Comparison of CO-dependent H₂ production with strong promoters in Thermococcus onnurineus NA1

To overproduce biotechnologically valuable products, the expression level of target genes has been modulated by using strong promoters. In a hyperthermophilic archaeon Thermococcus onnurineus NA1, two promoters, P(TN0413) and P(TN0157), which drive expression of the genes encoding the S-layer protein and glutamate dehydrogenase were inserted in front of a gene cluster encoding a carbon monoxide dehydrogenase, a hydrogenase and a Na⁺/H⁺ antiporter. Two promoters exhibited strong activity by increasing the transcription and translation levels of the gene cluster in the mutant strains by 2.5- to 49-folds and 1.4- to 3.3-folds, respectively, than the native promoter in the wild-type strain. While KS0413 with P(TN0413) promoter exhibited 2.7 to 4.7 times higher transcript level than KS0157 with P(TN0157) promoter, the levels of proteins were a little different between them. The biomass concentrations and H₂ production rates of two mutants were 2- to 3-fold higher than those of the wild-type strain in a bioreactor where CO was supplied at a flow rate of 120 ml min⁻¹. Two mutants showed differential response to the higher CO flow rate, 240 ml min⁻¹, in terms of growth pattern and product formation, indicating two promoters were regulated by culture conditions. The results demonstrate that not only promoter strength but also product-forming conditions should be considered in promoter engineering.