Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis

Abstract

Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.

Keywords: biocatalysis; biosynthesis; combinatorial biosynthesis; cyclizations; terpenes.

Scheme 1

The modular nature of diterpene biosynthesis. Overview of class II and class I diTPSs and their products. The decalin core is shown in bold. Zm=Zea mays; Cf=Coleus forskohlii; Ss=Salvia sclarea; Os=Oryza sativa; Mv=Marrubium vulgare, Tw=Tripterygium wilfordii.

Figure 1

The diterpene chemoscape. A) Levels of diterpenes accumulating in the N. benthamiana/Agrobacterium system (n=3). B) GC–MS chromatograms of extracts from the N. benthamiana control, CfTPS1/SsSCS expressed in N. benthamiana or in S. cerevisiae, the in vitro assay, and an authentic standard of (9S,1(0S‐manool (23 a). Total ion current (TIC) and extracted‐ion chromatogram (EIC) levels are not to scale. C) 2D representation of diterpenes detected in N. benthamiana expressing either a single diTPS or combinations of diTPSs from the two classes (y axis). Diterpenes are sorted on the x axis by their Kovats retention indices. Cf=C. forskohlii; Ss=S. sclarea; Mv=M. vulgare; Ep=Euphorbia peplus; Os=O. sativa; Tw=T. wilfordii; Zm=Z. mays. Diterpenes in samples expressing solely class II diTPSs are likely the result of endogenous phosphatase activity and non‐specific rearrangement (Figure S7).6a, 7

Figure 2

Combinatorial wheel. Formation of diterpenes by natural and engineered diTPS combinations. Bonds in a specified stereochemical configuration formed through stereoselective controlled synthesis are highlighted in red. Bonds in a specified stereochemical configuration for which there is no biosynthetic access to the other configuration are shown in black. The a and b suffixes indicate (+) and ent configurations, respectively, as dictated by the class II diTPS. Diterpenes with unknown structure are shaded in grey. * indicates clerodane diterpenes.