Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824.
Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added.
Wardwell
S A
SA
Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, Houston, TX 77005-1892, USA.
Yang
Y T
YT
Chang
H Y
HY
San
K Y
KY
Rudolph
F B
FB
Bennett
G N
GN
D000093:Acetoin / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002072:Butylene Glycols / Q000378:metabolism; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D017422:Sequence Analysis, DNA
D002072:Butylene Glycols; D003470:Culture Media; C026978:2,3-butylene glycol; D000093:Acetoin; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase