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

Journal Article · Fri Jun 01 00:00:00 EDT 1990 · Applied and Environmental Microbiology; (USA)

OSTI ID:6875622

Cary, J W; Petersen, D J; Bennett, G N ^[1]; Papoutsakis, E T ^[2]

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1. Rice Univ., Houston, TX (USA)
2. Northwestern Univ., Evanston, IL (USA)

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 genes encoding the two subunits of this enzyme have been cloned and 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 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 M{sub r} 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. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.

• OSTI ID: 6875622
• Journal Information: Applied and Environmental Microbiology; (USA), Journal Name: Applied and Environmental Microbiology; (USA) Vol. 56:6; ISSN 0099-2240; ISSN AEMID
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
ACETATES
BACTERIA
BUTYRIC ACID
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CLONING
CLOSTRIDIUM
CLOSTRIDIUM ACETOBUTYLICUM
COENZYMES
DNA
DNA HYBRIDIZATION
DNA-CLONING
ENZYMES
ESCHERICHIA COLI
GENE OPERONS
GENETIC ENGINEERING
HYBRIDIZATION
METHANOGENIC BACTERIA
MICROORGANISMS
MONOCARBOXYLIC ACIDS
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PLASMIDS
RECOMBINANT DNA
TRANSFERASES