A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis

Abstract

Significance Plants have evolved to produce an abundance of neuroactive small molecules. Many major classes of these compounds are derived from the amino acid lysine, including the Lycopodium alkaloids, which are produced by club moss species that are traditional herbal medicines in several cultures. In this work, we describe a likely metabolic regulon, or transcriptionally coregulated group of metabolic genes, for the Lycopodium alkaloids in the club moss Phlegmariurus tetrastichus, which we used to identify six enzymes within the biosynthesis of huperzine A (HupA), an acetylcholinesterase inhibitor with clinical interest as a treatment for neurological disease. Our results demonstrate precise transcriptional coregulation of biosynthetic steps within Lycopodium alkaloid metabolism and further uncover diverse enzymatic transformations for lysine-derived alkaloid biosynthesis in plants. Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer’s disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate–dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.