A Novel CO-Responsive Transcriptional Regulator and Enhanced H ₂ 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 H 2 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 ⌬CorR strain complemented with integrated corQR (⌬CorR/corQR 1 ) compared with those in the wild-type strain. In addition, the ⌬CorR/corQR 1 strain exhibited a much higher H 2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H 2 production rate (191.9 mmol liter ؊1 h ؊1 ) and the specific H 2 production rate (249.6 mmol g ؊1 h ؊1 ) of this strain were extremely high compared with those of CO-dependent H 2 -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 H 2 production. C arbon monoxide (CO) serves as a central metabolic intermediate in anaerobic metabolism (1), as an enzyme metallocenter ligand (2, 3), as a physiologically significant signal in higher organisms (4), and as a speculative component in an early mode of metabolism and the origin of life (5). CO can be utilized as carbon and energy sources for growth by numerous microorganisms containing carbon monoxide dehydrogenase (CODH), a key enzyme in CO metabolism. CODH oxidizes CO to carbon dioxide (CO 2 ), and the electrons generated by the process are coupled to diverse reactions, such as oxygen reduction, desulfurification, hydrogenogenesis, acetogenesis, and methanogenesis (6). When CO is aerobically oxidized, as in Pseudomonas thermocarboxydovorans and Oligotropha carboxidovorans, the reducing equivalents are transferred to oxygen through a CO-insensitive respiratory chain. Under anaerobic conditions, CO oxidation is linked to acetate production through the reductive acetyl coenzyme A (acetyl-CoA) or Wood-Ljungdahl pathway in acetogenic bacteria and to methane production as a substrate of CODH/ acetyl-CoA synthase in methanogenic archaea. Carboxydotrophic hydrogenogens like Rhodospirillum rubrum and Carboxydothermus hydrogenoformans oxidize CO through a water-gas shift reaction, CO ϩ H 2 O ¡ CO 2 ϩ H 2 (⌬G°= ϭ Ϫ20 kJ/mol), which produces hydrogen gas (7-9).There are several distinct CO regulation systems known for aerobic and anaerobic CO oxidation (1, ...

The two CO-dehydrogenases of Thermococcus sp. AM4

“…The CODH encoding gene is cooS2. This cluster is homologous to that found in other Thermococcus species [7]. We rename cooC2 the gene TAM4_1059, which encodes for a putative accessory nickel-insertion protein CooC.…”

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

“…Based on EMSA data, TON_1525 seems likely to play a role as a transcriptional repressor on the CODH promoter. Previously, a CO-responsive regulator, CorQR, was reported to activate the expression of the CODH gene cluster 15 . We did not detect any mutations in CorQR during the adaptation process.…”

“…Briefly, we designed primer sets for base-pair substitutions and mutated the TON_1525 and TON_0820 genes by site-directed mutagenesis. Each mutated gene and its flanking regions were ligated by one-step sequence- and ligation-independent cloning (SLIC) 46 , and subsequent mutants were generated through homologous recombination using an unmarked in-frame deletion 15 method and a modified gene disruption system that has been previously used for Thermococcus kodakarensis KOD1 47 . The mutations were verified by PCR using the primers listed in Supplementary Table 3 for Sanger sequencing.…”

“…CO-driven H 2 production is mediated by an enzymatic system encoded in the CODH gene cluster, composed of a carbon monoxide dehydrogenase (CODH), a hydrogenase and a Na + /H + antiporter 12 13 14 . We have enhanced the H 2 production rate of the strain through knowledge-based rational design, such as by overexpressing genes in the CODH gene cluster by promoter engineering or by CO regulator engineering 14 15 16 17 . Although the design was successful, the resulting mutant strains could not grow in a fermentor supplied with a CO flow rate above 800 ml min −1 .…”

Citrobacter amalonaticus Y19 for constitutive expression of carbon monoxide-dependent hydrogen-production machinery

“…Most of these microorganisms require high temperatures and lavish nutrients (e.g., hyperthermophilic bacteria such as Thermococcus onnurineus and Morella thermoacetica ) or sunlight (e.g., photosynthetic bacteria such as Rhodospirillum rubrum , Rubrivivax gelatinosa, and  Rhodopseudomonas palustris ) for growth and expression of WGSR enzymes [2–4]. Moreover, they usually grow only slowly and to a low cell density.…”