NADP or CO reduction by -encoded hydrogenase through interaction with formate dehydrogenase 3 in the hyperthermophilic archaeon NA1.

The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the -encoded hydrogenase of NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P) is significant in that the enzyme can function to supply reducing equivalents, just as F-reducing hydrogenases in methanogens use coenzyme F as an electron carrier. Additionally, it was identified that NA1 could produce formate from H and CO by the concerted action of -encoded hydrogenase and formate dehydrogenase Fdh3.

Direct Electron Transfer between the -Encoded Hydrogenase and Thioredoxin Reductase in the Nonmethanogenic Archaeon NA1.

To date, NAD(P)H, ferredoxin, and coenzyme F have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon NA1, the -encoded hydrogenase, a homolog of the F-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and pulldown assays.

Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning.

Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature.

Redox regulation of SurR by protein disulfide oxidoreductase in Thermococcus onnurineus NA1.

Protein disulfide oxidoreductases are redox enzymes that catalyze thiol-disulfide exchange reactions. These enzymes include thioredoxins, glutaredoxins, protein disulfide isomerases, disulfide bond formation A (DsbA) proteins, and Pyrococcus furiosus protein disulfide oxidoreductase (PfPDO) homologues. In the genome of a hyperthermophilic archaeon, Thermococcus onnurineus NA1, the genes encoding one PfPDO homologue (TON_0319, Pdo) and three more thioredoxin- or glutaredoxin-like proteins (TON_0470, TON_0472, TON_0834) were identified.

Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased.

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.