Structural Elucidation and Revised Biosynthetic Pathway of the Membrane Vesicle–Associated Antifungal Compound AFC-BC11

Abstract

Bacterial membrane vesicles (MVs) serve as delivery vehicles for hydrophobic and membrane-associated secondary metabolites, enhancing their solubility, stability, and bioactivity. Here, we show that the antifungal compound AFC-BC11, produced by Burkholderia cenocepacia K56–2, is released via MVs. Using HR-MS/MS, NMR, chemical degradation, and stable isotope feeding experiments, we determined the chemical structure of this compound and analyzed its biosynthesis. Our results confirm that the structure largely matches the recent report by Zhong et al., with a key difference: the double bond in the fatty acid moiety is positioned between C11 and C12. We provide compelling evidence that this constitution reflects the direct incorporation of cis-vaccenic acid, one of the most abundant fatty acids in B. cenocepacia, rather than a tailoring modification. Comparative analysis of afcU, afcF, and afcS mutants suggests a biosynthetic route involving ω-modification of cis-vaccenic acid, which would revise previously proposed pathways and open avenues for acyl chain engineering. Together, these findings establish AFC-BC11 as an MV-associated antifungal metabolite, provide a refined structural and biosynthetic model, and highlight the role of MVs in the dispersal of hydrophobic bioactive compounds.