Discovery of the leinamycin family of natural products by mining actinobacterial genomes

Abstract

Nature's ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF-SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF-SH didomains. Further analysis of the representative genomes from each of the clades identified 28 -type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature's rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature's biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

A 3‐hydroxy‐3‐methylglutaryl‐CoA synthase‐based probe for the discovery of the acyltransferase‐less type I polyketide synthases

“…Six HCSs from CB00144, CB00158, CB01229, CB01357, CB01456 and CB01509, are in the same cluster with SnaI (CBW45745.1) and VirC (BAF50725.1) involved in the biosynthesis of streptogramin A‐type antibiotics. Four HCSs from CB01828, CB01882, CB01883 and CB02980 are similar to LnmM, suggesting their involvement in the biosynthesis of the recently identified LNM‐family of natural products, in which a novel polyketide weishanmycin was identified from CB01883 using an alternative probe of DUF‐SH domain (Pan et al ., ). The HCS from CB02999 shares 98% sequence similarity with BaeG, while HCSs from CB00111 and CB01881 form a unique sub‐branch.…”

“…A recent metagenomic analysis suggested that AT‐less type I PKSs may constitute about 38% of modular PKSs among the sequenced bacterial genomes (O'Brien et al ., ). Our discovery of the LNM family of natural products through in silico and genome mining also revealed that there is huge potential for structure diversity from AT‐less type I PKSs (Pan et al ., ). Although KSs in either type I or II PKSs have been used to survey the structure diversity of PKSs in different geographic locations (Charlop‐Powers et al ., ; Charlop‐Powers et al ., ), the diversity of AT‐less type I PKSs in the environment, to our knowledge, has not been explored in a systematic manner.…”

P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities

“…3 Each lnm -type gene cluster contains at least one P450. Interestingly, the GNMs and WSMs do not have 1,3-dioxo-1,2-dithiolane rings or hydroxyl groups at C-8 or C-4′ (Figure S5), suggesting alternate or cryptic roles for these P450s.…”

“…With the recent discovery of 28 lnm -type gene clusters, 3 it is fascinating to consider the potential biosynthetic and biological implications of LNM-like natural products that are structurally distinct to 1 . For example, the GNMs and WSMs, which do not possess the 1,3-dioxo-1,2-dithiolane rings or hydroxyl groups at C-8 or C-4′, are likely significantly less active than 1 .…”

“…Recent efforts in mining actinobacterial genomes, however, revealed that the structural diversity for the LNM family of natural products is underappreciated. 3 LNM possesses potent DNA cleavage activity via an unprecedented mode of action. Specifically, upon the attack of cellular thiol agents at the 1,3-dioxo-1,2-dithiolane moiety, 1 undergoes a tandem rearrangement to form an episulfonium ion intermediate that can efficiently alkylate the N-7 position of deoxyguanosine bases of DNA (Figure S1A).…”