Introduction of Glyoxylate Bypass Increases Hydrogen Gas Yield from Acetate and L-Glutamate in Rhodobacter sphaeroides

Rhodobacter sphaeroides produces hydrogen gas (H-2) from organic compounds via nitrogenase under anaerobic-light conditions in the presence of poor nitrogen sources, such as L-glutamate. R. sphaeroides utilizes the ethylmalonyl-coenzyme A (EMC) pathway for acetate assimilation, but its H-2 yield from acetate in the presence of L-glutamate has been reported to be low. In this study, the deletion of ccr encoding crotonyl-coenzyme A (crotonyl-CoA) carboxylase/reductase, a key enzyme for the EMC pathway in R. sphaeroides, revealed that the EMC pathway is essential for H-2 production from acetate and L-glutamate but not for growth and acetate consumption in the presence of L-glutamate. We introduced a plasmid expressing aceBA from Rhodobacter capsulatus encoding two key enzymes for the glyoxylate bypass into R. sphaeroides, which resulted in a 64% increase in H-2 production. However, compared with the wild-type strain expressing heterologous aceBA genes, the strain with aceBA introduced in the genetic background of an EMC pathway-disrupted mutant showed a lower H-2 yield. These results indicate that a combination of the endogenous EMC pathway and a heterologously expressed glyoxylate bypass is beneficial for H-2 production. In addition, introduction of the glyoxylate bypass into a polyhydroxybutyrate (PHB) biosynthesis-disrupted mutant resulted in a delay in growth along with H-2 production, although its H-2 yield was comparable to that of the wild-type strain expressing heterologous aceBA genes. These results suggest that PHB production is important for fitness to the culture during H-2 production from acetate and L-glutamate when both acetate-assimilating pathways are present. IMPORTANCE As an alternative to fossil fuel, H-2 is a promising renewable energy source. Although photofermentative H-2 production from acetate is key to developing an efficient process of biohydrogen production from biomass-derived sugars, H-2 yields from acetate and L-glutamate by R. sphaeroides have been reported to be low. In this study, we observed that in addition to the endogenous EMC pathway, heterologous expression of the glyoxylate bypass in R. sphaeroides markedly increased H-2 yields from acetate and L-glutamate. Therefore, this study provides a novel strategy for improving H-2 yields from acetate in the presence of L-glutamate and contributes to a clear understanding of acetate metabolism in R. sphaeroides during photofermentative H-2 production.