Carbon Monoxide Autotrophy Metabolism for Enhanced Biochemical Production

Abstract

The production of biofuels and biochemicals through microbial fermentation is receiving attention as a technology to address concerns related to the depletion of fossil fuel resources and climate change. However, the economic viability of feedstocks for bioconversion remains an issue that needs to be addressed. C1 gas including carbon monoxide (CO), carbon dioxide (CO2), and methane (CH4) serves as a renewable and sustainable feedstock, but its low energy content and limited solubility make it challenging for the efficient production of biofuels and biochemicals. To produce the desired products through microbial fermentation, optimization of microbial strains and a thorough understanding of metabolism are essential. In this study, Eubacterium callanderi KIST612 strain, an acetogen that utilizes C1 gas, was used as a model strain to investigate the metabolic characteristics of CO gas as a substrate. E. callanderi KIST612 mainly produces acetate as well as butyrate, depending on the carbon and electron sources. The butyrate formation pathway of E. callanderi KIST612 was suggested by the previously studied transcriptome and proteome. The key enzymes for butyrate formation pathway are confirmed through enzyme activities in this study. Especially, acetate assimilatory butyrate formation was experimentally verified under CO growth. It is suggested that acetate assimilatory butyrate formation occurs to facilitate the decomposition of reducing equivalents for entire metabolic energy cycle. Moreover, acetate assimilation was more clearly identified in reprogrammed ethanol-producing strains, which have an electron sink pathway at the acetyl-CoA branch point in E. callanderi strain. It implies that there are physiological benefits in promoting the metabolic energy cycle through the regeneration of reducing equivalents in autotrophic condition. Interestingly, this study found that CO substrate consumption increased 10- folds higher than the wild-type with the end-product production, which is linked to the decomposition of reducing equivalents. As the key enzyme for consumption of CO gas substrates is CO dehydrogenase, this study constructed the mutant strains by enhancing the expression level of CO dehydrogenase to increase the CO gas consumption of E. callanderi strain. As a result, the heterologous expression of the Cc_AcsA gene (Ccar_18845) in E. callanderi KIST612 resulted in a 1.4-fold increase in enzyme activity of CO oxidation and a 1.7-fold increase in CO consumption rate compared to the wild-type. Furthermore, the improved CO oxidation rate led to a 3-fold increase in the maximum productivity of acetate and a 2.3-fold increase in the maximum productivity of butyrate, as compared to the wild-type. In conclusion, the production of reduced products such as butyrate and ethanol is closely linked in metabolism of CO autotrophy. This highlights the importance of promoting the metabolic energy cycle in facilitating product formation. In summary, understanding CO autotrophic metabolism is crucial in syngas bacterial fermentation for biofuel and biochemical production. This study provides insights into pathways and key enzymes for enhanced product formation under CO growth in E. callanderi KIST612. These findings contribute to the metabolic engineering of acetogens for bio-compound production using C1 gas.