Long Mengfei, Xu Meijuan, Ma Zhenfeng, Pan Xuewei, You Jiajia, Hu Mengkai, Shao Yu, Yang Taowei, Zhang Xian, Rao Zhiming
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Sci Adv. 2020 May 22;6(21):eaba2383. doi: 10.1126/sciadv.aba2383. eCollection 2020 May.
4-hydroxy-l-proline is produced by -proline-4-hydroxylase with l-proline through glucose fermentation. Here, we designed a thorough "from A to Z" strategy to significantly improve -4-hydroxy-l-proline production. Through rare codon selected evolution, M1 produced 18.2 g L l-proline. Metabolically engineered M6 with the deletion of , , , and , and mutation focused carbon flux to l-proline and released its feedback inhibition. It produced 15.7 g L -4-hydroxy-l-proline with 10 g L l-proline retained. Furthermore, a tunable circuit based on quorum sensing attenuated l-proline hydroxylation flux, resulting in 43.2 g L -4-hydroxy-l-proline with 4.3 g L l-proline retained. Finally, rationally designed l-proline hydroxylase gave 54.8 g L -4-hydroxy-l-proline in 60 hours almost without l-proline remaining-the highest production to date. The de novo engineering carbon flux through rare codon selected evolution, dynamic precursor modulation, and metabolic engineering provides a good technological platform for efficient hydroxyl amino acid synthesis.
4-羟基-L-脯氨酸是由脯氨酸-4-羟化酶通过葡萄糖发酵作用将L-脯氨酸转化生成的。在此，我们设计了一套全面的“从A到Z”策略，以显著提高4-羟基-L-脯氨酸的产量。通过稀有密码子选择进化，M1产生了18.2 g/L的L-脯氨酸。代谢工程改造的M6缺失了 、 、 、 和 ，且 突变使碳通量集中于L-脯氨酸并解除了其反馈抑制。它产生了15.7 g/L的4-羟基-L-脯氨酸，同时保留了10 g/L的L-脯氨酸。此外，基于群体感应的可调回路减弱了L-脯氨酸的羟化通量，从而产生了43.2 g/L的4-羟基-L-脯氨酸，同时保留了4.3 g/L的L-脯氨酸。最后，合理设计的L-脯氨酸羟化酶在60小时内几乎没有剩余L-脯氨酸的情况下产生了54.8 g/L的4-羟基-L-脯氨酸——这是迄今为止的最高产量。通过稀有密码子选择进化、动态前体调节和代谢工程进行的从头工程碳通量为高效羟基氨基酸合成提供了一个良好的技术平台。
