Genome Mining Reveals a Minimum Gene Set for the Biosynthesis of 32-Membered Macrocyclic Thiopeptides Lactazoles

Abstract

Although >100 thiopeptides have been discovered, the number of validated gene clusters involved in their biosynthesis is lagging. We use genome mining to identify a silent thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles. Lactazoles are structurally unique thiopeptides with a 32-membered macrocycle and a 2-oxazolyl-6-thiazolyl pyridine core. We demonstrate that lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazA to lazF). We show that lazC is involved in the macrocyclization process, leading to central pyridine moiety formation. Substitution of the endogenous promoter with a strong promoter results in an approximately 30-fold increase in lactazole A production and mutagenesis of lazC precursor gene in production of two analogs. Lactazoles do not exhibit antimicrobial activity but may modulate signaling cascades triggered by bone morphogenetic protein. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development.

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…Gene deletions, heterologous expression, and identification of intermediates, have allowed the reconstruction of putative thiopeptide biosynthetic pathways (Hayashi et al, 2014, Malcolmson et al, 2013). …”

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

“…(iii) It opens the door to the rational design and large-scale production of new thiopeptide analogs with improved pharmacological or bioactivity properties. Attempts to heterologously express diverse thiopeptide gene clusters have been met with mixed success (7,15,(38)(39)(40). In most cases, Streptomyces species, such as Streptomyces lividans and Streptomyces coelicolor, have been employed.…”

“…Thiopeptides, characterized by posttranslationally formed sulfurand nitrogen-containing heterocycles, constitute a rapidly expanding class of RiPP antibiotics (5). Over 100 different thiopeptides have been chemically identified from numerous culturable bacterial producers, and the mining of genomic and metagenomic data promises to uncover many more chemical species that have eluded discovery by conventional means (6)(7)(8).…”

“…1A) that consist of approximately 6 to 25 protein-coding genes (7). These include a gene encoding the precursor peptide that is modified to become the thiopeptide product as well as an assemblage of posttranslational modification genes that are required for thiopeptide maturation and genes for host immunity and other functions.…”

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

“…Their ribosomal synthesis, however, makes them attractive targets for genetic engineering of both the core peptide and the biosynthetic enzymes to create diverse thiopeptide chemical libraries. Previous efforts to explore pathway promiscuity and define enzyme function in vivo have coupled expression of core-peptide variants and/or gene deletions with organic extraction of pathway products (4)(5)(6)(7)(8)(9)(10). These studies succeed in identifying extractable products and intermediates that have been prematurely cleaved from the leader peptide, but overlook peptide intermediates that are chemically very different from product molecules.…”