E.i. Dupont De Nemours and Company

Butamax Advanced Biofuels LLC

Butamax (tm) Advanced Biofuels LLC

Der-Ing Liao

Yougen Li

Mark J Nelson

Daniel P Okeefe

Xiao-Song Tang

Katharine Janet Gibson

Quinn Qun Zhu

Howard Glenn Damude

Hongxian He

Jeremy S Minshull

Sridhar Govindarajan

Michael G Bramucci

Daniel P O'Keefe

Steven Cary Rothman

Alexander Vincent Tobias

Jean-Francois Tomb

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.

Ketol-acid reductoisomerase enzymes and methods of use

Methods for the fermentative production of isobutanol is provided by the fermentative growth of a recombinant microorganism expressing a highly active ketol-acid reductoisomerase enzyme in addition to other enzymes required for conversion of glucose to isobutanol.

Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes

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

Ketol-acid reductoisomerase using NADH

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.

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

Methods for the evolution of NADPH binding ketol-acid reductoisomerase enzymes to acquire NADH binding functionality are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated fromthat have undergone co-factor switching to bind NADH are described.

Sequences of B-dependent dehydratases with improved reaction kinetics are presented. Use of these B-dependent dehydratases reduce the rate of the enzyme's suicide inactivation in the presence of glycerol and 1,3-propanediol. The enzymes were created using error-prone PCR and oligonucleotide-directed mutagenesis to target the DhaB1 gene, which encodes the &#x3b1;-subunit of glycerol dehydratase. Mutants with improved reaction kinetics were rapidly identified using high throughput assays.

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The present invention relates to mutant &#x394;8 desaturase genes, which have the ability to convert eicosadienoic acid [20:2 &#x3c9;-6, EDA] to dihomo-&#x3b3;-linolenic acid [20:3, DGLA] and/or eicosatrienoic acid [20:3 &#x3c9;-3, ETrA] to eicosatetraenoic acid [20:3 &#x3c9;-3, ETA]. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding &#x394;8 desaturase along with methods of making long-chain polyunsaturated fatty acids (PUFAs) using these mutant &#x394;8 desaturases in plants and oleaginous yeast are disclosed.

Mutant &#x394;8 desaturase genes engineered by targeted mutagenesis and their use in making polyunsaturated fatty acids

The present invention relates to mutant delta-8 desaturase genes, which have the ability to convert eicosadienoic acid [20:2 omega-6, EDA] to dihomo-&#x3b3;-linolenic acid [20:3, DGLA] and/or eicosatrienoic acid [20:3 omega-3, ETrA] to eicosatetraenoic acid [20:3 omega-3, ETA]. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding delta-8 desaturase along with methods of making long-chain polyunsaturated fatty acids (PUFAs) using these mutant delta-8 desaturases in plants and oleaginous yeast are disclosed.

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Wilmington, DE, United States of America

Der-Ing Liao