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. 2021 Nov 25;12:6916. doi: 10.1038/s41467-021-27290-9

Fig. 6. Production enhancement of mevalonate via SpdNG-LWQT mediated CRISPR interference (CRISPRi).

Fig. 6

a The metabolic pathway of mevalonate from glucose. The pathway consists of three enzymes: thiolase (thl) from C. difficile, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) synthase (mvaS) from L. casei, and HMG-CoA reductase (mvaA) from R. pomeroyi. Three competing essential genes (shown in red) from E. coli are: gltA encoding citrate synthase, accA encoding the subunit α of acetyl-CoA carboxyltransferase, and fabD encoding malonyl-CoA-acyl carrier protein transacylase. b The influence of CRISPRi of gltA, accA, or fabD on cell fitness of E. coli BW25113(F′)::MVA. The dCas9s were expressed on pCS27, and sgRNAs were expressed on pZE12-luc. All sgRNAs were targeting coding sequence on nontemplate DNA strand. SpdCas9wt was coexpressed with sgRNAs targeting 5′-NGG-3′ PAM while SpdRY and SpdNG-LWQT were coexpressed with sgRNAs targeting 5′-CAT-3′ PAM at the ATG start codons. Cells were plated on Luria-Bertani (LB) plates containing 0.5 mM IPTG and appropriate antibiotics. Representative image from two independent repeats. c The effects of SpdCas9wt or its variants mediated CRISPRi on mevalonate production in E. coli host BW25113(F′)::MVA. The host strain with empty pCS27 and pZE12-luc was applied as a negative control (NC). All shake flasks were performed in M9 minimal medium containing 20 g/L glucose and 5 g/L yeast extract, and samples were taken at 48 h. Data indicated the mean ± standard deviation (n = 3 independent biological replicates). Source data are provided as a Source Data file.