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

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 (Solanum lycopersicum) gene cluster in yeast (Saccharomyces cerevisiae), 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 p-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 S. lycopersicum and Arabidopsis thaliana. Our work demonstrates yeast as a platform for characterizing putative plant gene clusters with the potential for compound structure and enzymatic activity discovery.