Chemical Diversification Based on Substrate Promiscuity of a Standalone Adenylation Domain in a Reconstituted NRPS System

Abstract

A nonribosomal peptide synthetase (NRPS) assembly line ( sfa) in Streptomyces thioluteus that directs the formation of the diisonitrile chalkophore SF2768 (1) has been characterized by heterologous expression and directed gene knockouts. Herein, differential metabolic analysis of the heterologous expression strain and the original host led to the isolation of an SF2768 analogue (2, a byproduct of sfa) that possesses N-isovaleryl rather than 3-isocyanobutyryl side chains. The proposed biosynthetic logic of sfa and the structural difference between 1 and 2 suggested substrate promiscuity of the adenylate-forming enzyme SfaB. Further substrate scope investigation of SfaB and a successfully reconstituted NRPS system including a four-enzyme cascade enabled incorporation of diverse carboxylic acid building blocks into peptide scaffolds, and 30 unnatural products were thus generated. This structural diversification strategy based on substrate flexibility of the adenylation domain and in vitro reconstitution can be applied to other adenylation-priming pathways, thus providing a supplementary method for diversity-oriented total synthesis. Additionally, the biocatalytic process of the putative lysine δ-hydroxylase SfaE was validated through the derivatization of two key aldehyde intermediates (2a and 2b), thereby expanding the toolkit of enzymatic C-H bond activation.