Deciphering Methanol Metabolism of Syngas-Utilizing Acetogen, Eubacterium limosum KIST612

Abstract

Since the biological conversion of syngas by microorganisms is environmentally friendly and cost-effective, it can be considered the next-generation energy source. Acetogens, which maintain a reductive acetyl-CoA pathway, represent one excellent option for desired biocatalysts, as their universal microbial machinery allowing for the uptake of all major gas components in syngas (e.g., CO, H2, and CO2) without any purification requirements; however, low water-solubility of the gas components (H2, CO, and CO2) cause decreasing the syngas conversion efficiency. Here, the methanol-based syngas conversion strategy was proposed, and its feasibility was confirmed through the following: (1) searching methanol conversion system based on genomic and proteomic analyses; (2) identifying features of methanol metabolism based on flux analysis; (3) confirming methanol effect on syngas fermentation based on proteomic and physiological analyses. As a methylotrophic acetogen with previously reported high syngas reaction rates and metabolic information, the Eubacterium limosum KIST612 strain was selected for the study. E. limosum KIST612 could integrate and utilize methanol into the reductive acetyl-CoA pathway using the methyltransferase system (encoded by the mta operon) that is highly expressed by methanol. However, due to balancing reducing equivalents, the strain cannot utilize methanol as the sole carbon and energy source. Methanol utilization based on the mta system required an electron acceptor. Although CO2 or formate can be used as an electron acceptor, the main product differed depending on the acceptor, indicating metabolic differences. Acetate was the only product under the methanol (30 mM)/formate (30 mM) condition, whereas butyrate was the main product under the methanol (30 mM)/CO2 (0.2 atm) condition. The result of flux analysis showed that the “butyrogenesis” might be determined depending on the required amount of Fd2-. Under the Fd2--necessary condition, methanol metabolism showed unusual characteristics from other autotrophic metabolisms. It was thought that obtaining and balancing reducing equivalents cause the reverse reactions of enzymes involved in the methyl branch of the reductive acetyl-CoA pathway and energy metabolism as well as butyrogenesis. It means that enzymes related to autotrophic metabolism (including CODH/ACS) could be highly expressed by methanol. Since CO inhibited methanol consumption, the proposed methanol-based strategy could be confirmed under the only “CO2-consumed phase” of syngas fermentation. The expression levels of the proteins related to autotrophic metabolism under the methanol condition were sufficient to meet the H2/CO2 metabolism requirement. Additionally, all genetic components were similarly regulated. Finally, it was confirmed that the H2 consumption rate and the degree of product reduction were increased by methanol. Notably, some acetogens are capable of methanol utilization, which can be directly incorporated into the pathway via methyltransferase like E. limosum KIST612. Further, the universality of this enzymatic reaction for methylotrophic acetogens could be deciphered based on the results of genome analyses and cultivation tests. The findings here have strong implications for enhancing the cyclical economy, sustainability, and availability of the syngas fermentation process during practical operations.