Verlamelin

A fungal strain (JC-1) of Simplicillium was isolated from a pod of oilseed rape (Brassica napus) infested with the blackleg pathogen Leptosphaeria biglobosa. This study was done to clarify its taxonomic identity using morphological and molecular approaches, to characterize its antifungal activity through bioassays and genome-based identification of antifungal metabolites, and to determine its efficacy in inducing systemic resistance (ISR) in oilseed rape. The results showed that JC-1 belongs to Simplicillium lamellicola. It displayed a strong antagonistic relationship with L. biglobosa, Botrytis cinerea (gray mold) and Sclerotinia sclerotiorum (stem rot). The cultural filtrates of JC-1 showed a high efficacy in suppressing infection by S. sclerotiorum on detached leaves of oilseed rape. Genome analysis indicated that JC-1 has the capability of producing multiple antifungal metabolites, including aureobasidin A1, squalestatin S1 and verlamelin. Inoculation of JC-1 on seeds of oilseed rape caused a suppressive effect on infection by L. biglobosa on the cotyledons of the resulting seedlings, suggesting that JC-1 can trigger ISR. Endophytic growth, accumulation of anthocyanins, up-regulated expression of CHI (for chalcone isomerase) and PR1 (for pathogenesis-related protein 1), and down-regulated expression of NECD3 (for 9-cis-epoxycarotenoid dioxygenase) were detected to be associated with the ISR. This study provided new insights into the biocontrol potential and modes of action of S. lamellicola.

Verlamelin and its new derivative (verlamelin B) were isolated from fermentation broth of entomopathogenic fungus Lecanicillium sp. HF627. As the structural elucidation of verlamelin so far was only preliminary, we studied and determined the absolute structure of these two compounds to be cyclo(5S-hydroxytetradecanoic acid-D-alloThr/Ser-D-Ala-L-Pro-L-Gln-D-Tyr-L-Val). This is the first study that precisely analyzed the structure of verlamelin.

Only limited studies are available on the molecular-level biosynthesis of cyclic lipopeptides (cyclic and hybrid molecules consisting of peptide and fatty acid moieties) in filamentous fungi. Here, we identified and characterized biosynthetic genes of the cyclic lipopeptides, known as verlamelins. Only four genes, coding for non-ribosomal peptide synthetase (NRPS), fatty acid hydroxylase, thioesterase, and AMP-dependent ligase, were found to be involved in verlamelin biosynthesis by the analysis of corresponding gene knockouts. Surprisingly, no gene(s) coding for fatty acid synthase or polyketide synthase was present in the cluster, while verlamelin A/B contained a 5-hydroxytetradecanoic acid moiety. Precursor feeding experiment indicated that both fatty acid hydroxylase and thioesterase are involved to supply 5-hydroxytetradecanoic acid. The results suggested that 5-hydroxytetradecanoic acid was supplied from primary metabolism via fatty acid hydroxylase and loaded onto NRPS. Elongation of the peptide and final cyclization were accomplished by NRPS. The knowledge obtained through this study should provide new insight into fungal lipopeptide biosynthesis.