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.

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Most researchers proved that adding PMMA in PVDF can reduce the crystal size of β-phase and improve the discharge efficiency. However, we found that PMMA could facilitate the formation of a new crystal variety (γ-2b), which contributes more to the improvement of discharge efficiency.