Hydrogen evolution under photoheterotrophic and dark fermentative conditions by recombinant Rhodobacter sphaeroides containing the genes for fermentative pyruvate metabolism of Rhodospirillum rubrum

Abstract

Rhodobacter sphaeroides KCTC 12085 is a natural isolate which shows a higher ability of photoheterotrophic H 2 production compared with a laboratory strain R. sphaeroides 2.4.1. The H 2 production by R. sphaeroides is observed only under light conditions. In order to render R. sphaeroides to produce H 2 fermentatively as well, a 4.4-kb Rhodospirillum rubrum DNA encompassing a gene coding for pyruvate-formate lyase (PFL) with its putative maturation protein, and a 29.4-kb R. rubrum gene cluster including formate-hydrogen lyase (FHL) complex, its maturation proteins, a transcriptional activator for FHL, and Fe-only hydrogenase with its maturation proteins were cloned and mobilized into R. sphaeroides KCTC 12085. The recombinant R. sphaeroides evolved H 2 during dark fermentative growth. The H 2 production by the recombinant R. sphaeroides under photoheterotrophic conditions was elevated twofold to show 4 mol H 2/mol of glucose compared with 2 mol H 2/mol of glucose by its parental strain carrying empty vector. Interestingly, addition of hypophosphite, an inhibitor of PFL, to the recombinant R. sphaeroides under photoheterotrophic conditions still resulted in 3 mol H 2/mol of glucose, suggestive of active H 2 production not only by nitrogenase but also by Fe-only hydrogenase, which are insensitive to hypophosphite. The results further suggest the photoheterotrophic H 2 production by FHL unless hypophosphite is present. Thus, the H 2 production by the recombinant R. sphaeroides under photoheterotrophic conditions appears to be mediated by the concerted actions of FHL, Fe-only hydrogenase, and nitrogenase. The H 2-evolving activity by the recombinant R. sphaeroides containing Fe-only hydrogenase of R. rubrum appears to depend on the presence its maturation proteins, suggestive of lack of such proteins in R. sphaeroides.