Extended Data Fig. 2. Characterization of pathway intermediates from overexpression of cytOsCPS4 + cytOsKSL4 + CYP99A3 via MEV engineering.

GC-MS total ion chromatogram (TIC) and MS spectra of products from overexpression of HMGR, cytGGPPS, cytOsCPS4, cytOsKSL4, and CYP99A3 in N. benthamiana. Representative TICs are shown for the experimental sample and controls. Three products with m/z corresponding to diterpene derived scaffolds were observed only upon expression of pathway genes. The major product identified corresponded to 2 and the two minor products corresponded to syn-pimaradien-19-al and syn-pimaradien-19-ol²⁹. For m/z corresponding to syn-copalyl alcohol see Supplementary Figure 9. Putative ion structures for 2 were assigned as reference. Putative structures are preliminary and based on predicted chemical formulas combined with analysis of the fragment ions observed in the MS spectra. An endogenous metabolite co-eluting with 2 was identified (* in the TIC). (b) MS spectra of endogenous metabolite. (c) GC-MS extracted ion chromatogram (EIC) using m/z = 241.2, corresponding to an abundant fragment found in the MS of 2, confirmed that 2 is only present upon expression of the requisite diTPSs and CYP99A3.