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), Vol. 56:6; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

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