ANALYSIS OF DEGENERATE VARIANTS OF CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824

It is known that degenerate mutants of the solvent producing Clostridi um acetobutylicum will spontaneously develop during repeated subcultur ing or continuous fermentation. Several hypotheses have been proposed as to what causes this spontaneous degeneration. One proposed explanat ion is that aberrations in regulatory proteins result in the failure o f the organism to respond to influences causing the switch from the pr oduction 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 product ion of the enzymes involved in solventogenesis. To further investigate the processes altered in degenerate mutants, a set of degenerate vari ants was obtained. This set includes strains obtained from repeated su bculturing, and from chemical mutagenesis. All of the strains show dec reased or no production of acetone and/or butanol by gas chromatograph y analysis, and the loss of enzyme activity of one or more of the enzy mes involved in solvent production. Experiments indicate that a geneti c region encoding an aldehyde/alcohol dehydrogenase (aad, adhE), the a cetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (ctfAB), and the acetoacetate decarboxylase (adc) is lost during the degenerat ion process. This result coupled with previous complementation studies of degenerate mutants with plasmids containing solvent formation gene s which yielded a restoration of solvent formation suggest that in ful l degenerates the defect is not simply a result of a loss of an essent ial expression factor, but is due to a defect in that region of the ch romosome encoding the solvent genes. (C) 1996 Academic Press