A Previously Unknown Biosynthetic Capacity of Chalcone Synthase Discovered By Heterologous Expression of a Putative Plant Biosynthetic Gene Cluster in Yeast

Plant biosynthetic gene clusters (BGCs) are operon-like organization pattern of genes in plant genome. Although BGCs are less abundant in plants than in microorganisms, more than 30 plant biosynthetic pathways, mainly in plant specialized metabolism, have been identified as BGCs in the past decades from diverse plants. As with the successful application of microbial BGCs in discovering novel microbial biosynthetic pathway, plant BGCs have been a promising approach to accelerate novel plant biosynthetic pathway and plant natural product discovery. For example, investigation of the known plant BGCs have led to the discovery of unexpected, novel pathway components (e.g., sugar transferases, transporters, and regulators) that cluster together with known pathway genes. However, the discovery of novel biosynthetic pathways and biosynthetic capabilities from putative BGCs is still in its infancy.

Here, we firstly identified a previously unknown amide biosynthetic pathway derived from tomato (Solanum lycopersicum) from predicted tomato BGC candidates. By expressing a putative tomato gene cluster in yeast (Saccharomyces cerevisiae), we identified two functionally related enzymes catalyzing cascaded amide biosynthetic reactions. The newly discovered pathway consists of a carboxyl methyltransferase (SlMT2), which methylates the primary metabolite 3-hydroxyanthranilic acid (3-HAA) to form a methyl ester, and a naringenin chalcone synthase (SlCHS), which catalyzes the condensation of 3-HAA methyl ester and p-coumaroyl–coenzyme A (CoA) through formation of an amide bond. The newly discovery nitrogen-carbon bond formation biocatalytic capacity of CHS is different from its well-characterized Polyketide Synthase activity that canonically catalyzes carbon-carbon bond formation through iterative decarboxylative Claisen condensation. We further demonstrated 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.