Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones

Abstract

Summary Cytochromes P450 (CYPs) play 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, that act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen.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 demonstrate 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 in diterpenoids metabolic engineering, and a continuing trend of CYPs promiscuity generating complex networks in terpenoid biosynthesis.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

“…Thus, it is likely that the ability of CYP76AHs to catalyze 11-oxo-13 R -manoyl oxide has evolved convergently in these plants (Figure 11).

10.7554/eLife.23001.023Figure 11.Phylogeny of known full-length CYP76AHs.The enzymes used are listed below with their accession numbers or source of publication: Cf CYP76AH15, KT382358; Cf CYP76AH17, KT382360; Cf CYP76AH8, KT382348; Cf CYP76AH11, KT382349; Cf CYP76AH16, KT382359; Cf CYP76AH9, KT382347; Cf CYP76AH10, KT382346; Cf CYP71D381, KT382342; Ro FS1, AJQ30187 (Božić et al, 2015); Sm CYP76AH3, KR140168 (Guo et al, 2016); Ro FS2, AJQ30188 (Božić et al, 2015); Sf FS, AJQ30186 (Božić et al, 2015); Ro CYP76AH4, (Zi and Peters, 2013); Ro CYP76AH5v1, (Zi and Peters, 2013); Ro CYP76AH5v2, (Zi and Peters, 2013); Ro CYP76AH6, (Zi and Peters, 2013); Ro CYP76AH7, (Zi and Peters, 2013); Sm CYP76AH1, AGN04215 (Guo et al, 2013); Sp CYP76AH24, ALM25796 (Ignea et al, 2016a). Coleus forskohlii enzymes are indicated by a solid black triangle.

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Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza

“…Subsequently, three CYP76 subfamily genes, CYP76AH1 13, CYP76AH3 20, and CYP76AK1 20, transform miltiradiene to seven oxygenated diterpenoids (Fig. 1).…”

RNA interference targeting CYP76AH1 in hairy roots of Salvia miltiorrhiza reveals its key role in the biosynthetic pathway of tanshinones

“…Down-regulation of SmCPS1 by RNAi resulted in substantial reduction of tanshinones, and metabolomics analysis revealed 21 potentially affected intermediates, indicating a complex network for tanshinone metabolism defined by certain key biosynthetic steps [10] (Fig.1). RNAi results indicated that CYP76AK1 plays a key role in the production of tanshinones [12]. On the other hand, the reduction of the contents of tanshinones was not statistically M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 significant in CYP76AH3 RNAi lines, implying that the reactions catalyzed by CYP76AH3 most likely is not rate limiting for tanshinone biosynthesis.…”

“…Based on co-expression analysis, two new CYPs, CYP76AH3 and CYP76AK1, were found and functionally characterized. They might work sequentially to catalyze the conversion of ferruginol into the metabolic intermediate of tanshinones [12]. RNASeq data of YE & Ag + -induced hairy roots of Danshen led to the detection of a total of 125 expressed CYPs belonging to 8 clans and 31 families, of which 40 were upregulated during the accumulation of tanshinones [13].…”