A Cytochrome P450‐Mediated Intramolecular Carbon–Carbon Ring Closure in the Biosynthesis of Multidrug‐Resistance‐Reversing Lathyrane Diterpenoids

Abstract

The Euphorbiaceae produce a wide variety of bioactive diterpenoids. These include the lathyranes, which have received much interest due to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potential applications in the treatment of pain, HIV and cancer. We report here a gene cluster from Jatropha curcas that contains the genes required to convert geranylgeranyl pyrophosphate into a number of diterpenoids, including the lathyranes jolkinol C and epi‐jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon–carbon ring closure. This requires the activity of two cytochrome P450s that we propose form a 6‐hydroxy‐5,9‐diketocasbene intermediate, which then undergoes an aldol reaction. The discovery of the P450 genes required to convert casbene to lathyranes will allow the scalable heterologous production of these potential anticancer drugs, which can often only be sourced in limited quantities from their native plant.

High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae

“…We have constructed a strain that synthesizes the lathyrane backbone casbene at high titer. We verified the ability of E. lathyris and J. curcas P450s to oxidize casbene, and resolved some discrepancies between the two pathways previously proposed (Luo et al, 2016;King et al, 2016). Finally, we optimized the expression of these P450s and an ADH to generate jolkinol C at high titers producing a strain with all pathway genes stably integrated as a chassis for additional gene discovery.…”

“…Additionally, we decided to test the CYP gene adjacent to the C9OX2 in the published J. curcas jolkinol C biosynthetic gene cluster ( Supplementary Fig. 1) (King et al, 2016). This enzyme, JcC9OX1p, whose activity was not previously reported, showed low-level activity on casbene at the C-9 position when expressed in our casbene-producing yeast strain.…”

“…We examined CYPs known and hypothesized to act on casbene in our high-titer casbene-producing yeast strain JWY509. CYPs from J. curcas and E. lathyris reported to act on C-9 of casbene (hereafter referred to as C9OX CYPs) and ones that act on C-5 and C-6 (hereafter referred to as C5OX CYPs) were cointegrated with a CPR-required for channelling electrons from NADPH to the CYP heme domain-found in the transcriptome of J. curcas, hereafter referred to as JcCPR1 (Luo et al, 2016;King et al, 2016). We used JcCpr1p for both the E. lathyris and J. curcas CYPs because it has been shown that plant P450-CPR interacting domains are highly conserved across plants species, and previous studies have had success using non-cognate CPRs within Euphorbiaceae for functional testing of casbene C9OX and C5OX CYPs (Luo et al, 2016;Jensen and Møller, 2010).…”

“…One of the C5OX CYPs, JcC5OX1p, has been shown to localize to the plastidial membrane rather than the endoplasmic reticulum when heterologously expressed in N. benthamiana (King et al, 2016). To test the expression of this CYP in yeast, we designed a chimeric version of the enzyme by replacing the predicted N-terminus plastidial transit sequence with the beginning of the ER-targeting ElC5OX1p sequence.…”

“…Different groups have debated the involvement of the ADH in the formation of jolkinol C; Luo and colleagues found that, in addition to the CYPs required to oxidized casbene, an Euphorbia lathyris ADH (ElAdh1) was necessary for jolkinol C formation in N. benthamiana and in vitro (Luo et al, 2016). In contrast, King and colleagues never mentioned the use of an ADH for the production of jolkinol C, but saw production of jolkinol C from coexpression of casbene synthase and two Jatropha curcas CYPs (King et al, 2016).…”