Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Biochemistry

Background:

• Jatropha curcas L. is a valuable source of oil for biofuels, but its seed cake contains toxic phorbol esters (PEs).
• These PEs limit the use of Jatropha seed cake in animal nutrition and pose safety concerns.
• Identifying the biosynthesis pathway of PEs is crucial for developing safer Jatropha varieties.

Purpose of the Study:

• To investigate the role of casbene synthase in phorbol ester biosynthesis in Jatropha.
• To develop a strategy for reducing phorbol ester content in Jatropha seeds.
• To assess the potential of low-PE Jatropha seed cake for animal nutrition.

Main Methods:

• Cloned two casbene synthase gene homologues (JcCASA163 and JcCASD168) from Jatropha.
• Performed in vitro functional analysis to confirm casbene synthase activity.
• Utilized RNA interference (RNAi) with a seed-specific promoter to down-regulate gene expression.

Main Results:

• Casbene synthase activity was confirmed for JcCASA163, converting geranylgeranyl diphosphate to casbene.
• Casbene synthase was implicated as a key enzyme in Jatropha phorbol ester biosynthesis.
• RNAi-mediated down-regulation of casbene synthase reduced seed PE levels by up to 85% without adverse effects.

Conclusions:

• Casbene is a direct precursor to phorbol esters in Jatropha.
• Down-regulating casbene synthase effectively reduces phorbol ester biosynthesis in Jatropha seeds.
• Engineered low-PE Jatropha seeds are heritable and offer a viable solution for safe animal feed development.