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

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.