Harnessing cell biomass generated during formate-driven H₂ production for sequential protein production in a...

Organic waste is generally discarded in bioprocesses, which limits sustainability and emphasizes the need for effective recycling strategies. This study presents a sequential protein production bioprocess using the hyperthermophilic archaeon Thermococcus onnurineus NA1, coupling H₂ production from formate with recycling of waste cell biomass. Based on transcriptomic analysis, the 125-bp promoter region of the TON_1795 gene (P1795), encoding the ABC-type maltodextrin transport system, was selected as the promoter for protein expression. This gene was found to be substrate-responsive, showing upregulation when shifting from formate to sugar conditions. The 350TF1 strain was constructed in the WTF350T strain by introducing a formate dehydrogenase tungsten-containing catalytic subunit (fdh3A) gene under the control of the P1795 promoter, and its sugar-dependent expression was confirmed by western blot analysis. During formate fermentation, the 350TF1 strain exhibited cell growth, H₂ production, biomass productivity, and a maximum cell density ranging from 0.89 ± 0.06- to 1.17 ± 0.23-fold relative to those of the WTF350T strain. After fermentation under formate conditions, the 350TF1 cells were harvested, and the protein was extracted. The extracted FDH3A expression was prominent in the presence of glucose, exhibiting a specific activity of 100.16 ± 33.18 U/mg after 6 h incubation, whereas the absence of glucose yielded an unstable form of the FDH3A with lower specific activity (25.52 ± 10.57 U/mg). These results demonstrated a formate-based bioprocess platform that enables the conversion of fermentation-derived waste biomass into value-added products, improving the sustainability of hyperthermophilic biofactories.