Fig. 6: Characterisation of pot marigold C16 hydroxylases.

A Total ion chromatograms of extracts of N. benthamiana leaves transiently co-expressing CoTXSS with CoCYP716A392 or CoCYP716A393. Ca Calendula arvensis (field marigold); Co Calendula officinalis (pot marigold). B Structural model illustrating the predicted position of Ψ-taraxasterol in the active site of CoCYP716A392. Residues selected for mutagenesis are highlighted in grey. Phylogenetic tree of CYP716A and alignment of residues in the active sites; taxa that are predominantly use Ψ-taraxasterol as a substrate are highlighted in orange and those use β-amyrin are highlighted in blue. The grey triangle represents a collapsed clade of 26 characterised CYPs from the CYP716A subfamily that hydroxylate residues of β-amyrin other than C16. C Extracted ion chromatograms and peak area analysis of extracts of N. benthamiana leaves transiently co-expressing CoTXSS with wild type and mutated CoCYP716A392. Control = HMGR + P19. D Quantification of triterpenes produced by transiently expressing CoTXSS with wild type and mutated CoCYP716A392. Error bars indicate the mean and standard error of 6 biological replicates (independent infiltrations). Significant differences in total Ψ-taraxasterol/β-amyrin content compared to wild type CoCYP716A392 (black lowercase letters), and significant differences in taraxasterol/β-amyrin ratio compared to wild type CoCYP716A392 (blue lowercase letters) were analysed using a Kruskal-Wallis test followed by post-hoc Wilcoxon rank sum test with a Benjamini-Hochberg correction (Supplementary Data 7). Samples that do not share the same lower-case letter are significantly different from each other (p < 0.05). P values are provided in Supplementary Data 9.