Fig. 3.

Diterpenoid product profiles from the CYP76AH15 mutant variants, in comparison to the native enzyme when expressed in yeast cells producing 13R-manoyl oxide (MO). a GC–MS chromatograms of the control strain MO (−) with CfPOR only, and strains from CYP76AH15 mutant-variants at the SRS1, SRS3 and SRS5 regions showing enhanced production of 11-oxo-13R-manoyl oxide (2). b Relative yields of the diterpenes 1 (red bars), 2 (green bars), 3 (light purple bars) and unknown 7 (light pink bars). The yeast strains expressing the SRS1, SRS3 and SRS5 variants of CYP76AH15 produced significantly higher levels of 2 and 3 and lower levels of 7, in comparison to the native enzyme. c Chemical structures of the identified products formed by yeast. d GC–MS chromatograms of the control strain MO (−) with CfPOR only, native CYP76AH15, SRS5 and SRS6 mutated variants, and 11β-hydroxy-MO as internal standard (STD). e MS spectrum of (7) with parent ion m/z 320. IS internal standard (1 mg/L 1-eicosene), MO 13R-manoyl oxide, TIC total ion chromatogram. Error bars indicate standard deviation from three biological replicates