Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany; Institute for Biology-Microbiology, Freie Universität Berlin, Königin-Luise-Str. 12-16, 14195 Berlin, Germany.
Metab Eng. 2018 May;47:31-41. doi: 10.1016/j.ymben.2018.02.015. Epub 2018 Mar 6.
Evolutionary approaches are often undirected and mutagen-based yielding numerous mutations, which need elaborate screenings to identify relevant targets. We here apply Metabolic engineering to Guide Evolution (MGE), an evolutionary approach evolving and identifying new targets to improve microbial producer strains. MGE is based on the idea to impair the cell's metabolism by metabolic engineering, thereby generating guided evolutionary pressure. It consists of three distinct phases: (i) metabolic engineering to create the evolutionary pressure on the applied strain followed by (ii) a cultivation phase with growth as straightforward screening indicator for the evolutionary event, and (iii) comparative whole genome sequencing (WGS), to identify mutations in the evolved strains, which are eventually re-engineered for verification. Applying MGE, we evolved the PEP and pyruvate carboxylase-deficient strain C. glutamicum Δppc Δpyc to grow on glucose as substrate with rates up to 0.31 ± 0.02 h which corresponds to 80% of the growth rate of the wildtype strain. The intersection of the mutations identified by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in three independently evolved mutants. Upon re-engineering in C. glutamicum Δppc Δpyc, the identified mutations led to diminished ICD activities and activated the glyoxylate shunt replenishing oxaloacetate required for growth. Intracellular relative quantitative metabolome analysis showed that the pools of citrate, isocitrate, cis-aconitate, and L-valine were significantly higher compared to the WT control. As an alternative to existing L-valine producer strains based on inactivated or attenuated pyruvate dehydrogenase complex, we finally engineered the PEP and pyruvate carboxylase-deficient C. glutamicum strains with identified ICD mutations for L-valine production by overexpression of the L-valine biosynthesis genes. Among them, C. glutamicum Δppc Δpyc ICD (pJC4ilvBNCE) produced up to 8.9 ± 0.4 g L-valine L, with a product yield of 0.22 ± 0.01 g L-valine per g glucose.
进化方法通常是无方向的和基于诱变的，产生许多突变，需要精心筛选才能确定相关的靶点。在这里，我们应用代谢工程指导进化（MGE）来进化和鉴定新的靶点，以改善微生物生产菌株。MGE 的基础是通过代谢工程损害细胞的新陈代谢，从而产生有针对性的进化压力。它由三个不同的阶段组成：（i）代谢工程，在应用菌株上产生进化压力，随后进行（ii）培养阶段，以生长作为进化事件的直接筛选指标，以及（iii）比较全基因组测序（WGS），以鉴定进化菌株中的突变，最终对这些突变进行重新工程设计以进行验证。应用 MGE，我们使 PEP 和丙酮酸羧化酶缺陷型 C. glutamicum Δppc Δpyc 菌株以葡萄糖为底物生长，其生长速率高达 0.31±0.02 h，相当于野生型菌株生长速率的 80%。通过 WGS 鉴定的突变的交集揭示了异柠檬酸脱氢酶（ICD）是三个独立进化的突变体中的一致靶点。在 C. glutamicum Δppc Δpyc 中重新工程设计后，鉴定的突变导致 ICD 活性降低，并激活乙醛酸支路，补充生长所需的草酰乙酸。细胞内相对定量代谢组学分析表明，与 WT 对照相比，柠檬酸、异柠檬酸、顺乌头酸和 L-缬氨酸的池明显更高。作为现有基于失活或减弱的丙酮酸脱氢酶复合物的 L-缬氨酸生产菌株的替代方案，我们最终通过过量表达 L-缬氨酸生物合成基因，对鉴定出的 ICD 突变的 PEP 和丙酮酸羧化酶缺陷型 C. glutamicum 菌株进行工程设计，用于 L-缬氨酸生产。其中，C. glutamicum Δppc Δpyc ICD（pJC4ilvBNCE）生产高达 8.9±0.4 g L-缬氨酸 L-，产物得率为 0.22±0.01 g L-缬氨酸每克葡萄糖。
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