Abstract
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.
Highlights
Industrial biomanufacturing that converts biomass-derived carbon sources into target chemicals holds promise for addressing global concerns over limited fossil resources and environmental problems
Genome editing tools based on both CRISPR/Cas[931–34] and CRISPR/Cas12a35–38 have been developed for C. glutamicum
CRISPR/Cas9-assisted rational flux-tuning and CRISPRimediated comprehensive transporter discovery were applied to transfer the wild-type C. glutamicum strain to an industrialstrength L-proline producer
Summary
Industrial biomanufacturing that converts biomass-derived carbon sources into target chemicals holds promise for addressing global concerns over limited fossil resources and environmental problems. Microbial strains are considered as the key biocatalysts of biomanufacturing[1] It is still challenging and time-consuming to develop microbial strains that meet the requirements of industrialization and commercialization, such as high production level (titer, yield, and productivity), free of plasmid, inducer, and antibiotic, and stable performance in scale-up fermentation[2,3,4]. Corynebacterium glutamicum is a major workhorse in industrial biomanufacturing[17] It is used for the industrial production of over six million tons of amino acids (such as L-lysine and L-glutamate) per year. This microorganism has been engineered to produce many more compounds ranging from alcohols, organic acids, and plant secondary metabolites[18]. Compared with the fermentative production of amino acids like L-lysine and L-glutamate with a high yield of ~0.70 g product/g glucose[21,22], the L-proline production has a relatively low yield of ~0.20 g/g23 (Supplementary Table 1)
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