Biological process for coproduction of hydrogen and thermophilic enzymes during CO fermentation

To develop a thermophilic cell factory system that uses CO gas, we attempted to engineer a hyperthermophilic carboxydotrophic hydrogenic archaeon Thermococcus onnurineus NA1 to be capable of producing thermophilic enzymes along with hydrogen (H2). The mutant strains 156T-AM and 156T-POL were constructed to have another copy of a gene encoding α-amylase or DNA polymerase, respectively, and exhibited growth rates and H2 production rates distinct from those of the parental strain, 156T, in gas fermentation using 100% CO or coal-gasified syngas. Purified α-amylase displayed starch-hydrolyzing activity, and whole-cell extracts of 156T-AM showed saccharifying activity for potato peel waste. PCR amplification was used to demonstrate that purified DNA polymerase was free from bacterial DNA contamination, in contrast to commercial bacteria-made enzymes. This study demonstrated that this archaeal strain could coproduce enzymes and H2 using CO-containing gas, providing a basis for cell factories to upcycle industrial waste gas.