Fig. 6: Synergistic multi-target engineering for improvement of daptomycin production.

a Schematic of rational multi-target combination based on pairwise synergetic screening. b Interactions of 17 repressors involved in daptomycin synthesis. The synergy coefficient q value represents the ratio of the actual measured value of indigoidine to the expected value (assuming that the effects of the two genes on yield are independent events). When q < 0.85, indicates antagonism; 0.85 ≤ q < 1.15, indicates additivity; and q ≥ 1.15, indicates synergism. c Interactions network between targets based on synergy coefficient. Red dots indicate four targets that have a synergistic relationship with each other. Other targets are indicated by blue dots. d Daptomycin titer of WT (column 1), WT^- (column 2), and CRISPRi array-mediated strains. “+“ indicates the use of CRISPRi to inhibit the corresponding gene, while “−“ indicates no inhibition. Two-tailed Student’s t test (n.s. not significant (p >  0.05); **p  <  0.01; ***p  <  0.001). [p = 0.900351215, 0.000191464, 0.001315463, 0.000379374, 0.000513844, 0.001715257, 0.003379535, 7.1047E−05, 5.99658E−05, 0.000115786, 0.000141011, 1.92672E−05 (left to right)]. e Growth and daptomycin production curves of WT and Sr11 in a 7.5L fermenter. Biomass is quantified as dry cell weight. f Heat map of dptE operon transcription levels in WT^idt3i, WT^idt5i, WT^idt9i, WT^idt15i, and Sr11. g Transcription profile of daptomycin BGC in WT^idt3i, WT^idt5i, WT^idt9i, WT^idt15i, and Sr11 grown in fermentation medium. Here we selected dptE gene for quantification using RT-qPCR. Data in (d, e, g) are shown as the mean ± SD (n = 3 biological replicates).