Metab Eng. 2018 Sep;49:153-163. doi: 10.1016/j.ymben.2018.08.004. Epub 2018 Aug 11.
Directed strain evolution restructures metabolism for 1-butanol production in minimal media.
Pontrelli S1, Fricke RCB1, Sakurai SSM2, Putri SP2, Fitz-Gibbon S3, Chung M1, Wu HY4, Chen YJ5, Pellegrini M6, Fukusaki E2, Liao JC7.
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles (UCLA), United States.
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Institute of Genomics and Proteomics, University of California, Los Angeles, United States.
Instrumentation Center, National Taiwan University, Taipei, Taiwan.
Institute of Chemistry, Academia Sinica, Taipei, Taiwan.
Institute of Genomics and Proteomics, University of California, Los Angeles, United States; Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, United States.
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Electronic address: liaoj@gate.sinica.edu.tw.
Engineering a microbial strain for production sometimes entails metabolic modifications that impair essential physiological processes for growth or production. Restoring these functions may require amending a variety of non-obvious physiological networks, and thus, rational design strategies may not be practical. Here we demonstrate that growth and production may be restored by evolution that repairs impaired metabolic function. Furthermore, we use genomics, metabolomics and proteomics to identify several underlying mutations and metabolic perturbations that allow metabolism to repair. Previously, high titers of butanol production were achieved by Escherichia coli using a growth-coupled, modified Clostridial CoA-dependent pathway after all native fermentative pathways were deleted. However, production was only observed in rich media. Native metabolic function of the host was unable to support growth and production in minimal media. We use directed cell evolution to repair this phenotype and observed improved growth, titers and butanol yields. We found a mutation in pcnB which resulted in decreased plasmid copy numbers and pathway enzymes to balance resource utilization. Increased protein abundance was measured for biosynthetic pathways, glycolytic enzymes have increased activity, and adenosyl energy charge was increased. We also found mutations in the ArcAB two-component system and integration host factor (IHF) that tune redox metabolism to alter byproduct formation. These results demonstrate that directed strain evolution can enable systematic adaptations to repair metabolic function and enhance microbial production. Furthermore, these results demonstrate the versatile repair capabilities of cell metabolism and highlight important aspects of cell physiology that are required for production in minimal media.