Adaptive evolution of a hyperthermophilic archaeon Thermococcus onnurineus NA1 during growth on formate

Abstract

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been reported to grow on formate coupled with H&not 2 production and ATP generation (HCOO- + H2O → HCO3- + H2, ΔG80oC = -2.6 kJ/mol). In an attempt to develop the strain for the enhanced H2 production on formate, adaptive evolution was adopted. T. onnurineus NA1 was grown to stationary phase to trigger spontaneous mutation and then transferred to a fresh medium containing formate. Through serial transfers of batch cultures, physiological changes were monitored and gradual increases in cell density, H2 production rate and formate consumption rate were observed. After 156 transfers, the evolved strain showed 1.4-, 1.3- and 1.3-fold higher values in maximum cell density, H2 production rate and formate consumption rate, respectively, in comparison with those of parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution on formate, genome sequencing was performed using the PacBio Single Molecule Real-Time (SMRT) sequencing technology. As a result, we discovered eleven mutation sites, including insertion (2 sites), deletion (2 sites) and substitution (7 sites) either at the intergenic (2 sites) or coding regions (9 sites) in a DNA sequence. Further mutational studies would be required to assess the contribution of gene mutations to the phenotypic changes during evolution. The potential of the enginehe enhanced H2 production on formate, adaptive evolution was adopted. T. onnurineus NA1 was grown to stationary phase to trigger spontaneous mutation and then transferred to a fresh medium containing formate. Through serial transfers of batch cultures, physiological changes were monitored and gradual increases in cell density, H2 production rate and formate consumption rate were observed. After 156 transfers, the evolved strain showed 1.4-, 1.3- and 1.3-fold higher values in maximum cell density, H2 production rate and formate consumption rate, respectively, in comparison with those of parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution on formate, genome sequencing was performed using the PacBio Single Molecule Real-Time (SMRT) sequencing technology. As a result, we discovered eleven mutation sites, including insertion (2 sites), deletion (2 sites) and substitution (7 sites) either at the intergenic (2 sites) or coding regions (9 sites) in a DNA sequence. Further mutational studies would be required to assess the contribution of gene mutations to the phenotypic changes during evolution. The potential of the engine