Metabolic pathway analysis of Mannheimia succiniciproducens and Escherichia coli for industrial applications

Abstract

With the advent of multi omics data, systems metabolic engineering based on system level has been developed to overcome limitations of previous approaches. Systems metabolic engineering that combines metabolic engineering with synthetic biology, systems biology engineering has provided accurate target genes for increasing productivity or yield as well as synthetic pathways to produce natural and non-natural chemicals. For better understanding of metabolism of micro-organism, metabolic pathway analysis based on in silico simulation was applied to analyze Mannheimia succiniciproducens which are known for efficient succinic acid producer and Escherichia coli which has been frequently used and studied research and industrial application. With regard to utilizing metabolic pathway analysis, we newly designed simple strategy called pathway clustering analysis for simple strain engineering and comparative analysis based on clustering method. This strategy was successfully applied to M. succiniciproducens and E. coli to give systemic perspectives in comparison. Additionally, methodology of $^{13}C$-metabolic flux analysis was constructed for quantification of metabolic fluxes of micro-organism. During construction process, we developed GC/MS data processing program and determined reliable amino acid fragments. With methodology of $^{13}C$-metabolic flux analysis, E. coli and M. succiniciproducens was investigated in terms of fluxes. Especially, the metabolic fluxes of three different E. coli strains K-12, B, and W were compared each other. Interestingly, $^{13}C$-metabolic flux analysis indicated that the fluxes of glyoxylate shunt and TCA cycle were highly active in B strains. The enhanced TCA cycle fluxes also resulted in higher fluxes into amino acid such as glutamate, aspartate and arginine in B strains. We also observed higher fluxes in glycolytic fluxes of W strains compared with K-12 and B strain. Furthermore, the activity of glyoxylate shunt and the usage of pentose phosphate pathway in engineered M. succiniciproducens were determined by using fluxome profiling technique. In summary, metabolic pathway analysis provided comprehensive insight in micro-organism