Recombination-Induced Variants of Clostridium acetobutylicum ATCC 824 with Increased Solvent Production

Abstract.

Three sporulation-specific genes (orfA, sigE, sigG) from Clostridium acetobutylicum ATCC 824 are arranged in a cluster, encoding the putative σ^E-processing enzyme, σ^E, and σ^G respectively. When they were transformed into Clostridium acetobutylicum while on a plasmid functional in this organism, transformants did not survive. Three kinds of recombinations were then attempted with nonreplicative plasmids: duplication of orfA and sigE, replacement of all of the three genes, and inactivation of orfA. While the wild-type strain ceased to grow and produce solvents in batch cultures after approximately 24 h, mutant strains were isolated that showed sustained growth for a much longer time and produced a threefold increase in acetone and butanol in test tube cultures. In addition, one of the derived strains showed a significantly higher growth rate. Features of the restriction maps of the recombinants did not correlate with expected maps, indicating possible complications occurring during the recombination events.