Characterization of a Cryptic NRPS Gene Cluster in Bacillus velezensis FZB42 Reveals a Discrete Oxidase Involved in Multithiazole Biosynthesis

Abstract

Polythiazole-containing nonribosomal peptides (NRPs) represent a rare and important group of natural products with intriguing structures and promising bioactivities. However, the biosynthetic mechanism of polythiazole in NRPS pathways remains poorly understood. Genome mining of the model biocontrol bacterium Bacillus velezensis FZB42 revealed that a cryptic NRPS gene cluster nrs might be involved in the synthesis of polyheterocycle family products. Heterologous expression of this gene cluster led to the identification of rare trithiazole-containing products, bacillothiazols A–N (1–14), with potent inhibitory activity against protein tyrosine phosphatase PTP1B. Unlike the general incis manner in which the oxidation of polythiazole in NRPs is canonically catalyzed by integrated oxidase (Ox) domains, our gene deletion and biochemical characterization revealed that a stand-alone oxidase NrsB in trans catalyzed the iterative oxidation of multithiazoline to multithiazole in bacillothiazol biosynthesis, which implied an unprecedented pathway of polythiazole formation in microbial NRPs. Notably, rather than the integrated NRPS Ox domains, the catalytic residues in NrsB were seemingly the same as ribosomally synthesized and post-translationally modified peptide (RiPP) dehydrogenases, which can catalyze iterative oxidation reactions, suggesting that NrsB might represent an interesting case in the evolution of integrated NRPS Ox domains and RiPP dehydrogenases. These findings not only resolve the long-standing puzzle of the natural products from the model strain B. velezensis FZB42 but also provide insight into the versatility of polythiazole biosynthesis.

Novel Plant-Associated Brevibacillus and Lysinibacillus Genomospecies Harbor a Rich Biosynthetic Potential of Antimicrobial Compounds

“…The cryptic nrs gene cluster of the model biocontrol bacterium Bacillus velezensis FZB42 also harbors three modules encoding for cysteine. Recently, trithiazole was identified as the product of the nrs gene cluster, and NrsB as the oxidizing enzyme of the thiazoline precursor [ 78 ]. However, a more careful comparison of the domain structure of both gene clusters revealed significant differences between both BGCs, excluding the possibility that the final product can be a polythiazole ( Figure 10 ).…”

Bathiapeptides: Polythiazole-Containing Peptides from a Marine Biofilm-Derived Bacillus sp.

“…Given the above evidence, we proposed the biosynthesis pathway of bathiapeptides produced by this BGC (Figure ). The chain elongation followed the canonical NRPS assembly logic for the five modules in BatA, BatB, and BatC, and the predicted dehydrogenase BatE was inferred to oxidize the thiazoline generated by the Cy domains, similar to oxidase NrsB in the biosynthesis of bacillothiazols, as two proteins shared the same conserved domains. The regular macrolactamization mediated by the first thioesterase localized in BatC led to the production of compound 6 .…”

“…Bathiapeptides are structurally related to marthiapeptide A and recently reported bacillothiazols, which share a rare tristhiazole functionality. The crystal structures reveal a planar hydrophilic pocket (Figure B) that may also be able to bind metal ions.…”

Hot off the Press

“…Genome mining of the bacterium Bacillus velezensis FZB42 and subsequent heterologous expression of a cryptic NRPS gene cluster has revealed a family of tri-thiazole natural products, including bacillothiazol A 25 . 25 Gene deletion and biochemical studies have identified the biosynthetic enzymes of the bacillothiazol pathway, including a flavin mononucleotide-dependent oxidase NrsB, which iteratively oxidises the thiazoline units to thiazoles. Varlaxins 1046A and 1022A 26 have been isolated from Nostoc species UHCC 0870 and been shown to inhibit human trypsin isoenzymes at sub-nanomolar concentrations.…”