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 c loned and expressed: in Escherichia coli and Clostridium acetobutylicum ATC C 824. The nucleotide sequence of the gene encoding the enzyme was determin ed to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gen e in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analy sis. 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 pr oduct of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction o f the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not s ufficient for 2,3-butanediol production even though the cultures were produ cing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added.