P123: Identification of Acetoin reductases involved in 2,3-butanediol pathway in Klebsiella oxytoca

Acetoin reductase (BudC) of Klebsiella oxytoca convert acetoin into 2,3-BDO during sugar fermentation. The budC deletion in K. oxytoca still permits low levels of 2,3-BDO production indicates the existence of another budC like gene. There annoted a gene AR (dar; acetoin reductase/diacetyl reductase) that shared high homology with budC in K. oxytoca. In the present study, the role of dar was investigated by cloning and expressing the gene in E. coli along with budB (acetolactate synthase) and budA (acetolactate decarboxylase). Furthermore, dar deletion mutants of K. oxytoca were developed and fermentation profiles were analyzed. The recombinant E. coli_budBAC and E. coli_budBA-dar produced 2,3-BDO from glucose and not E. coli_budBA which produced acetoin alone. This proved that Dar functions similar to BudC. Deletion of budC and dar in K. oxytoca_ΔldhA (lactate dehydrogenase) was carried out (K. oxytoca_ΔldhA_ΔbudC_Δdar) and subjected for glucose fermentation. The mutant showed more than >85% and 70% reduction in 2,3-BDO concentration than K. oxytoca_ΔldhA and K. oxytoca_ΔldhA_ΔbudC, respectively. Interestingly, K. oxytoca_ΔldhA_Δdar resulted a 50% reduction in the reversible conversion of 2,3-BDO into acetoin than K. oxytoca_ΔldhA which was observed from glucose depleted fermentation culture. Moreover, there could be another possible gene product exist in K. oxytoca apart from budC and dar for the conversion of 2,3-BDO.