Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, 9 Section 4, Renmin Road South, Chengdu 610041, People's Republic of China; College of Pharmacy and Biological Engineering, Chengdu University, 2025 Chengluo Avenue, Chengdu 610106, People's Republic of China.
Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, 9 Section 4, Renmin Road South, Chengdu 610041, People's Republic of China; University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, People's Republic of China.
J Biotechnol. 2021 Mar 10;329:104-117. doi: 10.1016/j.jbiotec.2021.01.015. Epub 2021 Feb 1.
Non-proteinogenic trans-4-hydroxy-l-proline (t4HYP), a crucial naturally occurred amino acid, is present in most organisms. t4HYP is a regio- and stereo-selectively hydroxylated product of l-proline and a valuable building block for pharmaceutically important intermediates/ingredients synthesis. Microbial production of t4HYP has aroused extensive investigations because of its low-cost and environmentally benign features. Herein, we reported metabolic engineering of endogenous l-proline biosynthetic pathway to enhance t4HYP production in trace l-proline-producing Escherichia coli BL21(DE3) (21-S0). The genes responsible for by-product formation from l-proline, pyruvate, acetyl-CoA, and isocitrate in the biosynthetic network of 21-S0 were knocked out to channel the metabolic flux towards l-proline biosynthesis. PdhR was knocked out to remove its negative regulation and aceK was deleted to ensure isocitrate dehydrogenase's activity and to increase NADPH/NADP level. The other genes for l-proline biosynthesis were enhanced by integration of strong promoters and 5'-untranslated regions. The resulting engineered E. coli strains 21-S1 ∼ 21-S9 harboring a codon-optimized proline 4-hydroxylase-encoding gene (P4H) were grown and fermented. A titer of 4.82 g/L of t4HYP production in 21-S6 overexpressing P4H was obtained at conical flask level, comparing with the starting 21-S0 (26 mg/L). The present work paves an efficient metabolic engineering way for higher t4HYP production in E. coli.
非蛋白源反式-4-羟基-L-脯氨酸（t4HYP）是一种重要的天然存在的氨基酸，存在于大多数生物中。t4HYP 是 L-脯氨酸区域和立体选择性羟化产物，也是合成具有重要药用价值的中间体/成分的宝贵构建模块。由于微生物生产 t4HYP 具有低成本和环境友好的特点，因此引起了广泛的研究。本文报道了对痕量 L-脯氨酸产生菌大肠杆菌 BL21(DE3)（21-S0）内源 L-脯氨酸生物合成途径进行代谢工程改造，以提高 t4HYP 的产量。敲除生物合成网络中 L-脯氨酸、丙酮酸、乙酰辅酶 A 和异柠檬酸的副产物形成相关基因，以将代谢通量导向 L-脯氨酸生物合成。敲除 PdhR 以消除其负调控作用，敲除 aceK 以确保异柠檬酸脱氢酶的活性并增加 NADPH/NADP 水平。通过整合强启动子和 5'-非翻译区增强其他 L-脯氨酸生物合成基因。携带密码子优化的脯氨酸 4-羟化酶编码基因（P4H）的工程大肠杆菌菌株 21-S1∼21-S9 进行培养和发酵。在摇瓶水平上，过表达 P4H 的 21-S6 菌株的 t4HYP 产量达到 4.82g/L，比起始菌株 21-S0（26mg/L）提高了 179 倍。本研究为大肠杆菌中 t4HYP 的高产提供了一种有效的代谢工程方法。
