(398d) A Sense of Balance: Exploring the Role of Metabolic Pathway Modularization in the Microbial Production of Chemicals

Efforts in designing synthetic cells for metabolic engineering applications have been almost exclusively based on redirecting fluxes towards product synthesis by enzyme overexpression. However, such strategies result in draining large amounts of carbon precursors and energy resources (such as ATP and NAD(P)H) from the host. The imposed metabolic burden can cause unexpected physiological changes and dramatically reduces cell performance. For example, our simulations show that cell catabolism is abundant in wild type strains so that they can support a certain amount of metabolic burden without having apparent biosynthesis deficiency (e.g., without showing a slower growth after mutations). However, cell burden usually increases when genetic modifications are introduced or fermentative products are accumulated. When cell catabolism and respiration is unable to support the increasing ATP expenditure, the biosynthesis yield will suddenly drop, forming a “cliff” (“the straw that broke the camel’s back”).

In order to address the issue of metabolic burden, our laboratory has focused on developing novel approaches that allow metabolic pathway balancing and division of labor (DoL) in order to mitigate and if possible eliminate the negative effects of metabolic burden on cellular physiology. In one example, we have modularized the fatty acid biosynthetic pathway in E.coli, and improved the final titer as well as the production rate by balancing the different modules using different plasmid copy numbers and ribosome-binding sites of different strengths. A similar approach was used to balance a synthetic metabolic pathway that allows conversion of single-carbon (C1) substrates to multi-carbon natural products in a recombinant E.coli strain. In more recent research, we have used co-culture and poly-culture approaches where multistep pathways for producing phytochemicals have been split into different production strains in order to alleviate metabolic burden, allowing the de novo production of high-value chemicals from recombinant microorganisms for the first time. In this talk, I will highlight our recent work on engineering such strategies and its implication for industrial biomanufacturing.