Systematic Analysis of Bottlenecks in a Multibranched and Multilevel Regulated Pathway: The Molecular Fundamentals of l-Methionine Biosynthesis in Escherichia coli

To produce chemicals and fuels from renewable resources, various strategies and genetic tools have been developed to redesign pathways and optimize the metabolic flux in microorganisms. However, in most successful cases, the target chemicals are synthesized through a linear pathway, and regular methodologies for the identification of bottlenecks and metabolic flux optimization in multibranched and multilevel regulated pathways, such as the l-methionine biosynthetic pathway, have rarely been reported. In the present study, a systematic analysis strategy was employed to gradually reveal and remove the potential bottlenecks limiting the l-methionine biosynthesis in E. coli. 80 genes in central metabolism and selected amino acids biosynthetic pathways were first repressed or upregulated to probe their effects on l-methionine accumulation. The l-methionine biosynthetic pathway was then modularized and iteratively genetic modifications were performed to uncover the multiple layers of limitations and stepwise im...