4-Hydroxybenzoic acid is a diffusible factor that connects metabolic shikimate pathway to the biosynthesis of a unique antifungal metabolite in Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF) produced by Lysobacter enzymogenes is a potential lead compound for developing new antibiotics. Yet, how L. enzymogenes regulates the HSAF biosynthesis remains largely unknown. Here, we show that 4-hydroxybenzoic acid (4-HBA) serves as a diffusible factor for regulating HSAF biosynthesis. The biosynthesis of 4-HBA involved an oxygenase, LenB2, and mutation of lenB2 almost completely abolished 4-HBA production, leading to significantly impaired HSAF production. Introduction of a heterologous gene coding for 4-HBA biosynthetic enzyme into the lenB2 mutant restored the production of 4-HBA and HSAF to their corresponding wild-type levels. Exogenous addition of 0.5-1 µM 4-HBA was sufficient to restore HSAF production in the lenB2 mutant. Furthermore, the shikimate pathway was found to regulate the biosynthesis of HSAF via 4-HBA. Finally, we identified a LysR-family transcription factor (LysRLe ) with activity directed to HSAF production. LysRLe could bind to the HSAF promoter and, as a result, regulates expression of HSAF biosynthesis genes. The 4-HBA could bind to LysRLe and appeared to partly enhance formation of the LysRLe -DNA complex. Collectively, our findings suggest that L. enzymogenes produces 4-HBA to serve as an adaptor molecule to link the shikimate pathway to the biosynthesis of a unique antifungal metabolite (HSAF).

Identification of a small molecule signaling factor that regulates the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes.

Lysobacter species are emerging as new sources of antibiotics. The regulation of these antibiotics is not well understood. Here, we identified a small molecule metabolite (LeDSF3) that regulates the biosynthesis of the antifungal antibiotic heat-stable antifungal factor (HSAF), a polycyclic tetramate macrolactam with a structure and mode of action distinct from the existing antifungal drugs. LeDSF3 was isolated from the culture broth of Lysobacter enzymogenes, and its chemical structure was established by NMR and MS. The purified compound induced green fluorescence in a reporter strain of Xanthomonas campestris, which contained a gfp gene under the control of a diffusible signaling factor (DSF)-inducible promoter. Exogenous addition of LeDSF3 in L. enzymogenes cultures significantly increased the HSAF yield, the transcription of HSAF biosynthetic genes, and the antifungal activity of the organism. The LeDSF3-regulated HSAF production is dependent on the two-component regulatory system RpfC/RpfG. Moreover, LeDSF3 upregulated the expression of the global regulator cAMP receptor-like protein (Clp). The disruption of clp led to no HSAF production. Together, the results show that LeDSF3 is a fatty acid-derived, diffusible signaling factor positively regulating HSAF biosynthesis and that the signaling is mediated by the RfpC/RpfG-Clp pathway. These findings may facilitate the antibiotic production through applied genetics and molecular biotechnology in Lysobacter, a group of ubiquitous yet underexplored microorganisms.