Elucidation and heterologous reconstitution of plant triterpene biosynthetic pathways

Abstract

Triterpenoids are a diverse and important class of natural products found throughout the plant kingdom. Their structural complexity, derived from the cyclization of oxidosqualene, provides a wide range of biological activities, including roles in plant growth, defense, and membrane stability. In addition, triterpenoids hold immense pharmacological potential, exhibiting anticancer, antiviral, anti-inflammatory, and cardioprotective properties, making them excellent candidates for drug development. This thesis investigates triterpenoid biosynthetic pathways, focusing first on oxidosqualene cyclases (OSCs). Using a sequence similarity network (SSN) approach to analyze large plant transcriptomic datasets, novel OSCs were identified and functionally characterized in Nicotiana benthamiana. From six tested OSCs, this work revealed three OSCs that produce rare triterpenes malabaricadiendiol and 19-epi-lupeol, as well as a novel triterpene, protostahopenol, a hopanoid stereoisomer. Site-directed mutagenesis identified key residues required for OSC activity, shedding light on essential structural elements necessary for enzymatic function. Understanding these residues provides a foundation for future studies aimed at modifying enzyme selectivity, improving catalytic efficiency, or engineering OSCs for the production of novel triterpenoid structures. This knowledge can be applied in metabolic engineering approaches to optimize triterpenoid biosynthesis for pharmaceutical and industrial applications. The second focus of this work is the withanolide biosynthetic pathway in Withania somnifera. Withanolides, a subclass of triterpenoids, have attracted attention due to their diverse bioactive properties and potential therapeutic applications. A key structural feature of many bioactive withanolides is the δ-lactone ring, which has been implicated in their biological activity by contributing to molecular interactions with target proteins. Understanding the enzymatic basis of withanolide biosynthesis is crucial for future biotechnological applications and pathway engineering efforts. Genome sequencing by collaboration partners revealed a conserved withanolide biosynthetic gene cluster in Solanaceae plants. Functional studies using metabolic engineering in Nicotiana benthamiana led to the identification of three cytochrome P450 monooxygenases (CYP87G1, CYP88C7, and CYP749B2) responsible for lactone ring formation in withanolides. As part of this pathway characterization, two shunt products were isolated and structurally elucidated. These intermediates provided crucial insights into the biosynthetic route by helping to infer the true pathway intermediates. The identification of these key enzymatic steps and pathway intermediates enhances the understanding of withanolide biosynthesis and facilitates future efforts in pathway engineering for the targeted production of bioactive withanolides. This work provides new insights into the enzymatic diversity and biosynthetic mechanisms governing triterpenoid metabolism. By identifying novel OSCs, characterizing key CYP enzymes in withanolide biosynthesis, and uncovering crucial intermediates, this study lays the foundation for future applications in synthetic biology and metabolic engineering