Production of Bioactive Diterpenoids in the Euphorbiaceae Depends on Evolutionarily Conserved Gene Clusters    

Abstract

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

“…The spectral data of 9 (SI Appendix, Fig. S1B) matches the previously reported 5-ketocasbene (18,26). Corroborating its ability to catalyze dehydrogenation of the 5-hydroxyl group, ADH1 also dehydrogenated 4 yielding the casbene dione derivative 10 (m/z 301.2157, [M+H] + ) ( Fig.…”

“…Recently, a Euphorbiaceae-specific expansion of P450s in the CYP71 clan was reported (16). Although the founding members of the subfamily were shown to catalyze epoxidation of fatty acids in Euphorbia lagascae Spreng (17), several P450s of the CYP726 subfamily in castor bean had activity toward C5 of casbene, which is a characteristic of the casbene-type diterpenoids present in castor bean and related species (18). In addition to P450s, members of the short-chain dehydrogenase∕reductase superfamily are also involved in terpenoid metabolism in many plants.…”

Investigation of terpene diversification across multiple sequenced plant genomes

“…Notably, the production of 5-keto-6-hydroxy-casbene is consistent with the presence of matching oxy groups in a previously isolated castor bean diterpenoid (53). Production of 5-keto-casbene by these CYPs and further investigation of this terpenoid biosynthetic gene cluster also has just been reported elsewhere (54). The ability of CYP71D subfamily members to operate in diterpenoid biosynthesis is consistent with recent work demonstrating functional pairing of CYP71D51 with a diterpene TPS (albeit from the TPS-e/f subfamily) from a tomato (Solanum lycopersicum) terpenoid biosynthetic gene cluster (55).…”

Comparative transcriptomics unravel biochemical specialization of leaf tissues of Stevia (Stevia rebaudiana) for diterpenoid production

“…Recently, functional diterpenoids with biological activities for treatment of a wide range of medical conditions have been reported from diverse medicinal plants (Guo et al, 2013;Zerbe et al, 2013;King et al, 2014;Pateraki et al, 2014). Although manoyl oxide itself is known to be an anticancer compound, it is considered as the molecular core for a large series of bioactive derivatives (Pateraki et al, 2014).…”