Fig. 5
Heterologous expression of the grh BGC from Streptomyces sp. LS-1 in the chassis strain S. coelicolor A3(2)-2023. (A) An illustration of the integration of the grh BGC into the chassis strain S. coelicolor A3(2)-2023. Firstly, the Streptomyces sp. LS-1 genome was analyzed by antiSMASH, and the 90 kb BGC grh was cloned into a pBAC vector by ExoCET. Secondly, Vika-based RMCE cassettes or the oriT-phiC31-apra cassette was inserted into pBAC-cm-grh by Red recombination in E. coli GB05-recET-tra-αβγ. Finally, BGC grh was integrated into the chromosome of the chassis strain and expressed. (B) Base peak chromatogram (BPC) analysis of fermentation extracts from S. coelicolor A3(2)-2023-grh-attBphiC31 (red) and S. coelicolor A3(2)-grh-attBphiC31 (blue): compound 1a/1b (BPC 919.22 + all MS), compound 2 (BPC 475.09 + all MS), compound 3 (BPC 6 46.11 + all MS), compound 4 (BPC 511.08 + all MS), and compound 5 (BPC 527.07 + all MS). (C) Production of compound 2 at four time-points in S. coelicolor A3(2)-grh-attBphiC31 (A3(2)-attBphiC31), S. coelicolor A3(2)-2023-grh-attBphiC31 (A3(2)-2023-attBphiC31), and S. coelicolor A3(2)-2023-vox-grh-vox2261 (A3(2)-2023-vox-vox2261). (D) Chemical structure of compound 3. Error bars indicate SD; n = 3; ns, p > 0.05; ***p < 0.001; ****p < 0.0001