FIG 3.

Gene knockdown supports the involvement of bal BGC in balmoralmycin production. (A) The balA1_KD, balA7_KD, and balA8_KD mutants generated using the CRISPR/dCas9 method exhibited different colony morphology on PM3 agar plates. Control, P01 with an empty CRISPR/dCas9 vector; WT, P01 wild-type. The dark red and orange colors exhibited by the WT and the control plates were not observed for the three mutant strains, likely due to the reduced production of the colored compounds 1 − 8. (B) High-performance liquid chromatography (HPLC) analysis of the organic extract of the strains suggested that the CRISPR/dCas9-assisted knockdown of balA1, A7, or A8 drastically decreased the production of balmoralmycin (compound 1) and the shunt products (compounds 2−8). The HPLC detector was set at 260 nm wavelength.