CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts

Abstract

Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.

Keywords: gene discovery; metabolic engineering; phytoterpenoids biosynthesis; synthetic pathway.

Fig. 1.

Partial pathways for tanshinones biosynthesis and structures for some representative tanshinones. Solid arrows indicate the established relationships, and dashed arrows indicate hypothetical relationships.

Fig. 2.

Results of stable-isotope labeling experiments. Electrical ionization mass spectra of miltiradiene (A and B), ferruginol (C and D), and cryptotanshinone (E) from dashen hairy roots fed with unlabeled (A and C) or ¹³C-labeled miltiradiene (B, D, and E).

Fig. 3.

Results of in vitro turnover of miltiradiene by CYP76AH1. (A) GC–MS chromatogram of extracts from the reaction containing CYP76AH1 microsomes with NADPH (Upper trace) and control (Lower trace). (B) Mass spectra of the reaction product compared with that of authentic ferruginol.

Fig. 4.

Kinetic profile of CYP76AH1. Experimental details are included in Materials and Methods.

Fig. 5.

Correlation of gene expression of CYP76AH1 and ferruginol accumulation. (A) Accumulation of ferruginol in hairy roots responding to Ag⁺ induction. (B) Real-time PCR analysis of CYP76AH1 mRNA level in hairy roots of danshen after exposure to Ag⁺.

Fig. 6.

Results of the assembled ferruginol production pathways. Yeast strains were cultivated in a ZWY-1102 shaking incubator (Shanghai Zhicheng) with 100 mL YPD media at 30 °C, 200 rpm for 48 h. All data represent the averages ± SDs of three independent clones.