Fig. 6: CRISPRi screening and characterization of l-proline exporter in C. glutamicum.
a Workflow of screening l-proline exporter by constructing an arrayed CRISPRi library targeting the potential transporter genes. A pair of oligos were synthesized and annealed to generate dsDNAs harboring a spacer sequence. Totally 397 CRISPRi plasmids were constructed using the dsDNAs and pdCas9gRNA-ccdB. Each CRISPRi plasmid targeted a potential transporter gene of C. glutamicum. The CRISPRi plasmids were individually transformed into an l-proline producing C. glutamicum PRO-CRISPRi expressing a deregulated ProBG149D variant24. The resultant 392 strains were cultivated in 96-deep-well plates for the first round of screening. Strains with significantly reduced l-proline production were selected for a second round of screening by cultivation in 24-deep-well plates. The transporter genes whose repression significantly reduced l-proline production were considered as potential l-proline exporters for characterization. b Volcano plot of differential l-proline production levels caused by CRISPRi repression of transporter genes. The results of the first round of screening are analyzed and shown. Three parallel l-proline production experiments were conducted in 96-deep-well plates for all the 392 strains. Strain PRO-CRISPRi harboring a nontargeting CRISPRi system was used as a control. Gene repressions causing significant increases and decreases in l-proline production are indicate in red and blue dots, respectively (P < 0.05, Student’s two-tailed t-test). Grey dots represent those with non-significant changes in l-proline production level. Twenty-one exporters (blue dots) were selected for a second round of screening. c The second round of screening of the 21 l-proline exporter candidates by cultivation in 24-deep-well plates. Data are presented as mean values +/− SD (n = 3 independent experiments). All Student’s two-tailed t-tests compare the l-proline production levels of strains expressing a gene-targeting CRISPRi system with the control strain expressing a nontargeting CRISPRi system (***P = 0.0007 for Cgl2622, **P = 0.0027 for Cgl1436, ***P = 0.00005 for Cgl1933, ***P = 3 × 10−8 for Cgl2348, ***P = 4 × 10−7 for Cgl1360, ***P = 0.0001 for Cgl1937). d Effects of deletion of cgl2622, cgl1436, and cgl2348 on l-proline production. Strains were cultivated in 24-deep-well plates for l-proline production. Data are presented as mean values +/− SD (n = 3 independent experiments). ***P = 0.00003, Student’s two-tailed t-test. e Effects of deletion, complementation, and overexpression of cgl2622 on growth and extracellular and intracellular accumulation of l-proline. Complementation was conducted by expressing cgl2622 via a plasmid under the control of IPTG-inducible Ptrc in the cgl2622-deleted PRO-CRISPRi strain. Strains were cultivated in shake flasks to obtain enough cells for the measurement of intracellular l-proline. Data are presented as mean values +/− SD (n = 3 independent experiments). Source data underlying Fig. 6b–e are provided as a Source Data file.
