Department of Chemical and Biomolecular Engineering, University of California, Los Angeles (UCLA), United States.
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Metab Eng. 2018 Sep;49:153-163. doi: 10.1016/j.ymben.2018.08.004. Epub 2018 Aug 11.
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.
工程菌株的生产有时需要进行代谢修饰，这些修饰会损害生长或生产所必需的生理过程。恢复这些功能可能需要修改各种不明显的生理网络，因此，合理的设计策略可能并不实用。在这里，我们证明通过修复受损代谢功能的进化可以恢复生长和生产。此外，我们使用基因组学、代谢组学和蛋白质组学来鉴定允许代谢修复的几个潜在突变和代谢扰动。此前，在所有天然发酵途径被删除后，大肠杆菌使用一种与生长偶联的、改良的梭菌 CoA 依赖性途径来实现高丁醇产量，但仅在丰富的培养基中观察到生产。宿主的天然代谢功能无法在最小培养基中支持生长和生产。我们使用定向细胞进化来修复这种表型，并观察到生长、产量和丁醇得率的提高。我们发现 pcnB 中的一个突变导致质粒拷贝数减少和途径酶的减少，以平衡资源利用。生物合成途径的蛋白质丰度增加，糖酵解酶的活性增加，腺苷能量电荷增加。我们还发现 ArcAB 双组分系统和整合宿主因子 (IHF) 的突变，这些突变可以调节氧化还原代谢，改变副产物的形成。这些结果表明，定向菌株进化可以使细胞适应代谢功能的修复，并增强微生物的生产能力。此外，这些结果还表明了细胞代谢具有广泛的修复能力，并强调了在最小培养基中生产所需的细胞生理学的重要方面。
