UCLA

Harnessing the adaptive nature of cell metabolism presents an opportunity to understand the function of biological systems, how they adapt, and how they may respond when challenged. It also stands as a tool that can aid in restoring impaired metabolic function caused by engineering microbial production. Many studies have demonstrated the ability of the cell to overcome metabolite auxotrophies and have elucidated underlying mechanisms. However, these studies have primarily focused on mechanisms that directly replace mutant function. In this work, we first aim to expand this view by evolving and elucidating more complex adaptive mechanisms. As an example, we used a ΔpanD strain of E. coli, a β-alanine auxotroph, to demonstrate that entire metabolic pathways can evolve to repair auxotrophy. Using directed strain evolution, we showed that E. coli successively evolved three distinct metabolic pathways to synthesize β-alanine. The first involved significant rewiring and repurposing of the uracil synthesis and degradation pathways. The second relied on a gain-of-function mutation in ornithine decarboxylase (SpeC) which altered substrate and reaction specificity. The third pathway emerged that relies on synthesis of polyamines.

This work also serves as a demonstration of how metabolism can be evolved to overcome impaired metabolic function that may be incurred through engineering microbes for production. As an example, we focused on a modified strain of E. coli that is capable of producing high titers of butanol in rich media using an anaerobic, growth-coupled, modified Clostridial CoA-dependent pathway. For unknown reasons, strain modifications impaired metabolic function. Using directed strain evolution, a strain was acquired that has improved growth, titers and butanol yields. We further identified several mutations that adapted energy and carbon

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metabolism and optimized expression of pathway enzymes. These works collectively demonstrate the elucidation of adaptive mechanisms of cell metabolism and further, they demonstrate applications in strain engineering.