Fig. 6.

Production of forskolin and intermediates in yeast strains expressing native CYP76AH15 or the A99I  mutant  variant. a GC–MS chromatograms of the control strain MO (−) expressing CfPOR only, FORSK AH15 and FORSK A99I yeast strains expressing either native CYP76AH15 or A99I, respectively, together with the rest of the forskolin biosynthetic genes (CYP76AH11, CYP76AH16, CfACT1-8 and CfPOR). FORSK A99I showed increased accumulation of 2, 3 and the unidentified intermediates b and c, whereas production of 1, 8 and a was lowered compared to FORSK AH15. b Relative extracted ion chromatograms corresponding to [M+H]⁺ of forskolin at m/z 411.2381 ± 0.05 by LC–MS analysis. The analysis showed lowered production of forskolin in the strain FORSK A99I compared to FORSK AH15. c Identified products on GC–MS and LC–MS. IS internal standard (10 mg/L 1-eicosene), MO 13R-manoyl oxide, TIC total ion chromatogram, EIC extracted ion chromatograms