Abstract: The present invention relates to recombinant microorganisms comprising biosynthetic pathways and methods of using said recombinant microorganisms to produce various beneficial metabolites. In various aspects of the invention, the recombinant microorganisms may further comprise one or more modifications resulting in the reduction or elimination of 3 keto-acid (e.g., acetolactate and 2-aceto-2-hydroxybutyrate) and/or aldehyde-derived by-products. 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.

Abstract: There is disclosed a method of producing isobutanol. In an embodiment, the method includes providing a microorganism transformed with an isobutanol producing pathway containing at least one exogenous gene. The microorganism is selected to produce isobutanol from a carbon source at a yield of at least 10 percent theoretical. The method includes cultivating the microorganism in a culture medium containing a feedstock providing the carbon source, until isobutanol is produced. The method includes recovering the isobutanol. In one embodiment, the microorganism is a yeast with a Crabtree-negative phenotype. In another embodiment, the microorganism is a yeast microorganism with a Crabtree-positive phenotype. There is disclosed a microorganism for producing isobutanol. In an embodiment, the microorganism includes an isobutanol producing pathway containing at least one exogenous gene, and is selected to produce a recoverable quantity of isobutanol from a carbon source at a yield of at least 10 percent theoretical.

Abstract: The present invention provides recombinant microorganisms comprising isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol, wherein said recombinant microorganism is selected to produce isobutanol from a carbon source. Methods of using said recombinant microorganisms to produce isobutanol are also provided. In various aspects of the invention, the recombinant microorganisms may comprise a cytosolically active isobutanol pathway enzymes. In some embodiments, the invention provides mutated, modified, and/or chimeric isobutanol pathway enzymes with cytosolic 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.

Abstract: 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.

Abstract: Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. Mutant 9-10A-A328V hydroxylates octane primarily at the 2-positio to form S-2-octanol (40% ee). Another mutant, 1-12G, hydroxylates alkanes larger than hexane primarily at the 2-position, but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active for alkane substrates and support thousands of product turnovers. These regio- and enantio-selectivities are retained in whole-cell biotransformations with E. coli, where the engineered P450s can be expressed at high levels and the expensive cofactor is supplied endogenously.

Abstract: The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

Abstract: There are disclosed metabolically-engineered yeast and methods of producing n-butanol. In an embodiment, metabolically-engineered yeast is capable of metabolizing a carbon source to produce n-butanol, at least one pathway produces increased cytosolic acetyl-CoA relative to cytosolic acetyl-CoA produced by a wild-type yeast, and at least one heterologous gene encodes and expresses at least one enzyme for a metabolic pathway capable of utilizing NADH to convert acetyl-CoA to n-butanol. In another embodiment, a method of producing n-butanol includes (a) providing metabolically-engineered yeast capable of metabolizing a carbon source to produce n-butanol, at least one pathway produces increased cytosolic acetyl-CoA relative to cytosolic acetyl-CoA produced by a wild-type yeast, and at least one heterologous gene encodes and expresses at least one enzyme for a metabolic pathway utilizing NADH to convert acetyl-CoA to n-butanol; and (b) culturing the yeast to produce n-butanol. Other embodiments are also disclosed.

Abstract: Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. Mutant 9-10A-A328V hydroxylates octane primarily at the 2-positio to form S-2-octanol (40% ee). Another mutant, 1-12G, hydroxylates alkanes larger than hexane primarily at the 2-position, but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active for alkane substrates and support thousands of product turnovers. These regio-and enantio-selectivities are retained in whole-cell biotransformations with E. coli, where the engineered P450s can be expressed at high levels and the expensive cofactor is supplied endogenously.

Abstract: Methods and compositions for the production of bio-based material precursors are provided.

Abstract: There is disclosed a method of producing isobutanol. In an embodiment, the method includes providing a microorganism transformed with an isobutanol producing pathway containing at least one exogenous gene. The microorganism is selected to produce isobutanol from a carbon source at a yield of at least 10 percent theoretical. The method includes cultivating the microorganism in a culture medium containing a feedstock providing the carbon source, until isobutanol is produced. The method includes recovering the isobutanol. In one embodiment, the microorganism is a yeast with a Crabtree-negative phenotype. In another embodiment, the microorganism is a yeast microorganism with a Crabtree-positive phenotype. There is disclosed a microorganism for producing isobutanol. In an embodiment, the microorganism includes an isobutanol producing pathway containing at least one exogenous gene, and is selected to produce a recoverable quantity of isobutanol from a carbon source at a yield of at least 10 percent theoretical.

Abstract: Methods for producing a biofuel are provided. Also provided are biocatalysts that convert a feedstock to a biofuel.

Abstract: Methods for producing a biofuel precursor are provided. Also provided are biocatalysts that convert a feedstock to a biofuel precursor.

Abstract: A recombinant microorganism expressing at least a heterologous enzyme of an NADH-dependent pathway for conversion of a carbon source to n-butanol, metabolic intermediate and/or a derivative thereof and capable of producing n-butanol, a metabolic intermediate and/or a derivative thereof at a high yield and related methods. The recombinant microorganism engineered to inactivate a native enzyme of one or more pathways that compete with NADH-dependent heterologous pathway, and/or to balance the NADH-dependent heterologous pathway with respect to NADH production and consumption.

Abstract: Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. Mutant 9-10A-A328V hydroxylates octane primarily at the 2-positio to form S-2-octanol (40% ee). Another mutant, 1-12G, hydroxylates alkanes larger than hexane primarily at the 2-position, but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active for alkane substrates and support thousands of product turnovers. These regio- and enantio-selectivities are retained in whole-cell biotransformations with E. coli, where the engineered P450s can be expressed at high levels and the expensive cofactor is supplied endogenously.

Abstract: There are disclosed recombinant microorganisms engineered to increase product yield in a biotransformation. In an embodiment, the microorganisms are engineered to increase the amount of NAD(P)H available for a NAD(P)H-requiring oxidoreductase involved in a biotransformation. There are also disclosed methods and systems for using recombinant microorganisms engineered to increase the amount of NAD(P)H available for a NAD(P)H-requiring oxidoreductase involved in a biotransformation. Other embodiments are also disclosed.

Abstract: In an embodiment, there is disclosed a recombinant microbial host cell having each of the DNA molecules encoding a polypeptide or group of polypeptides that catalyze the conversion: (i) Acetyl-CoA to Acetate and CoA ??(conversion 1) (ii) Acetyl-CoA to Acetoacetyl-CoA and CoA ??(conversion 2) (iii) Acetoacetyl-CoA and Acetate to Acetoacetate and Acetyl-CoA ??(conversion 3.1) (iv) Acetoacetate to Acetone and CO2 ??(conversion 4) (v) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ ??(conversion 5) wherein at least one DNA molecule is heterologous to the microbial host cell and wherein the microbial host cell produces isopropanol. In another embodiment, a method is disclosed for the production of isopropanol including providing a recombinant microbial host cell, the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced, and recovering the isopropanol.

Abstract: This invention relates to modified hydroxylases. The invention further relates to cells expressing such modified hydroxylases and methods of producing hydroxylated alkanes by contacting a suitable substrate with such cells.

Abstract: Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. Mutant 9-10A-A328V hydroxylates octane primarily at the 2-positio to form S-2-octanol (40% ee). Another mutant, 1-12G, hydroxylates alkanes larger than hexane primarily at the 2-position, but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active for alkane substrates and support thousands of product turnovers. These regio- and enantio-selectivities are retained in whole-cell biotransformations with E. coli, where the engineered P450s can be expressed at high levels and the expensive cofactor is supplied endogenously.