Elucidation of novel turnagainolides and their biosynthetic gene cluster in Bacillus subtilis.

Turnagainolides represent a unique class of cyclic depsipeptides characterized by the presence of a rare (E)-3-hydroxy-5-phenylpent-4-enoic acid (Hppa) residue, exhibiting diverse bioactivities. While previous studies have identified turnagainolides and their congeners from various microorganisms, including Microascus, Bacillus, Arthrobacter, and Streptomyces, their biosynthetic gene cluster and pathways remained elusive. Here, we uncovered four novel compounds, turnagainolide congeners D-G (3-6), and two known compounds, turnagainolides A-B (1, 2), from Bacillus subtilis LP. Their chemical structures were elucidated through a combination analysis of LC-MS analysis, NMR spectroscopy, and the Mosher derivatization technique. To investigate their biosynthetic gene cluster, comprehensive genome sequencing, phylogenetic analysis, and anti-SMASH-based prediction were conducted, and gene knockout experiments confirmed the correlation between the tur-BGC and the biosynthesis of these compounds. The alignment of protein sequences encoded by tur-BGC against public protein databases revealed homologous proteins exclusively in Bacillus species. These findings not only expand the chemical diversity of cyclic peptides in Bacillus but also provide critical insights into the biosynthetic pathway of turnagainolides and their evolutionary lineage.

Importance: Microbial natural products represent an invaluable resource in drug discovery, providing a vast reservoir of structurally and functionally diverse compounds with promising therapeutic potential. A comprehensive understanding of natural product biosynthesis not only deepens our knowledge of their chemical complexity but also drives advancements in chemical synthesis and metabolic engineering, paving the way for the generation of novel bioactive compounds. In this study, we report that a marine axenic culture of B. subtilis LP synthesizes six turnagainolides (1-6), which exhibit both biofilm-inhibitory and cytotoxic activities. These findings expand our understanding of the structure-activity relationships of turnagainolides and offer new insights into their potential biological roles. Moreover, the identification of biosynthetic gene clusters and the proposed biosynthetic pathway provide a valuable framework for elucidating turnagainolide biosynthesis, laying the groundwork for future efforts to optimize their production and explore their applications in drug development.