Discovery and Biosynthesis of Pepticinnamins G–M Featuring Three Enzymes-Catalyzed Nonproteinogenic Amino Acid Formation

Abstract

Since pepticinnamin E was discovered almost 30 years ago, no other pepticinnamin family of natural products have been reported to date. We here report the discovery of pepticinnamins G-I (1-3) from a marine Streptomyces sp. PKU-MA01144 and pepticinnamins J-M (4-7) from several mutants, and these new compounds contain different N-methyl-L-alanine and L-tyrosine residues to pepticinnamin E. Genome sequencing, heterologous expression, gene-deletion and reconstitution of enzymatic reaction in vitro identified the biosynthetic gene cluster of 1-7, and first experimentally established the biosynthesis of nonproteinogenic 2-chloro-3-hydroxy-4-methoxy-L-phenylalanine residue by a biopterin-dependent hydroxylase Pep10, an O-methyltransferase Pep9 and a flavin-dependent halogenase Pep1. The biosynthetic research and the heterologous expression system in this study set the stage for pathway engineering for more pepticinnamins generation in the future.