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Brettanomyces genomics: catching up with Saccharomyces

All in all, our knowledge of Brettanomyces is still very limited compared with the vast number of data available for S. cerevisiae, the model fungus par excellence. However, several research groups currently are trying to bridge this gap and unravel the peculiar genomic properties of this industrially highly relevant yeast. However, several important challenges remain.

First, despite the fact that some research groups reported ascospore formation (albeit at low frequency) of B. bruxellensis (e.g. CBS74 and CBS4914; Van der Walt et al., 1964) and B. anomalus (e.g. CBS8139; Th. Smith and van Grinsven, 1984) strains, it still remains unclear to what extent Brettanomyces can reproduce sexually, how often this occurs, and what the influence of these events is on the evolution of the species. The extreme karyotypic variability and frequently observed allotriploidy might hint towards the lack (or at least the rarity) of sexual reproduction. In line with this theory, an extensive genetic investigation of the Brettanomyces population within a winery (spanning several vintages) revealed that the same persistent clonal Brettanomyces population was responsible for wine spoilage in this winery for over half a century (Albertin et al., 2014).

Second, the genetic and molecular toolbox for Brettanomyces should be expanded in order to facilitate more in-depth genetic analysis. For example, an optimal transformation protocol is still lacking, auxotrophic mutants are scarce, and only a limited amount of dedicated commercial tools, such as plasmid vectors or gene arrays, are available (Schifferdecker et al., 2014). However, some initial attempts to develop molecular tools and protocols were conducted. Miklenic and coworkers developed various transformation protocols (based on spheroplast transformation, electroporation, and the LiAc/PEG method) and described the first (reported) genetic transformation of B. bruxellensis, using a kanMX4 marker cassette that allowed for selection on agar plates containing geneticin (Miklenic et al., 2013). Additionally, Schifferdecker and colleagues developed a URA3-deficient mutant strain and a plasmid (named P892, derived from pUC57) containing a functional URA3 gene of CBS2499 (Schifferdecker et al., 2014). Using these tools, an auxotrophic transformation system and an expression vector was developed, allowing overexpression of individual genes with a constitutive TEF1 promoter (Schifferdecker et al., 2016). While commercial microarray kits for Brettanomy- ces are still lacking, a first large-scale transcriptomic analysis of B. bruxellensis was recently performed (Tiukova et al., 2013). Using whole transcriptome sequencing, the authors managed to detect the expression of 3715 out of the 4861 annotated genes of CBS 11270, and the results elucidated survival strategies of this yeast in harsh environments. For example, they observed a low expression of genes involved in glycerol production and a high number of expressed sugar transporter genes, two mechanisms possibly accounting for the high competitiveness of B. bruxellensis in oxygen-limited and nutrient-deprived environments (Tiukova et al., 2013).

Third, to fully grasp the Brettanomyces population structure and evolutionary history, analysis of a broader collection of ecologically and geographically diverse strains is necessary. While the main focus thus far is put on wine spoilers (mainly originating from Australia), preliminary investigation of strains from different niches and geographical origins already revealed a large degree of intraspecific (phenotypic and genetic) diversity, and sometimes revealed links between phenotypic behaviour and source of isolation (Crauwels et al., 2014, 2015b). While some of these correlations might be due to genetic drift and shared population histories of strains originating from the same environment, some might reflect adaptive mechanisms developed in industrial niches. However, more elaborate studies, targeting the genetic and phenotypic characterization of large collections of diverse strains, will yield more information on some fundamental questions, and shed light on the currently obscure Brettanomyces evolutionary history. For example, it will be interesting to see if (and to what extent) Brettanomyces is domesticated, and how the Brettanomyces population is structured.

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