Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

Abstract

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase [butyrate-acetoacetate CoA-transferase] [EC 2.8.3.9]) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The purification and properties of the enzyme have recently been described (D. P. Weisenborn, F. B. Rudolph, and E. T. Papoutsakis, Appl. Environ. Microbiol. 55:323-329, 1989). The genes encoding the two subunits of this enzyme have been cloned by using synthetic oligodeoxynucleotide probes designed from amino-terminal sequencing data from each subunit of the CoA-transferase. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by using these probes. Subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of Mr of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E. coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)