The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis

Abstract

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids.

Figure 1. C. asiatica sapogenins and in vivo functional analysis of C. asiatica CYP716s.

(a) Examples of oleanane- and ursane-type sapogenins found in C. asiatica. (b) Overlay of GC–MS total ion current chromatograms showing accumulation of standard compounds and triterpenoids produced in yeast strains expressing single C. asiatica CYP716s. (c) Overlay of GC–MS total ion current chromatograms showing accumulation of standard compounds and triterpenoids produced in yeast strains expressing C. asiatica CYP716s in combination with CYP716A83. Annotated triterpenoid peaks are indicated with numbers: (1) β-amyrin, (2) erythrodiol, (3) oleanolic acid, (4) oleanolic aldehyde (which co-elutes with a nonspecific peak), (5) 6β-hydroxy β-amyrin, (6) maslinic acid, (7) putative incompletely derivatized 6β-hydroxy oleanolic acid and (8) 6β-hydroxy maslinic acid. (d) Proposed biosynthetic pathway of sapogenins in C. asiatica. Arrows of higher weight reflect the preferred order of reactions in the biosynthetic pathway, as supported by the semi-quantitative analysis shown in Table 1.

Figure 2. P. grandiflorus sapogenins and in vivo functional analysis of P. grandiflorus CYP716s.

(a) Examples of P. grandiflorus sapogenins. (b) Overlay of GC–MS total ion current chromatograms (TICs) showing accumulation of standard compounds and triterpenoids produced in yeast strains expressing single P. grandiflorus CYP716s. Annotated triterpenoid peaks are indicated with numbers: (1) β-amyrin, (2) 16β-hydroxy β-amyrin, (3) putative hydroxy β-amyrin, (4) putative hydroxy β-amyrin, (5) erythrodiol, (6) oleanolic acid and (7) 12,13α-epoxy β-amyrin. (c) Overlay of GC–MS TIC showing accumulation of standard compounds and triterpenoids produced in yeast strains expressing P. grandiflorus CYP716s in combination with CYP716A140. Annotated triterpenoid peaks are indicated with numbers: (1) β-amyrin, (2) 16β-hydroxy β-amyrin, (3) putative hydroxy β-amyrin, (4) erythrodiol, (5) unknown triterpenoid, (6) oleanolic acid, (7) putative 16β-hydroxy oleanolic acid (co-elutes with an impurity in common for all samples), (8) unknown triterpenoid and (9) 12,13α-epoxy β-amyrin. (d) LC-APCI-FT-ICR-MS analysis of the yeast strains expressing GgBAS with CYP716A140 and CYP716S5 (1) and GgBAS with CYP716A140 only (2), and authentic standards of 12α-hydroxy β-amyrin-13,28β-lactone (3) and 12,13α-epoxy oleanolic acid (4). Chromatograms represent a mass range of m/z 473.3622–473.3625 Da. (e) Comparison of the MS (473.36→427) fragmentation of the peak at retention time 12.61 min compared with the MS fragmentation of an authentic 12α-hydroxy β-amyrin-13,28β-lactone standard. (f) The proposed biosynthetic pathway of triterpene saponins in P. grandiflorus. Arrows of higher weight reflect the preferred order of reactions in the biosynthetic pathway, as supported by the semi-quantitative analysis shown in Table 1.

Figure 3. Sapogenins from the Aquilegia genus and new triterpenoid compounds produced by A. coerulea CYP716s.

(a) Examples of sapogenins from A. vulgaris including three heterocyclic cycloartanes. (b) Overlay of GC–MS total ion current chromatograms showing accumulation of triterpenoids produced in yeast strains expressing SlCAS together with CYP716A113 (red), SlCAS only (green), or CYP716A113 only (blue), compared with a control (empty) strain (black). (c) Overlay of GC–MS total ion current chromatograms showing accumulation of standard compounds and triterpenoids produced in yeast strains expressing single A. coerulea CYP716s in combination with GgBAS. Candidate-specific peaks are indicated with numbers (1) β-amyrin, (2) 16β-hydroxy β-amyrin, (3) erythrodiol and (4) oleanolic acid.

Figure 4. Maximum likelihood phylogenetic analysis of the CYP716 family.

(a) CYP716 members form distinct subgroups and can be divided in eudicot, angiosperm and ancient CYP716s. (b) CYP716 subgroups distributed per plant order, showing the point of emergence of different subgroups and suggesting diversification of the CYP716 family in early eudicots. Plant orders are depicted according to the APG III system (angiosperms) and the tree of life project (the rest, https://tree.opentreeoflife.org/) taxonomy.