Significantly enhancing production of trans-4-hydroxy-L-proline by integrated system engineering in Escherichia coli
Significantly enhancing production of trans-4-hydroxy-L-proline by integrated system engineering in Escherichia coli
复制标题
通过集成系统工程在大肠杆菌中显着提高反式-4-羟基-L-脯氨酸的产量
DOI：
10.1126/sciadv.aba2383
复制
发表时间：
2020-05-01
期刊：
影响因子：
13.6
通讯作者：
Rao, Zhiming
中科院分区：
文献类型：
--
作者：
Long, Mengfei;Xu, Meijuan;Rao, Zhiming
反式-4-羟基-L-脯氨酸是由L-脯氨酸经葡萄糖发酵产生的反式-4-羟化酶。在这里，我们设计了一个彻底的“从A到Z”的策略，以显着提高反式-4-羟基-L-脯氨酸的生产。通过稀有密码子选择进化，大肠杆菌M1产生18.2 g L-1 L-脯氨酸。代谢工程改造的M6与putA，proP，putP，和aceA的缺失，和proB突变集中碳通量的L-脯氨酸和释放其反馈抑制。该菌产反式-4-羟基-L-脯氨酸15.7g/L，保留脯氨酸10 g/L。此外，基于群体感应的可调谐电路衰减L-脯氨酸羟基化通量，导致43.2 g L-1反式-4-羟基-L-脯氨酸与4.3 g L-1 L-脯氨酸保留。最后，合理设计的L-脯氨酸羟化酶在60小时内几乎没有L-脯氨酸降解的情况下产生54.8 g L-1反式-4-羟基-L-脯氨酸-迄今为止的最高产量。通过稀有密码子选择进化、动态前体调控和代谢工程等手段，从头工程化碳通量为羟基氨基酸的高效合成提供了良好的技术平台。
Trans-4-hydroxy-L-proline is produced by trans-proline-4-hydroxylase with L-proline through glucose fermentation. Here, we designed a thorough "from A to Z" strategy to significantly improve trans-4-hydroxy-L-proline production. Through rare codon selected evolution, Escherichia coli M1 produced 18.2 g L-1 L-proline. Metabolically engineered M6 with the deletion of putA, proP, putP, and aceA, and proB mutation focused carbon flux to L-proline and released its feedback inhibition. It produced 15.7 g L-1 trans-4-hydroxy-L-proline with 10 g L-1 L-proline retained. Furthermore, a tunable circuit based on quorum sensing attenuated L-proline hydroxylation flux, resulting in 43.2 g L-1 trans-4-hydroxy-L-proline with 4.3 g L-1 L-proline retained. Finally, rationally designed L-proline hydroxylase gave 54.8 g L-1 trans-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.