producing a large amount of steviol monoside as a sweetener component of stevia. Therefore, when the recombinant microorganism of the present invention is used, the metabolic pathway is manipulated by the recombinant gene introduced into the microorganism, rather than directly isolating the steviol monoside from the extract of Stevia, so that the steviol monoside can be mass- And can be usefully used in food industries such as natural sweeteners and beverages using the same.

Seed-preferred regulatory elements and uses thereof

The present invention provides compositions and methods for regulating expression of isolated nucleotide sequences in a plant. The compositions are novel nucleic acid sequences for callus-tissue and seed embryo-preferred regulatory sequences. Methods for expressing an isolated nucleotide sequence in a plant or callus tissue using the regulatory sequences are also provided. The methods comprise transforming a plant cell to contain an isolated nucleotide sequence operably linked to the regulatory sequences of the present invention and regenerating a stably transformed plant or callus tissue from the transformed plant cell.

Escherichia coli recombinant bacterium capable of efficiently utilizing glucose to synthesize inositol, and construction method and application thereof

本发明公开了一种高效利用葡萄糖合成肌醇的大肠杆菌重组菌及其构建方法与应用。本发明所提供的产肌醇的大肠杆菌重组菌构建方法包括：将肌醇‑3‑磷酸合成酶基因和肌醇单磷酸酶基因导入宿主菌，得到产肌醇的大肠杆菌重组菌；所述宿主菌为野生型大肠杆菌或突变型大肠杆菌；所述突变型大肠杆菌为将突变型大肠杆菌SG104基因组中的葡萄糖‑6‑磷酸异构酶基因或磷酸葡萄糖变位酶基因或葡萄糖‑6‑磷酸脱氢酶基因敲除后得到的突变型大肠杆菌。利用本发明所构建的产肌醇的大肠杆菌重组菌可以高效利用葡萄糖合成肌醇，肌醇产量最高可达375mM(67.5g/L)。本发明对于肌醇生产来说，将产生巨大的经济效益，具有重大的推广应用价值。

Xanthohumol-related protein and application thereof in stabilizing product in xanthohumol synthesis pathway

本发明公开了黄腐醇相关蛋白及其在稳定黄腐醇合成途径中产物中的应用。本发明公开的黄腐醇相关蛋白为如下A1)、A2)或A3)：A1)氨基酸序列是序列1的蛋白质；A2)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。实验证明，本发明的黄腐醇相关蛋白具有稳定黄腐醇合成途径中产物的作用，在黄酮类化合物和脱甲基黄腐醇合成生物学应用以及人类健康保健等领域有广泛的应用前景。

Enzyme expressed in saccharomyces cerevisiae, genetic engineering bacteria for high yield of alpha-and gamma-tocotrienols and construction method thereof

本发明公开了一种在酿酒酵母中表达的关键酶及高产α‑和γ‑生育三烯酚的基因工程菌及其构建方法。本发明通过基因克隆和密码子优化，获得五个可以在酿酒酵母中成功表达的酶，分别为HPPD、HPT、MPBQMT、TC和γ‑TMT；并将这五个酶逐步整合到酿酒酵母基因组上，进一步通过切除叶绿体转运肽和过表达限速酶，构建得到高产α‑和γ‑‑生育三烯酚的基因工程酵母菌株，命名为：YS‑356c，保藏编号为：CCTCC NO：M2019572。其总生育三烯酚产量达到2.09mg/g细胞干重。本发明的工程菌株可经发酵培养，直接从菌体中获得α‑和γ‑生育三烯酚，具有良好的市场前景和开应用价值。

Optimized recombination and application of 4-chlorocatechol degradation gene cluster

本发明公开了一种优化后适用于大肠杆菌表达的4‑氯邻苯二酚降解相关的基因簇及其应用。该基因簇包含四个基因，每个基因都由独立的T7启动子和终止子控制，其核苷酸序列分别如SEQ ID No 1、SEQ ID No 2、SEQ ID No 3和SEQ ID No 4所示，其编码的蛋白质的氨基酸序列分别如SEQ ID No 5、SEQ ID No 6、SEQ ID No 7和SEQ ID No 8所示。本发明优化合成的基因能够在大肠杆菌中成功表达，表达这个基因簇的大肠杆菌不仅能够降解4‑氯邻苯二酚，还能够降解邻苯二酚和3‑氯邻苯二酚，因此该基因簇可用于制备降解邻苯二酚，3‑氯邻苯二酚和4‑氯邻苯二酚的微生物。

Recombinant microoganisms for producing steviolmonoside and method for steviolmonoside using the same

본 발명은 재조합 벡터로 형질전환된 스테비올모노사이드 생산용 재조합 미생물 및 상기 재조합 미생물을 이용한 스테비올모노사이드의 생산방법에 관한 것이다. 본 발명에 따른 재조합 벡터로 형질전환된 재조합 미생물은 스테비아의 감미 성분으로서 스테비올의 배당체인 스테비올모노사이드를 다량으로 생산하는 우수한 효과가 있다. 따라서, 본 발명의 재조합 미생물을 이용할 경우, 스테비아의 추출물로부터 직접 스테비올모노사이드를 분리하는 것이 아닌, 미생물에 도입된 재조합 유전자에 의해 대사경로를 조작함으로써, 간단한 공정만으로도 스테비올모노사이드를 대량으로 제공할 수 있어, 이를 이용하는 천연 감미료, 음료 등의 식품 산업에 유용하게 이용될 수 있다.

Chalcone isomerase gene of asparaguses, protein encoded by chalcone isomerase gene and application of chalcone isomerase gene

本发明提供芦笋查尔酮异构酶基因及其编码的蛋白与应用。芦笋查尔酮异构酶基因AoCHI1的CDS序列如SEQ ID NO:1所示，其编码蛋白的氨基酸序列如SEQ ID NO:2所示。本发明首次从芦笋中克隆得到查尔酮异构酶基因AoCHI1，该基因为植物类黄酮合成路径中的关键基因之一，采用基因工程的方法，将基因AoCHI1转化到目标植株中，可促进转基因植株中总黄酮含量的增加，为今后利用基因工程技术改良植物品质，获得具有高抗氧化性的药物或食物提供了重要的理论依据，具有广阔的应用前景和极大的经济价值。

Lce mutant of key gene in wheat carotenoid synthetic pathway and application thereof

本发明公开了小麦类胡萝卜素合成途径关键基因Lcye突变体及其应用。本发明提供了如下蛋白质：将Lcye‑D1蛋白的第253位的丝氨酸替换为苯丙氨酸后得到的蛋白质，或将其经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同能力的衍生蛋白质，或与其所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质，或在其N端和/或C端连接标签后得到的融合蛋白。本发明不仅验证Lcye基因功能，也为面粉及其制品颜色性状改良提供了理论依据和种质资源。

Cellular production of glucaric acid

Method for producing myo-inositol and myo-inositol derivative

[과제] 유전자 재조합 기술과 합성 생물학적 수법의 적용에 적합한, 대장균 등의 내인성 미오이노시톨 생합성 경로를 갖지 않는 숙주 미생물에 대하여, 유의적으로 개선된 미오이노시톨 생산능을 부여하는 것. [해결수단] 내인성 미오이노시톨 생합성 경로를 갖지 않는 숙주 미생물 내에 미오이노시톨 생합성 경로를 도입하여 얻어지는 형질 전환체에 있어서, 이노시톨 모노포스파타아제 활성을 강화한다. [PROBLEMS] To provide a significantly improved myoinositol producing ability for a host microorganism which does not have an endogenous myoinositol biosynthetic pathway such as Escherichia coli, which is suitable for application of genetic recombination technology and synthetic biological techniques. [Solution] Inositol monophosphatase activity is enhanced in a transformant obtained by introducing a myoinositol biosynthetic pathway into a host microorganism having no endogenous myoinositol biosynthetic pathway.

Inositol preparation method

Provided is an inositol preparation method. By using starch and cellulose or substrates thereof as substrates, adding an enzyme capable of promoting hydrolysis of the starch or the cellulose and by using an enzyme of a by-product glucose, a multienzyme reaction system is established, and the substrates are converted into inositol. The conversion rates of raw materials and the yield of the inositol are improved.

Diterpene production in yarrowia

Production of flavonoids by recombinant microorganisms

Methods and compositions are provided for production of flavonoids in microbial hosts. The compositions comprises a set of genes which encode for enzymes involved in one or more steps in the biosynthetic pathway for the conversion of phenylpropanoids to various flavonoids. The method comprises the steps of introducing the set of genes into a heterologous host cell, allowing growth of the cells in a suitable medium such that the expression of the genes results in production of enzymes. When specific substrate(s) is/are provided to the transformed cell, the enzymes act on the substrate(s) to produce the desired flavonoids.

Recombinant yeast with artificial cellular organelles and producing method for isoprenoids with same

본 발명은 유전자 조작을 통하여 세포 소기관이 변이된 재조합 효모 및 이를 이용한 다양한 아이소프레노이드 생산 방법에 관한 것이다. 유전자 재조합을 통해 세포 소기관인 퍼옥시좀의 개수 또는 크기가 증가된 변이된 효모에 유용산물인 아이소프레노이드 대사 경로를 도입하는 경우, 세포 소기관 변이가 없는 효모에 비하여 아이소프레노이드 생산량을 현저하게 증대시킬 수 있어 아이소프레노이드의 대량 생산이 가능하며, 이를 통해 아이소프레노이드가 적용되는 다양한 산업 분야에 유용하게 활용 가능하다. The present invention relates to a recombinant yeast in which organelles have been mutated through genetic manipulation, and a method for producing various isoprenoids using the same. When introducing the isoprenoid metabolic pathway, which is a useful product, to mutated yeast whose number or size of peroxisomes, which are organelles, is increased through genetic recombination, the amount of isoprenoid production is significantly increased compared to yeast without cell organelle mutations. This enables mass production of isoprenoids, which can be usefully used in various industrial fields to which isoprenoids are applied.

Transgenic chlamydomonas for improving lutein content of chlamydomonas reinhardtii as well as construction method and application of transgenic chlamydomonas

本发明提供一种用于提高莱茵衣藻叶黄素含量的转基因衣藻及其构建方法、应用，所述构建方法通过扩增得到普通小球藻的番茄红素ε‑环化酶基因，构建所述番茄红素ε‑环化酶基因重组表达载体；将所述番茄红素ε‑环化酶基因重组表达载体转化至莱茵衣藻细胞中，得到转番茄红素ε‑环化酶基因莱茵藻。所得到的转基因衣藻能够高产叶黄素，利用藻类无性繁殖、生长快速的特点，因此可以低成本，快速地获取叶黄素。

Extracellular diterpene production

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism in a suitable fermentation medium, wherein the microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity; a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol, whereby a diterpene or glycosylated diterpene is produced extracellularly in the fermentation medium; and b. recovering the diterpene or glycosylated diterpene from the fermentation medium.

Methods and compositions for determining isomerase enzymatic activity

Tulip chalcone isomerase TfCHI protein and encoding gene thereof

本发明涉及一种郁金香查尔酮异构酶TfCHI蛋白及其编码基因，所述蛋白质为由如SEQ ID NO.4所示的氨基酸序列组成的蛋白质或如SEQ ID NO.4所示的氨基酸序列经过取代、缺失或者添加一个或几个氨基酸且具有郁金香查尔酮异构酶活性的蛋白质。本发明还提供了一种编码上述蛋白质的如SEQ ID NO.3所示的核酸序列；利用本发明的郁金香查尔酮异构酶基因，通过各种常规筛选方法，能筛选出与查尔酮异构酶TfCHI相关发生相互作用的物质、受体、抑制剂或拮抗剂等。利用本发明的郁金香查尔酮异构酶基因，为通过基因工程改变花色，创造新花色提供了一种新途径。

Method for generating new gene in organism and use thereof

Provided is a method for creating a new gene in an organism in the absence of an artificial DNA template, and a use thereof. The method comprises simultaneously generating DNA breaks at two or more different specific sites in the organism's genome, wherein the specific sites are genomic sites capable of separating different gene elements or different protein domains, and the DNA breaks are ligated to each other through non-homologous end joining (NHEJ) or homologous repair to generate a new combination of the different gene elements or different protein domains that is different from the original genome sequence, thereby creating a new gene. The new gene can change the growth, development, resistance, yield and other traits of the organism.

Inositol-3-phosphate synthase mutant and application thereof in constructing corynebacterium glutamicum capable of producing glutamine at high yield

本发明涉及生物工程领域，具体涉及一种肌醇‑3‑磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用。本发明发现，肌醇‑3‑磷酸合酶第84位丝氨酸突变为其他氨基酸后，有助于在谷氨酸棒杆菌中增强其产生谷氨酰胺的能力。与未发生上述突变的野生型菌株相比，本发明中谷氨酸棒杆菌产生谷氨酰胺的能力得到增强，谷氨酰胺产量从28.5g/L提高到30.7g/L，产酸提高7.7％，有利于对谷氨酰胺高产菌株的筛选和构建。

Extracellular diterpene production

本发明涉及一种生产二萜或糖基化二萜的方法，所述方法包括：a.使重组微生物在合适的发酵培养基中发酵，其中所述微生物包含编码：具有内根-柯巴基焦磷酸合酶活性的多肽；具有内根-贝壳杉烯合酶活性的多肽；具有内根-贝壳杉烯氧化酶活性的多肽；和具有贝壳杉烯酸13-羟化酶活性的多肽的一个或多个核苷酸序列，并且其中所述核苷酸序列的表达赋予所述微生物生产至少甜菊醇的能力，其中在所述发酵培养基中在细胞外生产二萜或糖基化二萜；以及b.从所述发酵培养基回收二萜或糖基化二萜。

Polypeptides with diterpene synthase activity

The present invention relates to polypeptides with diterpene synthase activity. The invention further relates to polynucleotides encoding such polypeptides. Also provided are vectors for expression of the polypeptides and host cells expressing the polypeptides.

Method for producing myo-inositol and myo-inositol derivative

[과제] 유전자 재조합 기술과 합성 생물학적 수법의 적용에 적합한, 대장균 등의 내인성 미오이노시톨 생합성 경로를 갖지 않는 숙주 미생물에 대하여, 유의적으로 개선된 미오이노시톨 생산능을 부여하는 것. [해결수단] 내인성 미오이노시톨 생합성 경로를 갖지 않는 숙주 미생물 내에 미오이노시톨 생합성 경로를 도입하여 얻어지는 형질 전환체에 있어서, 이노시톨 모노포스파타아제 활성을 강화한다.

Steviol glycoside transport

A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto.

Tripterygium wilfordii TwKS and applications of the TwCPS3 in Kaurane diterpine compound is prepared

本发明涉及雷公藤柯巴基焦磷酸合酶TwCPS3和雷公藤贝壳杉烯合酶TwKS在制备贝壳杉烷型二萜中的应用。通过对雷公藤柯巴基焦磷酸合酶基因TwCPS3和雷公藤贝壳杉烯合酶基因TwKS进行重组表达，在重组蛋白的催化下，成功的以GGPP为底物合成了赤霉素的前体衍生物贝壳杉烷型二萜化合物。本发明对赤霉素的生物合成、以及雷公藤甲素等二萜化合物的合成调控具有重要的意义。

Production of manool

本文提供了生产(+)‑迈诺醇的方法，包括：将香叶基香叶基二磷酸与柯巴基二磷酸(CPP)合酶接触以形成(9S,10S)‑柯巴基二磷酸，其中该CPP合酶包含与从SEQ ID NO:1和SEQ ID NO:2中选出的多肽具有至少90％、95％、98％、99％和100％序列同一性的氨基酸序列；并使CPP与香紫苏醇合酶接触以形成(+)‑迈诺醇。

manool production

Method of production of myo-inositol and myo-inositol derivative

【課題】適用於基因重組技術及合成生物學的方法，對於大腸菌等未具有內因性肌肉肌醇生合成路徑之宿主微生物賦予顯著改善之肌肉肌醇生產能。【解決方法】在未具有內因性肌肉肌醇生合成路徑之宿主微生物內導入肌肉肌醇生合成路徑獲得之形質轉化體，強化肌醇單磷酸酶活性。

Promoter for regulating and controlling expression of yeast exogenous gene, regulation and control method and application thereof

本发明提供了调控酵母外源基因表达的启动子，调控方法及其应用。本发明通过对酵母中GAL调控系统进行改造，从而达到不需半乳糖诱导只通过调节发酵液中的葡萄糖浓度即可控制GAL调控系统中GAL启动子所控制的基因表达的目的。本发明只采用葡萄糖激活型启动子HXT1替换GAL80启动子即可达到上述目的。本发明还将该方法用于酿酒酵母β‑胡萝卜素生物合成的调控，验证了所述调控系统的有效性。

Seed-preferred regulatory elements and uses thereof

The present invention provides compositions and methods for regulating expression of isolated nucleotide sequences in a plant. The compositions are novel nucleic acid sequences for seed-preferred regulatory sequences. Methods for expressing an isolated nucleotide sequence in a plant using the regulatory sequences are also provided. The methods comprise transforming a plant cell to contain an isolated nucleotide sequence operably linked to the seed-preferred regulatory sequences of the present invention and regenerating a stably transformed plant from the transformed plant cell.

Function and application of SiLCYE for regulating and controlling anabolism of zeaxanthin and other millet carotenoids

本发明公开了提高谷子玉米黄质或/和叶黄素硬脂酸酯或/和花药黄质含量的方法，所述方法包括通过抑制或降低谷子中蛋白质编码基因的表达量，和/或，抑制或降低谷子中所述的蛋白质的活性和/或含量，来提高谷子中玉米黄质或/和叶黄素硬脂酸酯或/和花药黄质的含量；所述蛋白质为SiLCYE蛋白。通过同源克隆技术克隆了SiLCYE。SiLCYE氨基酸序列如SEQ ID NO.1所示，其编码序列如SEQ ID NO.2所示。通过CRISPR‑Cas9编辑技术获得SiLCYE基因的编辑株系，通过代谢组学分析明确了这两个基因在谷子类胡萝卜素代谢反应中的调控作用，为揭示谷子类胡萝卜素代谢途径调控机制及培育富含类胡萝卜素高营养品质的谷子新种质提供了实验基础和基因资源。

ABC transporter for efficient production of rebaudiosides

本发明提供了用于改进甜菊糖苷生成量的经遗传修饰的宿主细胞、组合物和方法。在一些实施方案，所述宿主细胞经遗传修饰以包含异源核酸表达盒，所述异源核酸表达盒表达能够将甜菊糖苷转运至细胞外空间或转运至细胞内细胞器的腔空间的ABC转运蛋白。在一些实施方案，所述宿主细胞进一步包含一种或多种异源核苷酸序列，所述一种或多种异源核苷酸序列编码能够在所述宿主细胞中生成一种或多种甜菊糖苷的途径的其他酶。本发明所述的宿主细胞、组合物和方法提供了异源生成甜菊糖苷(包括但不限于，瑞鲍迪苷D和瑞鲍迪苷M)的有效途径。

The recombinant production of steviol glycoside

本文公开了经过改造能够表达编码甜菊醇生物合成酶和UDP-糖基转移酶(UGTs)的新型重组基因的重组微生物、植物和植物细胞。这些微生物、植物或植物细胞可以产生甜菊醇或甜菊糖苷(例如甜叶悬钩子苷或莱鲍迪苷A)，作为食品中的天然甜味剂和膳食补充剂。

Optimized recombination and application of 3-chlorocatechol degradation gene cluster

本发明公开了一种优化后适用于大肠杆菌表达的3‑氯邻苯二酚降解基因簇及其应用。该基因簇包含四个相关基因，每个基因都由独立的T7启动子和终止子控制，其核苷酸序列分别如SEQ ID No 1、SEQ ID No 2、SEQ ID No 3和SEQ ID No 4所示，其编码的蛋白质的氨基酸序列分别如SEQ ID No 5、SEQ ID No 6、SEQ ID No 7和SEQ ID No 8所示。本发明优化合成的基因能够在大肠杆菌中成功表达，发现阳性株系不仅能够降解3‑氯邻苯二酚，还可以降解邻苯二酚和4‑氯邻苯二酚，因此本发明所优化的基因簇可用于制备降解3‑氯邻苯二酚、邻苯二酚和4‑氯邻苯二酚的微生物。

Pichia pastoris engineering bacteria for producing inositol and fermentation method

本发明属于生物技术领域，具体涉及一种生产肌醇的毕赤酵母工程菌及发酵方法。本发明通过在毕赤酵母中过表达内源和外源的肌醇合成关键酶基因，并敲除肌醇转运蛋白基因，提升毕赤酵母积累肌醇的能力；通过敲除果糖‑6‑磷酸激酶2（Pfk2）基因，并利用甘油诱导启动子调控表达葡萄糖‑6‑磷酸异构酶（Pgi）基因、果糖‑6‑磷酸激酶1（Pfk1）基因和葡萄糖‑6‑磷酸脱氢酶（Zwf）基因，实现在以葡萄糖为碳源时弱化糖酵解和磷酸戊糖途径的目的，从而提高肌醇合成前体物葡萄糖‑6‑磷酸的供给。在高密度发酵中，利用甘油和葡萄糖在培养基中的添加时间来控制菌体生长及肌醇的生产，大大提高了毕赤酵母工程菌株肌醇的生产能力。

Function and application of SiLCYE for regulating and controlling anabolism of zeaxanthin and other millet carotenoids

本发明公开了提高谷子玉米黄质或/和叶黄素硬脂酸酯或/和花药黄质含量的方法，所述方法包括通过抑制或降低谷子中蛋白质编码基因的表达量，和/或，抑制或降低谷子中所述的蛋白质的活性和/或含量，来提高谷子中玉米黄质或/和叶黄素硬脂酸酯或/和花药黄质的含量；所述蛋白质为SiLCYE蛋白。通过同源克隆技术克隆了SiLCYE。SiLCYE氨基酸序列如SEQ ID NO.1所示，其编码序列如SEQ ID NO.2所示。通过CRISPR‑Cas9编辑技术获得SiLCYE基因的编辑株系，通过代谢组学分析明确了这两个基因在谷子类胡萝卜素代谢反应中的调控作用，为揭示谷子类胡萝卜素代谢途径调控机制及培育富含类胡萝卜素高营养品质的谷子新种质提供了实验基础和基因资源。

Method of producing terpenes or terpenoids

The present invention relates to arecombinant Deinococcus bacterium exhibiting enhanced 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DXP) pathway, and its use for producing terpene or terpenoid compounds.

Immobilized multienzyme system for producing inositol and method for producing inositol

本发明涉及一种用于生产肌醇的固定化多酶体系，其由多孔多巴胺微球与用于生产肌醇的多酶混合物均匀混合而成。本发明将淀粉转化为肌醇的酶法催化路径中的全部4个酶采用多孔微球同时进行固定化从而获得固定化多酶体系，利用该固定化多酶体系催化淀粉转化为肌醇，可以实现酶的可回收利用，从而大大降低了肌醇制备所需投入的酶用量，减少了生产成本。

Directional design and biosynthesis method of high-activity phenylpropanoid derivatives

本发明公开了一种高活性苯丙素类衍生物的定向设计及其生物合成方法。该设计方法利用氨基肉桂酸作为苯丙素类化合物的合成前体，并利用计算机辅助设计对所获得衍生物与靶蛋白进行对接来评价其生物活性高低。本发明主要涉及三个高活性苯丙素类衍生物，氨基双去甲氧基姜黄素(C 19 H 18 N 2 O 2 )、氨基白藜芦醇(C 14 H 13 NO 2 )及氨基柚皮素(C 15 H 13 NO 4 )，结构分别为： 本发明构建了可生产该类衍生物的大肠杆菌工程菌，并进行发酵及分离纯化。本发明中所采用的设计策略为天然产物的定向设计及生物合成提供了借鉴。

Process for the biotechnological production of dihydrochalcones

Beschrieben werden ein Verfahren zur Herstellung eines Dihydrochalkons, insbesondere von Phloretin, unter Verwendung eines transgenen Mikroorganismus, enthaltend einen Nukleinsäure-Abschnitt (a), umfassend oder bestehend aus einem für eine bakterielle Chalconisomerase kodierenden Gen, und/oder einen Nukleinsäure-Abschnitt (a‘), umfassend oder bestehend aus einem für eine pflanzliche Chalconisomerase kodierenden Gen, sowie einen Nukleinsäure-Abschnitt (b), umfassend oder bestehend aus einem für eine bakterielle Enoat-Reduktase kodierenden Gen, entsprechende transgene Mikroorganismen, enthaltend einen Nukleinsäure-Abschnitt (a), umfassend oder bestehend aus einem für eine bakterielle Chalconisomerase kodierenden Gen, und/oder einen Nukleinsäure-Abschnitt (a‘), umfassend oder bestehend aus einem für eine pflanzliche Chalconisomerase kodierenden Gen, und/oder einen Nukleinsäure-Abschnitt (b), umfassend oder bestehend aus einem für eine bakterielle Enoat-Reduktase kodierenden Gen, sowie Wirtszellen, die ein oder mehrere identische oder unterschiedliche solche Vektoren enthalten. Described are a method for producing a dihydrochalcone, in particular phloretin, using a transgenic microorganism containing a nucleic acid section (a) comprising or consisting of a gene coding for a bacterial chalconisomerase, and / or a nucleic acid section (a ' ), comprising or consisting of a gene coding for a plant chalconisomerase, and a nucleic acid section (b), comprising or consisting of a gene coding for a bacterial enoate reductase, corresponding transgenic microorganisms containing a nucleic acid section (a), comprising or consisting of a gene coding for a bacterial chalconisomerase, and / or a nucleic acid section (a ') comprising or consisting of a gene coding for a plant chalconisomerase, and / or a nucleic acid section (b) comprising or consisting from a coding for a bacterial enoate reductase n gene, as well as host cells containing one or more identical or different such ...

Recombinant microorganism with kauronic acid production ability and method for preparing kauronic acid using the same

본 발명은 스테비오사이드의 전구체인 카우린산 생성능을 가지는 재조합 미생물 및 이를 이용한 카우린산의 제조 방법에 관한 것이다. 본 발명에 따르면 스테비아의 추출물로부터 직접 분리하는 것이 아닌, 미생물에 도입된 재조합 유전자에 의해 대사경로를 조작함으로써, 간단한 공정만으로도 천연 감미료인 스테비오사이드의 전구체인 카우린산을 다량으로 제공할 수 있다.

Method of producing α-ionone by fermentation

Method for enzymatically preparing highly concentrated myo-inositol

The present disclosure relates to a method for preparing myo -inositol using myo -inositol monophosphate synthase consisting of an amino acid sequence of SEQ ID NO: 1 and/or myo -inositol monophosphate phosphatase consisting of an amino acid sequence of SEQ ID NO: 3.

Method for producing myo-inositol and myo-inositol derivative

Compositions and methods of biosynthesizing carotenoids and their derivatives

The present invention relates to compositions and methods of producing carotenoids and carotenoid derivatives.