Functional and evolutionary characterization of a secondary metabolite gene cluster in budding yeasts

Abstract

Secondary metabolites are key in how organisms from all domains of life interact with their environment and each other. The iron-binding molecule pulcherrimin was described a century ago, but the genes responsible for its production in budding yeasts have remained uncharacterized. Here, we used phylogenomic footprinting on 90 genomes across the budding yeast subphylum Saccharomycotina to identify the gene cluster associated with pulcherrimin production. Using targeted gene replacements in Kluyveromyces lactis, we characterized the four genes that make up the cluster, which likely encode two pulcherriminic acid biosynthesis enzymes, a pulcherrimin transporter, and a transcription factor involved in both biosynthesis and transport. The requirement of a functional putative transporter to utilize extracellular pulcherrimin-complexed iron demonstrates that pulcherriminic acid is a siderophore, a chelator that binds iron outside the cell for subsequent uptake. Surprisingly, we identified homologs of the putative transporter and transcription factor genes in multiple yeast genera that lacked the biosynthesis genes and could not make pulcherrimin, including the model yeast Saccharomyces cerevisiae. We deleted these previously uncharacterized genes and showed they are also required for pulcherrimin utilization in S. cerevisiae, raising the possibility that other genes of unknown function are linked to secondary metabolism. Phylogenetic analyses of this gene cluster suggest that pulcherrimin biosynthesis and utilization were ancestral to budding yeasts, but the biosynthesis genes and, subsequently, the utilization genes, were lost in many lineages, mirroring other microbial public goods systems that lead to the rise of cheater organisms.

Pulcherrimin formation controls growth arrest of theBacillus subtilisbiofilm

“…The patchy distribution of the genes both within a single species and between closely related bacteria suggests loss of the genes overtime, rather than reoccurring acquisition. This pattern of gene loss and distribution is consistent with the profile observed in yeast (46). However, given the knowledge that at least two routes have evolved for pulcherriminic acid production (46,47), we cannot exclude the possibility that other species also synthesize pulcherriminic acid using an as yet unidentified pathway.…”

“…Pulcherrimin has been observed to be made by both eukaryotic and prokaryotic microorganisms (27,45). It is, however, only more latterly that the biosynthetic pathways involved have been elucidated (46,47). Our comparative genomic analysis revealed that the genes needed for pulcherriminic acid synthesis and secretion (yvmC, cypX and yvmA) were found in the genome or on plasmids of species beyond B. subtilis.…”

“…Consistent with this, pulcherrimin production has been associated with biocontrol processes (49). However, this long held interpretation of how pulcherrimin functions has been challenged by the recent classification of pulcherriminic acid as a siderophore in the yeast Kluyveromyces lactis (46). In K. lactis a membrane bound protein encoded by the gene PUL3 is linked with utilisation of pulcherrimin via uptake.…”

“…This opens up the possibility that pulcherrimin producers across both bacterial and eukaryotic microbes are exploited by neighbouring "cheater" cells. In this context, the non-producing cells could make use of the chelated iron but would not contribute to its deposition (46). In nature, the routes used for iron acquisition are highly diverse and competition between ecologically distant species is common.…”

Snf2 controls pulcherriminic acid biosynthesis and connects pigmentation and antifungal activity of the yeastMetschnikowia pulcherrima

“…Pulcherriminic acid is formed by the oxidation of cyclodileucine (cyclo(Leu-Leu) (24,25). Pulcherrimin was first described in yeasts more than 60 years ago, but only very recently the four genes involved in its biosynthesis and transport in Kluyveromyces lactis have been identified (26), and the structure of the molecule synthesized by yeasts has not been determined by modern analytical methods. However, pulcherrimin is also produced by certain bacteria such as Bacillus licheniformis, where the two genes yvmC and cypX, coding for a cyclodipeptide synthase and cytochrome P450 oxidase, respectively, are responsible for its biosynthesis (27)(28)(29).…”