“Hyphae Intertwined, Biomolecules Co-Born”—New Polyketides Induction by Co-Culture of the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48 and Pestalotiopsis sp. HHL-101 at Both Volatile and Non-Volatile Levels

The co-culture technique, mimicking natural microbial interactions, has proven to be successful at activating silent biosynthetic gene clusters (BGCs) to produce novel metabolites or enhance the yield of specific metabolites. To effectively decode induction processes, it is critical to have a comprehensive understanding of intermicrobial interactions across both volatile and non-volatile metabolomes. As part of our attempt to uncover structurally unique and biologically active natural products from mangrove endophytic fungi, <i>Phomopsis asparagi</i> DHS-48 was co-cultured with another mangrove fungal strain, <i>Pestalotiopsis</i> sp. HHL-101. The competition interaction of the two strains was investigated using morphology and scanning electron microscopy (SEM), and it was discovered that the mycelia of the DHS-48 and HHL-101 compressed and tangled with each other in the co-culture system, forming an interwoven pattern. To profile volatile-mediated chemical interactions during fungal co-culture, headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) coupled with orthogonal partial least squares-discriminant analysis (OPLS-DA) was adopted. Meanwhile, non-volatile metabolites from both liquid and solid small-scale co-cultures were profiled via HPLC. Two new polyketides, named phaseolorin K (<b>1</b>) and pestaphthalide C (<b>7</b>), together with 11 known compounds (<b>2</b>-<b>6</b>, <b>8</b>-<b>13</b>), were characterized from solid-state co-cultivation extracts of these two titled strains. Their planar structures were established by analysis of HRMS, MS/MS, and NMR spectroscopic data, while absolute configurations were assigned using ECD calculations. Co-culture feeding experiments demonstrated that DHS-48 exerts antagonistic activity against HHL-101 through altering its hyphal morphology, which mediated enhanced biosynthesis of non-volatile antimicrobial metabolites <b>5</b> and <b>6</b>. Biological assays revealed that compounds <b>4</b>-<b>6</b> exhibited potent in vitro cytotoxicity against human cancer cell lines HeLa and HepG2, compared to the positive controls adriamycin and fluorouracil. Compound <b>2</b> moderately inhibited the proliferation of ConA-induced T and LPS-induced B murine spleen lymphocytes.