Seong Hyuk Lee

A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain.

ABSTRACT Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and “Candidatus Korarchaeum cryptofilum” OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR ↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR ↑ strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter−1 h−1) and the specific H2 production rate (249.6 mmol g−1 h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production.

Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon.

A strong promoter increases transcription of the genes of interest and enhances the production of various valuable substances. For a hyperthermophilic archaeon Thermococcus onnurineus NA1, which can produce H2 from carbon monoxide oxidation, we searched for a novel endogenous strong promoter by transcriptome analysis using high-throughput RNA sequencing. Based on the relative transcript abundance, we selected one promoter to encode a hypothetical gene, of which homologs were found only in several Thermococcales strains. This promoter, PTN0510, was introduced into the front of CO-responsible hydrogenase gene cluster encoding a carbon monoxide dehydrogenase (CODH), a hydrogenase, and a Na+/H+ antiporter. In the resulting mutant strain, KS0510, transcription and translation level of the gene cluster increased by 4- to 14-folds and 1.5- to 1.9-folds, respectively, in comparison with those of the wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of the wild-type strain. The PTN0510 was identified to be much stronger than the well-known two strong promoters, gdh and slp promoters from Thermococcus strains, through RT-qPCR and Western blotting analyses and kinetics of H2 production. In this study, we demonstrated that the RNA-seq approach is a good strategy to mine novel strong promoters of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce valuable products such as enzymes and metabolites through metabolic engineering.

Characterization of the frhAGB-encoding hydrogenase from a non-methanogenic hyperthermophilic archaeon

The F420-reducing hydrogenase has been known as a key enzyme in methanogenesis. Its homologs have been identified in non-methanogenic hyperthermophilic archaea, including Thermococcus onnurineus NA1, but neither physiological function nor biochemical properties have been reported to date. The enzyme of T. onnurineus NA1 was distinguished from those of other methanogens and the members of the family Desulfurobacteriaceae with respect to the phylogenetic distribution of the α and β subunits, organization of frhAGB genes and conservation of F420-coordinating residues. RT-qPCR and Western blot analyses revealed frhA gene is not silent but is expressed in T. onnurineus NA1 grown in the presence of sulfur, carbon monoxide, or formate. The trimeric enzyme complex was purified to homogeneity via affinity chromatography from T. onnurineus NA1 and exhibited catalytic activity toward the electron acceptors such as viologens and flavins but not the deazaflavin coenzyme F420. This is the first biochemical study on the function of the frhAGB-encoding enzyme from a non-methanogenic archaea.