Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308, China.
Nat Commun. 2022 Feb 16;13(1):891. doi: 10.1038/s41467-022-28501-7.
Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with medicine, feed, and food applications. To facilitate L-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards L-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an L-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces L-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.
开发高产菌株对于生物制造生化产品和生物燃料非常重要，但需要大量的工程细胞代谢和发现功能组件的工作。在此，我们优化并使用 CRISPR 辅助编辑和 CRISPRi 筛选方法，将野生型谷氨酸棒杆菌转化为 L-脯氨酸的高产菌株，L-脯氨酸在医药、饲料和食品方面具有应用价值。为了促进 L-脯氨酸的生产，使用 CRISPR 辅助的单链 DNA 重组酶筛选反馈调节的关键生物合成酶 γ-谷氨酰激酶的变体。为了增加碳通量流向 L-脯氨酸生物合成，使用定制的启动子文库对通过计算机分析预测的通量控制基因进行微调。最后，构建了一个针对所有 397 种转运蛋白的靶向 CRISPRi 文库，以发现 L-脯氨酸外排蛋白 Cgl2622。最终的无质粒、抗生素和诱导剂的菌株以 142.4 g/L、2.90 g/L/h 和 0.31 g/g 的水平生产 L-脯氨酸。CRISPR 辅助的菌株开发策略可用于工程化工业强度菌株以实现高效的生物制造。
