期刊
NATURE COMMUNICATIONS
卷 13, 期 1, 页码 -出版社
NATURE PORTFOLIO
DOI: 10.1038/s41467-022-28501-7
关键词
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资金
- National Key R&D Program of China [2018YFA0901400]
- National Natural Science Foundation of China [31800038, 31870044, 21908239]
- Key R&D Program of Shandong Province [2021CXGC010602]
- International Partnership Program of Chinese Academy of Sciences [153D31KYSB20170121]
- Youth Innovation Promotion Association of Chinese Academy of Sciences [2021177]
- Tianjin Project+Team Key Training Program [XC202038]
- Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project [TSBICIP-KJGG-005]
In this study, the authors use CRISPR technology and CRISPRi screening to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with various applications. By optimizing the biosynthetic enzyme and regulating the carbon flux, they achieved efficient production of L-proline.
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 gamma-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. Corynebacterium glutamicum is a major workhorse in industrial biomanufacturing of amino acids. Here, the authors employ CRISPR-assisted rational flux-tuning and CRISPRi screening of a L-proline exporter to covert a wild-type C. glutamicum to a hyperproducer of L-proline.
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