Hydrogen production and fermentation in heliobacteria

Heliobacteria are the only phototrophic members of the gram-positive bacteria. Heliomicrobium modesticaldum can grow chemotrophically in the dark using pyruvate as a carbon source. It can grow phototrophically only on acetate (plus CO 2 ). We found that dihydrogen production occurred only when fixing N 2 and increased ~5-fold upon replacing N 2 with argon. Introduction of genes encoding a [FeFe] hydrogenase and maturases ( hydAEFG ) from Acetivibrio thermocellus conferred the ability to produce H 2 at much higher levels. The modified cells made the most H 2 when grown chemotrophically, less when grown mixotrophically, and none when grown phototrophically, suggesting that only excess electrons were available for H 2 production. We tested several molecules as potential reduced fermentation products by this species. Only propanoate was observed, and its extracellular concentration could reach several millimolar. There was an inverse correlation between light intensity and propanoate production when the cells were grown on pyruvate, and phototrophic cultures produced no detectable propanoate. We conclude that propanoate is the major reduced fermentation product made by Hmb. modesticaldum, and we propose a pathway to produce it that is essentially a mini-cycle within the reductive TCA (rTCA) cycle, combining the first four reactions of the rTCA cycle with methylmalonyl-CoA mutase and epimerase, and methylmalonyl-CoA:pyruvate carboxytransferase. This pathway would, in effect, dispose of four reducing equivalents while driving the conversion of pyruvate to propanoate. The genes for the last six enzymes of this pathway seem to be part of an operon; the genes and potential operon are conserved in other heliobacteria, suggesting that this pathway exists in all or most heliobacteria. IMPORTANCE This study establishes that heliobacteria, the only phototrophic members of the Bacillota , produce propanoic acid as the reduced fermentation product when growing chemotrophically. The pathway used to make it resembles that of the Propionibacteria , except in the context of a mini-cycle within the reductive TCA (rTCA) cycle. This work confirms that the Heliomicrobium genus lacks a functional hydrogenase to make hydrogen as a way to dispose of excess electrons. Finally, it is shown that these organisms can be engineered to make dihydrogen as a reduced fermentation product if the genes for a clostridial hydrogenase and assembly proteins are expressed in Heliomicrobium modesticaldum .