(174e) Discovery and Biosynthesis of Oxazolismycins, a New Class of Angiotensin-Converting Enzyme Inhibitors

Natural products (NPs) possess various biological activities and have long served as potential drug candidates. Among these, pyridine derivatives have garnered significant attention due to their biological significance, exemplified by FDA-approved drugs such as Omeprazole and Netupitant. NPs containing pyridine structures are thus of considerable interest. This study presents the discovery of a novel pyridine derivative NP, oxazolismycin, characterized by an oxazole and fully substituted pyridine structure. Oxazolismycin exhibited a moderate angiotensin-converting enzyme (ACE) inhibitory effect (IC₅₀ = 3.66 μM), suggesting its potential therapeutic application for ACE-related diseases.

Oxazolismycin was produced by heterologous expression of a nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid biosynthetic gene cluster (BGC). The hybrid BGC, initially identified in the genome of Streptomyces griseochromogenes by antiSMASH analysis, contains a PKS-NRPS hybrid gene and an NRPS that possess high sequence similarity compared to the corresponding genes responsible for synthesizing the pyridine ring of caerulomycin and collismycin. The 56kb BGC was cloned using the CAPTURE (Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination) direct cloning method and was transferred to the heterologous host Streptomyces lividans TK24 via intergeneric conjugation for heterologous expression. Oxazolismycin was detected as a new product in the crude extract by HPLC, and the structure was determined using NMR and X-ray crystallography.

To delineate the biosynthetic boundary, the BGC was subcloned into a 30kb construct harboring essential biosynthetic elements. Remarkably, oxazolismycin production persisted in the heterologous host, indicating that the 30kb BGC contained the requisite enzymatic machinery for biosynthesis. Further exploration involving gene knockouts and metabolite analysis of mutant strains unveiled the biosynthetic pathway of oxazolismycin.

Interestingly, oxazolismycin shares a structural similarity with another ACE inhibitor karnamicin. The structural difference can be attributed to different substrates and substrate selection discrepancies. We propose that by combining the different enzymes, the final products can be engineered to create a diverse library of ACE inhibitors. These findings collectively contribute to unraveling the biosynthetic pathway of oxazolismycin, offering potential therapeutic applications, and deepening our understanding of natural product biosynthesis.