Characterization of the casbene synthase homolog from Jatropha (<i>Jatropha curcas</i> L.)

Abstract

Abstracte high oil content of Jatropha (Jatropha curcas L.) seeds makes Jatropha an attractive resource for the production of sustainable bioenergy. However, the Jatropha seed kernels also contain antinutrients and various toxins that persist in the oil and seed cakes and pose a safety risk. Since phorbol esters (PEs) are the major contributor to toxicity, a better understanding of PE biosynthesis is expected to elucidate an e ective strategy for the utilization of Jatropha plants. In this study, a Jatropha curcas casbene synthase homolog (JcCSH) with high sequence similarity to casbene synthases (CSs) from Ricinus communis, Euphorbia esula, and Sapium sebiferum was cloned from Jatropha leaf tissue. CS has been hypothesized to catalyze the rst step of phorbol biosynthesis. JcCSH encodes a protein that contains a chloroplast transit peptide and a DDXXD motif that is conserved among known terpene cyclases. JcCSH was expressed in seedlings, mature leaves, and the esh of developing fruits, but not in developing seeds. Our results suggest that JcCSH is widely involved in casbene biosynthesis in various tissues other than seeds. Key words:Casbene synthase, Jatropha (Jatropha curcas L.), phorbol ester.Recently, Jatropha (Jatropha curcas L.) has attracted much attention as a potential resource for the production of sustainable bioenergy, following concerns about the depletion of fossil fuels and global warming. Jatropha, a perennial shrub that originated in Mexico and Central America and belongs to the Euphorbiaceae family, is widely distributed between the tropics of Cancer and Capricorn.e seed kernels are rich in oil (44-62%) and proteins (22-35%), making this plant a favorable feedstock not only for biodiesel but also for protein (Makkar et al. 1998). However, it is hard to utilize the Jatropha seed cakes for animal feed a er extracting the oil, because several antinutrients and various toxins, such as curcin, trypsin inhibitors, and phorbol esters (PEs), are contained in the seed kernels (Makkar et al. 1998). Among these harmful substances, the major contributor to toxicity is PEs (Makkar et al. 1998). PEs are tetracyclic diterpenoids that occur in the seeds, sap, and latex of plants of the ymelaeaceae and Euphobiaceae families. Six PE derivatives were identified from Jatropha oil and their structures were solved by nuclear magnetic resonance (NMR) analysis. They were all found to contain 12-hydroxy-16-deoxyphorbol (Adolf et al. 1984;Haas et al. 2002). Since a PE isolated from Jatropha is known to have tumor-promoting activity (Hirota et al. 1988), it is critical to eliminate the risk of PEs toxicity if Jatropha is to be used commercially. One e ective means of doing so is to shut down and/or modify the biosynthesis of PEs at the molecular level.PEs are composed of a tetracyclic diterpene, named tiglian, and two fatty acids, which are esterified to C13 and C16 of the tiglian skeleton (Haas et al. 2002). The biosynthetic pathway of PEs is currently poorly understood. The only presumed step is the first cyclization of...

Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

“…diterpene synthases demonstrated a common H-bond network (SI Appendix, Fig. S1 D and E) (27,29,(33)(34)(35)(36)(37)(38)(39)(40)(41). This suggests that a conserved basic amino acid-rich triad sets and holds PP i in place during the complete catalytic cascade.…”

Novel family of terpene synthases evolved from trans -isoprenyl diphosphate synthases in a flea beetle

“…The reaction cascade is either initiated by a metal iondependent ionization of the diphosphate moiety or a protonation of the substrate, and can be terminated by proton abstraction or water addition (11,13,15). Because the reaction cascade may be branched and termination may occur at multiple levels, many TPSs are multiproduct enzymes forming complex mixtures of compounds (16)(17)(18)(19). Moreover, some TPSs also accept multiple substrates to produce monoterpenes, sesquiterpenes, and diterpenes (11).…”

Reverse Transcription-PCR analysis of geranylgeranyl diphosphate synthase (JcGGPPS) in Jatropha curcas L. and in silico analysis of Casbene Synthase (JcCS) among Euphorbiaceae

“…Little information about the biosynthetic pathway of phorbol ester has been elucidated. However, a recent study reported that Casbene Synthase (CS) might be involved in the first step of tiglian cyclization [15]. This report suggests that CS might be an essential enzyme, which regulates the biosynthetic of phorbol esters.…”

“…J. curcas Casbene Synthase (JcCS) has been identified and reported that JcCS is expressed in seedlings, mature leaves and in the mesocarp of developing fruits. Nevertheless, it is not expressed in developing seeds [15]. For a better understanding of the CS of several Euphorbiaceae, we analyzed the sequence of CS of six Euphorbiaceae plants, including J. curcas (XP_012066843), Ricinus communis (XP_002513340), Triadica sebifera (ADB90272), Euphorbia fischeriana (AFQ98015), Euphorbia esula (ADB90273) and Euphorbia peplus (AGN70884).…”

“…Despite their toxicity, phorbol esters also offer many interesting activities including insecticidal, molluscicidal and anti-mycobacterial [12][13][14]. A recent study has demonstrated that Casbene Synthase plays a significant role in the synthesis of phorbol esters [15].…”