Characterization of metabolites and genes in the fermentation pathway of Clostridium acetobutylicum ATCC824
Boynton, Zhuang Luo
Rudolph, Frederick B.
Doctor of Philosophy
In Clostridium acetobutylicum, central-fermentation-pathway enzymes involved in butyryl-CoA synthesis play key roles in acid and solvent production, whereas the acetate production is an important element during acidogenesis and generates ATP. Genes encoding enzymes involved in acetate- and butyryl-CoA-synthesis were cloned, sequenced and expressed. CoA and its derivatives, which are intermediate metabolites for these pathways, were isolated and analyzed to identify regulatory factors for the enzymes involved in such fermentation. Five genes: crt, bcd, etfB, etfA and hbd, which encode the central clostridial pathway enzymes crotonase, butyryl-CoA dehydrogenase (BCD), putative electron-transfer flavoprotein (ETF) $\beta$- and $\alpha$-subunits, and $\beta$-hydroxybutyryl-CoA dehydrogenase, respectively, were cloned and shown to be clustered in the chromosome. These genes are co-transcribed and form an operon, suggesting that BCD in clostridia might interact with ETFs in its redox function. Cloning and sequencing studies reveal that pta and ack, encoding acetate-production-pathway enzymes phosphotransacetylase (PTA) and acetate kinase (AK), respectively, are adjacent in the chromosome. Primer extension analysis suggests an operon arrangement for these tandem genes. Overexpression of ack and pta in C. acetobutylicum shows that the final ratios of acetate to other major products were higher and also results in a greater proportion of two- versus four-carbon-derived products. Formation of a mutant strain by inactivation of the chromosomal pta gene decreased acetate formation. The PTA and AK activities of such a mutant were correspondingly reduced. Intracellular levels of CoA and its derivatives involved in the metabolic pathways were analyzed using reverse-phase high performance liquid chromatography. During the acidogenic-to-solventogenic shift, butyryl-CoA concentration increased rapidly, whereas those for free CoA and acetyl-CoA decreased. These observations were accompanied by a rapid increase of solvent-pathway enzyme activity and a decrease of acid-pathway enzyme activity. Levels of acetoacetyl-CoA, $\beta$-hydroxy-butyryl-CoA and crotonyl-CoA in crude cell extracts were below detectability. The possible roles of CoA and its derivatives in regulating specific enzyme activity were evaluated.
Microbiology; Molecular biology