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Directed Evolution of Ornithine Cyclodeaminase Using an EvolvR-Based Growth-Coupling Strategy for Efficient Biosynthesis of l-Proline.
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2020-06-18 , DOI: 10.1021/acssynbio.0c00198
Mengfei Long 1 , Meijuan Xu 1 , Zhina Qiao 1 , Zhenfeng Ma 1 , Tolbert Osire 1 , Taowei Yang 1 , Xian Zhang 1 , Minglong Shao 1 , Zhiming Rao 1
Affiliation  

l-Proline takes a significant role in the pharmaceutical and chemical industries as well as graziery. Typical biosynthesis of l-proline is from l-glutamate, involving three enzyme reactions as well as a spontaneous cyclization. Alternatively, l-proline can be also synthesized in l-ornithine and/or l-arginine producing strains by an ornithine aminotransferase (OCD). In this study, a strategy of directed evolution combining rare codon selection and pEvolvR was developed to screen OCD with high catalytic efficiency, improving l-proline production from l-arginine chassis cells. The mutations were generated by CRISPR-assisted DNA polymerases and were screened by growth-coupled rare codon selection system. OCDK205G/M86K/T162A from Pseudomonas putida was identified with 2.85-fold increase in catalytic efficiency for the synthesis of l-proline. Furthermore, we designed and optimized RBS for the BaargI and Ppocd coupling cascade using RedLibs, as well as sRNA inhibition of argF to moderate l-proline biosynthesis in l-arginine overproducing Corynebacterium crenatum. The strain PS6 with best performance reached 15.3 g/L l-proline in the shake flask and showed a titer of 38.4 g/L in a 5 L fermenter with relatively low concentration of residual l-ornithine and/or l-arginine.

中文翻译:

使用基于EvolvR的生长偶联策略有效地合成l-脯氨酸的鸟氨酸环氧化酶的定向进化。

l-脯氨酸在制药和化学工业以及放牧中起着重要作用。1-脯氨酸的典型生物合成来自1-谷氨酸,涉及三个酶反应以及自发环化。可选地,-脯氨酸也可以在合成鸟氨酸和/或精氨酸产生菌的鸟氨酸氨基转移酶(OCD)。在这项研究中,定向进化相结合稀有密码子的选择和pEvolvR战略的开发与催化效率高屏幕OCD,提高从脯氨酸生产-精氨酸底盘细胞。突变是由CRISPR辅助的DNA聚合酶产生的,并通过生长偶联稀有密码子选择系统进行筛选。鉴定恶臭假单胞菌的OCD K205G / M86K / T162A,其对1-脯氨酸的合成的催化效率提高了2.85倍。此外,我们设计和优化RBS用于BaargIPpocd使用RedLibs耦合级联,以及对的sRNA抑制ARGF至中度-脯氨酸的生物合成在精氨酸过量产生钝齿棒杆菌。性能最佳的PS6菌株达到15.3 g / L l-摇瓶中的脯氨酸的滴定度,在5 L发酵罐中的滴定度为38.4 g / L,残留的1-鸟氨酸和/或1-精氨酸浓度相对较低。
更新日期:2020-07-17
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