The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase

Alice Di Girolamo,Janani Durairaj,Adèle Van Houwelingen,Francel Verstappen,Dirk Bosch,Katarina Cankar,Harro Bouwmeester,Dick De Ridder,Aalt D.J Van Dijk,Jules Beekwilder

doi:10.1016/j.abb.2020.108647

Abstract

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, β-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

Highlights

Plant species have highly specific chemical profiles, which are often determined by the presence of different terpenes and other secondary metabolites
We demonstrate how few residue positions are responsible for sub strate specificity, allowing a monoterpene synthase (monoTPS) to acquire sesquiTPS activity, without losing its original function
The concentration of santalenes was highest in the roots, this tissue was further selected for RNA extraction and cDNA sequencing

Summary

Introduction

Plant species have highly specific chemical profiles, which are often determined by the presence of different terpenes and other secondary metabolites These profiles serve the plant to function in its ecological niche, but have been widely employed in human applications, including pharmaceuticals, nutraceuticals, food and cosmetics [1]. In some cases, such applications lead to a high demand for the natural source of the metabolites and put increasing pressure on the conserva tion of the plant species from which they are derived [2,3,4].

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