Paul A. Henschke

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene.

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3‐phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild‐type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild‐type and GPD2 overexpressing strains (GPD2‐OP) decreased acetic acid production by three‐ and four‐fold, respectively. In conjunction with reduced acetic acid production, the GPD2‐OP ald6Δ strain produced more glycerol and less ethanol than the wild‐type. The growth rate and fermentation rate were similar for the modified and wild‐type strains, although the fermentation rate for the GPD2 ald6Δ strain was slightly less than that of the other strains from 24 h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour‐active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains. Copyright

AFLP fingerprinting for analysis of yeast genetic variation

Amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains, type strains and winery isolates from a number of yeast species. AFLP was shown to be effective in discriminating closely related strains. Furthermore, sufficient similarity in the fingerprints produced by yeasts of a given species allowed classification of unknown isolates. The applicability of the method for determining genome similarities between yeasts was investigated by performing cluster analysis on the AFLP data. Results from two species, Saccharomyces cerevisiae and Dekkera bruxellensis, illustrate that AFLP is useful for the study of intraspecific genetic relatedness. The value of the technique in strain differentiation, species identification and the analysis of genetic similarity demonstrates the potential of AFLP in yeast ecology and evolutionary studies.

Differentiation and species identification of yeasts using PCR

A PCR-based method has been developed that permits both intraspecies differentiation and species identification of yeast isolates. Oligonucleotide primers that are complementary to intron splice sites were used to produce PCR fingerprints that display polymorphisms between different species of indigenous wine yeasts. Although polymorphisms existed between isolates of the same species, the banding patterns shared several amplification products that allowed species identification. Importantly, the method was able to distinguish between species of the closely related Saccharomyces sensu stricto yeasts. In two cases where isolates could not be positively identified there was discrepancy between the phenetic and phylogenetic species concept. The method has applications in yeast ecological studies, enabling the rapid grouping of isolates with related genomes and the investigation of population dynamics of strains of the same species.

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation.

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer‐sensitive interactions were observed at pH 4·5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3·5, the incidence of killer‐sensitive interactions was reduced by 70% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.