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.

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.sub.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.

The biological production of beta-hydroxyisovalerate (?HIV) using at least one non-natural enzyme. The non-natural enzyme for the biologically-derived ?HIV provides more beta-hydroxyisovalerate synthase activity than the wild-type parent. The non-natural enzyme having one or more modifications of substrate-specificity positions. The non-natural enzyme can be expressed in a microorganism, such as a yeast or bacteria, wherein the microorganism comprises an active ?HIV metabolic pathway for the production of ?HIV. Alternatively, the non-natural enzyme can be a ?HIV synthase used to produce ?HIV in a cell-free environment. The biological derivation of ?HIV eliminates toxic by-products and impurities that result from the chemical production of ?HIV, such that ?HIV produced by a non-natural enzyme prior to any isolation or purification process has not been in substantial contact with any halogen-containing component.

Genetically modified microorganisms that have the ability to convert carbon substrates into chemical products such as isobutanol are disclosed. For example, genetically modified methanotrophs that are capable of generating isobutanol at high titers from a methane source are disclosed. Methods of making these genetically modified microorganisms and methods of using them are also disclosed.

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.

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.

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.

Genetically modified microorganisms that have the ability to convert carbon substrates into chemical products such as isobutanol are disclosed. For example, genetically modified methanotrophs that are capable of generating isobutanol at high titers from a methane source are disclosed. Methods of making these genetically modified microorganisms and methods of using them are also disclosed.

The present invention provides a genetically engineered pantoic acid-producing strain having or having an enhanced NADH-dependent acetohydroxy acid reductoisomerase, a method for producing the strain, a method for producing D-pantoic acid using the strain, and use thereof in production of D-pantoic acid.

This disclosure provides self-assembling Ketol-acid reductoisomerase (KARI) nanoparticles that are capable of displaying multiple copies of antigens, antibodies and/or proteins or peptides on its surface; as well as nucleic acids encoding recombinant KARI molecules, vectors expressing recombinant KARI molecules, immunogenic polypeptides comprising the self-assembling KARI nanoparticles, methods of producing the self-assembling KART nanoparticles, and methods for eliciting an immune response against an antigen in a subject comprising the self-assembling KARI nanoparticles.