Adaptive evolution reveals a mutation of hydrogenase effective to enhance formate-dependent hydrogen production in Thermococcus onnurineus NA1

Abstract

The hyperthermophilic archaeon, Thermococcus onnurineus NA1 could grow on formate, coupled with hydrogen production. Formate conversion to hydrogen was never thought to be energetic enough to sustain cell growth. T. onnurineus NA1 was the first example that a single strain can grow on formate, producing hydrogen. The formate-dependent hydrogen production in the strain was mediated by a formate-hydrogen lyase (FHL) complex comprised of formate dehydrogenase module, hydrogenase module and Na+/H+ antiporter module. We also reported that significant changes of cell growth and hydrogen production could be observed in 156 times serially-transferred strain (WTF-156T) on formate-containing medium. While no mutation in WTF-156T was found in the genes encoding FHL complex, a mutation at formate transporter (TON_1573) caused significant increase in formate consumption and H2 production. In this study, the WTF-156T strain was further serially-transferred over another 200 times. The resulting strain, WTF-350T strain, exhibited much faster consumption rate of formate and higher growth rate than those of WTF-156T strain. Genomic analysis of WTF-350T strain revealed additional 16 mutations on top of the 11 mutations identified in the WTF-156T strain. We focused on investigating the effect of two mutations found in formate consumption machinery: H339N mutation at the gene encoding a membrane subunit (TON_1565) of the multimeric membrane-bound hydrogenase of the FHL complex and additional mutation (S104N) at formate transporter (TON_1573). We introduced each mutation into the parent strain, and then evaluated formate consumption rate using resting cells of each mutant. The resulting mutants harboring each mutation at TON_1565 (H339N) or TON_1573 (S104N) showed higher formate consumption rate of 12% and 14% at pH 6.5 and 34% and 32% at pH 7.8, respectively, than the parent strain. The underlying mechanism to explain the change will be discussed further.