Chemical profiling of extracts from a mud dauber wasp-associated fungus, Aspergillus sp. (CMB-W031), revealed a remarkably diverse array of secondary metabolites, with many biosynthetic gene clusters being transcriptionally responsive to specific culture conditions. Chemical fractionation of a jasmine rice cultivation yielded many known fungal metabolites, including the highly cytotoxic (-)-stephacidin B and an unprecedented nonribosomal peptide synthase derived nitro depsi-tetrapeptide diketopiperazine, waspergillamide A (1). All structures were assigned by detailed spectroscopic analysis and, where appropriate, chemical degradation and Marfey's analysis.
We report on a preliminary investigation of the use the Gram-negative bacterial cell wall constituent lipopolysaccharide (LPS) as a natural chemical cue to stimulate and alter the expression of fungal secondary metabolism. Integrated high-throughput micro-cultivation and micro-analysis methods determined that 6 of 40 (15%) of fungi tested responded to an optimal exposure to LPS (0.6 ng/mL) by activating, enhancing or accelerating secondary metabolite production. To explore the possible mechanisms behind this effect, we employed light and fluorescent microscopy in conjunction with a nitric oxide (NO)-sensitive fluorescent dye and an NO scavenger to provide evidence that LPS stimulation of fungal secondary metabolism coincided with LPS activation of NO. Several case studies demonstrated that LPS stimulation can be scaled from single microplate well (1.5 mL) to preparative (>400 mL) scale cultures. For example, LPS treatment of Penicillium sp. (ACM-4616) enhanced pseurotin A and activated pseurotin A1 and pseurotin A2 biosynthesis, whereas LPS treatment of Aspergillus sp. (CMB-M81F) substantially accelerated and enhanced the biosynthesis of shornephine A and a series of biosynthetically related ardeemins and activated production of neoasterriquinone. As an indication of broader potential, we provide evidence that cultures of Penicillium sp. (CMB-TF0411), Aspergillus niger (ACM-4993F), Rhizopus oryzae (ACM-165F) and Thanatephorus cucumeris (ACM-194F) were responsive to LPS stimulation, the latter two examples being particular noteworthy as neither are known to produce secondary metabolites. Our results encourage the view that LPS stimulation can be used as a valuable tool to expand the molecular discovery potential of fungal strains that either have been exhaustively studied by or are unresponsive to traditional culture methodology.
Chemical analysis of an Australian mud dauber wasp-associated fungus, Talaromyces sp. (CMB-W045), yielded five new coprogen siderophores, talarazines A-E (1-5), together with dimerumic acid (6), desferricoprogen (7), and elutherazine B (8). Structures inclusive of absolute configuration were assigned on the basis of detailed spectroscopic analysis and application of the C Marfey's method. We report on the noncytotoxic Fe(III) chelation properties of 1-8 and demonstrate that biosynthesis is regulated by available Fe(III) in culture media. We demonstrate a magnetic nanoparticule approach to extracting high-affinity Fe(III) binding metabolites (i.e., 8) from complex extracts.
Cultures of the estuarine fungus Penicillium roseopurpureum (CMB-MF038) yielded a diverse array of polyketides, many of which were related via a highly convergent biosynthetic pathway. In addition to revising and assigning structures, and documenting chemical and biological properties, pro-drug cytotoxic properties were attributed to roseopurpurins H (10) and I (11) on the basis of in situ reverse Michael addition to a cytotoxic Michael acceptor (12).
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