Identification of Pyrrolizilactone Biosynthetic Gene Cluster with Unique Short-Chain Dehydrogenase Gene for 3-Methylproline Formation

Natural products often incorporate nonproteogenic amino acids for diversifying their chemical structures and biological activities. Methylproline is one such building block found in bioactive natural products. Among these, tricyclic pyrrolizidinone compounds, including UCS1025A, CJ-16,264, and pyrrolizilactone, possess complex structures and distinct stereochemistry. The pyrrolizilactone biosynthetic gene cluster is identified in Pleosporales sp. RKB3564 through genome and transcriptome analyses. By comparing the absolute configuration and biosynthetic gene (pzl) cluster of pyrrolizilactone with those of UCS1025A, the biosynthetic pathway of pyrrolizilactone, including its stereochemical divergent steps, is proposed. The pzl cluster contains enzymes-encoding genes, including those of the short-chain dehydrogenase/reductase PzlF and α-ketoglutarate dioxygenase PzlG, responsible for the formation of 3-methylproline, a building block of the tricyclic pyrrolizidinone structure. Functional analyses using recombinant PzlF and PzlG demonstrates the conversion of l-isoleucine to 3-methylproline. Unlike known methylproline biosynthetic pathways, the standalone enzyme PzlF, which is derived from the reductase domain of a polyketide synthase-nonribosomal peptide synthetase hybrid enzyme, functions as a pyrroline carboxylate reductase. This article provides new insights into the biosynthesis of complex natural products and highlights the potential for discovering new enzymes through the analysis of biosynthetic pathways of natural products with unique chemical structures.