RECOMBINATION-INDUCED VARIANTS OF CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824WITH INCREASED SOLVENT PRODUCTION

Three sporulation-specific genes (orfA, sigE, sigG) from Clostridium a cetobutylicum ATCC 824 are arranged in a cluster, encoding the putativ e sigma(E)-processing enzyme, sigma(E), and sigma(G) respectively. Whe n they were transformed into Clostridium acetobutylicum while on a pla smid functional in this organism, transformants did not survive. Three kinds of recombinations were then attempted with nonreplicative plasm ids: duplication of orfA and sigE, replacement of all of the three gen es, and inactivation of orfA. While the wild-type strain ceased to gro w and produce solvents in batch cultures after approximately 24 h, mut ant strains were isolated that showed sustained growth for a much long er time and produced a threefold increase in acetone and butanol in te st tube cultures. In addition, one of the derived strains showed a sig nificantly higher growth rate, Features of the restriction maps of the recombinants did not correlate with expected maps, indicating possibl e complications occurring during the recombination events.