Metabolism of acetoin in Clostridium acetobutylicum ATCC 824
Wardwell, Stephanie Anne
Bennett, George N.
Doctor of Philosophy
Acetoin is a product of fermentation by C. acetobutylicum, and a component of several natural and artificial flavors, including butter flavor. Acetolactate synthase (ALS) catalyzes the production of acetolactate from pyruvate. Acetolactate is converted to acetoin enzymatically or chemically. A putative catabolic acetolactate synthase was cloned from C. acetobutylicum . The gene is 1692 nucleotides long. His-tagged ALS protein was purified from E. coli. In vivo experiments in E. coli and C. acetobutylicum and in vitro experiments failed to detect activity for the acetolactate synthase, though it probably is involved in Clostridial acetoin production. Introduction of a B. subtilis ALS under control of its own promoter into C. acetobutylicum cells was also found to have no effect on solvent production. Acetoin reductase (AR) catalyzes the production of 2,3-butanediol from acetoin. 2,3-butanediol is a compound of commercial interest for use in making polyurethane foams, moistening agents, carriers for pharmaceuticals, and liquid fuel, among other products. The K. pneumoniae CG21 acetoin reductase gene was cloned into a Clostridial/E. coli shuffle vector and expressed in both hosts. The nucleotide sequence of the gene is 768 bp long. SDS-PAGE analysis of expressed protein indicates a molecular weight of 31,000 Da. Activity of the K. pneumoniae acetoin reductase gene in C. acetobutylicum, did not result in 2,3-butanediol synthesis during fermentation, as determined by analysis of culture supernatants. However, addition of racemic acetoin to Clostridial cells expressing the ar , yielded small amounts of 2,3-butanediol. The stereoisomer of acetoin produced by C. acetobutylicum may be incompatible with the acetoin reductase or enzyme activity in cells may be too low. A B. subtilis oxidoreductase gene, ywrO, was cloned and analyzed as a putative acetoin reductase. In vitro and in vivo experiments conducted in E. coli suggest it is not an acetoin reductase. E. coli expressing the ar of K. pneumoniae grown in the presence of racemic acetoin make 2,3-butanediol. Expression of the K. pneumoniae als and ar from a Clostridial promoter was examined, but did not promote 2,3-butanediol production in E. coli and C. acetobutylicum.
Molecular biology; Microbiology