Copper starvation induces antimicrobial isocyanide integrated into two distinct biosynthetic pathways in fungi

Abstract

The genomes of many filamentous fungi, such as Aspergillus spp., include diverse biosynthetic gene clusters of unknown function. We previously showed that low copper levels upregulate a gene cluster that includes crmA, encoding a putative isocyanide synthase. Here we show, using untargeted comparative metabolomics, that CrmA generates a valine-derived isocyanide that contributes to two distinct biosynthetic pathways under copper-limiting conditions. Reaction of the isocyanide with an ergot alkaloid precursor results in carbon-carbon bond formation analogous to Strecker amino-acid synthesis, producing a group of alkaloids we term fumivalines. In addition, valine isocyanide contributes to biosynthesis of a family of acylated sugar alcohols, the fumicicolins, which are related to brassicicolin A, a known isocyanide from Alternaria brassicicola. CrmA homologs are found in a wide range of pathogenic and non-pathogenic fungi, some of which produce fumicicolin and fumivaline. Extracts from A. fumigatus wild type (but not crmA-deleted strains), grown under copper starvation, inhibit growth of diverse bacteria and fungi, and synthetic valine isocyanide shows antibacterial activity. CrmA thus contributes to two biosynthetic pathways downstream of trace-metal sensing.

Fig. 1. The crm BGC and associated metabolites from A. fumigatus.

a Comparison of crm BGCs in A. fumigatus, P. commune, and C. heterostrophus. Homologous genes are marked with colors based on their corresponding functions. b Volcano plot of differential metabolites in wild type upregulated by copper starvation (red dots represent metabolites 20-fold upregulated at p < 0.05 as calculated by unpaired two-sided t-test, unadjusted for the number of comparisons, based on 6 independent replicates). c Total number of crmA-dependent metabolites in wildtype upregulated by copper starvation or crmA overexpression compared to the crmA deletion mutant and Venn diagram showing overlap of metabolites induced in WT A. fumigatus under copper-limited relative to copper-replete conditions, metabolites abolished in crmA deletion mutants, and metabolites induced in crmA overexpression mutants relative to WT grown under copper-limited conditions. d, e Structures, MS2 spectra, ESI + ion chromatograms, and HMBC correlations of fumivaline A (1) and fumicicolin A (3). Part of the structure highlighted in blue represents festuclavine (2). f, g Structures and ESI- ion chromatograms for N-formylvaline (4) and (S)−2-isocyanoisovaleric acid (valine isocyanide, 5). Source data are provided as a Source Data file.

Fig. 2. Comparison of fumivaline BGCs and ergot alkaloids production.

a Ergot alkaloid biosynthesis pathways in A. fumigatus and P. commune. l-Tryptophan (8) and dimethylallyl pyrophosphate are converted into chanoclavine-1 aldehyde (7) by DmaW and EasC-F, which is then converted into festuclavine (2) by EasA and EasG in A. fumigatus and P. commune. Valine (9) is converted into valine isocyanide (5) by CrmA, which then reacts with the imine intermediate of festuclavine (6), resulting in formation of the amide bond in fumivaline A following hydration. In addition, hydration of valine isocyanide (5) produces N-formylvaline (4). b Comparison of crm BGCs of A. fumigatus and P. commune. Homologous genes are marked using the same colors as in a. A and C-G represent easA and easC-G respectively. W represents dmaW. c Relative abundances of N-formylvaline (4), fumivaline A (1), and festuclavine (2) and pyroclavine in A. fumigatus, P. commune, and P. expansum (gray, red, and blue, respectively) grown without copper (-) or with copper. Bars represent mean ± s.e.m. with six independent biological replicates for A. fumigatus wildtype and three for the other strains. p values were calculated by unpaired, two-sided t-test with Welch’s correction, ****P < 0.0001. d Relative abundances of N-formylvaline (4) and [7-²H]-N-formylvaline (4a) in extracts of wildtype A. fumigatus (grown without copper) extracted with deuterated or non-deuterated solvents. Source data are provided as a Source Data file.

Fig. 3. Identification of fumicicolin A and related metabolites.

a Partial representation of MS2 network (cosine >0.7) for WT C. heterostrophus in ESI+, showing the cluster containing fumicicolin A (3) and the heterocicolins (10–13). Shown nodes are strongly upregulated in wildtype grown without copper (see Supplementary Fig. 4 for full network). b Relative abundance of fumicicolin A (3) in A. fumigatus, C. heterostrophus, and Penicillium spp., and its structural similarity with known d-mannitol derivatives, maculansin A (16) and brassicicolin A (17). c ESI + ion chromatograms of heterocicolins A (10), C (12), E (14), and F (15) in WT C. heterostrophus grown with (blue) or without copper (red) and ΔcrmA (green) grown without copper. Dashed arrows indicate fragmentation in MS2 spectra. In b, bars represent mean ± s.e.m. with six independent biological replicates for A. fumigatus and C. heterostrophus wildtype under copper-limited conditions and three for the other strains/conditions. Source data are provided as a Source Data file.

Fig. 4. Putative biosynthesis of CrmA-derived metabolites in diverse fungi and antimicrobial activities.

a Fumivaline biosynthesis was observed in A. fumigatus and P. commune, which has the ergot alkaloid BGC and a homolog for crmA, whereas fumicicolin biosynthesis was observed in A. fumigatus, Penicillium spp. and C. heterostrophus, all of which harbor crmA homologs. b Growth of Listeria monocytogenes, Escherichia coli, Pencillium expansum, and Alternaria brassicicola is inhibited when challenged with extracts from WT but not ΔcrmA A. fumigatus grown without copper supplementation. Extracts from copper supplemented cultures do not inhibit microbial growth. c Valine isocyanide (5) significantly inhibits the growth of Staphylococcus aureus at all concentrations tested and inhibits the growth of E. coli at 125 μM and higher. MIC₅₀, minimum inhibitory concentration to inhibit 50% growth. d Valine isocyanide (5) and N-formylvaline (4) show synergistic antifungal activity against Candida auris at 36 h. In c and d, bars represent mean ± s.e.m. with three independent biological replicates. One-way ANOVA with Dunnett’s multiple comparisons test was performed to assess if the differences in survival at the range of concentrations were statistically significant (at p < 0.05) from survival with solvent only (0 µM), ****p < 0.0001, NS, not significant. Source data are provided as a Source Data file.

Fig. 5. Phylogenetic tree and proposed functions of CrmA-derived metabolites in fungal ecology.

a CrmA proteins are found in three fungal taxa (for details, see Supplementary Data 1). CrmA is primarily found in pathogenic fungi including the insect pathogens Metarhizium, Cordyceps and Beauveria and the fungal parasite Trichoderma (Sordariomycetes), leaf blight pathogens Bipolaris(e.g., Cochliobolus) and Alternaria (Dothideomycetes) as well as in opportunistic pathogens and saprophytic Aspergillus and Pencillium spp. and the dermatophytic genus Trichophyton (Eurotiales). Our model proposes that CrmA metabolites are synthesized under copper starvation, where they act as virulence factors facilitating copper acquisition during fungal/host encounters or as antimicrobial compounds during competition in copper-limited niches. b Proposed rhabdoplanin A biosynthesis via an Ugi-like reaction in bacteria. Panel (a) was created with BioRender.com.