METHOD FOR THE ENZYMATIC PRODUCTION OF 3-BUTEN-2-ONE

Described is a method for the production of 3-buten-2-one comprising the enzymatic conversion of 4-hydroxy-2-butanone into 3-buten-2-one by making use of an enzyme catalyzing 4-hydroxy-2-butanone dehydration, wherein said enzyme catalyzing 4-hydroxy-2-butanone dehydration is (a) a 3-hydroxypropiony-CoA dehydratase (EC 4.2.1.116), (b) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), (c) an enoyl-CoA hydratase (EC 4.2.1.17), (d) a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59), (e) a crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58), (f) a 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.60), (g) a 3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.61 ), (h) a long-chain-enoyl-CoA hydratase (EC 4.2.1.74), or (i) a 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18). The produced 3-buten-2-one can be further converted into 3-buten-2-ol and finally into 1,3-butadiene.

RECOMBINANT N-PROPANOL AND ISOPROPANOL PRODUCTION

The present invention relates to methods of producing n-propanol, isopropanol, and coproducing n-propanol with isopropanol. The present invention also relates to methods for producing propylene, as well as host cells capable of n-propanol and isopropanol production.

CONTINUOUS FLOW METHOD FOR PREPARING (R)-3-HYDROXY-5-HEXENOATE

Disclosed herein relates to biopharmaceuticals, and more particularly to a continuous flow method for preparing (R)-3-hydroxy-5-hexenoate. Carbonyl reductase and isopropanol dehydrogenase are co-immobilized onto an inert solid medium simultaneously to prepare a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, which is then filled into a microchannel reactor of the micro reaction system. A solution containing substrate 3-carbonyl-5-hexenoate is subsequently pumped into the microchannel reactor to perform an asymmetric carbonyl reduction reaction to obtain (R)-3-hydroxy-5-hexenoate. 2. The method of claim 1 , wherein in step (1) claim 1 , the inert solid medium is a composite material of polyvinyl alcohol and polyethylene glycol; and the step of “co-immobilizing a carbonyl reductase and an isopropanol dehydrogenase onto an inert solid medium simultaneously to prepare the co-immobilized catalyst” comprises:(a) preparing an aqueous solution of the polyvinyl alcohol and the polyethylene glycol; heating the aqueous solution until the aqueous solution becomes clear; and cooling the aqueous solution to 50° C. or less to obtain a first solution;(b) adding a crude carbonyl reductase solution and a crude isopropanol dehydrogenase solution into the first solution followed by uniform mixing to obtain a second solution; and(c) dropwise adding the second solution onto a polyethylene film; drying the polyethylene film at 35-40° C. for 0.5-1 hour to obtain the co-immobilized catalyst; and storing the co-immobilized catalyst at 4° C. for later use;wherein an amino acid sequence of the carbonyl reductase is shown in SEQ ID NO: 1; and an amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO: 2;a weight ratio of the polyvinyl alcohol to the polyethylene glycol is 5:1-3;the crude carbonyl reductase solution and the crude isopropanol dehydrogenase solution both have an initial concentration of 10%-30% (w/v); andin step (b), a volume ratio of the ...

BIOSYNTHESIS OF 1,3-BUTANEDIOL

This document describes biochemical pathways for producing 1,3-butanediol using a polypetide having β-ketothiolase activity to form a 3-oxo-5-hydroxypentanoyl-CoA intermediate that can be enzymatically converted to 1,3-butanediol, as well as recombinant hosts producing 1,3-butanediol. 1: A method of producing 3-oxo-5-hydroxypentanoyl-CoA , said method comprising enzymatically converting 3-hydroxypropionyl-CoA to 3-oxo-5-hydroxypentanoyl-CoA using a polypeptide having β-ketothiolase activity classified under EC. 2.3.1.-.2: The method of claim 1 , wherein said polypeptide having β-ketothiolase activity is classified under EC 2.3.1.16 or EC 2.3.1.174 and/or has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 claim 1 , 2 claim 1 , 7 or 13.34-. (canceled)5: The method of claim 1 , further comprising enzymatically converting 3-oxo-5-hydroxypentanoyl-CoA to 1 claim 1 ,3-butanediol using a thioesterase or a CoA transferase claim 1 , a decarboxylase claim 1 , and a secondary alcohol dehydrogenase.6: The method of claim 5 , wherein said thioesterase is classified under EC 3.1.2.- and/or has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 or 14.7. (canceled)8: The method of claim 5 , wherein said CoA transferase is classified under EC 2.8.3.- claim 5 , said decarboxylase is classified under EC 4.1.1.4 claim 5 , and said secondary alcohol dehydrogenase is classified under EC 1.1.1.B3 claim 5 , EC 1.1.1.B4 claim 5 , or EC 1.1.1.80.9: The method of claim 8 , wherein said CoA transferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or 6 claim 8 , said decarboxylase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 10 claim 8 , and said secondary alcohol dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 claim 8 , 9 claim 8 , or 11.1013-. (canceled)14: A method for ...

RECOMBINANT N-PROPANOL AND ISOPROPANOL PRODUCTION

The present invention relates to methods of producing n-propanol, isopropanol, and coproducing n-propanol with isopropanol. The present invention also relates to methods for producing propylene, as well as host cells capable of n-propanol and isopropanol production.

PREPARATION METHOD FOR (R)-3-HYDROXYL-5-HEXENOATE

The present disclosure relates to the technical field of biochemical engineering and particularly discloses a preparation method for (R)-3-hydroxyl-5-hexenoate. In the method of the present disclosure, the (R)-3-hydroxyl-5-hexenoate is prepared by catalytic reduction of 3-carbonyl-5-hexenoate by ketoreductase with 3-carbonyl-5-hexenoate as the substrate. The amino acid sequence of ketoreductase is shown in SEQ ID NO.1. In the present disclosure, the (R)-3-hydroxyl-5-hexenoate having a very high chiral purity is obtained by asymmetric reduction by ketoreductase as the biocatalyst. The present disclosure has the advantages of easy operation, mild reaction conditions, high reaction yield and good practical industrial application value.

Recombinant n-propanol and isopropanol production

The present invention relates to methods of producing n-propanol, isopropanol, and coproducing n-propanol with isopropanol. The present invention also relates to methods for producing propylene, as well as host cells capable of n-propanol and isopropanol production.

Recombinant n-propanol and isopropanol production

The present invention relates to methods of producing n-propanol, isopropanol, and coproducing n-propanol with isopropanol. The present invention also relates to methods for producing propylene, as well as host cells capable of n-propanol and isopropanol production.

L-이소루신 생산능을 가지는 코리네박테리움 속 미생물 및 이를 이용하여 L-이소루신을 생산하는 방법

... 본 발명은 시트라말레이트 신타아제(citramalate synthase) 활성을 가지는 단백질을 포함하는 L-이소루신 생산능을 가지는 코리네박테리움(Corynebacterium) 속 미생물, 및 이를 이용하여 L-이소루신을 생산하는 방법에 관한 것이다.

Biosynthesis of 1,3-butanediol

This document describes biochemical pathways for producing 1,3-butanediol using a polypetide having β-ketothiolase activity to form a 3-oxo-5-hydroxypentanoyl-CoA intermediate that can be enzymatically converted to 1,3-butanediol, as well as recombinant hosts producing 1,3-butanediol.

CORYNEBACTERIUM SP. MICROORGANISM HAVING L-ISOLEUCINE PRODUCTIVITY, AND PRODUCTION METHOD FOR L-ISOLEUCINE USING SAME

The present invention relates to a Corynebacterium sp. microorganism having L-isoleucine productivity, which includes protein with citramalate synthase activity; and to a production method for L-isoleucine using the same. COPYRIGHT KIPO 2017 ...

RECOMBINANT N-PROPANOL AND ISOPROPANOL PRODUCTION

Method for the production of isoamyl alcohol

Described is a method for the production isoamyl alcohol (3-methylbutan-1-ol) comprising the enzymatic conversion of 3-methylbutyryl-CoA (isovaleryl-CoA) into isoamyl alcohol comprising: (a) two enzymatic steps comprising (i) first the enzymatic conversion of 3-methylbutyryl-CoA into 3-methylbutyraldehyde (3-methylbutanal or isovaleraldehyde); and (ii) then enzymatically converting the thus obtained 3-methylbutyraldehyde into said isoamyl alcohol; or (b) a single enzymatic reaction in which 3-methylbutyryl-CoA is directly converted into isoamyl alcohol by making use of an alcohol-forming short chain acyl-CoA dehydrogenase/fatty acyl-CoA reductase or an alcohol-forming fatty acyl-CoA reductase (long-chain acyl-CoA:NADPH reductase) (EC 1.2.1.84). Further, described is the above method wherein the 3-methylbutyryl-CoA can be provided by the enzymatic conversion of 3-methylcrotonyl-CoA into said 3-methylbutyryl-CoA. It is also described that the thus obtained isoamyl alcohol can be further enzymatically ...

METHODS AND CELLS FOR PRODUCTION OF VOLATILE COMPOUNDS

The present invention relates to thermophilic cells and methods for the microbial production of volatile compounds, including acetone, butanone and isopropanol. Also provided are nucleic acid constructs, vectors and host cells useful in such methods.

Engineered bacterial strain and application of same to preparation of (R)-3-hydroxy-5-hexenoate

ISOPROPANOL PRODUCTION BY BACTERIAL HOSTS

Provided herein are Lactobacillusmutants having a disruption to an endogenous gene encoding an acetate kinase, wherein the mutants produce increased amounts of recombinant isopropanol. Also described aremethods forproducing the mutants and methods of using the mutants to produce isopropanol.

METHOD FOR THE PRODUCTION OF ISOAMYL ALCOHOL

Described is a method for the production isoamyl alcohol (3-methylbutan-1-ol) comprising the enzymatic conversion of 3-methylbutyryl-CoA (isovaleryl-CoA) into isoamyl alcohol comprising: (a) two enzymatic steps comprising (i) first the enzymatic conversion of 3-methylbutyryl-CoA into 3-methylbutyraldehyde (3-methylbutanal or isovaleraldehyde); and (ii) then enzymatically converting the thus obtained 3-methylbutyraldehyde into said isoamyl alcohol; or (b) a single enzymatic reaction in which 3-methylbutyryl-CoA is directly converted into isoamyl alcohol by making use of an alcohol-forming short chain acyl-CoA dehydrogenase/fatty acyl-CoA reductase or an alcohol-forming fatty acyl-CoA reductase (long-chain acyl-CoA:NADPH reductase) (EC 1.2.1.84). Further, described is the above method wherein the 3-methylbutyryl-CoA can be provided by the enzymatic conversion of 3-methylcrotonyl-CoA into said 3-methylbutyryl-CoA. It is also described that the thus obtained isoamyl alcohol can be further enzymatically converted into 3-methylbutyl acetate (isoamyl acetate) as described herein. Described are also recombinant organisms or microorganisms which are capable of performing the above enzymatic conversions. Furthermore, described are uses of enzymes and enzyme combinations which allow the above enzymatic conversions. 1. A method for the production of isoamyl alcohol (3-methylbutan-1-ol) comprising the enzymatic conversion of 3-methylbutyryl-CoA into isoamyl alcohol comprising: (i) first the enzymatic conversion of 3-methylbutyryl-CoA into 3-methylbutyraldehyde; and', '(ii) then enzymatically converting the thus obtained 3-methylbutyraldehyde into said isoamyl alcohol; or, '(a) two enzymatic steps comprising'}(b) a single enzymatic reaction in which 3-methylbutyryl-CoA is directly converted into isoamyl alcohol by making use of an alcohol-forming short chain acyl-CoA dehydrogenase/fatty acyl-CoA reductase or an alcohol-forming fatty acyl-CoA reductase (long-chain acyl-CoA:NADPH ...

METHOD FOR PRODUCING 2-BUTANOL AND RECOMBINANT MICROORGANISM HAVING 2-BUTANOL PRODUCTION CAPACITY

This invention is intended to produce 2-butanol with excellent productivity via a fermentation process. Recombinant microorganisms into which the acetoacetyl-CoA synthase gene and a group of genes (i.e., genes involved in 2-propanol synthesis) encoding a set of enzymes synthesizing 2-propanol from acetoacetyl-CoA have been introduced are cultured, so that, in addition to 2-propanol, 2-butanol is produced at a high level in a medium.

Preparation method for (R)-3-hydroxyl-5-hexenoate

The present disclosure relates to the technical field of biochemical engineering and particularly discloses a preparation method for (R)-3-hydroxyl-5-hexenoate. In the method of the present disclosure, the (R)-3-hydroxyl-5-hexenoate is prepared by catalytic reduction of 3-carbonyl-5-hexenoate by ketoreductase with 3-carbonyl-5-hexenoate as the substrate. The amino acid sequence of ketoreductase is shown in SEQ ID NO.1. In the present disclosure, the (R)-3-hydroxyl-5-hexenoate having a very high chiral purity is obtained by asymmetric reduction by ketoreductase as the biocatalyst. The present disclosure has the advantages of easy operation, mild reaction conditions, high reaction yield and good practical industrial application value.

Method for producing 2-butanol and recombinant microorganism having 2-butanol production capacity

This invention is intended to produce 2-butanol with excellent productivity via a fermentation process. Recombinant microorganisms into which the acetoacetyl-CoA synthase gene and a group of genes (i.e., genes involved in 2-propanol synthesis) encoding a set of enzymes synthesizing 2-propanol from acetoacetyl-CoA have been introduced are cultured, so that, in addition to 2-propanol, 2-butanol is produced at a high level in a medium.

célula hospedeira recombinante de lactobacillus, e, métodos para produzir isopropanol, isopropanol e n-propanol, e propileno

METHOD OF PRODUCING 2-BUTANOL AND RECOMBINANT MICROORGANISM HAVING 2-BUTANOL PRODUCTION CAPACITY

PROBLEM TO BE SOLVED: To produce 2-butanol using a fermentation process with high productivity. SOLUTION: There is provided a method of producing 2-butanol with high productivity in a medium, in addition to 2-propanol, by culturing recombinant microorganisms into which an introduced acetoacetyl-CoA synthase gene and a group of genes, i.e. genes involved in 2-propanol synthesis, encoding a set of enzymes synthesizing 2-propanol from acetoacetyl-CoA have been introduced. COPYRIGHT: (C)2012,JPO&INPIT ...

método para produzir 3-oxo-5-hidroxipentanoil-coa, método para biossintetizar 1,3-butanodiol, hospedeiro recombinante, produto bioderivado, produto biobaseado ou produto derivado de fermentação, retícula bioquímica de ocorrência não natural, construto de ácido nucleico ou vetor de expressão e composição

Continuous flow method for preparing (R)-3-hydroxy-5-hexenoate

Disclosed herein relates to biopharmaceuticals, and more particularly to a continuous flow method for preparing (R)-3-hydroxy-5-hexenoate. Carbonyl reductase and isopropanol dehydrogenase are co-immobilized onto an inert solid medium simultaneously to prepare a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, which is then filled into a microchannel reactor of the micro reaction system. A solution containing substrate 3-carbonyl-5-hexenoate is subsequently pumped into the microchannel reactor to perform an asymmetric carbonyl reduction reaction to obtain (R)-3-hydroxy-5-hexenoate.

Biosynthesis of 1,3-Butanediol

This document describes biochemical pathways for producing 1,3-butanediol using a polypetide having β-ketothiolase activity to form a 3-oxo-5-hydroxypentanoyl-CoA intermediate that can be enzymatically converted to 1,3-butanediol, as well as recombinant hosts producing 1,3-butanediol. 1. A method of producing 3-oxo-5-hydroxypentanoyl-CoA , said method comprising:enzymatically converting 3-hydroxypropionyl-CoA to 3-oxo-5-hydroxypentanoyl-CoA using a polypeptide having β-ketothiolase activity classified under EC. 2.3.1.-.2. The method of claim 1 , whereinsaid polypeptide having β-ketothiolase activity is classified under EC 2.3.1.16. or EC 2.3.1.174.3. (canceled)4. The method of claim 1 , wherein said polypeptide having β-ketothiolase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 claim 1 , 2 claim 1 , 7 or 13.5. The method of claim 1 , further comprising:enzymatically converting 3-oxo-5-hydroxypentanoyl-CoA to 1,3-butanediol using a thioesterase or a CoA transferase, a decarboxylase, and a secondary alcohol dehydrogenase.6. The method of claim 5 , wherein said thioesterase is classified under EC 3.1.2.- claim 5 , said CoA transferase is classified under EC 2.8.3.- claim 5 , said decarboxylase is classified under EC 4.1.1.4 or said secondary alcohol dehydrogenase is classified under EC 1.1.1.B3 claim 5 , EC 1.1.1.B4 claim 5 , or EC 1.1.1.80.7. The method of claim 5 , wherein said thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 or 14; said CoA transferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or 6; said decarboxylase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 10; or said secondary alcohol dehydrogenase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 claim 5 , 9 claim 5 , or 11.820-. (canceled)21. The method of claim 1 , wherein ...

Recombinant n-propanol and isopropanol production

The present invention relates to methods of producing n-propanol, isopropanol, and coproducing n-propanol with isopropanol. The present invention also relates to methods for producing propylene, as well as host cells capable of producing n-propanol and isopropanol.

Biosynthesis of 1,3-butanediol

Yeast organism producing isobutanol at a high yield

The present invention provides recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and/or glycerol-3-phosphate dehydrogenase activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

Hmg-coa reductase derived peptide and cosmetic or pharmaceutical composition containing same

The present invention relates to a peptide of general formula (I): R 1 -(AA) n -X 1 -Gly-Glu-Leu-Ser-X 2 -X 3- (AA) p -R 2 , derived from human HMG-CoA reductase. The present invention also relates to a cosmetic or pharmaceutical composition comprising at least one peptide of general formula (I), in a physiologically suitable medium. The present invention further relates to the use of this novel peptide as an active principle that activates human HMG-CoA reductase in a cosmetic composition intended to strengthen the barrier function of the skin and to stimulate epidermal differentiation. The invention further applies to a cosmetic treatment method intended to prevent and/or combat the external stresses and signs of cutaneous aging.

Fermentive production of four carbon alcohols

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway.

Acetate supplemention of medium for butanologens

The invention relates to the fields of industrial microbiology and alcohol production. More specifically, the invention relates to improved production of butanol isomers by recombinant microorganisms containing an engineered butanol pathway and disrupted activity of the genes in pathways for the production of by-products during the fermentation when the microorganisms are grown in a fermentation medium containing acetate. In embodiments, recombinant microorganisms have an increased growth rate in a fermentation medium containing acetate as a C2 supplement.

Ketol-acid reductoisomerase enzymes and methods of use

Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

Fermentive Production of Four Carbon Alcohols

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway.

Plants with enhanced yield and methods of construction

Transgenic plants having enhanced yield and having enhanced seed yield are disclosed. The transgenic plants are transformed with a transgenic polynucleotide encoding one or more metabolic enzymes. The metabolic enzymes can be from any biological source. The transgenic polynucleotide(s) comprises a nucleic acid sequences encoding the metabolic enzymes under the control of functional plant promoters, the one or more metabolic enzymes are targeted to the plastids by the addition of plastid targeting signals. Optionally the functional plant promoters are seed specific promoters and the metabolic enzymes are targeted to the plastids by the addition of plastid targeting peptide heterologous to the metabolic enzymes. Methods of making the transgenic plants and transgenic polynucleotides are disclosed. The magnitude of the increases in seed yield achieved with these transgenic plants are unprecedented.

Discovery of enzymes from the alpha-keto acid decarboxylase family

2-ketoacid decarboxylase enzymes, compositions encoding for 2 ketoacid decarboxylase enzymes, and host cells comprising such enzymes or compositions are provided

Microorganisms of the genus corynebacterium having l-isoleucine producing ability and methods for producing l-isoleucine using the same

The present application relates to a microorganism of the genus Corynebacterium having L-isoleucine producing ability which comprises a protein having an activity of citramalate synthase, and a method for producing L-isoleucine using the same.

Novel Host Cells and Methods for Producing Isopentenol from Mevalonate

The present invention provides for a genetically modified host cell capable of producing isopentenol and/or 3-methyl-3-butenol, comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD) (b) an increased expression of a phosphatase capable of converting isopentenol into 3-methyl-3-butenol, (c) optionally the genetically modified host cell does not express, or has a decreased expression of one or more of NudB, phosphomevalonate kinase (PMK), and/or PMD, and (d) optionally one or more further enzymes capable of converting isopentenol and/or 3-methyl-3-butenol into a third compound, such as isoprene. 1. A polypeptide having a phosphomevalonate decarboxylase (PMD) enzymatic activity , and encoding an amino acid sequence comprising (a) at least 70% identity with SEQ ID NO:1 , and (b) a histidine at position 74 , a phenylalanine at position 145 , a histidine at position 74 , or a phenylalanine at position 145 , corresponding to the numbering of SEQ ID NO:1.2. The polypeptide of claim 1 , wherein the amino acid sequence comprises (i) a histidine at position 74 claim 1 , (ii) a phenylalanine at position 145 claim 1 , or (iii) a histidine at position 74 and a phenylalanine at position 145.3. The polypeptide of claim 1 , wherein the amino acid sequence comprises the following amino acid residues: E at position 71 claim 1 , S at position 108 claim 1 , N at position 110 claim 1 , A at position 119 claim 1 , S at position 120 claim 1 , S at position 121 claim 1 , A at position 122 claim 1 , S at position 155 claim 1 , R at position 158 claim 1 , S at position 208 claim 1 , and D at position 302 corresponding to SEQ ID NO:1.4. The polypeptide of claim 1 , wherein the amino acid sequence comprises at least 80% identity with SEQ ID NO:1.5. The polypeptide of claim 4 , wherein the amino acid sequence comprises at least 90% identity with SEQ ID NO:1.6. The polypeptide of claim 5 , wherein the amino acid sequence comprises at least 95% identity with SEQ ID NO:1.7. ...

GENETICALLY ENGINEERED MICROORGANISMS AND PROCESSES FOR THE PRODUCTION OF CANNABINOIDS FROM A CARBON SOURCE PRECURSOR

A method is provided for biosynthetic production of cannabinoids in microorganisms from a carbon source precursor. This method describes the genetic modifications needed to engineer microorganisms to produce cannabinoids as well as a method for identifying and quantifying cannabinoids from fermentation broth. A system is also provided for tuning the method to produce different cannabinoids of interest by systematically modulating the enzymes encoded by the genetic modifications introduced in the microorganism. 1. A method for producing at least one cannabinoid from a carbon source precursor , comprising:genetically modifying a microorganism to express enzymes for converting the carbon source precursor into at least one cannabinoid within the genetically modified bacterial strain.2. The method according to claim 1 , wherein the carbon source precursor is glucose and the method further comprises converting the glucose to hexanoate.3. The method according to claim 2 , wherein the at least one cannabinoid comprises cannabigerolic acid.4E. coli.. The method according to claim 3 , wherein the microorganism is a bacterial strain5. The method according to claim 4 , wherein genetically modifying the bacterial strain comprises recombinantly incorporating a mutated FadD gene at the genomic location of a FadE gene of the bacterial strain to express the mutated FadD enzyme and simultaneously knock out the FadE gene of the bacterial strain.6. The method according to claim 5 , wherein the mutated FadD gene comprises a nucleotide sequence of SEQ ID NO: 10.7. The method according to claim 4 , wherein genetically modifying the bacterial strain comprises transforming the bacterial strain to express olivetol synthase claim 4 , olivetolic acid cyclase claim 4 , and CsPT1.8. The method according to claim 7 , wherein the olivetol synthase comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2 claim 7 , wherein the olivetolic acid cyclase comprises a ...

Fermentive Production of Four Carbon Alcohols

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway. 182-. (canceled)84. The method of claim 83 , further comprising recovering the bioproduced isobutanol.85. The method of claim 84 , further comprising removing solids from the fermentation medium.86. The method of claim 84 , wherein the recovering is by distillation claim 84 , liquid-liquid extraction claim 84 , adsorption claim 84 , decantation claim 84 , pervaporation claim 84 , or combinations thereof.87. The method of claim 85 , wherein the removing is by centrifugation claim 85 , filtration claim 85 , or decantation.88. The method of claim 85 , wherein the removing occurs before the recovering. This application is a continuation of and claims priority to U.S. patent application Ser. No. 14/715,992, filed May 19, 2015 which is a continuation of and claims priority to U.S. patent application Ser. No. 13/539,125, now U.S. Pat. No. 9,068,190, filed on Jun. 29, 2012 which is a continuation of and claims priority to U.S. patent application Ser. No. 12/939,284, now U.S. Pat. No. 8,283,144, filed on Nov. 4, 2010 which is a continuation of and claims priority to U.S. patent application Ser. No. 11/586,315, now U.S. Pat. No. 7,851,188, filed on Oct. 25, 2006, which claims priority under 35 U.S.C. §119 from U.S. Provisional Application Ser. No. 60/730,290, filed Oct. 26, 2005.The invention relates to the field of industrial microbiology and the production of alcohols. More specifically, isobutanol is produced via industrial fermentation of a recombinant microorganism.Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need ...

Ketol-Acid Reductoisomerase Enzymes and Methods of Use

Provided herein are polypeptides having ketol-acid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways. 132-. (canceled)33. A polypeptide having ketol-acid reductoisomerase activity , wherein the polypeptide comprisesa. the amino acid sequence of SEQ ID NO: 42;b. at least 99% identity to SEQ ID NO: 42;c. at least 99% identity to SEQ ID NO: 42 and at least one of the following substitutions: M3011, Y239H, Y113F, S322A, A71K, N114G, A329R, A69T, N114S, G72W, A295V, E264K, R280D, A329E, S157T, M238I, Q272T, K335M, R280H, and a combination thereof; ord. an active fragment of any one of (a) to (c).34. The polypeptide of further comprising a substitution at at least one of position L33 claim 33 , P47 claim 33 , F50 claim 33 , F61 claim 33 , 180 claim 33 , and V156.35. The polypeptide of further comprising at least one of the following substitutions: I13L claim 33 , P47Y claim 33 , F50A claim 33 , V53A claim 33 , S86A claim 33 , A268E claim 33 , V76I claim 33 , L88V claim 33 , and a combination thereof.36. A recombinant microbial host cell comprising the polypeptide of .37. The recombinant microbial host cell of claim 36 , wherein the recombinant microbial host cell further comprises reduced or eliminated acetolactate reductase activity.38. The recombinant microbial host cell of claim 37 , wherein the recombinant microbial host cell further comprises at least one deletion claim 37 , mutation claim 37 , and/or substitution in fra2.39. The recombinant microbial host cell of further comprising the substrate to product conversions:a. pyruvate to acetolactateb. 2,3-dihydroxyisovalerate to α-ketoisovaleratec. α-ketoisovalerate to isobutyraldehyde; andd. isobutyraldehyde to isobutanol.40. The recombinant microbial host cell of claim 36 , wherein the recombinant microbial host cell is a yeast host cell. ...

Polypeptides with Ketol-Acid Reductoisomerase Activity

Polypeptides having ketol-acid reductoisomerase activity are provided. Also disclosed are recombinant host cells comprising isobutanol biosynthetic pathways employing such polypeptides. Methods for producing isobutanol employing host cells comprising the polypeptides having ketol-acid reductoisomerase activity are also disclosed. 1. A recombinant host cell comprising an isobutanol biosynthetic pathway anda. a heterologous polypeptide with ketol-acid reductoisomerase activity having at least about 85%, at least about 90% identity, at least about 95%, or at least about 98% identity to one of the following: K9JM2 (SEQ ID NO: 193), K9JM3 (SEQ ID NO: 194), K9JM4 (SEQ ID NO: 195), K9JM5 (SEQ ID NO: 196), K9JM6 (SEQ ID NO: 197), K9JM7 (SEQ ID NO: 198), K9JM8 (SEQ ID NO: 199), K9JM9 (SEQ ID NO: 200), K9JM10 (SEQ ID NO: 201), K9JM11 (SEQ ID NO: 202), K9JM12 (SEQ ID NO: 203), K9JM13 (SEQ ID NO: 204), K9JM14 (SEQ ID NO: 205), K9JM15 (SEQ ID NO: 206), K9JM16 (SEQ ID NO: 207), K9JM17 (SEQ ID NO: 208), K9JM18 (SEQ ID NO: 209), K9JM19 (SEQ ID NO: 210), K9JM20 (SEQ ID NO: 211), K9JM21 (SEQ ID NO: 212), K9JM22 (SEQ ID NO: 213), K9JM23 (SEQ ID NO: 214), K9JM24 (SEQ ID NO: 215), K9JM25 (SEQ ID NO: 216), K9JM26 (SEQ ID NO: 217), K9JM27 (SEQ ID NO: 218), K9JM28 (SEQ ID NO: 219), K9JM29 (SEQ ID NO: 220), K9JM30 (SEQ ID NO: 221), K9JM31 (SEQ ID NO: 222), JM32 (SEQ ID NO: 223), JM33 (SEQ ID NO: 224), JM34 (SEQ ID NO: 225), JM35 (SEQ ID NO: 226), JM36 (SEQ ID NO: 227), JM37 (SEQ ID NO: 228), JM38 (SEQ ID NO: 229), JM39 (SEQ ID NO: 230), JM40 (SEQ ID NO: 231), JM42 (SEQ ID NO: 232), JM43 (SEQ ID NO: 233), JM44 (SEQ ID NO: 234), K9SB2 (SEQ ID NO: 235), K9_DAVID_SH (SEQ ID NO: 236), K9ALL3 (SEQ ID NO: 237), K9_URSALA (K9SB2+A56V) (SEQ ID NO: 239), JM41 (SEQ ID NO: 240), K9ALL148 (SEQ ID NO: 241), K9JM148 (SEQ ID NO: 242), K9ALL156 (SEQ ID NO: 243), K9JM156 (SEQ ID NO: 244), K9ALL191 (SEQ ID NO: 245), K9JM191 (SEQ ID NO: 246), K9ALL254 (SEQ ID NO: 247), K9ALL278 (SEQ ID NO: 248), K9ALL37 (SEQ ...

METHOD FOR REDUCING MISINCORPORATION OF NON-CANONICAL BRANCHED-CHAIN AMINO ACIDS

The present invention relates to a method for producing a recombinant polypeptide of interest in a microbial host cell, comprising (a) introducing a polynucleotide encoding the polypeptide of interest into a microbial host cell which has been modified such that an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase activity (EC 4.3.1.19) is modulated in said microbial host cell as compared to the enzymatic activity in an unmodified microbial host cell, and (b) expressing said polypeptide of interest in said microbial host cell. Moreover, the present invention relates to a method for reducing misincorporation of at least one non-canonical branched-chain amino acid into a recombinant polypeptide of interest expressed in a microbial host cell.

FUNCTIONALIZED NANOPARTICLES FOR ENHANCED AFFINITY PRECIPITATION OF PROTEINS

The present invention provides a nanoparticle capable of binding specifically to a target protein in a solution and precipitating with the target protein out of the solution upon addition of the target protein to the solution. The precipitation may be reversed release the target protein from the nanoparticle, which may be reused for precipitating the target protein. Also provided are a method for purifying a target protein by affinity precipitation using the nanoparticle without chromatography and a method for preparing the nanoparticle. 1. A nanoparticle comprising a fusion protein and a scaffolding domain , wherein the fusion protein is covalently bound to the scaffolding domain , wherein the fusion protein comprises an affinity domain specific for a target protein and a stimuli responsive precipitation domain , wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm , wherein the nanoparticle is soluble in a first solution in the absence of the target protein , wherein the nanoparticle is capable of binding specifically to the target protein in the first solution and precipitating with the target protein out of the first solution in response to a first stimulus , and wherein the first stimulus comprises addition of the target protein to the first solution.2. The nanoparticle of claim 1 , wherein the precipitated nanoparticle is capable of being solubilized in a second solution to release the target protein from the nanoparticle in the second solution in response to a second stimulus.3. The nanoparticle of claim 1 , wherein the first stimulus consists of addition of the target protein to the first solution claim 1 , wherein the first solution has a temperature of 15-25° C. claim 1 , a salt concentration of 50-200 mM and a pH of pH 6-9 claim 1 , and wherein the molar ratio of the affinity domain to the target protein in the first solution is in the range of 3:1-6:1.4. The nanoparticle of claim 1 , wherein the first ...

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 ...

MICROBIAL FERMENTATION FOR THE PRODUCTION OF TERPENES

The invention provides a method for producing a terpene or a precursor thereof by microbial fermentation. Typically, the method involves culturing a recombinant bacterium in the presence of a gaseous substrate whereby the bacterium produces a terpene or a precursor thereof, such as mevalonic acid, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, geranyl pyrophosphate, farnesyl pyrophosphate, and/or farnesene. The bacterium may comprise one or more exogenous enzymes, such as enzymes in mevalonate, DXS, or terpene biosynthesis pathways. 1ClostridiumMoorella.. A recombinant C1-fixing bacteria capable of producing mevalonic acid , or a terpene precursor , from a carbon source comprising a nucleic acid encoding a group of exogenous enzymes comprising thiolase , 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase , and HMG-CoA reductase , wherein the bacteria are or2. The bacteria according to claim 1 , further comprising a nucleic acid encoding a group of enzymes comprising mevalonate kinase claim 1 , phosphomevalonate kinase claim 1 , and mevalonate diphosphate decarboxylase.3. The bacteria according to claim 2 , wherein the terpene precursor is isopentenyl diphosphate.4. The bacteria according to claim 2 , further comprising a nucleic acid encoding an exogenous enzyme selected from the group consisting of isopentenyl diphosphate isomerase and geranyltranstransferase.5. The bacteria according to claim 2 , further comprising a nucleic acid encoding both exogenous enzymes isopentenyl diphosphate isomerase and geranyltranstransferase.6. The bacteria according to claim 4 , wherein the terpene precursor is dimethylallyl pyrophosphate or geranyl pyrophosphate.7. The bacteria according to claim 4 , further comprising a nucleic acid encoding an exogenous enzyme comprising isoprene synthase.8. The bacteria according to claim 4 , wherein the terpene precursor is farnesyl pyrophosphate.9. The bacteria according to claim 4 , further comprising a nucleic acid ...

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 ...

Novel Host Cells and Methods for Producing Isopentenol from Mevalonate

The present invention provides for a genetically modified host cell capable of producing isopentenol and/or 3-methyl-3-butenol, comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD) (b) an increased expression of a phosphatase capable of converting isopentenol into 3-methyl-3-butenol, (c) optionally the genetically modified host cell does not express, or has a decreased expression of one or more of NudB, phosphomevalonate kinase (PMK), and/or PMD, and (d) optionally one or more further enzymes capable of converting isopentenol and/or 3-methyl-3-butenol into a third compound, such as isoprene. 1. A genetically modified host cell capable of producing isopentenol and/or 3-methyl-3-butenol , comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD) (b) an increased expression of a phosphatase capable of converting isopentenol into 3-methyl-3-butenol , (c) optionally the genetically modified host cell does not express , or has a decreased expression of one or more of NudB , phosphomevalonate kinase (PMK) , and/or PMD , and (d) optionally one or more further enzymes capable of converting isopentenol and/or 3-methyl-3-butenol into a third compound.2. The genetically modified host cell of claim 1 , wherein the decreased expression is a disruption of the promoter or knock out of the gene encoding NudB claim 1 , PMK claim 1 , and/or PMD.3. The genetically modified host cell of claim 1 , wherein the third compound is isoprene.4. The genetically modified host cell of claim 1 , further comprising an increased expression of one or more of AtoB claim 1 , hydroxymethylglutaryl-CoA synthase (HMGS) claim 1 , hydroxymethylglutaryl-CoA reductase (HMGR) claim 1 , and/or mevalonate kinase (MK).5. The genetically modified host cell of claim 1 , wherein the PMD is encoded on a nucleotide sequence which is in nucleic acids which is transformed into the genetically modified host cell claim 1 , or host cell prior to genetic modification.6. The ...

MUTANT ENZYME AND PRODUCTION METHOD FOR TERPENOID USING SAID MUTANT ENZYME

The object of the present invention is to efficiently produce useful terpenoid compounds, and specifically, to provide a method for preparing squalene, which is an important intermediate of terpenoid. The object can be solved by a hydroxymethylglutaryl CoA reductase (HMGR) comprising:(a) an amino acid other than alanine (A) at the 10th position in an Sα2 amino acid sequence of HMGR, (b) an amino acid other than proline (P) at the 1st position from the carboxyl terminal in the DKK region of the HMG-CoA binding site of HMGR, (c) an amino acid other than alanine (A) at the 1st position in an Lα2 amino acid sequence of HMGR, and (d) an amino acid other than glutamic acid (E) at the 6th position in an Lα2 amino acid sequence of HMGR of the present invention. 1. A hydroxymethylglutaryl CoA reductase (HMGR) comprising:(a) an amino acid other than alanine (A) at the 10th position in an Sα2 amino acid sequence of HMGR,(b) an amino acid other than proline (P) at the 1st position from the carboxyl terminal in the DKK region of the HMG-CoA binding site of HMGR,(c) an amino acid other than alanine (A) at the 1st position in an Lα2 amino acid sequence of HMGR, and(d) an amino acid other than glutamic acid (E) at the 6th position in an Lα2 amino acid sequence of HMGR.2. (canceled)3. The hydroxymethylglutaryl CoA reductase according to claim 1 , further comprising one or more amino acids selected from the group consisting of:(e) an amino acid other than isoleucine (I) at the 7th position in an Sα2 amino acid sequence of HMGR,(f) an amino acid other than isoleucine (I) at the 5th position in an Lα2 amino acid sequence of HMGR, and(g) an amino acid other than alanine (A) at the 6th position in an Sα2 amino acid sequence of HMGR.4. The hydroxymethylglutaryl CoA reductase according to claim 3 , wherein the 10th amino acid in an Sα2 amino acid sequence is serine (S) claim 3 , the 1st amino acid from the carboxyl terminal in the DKK region of the HMG-CoA binding site is alanine (A) claim ...

NEW ENZYMATIC PROCESS FOR PRODUCTION OF MODIFIED HOP PRODUCTS

The present invention relates to a process for producing a beer bittering agent via enzyme catalyzed bioconversion of hop-derived isoalpha acids to dihydro-(rho)-isoalpha acids and to the novel enzyme catalysts which may be employed in such a process. 1. A process for the preparation of dihydro-(rho)-isoalpha acids , comprising treating isoalpha acids with a ketoreductase enzyme or a microorganism expressing a gene that encodes the ketoreductase.2. The process according to claim 1 , wherein the process is carried out in an aqueous system.3. The process according to claim 2 , wherein the process is carried out under mild temperature and pH conditions.4. The process according to claim 1 , comprising adding the ketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids followed by incubation.5. The process according to claim 1 , comprising adding the ketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids in the presence of isopropanol for cofactor recycling claim 1 , followed by incubation.6. The process according to claim 5 , wherein the concentration of isoalpha acids claim 5 , i.e. the substrate claim 5 , is maximized to increase the volumetric productivity of the bioconversion.7. The process according to claim 5 , wherein the concentration of the cofactor NADPH or NADP in the mixture is minimized to improve the economics of the bioconversion.8. The process according to claim 1 , comprising adding the ketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids in the presence of another enzyme for cofactor recycling claim 1 , followed by incubation.9. The process according to claim 1 , comprising adding a whole cell biocatalyst claim 1 , wherein the whole cell biocatalyst is an immobilized microorganism expressing the gene which encodes a ketoreductase claim 1 , to a mixture of isoalpha acids followed by incubation.10. The process according to claim 1 , comprising culturing a microorganism expressing the gene which encodes the ...

Bacteria engineered to treat disorders involving the catabolism of a branched chain amino acid

The present disclosure provides recombinant bacterial cells that have been engineered with genetic circuitry which allow the recombinant bacterial cells to sense a patient's internal environment and respond by turning an engineered metabolic pathway on or off. When turned on, the recombinant bacterial cells complete all of the steps in a metabolic pathway to achieve a therapeutic effect in a host subject. These recombinant bacterial cells are designed to drive therapeutic effects throughout the body of a host from a point of origin of the microbiome. Specifically, the present disclosure provides recombinant bacterial cells comprising a heterologous gene encoding a branched chain amino acid catabolism enzyme. The disclosure further provides pharmaceutical compositions comprising the recombinant bacteria, and methods for treating disorders involving the catabolism of branched chain amino acids using the pharmaceutical compositions disclosed herein.

Host Cells and Methods for Production of Isobutanol

The invention relates to recombinant host cells having at least one integrated polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway, e.g., pyruvate to acetolactate conversion. The invention also relates to methods of increasing the biosynthetic production of isobutanol, 2,3-butanediol, 2-butanol or 2-butanone using such host cells. 1. A recombinant host cell comprising:{'i': Bacillus subtilis, Klebsiella pneumonia, Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, Streptococcus thermophiles, Vibrio angustum', 'Bacillus cereus;, '(a) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of pyruvate to acetolactate wherein the polypeptide is an acetolactate synthase from , or'}(b) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate wherein the polypeptide is a ketol-acid reductoisomerase and the ketol-acid reductoisomerase has at least 95% identity to SEQ ID NO: 224;{'i': Escherichia coli, Bacillus subtilis, Methanococcus maripaludis', 'Streptococcus mutans;, '(c) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate wherein the polypeptide is a dihydroxyacid dehydratase from , or'}(d) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde wherein the polypeptide is a branched-chain α-keto acid decarboxylase and the branched-chain α-keto acid decarboxylase has at least 95% identity to SEQ ID NO: 48; and{'i': Achromobacter xylosoxidans', 'Beijerinkia indica,, '(e) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol wherein the polypeptide is an alcohol dehydrogenase from or'}wherein expression of pyruvate decarboxylase in the ...

KETOL-ACID REDUCTOISOMERASE USING NADH

Methods for the evolution of NADPH specific ketol-acid reductoisomerase enzymes to acquire NADH specificity are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from that have undergone co-factor switching to utilize NADH are described. 1. A mutant ketol-acid reductoisomerase enzyme comprising the amino acid sequence as set forth in SEQ ID NO: 29.2. A nucleic acid molecule encoding the mutant ketol-acid reductoisomerase enzyme of .3. A nucleic acid molecule encoding a mutant ketol-acid reductoisomerase enzyme having the amino acid sequence as set forth in SEQ ID NO:19.4. A mutant ketol-acid reductoisomerase enzyme as set for in SEQ ID NO:195. A recombinant cell comprising the mutant ketol-acid reductoisomerase enzyme of .6. A mutant ketol-acid reductoisomerase enzyme as set forth in SEQ ID NO:17 comprising at least one mutation at a residue selected from the group consisting of 24 claim 1 , 33 claim 1 , 47 claim 1 , 50 claim 1 , 52 claim 1 , 53 claim 1 , 61 claim 1 , 80 claim 1 , 115 claim 1 , 156 claim 1 , 165 claim 1 , and 170.7. A mutant ketol-acid reductoisomerase enzyme according to wherein:a) the residue at position 47 has an amino acid substation selected from the group consisting of A, C, D, F, G, I, L, N, P, H, T, E and Y;b) the residue at position 50 has an amino acid substitution selected from the group consisting of A, C, D, E, F, G, M, N, V, W and I;c) the residue at position 52 has an amino acid substitution selected from the group consisting of A, C, D, G, H, N, Y, and S;d) the residue at position 53 has an amino acid substitution selected from the group consisting of A, H, I, W, Y, C, and R;e) the residue at position 156 has an amino acid substitution of V;f) the residue at position 165 has an amino acid substitution of M;g) the residue at position 61 has an amino acid substitution of F;h) the residue at position 170 has an amino acid substitution of A;i) the residue at position 24 has an amino acid substitution of F;j) the ...

Recombinant yeast and method for producing ethanol using the same

The invention is intended to improve xylose assimilation ability and ethanol fermentation ability in a xylose-assimilating yeast into which a xylose isomerase gene has been introduced. The amount of NADH produced by the recombinant yeast into which the xylose isomerase gene had been introduced as a result of the enzymatic reaction of acetohydroxy acid reductoisomerase is lowered.

Integration of a Polynucleotide Encoding a Polypeptide that Catalyzes Pyruvate to Acetolactate Conversion

The invention relates to recombinant host cells having at least one integrated polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway, e.g., pyruvate to acetolactate conversion. The invention also relates to methods of increasing the biosynthetic production of isobutanol, 2,3-butanediol, 2-butanol or 2-butanone using such host cells. 147-. (canceled)48. A recombinant host cell comprising:{'i': Bacillus subtilis, Klebsiella pneumonia, Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, Streptococcus thermophiles, Vibrio angustum', 'Bacillus cereus., '(a) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of pyruvate to acetolactate wherein the polypeptide is an acetolactate synthase from , or'}{'i': Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa, Pseudomonas fluorescens', 'Anaerostipes caccae., '(b) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate wherein the polypeptide is a ketol-acid reductoisomerase from , or'}{'i': Escherichia coli, Bacillus subtilis, Methanococcus maripaludis', 'Streptococcus mutans, '(c) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate wherein the polypeptide is a dihydroxyacid dehydratase from , or ; and'}{'i': Listeria grayi, Lactococcus lactis', 'Macrococcus caseolyticus., '(d) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde wherein the polypeptide is a branched-chain α-keto acid decarboxylase from , or'}49Achromobacter xylosoxidansBeijerinkia indica.. The recombinant host cell of further comprising a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol wherein the polypeptide ...

ENGINEERING PLANTS WITH RATE LIMITING FARNESENE METABOLIC GENES

The disclosed invention provides methods and compositions for increasing terpenoid production, such as sesquiterpenoids, such as farnesene, in plant cells. 1. A plant cell having increased production of at least one terpenoid native to a plant , the method comprising expressing in a plant cell a heterologous nucleic acid encoding for (a) HMG-CoA reductase , (b) 1-deoxy-D-xylulose-5-phosphate synthase , (c) farnesyl pyrophosphate synthase , and (d) β-farnesene synthase , wherein production of the at least one terpenoid is significantly increased when compared to a wild-type plant cell not encoding the heterologous nucleic acids.2. The method of claim 1 , wherein{'i': Arabidopsis, Oryza, Saccharomyces', 'Hevea, 'a. the HMG-CoA reductase is an , or HMG-CoA reductase;'}{'i': Arabidopsis, Oryza, Saccharomyces', 'Zea, 'b. the 1-deoxy-D-xyululose-5-phophate is an , or 1-deoxy-D-xyululose;'}{'i': Arabidopsis, Oryza', 'Solanum, 'c. the farnesyl pyrophosphate synthase is an , or farnesyl pyrophosphate; or'}{'i': Arabidopsis, Oryza', 'Artemisia, 'd. the β-farnesene synthase is an , or β-farnesene synthase.'}3. The method of claim 2 , wherein{'i': Arabidopsis thaliana, Oryza sativa, Saccharomyces cerevisiae', 'Hevea, 'a. the HMG-CoA reductase is an , or HMG-CoA reductase;'}{'i': Arabidopsis thaliana, Oryza sativa, Saccharomyces cerevisiae', 'Zea mays, 'b. the 1-deoxy-D-xyululose-5-phophate is an , or 1-deoxy-D-xyululose;'}{'i': Arabidopsis thaliana, Oryza sativa', 'Solanum lycopersicon, 'c. the farnesyl pyrophosphate synthase is an , or farnesyl pyrophosphate;'}{'i': Arabidopsis thaliana, Oryza sativa', 'Artemisia annua β, 'd. the β-farnesene synthase is an , or -farnesene synthase.'}4. The method of claim 3 , wherein at least one nucleic acid is codon-optimized for expression in a plant.5. The method of claim 3 , whereina. the HMG-CoA reductase is encoded by a polynucleotide having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of ...

METHOD FOR THE PREPARATION OF 2,4-DIHYDROXYBUTYRATE

A method for the preparation of 2,4-dihydroxybutyric acid from homoserine includes a first step of conversion of the primary amino group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate, and a second step of reduction of the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate. 1. A method for the preparation of 2 ,4-dihydroxybutyrate (identical to 2 ,4-dihydroxybutyric acid) from homoserine comprising a two steps pathway:a first step of conversion of the primary amino group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate, anda second step of reduction of the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate.2. The method of claim 1 , wherein the first and/or second step(s) is catalyzed by an enzyme encoded by an endogenous or a heterologous gene.3. The method of wherein the first step is catalyzed by an enzyme having homoserine transaminase claim 1 , homoserine dehydrogenase claim 1 , or homoserine oxidase activity claim 1 , respectively.4. The method of wherein the enzyme having homoserine transaminase activity is encoded by gene ilvE claim 3 , tyrB claim 3 , aspC claim 3 , araT claim 3 , bcaT claim 3 , AR08.5. The method of wherein the enzyme having homoserine transaminase activity is encoded by sequence set forth in SEQ ID No.59 claim 4 , SEQ ID No.61 claim 4 , SEQ ID No.63 claim 4 , SEQ ID No.65 claim 4 , SEQ ID No. 67 or SEQ ID No.69 or any sequence sharing a homology of at least 50% with said sequences or corresponds to SEQ ID No.60 claim 4 , SEQ ID No.62 claim 4 , SEQ ID No.64 claim 4 , SEQ ID No.66 claim 4 , SEQ ID No.68 claim 4 , SEQ ID No.70 or any sequence sharing a homology of at least 50% with said sequences.6. The method of wherein the second step is catalysed by an enzyme having OHB reductase activity.7. The method of wherein the enzyme having OHB reductase activity is lactate dehydrogenase claim 6 , or malate dehydrogenase claim 6 , or branched-chain 2-hydroxyacid dehydrogenase. ...

METHOD FOR THE PREPARATION OF 2,4-DIHYDROXYBUTYRATE

A method for the preparation of 2,4-dihydroxybutyric acid from homoserine includes a first step of conversion of the primary amino group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate, and a second step of reduction of the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate. 122-. (canceled)23. A method for the preparation of 2 ,4-dihydroxybutyrate (2 ,4-DHB) from homoserine comprising:deaminating homoserine to form 2-oxo-4-hydroxybutyrate (OHB), where the deamination of homoserine is catalyzed by an enzyme having homoserine transaminase activity, wherein the enzyme having homoserine transaminase activity is produced via a transformed host microorganism that comprises a first chimeric gene including a first nucleic acid sequence encoding the enzyme having homoserine transaminase activity for converting the primary amino acid group of homoserine to a carbonyl group to obtain OHB; andreducing the OHB to form 2,4-DHB, where the reduction of OHB is catalyzed by an enzyme having OHB reductase activity, wherein the enzyme having OHB reductase activity is produced via the transformed host microorganism, which further comprises a second chimeric gene including a second nucleic acid sequence encoding the enzyme having OHB reductase activity for reducing OHB to 2,4-DHB.24. The method of claim 23 , wherein the enzyme having homoserine transaminase activity is selected from the group consisting of enzymes classified in E.C. 2.6.1.1 claim 23 , E.C. 2.6.1.2 claim 23 , E.C. 2.6.1.42 claim 23 , E.C. 2.6.1.57 or E.C. 2.6.1.88.26. The method of claim 23 , wherein the enzyme having OHB reductase activity is selected from the group consisting of lactate dehydrogenases classified in E.C.1.1.1.27 or E.C.1.1.1.28 claim 23 , malate dehydrogenases classified in E.C.1.1.1.37 claim 23 , E.C.1.1.1.82 or E.C.1.1.1.299 claim 23 , or branched-chain 2-hydroxyacid dehydrogenases classified in E.C.1.1.1.272 or E.C.1.1.1.345.27. The method of claim 26 , wherein the ...

Biological Production of Multi-Carbon Compounds from Methane

Multi-carbon compounds such as ethanol, n-butanol, sec-butanol, isobutanol, tert-butanol, fatty (or aliphatic long chain) alcohols, fatty acid methyl esters, 2,3-butanediol and the like, are important industrial commodity chemicals with a variety of applications. The present invention provides metabolically engineered host microorganisms which metabolize methane (CH) as their sole carbon source to produce multi-carbon compounds for use in fuels (e.g., bio-fuel, bio-diesel) and bio-based chemicals. Furthermore, use of the metabolically engineered host microorganisms of the invention (which utilize methane as the sole carbon source) mitigate current industry practices and methods of producing multi-carbon compounds from petroleum or petroleum-derived feedstocks, and ameliorate much of the ongoing depletion of arable food source “farmland” currently being diverted to grow bio-fuel feedstocks, and as such, improve the environmental footprint of future bio-fuel, bio-diesel and bio-based chemical compositions. 191-. (canceled)92. A genetically modified methanotroph comprising a heterologous polynucleotide encoding for an alcohol dehydrogenase (ADH) , wherein the alcohol dehydrogenase can catalyze the conversion of isobutyraldehyde to isobutanol and comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 10 , and wherein said methanotroph is capable of converting formaldehyde to pyruvate through a type I RuMP pathway or a type II serine pathway.93. The methanotroph of claim 92 , wherein the methanotroph further comprises a heterologous polynucleotide encoding a ketoacid decarboxylase (KDC) claim 92 , wherein the ketoacid decarboxylase can catalyze the conversion of ketoisovalerate to isobutryaldehyde.94. The methanotroph of claim 93 , wherein the methanotroph further comprises a heterologous polynucleotide encoding an acetolactate synthase (ALS) claim 93 , a heterologous polynucleotide encoding a ketol-acid reductoisomerase (KARI) claim 93 , ...

Microbial fermentation for the production of terpenes

The invention provides a method for producing a terpene or a precursor thereof by microbial fermentation. Typically, the method involves culturing a recombinant bacterium in the presence of a gaseous substrate whereby the bacterium produces a terpene or a precursor thereof, such as mevalonic acid, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, geranyl pyrophosphate, farnesyl pyrophosphate, and/or farnesene. The bacterium may comprise one or more exogenous enzymes, such as enzymes in mevalonate, DXS, or terpene biosynthesis pathways.

METHODS, HOSTS, AND REAGENTS RELATED THERETO FOR PRODUCTION OF UNSATURATED PENTAHYDROCARBONS, DERIVATIVES AND INTERMEDIATES THEREOF

This application describes methods, including non-naturally occurring methods, for biosynthesizing unsaturated pentahydrocarbons, such as isoprene and intermediates thereof, via the mevalonate pathway, as well as non-naturally occurring hosts for producing isoprene. 1. A method for synthesizing isoprene in a chemolithotrophic host comprising:enzymatically converting acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA using a polypeptide having the activity of a hydroxymethylglutaryl-CoA synthase enzyme having the amino acid sequence set forth in SEQ ID No: 2 or 10 or a functional fragment of said enzyme; andenzymatically converting 3-hydroxy-3-methylglutaryl-CoA to (R)-mevalonate using a polypeptide having the activity of a hydroxymethylglutaryl Co-A reductase enzyme having the amino acid sequence set forth in SEQ ID No: 3 or 9 or a functional fragment of said enzyme.2. (canceled)3. The method of claim 1 , further comprising at least one of:enzymatically converting acetyl-CoA to acetoacetyl-CoA using a polypeptide having the activity of an acetyl-CoA acetyltransferase enzyme having the amino acid sequence set forth in SEQ ID No: 1 or 9 or a functional fragment of said enzyme;enzymatically converting (R)-mevalonate to (R)-5-phosphomevalonate using a polypeptide having the activity of a mevalonate-kinase enzyme having the amino acid sequence set forth in SEQ ID No: 4 or 11 or a functional fragment of said enzyme;enzymatically converting (R)-5-phosphomevalonate to (R)-5-diphosphomevalonate using a polypeptide having the activity of a phosphomevalonate kinase enzyme having the amino acid sequence set forth in SEQ ID No: 5 or 12 or a functional fragment of said enzyme;enzymatically converting (R)-5-diphosphomevalonate to isopentenyl diphosphate using a polypeptide having the activity of a diphosphomevalonate decarboxylase enzyme having the amino acid sequence set forth in SEQ ID No: 6 or 13 or a functional fragment of said enzyme;enzymatically converting isopentenyl ...

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 ...

Biological Production of Multi-Carbon Compounds from Methane

Multi-carbon compounds such as ethanol, n-butanol, sec-butanol, isobutanol, tert-butanol, fatty (or aliphatic long chain) alcohols, fatty acid methyl esters, 2,3-butanediol and the like, are important industrial commodity chemicals with a variety of applications. The present invention provides metabolically engineered host microorganisms which metabolize methane (CH 4 ) as their sole carbon source to produce multi-carbon compounds for use in fuels (e.g., bio-fuel, bio-diesel) and bio-based chemicals. Furthermore, use of the metabolically engineered host microorganisms of the invention (which utilize methane as the sole carbon source) mitigate current industry practices and methods of producing multi-carbon compounds from petroleum or petroleum-derived feedstocks, and ameliorate much of the ongoing depletion of arable food source “farmland” currently being diverted to grow bio-fuel feedstocks, and as such, improve the environmental footprint of future bio-fuel, bio-diesel and bio-based chemical compositions.

2-isopropylmalate synthetase and engineering bacteria and application thereof

The invention relates to a 2-isopropyl malate synthase, a genetically engineered bacterium for producing L-leucine and application thereof and belongs to the field of metabolic engineering. The genetically engineered bacterium is obtained by overexpressing an isopropyl malate synthase coding gene leuA M for relieving feedback inhibition by L-leucine, an acetohydroxy acid synthase coding gene ilvBN M for relieving feedback inhibition by L-isoleucine, a 3-isopropyl malate dehydrogenase coding gene leuB and a 3-isopropyl malate dehydratase coding gene leuCD in host cells. The genetically engineered bacterium for producing the L-leucine is free from nutritional deficiency, rapid in growth, short in fermentation period, high in yield and high in conversion rate.

Fermentive Production of Four Carbon Alcohols

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway. 182-. (canceled)84. The recombinant microbial host cell of claim 83 , wherein the substrate to product conversion of pyruvate to acetolactate is performed by a recombinantly expressed acetolactate synthase.85. The recombinant microbial host cell of claim 83 , wherein the substrate to product conversion of acetolactate to 2 claim 83 ,3-dihydroxyisovalerate is performed by a recombinantly expressed acetohydroxy acid isomeroreductase.86. The recombinant microbial host cell of claim 83 , wherein the substrate to product conversion of 2 claim 83 ,3-dihydroxyisovalerate to α-ketoisovalerate is performed by a recombinantly expressed acetohydroxy acid dehydratase.87. The recombinant microbial host cell of claim 85 , wherein the substrate to product conversion of isobutryaldehyde to isobutanol is performed by a recombinantly expressed branched-chain alcohol dehydrogenase.88. The recombinant microbial host cell of claim 83 , wherein the substrate to product conversion of α-ketoisovalerate to isobutyryl-CoA is performed by a recombinantly expressed branched-chain keto acid dehydrogenase.89. The recombinant microbial host cell of claim 83 , wherein the substrate to product conversion of to isobutyryl-CoA to isobutyraldehyde is performed by a recombinantly expressed acylating aldehyde dehydrogenase.90. The recombinant microbial host cell of claim 83 , wherein the cell is selected from the group consisting of: bacterium claim 83 , cyanobacterium claim 83 , filamentous fungus claim 83 , and yeast.91. The recombinant microbial host cell of further comprising an inactivated gene thereby reducing yield loss from competing pathways for carbon flow.92Bacillus subtilis, Klebsiella pneumoniaeLactococcus lactis.. The recombinant microbial host cell of claim ...

YEAST ORGANISM PRODUCING ISOBUTANOL AT A HIGH YIELD

The present invention provides recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and/or glycerol-3-phosphate dehydrogenase activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms. 1. A method of producing isobutanol , comprising:a) providing a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein the recombinant microorganism has been engineered to contain one or more modifications in a transcriptional regulator of a PDC gene;b) cultivating the microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of the isobutanol is produced; andc) recovering the isobutanol.2. The method of claim 1 , wherein the microorganism comprises an isobutanol producing metabolic pathway comprising the following substrate to product conversions:(i) pyruvate to acetolactate;(ii) acetolactate to 2,3-dihydroxyisovalerate;(iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate;(iv) α-ketoisovalerate to isobutyraldehyde; and(v) isobutyraldehyde to isobutanol.3. The method of claim 1 , wherein the microorganism expresses(a) an acetolactate synthase to catalyze the conversion of pyruvate to acetolactate;(b) a ketol-acid reductoisomerase to catalyze the conversion of acetolactate to 2,3-dihydroxyisovalerate;(c) a dihydroxyacid dehydratase to catalyze the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate;(d) an α-ketoisovalerate decarboxylase to catalyze the ...

Microorganisms and methods for producing cannabinoids and cannabinoid derivatives

The present disclosure provides genetically modified host cells that produce a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative. The present disclosure provides methods of synthesizing a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative.

Protein Crystals

A method for producing an ordered protein lattice, the method comprising: (a) providing a first component comprising a subunit of a homooligomeric protein assembly fused to a first subunit of a heterooligomeric protein assembly, and a second component comprising a second subunit of the heterooligomeric protein assembly, wherein the homooligomeric protein assembly and the heterooligomeric protein assembly are each symmetrical in two or three dimensions and share a rotational symmetry axis of the same order; (b) mixing said first monomer and said second monomer to produce a mixture; and (c1) (i) heating the mixture to a temperature about 2° C. to about 10° C. below the visible dissociation temperature; (ii) cooling the mixture by about 10° C. to about 20° C.; and (iii) repeating steps (i) and (ii) at least 10 times; or (c2) (i) heating the mixture to a temperature about 2° C. to about 30° C. or more below the visible dissociation temperature; and (ii) holding the mixture at a temperature about 2° C. to about 30° C. or more below the melt temperature, thereby producing an ordered protein lattice. 2. A method according to claim 1 , wherein the homooligomeric protein is thermostable.3. A method according to claim 1 , wherein each cycle of steps (c1) (i) and (ii) is 5 seconds or longer.4. A method according to claim 1 , wherein in step (c2) the temperature is kept constant for from about 1 hour to about 3 months.5. A method according to claim 1 , wherein the mixture comprises one or more additives that reduce the melt temperature.6. A method according to claim 1 , wherein the mixture has a protein concentration of from about 1 to 100 mg/ml.7. A method according to claim 1 , wherein in step (b) the first component and second component are mixed at a ratio of from 8:1 to 1:8.8. An ordered protein lattice comprising:a first component comprising a subunit of a homooligomeric protein assembly fused to a first subunit of a heterooligomeric protein assembly; and(ii) a second ...

METHODS AND COMPOSITIONS FOR THE AUGMENTATION OF PYRUVATE AND ACETYL-COA FORMATION

The present disclosure identifies methods and compositions for modifying photoautotrophic organisms as hosts, such that the organisms efficiently convert carbon dioxide and light into pyruvate or acetyl-CoA, and in particular the use of such organisms for the commercial production of molecules derived from these precursors, e.g., ethanol. 1. An engineered photosynthetic microbe , wherein said engineered photosynthetic microbe comprises a recombinant MdhP enzyme.2Pisum sativum. The engineered photosynthetic microbe of claim 1 , wherein said recombinant MdhP enzyme is a MdhP enzyme.3. The engineered photosynthetic microbe of claim 1 , wherein said recombinant MdhP enzyme is at least 95% identical to SEQ ID NO: 1.4. The engineered photosynthetic microbe of claim 1 , wherein said recombinant MdhP enzyme is at least 95% identical to SEQ ID NO: 2.5. The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe comprises an additional mutation which reduces the expression or activity of its endogenous Mdh enzyme.6. The engineered photosynthetic microbe of claim 5 , wherein said mutation is a knockout of the gene encoding said endogenous Mdh enzyme.7. The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe further comprises a recombinant phosphoenol pyruvate carboxylase.8. The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe further comprises a recombinant NADPH-linked malic enzyme.9. The engineered photosynthetic microbe of claim 1 , wherein said engineered photosynthetic microbe further comprises a recombinant phosphoenol pyruvate carboxylase and a recombinant NADPH-linked malic enzyme.10. The engineered photosynthetic microbe of or claim 1 , wherein said recombinant phosphoenol pyruvate carboxylase is the S8D mutant phosphoenol pyruvate carboxylase.11Sorghum. The engineered photosynthetic microbe of claim 10 , wherein said S8D mutant phosphoenol ...

PREPARATION OF 7-DEHYDROCHOLESTEROL AND/OR THE BIOSYNTHETIC INTERMEDIATES AND/OR SECONDARY PRODUCTS THEREOF IN TRANSGENIC ORGANISMS

The present invention relates to a method for preparing 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof by culturing organisms, in particular yeasts. Furthermore, the invention relates to the preparation of the nucleic acid constructs required for preparing the genetically modified organisms and to said genetically modified organisms, in particular yeasts, themselves. 1. A method for preparing 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof comprising the step of culturing organisms which , compared to the wild type , have an increased activity of at least one of the activities selected from the group consisting of Δ8-Δ7-isomerase activity , Δ5-desaturase activity and Δ24-reductase activity.2. The method of claim 1 , wherein the organisms claim 1 , compared to the wild type claim 1 , have an increased activity of at least two of the activities selected from the group consisting of Δ8-Δ7-isomerase activity claim 1 , Δ5-desaturase activity and Δ24-reductase activity.3. The method of claim 1 , wherein the organisms claim 1 , compared to the wild type claim 1 , have an increased Δ8-Δ7-isomerase activity claim 1 , Δ5-desaturase activity and Δ24-reductase activity.4. The method of claim 1 , further comprising the step of increasing the Δ8-Δ7-isomerase activity by increasing claim 1 , compared to the wild type claim 1 , gene expression of a nucleic acid encoding a Δ8-Δ7-isomerase.5. The method of claim 4 , wherein the gene expression is increased by introducing into the organism one or more nucleic acids encoding a Δ8-Δ7-isomerase.6. The method of claim 5 , further comprising the step of introducing the nucleic acids claim 5 , which encode proteins comprising the amino acid sequence SEQ. ID. NO. 2 or a sequence derived from this sequence by substitution claim 5 , insertion or deletion of amino acids claim 5 , which is at least 30% identical at the amino acid level with the sequence ...

BACTERIA ENGINEERED FOR ESTER PRODUCTION

The present disclosure provides recombinant bacteria with elevated 2-keto acid decarboxylase and alcohol transferase activities. Some recombinant bacteria further have elevated aldehyde dehydrogenase activity. Some recombinant bacteria further have reduced alcohol dehydrogenase and/or aldehyde reductase activity. Methods for the production of the recombinant bacteria, as well as for use thereof for production of various esters are also provided. 1. A bacterium comprising a recombinant polynucleotide encoding an alcohol transferase (ATF) and either a 2-keto acid decarboxylase (KDC) or a 2-ketoisovalerate dehydrogenase enzyme complex (KIVDH) , wherein expression of the ATF and either the KDC or the KIVDH results in an increase in production of an ester as compared to a corresponding bacterium lacking the recombinant polynucleotide.2. The bacterium of claim 1 , wherein the recombinant polynucleotide further encodes an aldehyde dehydrogenase (ALDH) claim 1 , wherein expression of the ALDH claim 1 , the ATF claim 1 , and either the KDC or the KIVDH results in an increase in production of an ester as compared to a corresponding bacterium lacking the recombinant polynucleotides.3. The bacterium of claim 2 , wherein the recombinant polynucleotide comprises one claim 2 , two or three recombinant polynucleotides.4. The bacterium of claim 3 , wherein the ATF is ATF1.5. The bacterium of claim 4 , wherein the ester comprises an acetate ester.6. The bacterium of claim 5 , wherein the acetate ester comprises one or more of ethyl acetate claim 5 , isobutyl acetate claim 5 , isoamyl acetate claim 5 , propyl acetate claim 5 , amyl acetate claim 5 , and 2-pheneethyl acetate.7. The bacterium of claim 3 , wherein the ATF is EHT1.8. The bacterium of claim 7 , wherein the ester comprises an isobutyrate ester.9. The bacterium of claim 8 , wherein the isobutyrate ester comprises one or more of ethyl isobutyrate claim 8 , isobutyl isobutyrate claim 8 , 3-methylbutyl isobutyrate claim 8 , and ...

METHODS OF PRODUCING 7-CARBON CHEMICALS VIA C1 CARBON CHAIN ELONGATION ASSOCIATED WITH COENZYME B SYNTHESIS

This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming one or two terminal functional groups, each comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the C1 elongation enzymes or homolog associated with coenzyme B biosynthesis. 132.-. (canceled)33. A recombinant host comprising at least one exogenous nucleic acid encoding (i) a (homo)citrate synthase classified under EC 2.3.3.14 or EC 2.3.3.13 , (ii) a (homo)citrate dehydratase and a (homo)aconitate hydratase classified under EC 4.2.1.114 , EC 4.2.1.36 , or EC 4.2.1.33 , (iii) an iso(homo)citrate dehydrogenase classified under EC 1.1.1.85 , EC 1.1.1.87 , or EC 1.1.1.286 , and/or (iv) an indolepyruvate decarboxylase classified under EC 4.1.1.43 or EC 4.1.1.74 , or a 2-oxoglutarate dehydrogenase complex comprising one or more enzymes classified under EC 1.2.4.2 , EC 1.8.1.4 , and EC 2.3.1.61 , said host producing pimeloyl-CoA or pimelate semialdehyde.34. The recombinant host of claim 33 , further comprising at least one exogenous nucleic acid encoding one or more of a thioesterase classified under EC 3.1.2.- claim 33 , an aldehyde dehydrogenase classified under EC 1.2.1.3 claim 33 , a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.- claim 33 , a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.- claim 33 , a glutaconate CoA-transferase classified under EC 2.8.3.12 claim 33 , a reversible succinyl-CoA-ligase classified under EC 6.2.1.5 claim 33 , an acetylating aldehyde dehydrogenase classified under EC 1.2.1.10 claim 33 , or a carboxylate reductase classified under EC 1.2.99.6 claim 33 , said host producing pimelic acid or pimelate semialdehyde.35. The recombinant host of claim 34 , further comprising at least one exogenous nucleic acid encoding a ω-transaminase ...

CELL-FREE AND MINIMIZED METABOLIC REACTION CASCADES FOR THE PRODUCTION OF CHEMICALS

Provided are enzymatic processes for the production of chemicals like ethanol from carbon sources like glucose, in particular, a process for the production of a target chemical is disclosed using a cell-free enzyme system that converts carbohydrate sources to the intermediate pyruvate and subsequently the intermediate pyruvate to the target chemical wherein a minimized number of enzymes and only one cofactor is employed. 2. The process of claim 1 , wherein the temperature of the process is maintained in a range from 40-80° C.3. The process of claim 1 , wherein the process is maintained at the given temperature for at least 1 hour.4. The process of claim 1 , step (1) further comprising the use of a single dehydrogenase for the oxidation of glucose and/or galactose to gluconate and/or galactonate.5. The process of claim 1 , step (2) further comprising the conversion of gluconate to 2-keto-3-deoxygluconate and of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate and/or the conversion of galaconate to 2-keto-3-deoxygalactonate and of 2-keto-3-deoxygalactonate to glyceraldehyde and pyruvate.6. The process of claim 1 , step (2) further comprising the use of dehydroxy acid dehydratase and keto-3-deoxygluconate aldolase.7. The process of claim 1 , step (3) further comprising the use of a dehydrogenase for the oxidation of glyceraldehyde to glycerate.8. The process of claim 1 , wherein steps (1) and (3) are carried out with the use of a single dehydrogenase.9. The process of claim 1 , wherein no net production of ATP occurs.10. The process of claim 1 , wherein no ATPase or arsenate is added.11. The process of claim 1 , wherein said process occurs without ATP and/or ADP as cofactors.12. The process of claim 1 , wherein the single cofactor is selected from the group consisting of NAD/NADH claim 1 , NADP/NADPH claim 1 , and FAD/FADH2.13. The process of claim 11 , wherein the single cofactor is NAD/NADH.14. The process of claim 1 , wherein the enzyme activity of each ...

Yeast Organism Producing Isobutanol at a High Yield

The present invention provides recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and/or glycerol-3-phosphate dehydrogenase activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the , Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms. 2. The recombinant yeast microorganism of claim 1 , wherein all endogenous PDC genes and all endogenous GPD genes are disrupted claim 1 , mutated claim 1 , or deleted.3. The recombinant yeast microorganism of claim 1 , wherein said one or more endogenous PDC genes is selected from the group consisting of PDC1 claim 1 , PDC2 claim 1 , PDC5 claim 1 , and PDC6.4. The recombinant yeast microorganism of claim 1 , wherein said one or more endogenous GPD genes is selected from the group consisting of GPD1 and GPD2.5. The recombinant yeast microorganism of claim 1 , wherein said ALS is a cytosolically-localized ALS.6Lactococcus lactis. The recombinant yeast microorganism of claim 5 , wherein said cytosolically-localized ALS is encoded by the alsS gene.7Bacillus subtilis. The recombinant yeast microorganism of claim 5 , wherein said cytosolically-localized ALS is encoded by the alsS gene.8. The recombinant yeast microorganism of claim 1 , wherein said recombinant yeast microorganism has been engineered to disrupt or delete an endogenous pyruvate dehydrogenase (PDH) gene.9. The recombinant yeast microorganism of claim 1 , wherein said recombinant yeast microorganism further expresses a heterologous gene encoding a ketol-acid reductoisomerase.10. The recombinant ...

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 ...

DHAD Variants and Methods of Screening

Methods of screening for dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed, along with DHAD variants identified by these methods. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol. 1136-. (canceled)137Streptococcus mutans. A recombinant host cell comprising an isolated polypeptide having dihydroxy-acid dehydratase (DHAD) activity , wherein the polypeptide is at least 95% identical to a DHAD and comprises one or more amino acid substitutions selected from:(a) aspartic acid at a position corresponding to position 33 of SEQ ID NO: 168;(b) glutamic acid at a position corresponding to position 62 of SEQ ID NO: 168;(c) valine at a position corresponding to position 115 of SEQ ID NO: 168;(d) glutamic acid at a position corresponding to position 116 of SEQ ID NO: 168;(e) serine at a position corresponding to position 119 of SEQ ID NO: 168;(f) arginine at a position corresponding to position 158 of SEQ ID NO: 168;(g) glutamine at a position corresponding to position 176 of SEQ ID NO: 168;(h) leucine at a position corresponding to position 179 of SEQ ID NO: 168;(i) arginine at a position corresponding to position 322 of SEQ ID NO: 168;(j) serine at a position corresponding to position 425 of SEQ ID NO: 168;(k) glycine at a position corresponding to position 524 of SEQ ID NO: 168;(l) valine or leucine at a position corresponding to position 562 of SEQ ID NO: 168;(m) arginine, cysteine, or glycine at a position corresponding to position 563 of SEQ ID NO: 168;(n) glutamic acid at a position corresponding to position 564 of SEQ ID NO: 168; and(o) aspartic acid at a position corresponding to position 567 of SEQ ID NO: 168.1381. The recombinant host cell of claim , wherein the polypeptide comprises one or more ...

Engineered Microorganisms for Production of Commodity Chemicals and Cellular Biomass

The present disclosure provides methods of producing commodity products, the methods involving culturing a host cell that is genetically modified to produce a uronate dehydrogenase (UDH) that converts a sugar acid to its corresponding 1,5-aldonolactone, that uses NADP+ or NAD+ as a cofactor, and that produces NADPH or NADH, respectively, where the host cell coexpresses an endogenous or a heterologous reductase that utilizes the produced NADPH or NADH to generate the commodity product or a precursor thereof. The present disclosure provides a method of producing downstream products of glycerol and pyruvate in a genetically modified microbial host cell, the method involving culturing a genetically modified microbial host cell of the present disclosure in a culture medium comprising D-galacturonic acid. The present disclosure provides variant UDH polypeptides that utilize NADP+, nucleic acids encoding the variant UDH polypeptides; and host cells genetically modified with the nucleic acids.

BACTERIA ENGINEERED TO TREAT DISORDERS INVOLVING THE CATABOLISM OF A BRANCHED CHAIN AMINO ACID

The present disclosure provides recombinant bacterial cells that have been engineered with genetic circuitry which allow the recombinant bacterial cells to sense a patient's internal environment and respond by turning an engineered metabolic pathway on or off. When turned on, the recombinant bacterial cells complete all of the steps in a metabolic pathway to achieve a therapeutic effect in a host subject. These recombinant bacterial cells are designed to drive therapeutic effects throughout the body of a host from a point of origin of the microbiome. Specifically, the present disclosure provides recombinant bacterial cells comprising a heterologous gene encoding a branched chain amino acid catabolism enzyme. The disclosure further provides pharmaceutical compositions comprising the recombinant bacteria, and methods for treating disorders involving the catabolism of branched chain amino acids using the pharmaceutical compositions disclosed herein. 1. A bacterium comprising gene sequence(s) encoding one or more branched chain amino acid catabolism enzyme(s) operably linked to a directly or indirectly inducible promoter that is not associated with the branched chain amino acid catabolism enzyme gene in nature.2. The bacterium of claim 1 , wherein the bacterium further comprises gene sequence(s) encoding one or more transporter(s) of a branched chain amino acid operably linked to a promoter that is not associated with the transporter gene in nature.3. The bacterium of claim 2 , wherein the promoter is a directly or indirectly inducible promoter.4. The bacterium of or claim 2 , wherein the bacterium further comprises a genetic modification that reduces export of a branched chain amino acid from the bacterium.5. The bacterium of or claim 2 , wherein the bacterium further comprises a genetic modification that reduces endogenous biosynthesis of a branched chain amino acid in the bacterium.6. The bacterium of or claim 2 , wherein the bacterium further comprises gene sequence ...

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.

Mutant Host Cells For The Production Of 3-Hydroxypropionic Acid

Provided herein are recombinant host cells having an active 3-Hydroxypropionic Acid (3-HP) pathway wherein the host cells comprise a disruption to an endogenous gene that encodes for a pyruvate reductase. Also described are methods of making the host cells, and methods using the cells to produce 3-HP and derivatives of 3-HP (e.g., acrylic acid). 1. A recombinant yeast cell , comprising (1) an active 3-HP pathway capable of producing 3-HP and (2) a disruption to an endogenous gene that encodes for a pyruvate reductase , wherein:(a) the pyruvate reductase has at least 80% sequence identity to SEQ ID NO: 205;(b) the coding sequence of the endogenous gene encoding the pyruvate reductase hybridizes under at least high stringency conditions with the full-length complementary strand of SEQ ID NO: 204; or(c) the coding sequence of the endogenous gene encoding the pyruvate reductase has at least 80% sequence identity to SEQ ID NO: 204.2. The recombinant cell of claim 1 , wherein the pyruvate reductase has at least 90% sequence identity to SEQ ID NO: 205.3. The recombinant cell of claim 1 , wherein the pyruvate reductase differs by no more than ten amino acids from SEQ ID NO: 205.4. The recombinant cell of claim 1 , wherein the cell comprises a disruption to an endogenous gene that encodes for a pyruvate reductase comprising or consisting of SEQ ID NO: 205.5. The recombinant cell of claim 1 , wherein the coding sequence of the endogenous gene encoding the pyruvate reductase has at least 90% sequence identity to SEQ ID NO: 204.6. The recombinant cell of claim 1 , wherein the coding sequence of the endogenous gene encoding the pyruvate reductase comprises or consists of SEQ ID NO: 204.7. The recombinant cell of claim 1 , wherein cell produces less D-lactate compared to the parent strain that lacks disruption of the endogenous gene encoding for the pyruvate reductase claim 1 , when cultivated under identical conditions.8. The recombinant cell of claim 1 , wherein the cell ...

FERMENTIVE PRODUCTION OF FOUR CARBON ALCOHOLS

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway. 1. A vector comprising a first heterologous gene encoding an acetolactate synthase , a second heterologous gene encoding an acetohydroxy acid reductoisomerase , a third heterologous gene encoding an acetohydroxy acid dehydratase , and a fourth heterologous gene encoding a branched-chain α-keto acid decarboxylase , wherein each heterologous gene is operably linked to a promoter and lacks a mitochondrial targeting sequence.2. The vector of claim 1 , wherein the vector is a chromosomal integration vector.3. The vector of claim 1 , wherein the vector is an autonomous replication vector.4. The vector of claim 1 , further comprising a fifth heterologous gene encoding an alcohol dehydrogenase.5. A set of vectors comprising a first vector and a second vector claim 1 , wherein each vector comprises one or more heterologous genes encoding an enzyme that catalyzes a reaction in an engineered isobutanol biosynthetic pathway and wherein one of the one or more heterologous genes encodes a branched-chain α-keto acid decarboxylase claim 1 , wherein each heterologous gene is operably linked to a promoter and lacks a mitochondrial targeting sequence.6. The set of vectors of claim 5 , wherein the engineered isobutanol biosynthetic pathway comprises an acetolactate synthase (ALS) enzyme claim 5 , a ketol-acid reductoisomerase (KARI) enzyme claim 5 , a dihydroxy-acid dehydratase (DHAD) enzyme claim 5 , a branched chain keto acid decarboxylase enzyme claim 5 , and an alcohol dehydrogenase (ADH) enzyme.7. The set of vectors of claim 5 , wherein one or more of the vectors is a chromosomal integration vector.8. The set of vectors of claim 5 , wherein one or more of the vectors is an autonomous replication vector.9. The set of vectors of claim 5 , wherein one ...

DECARBOXYLASE PROTEINS WITH HIGH KETO-ISOVALERATE DECARBOXYLASE ACTIVITY

The present invention relates to recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise at least one nucleic acid molecule encoding a polypeptide with keto-isovalerate decarboxylase (KIVD) activity, wherein said polypeptide is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a polypeptide selected from SEQ ID NOs: 1-214. Also provided are modified decarboxylases exhibiting an improved ability to utilize α-ketoisovalerate as a substrate in various beneficial enzymatic conversions. 1101.-. (canceled)102L. lactisL. lactisL. lactisL. lactisL. lactisL. lactisL. lactis. A recombinant microorganism comprising at least one nucleic acid molecule encoding a modified decarboxylase enzyme , wherein the modified decarboxylase enzyme has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) serine 286 of the KIVD (SEQ ID NO: 197); (b) glutamine 377 of the KIVD (SEQ ID NO: 197); (c) phenylalanine 381 of the KIVD (SEQ ID NO: 197); (d) valine 461 of the KIVD (SEQ ID NO: 197); (e) isoleucine 465 of the KIVD (SEQ ID NO: 197); (f) methionine 538 of the KIVD (SEQ ID NO: 197); and (g) phenylalanine 542 of the KIVD (SEQ ID NO: 197).103L. lactis. The recombinant microorganism of claim 102 , wherein the residue corresponding to position 286 of the KIVD (SEQ ID NO: 197) is replaced with a residue selected from serine claim 102 , threonine claim 102 , asparagine claim 102 , glycine claim 102 , alanine claim 102 , proline claim 102 , glutamine claim 102 , and aspartic acid.104L. lactis. The recombinant microorganism of claim 102 , wherein the residue corresponding to position 377 of the KIVD (SEQ ID NO: 197) is replaced with a residue selected from glutamine claim 102 , serine claim 102 , threonine claim 102 , and ...

PROCESSES TO PREPARE ELONGATED 2-KETOACIDS AND C5-C10 COMPOUNDS THEREFROM VIA GENETIC MODIFICATIONS TO MICROBIAL METABOLIC PATHWAYS

Genetically modified isopropylmalate synthases, processes for preparing a C-C2-ketoacids utilizing genetically modified isopropylmalate synthases, and microbial organisms including genetically modified isopropylmalate synthases are described. The genetically modified isopropylmalate synthases, processes for preparing a C-C2-ketoacids, and microbial organisms including genetically modified isopropylmalate synthases can be particularly useful for producing corresponding Caldehydes, alcohols, carboxylic acids, and Calkanes both in vivo and in vitro. 121-. (canceled)22. A process for preparing a C-C2-ketoacid , the process comprising: (I) providing at least one of a C-C2-ketoacid substrate , with:(a) a genetically modified isopropylmalate synthase (IPMS) comprising at least one of:(i) an amino acid sequence comprising SEQ ID NO: 1 and comprising the mutations H97A, S139G, N167G, P169A, G181A, A182G, G210A, A214S, and G462D and having IPMS activity;(ii) an amino acid sequence comprising SEQ ID NO: 2 and comprising the mutations H97A, S139G, N167G, P169A, M255L, R260A, N264Q, and G462D and having IPMS activity; or(iii) an amino acid sequence comprising SEQ ID NO: 2 and comprising the mutations H97A, S139G, N167G, P169A, D348E, D350E, M353L, Q355N, and G462D and having IPMS activity;(b) a isopropylmalate isomerase having isopropylmalate isomerase activity; and{'sub': 4', '10', '7', '11', '4', '10', '7', '11, '(c) a isopropylmalate dehydrogenase having isopropylmalate dehydrogenase activity; under conditions that the at least one of the C-C2-ketoacid substrate is converted to the C-C2-ketoacid; and wherein the conversion of the at least one of the C-C2-ketoacid substrate to the C-C2-ketoacid occurs via one or more biochemical reactions.'}23. The process according to claim 22 , wherein the at least one of the C-C2-ketoacid substrate comprises 2-ketobutyrate.24. The process according to claim 22 , wherein the at least one of the C-C2-ketoacid substrate comprises 2- ...

MICROORGANISMS AND METHODS FOR PRODUCING CANNABINOIDS AND CANNABINOID DERIVATIVES

The present disclosure provides genetically modified host cells that produce a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative. The present disclosure provides methods of synthesizing a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative. 181-. (canceled)82. A genetically modified host cell that produces a cannabinoid or cannabinoid derivative , wherein the host cell is genetically modified with:a) one or more integrated heterologous nucleic acids encoding a tetraketide synthase (TKS) polypeptide;b) one or more integrated heterologous nucleic acids encoding an olivetolic acid cyclase (OAC) polypeptide;c) one or more integrated heterologous nucleic acids encoding an acyl-activating enzyme (AAE) polypeptide; andd) one or more integrated heterologous nucleic acids encoding a geranyltransferase (GOT) polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 110 or at least 90% sequence identity to SEQ ID NO: 100.83. The genetically modified host cell of claim 82 , wherein the cell is genetically modified to comprise between two and four copies of the heterologous nucleic acid encoding a tetraketide synthase (TKS).84. The genetically modified host cell of claim 82 , wherein the cell is genetically modified to comprise between two and four copies of the heterologous nucleic acid encoding an olivetolic acid cyclase (OAC).85. The genetically modified host cell of claim 82 , wherein the host cell is further genetically modified with:a) one or more heterologous nucleic acids encoding a tetrahydrocannabinolic acid (THCA) synthase polypeptide or a cannabidiolic acid (CBDA) synthase polypeptide;b) one or more heterologous nucleic acids encoding one or more polypeptides that generate geranyl pyrophosphate;c) one or more heterologous nucleic acids encoding one or more polypeptides that generate hexanoyl-CoA or derivatives thereof; andd) ...

Microorganism having carbon dioxide fixation pathway introduced thereinto

An acetyl-CoA-producing microorganism, which is capable of efficiently synthesizing acetyl-CoA using carbon dioxide, and a substance production method using the same are provided. An acetyl-CoA-producing microorganism including an acetyl-CoA production cycle obtained by imparting at least one type of enzymatic activity selected from the group consisting of malate thiokinase, malyl-CoA lyase, glyoxylate carboligase, 2-hydroxy-3-oxopropionate reductase, and hydroxypyruvate reductase, to a microorganism.

ENZYME SCAFFOLDS AND METHODS OF USE

Polypeptide scaffolds comprising enzymatic proteins are provided. The enzymatic polypeptide scaffolds comprise heterologous enzymes to form a heterologous metabolic pathway, and can be targeted to a substrate through a surface anchoring domain. The enzymatic polypeptide scaffolds leverage the high specificity and affinity protein/protein interaction between the cohesins and dockerins of microorganismal cellulosomes to form custom enzymatic arrays. 1. An enzymatic polypeptide scaffold , comprising:a first linker domain, a first cohesin domain, and a second cohesin domain, wherein the linker domain interconnects the first and second cohesin domains;a first recombinant polypeptide comprising a first dockerin domain and a glycerol dehydrogenase catalytic domain, wherein the first dockerin domain selectively binds to the first cohesin domain; anda second recombinant polypeptide comprising a second dockerin domain and a aldehyde dehydrogenase catalytic domain, wherein the second dockerin domain selectively binds to the second cohesin domain.2. The enzymatic polypeptide scaffold of claim 1 , further comprising:a second linker domain and a third cohesin domain, wherein the second linker domain interconnects the second and third cohesin domains; anda third recombinant polypeptide comprising a third dockerin domain and an NADH oxidase catalytic domain, wherein the third dockerin domain selectively binds to the third cohesin domain.3. The enzymatic polypeptide scaffold of claim 1 , further comprising a surface anchoring domain and an anchoring linker domain claim 1 , wherein the anchoring linker domain interconnects the surface anchoring domain and the first cohesin domain.4. The enzymatic polypeptide scaffold of claim 2 , further comprising:a first polypeptide linker between the first dockerin domain and the glycerol dehydrogenase catalytic domain,a second polypeptide linker between the second dockerin domain and the aldehyde dehydrogenase catalytic domain, anda third ...

CELL-FREE PRODUCTION OF BUTANOL

Provided herein, in some aspects, are methods and compositions for producing large-scale quantities of butanol, including normal butanol (n-butanol), isobutanol, and 2-butanol using a cell-free system. 1. A method of producing a cell lysate for producing n-butanol , the method comprising:(a) culturing engineered cells that express at least one enzyme of a n-butanol biosynthetic pathway selected from the group consisting of: glucokinase, phosphoglucose isomerase, phosphofructokinase, fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate dehydrogenase complex, acetyl-CoA acetyltransferase, hydroxybutyrl-CoA dehydrogenase, enoyl-CoA hydratase, crotonyl-CoA reductase, butyraldehyde dehydrogenase, and alcohol dehydrogenase, wherein the cells are cultured under conditions that result in expression of enzymes; and(b) lysing engineered cells cultured in step (a), thereby producing a cell lysate that comprises at least one enzyme of the n-butanol biosynthetic pathway.2. The method of claim 1 , wherein the engineered cells express at least 2 enzymes of the n-butanol biosynthetic pathway selected from the group consisting of: glucokinase claim 1 , phosphoglucose isomerase claim 1 , phosphofructokinase claim 1 , fructose bisphosphate aldolase claim 1 , triose phosphate isomerase claim 1 , glyceraldehyde 3-phosphate dehydrogenase claim 1 , phosphoglycerate kinase claim 1 , phosphoglycerate mutase claim 1 , enolase claim 1 , pyruvate kinase claim 1 , pyruvate dehydrogenase complex claim 1 , acetyl-CoA acetyltransferase claim 1 , hydroxybutyrl-CoA dehydrogenase claim 1 , enoyl-CoA hydratase claim 1 , crotonyl-CoA reductase claim 1 , butyraldehyde dehydrogenase claim 1 , and alcohol dehydrogenase.3. The method of claim 2 , wherein the engineered cells express 2 to 17 enzymes of the n-butanol biosynthetic pathway selected from the group consisting of: ...

PLANTS WITH ENHANCED YIELD AND METHODS OF CONSTRUCTION

Transgenic plants having enhanced yield and having enhanced seed yield are disclosed. The transgenic plants are transformed with a transgenic polynucleotide encoding one or more metabolic enzymes. The metabolic enzymes can be from any biological source. The transgenic polynucleotide(s) comprises a nucleic acid sequences encoding the metabolic enzymes under the control of functional plant promoters, the one or more metabolic enzymes are targeted to the plastids by the addition of plastid targeting signals. Optionally the functional plant promoters are seed specific promoters and the metabolic enzymes are targeted to the plastids by the addition of plastid targeting peptide heterologous to the metabolic enzymes. Methods of making the transgenic plants and transgenic polynucleotides are disclosed. The magnitude of the increases in seed yield achieved with these transgenic plants are simply unprecedented. 1. A transgenic plant comprising a one or more heterologous enzymes , each heterologous enzyme encoded by a transgene ,wherein the one or more heterologous enzymes are selected from the group consisting of: an oxygen tolerant pyruvate oxidoreductase (Por), a pyruvate carboxylase (Pyc), a malate synthase (AceB), a malate dehydrogenase (Mdh), a malate thiokinase (SucC and SucD), a malyl-CoA Lyase (Mcl), isocitrate lyase (Id), fumarate hydratase (FumC), NADH-dependent fumarate reductase (FRDg), aconitase hydratase 1 (AcnA), ATP-citrate lyase A-1 (AclA), and ATP-citrate lyase subunit B2 (AclB),wherein the transgenic plant has one or more increased properties selected from yield, seed yield, and seed oil content compared to a plant of the same species not comprising the one or more heterologous enzymes.2. The transgenic plant of claim 1 , wherein the transgenic plant wherein the one or more heterologous enzymes are selected from the group consisting of:an oxygen tolerant pyruvate oxidoreductase (Por), pyruvate carboxylase (Pyc), malate synthase (AceB), malate dehydrogenase ...

Methods, hosts, and reagents related thereto for production of unsaturated pentahydrocarbons, derivatives and intermediates thereof

This application describes methods, including non-naturally occurring methods, for biosynthesizing unsaturated pentahydrocarbons, such as isoprene and intermediates thereof, via the mevalonate pathway, as well as non-naturally occurring hosts for producing isoprene.

Fermentive Production of Isobutanol Using Highly Effective Ketol-Acid Reductoisomerase Enzymes

Ketol-acid reductoisomerase enzymes have been identified that provide high effectiveness in vivo as a step in an isobutanol biosynthetic pathway in bacteria and in yeast. These KARIs are members of a clade identified through molecular phylogenetic analysis called the SLSL Clade. 118-. (canceled)19. A recombinant yeast cell comprising at least one nucleic acid molecule encoding a polypeptide having ketol-acid reductoisomerase (KARI) activity wherein the polypeptide having KARI activity has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 , 36 , 38 , 40 , 56 , 60 , or 68 and wherein the yeast cell comprises:(i) at least one inactivated pyruvate decarboxylase (PDC) gene,(ii) a modification to reduce glycerol-3-phosphate dehydrogenase activity, and(iii) a heterologous polynucleotide encoding a polypeptide with phosphoketolase activity.20. The recombinant yeast cell of claim 19 , wherein the inactivated pyruvate decarboxylase (PDC) gene is PDC1 claim 19 , PDC5 claim 19 , or PDC6.21. The recombinant yeast cell of claim 19 , wherein the recombinant yeast cell further comprises at least one deletion claim 19 , mutation claim 19 , or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis.22. The recombinant yeast cell of claim 19 , wherein the yeast cell expresses an engineered isobutanol biosynthetic pathway.23. The recombinant yeast cell of claim 22 , wherein the engineered isobutanol biosynthetic pathway comprises the following substrate to product conversions:(i) pyruvate to acetolactate catalyzed by acetolactate synthase,(ii) 2,3-dihydroxyisovalerate to a-ketoisovalerate catalyzed by dihydroxy-acid dehydratase, and(iii) α-ketoisovalerate to isobutyraldehyde catalyzed by branched-chain a-keto acid decarboxylase.24. The recombinant yeast cell of claim 23 , wherein the acetolactate synthase has an EC number 1.1.1.86.25. The recombinant yeast cell of claim 23 , wherein the dihydroxy-acid ...

MICROORGANISMS AND METHODS FOR PRODUCING CANNABINOIDS AND CANNABINOID DERIVATIVES

The present disclosure provides genetically modified host cells that produce a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative. The present disclosure provides methods of synthesizing a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative. 181.-. (canceled)82. A genetically modified yeast cell for producing a cannabinoid or a cannabinoid derivative , the genetically modified yeast cell comprising one or more heterologous nucleic acids encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO:110 or SEQ ID NO:100.83. The genetically modified yeast cell of claim 82 , wherein the genetically modified yeast cell further comprises one or more heterologous nucleic acids encoding a tetraketide synthase (TKS) polypeptide and one or more heterologous nucleic acids encoding an olivetolic acid cyclase (OAC) polypeptide claim 82 , or one or more heterologous nucleic acids encoding a fusion TKS and OAC polypeptide.84. The genetically modified yeast cell of claim 83 , wherein the TKS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:11 and the OAC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:10.85. The genetically modified yeast cell of claim 82 , wherein the genetically modified yeast cell further comprises one or more of the following:a) one or more heterologous nucleic acids encoding a polypeptide that generates an acyl-CoA compound or an acyl-CoA compound derivative; orb) one or more heterologous nucleic acids encoding a polypeptide that generates geranyl pyrophosphate.86. The genetically modified yeast cell of claim 85 , wherein the genetically modified yeast cell further comprises one or more heterologous nucleic acids encoding a polypeptide that generates an acyl-CoA ...

ISOBUTANOL TOLERANCE IN YEAST WITH AN ALTERED LIPID PROFILE

Provided herein are recombinant yeast host cells and methods for their use for production of fermentation products from an engineered pyruvate utilizing pathway. The yeast host cells provided herein comprise an altered lipid profile, which confers resistance to butanol. 163-. (canceled)64. A yeast microorganism comprising an engineered butanol biosynthetic pathway and an altered lipid profile , wherein the yeast microorganism comprises a different composition of fatty acids as compared to a wild-type yeast microorganism grown under standard fermentation conditions.651. The yeast microorganism of claim , wherein the yeast microorganism is engineered to express one or more enzymes selected from the group consisting of fatty acid desaturase , fatty acid elongase , cyclopropane fatty acid synthase , or combinations thereof.661. The yeast microorganism of claim , wherein the altered lipid profile comprises one or more of the following: (1) an increase in the concentration of C18:1 , C18:2 , and C18:3 fatty acids , (2) an increase in the ratio of unsaturated to saturated fatty acids , (3) an increase in the concentration of cyclopropane fatty acid , and (4) an increase in the C18 to C16 fatty acid concentration ratio , as compared to a microorganism that lacks an altered lipid profile.67. The yeast microorganism of claim 65 , wherein the fatty acid desaturase is selected from:a) a polypeptide that has at least 90% identity to any one or more of SEQ ID NOs: 1, 2, or 9;b) a polypeptide encoded by a nucleic acid sequence that has at least 90% identity to any one or more of SEQ ID NOs: 3, 4, or 10;c) a fatty acid desaturase having an EC number 1.14.19.1 or 1.14.19.6; and{'i': Yarrowia lipolytica, Fusarium moniliforme', 'Mortierella alpine., 'd) a fatty acid desaturase isolated from , or'}68. The yeast microorganism of claim 65 , wherein the fatty acid elongase is selected from:a) a polypeptide that has at least 90% identity to any one or more of SEQ ID NOs: 11, 15, or 16;b) a ...

RECOMBINANT CELLS AND METHOD FOR PRODUCING ISOPRENE OR TERPENE

To provide a recombinant cell being an anaerobic archaeon, including a gene encoding isoprene synthase, a gene encoding monoterpene synthase, a gene encoding sesquiterpene synthase, a gene encoding diterpene synthase, a gene encoding squalene synthase, or a gene encoding phytoene synthase as a first foreign gene, wherein the first foreign gene is expressed, and the recombinant cell is capable of producing isoprene or terpene having 10, 15, 20, 30, or 40 carbon atoms. 1. A recombinant cell being an anaerobic archaeon , comprising: a gene encoding isoprene synthase , a gene encoding monoterpene synthase , a gene encoding sesquiterpene synthase , a gene encoding diterpene synthase , a gene encoding squalene synthase , or a gene encoding phytoene synthase as a first foreign gene ,wherein the first foreign gene is expressed, and the recombinant cell produces isoprene or terpene having 10, 15, 20, 30, or 40 carbon atoms.2. The recombinant cell according to claim 1 , growing using at least one selected from the group consisting of carbon monoxide claim 1 , carbon dioxide claim 1 , methane claim 1 , methanol claim 1 , and acetic acid as a sole carbon source.3. The recombinant cell according to claim 1 , comprising carbon monoxide dehydrogenase.4. The recombinant cell according to claim 1 , having a function of synthesizing acetyl-CoA from methyltetrahydropterin claim 1 , carbon monoxide claim 1 , and CoA.5. The recombinant cell according to claim 1 , having a methane formation potential.6. The recombinant cell according to claim 1 , growing using carbon dioxide as a sole carbon source and hydrogen as an energy source.7. The recombinant cell according to claim 1 , growing using methanol as a sole carbon source.8. (canceled)9MethanosarcinaMethanococcusMethanothermococcusMethanothermobacterMethanothrixThermococcusThermofilumArchaeoglobus.. The recombinant cell according to claim 1 , belonging to the genus claim 1 , the genus claim 1 , the genus claim 1 , the genus claim 1 , ...

PROCESS TO PREPARE ELONGATED 2-KETOACIDS AND C-5-C10 COMPOUNDS THEREFROM VIA GENETIC MODIFICATIONS TO MICROBIAL METABOLIC PATHWAYS

Genetically modified LeuCD′ enzyme complexes, processes for preparing a C-C2-ketoacid utilizing genetically modified LeuCD′ enzyme complexes, and microbial organisms including modified LeuCD enzyme complexes are described. The instantly-disclosed genetically modified LeuCD′ enzyme complexes, processes for preparing a C-C2-ketoacid, and microbial organisms including modified LeuCD′ enzyme complexes can be particularly useful for producing C-Caldehydes, alkanes, alcohols, and carboxylic acids, both in vivo and in vitro. 125-. (canceled)26. A process for preparing a C-C2-ketoacid , the process comprising: (I) providing at least one of a C-C2-ketoacid substrate with:(A) at least one isopropylmalate synthase enzyme having isopropylmalate synthase activity;(B) at least one isopropylmalate dehydrogenase enzyme having isopropylmalate dehydrogenase activity; and (1) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 1 and at least one modification wherein alanine is substituted for Val-35 and a a native LeuD subunit comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 2;', '(2) a combination of a native LeuC subunit comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 2 and at least one modification wherein alanine is substituted for Leu-31;', '(3) a combination of a native LeuC subunit comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 2 and at least one modification wherein glycine is substituted for Leu-31;', '(4) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 1 and at least one modification wherein alanine is ...

RECOMBINANT CELL, METHOD FOR PRODUCING RECOMBINANT CELL, AND METHOD FOR PRODUCING ORGANIC COMPOUND

A recombinant cell having a function of synthesizing acetyl-CoA from methyltetrahydrofolate, carbon monoxide, and CoA, including: a gene that expresses an exogenous NAD(P)H consumption pathway, the gene being expressed in the recombinant cell, wherein expression in at least one of endogenous NAD(P)H consumption pathways of the recombinant cell is down-regulated, and the endogenous NAD(P)H consumption pathway is different from the exogenous NAD(P)H consumption pathway, and the recombinant cell produces an organic compound having 4 or more carbon atoms from at least one selected from the group consisting of carbon monoxide and carbon dioxide via the exogenous NAD(P)H consumption pathway. 1. A recombinant cell having a function of synthesizing acetyl-CoA from methyltetrahydrofolate , carbon monoxide , and CoA , comprising:a gene that expresses an exogenous NAD(P)H consumption pathway, the gene being expressed in the recombinant cell,wherein expression in at least one of endogenous NAD(P)H consumption pathways of the recombinant cell is down-regulated, and the endogenous NAD(P)H consumption pathway is different from the exogenous NAD(P)H consumption pathway, andthe recombinant cell produces an organic compound having 4 or more carbon atoms from at least one selected from the group consisting of carbon monoxide and carbon dioxide via the exogenous NAD(P)H consumption pathway.2ClostridiumMoorella. The recombinant cell according to claim 1 , being a bacterium or a bacterium.3. (canceled)4. The recombinant cell according to claim 2 , wherein the exogenous NAD(P)H consumption pathway is a mevalonate pathway.5. The recombinant cell according to claim 4 , wherein the mevalonate pathway is a mevalonate pathway of yeast claim 4 , prokaryote claim 4 , or actinomycete.6. (canceled)7. The recombinant cell according to claim 4 , wherein HMG-CoA reductase of the mevalonate pathway includes NADH-dependent HMG-CoA reductase.8. (canceled)9. The recombinant cell according to claim 4 , ...

Integration of a Polynucleotide Encoding a Polypeptide That Catalyzes Pyruvate to Acetolactate Conversion

The invention relates to recombinant host cells having at least one integrated polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway, e.g., pyruvate to acetolactate conversion. The invention also relates to methods of increasing the biosynthetic production of isobutanol, 2,3-butanediol, 2-butanol or 2-butanone using such host cells. 147-. (canceled)48. A recombinant host cell comprising:{'i': Bacillus subtilis, Klebsiella pneumonia, Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, Streptococcus thermophiles, Vibrio angustum,', 'Bacillus cereus;, '(a) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of pyruvate to acetolactate wherein the polypeptide is an acetolactate synthase from or'}(b) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate wherein the polypeptide is a ketol-acid reductoisomerase and the ketol-acid reductoisomerase has at least 95% identity to SEQ ID NO: 83;{'i': Escherichia coli, Bacillus subtilis, Methanococcus maripaludis,', 'Streptococcus mutans;, '(c) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate wherein the polypeptide is a dihydroxyacid dehydratase from or and'}(d) a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion of α-ketoisovalerate to isobutyraldehyde wherein the polypeptide is a branched-chain α-keto acid decarboxylase and the branched-chain α-keto acid decarboxylase has at least 95% identity to SEQ ID NO: 247.49Achromobacter xylosoxidansBeijerinkia indica.. The recombinant host cell of further comprising a polynucleotide encoding a polypeptide which catalyzes the substrate to product conversion isobutyraldehyde to isobutanol wherein the polypeptide is an alcohol dehydrogenase from or50. ...

PROCESSES TO PREPARE ELONGATED 2-KETOACIDS AND C6-C10 COMPOUNDS THEREFROM VIA GENETIC MODIFICATIONS TO MICROBIAL METABOLIC PATHWAYS

Modification of metabolic pathways includes genetically engineering at least one enzyme involved in elongating 2-ketoacids during leucine biosynthesis, and preferably at least isopropylmalate dehydrogenase or synthase (LeuB or LeuA in ), to include at least such non-native enzyme, enzyme complex, or combination thereof to convert 2-ketobutyrate or 2-ketoisovalerate to a C7-C11 2-ketoacid, wherein the production of such is at a higher efficiency than if a purely native pathway is followed. The C7-C11 2-ketoacid may then be converted, via a native or genetically engineered thiamin dependent decarboxylase, to form a C6-C10 aldehyde having one less carbon than the C7-C11 2-ketoacid being converted. In some embodiments the C6-C10 aldehyde may then be converted via additional native or genetically engineered enzymes to form other C6-C10 products, including alcohols, carboxylic acids, and alkanes. This genetic engineering offers the opportunity for commercial scale of in vivo biosynthetic processes that may be more cost-efficient than non-biobased approaches to produce the same products. 1. A process for preparing a C7-C11 2-ketoacid comprising contacting (1) a native or genetically modified LeuA enzyme;', [{'i': 'Escherichia coli', '(a) obtained from and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified in that alanine, glycine, valine or leucine is independently substituted for Leu-96, Val-198, or a combination thereof; or'}, '(b) the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (a; and, '(2) a genetically modified LeuB′ enzyme, wherein the enzyme is'}, '(3) a native or genetically modified LeuCD′ enzyme complex;, 'a substrate, selected from 2-ketobutyrate and 2-ketoisovalerate, and'}under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7- ...

RECOMBINANT MICROORGANISM HAVING HETEROLOGOUS GENES INTRODUCED THERETO AND METHOD FOR PRODUCING USEFUL MATERIAL FROM FORMIC ACID AND CARBON DIOXIDE USING SAME MICROORGANISM

The present invention relates to a recombinant microorganism having heterologous genes introduced thereto and a method for producing a useful material from formic acid and carbon dioxide using the microorganism. The present invention provides a novel microorganism having a cyclic metabolic pathway introduced thereto through which C3 or higher carbon organic compounds can be synthesized from formic acid and carbon dioxide, whereby carbon dioxide rich in nature and formic acid that is of low toxicity and suitable for anabolic reaction in view of reaction kinetics and which can be easily and rapidly synthesized from carbon dioxide can be used to effectively synthesize the C3 organic compound pyruvic acid from which various high-value added compound can be synthesized. 1. A recombinant microorganism having improved assimilation from formic acid and carbon dioxide , obtained by introducing a gene encoding an enzyme involved in a formic acid assimilation pathway or a recombinant vector containing the gene into a host microorganism having a central carbon assimilation pathway.2. The recombinant microorganism according to claim 1 , wherein the enzyme is at least one selected from the group consisting of formate-tetrahydrofolate ligase claim 1 , methenyl tetrahydrofolate cyclohydrolase and methylene-tetrahydrofolate dehydrogenase.3. The recombinant microorganism according to claim 2 , wherein the gene encoding formate-tetrahydrofolate ligase is a nucleic acid molecule represented by SEQ ID NO: 7 claim 2 ,the gene encoding methenyl tetrahydrofolate cyclohydrolase is a nucleic acid molecule represented by SEQ ID NO: 8 or SEQ ID NO: 19, andthe gene encoding methylene-tetrahydrofolate dehydrogenase is a nucleic acid represented by SEQ ID NO: 9.4. The recombinant microorganism according to claim 1 , wherein i) the host microorganism inherently possesses a central carbon assimilation pathway; or ii) a central carbon assimilation pathway is introduced into the host microorganism.5. ...

BIOFUEL PRODUCTION BY RECOMBINANT MICROORGANISMS

Provided herein are metabolically-modified microorganisms useful for producing biofuels. More specifically, provided herein are methods of producing high alcohols including isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from a suitable substrate. 141-. (canceled)42. A recombinant prokaryote comprising:(a) an α-isopropylmalate synthase polypeptide having at least 90% identity to SEQ ID NO:50 and having α-isopropylmalate synthase activity;(b) a β-isopropylmalate dehydrogenase polypeptide having at least 90% identity to SEQ ID NO:52 and having β-isopropylmalate dehydrogenase activity;(c) an α-isopropylmalate isomerase polypeptide having at least 90% identity to SEQ ID NO:54 and having α-isopropylmalate isomerase activity;(d) a 2-keto-acid decarboxylase polypeptide having at least 90% identity to SEQ ID NO:28 and having 2-keto acid decarboxylase activity; and(e) a NADH-dependent alcohol dehydrogenase having at least 90% identity to SEQ ID NO:38;and further comprising (f) or (f′): (i) an α-isopropylmalate isomerase polypeptide having at least 90% identity to SEQ ID NO:56 and having α-isopropylmalate isomerase activity;', '(ii) a threonine dehydratase polypeptide having at least 90% identity to SEQ ID NO:48 and having threonine dehydratase activity; and optionally', '(iii) a citramalate synthase polypeptide having at least 90% identity to SEQ ID NO:58 and having citramalate synthase activity; or, '(f) polypeptides comprising (i) an acetohydroxy acid synthase polypeptide having at least 90% identity to SEQ ID NO:42 and having acetohydroxy acid synthase activity;', '(ii) an acetohydroxy acid synthase polypeptide having at least 90% identity to SEQ ID NO:40 and having acetohydroxy acid synthase activity;', '(iii) an acetohydroxy acid isomeroreductase polypeptide having at least 90% identity to SEQ ID NO:44 and having acetohydroxy acid isomeroreductase activity; and', '(iv) a dihydroxy-acid dehydratase polypeptide having at least ...

BIOCHEMICAL UPGRADING OF HIGH-PROTEIN BIOMASS AND GRAIN PRODUCTS

The present invention relates to methods of upgrading biomass to provide useful chemical intermediates, fuels, amino acids, nutrients, etc. In particular examples, the biomass is a by-product of ethanol production and is mainly used as high-protein feed. Described herein are methods for upgrading such biomass, such as by implementing pre-treatment conditions and by employing fermentation conditions including modified organisms. 1. An isolated , genetically engineered organism comprising:a modified alcohol dehydrogenase having increased reactivity with nicotinamide adenine dinucleotide (NADH), as compared to a wild-type alcohol dehydrogenase; anda modified ketol-acid reductoisomerase having increased reactivity with NADH, as compared to a wild-type ketol-acid reductoisomerase.2. The isolated claim 1 , genetically engineered organism of claim 1 , wherein the modified alcohol dehydrogenase comprises at least one amino acid substitution claim 1 , as compared to the wild-type alcohol dehydrogenase.3. The isolated claim 1 , genetically engineered organism of claim 1 , wherein the modified ketol-acid reductoisomerase comprises at least one amino acid substitution claim 1 , as compared to the wild-type ketol-acid reductoisomerase.4. The isolated claim 1 , genetically engineered organism of claim 1 , wherein the modified alcohol dehydrogenase comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs:1-5 claim 1 , or a fragment thereof.5. The isolated claim 4 , genetically engineered organism of claim 4 , wherein the modified ketol-acid reductoisomerase comprises a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs:6-7 claim 4 , or a fragment thereof.6. The isolated claim 1 , genetically engineered organism of claim 1 , wherein the modified alcohol dehydrogenase has increased specificity for NADH over nicotinamide adenine dinucleotide phosphate (NADPH) claim 1 , as compared to the wild-type alcohol dehydrogenase; ...

RECOMBINANT HOST CELLS AND METHODS FOR THE PRODUCTION OF ISOBUTYRIC ACID

Methods and materials related to producing isobutyric acid are disclosed. Specifically, isolated nucleic acids, polypeptides, host cells, methods and materials for producing isobutyric by direct microbial fermentation from a carbon source are disclosed. 1. A recombinant host cell , comprising:an isobutyric acid biosynthetic pathway, comprising heterologous nucleic acids encoding an acetolactate synthase, a ketol-acid reductoisomerase, a dihydroxy-acid dehydratase, a branched-chain-2-oxoacid decarboxylase, and an isobutyraldehyde dehydrogenase;wherein the heterologous nucleic acids are expressed in sufficient amounts to produce isobutyric acid.2Pichia kudriazeviiSaccharomyces cerevisiae.. The recombinant host cell of claim 1 , which is selected from and3Escherichia coli, Corynebacterium glutamicum, Bacillus subtilisLactococcus lactis.. The recombinant host cell of claim 1 , which is selected from claim 1 , and4. The recombinant host cell of any one of to claim 1 , wherein the acetolactate synthase has at least 60% amino acid identity to SEQ ID NO: 19 or SEQ ID NO: 1.5. The recombinant host cell of claim 4 , wherein the amino acid sequence of the acetolactate synthase is represented by SEQ ID NO: 1.6. The recombinant host cell of any one of to claim 4 , wherein the ketol-acid reductoisomerase has at least 60% amino acid identity to SEQ ID NO: 20.7. The recombinant host cell of any one of to claim 4 , wherein the ketol-acid reductoisomerase has at least 60% amino acid identity to SEQ ID NO: 2 or at least 60% amino acid identity to SEQ ID NO: 21.8. The recombinant host cell of claim 7 , wherein the amino acid sequence of the ketol-acid reductoisomerase is represented by SEQ ID NO: 2 or SEQ ID NO: 21.9. The recombinant host cell of any one of to claim 7 , wherein the dihydroxy-acid dehydratase has at least 47% amino acid identity to SEQ ID NO: 22.10. The recombinant host cell of claim 9 , wherein the dihydroxy-acid dehydratase has at least 60% amino acid identity to SEQ ...

PRODUCTION OF CANNABINOIDS IN MICROORGANISMS FROM A CARBON SUGAR PRECURSOR

A method is provided for biosynthetic production of cannabinoids in microorganisms from a carbon source precursor. This method describes the genetic modifications needed to engineer microorganisms to produce cannabinoids as well as a method for identifying and quantifying cannabinoids from fermentation broth. A system is also provided for tuning the method to produce different cannabinoids of interest by systematically modulating the enzymes encoded by the genetic modifications introduced in the microorganism. 1. A method for producing at least one cannabinoid from a carbon source precursor , comprising:genetically modifying a bacterial strain to express enzymes for converting the carbon source precursor into the at least one cannabinoid within the genetically modified bacterial strain.2. The method according to claim 1 , wherein the carbon source precursor is glucose and the method further comprises converting the glucose to hexanoate.3. The method according to claim 2 , wherein the at least one cannabinoid comprises cannabigerolic acid.4E. coli.. The method according to claim 3 , wherein the bacterial strain is5. The method according to claim 1 , wherein genetically modifying the bacterial strain comprises recombinantly incorporating a mutated FadD gene to express a mutated FadD enzyme which knocks out a FadE gene of the bacterial strain.6. The method according to claim 5 , wherein the mutated FadD gene comprises a nucleotide sequence of SEQ ID NO. 10.7. The method according to claim 1 , wherein genetically modifying the bacterial strain comprises transforming the bacterial strain to express olivetol synthase claim 1 , olivetolic acid cyclase claim 1 , and CsPT1.8. The method according to claim 7 , wherein the olivetol synthase comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO. 2 claim 7 , wherein the olivetolic acid cyclase comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO. 4 claim 7 , and ...

Fermentive production of four carbon alcohols

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway.

Method for the production of 7-dehydrocholesterol and/or the biosynthetic intermediate or subsequent products thereof in transgenic organisms

The invention relates to a method for the production of 7-dehydrocholesterol and/or the biosynthetic intermediate or subsequent products thereof by the cultivation of organisms, in particular, yeasts which show an increased activity of at least one activity, selected from delta-8/delta-7 isomerisation activity, delta-5 desaturase activity or delta-24 reductase activity. The invention further relates to the nucleic acid constructs necessary for production of the genetically-modified organisms and the genetically-modified organisms, in particular, the yeasts themselves.

Fermentative production of tetracarbon alcohols

FIELD: chemistry. SUBSTANCE: method of producing isobutanol involves obtaining a recombinant microbial host cell which contains a fermentative isobutanol channel which contains DNA molecules which code a set of polypeptides which catalyse the following substrate conversions to a product: i) pyruvate to acetolactate; ii) acetolactate to 2,3-dihydroxyisovalerate; iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate; iv) α-ketoisovalerate to isobutyraldehyde; and v) isobutyraldehyde to isobutanol, and bringing the host cell into contact with the fermented carbon substrate in a fermentation medium under conditions at which isobutanol is produced. EFFECT: isobutanol with high efficiency. 54 cl, 1 dwg, 13 tbl, 22 ex РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) 2 394 913 (13) C2 (51) МПК C12P 7/16 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ, ПАТЕНТАМ И ТОВАРНЫМ ЗНАКАМ (12) ОПИСАНИЕ ИЗОБРЕТЕНИЯ К ПАТЕНТУ (24) Дата начала отсчета срока действия патента: 25.10.2006 (30) Конвенционный приоритет: 26.10.2005 US 60/730,290 2 3 9 4 9 1 3 R U (56) Список документов, цитированных в отчете о поиске: YOSHIMOTO H. et al. Genetic and physiological analysis of branched-chain alcohols and isoamyl acetate production in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2002 Aug; 59(4-5):501-8. Epub 2002 Jun 22. DICKINSON J.R. An investigation of the metabolism of valine to isobutyl alcohol in Saccharomyces cerevisiae. J Biol Chem. 1998 Oct 2; 273(40):25751-6. WO 0050624 A, 31.08.2000. RU 2215782 C2, 10.06.2003. (85) Дата перевода заявки PCT на национальную фазу: 26.05.2008 (86) Заявка PCT: US 2006/041602 (25.10.2006) (87) Публикация PCT: WO 2007/050671 (03.05.2007) Адрес для переписки: 129090, Москва, ул. Б.Спасская, 25, стр.3, ООО "Юридическая фирма Городисский и Партнеры", пат.пов. Е.Е.Назиной, рег. № 517 (54) ФЕРМЕНТАТИВНОЕ ПОЛУЧЕНИЕ ЧЕТЫРЕХУГЛЕРОДНЫХ СПИРТОВ (57) Реферат: Изобретение относится к биотехнологии и представляет собой способ получения изобутанола, включающий ...

Monacolin K Biosynthesis Genes

모나스커스에 의해 생산되는 콜레스테롤 저해제, 모나콜린 케이는 폴리케타이드의 2차 대사산물이다. 본 발명은 모나콜린 케이 생합성 유전자 클러스터에 특이적인 프로브를 제공한다. 가능한 모나콜린 케이 유전자 클러스터를 가지는 BAC 클론이 BAC (세균성 인공 염색체) 라이브러리로부터 스크린되어졌고, 서열분석과 주석달기가 이들 클론에 수행되어졌다. 결과에 의하면 2개의 폴리케타이드 합성효소 (PKS) 유전자와 모나콜린 케이 합성과 관련된 7개의 조절유전자가 얻어졌다. 이들 유전자의 전장 cDNA가 RT-PCR에 의해 얻어졌고, 이들 유전자의 발현용 발현벡터에 클론되어졌다. The cholesterol inhibitor produced by Monascus, Monacholine K, is a secondary metabolite of polyketide. The present invention provides probes specific for the monacoline k biosynthetic gene cluster. BAC clones with possible Monacoline K gene clusters were screened from BAC (bacterial artificial chromosome) libraries and sequencing and annotation were performed on these clones. The results show two polyketide synthase (PKS) genes and seven regulatory genes associated with monacoline k synthesis. Full length cDNAs of these genes were obtained by RT-PCR and cloned into expression vectors for expression of these genes.

一个影响烟草腋芽分化的NtIPMD基因

本申请属于烟草基因工程领域，具体涉及一个影响烟草腋芽分化的 NtIPMD 基因。该基因包括1218bp碱基，具体核苷酸序列SEQ ID NO.1所示，其中第68~403位核苷酸为特异性核酸片段。该基因所编码的异丙基苹果酸脱氢酶NtIPMD，其特征在于，异丙基苹果酸脱氢酶NtIPMD包括405个氨基酸。本申请针对 NtIPMD 基因的初步研究表明，该基因与植株的腋芽发育相关，将该基因沉默后，可促进侧枝发育，利用这一特性，可以通过基因沉默或超表达技术将基因进行沉默或超表达，从而实现分子水平上对于该植株株型的调整，同时对于烟草新品种培育具有重要影响，因而具有较为重要的实用价值。

Dehydrogenase for preparing (R) -4-chloro-3-hydroxybutanoate ethyl ester

本发明提供了一种利用脱氢酶制备(R)‑4‑氯‑3‑羟基丁酸乙酯的方法，包括如下步骤：以4‑氯乙酰乙酸乙酯为底物，使用异丙醇脱氢酶SEQ ID NO:1或者其突变体SEQ ID NO:3催化还原反应，得到(R)‑4‑氯‑3‑羟基丁酸乙酯。

Production of odd chain fatty acid derivatives in recombinant microbial cells

Recombinant microbial cells are provided which have been engineered to produce fatty acid derivatives having linear chains containing an odd number of carbon atoms by the fatty acid biosynthetic pathway. Also provided are methods of making odd chain fatty acid derivatives using the recombinant microbial cells, and compositions comprising odd chain fatty acid derivatives produced by such methods.

Method for constructing recombinant bacterium for producing citral, recombinant bacterium constructed by method and application of recombinant bacterium

本发明涉及一种构建生产柠檬醛的重组菌的方法，及由其构建的重组菌及其应用。在本发明中，构建了两个质粒，其中一个质粒包含用于表达生成IPP和DMAPP的各种酶的编码基因：atoB、HMGS、HMGR、MK、PMK、PMD和idi，另一个质粒包含用于以IPP和DMAPP为前体合成柠檬醛的酶的编码基因：GPPS、GES、geoA和idi。用上述两质粒共转染常规的BW25113菌可得到重组菌，该重组菌可在经典LB培养基中以高产量发酵生产柠檬醛。

Recombinant yarrowia lipolytica and construction method and application thereof

本发明提供一株重组解脂耶罗维亚酵母及其构建方法和应用，涉及生物工程领域。该重组解脂耶罗维亚酵母，是解脂耶罗维亚酵母（ Yarrowia lipolytica ）XJ‑8株，已在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC No.19064。本发明重组解脂耶罗维亚酵母的构建方法，操作简单、高效，能够高效合成β‑榄香烯和/或大根香叶烯A。

Recombinant yarrowia lipolytica and construction method and application thereof

本发明提供一株重组解脂耶罗维亚酵母及其构建方法和应用，涉及生物工程领域。该重组解脂耶罗维亚酵母，是解脂耶罗维亚酵母（ Yarrowia lipolytica ）XJ‑8株，已在中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC No.19064。本发明重组解脂耶罗维亚酵母的构建方法，操作简单、高效，能够高效合成β‑榄香烯和/或大根香叶烯A。

Yeast organism producing isobutanol at a high yield

The present invention provides recombinant mircoorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and/or glycerol-3-phosphate dehydrogenase activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

Yeast organism producing isobutanol at a high yield

The present invention provides recombinant mircoorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and/or glycerol-3-phosphate dehydrogenase activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

Yeast organism producing isobutanol at a high yield

The present invention provides recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and/or glycerol-3-phosphate dehydrogenase activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade , Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

Ketol-acid reductoisomerase using NADH

Methods for the evolution of NADPH specific ketol-add reductoisomerase enzymes to acquire NADH specificity are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from Pseudomonas that have undergone co-factor switching to utilize NADH are described.

Ketol-acid reductoisomerase enzymes and methods of use

Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

Polypeptides with ketol-acid reductoisomerase activity

Polypeptides having ketol-acid reductoisomerase activity are provided. Also disclosed are recombinant host cells comprising isobutanol biosynthetic pathways employing such polypeptides. Methods for producing isobutanol employing host cells comprising the polypeptides having ketol-acid reductoisomerase activity are also disclosed.

Ketol-acid reductoisomerase using NADH

Methods for the evolution of NADPH specific ketol-acid reductoisomerase enzymes to acquire NADH specificity are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from Pseudomonas that have undergone co-factor switching to utilize NADH are described.

Ketol-acid reductoisomerase using NADH

Methods for the evolution of NADPH specific ketol-acid reductoisomerase enzymes to acquire NADH specificity are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from Pseudomonas that have undergone co-factor switching to utilize NADH are described.

Ketol-acid reductoisomerase enzymes and methods of use

Provided herein are polypeptides having ketol-acid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

Polypeptides with ketol-acid reductoisomerase activity

Polypeptides having ketol-acid reductoisomerase activity are provided. Also disclosed are recombinant host cells comprising isobutanol biosynthetic pathways employing such polypeptides. Methods for producing isobutanol employing host cells comprising the polypeptides having ketol-acid reductoisomerase activity are also disclosed.

Mutant Strain with improved isoleucine production having reduced by-product

본 발명은 이소루이신 생산능 변이 균주, 이의 제조방법 및 이소루이신 생산방법을 제공한다. 본 발명의 이소루이신 생산능 변이 균주는 야생형 균주와 비교하여 노르발린(norvaline)은 전혀 생성하지 않고 발린 및 α-아미노부티릭산(α-aminobutyric acid)과 같은 부산물의 생성은 현저히 감소하여 이소루이신의 생산이 증가된 효과를 갖는다. 또한, 본 발명은 부산물의 생산능을 감소시킴으로써 이소루이신을 높은 순도로 정제할 수 있을 뿐 만 아니라 이소루이신의 회수율을 향상시켜 이소루이신의 제조 원가를 크게 낮출 수 있다. The present invention provides a mutant strain of isoleucine production, a method for producing the same, and a method for producing isoleucine. The isoleucine production ability mutant strain of the present invention produces no norvaline at all compared with the wild type strain and the production of by-products such as valine and -aminobutyric acid is remarkably reduced, God's production has an increased effect. In addition, the present invention not only can purify isoleucine with high purity by reducing the productivity of by-products, but also can improve the recovery rate of isoleucine and greatly reduce the manufacturing cost of isoleucine.

Recombinant strain for producing beta-elemene or germacrene A

本发明提供了一种重组菌，所述重组菌的出发菌株为酿酒酵母，所述重组菌中表达有吉马烯A合酶(GAS)，所述GAS来源于多变鱼腥藻(Anabaena variabilis)ATCC29413；本发明通过优化吉马烯A合成模块、MVA途径内源性改造模块、合酶突变改造模块以及外排通道模块，有效地提高了酵母合成吉马烯A的效率；重组酵母菌可以高效的用于β‑榄香烯和/或吉马烯A的生产。