Development of CO gas conversion system using high CO tolerance biocatalyst

• High CO tolerance Eubacterium limosum ECO2 strain via adaptive laboratory evolution. • ECO2 strain exhibited an increased CO gas conversion rate under high CO conditions. • ECO2 has an H636R mutation in acetyl-CoA synthase, which causes high CO tolerance. • Microbial biocatalysts that enable convert C1 to C4 were constructed using ECO2. • The engineered strain showed 4.5-fold higher productivity of C4 than the wild-type. Acetogenic bacteria (acetogens) are microorganisms with high potential as biocatalysts that can convert C1 gas, such as carbon dioxide (CO 2) or carbon monoxide (CO), into biomass or biochemicals; however, high CO concentrations cause growth retardation in these microorganisms. In this study, we performed adaptive evolution of Eubacterium limosum , an acetogen, for approximately 400 generations under high CO synthetic gas (syngas) conditions to obtain E. limosum ECO2, which can rapidly proliferate even under high CO conditions. A mutation was revealed in H636R in the Acetyl-CoA synthase (ACS) protein of the ECO2 strain through whole-genome re-sequencing, and the mutation increased the CO gas conversion rate. By analyzing the transcript profiling of the ECO2 strain under high CO syngas conditions, the transcript levels of the methyl branch in the Wood–Ljungdahl pathway and the energy conservation system increased at high CO concentration conditions. Finally, to convert CO into value-added biochemicals, 2,3-BDO (2,3-butanediol) biosynthesis pathways were constructed and transformed into the E. limosum ECO2 strain. This strain showed capability to produce 2.60 mg L -1 h -1 of 2,3-BDO through CO 66% syngas fed-batch fermentation. This was 4.6-fold higher than that of the wild type. We developed a microorganism with high CO tolerance, and this strain showed high application potential as a biocatalyst that can efficiently convert CO gas into value-added chemicals.