A heterologous expression platform in Aspergillus nidulans for enhanced production of a marine leucine-rich non-ribosomal peptid

The marine filamentous fungus Asteromyces cruciatus 763 (Pleosporaceae, Ascomycota), collected from the coast of La Jolla (San Diego, USA), produces a leucine-rich cyclic pentapeptide named lajollamide A. The chemical structure was previously determined by HPLC-MS and NMR (Gulder et al., 2012). Combination of whole genome sequencing and comprehensive bioinformatic analysis enabled the identification of the putative biosynthetic gene cluster (BGC) and a prediction-based proposal of the corresponding biosynthetic pathway. The BGC consists only of a single intron-containing non-ribosomal peptide synthetase (NRPS) gene. The NRPS catalyzes the synthesis of lajollamide A from l-leucine (3x), N-methyl-l-leucine (1x) and l-valine (1x). Leucine-rich cyclic pentapeptides, such as lajollamide A, are underexplored with regards to their biological activity. To fully explore the potential of lajollamide A, a sustainable production is required. The optimization of the natural producer does not represent a reasonable approach as several attempts to transform A. cruciatus 763 only led to unstable transformants. Consequently, increased production titers were attempted in a genetically manipulable heterologous host that generally enables a successful expression of fungal BGCs. In order to create a high-producing strain, the NRPS gene was cloned under a strong constitutive promoter and transferred into Aspergillus nidulans GR5 by PEG-mediated transformation. The transformants generated by multiple random ectopic integration of the introduced plasmid-DNA were subjected to a small-scale chemical screening process. The extracts obtained from the culture broth of the modified A. nidulans strains were analyzed by HPLC-MS. A total of 15 lajollamide A-producing strains were identified from 50 transformants. The successful reconstruction of the lajollamide A biosynthetic pathway demonstrates that A. nidulans can generally serve as a heterologous host for the expression of fungal gene clusters from the genus Asteromyces. The generation of this heterologous expression platform laid the foundation for a faster and more cost-effective microbial production of lajollamide A.