Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli

Journal Article · Sat Dec 01 00:00:00 EST 1990 · Applied and Environmental Microbiology; (USA)

OSTI ID:5948411

Contag, P R; Williams, M G; Rogers, P ^[1]

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1. Univ. of Minnesota, Minneapolis (USA)

A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids. pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grown anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pPC58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent M{sub r} of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.

• OSTI ID: 5948411
• Journal Information: Applied and Environmental Microbiology; (USA), Vol. 56:12; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ESCHERICHIA COLI
GENETIC ENGINEERING
LACTATE DEHYDROGENASE
DNA-CLONING
CLOSTRIDIUM ACETOBUTYLICUM
ENZYME ACTIVITY
MUTANTS
RECOMBINANT DNA
BACTERIA
CLONING
CLOSTRIDIUM
DNA
DNA HYBRIDIZATION
ENZYMES
HEMIACETAL DEHYDROGENASES
HYBRIDIZATION
METHANOGENIC BACTERIA
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
550200* - Biochemistry