The invention relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In certain embodiments, the invention includes a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. In this embodiment, this dual enzyme system may include thermostable Adenosyl Kinase, and/or Polyphosphate Kinase enzymes.

Genetically modified microorganisms that have the ability to convert carbon substrates into chemical products such as 2,3-BDO are disclosed. For example, genetically modified methanotrophs that are capable of generating 2,3-BDO at high titers from a methane source are disclosed. Methods of making these genetically modified microorganisms and methods of using them are also disclosed.

Signal sequences useful for targeting proteins and peptides to the surface of endospores produced by Paenibacillus family members and methods of using the same are provided. The display of heterologous molecules, such as peptides, polypeptides and other recombinant constructs, on the spore surface of Paenibacillus family members, using particular N-terminal targeting sequences and derivatives of the same, are also provided.

Non-naturally occurring Zymomonas strains useful for the production of 2,3-butanediol are provided.

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.

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.

Signal sequences useful for targeting proteins and peptides to the surface of endospores produced by Paenibacillus family members and methods of using the same are provided. The display of heterologous molecules, such as peptides, polypeptides and other recombinant constructs, on the spore surface of Paenibacillus family members, using particular N-terminal targeting sequences and derivatives of the same, are also provided.

Provided is a recombinant Saccharomyces cerevisiae for producing 2,3-butanediol, wherein all GPD1 and GPD2 genes involved in glycerol biosynthesis are removed and a gene encoding NADH oxidase is introduced, and wherein pyruvate decarboxylase activity is inactivated and Candida tropicalis PDC1 gene encoding Candida tropicalis pyruvate decarboxylase 1-is introduced, and wherein expression of the Candida tropicalis PDC1 gene is regulated by a GPD2 promoter.

In a method for producing acetoin, butanediol, or butanol from ethanol according to the present invention, a cell-free catalysis method was used by designing an artificial synthetic pathway so that proteins of NOX, EtDH, FLS, BDH, and DDH and variant proteins thereof exhibit cascade catalytic activity as enzymes. Compared to existing fermentation methods using microorganisms, the production method according to the present invention does not require cell growth and has a short synthetic pathway, a fast reaction rate, high yield and productivity, adjustment of targeted reaction conditions is convenient, and butanol may be effectively produced. Moreover, same may be reused numerous times by fixing the proteins to nano-particles, and are also effective for producing acetoin, butanediol, or butanol, thus being economical. Therefore, the production method may be usefully adopted in the relevant industries requiring acetoin, butanediol, or butanol.

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.