Analysis of degenerate variants of Clostridium acetobutylicum ATCC 824

It is known that degenerate mutants of the solvent producing Clostridium acetobutylicum will spontaneously develop during repeated subculturing or continuous fermentation. Several hypotheses have been proposed as to what causes this spontaneous degeneration. One proposed explanation is that aberrations in regulatory proteins result in the failure of the organism to respond to influences causing the switch from the production of acids (acetate and butyrate) to the production of solvents (acetone, butanol, and ethanol). Another possibility is a mutation or rearrangement of the region of the chromosome involved in the production of the enzymes involved in solventogenesis. To further investigate the processes altered in degenerate mutants, a set of degenerate variants was obtained. This set includes strains obtained from repeated subculturing, and from chemical mutagenesis. All of the strains show decreased or no production of acetone and/or butanol by gas chromatography analysis, and the loss of enzyme activity of one or more of the enzymes involved in solvent production. Experiments indicate that a genetic region encoding an aldehyde/alcohol dehydrogenase (aad, adhE), the acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (ctfAB), and the acetoacetate decarboxylase (adc) is lost during the degeneration process. This result coupled with previous complementation studies of degenerate mutants with plasmids containing solvent formation genes which yielded a restoration of solvent formation suggest that in full degenerates the defect is not simply a result of a loss of an essential expression factor, but is due to a defect in that region of the chromosome encoding the solvent genes. (C) 1996 Academic Press