Harnessing Natural Diversity to Probe Metabolic Pathways

Abstract

Analyses of cellular processes in the yeast Saccharomyces cerevisiae rely primarily upon a small number of highly domesticated laboratory strains, leaving the extensive natural genetic diversity of the model organism largely unexplored and unexploited. We asked if this diversity could be used to enrich our understanding of basic biological processes. As a test case, we examined a simple trait: the utilization of di/tripeptides as nitrogen sources. The capacity to import small peptides is likely to be under opposing selective pressures (nutrient utilization versus toxin vulnerability) and may therefore be sculpted by diverse pathways and strategies. Hitherto, dipeptide utilization in S. cerevisiae was solely ascribed to the activity of a single protein, the Ptr2p transporter. Using high-throughput phenotyping and several genetically diverse strains, we identified previously unknown cellular activities that contribute to this trait. We find that the Dal5p allantoate/ureidosuccinate permease is also capable of facilitating di/ tripeptide transport. Moreover, even in the absence of Dal5p and Ptr2p, an additional activity-almost certainly the periplasmic asparaginase II Asp3p-facilitates the utilization of dipeptides with C-terminal asparagine residues by a different strategy. Another, as-yet-unidentified activity enables the utilization of dipeptides with C-terminal arginine residues. The relative contributions of these activities to the utilization of di/tripeptides vary among the strains analyzed, as does the vulnerability of these strains to a toxic dipeptide. Only by sampling the genetic diversity of multiple strains were we able to uncover several previously unrecognized layers of complexity in this metabolic pathway. High-throughput phenotyping facilitates the rapid exploration of the molecular basis of biological complexity, allowing for future detailed investigation of the selective pressures that drive microbial evolution.Citation: Homann OR, Cai H, Becker JM, Lindquist SL (2005) Harnessing natural diversity to probe metabolic pathways. PLoS Genet 1(6): e80.

A novel fungal family of oligopeptide transporters identified by functional metatranscriptomics of soil eukaryotes

“…Environmental cDNAs encoding dipeptide transporter genes were selected by the functional complementation of the W303 (MATa ura3 can1-100 ho ptr2D dal5D) S. cerevisiae mutant strain unable to use most dipeptides as N sources (Homann et al, 2005). Yeast cells transformed by the cDNA library were plated on a yeast nitrogen base minimal medium lacking uracil and containing the three dipeptides Tyr-Ala, Ala-Leu and Ala-Tyr as sole N sources (0.25 mM each).…”

“…Transformants were plated onto a selective medium, containing three dipeptides as N sources, Tyr-Ala was used by Ptr2p but not by Dal5p, Ala-Leu was used by Dal5p but not by Ptr2p, and Ala-Tyr was neither used by Ptr2p nor by Dal5p in the W303 background (Homann et al, 2005). Among 7.7 Â 10 5 screened transformants, 25 grew on the selective medium.…”

“…Nevertheless, although the chemical diversity of these oligomers is necessarily higher than the diversity of the monomers, the number and diversity of the corresponding identified transporters is far lower. Indeed, concerning N sources assimilation, only two di/tripeptide transporter coding genes have been identified in the yeast S. cerevisiae, PTR2 and DAL5 (Homann et al, 2005;Cai et al, 2007), representing two of the three known families of eukaryotic dipeptide transporters (Frølund et al, 2010). In S. cerevisiae, each of these two transporters display a broad substrate specificity.…”

“…For example, when overexpressed in yeast cells, Ptr2p alone could transport more than 100 distinct dipeptides. However, translational and post-translational regulation mechanisms limit Ptr2p levels in wild-type yeasts and as a consequence also limit the actual diversity of dipeptides that these strains can ultimately use as N sources (Homann et al, 2005).…”

“…These libraries can be explored for novel genes by expression in a model microorganism. In our case, we performed the functional complementation of a S. cerevisiae mutant defective in dipeptide assimilation as a result of the deletion of the PTR2 and DAL5 genes (Homann et al, 2005).…”