Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones

Abstract

Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis.

Keywords: Salvia miltiorrhiza Bunge; cytochrome P450 (CYP) monooxygenases; diterpenoid biosynthesis; enzymatic promiscuity; metabolic pathways; synthetic biology.

Fig. 1

Tanshinones and partial biosynthetic pathway in S. miltiorrhiza. (a) Representative tanshinones found in S. miltiorrhiza. (b) Proposed partial biosynthetic pathway of tanshinones. The red arrow indicates the oxidation reaction catalyzed by the CYP76AH3 enzyme. The blue arrow indicates the oxidation reaction catalyzed by the CYP76AK1 enzyme.

Fig. 2

LC-MS analysis results of reaction mixtures of 5 catalyzed by yeast microsomes containing CYPs from S. miltiorrhiza. (a) The extracted ion current (EIC) chromatogram of 11-hydroxy ferruginol (6). (b) The EIC of sugiol (7). (c) The EIC of 11-hydroxy sugiol (8) and 10-hydroxymethyl tetrahydromiltirone (10). (d) The EICs of 11,20-dihydroxy ferruginol (9). (e) The EIC of 11,20-dihydroxy sugiol (11). The resulting daughter ion mass spectra are shown in Fig. S2, S5, S6, and S8 for compound 6, 9, 10, and 11, respectively. The green, blue and red lines represent the yeast harboring the plasmid pESC-His, pESC-His::CYP76AK1 and pESC-His::CYP76AH3/CYP76AK1, respectively.

Fig. 3

The relationship between the expression levels of CYP76AH3 and CYP76AK1 and the contents of different terpenoids products. (a) Expression levels of CYP76AH3 and CYP76AK1 in root, stem and leaf of in S. miltiorrhiza. (b) Expression levels of CYP76AH3 and CYP76AK1 in RNAi down-regulated in S. miltiorrhiza hairy roots. (c) Contents of tanshinones 1, 2 and 3 in RNAi down-regulated in S. miltiorrhiza hairy roots. (d) Contents of oxygenated terpenoids intermediates 5 – 11 in RNAi down-regulated in S. miltiorrhiza hairy roots. Expression levels were normalized using β-actin as an internal standard. The error bars represent the standard error of means from three independent replications for tissue expression analysis and from 5 to 6 lines for RNAi down-regulated hairy roots. *P < 0.05, ** P< 0.01 and *** P< 0.001 were determined by an unpaired t test using GraphPad Prism 6.

Fig. 4

Homology modeling and docking analysis of CYP76AK1 from S. miltiorrhiza. (a) Homology modeling of CYP76AK1. Docking poses of compound 5 (b), 6 (c), 7 (d), 8 (e). Compound structure is depicted as stick with carbons colored pink and oxygens red. Heme is depicted as stick with carbons colored yellow and iron blue. Distance between C20 and heme iron is indicated by dashed line with the length indicated in Å.

Fig. 5

Engineered yeasts for the production of oxygenated terpenoids intermediates. YJ35 was constructed previously (Guo et al., 2013). The YJ51 (a) and YJ61 (b) were constructed by transforming pESC-His::CYP76AH3 and pESC-His::CYP76AH3/CYP76AK1 into YJ35, respectively. YJ62 (c) was constructed by replacing the GAL1 and GAL10 promoter in pESC-His::CYP76AH3/CYP76AK1 with the constitutive promoter TEF1 and PGK1 and transforming into YJ35. The pie chart and its area represent the percentages of ferruginol derivatives (compounds 6, 7, 8, 9, 10, and 11) and the accumulation of diterpenoids produced in the YJ51 (a), YJ61 (b) and YJ62 (c) after 72 h of shake-flask fermentation at 250 rpm in YNB with galactose for YJ51 and YJ61, and YNB with glucose for YJ62. The data represent the mean value of three independent replications.