RESUMO
Severe oenological conditions, such as limited assimilable nitrogen and high sugar contents restrict yeast's ability to successfully complete fermentation. In the absence of a comprehensive commercially available deletion collection in a wine yeast background, a screening approach was applied to a transposon library in a wine yeast derivative to identify clones with superior fermentation performance. Five candidate genes, when disrupted by Ty insertion, were identified as enabling yeast to efficiently complete a model oenological fermentation with limited nitrogen availability. Analogous single gene disruptions were subsequently constructed in the haploid wine yeast strain C911D, and the performance of these during fermentation was analysed. Deletion of ECM33 resulted in the shortest fermentation (up to 31% reduction) in both synthetic medium and grape juice. Interestingly, no significant differences were found in nitrogen utilization, cell viability or biomass yield between ∆ecm33 and the wild type. ∆ecm33 did, however, display growth hypersensitivity to the dyes Calcofluor White and Congo Red, suggesting a link to cell wall integrity. Transcriptional profiling of ∆ecm33 during fermentation demonstrated the up-regulation of SLT2 and HOG1, encoding mitogen activated protein kinases involved in the cell wall integrity (CWI) and high osmolarity glycerol (HOG) pathways, respectively. CHS3 a major chitin synthase gene was also found to be upregulated, and the transcript abundance of key genes of central nitrogen metabolism, GLN1, GLT1, GDH1 and GDH2 in mutant ∆ecm33 were also altered. The findings highlight the complexity of the robust fermentation phenotype and provide clues for further improvement of industrial strains.