Two Different Adaptive Passages of a Hyperthermophile Pinpoint a Critical Residue at the Formate Transporter to Confer Higher Growth and H2 Production on Formate

Abstract

The hyperthermophilic archaeon Thermococcus onnurineus NA1 could grow on formate and produce H2. Formate conversion to hydrogen in the strain was mediated by a formate-hydrogen lyase (FHL) complex and was indeed a part of chemiosmotic coupling to ATP generation. In this study, we employed an adaptation approach to investigate molecular changes to enhance the cell growth on formate. As serial transfer on formate-containing medium at the serum vial continued, cell growth, H2 production and formate consumption increased remarkably. The whole-genome sequencing of the strain transferred 156 times (WTF-156T) revealed dozens of mutations. No mutation was found among the genes of FHL, but a point mutation was identified in the formate transporter. The mutation, when introduced into the genome of the parent strain, conferred increase in formate consumption and H2 production. Another adaptive passage carried out by culturing repeatedly in a bioreactor revealed that a mutation of formate transporter took place at the same residue, indicating that substitution of this residue at the formate tranporter may cause a substantial increase in the formate uptake and then enhance cell growth. Finally, the WTF-156T mutant was demonstrated to enhance H2 production using formate in a bioreactor.ing to ATP generation. In this study, we employed an adaptation approach to investigate molecular changes to enhance the cell growth on formate. As serial transfer on formate-containing medium at the serum vial continued, cell growth, H2 production and formate consumption increased remarkably. The whole-genome sequencing of the strain transferred 156 times (WTF-156T) revealed dozens of mutations. No mutation was found among the genes of FHL, but a point mutation was identified in the formate transporter. The mutation, when introduced into the genome of the parent strain, conferred increase in formate consumption and H2 production. Another adaptive passage carried out by culturing repeatedly in a bioreactor revealed that a mutation of formate transporter took place at the same residue, indicating that substitution of this residue at the formate tranporter may cause a substantial increase in the formate uptake and then enhance cell growth. Finally, the WTF-156T mutant was demonstrated to enhance H2 production using formate in a bioreactor.