Deletion of QDR genes in a bioethanol-producing yeast strain reduces propagation of contaminating lactic acid bacteria.

Bacterial contaminations in yeast fermentation tanks are a recurring problem for the bioethanol production industry. Lactic acid bacteria (LAB), particularly of the genus Lactobacillus, are the most common contaminants. Their proliferation can reduce fermentation efficiency or even impose premature shutdown for cleaning. We have previously reported that laboratory yeast strains naturally excrete amino acids via transporters of the Drug: H+ Antiporter-1 (DHA1) family. This excretion allows yeast to cross-feed LAB, which are most often unable to grow without an external amino acid supply. Whether industrial yeast strains used in bioethanol production likewise promote LAB proliferation through cross-feeding has not been investigated. In this study, we first show that the yeast strain Ethanol Red used in ethanol production supports growth of Lactobacillus fermentum in an amino-acid-free synthetic medium. This effect was markedly reduced upon homozygous deletion of the QDR3 gene encoding a DHA1-family amino acid exporter. We further show that cultivation of Ethanol Red in a nonsterile sugarcane-molasses-based medium is associated with an increase in lactic acid due to LAB growth. When Ethanol Red lacked the QDR1, QDR2, and QDR3 genes, this lactic acid production was not observed and ethanol production was not significantly reduced. Our results indicate that Ethanol Red cultivated in synthetic or molasses medium sustains LAB proliferation in a manner that depends on its ability to excrete amino acids via Qdr transporters. They further suggest that using mutant industrial yeast derivatives lacking DHA1-family amino acid exporters may be a way to reduce the risk of bacterial contaminations during fermentation.

Overlapping Roles of Yeast Transporters Aqr1, Qdr2, and Qdr3 in Amino Acid Excretion and Cross-Feeding of Lactic Acid Bacteria.

Microbial species occupying the same ecological niche or codeveloping during a fermentation process can exchange metabolites and mutualistically influence each other's metabolic states. For instance, yeast can excrete amino acids, thereby cross-feeding lactic acid bacteria unable to grow without an external amino acid supply. The yeast membrane transporters involved in amino acid excretion remain poorly known. Using a yeast mutant overproducing and excreting threonine (Thr) and its precursor homoserine (Hom), we show that excretion of both amino acids involves the Aqr1, Qdr2, and Qdr3 proteins of the Drug H+-Antiporter Family (DHA1) family. We further investigated Aqr1 as a representative of these closely related amino acid exporters. In particular, structural modeling and molecular docking coupled to mutagenesis experiments and excretion assays enabled us to identify residues in the Aqr1 substrate-binding pocket that are crucial for Thr and/or Hom export. We then co-cultivated yeast and Lactobacillus fermentum in an amino-acid-free medium and found a yeast mutant lacking Aqr1, Qdr2, and Qdr3 to display a reduced ability to sustain the growth of this lactic acid bacterium, a phenotype not observed with strains lacking only one of these transporters. This study highlights the importance of yeast DHA1 transporters in amino acid excretion and mutualistic interaction with lactic acid bacteria.

The Bul1/2 Alpha-Arrestins Promote Ubiquitylation and Endocytosis of the Can1 Permease upon Cycloheximide-Induced TORC1-Hyperactivation.

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.

Multiple nucleobase transporters contribute to boscalid sensitivity in Aspergillus nidulans.

The development of fungicide-resistant fungal populations represents a major challenge for the agrochemical and agri-food sectors, which threatens food supply and security. The issue becomes complex for fungi that cause quantitative and qualitative losses due to mycotoxin biosynthesis. Nonetheless, currently, the molecular details underlying fungicide action and fungal resistance mechanisms are partially known. Here, we have investigated whether plasma membrane transporters contribute to specific fungicide uptake in the model fungus Aspergillus nidulans. Independent physiological tests and toxicity screening of selected fungicides provided evidence that the antifungal activity of Succinate Dehydrogenase Inhibitors (SDHIs) is associated with the expression of several nucleobase-related transporters. In particular, it was shown that a strain genetically inactivated in all seven nucleobase-related transporters is resistant to the fungicide boscalid, whereas none of the single null mutants exhibited significant resistance level. By constructing and testing isogenic strains that over-express each one of the seven transporters, we confirmed that five of them, namely, UapC, AzgA, FycB, CntA, and FurA, contribute to boscalid uptake. Additionally, by employing metabolomics we have examined the effect of boscalid on the metabolism of isogenic strains expressing or genetically lacking boscalid-related nucleobase transporters. The results confirmed the involvement of specific nucleobase transporters in fungicide uptake, leading to the discovery of corresponding metabolites-biomarkers. This work is the first report on the involvement of specific transporters in fungicide uptake and toxicity and their impact on fungal metabolism regulation and results might be further exploited towards the deeper understanding of fungal resistance to fungicides.