Co-dependent H₂ production by genetically engineered Thermococcus onnurineus NA1

Hydrogenogenic CO oxidation (CO+H₂O → CO₂+H2) has the potential for H₂ production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H₂, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolismby gene disruption and transcriptional analysis. To develop a strain producing high levels of H₂, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na⁺/H⁺ antiporter and a 1.8-fold-higher specific activity for CO-dependent H₂ production than did the wild-type strain. The H₂ production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H₂ production rate of the engineered strain was severalfold higher than those of any other COdependent H₂-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H₂ production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.