Modification of hydrogenase activities in thermophilic bacteria to enhance ethanol production

A bacterium capable of fermenting a saccharification product of a carbohydrate-rich biomass substrate, wherein ldh and hydtrA genes are inactivated by genetic engineering.

A Thermoanaerobacterium saccharolyticum strain deposited under Patent Deposit Designation No. PTA-8897.

An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity with a polynucleotide sequence selected from the group consisting of SEQ ID NOS:

• 1-8.

An isolated polynucleotide molecule comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOS:

• 1-8.

A genetically engineered cell expressing a hydrogenase encoded by a gene having at least 90% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOS:

• 1-8, the expression of said hydrogenase being driven by a heterologous promoter.

A genetic construct comprising a coding sequence having at least 90% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOS:

• 1-8, said coding sequence being operably linked to a promoter capable of controlling transcription in a bacterial cell.

A method for producing ethanol, said method comprising:

• generating an organism with at least one gene encoding a hydrogenase that is inactivated;
• and
• incubating the organism in a medium containing at least one substrate selected from the group consisting of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, cellulose, pectin and combinations thereof to allow for production of ethanol from the substrate.

A method for producing ethanol, said method comprising:

• providing within a reaction vessel, a reaction mixture comprising a carbohydrate-rich biomass substrate, a cellulolytic material, and a fermentation agent, the fermentation agent comprising a bacterium that has been genetically modified to inactivate at least one hydrogenase gene endogenous to said bacterium,
• wherein the reaction mixture is incubated under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the carbohydrate-rich biomass substrate.

An isolated protein molecule having hydrogenase activity, said molecule comprising a polypeptide having an amino acid sequence having at least 90% sequence identity with a polypeptide selected from the group consisting of SEQ ID NOS:

• 9-16.

A bacterium capable of fermenting a saccharification product of a carbohydrate-rich biomass substrate, wherein at least one hydrogenase gene endogenous to said bacterium has been inactivated by genetic engineering.