The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
ACS Synth Biol. 2020 Jul 17;9(7):1855-1863. doi: 10.1021/acssynbio.0c00198. Epub 2020 Jul 8.
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. OCD from was identified with 2.85-fold increase in catalytic efficiency for the synthesis of l-proline. Furthermore, we designed and optimized RBS for the and coupling cascade using RedLibs, as well as sRNA inhibition of to moderate l-proline biosynthesis in l-arginine overproducing . 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.
l-脯氨酸在制药和化工行业以及畜牧业中都起着重要作用。l-脯氨酸的典型生物合成途径是从 l-谷氨酸开始，涉及三个酶反应和一个自发的环化反应。或者，l-脯氨酸也可以通过鸟氨酸转氨酶（OCD）在 l-鸟氨酸和/或 l-精氨酸生产菌株中合成。在这项研究中，开发了一种结合稀有密码子选择和 pEvolvR 的定向进化策略，以筛选具有高催化效率的 OCD，从而提高 l-精氨酸底盘细胞中 l-脯氨酸的产量。通过 CRISPR 辅助的 DNA 聚合酶产生突变，并通过生长偶联的稀有密码子选择系统进行筛选。从 鉴定出的 OCD 对 l-脯氨酸合成的催化效率提高了 2.85 倍。此外，我们使用 RedLibs 为 和 偶联级联设计和优化了 RBS，以及通过 sRNA 抑制 来适度调节 l-精氨酸过量产生的 l-脯氨酸生物合成。具有最佳性能的 PS6 菌株在摇瓶中达到 15.3 g/L 的 l-脯氨酸，在 5 L 发酵罐中达到 38.4 g/L 的浓度，残留的 l-鸟氨酸和/或 l-精氨酸浓度相对较低。
