Tripterygium wilfordii cytochrome P450s catalyze the methyl shift and epoxidations in the biosynthesis of triptonide

Abstract

The diterpenoid triepoxides triptolide and triptonide from Tripterygium wilfordii (thunder god wine) exhibit unique bioactivities with potential uses in disease treatment and as a non-hormonal male contraceptives. Here, we show that cytochrome P450s (CYPs) from the CYP71BE subfamily catalyze an unprecedented 18(4→3) methyl shift required for biosynthesis of the abeo-abietane core structure present in diterpenoid triepoxides and in several other plant diterpenoids. In combination with two CYPs of the CYP82D subfamily, four CYPs from T. wilfordii are shown to constitute the minimal set of biosynthetic genes that enables triptonide biosynthesis using Nicotiana benthamiana and Saccharomyces cerevisiae as heterologous hosts. In addition, co-expression of a specific T. wilfordii cytochrome b₅ (Twcytb₅-A) increases triptonide output more than 9-fold in S. cerevisiae and affords isolation and structure elucidation by NMR spectroscopic analyses of 18 diterpenoids, providing insights into the biosynthesis of diterpenoid triepoxides. Our findings pave the way for diterpenoid triepoxide production via fermentation.

Fig. 1. 18(4→3) abeo-abietane methyl shift.

a Hypothesized scheme for the rearrangement of methyl groups on the A-ring of labdane type diterpenoids and examples of diterpenoids with the 18(4→3) abeo-abietane core structure. Examples of labdane type diterpenoids from the Lamiaceae species Coleus forskohlii and the Celastracae species T. wilfordii with an abeo-abietane core structure are shown. The rings of the tricyclic core structure are denoted A, B, and C. b Maximum-likelihood phylogenetic analysis of selected CYP genes from the 71 clan, including plant CYPs from the CYP82 and CYP71 subfamily, and NtCYP51 as the root. Shown CYPs are involved in either phenylpropanoid (purple) or terpenoid (grey) biosynthesis (Supplementary Table 22, Supplementary Fig. 1). TwCYP genes functionally characterized in this work highlighted in bold. Nodes marked * are supported by bootstrap values >85%. c Schematic overview of the genome location of the Tw genes used for heterologous biosynthesis of triptonide in the T. wilfordii genome. Genes marked with * are found as tandem repeats with each copy having on average nucleotide sequence identity of 88.7% to the cDNA sequence of each of the genes used in this paper (Supplementary Table 23). TwCYP71BE84 and TwCYP71BE86 homologs are each found in tandem repeats in two different loci of the T. wilfordii genome.

Fig. 2. Biosynthesis of triptonide from miltiradiene.

a Heterologously expressed genes constituting the minimal set of biosynthetic components required for heterologous triptonide biosynthesis. b quantity (bars) of putative key intermediates in the biosynthetic pathway of triptonide (right), when established in vivo via heterologous gene expression in N. benthamiana. c, quantification of intermediates from engineered S. cerevisiae (strains listed in Supplementary Table 28). The combination of genes co-expressed with the miltiradiene biosynthetic genes is indicated by black dots. Quantification was based on peak areas of signature m/z values for the putative intermediates, normalized to the peak area of signature m/z values for the internal standard (IS) used in the GC-MS (compounds 3–5) and LC-qTOF-MS (compounds 6–8 & 2). Signature m/z values are denoted in the header of each bar diagram. Bars represent the average of n = 3 [N. benthamiana] or n = 4 [S. cerevisiae] biological replicates, with error bars representing standard error of the mean (SEM). Values from each replicate is marked by black diamond squares. White- and grey fill color of the bars distinguishes compound quantity in relation to no expression (grey) or co-expression (white) of Twcytb₅-A, respectively. Mass tolerance was ±0.1 m/z for GC-MS and ±0.005 m/z for LC-qTOF-MS for signature m/z values. Source data are provided as a Source Data file.

Fig. 3. Accumulation in fed-batch fermentation.

The accumulation of 8 (dotted black) and 2 (solid black) from NVJ8.5 (Supplementary Table 28) grown in a fed-batch fermentation. Density of the S. cerevisiae culture was based on the optical density of daily culture samples.

Fig. 4. Proposed biosynthetic pathway of triptonide (2) from 14-hydroxy-dehydroabietadiene (5) in S. cerevisiae.

a The biosynthetic pathway from 5 to 2 illustrating the formation of oxygenated miltiradiene derived intermediates catalyzed by the action of TwCYP82’s (blue arrows) and TwCYP71BE’s (red arrows) on the A- and C-ring of the abietane diterpene backbone, respectively. Underlined compounds or diastereomers hereof have previously been identified in plant extracts from Tripterygium species (Supplementary Table 1). b a hypothetical Wagner-Meerwein rearrangement reaction (W.-M.) to account for the methyl shift of C-18 to C-3 in the abietane carbon backbone. Cpd I and Cpd II represents states of the heme in the CYP catalytic cycle. Red line at heme bound hydroxyl symbolizes inhibition of oxygen rebound facilitating e^- transfer as proposed by for CYP mediated carbocation formation. Compound 13 and oxygenated compounds hereof including 14, 17, and 18, would be products caused by the failure to inhibit oxygen rebound and additional oxygenation likely to be catalyzed by the TwCYPs heterolgously expressed in S. cerevisiae. Alternatively, the C18(4→3) could be mediated via a mechanism relying on the radical formed by the Compound II (CpdII) state of the involved CYP enzyme (Supplementary Fig. 7).