Adaptive evolution of a hyperthermophilic archaeon pinpoints a formate transporter as a critical factor for the growth enhancement on formate

Abstract

Previously, we reported that the hyperthermophilic archaeon Thermococcus onnurineus NA1 could growon formate and produce H2. Formate conversion to hydrogen was mediated by a formate-hydrogenlyase complex and was indeed a part of chemiosmotic coupling to ATP generation. In this study, we employed an adaptation approach to enhance the cell growth on formate and investigated molecularchanges. As serial transfer continued on formate-containing medium at the serum vial, cell growth, H2 production and formate consumption increased remarkably. The 156 times transferred-strain, WTF-156T, was demonstrated to enhance H2 production using formate in a bioreactor. The whole-genomesequencing of the WTF-156T strain revealed eleven mutations. While no mutation was found amongthe genes encoding formate hydrogen lyase, a point mutation (G154A) was identified in a formatetransporter (TON_1573). The TON_1573 (A52T) mutation, when introduced into the parent strain,conferred increase in formate consumption and H2 production. Another adaptive passage, carried outby culturing repeatedly in a bioreactor, resulted in a strain, which has a mutation in TON_1573 (C155A) causing amino acid change, A52E. These results implicate that substitution of A52 residue of a formate transporter might be a critical factor to ensure the increase in formate uptake and cell growth.motic coupling to ATP generation. In this study, we employed an adaptation approach to enhance the cell growth on formate and investigated molecularchanges. As serial transfer continued on formate-containing medium at the serum vial, cell growth, H2 production and formate consumption increased remarkably. The 156 times transferred-strain, WTF-156T, was demonstrated to enhance H2 production using formate in a bioreactor. The whole-genomesequencing of the WTF-156T strain revealed eleven mutations. While no mutation was found amongthe genes encoding formate hydrogen lyase, a point mutation (G154A) was identified in a formatetransporter (TON_1573). The TON_1573 (A52T) mutation, when introduced into the parent strain,conferred increase in formate consumption and H2 production. Another adaptive passage, carried outby culturing repeatedly in a bioreactor, resulted in a strain, which has a mutation in TON_1573 (C155A) causing amino acid change, A52E. These results implicate that substitution of A52 residue of a formate transporter might be a critical factor to ensure the increase in formate uptake and cell growth.