Discovery of a previously unknown biosynthetic capacity of naringenin chalcone synthase by heterologous expression of a tomato gene cluster in yeast.

Abstract

Chalcone synthase (CHS) canonically catalyzes carbon-carbon bond formation through iterative decarboxylative Claisen condensation. Here, we characterize a previously unidentified biosynthetic capability of SlCHS to catalyze nitrogen-carbon bond formation, leading to the production of a hydroxycinnamic acid amide (HCAA) compound. By expressing a putative tomato (<i>Solanum lycopersicum</i>) gene cluster in yeast (<i>Saccharomyces cerevisiae</i>), we elucidate the activity of a pathway consisting of a carboxyl methyltransferase (SlMT2), which methylates the yeast primary metabolite 3-hydroxyanthranilic acid (3-HAA) to form a methyl ester, and a SlCHS, which catalyzes the condensation of 3-HAA methyl ester and <i>p</i>-coumaroyl-coenzyme A (CoA) through formation of an amide bond. We demonstrate that this aminoacylation activity could be a common secondary activity in plant CHSs by validating the activity in vitro with variants from <i>S. lycopersicum</i> and <i>Arabidopsis thaliana</i> Our work demonstrates yeast as a platform for characterizing putative plant gene clusters with the potential for compound structure and enzymatic activity discovery.