Abstract
ABSTRACTMicrobial secondary metabolites, including isocyanide moieties, have been extensively mined for their repertoire of bioactive properties. Although the first naturally occurring isocyanide (xanthocillin) was isolated from the fungus Penicillium notatum over half a century ago, the biosynthetic origins of fungal isocyanides remain unknown. Here we report the identification of a family of isocyanide synthases (ICSs) from the opportunistic human pathogen Aspergillus fumigatus. Comparative metabolomics of overexpression or knockout mutants of ICS candidate genes led to the discovery of a fungal biosynthetic gene cluster (BGC) that produces xanthocillin (xan). Detailed analysis of xanthocillin biosynthesis in A. fumigatus revealed several previously undescribed compounds produced by the xan BGC, including two novel members of the melanocin family of compounds. We found both the xan BGC and a second ICS-containing cluster, named the copper-responsive metabolite (crm) BGC, to be transcriptionally responsive to external copper levels and further demonstrated that production of metabolites from the xan BGC is increased during copper starvation. The crm BGC includes a novel type of fungus-specific ICS-nonribosomal peptide synthase (NRPS) hybrid enzyme, CrmA. This family of ICS-NRPS hybrid enzymes is highly enriched in fungal pathogens of humans, insects, and plants. Phylogenetic assessment of all ICSs spanning the tree of life shows not only high prevalence throughout the fungal kingdom but also distribution in species not previously known to harbor BGCs, indicating an untapped resource of fungal secondary metabolism.
Highlights
Microbial secondary metabolites, including isocyanide moieties, have been extensively mined for their repertoire of bioactive properties
We identified four isocyanide synthases (ICSs) parsed across three biosynthetic gene clusters (BGCs) (Fig. 1B) within the A. fumigatus genome: notably, three of the four ICSs reside in BGCs undetected by mbio.asm.org 3 current in silico BGC prediction algorithms
We further demonstrate the production of isocyanides and transcription of their biosynthetic enzymes in A. fumigatus to be copper responsive, and we show that ICS overexpression induces cellular copper starvation
Summary
Microbial secondary metabolites, including isocyanide moieties, have been extensively mined for their repertoire of bioactive properties. In addition to industrial and pharmaceutical applications, naturally occurring bacterial isocyanides such as the diisonitrile SF2768 from Streptomyces thioluteus [7] have been shown to function as chalkophores: natural products involved in copper chelation and extracellular copper uptake Due to their ability to form coordination complexes with various transition metals, there is growing evidence that isocyanides play an important role in pathogenesis of insect, plant, and human diseases [5, 8, 10]. Despite their broad range of applications and emerging impact on various hostpathogen interactions, relatively little is known about the biosynthesis or molecular switches governing the production of isocyanides, in contrast to the well-studied nonribosomal peptide and polyketide biosynthetic pathways. Two new biosynthetic routes to bacterial isocyanide production have been reported: one that requires the activity of two modifying enzymes, a thioesterase homologue (ScoD) and a nonheme iron(II)-dependent oxidase (ScoE), identified in Mycobacterium tuberculosis to produce isonitrile lipopeptides [8], in addition to another that requires the activity of a typical nonribosomal peptide synthetase (SfaD), identified in S. thioluteus to produce the diisonitrile SF2768 [7]
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