Metabolic perturbation by homologous expression of the CO dehydrogenase gene cluster in the CO utilizing acetogen Eubacterium limosum KCTC13263BP

Abstract

The bioconversion of the synthesis gas has been highlighted as a feasible option in industrial applications. The synthesis gas is a mixture gas of hydrogen (H2), carbon dioxide (CO2) and carbon monoxide (CO). Since the synthesis gas is a by-product of a variety of industries such as ironworks and powerplants, there have been extensive studies for utilizing the synthesis gas throughout the years. In the beginning, the synthesis gas had been utilized via direct combustion. But in the recent days, there are several enterprises successfully demonstrated the conversion processes from industrial by-products to biofuel and biochemicals. This approach is mainly performed by microbial fermentation since if more beneficial in terms of production of the diverse products with increased selectivity than the chemical-dependent processes. The microorganisms used for the synthesis bioconversion should have a high tolerance to the substrate CO and the productivity of acids. Therefore, metabolic engineering is necessary to produce microorganisms of interest. The most frequently attempted method for enhancing microorganisms is to perturb the metabolism within the microorganisms via expression level modification. Although synthesis gas bioconversion rate via expression level modification was not always successful, the method through metabolic perturbation for more accurate selection for target metabolisms (or reactions) is demanded. In this research, the bacterium Eubacterium limosum KCTC13263BP (formerly known as KIST612) was employed to find out the optimum protocols for enhancing CO conversion and acetate production rates. E. limosum KCTC13263BP is an acetogenic strain that can produce acetic and butyric acids by the consumption of CO as the sole carbon and energy sources. E. limosum KCTC13263BP is a Gram-positive, strictly anaerobic bacterium that fixes carbon via the Wood-Ljungdahl pathway (the W-L Pathway). The W-L pathway oxidates CO to acetyl-CoA as a reduced intermediate and either converts to acetate or incorporates into cellular biomass. This dissertation suggested the combined protocol for utilizing genome scale modeling, target gene selection and manipulation on expression levels of target proteins, to enhance CO consumption and acetate production rates. At the beginning of this research, it was surveyed the reconstruction of genome scale model, for selecting target genes in CO autotrophic growth condition of the strain. The flux stability analysis based on the reconstructed genome scale model had successfully screened the target genes for expression. The screened target genes were then adopted for constructing the E. limosum mutants carrying expression vectors. Among the five constructed mutants, the mutant ELM031 was observed an increment in specific CO oxidation and specific acetate production rates by 3.1-fold and 1.4-fold compared to the wild-type strain, respectively. The estimated fluxes established throughout the pathways within the mutant ELM031 compared with wild-type had been analyzed. It was suggested that redox cycles established between the W-L pathway and the Rhodobacter nitrogen fixation complex (the Rnf complex) roles the major governor for coupled response overall fluxes. This hypothesis was verified by the measurement of ratio in oxidized/reduced forms of nicotinamide adenine dinucleotide. Additionally, it was suggested that the tetrahydrofolate-related reactions could be a way to decompose reducing equivalent to facilitate redox cycle in the strain.