RESUMO
In winemaking, slow or stuck alcoholic fermentation can impact processing efficiency and wine quality. Residual fructose in the later stages of fermentation can leave the wine 'out of specification' unless removed, which requires reinoculation or use of a more fructophilic yeast. As such, robust, fermentation efficient strains are still highly desirable to reduce this risk. We report on a combined EMS mutagenesis and Directed Evolution (DE) approach as a 'proof of concept' to improve fructose utilization and decrease fermentation duration. One evolved isolate, Tee 9, was evaluated against the parent, AWRI 796 in defined medium (CDGJM) and Semillon juice. Interestingly, Tee 9 exhibited improved fermentation in CDGJM at several nitrogen contents, but not in juice. Genomic comparison between AWRI 796 and Tee 9 identified 371 mutations, but no chromosomal copy number variation. A total of 95 noncoding and 276 coding mutations were identified in 297 genes (180 of which encode proteins with one or more substitutions). Whilst introduction of two of these, Gid7 (E726K) or Fba1 (G135S), into AWRI 796 did not lead to the fermentation improvement seen in Tee 9, similar allelic swaps with the other mutations are needed to understand Tee 9's adaption to CDGJM. Furthermore, the 378 isolates, potentially mutagenized but with the same genetic background, are likely a useful resource for future phenotyping and genome-wide association studies.
Assuntos
Vitis , Vinho , Variações do Número de Cópias de DNA , Fermentação , Frutose/metabolismo , Estudo de Associação Genômica Ampla , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vitis/metabolismoRESUMO
When investigating yeast gene function in relation to fermentation, many screens rely on haploid yeast derivatives. This, however, is not representative of industrial strains, which are typically diploid. One such example is the disruption of ECM33, which was associated with improved fermentation in the haploid wine yeast C911D, but remains uncharacterised in a diploid industrial strain background. We report on the homozygous disruption of ECM33 in Lalvin EC1118 using CRISPR/Cas9. EC1118 ecm33 resulted in a reduction of fermentation duration in a defined medium with limiting and sufficient nitrogen (-20% and -13%, respectively) when shaken. Increased cell size and aggregation, a phenotype previously unidentified in ecm33∆ as haploid yeast tend to aggregate, was also observed. This phenotype led to premature settling thereby the yeast behaving similarly to EC1118 in wine-like semi-static fermentations in a chemically defined medium. Further assessment in semi-static Riesling and Chardonnay fermentations inoculated based on cell number or biomass resulted in no significant difference or significantly slower fermentation duration in comparison the EC1118, nullifying the benefits of this mutation unless agitation is applied. This study draws attention to phenotypes being condition-dependent, highlighting the need to characterise and verify fermentation efficiency mutations in industrial yeast.
Assuntos
Proteínas de Saccharomyces cerevisiae , Vinho , Diploide , Fermentação , Proteínas de Membrana , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vinho/análiseRESUMO
A deficiency of nitrogenous nutrients in grape juice can cause stuck and sluggish alcoholic fermentation, which has long been a problem in winemaking. Nitrogen requirements vary between wine yeast strains, and the ability of yeast to assimilate nitrogen depends on the nature and concentration of nitrogen present in the medium. In this study, a wine yeast gene deletion collection (1844 deletants in the haploid AWRI1631 background) was screened to identify genes whose deletion resulted in a reduction in the time taken to utilise all sugars when grown in a chemically defined grape juice medium supplemented with limited nitrogen (75 mg L-1 as a free amino acid mixture). Through micro-scale and laboratory-scale fermentations, 15 deletants were identified that completed fermentation in a shorter time than the wildtype (c.a. 15%-59% time reduction). This group of genes was annotated to biological processes including protein modification, transport, metabolism and ubiquitination (UBC13, MMS2, UBP7, UBI4, BRO1, TPK2, EAR1, MRP17, MFA2 and MVB12), signalling (MFA2) and amino acid metabolism (AAT2). Deletion of MFA2, encoding mating factor-a, resulted in a 55% decrease in fermentation duration. Mfa2Δ was chosen for further investigation to understand how this gene deletion conferred fermentation efficiency in limited nitrogen conditions.