Genetic manipulation of butyrate formation pathways in Clostridium butyricum

Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H2, it can produce acids (lactic, acetic and butyric acids) and CO2, as well as a small amount of ethanol. It has been proposed that elimination of competing pathways, such as the butyrate formation pathway, should increase H2 yields in Clostridium species. However, the application of this strategy has been hindered by the unavailability of genetic tools for these organisms. In this study, we successfully transferred a plasmid (pMTL007) to C. butyricum by inter-specific conjugation with Escherichia coli and disrupted hbd, the gene encoding β-hydroxybutyryl-CoA dehydrogenase in C. butyricum. Fermentation data showed that inactivation of hbd in C. butyricum eliminated the butyrate formation pathway, resulting in a significant increase in ethanol production and an obvious decrease in H2 yield compared with the wild type strain. However, under low partial pressure of H2, the hbd-deficient strain showed increased H2 production with the simultaneous decrease of ethanol production, indicating that H2 production by C. butyricum may compete for NADH with the ethanol formation pathway. Together with the discovery of a potential bifurcating hydrogenase, this study extends our understanding of the mechanism of H2 production by C. butyricum.

► A conjugation system for C. butyricum was developed. ► We successfully disrupt β-hydroxybutyryl-CoA dehydrogenase gene (hbd) in C. butyricum. ► We examine the effect of hbd disruption on the metabolism of C. butyricum. ► Partial genes encoding homologs of three subunits of a potential bifurcating hydrogenase were amplified from the genome of C. butyricum.