Future directions for breeding and genetic manipulation of brewing yeasts

Recent advances in DNA sequencing technologies, as well as novel applications of older genetic techniques - especially when such technologies are combined - hold much promise for the future development of brewing-related yeast strains. These methods, discussed below, include directed construction of hybrid yeasts, genome shuffling, ‘classic' genetic modification techniques, and recent techniques for minimally invasive genetic modifications of genomes.

The future of hybrid yeasts in brewing

Novel yeast strains created through both interspecific (mating between two strains of different species) and intraspecific (mating between two strains of the same species) yeast hybridizations have been commercialized in industries such as biofuels and wine; it does not appear that any such ‘laboratory-made' hybrids are currently used commercially in the brewing industry. Whether this is due to the potential perception (warranted or not) oflaboratory-made hybrids as ‘genetically modified organisms' (GMOs), a previous failure to obtain successful beer strains through hybridization, or for other intangible reasons, it is clear that recent discoveries from whole-genome sequencing, along with advances in genetic techniques, make it easier and possibly more rewarding than ever to carry out creative hybridizations using brewing yeasts. For example, in the wine arena, interspecific hybrids between many of the different members of the Sac- charomyces genus have been constructed via the rare mating method, using a diploid wine yeast as the S. cerevisiae parent; the resulting hybrids often provide unique and desirable sensory characteristics to wine, and some of the strains are now being used commercially (Bellon et al., 2011, 2013, 2015).

There is some movement towards these types of studies in the beer arena. With the recent discovery of free-living S. eubayanus as the non-S. cerevisiae component of the lager yeast genome (Libkind et al., 2011), novel lager-like yeast hybrids are beginning to be deliberately constructed to create strains with unique fermentation, flavour, and aroma characteristics, as discussed above in the ‘Direct mating' section (Krogerus et al., 2015; Mertens et al., 2016). Phenotypic screens of ale yeasts (see, for example, Steensels et al., 2014a), including ‘heirloom' strains (see, for example, Parker et al., 2014), has highlighted the diversity that exists for aroma profiles and other traits among these yeasts (Fig. 5.2A). The wide spectrum of different ale yeasts that can serve as the S. cerevisiae parent, along with the very recent isolations of several genetically diverse and geographically far-flung strains of S. eubayanus (Bing et al., 2014; Peris et al., 2014, 2015; Baker et al., 2015), points to an expanding universe of possible ‘lagerlike' hybrid combinations. Deliberate intraspecific hybridization (e.g. between different strains of S. cerevisiae yeasts; see discussion above of Steensels et al., 2014a, crosses of ale and sake yeasts, and Kvitek, unpublished results of crossing two different saison yeasts) appears to have been more rarely studied among brewing strains than interspecific hybridizations. This is possibly because lager beer is far and away the dominant beer style in the world, and thus experiments to optimize lager yeasts may be more common and/or able to be funded. Overall, experimental hybridization studies have not been thoroughly explored in a brewing context, and may provide increased genetic diversity that can subsequently be screened/selected for novel and desirable features (Figs. 5.3 and 5.4). Additionally, intergeneric yeast hybrids - mainly obtained through protoplast fusion - have been explored in other industrial applications (Morales and Dujon, 2012; Steensels et al., 2014b) but not in brewing yeasts; this approach may provide a wealth of strains with even more traits of interest.

Genome shuffling to combine traits and/or to discover the genetic basis of phenotypic traits in brewing yeasts

Other genetic techniques, based on combining and/or mixing the genomes of strains, offer new possibilities for brewing yeast strain construction. Because virtually all brewing strains are non-mating (due to being of diploid or even higher ploidy, or aneuploid), it is not possible to just merely mate two such strains together to mix their genomes (as one can do with stably haploid, HO-mutant laboratory strains). However, it is often possible to perform mass mating of spores, or mass cell fusion, to rapidly combine desired traits from two or more brewing yeast strains into a single strain. These techniques can also work for starting strains that are heterogeneous, such as mixed populations (for example, a mutagenized population of cells, or a pool of meiotically recombined cells). First, a very large number of cells (or spores, if the strains can produce viable spores) from each starting ‘parental' population are generated (note that there can be one, two, or even more starting populations); they are then all mixed together, allowing random mating (if haploid cells or spores) or random cell fusion (if asexual cells are made into protoplasts) to occur. This leads to a mixing of the genotypes of the various starting populations into single cells, often resulting in cells bearing the combined traits of interest. If selection or enrichment for cells carrying the desired combination of traits is possible, several rounds of mass mating (or mass fusion) followed by selection can be performed iteratively to give further refinement or stronger expression of the desired phenotypic traits. In evolutionary terms, this process consists of genetic recombination followed by natural selection of genotypes displaying advantageous phenotypes in the imposed environmental condition, repeated over several generations.

The goal in these typical mass-mating or mass- fusion methods has usually been to achieve a stable hybrid line that expresses the desired traits. Related techniques that result in ‘genome shuffling' may prove to be a valuable research tool to discover the genetic bases of phenotypic traits important in beer production, and may also be of use in generating novel strains that combine beneficial traits. Genome shuffling experiments are similar to the mass-mating or mass-fusion methods, but are performed such that genome recombination occurs at every round, eventually mixing the two starting genomes together into single cells, but in a very patchwork manner that varies from cell to cell, thus allowing different phenotypic combinations to be observed. Again, this can be used to bring together and enhance many positive traits from different backgrounds into one superior strain. But the recombined populations can also be used to map where the locations of genes controlling the traits reside. Brief descriptions of techniques and examples from the brewing industry (if available) are discussed below.

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