Structure-based engineering of the transcriptional regulator EnfR in a hyperthermophilic archaeon to enhance H2 production

Abstract

Structural insights from a previous study indicated that altering the DNA-interaction region of EnfR, a novel repressor crucial for expression of the codh gene cluster, could influence gene expression associated with H2 production in Thermococcus onnurineus NA1. In this study, a rational design based on the structure of EnfR was employed to enhance CO-dependent H2 production. Among several mutations predicted through structural modeling of EnfR, the R44A mutation was selected, leading to the construction of the MC21 strain (EnfR-R44A). The transcriptomic analysis of the MC21 strain under CO conditions revealed the upregulation of genes involved in H2 production from CO and sugar fermentation. To evaluate the influence on cellular performance, growth and H2 production of the MC21 strain were compared with those of the wild-type strain under diverse culture conditions. Interestingly, the MC21 strain exhibited a growth rate comparable to that of the wild-type strain. Additionally, it achieved 1.28-and 2.32-fold increases in biomass productivity and 1.79-and 2.26-fold higher maximum H2 production rates under CO and CO with maltodextrin, respectively. Our study emphasizes the potential of advancing H2-producing microbial cell factories through structure-based engineering of the transcriptional regulatory protein.