Fig. 3: Test of CRISPRi-based reporter system for screening in S. roseosporus.

a Phenotypes of WT, ∆bldD (bldD deletion mutant), WT^bldDi (bldD-inhibited mutant), and the control strain WT^- (WT with pSETdCas9Rg-2). These strains were grown on DA1 plates at 28 °C for 5 days. b Transcription levels of bldD and genes of daptomycin BGC in WT, WT^-, WT^bldDi, and ∆bldD. Samples were collected after 6 days growth on DA1 plate. [p = 0.253602325, 1.99842E−05, 5.07332E−07, 0.338845868, 2.80015E−06, 7.24141E−07, 0.926512747, 0.00016914, 4.31204E−05, 0.818549401, 4.86554E−07, 1.11713E−07, 0.218100115, 2.23403E−06, 5.25857E−07, 0.085602875, 0.00021213, 9.19636E−06 (left to right)]. c Daptomycin titer of WT, WT^-, WT^bldDi, and ∆bldD grown in fermentation medium for 10 days. [p = 0.621013707, 3.34048E−05, 2.02346E−05 (left to right)]. d Schematic for evaluating the compatibility between CRISPRi and our developed reporter system through introducing a single target or mini-librariy. e Indigoidine level in WT^-, Srdi, Srdi^- (Srdi with plasmid pSETdCas9Rg-2), and Srdi^wblAi (Srdi with inhibited wblA). [p = 4.46815E−06, 3.68173E−06, 7.32406E−06, 0.988813111, 0.000230028 (left to right)]. f Daptomycin titer in WT, WT^-, WT^wblAi, Srdi, Srdi^-, and Srdi^wblAi grown in fermentation medium for 10 days. [p = 0.703966652, 0.00359405, 0.906149569, 0.000788765 (left to right)]. g Indigoidine levels of transformants with the mini-library. Specifically, “−” denotes the control strain Srdi^-; tetR1 through tetR11 represents strains with CRISPRi-inhibited tetR1 through tetR11, respectively; wblA indicates Srdi^wblAi. Here the indigoidine levels of 100 transformants covering all the 11 targets were determined and showed in the violin plots. h Correlation between daptomycin titer and indigoidine level in Srdi derivatives Srdi^-, Srdi^tetR1i through Srdi^tetR11i, and Srdi^wblAi. i Transcription levels of daptomycin BGC genes and idgS in Srdi^-, Srdi^tetR1i, Srdi^tetR6i, and Srdi^tetR7i grown in seed medium for 2 days. [p = 0.078571011, 3.84465E−05, 0.000447016, 0.271714823, 7.81638E−05, 0.00051151, 0.814630767, 0.000153486, 0.000297848, 0.158558972, 0.000137847, 0.001065091, 0.195323921, 0.000152317, 1.79512E−05, 0.682506353, 9.25055E−05, 0.000782728, 0.377330586, 0.000588997, 0.001023336 (left to right)]. j Daptomycin titer of WT, ∆tetR6 (tetR6 deletion mutant), and ∆tetR7 (tetR7 deletion mutant). [p = 0.005056642, 0.019752034 (left to right)]. Data in (b, c, e, f, h, i, j) are shown as the mean ± SD (n = 3 biological replicates). Two-tailed Student’s t test was used in (b, c, e, f, i, j) to analyze the statistical significance (n.s., not significant (p >  0.05); *p  <  0.05; **p  <  0.01; ***p  <  0.001; ****p  <  0.0001).