The title of this post may sound a little confusing or complex for those not familiar with the nomenclature of the field, but I’ll try my best to explain our recent results to you. As I’ve written many times previously on this blog ([1], [2], [3], [4], and [5]), I have been working with and researching yeast hybrids and their use in brewing for my PhD thesis. I’ve been focusing especially on lager yeast hybrids, i.e. Saccharomyces cerevisiae × Saccharomyces eubayanus. Research, both our own and in other labs, has shown that generating new lager yeast hybrids by breeding strains of S. cerevisiae with S. eubayanus is possible, and that these new hybrids often possess desirable properties compared to the parent strains. These properties include improved fermentation rate, better stress tolerance and a more diverse formation of aroma compounds. However, one trait that has been plaguing all de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours (POF, i.e. the spicy, clove-like aroma that is characteristic of wheat beers and Belgian-style beers). This is an undesirable trait that is inherited from the Saccharomyces eubayanus parent. It doesn’t matter if the other parent produces phenolic off-flavours or not, the de novo lager hybrid will always produce it, as it inherits the relevant genes (more on this below) from the S. eubayanus parent. So with this in mind, and the idea that it would be cool if you could combine properties from more than two parent strains into a hybrid, we started thinking and experimenting.

One way that it is possible to remove unwanted traits from a yeast strain, is through sporulation and meiotic recombination (note, this only works if the strain is heterozygous at the loci responsible for the trait). Sporulation would also give us the opportunity to add in features from a third parent strain, as one could mate the hybrid spore clone (having a single mating type) with a third parent strain. However, interspecies hybrids tend to be sterile (this is also true for animal hybrids such as mules or ligers), which means that they don’t form any viable spores. Therefore, one would think that this is an impossible route to go down. What is interesting though, is that studies have shown that sterility mainly afflicts allodiploid hybrids (i.e. the hybrid has inherited one copy of each chromosome from both parents, just like us humans), and that allotetraploid hybrids (i.e. the hybrid has inherited both copies of each chromosome from both parents) tend to remain fertile. With this in mind, we constructed a set of five hybrids from three parents. Two of these hybrids contained DNA from all three parent strains, and from one of these two hybrids we successfully removed POF formation. These hybrids were constructed with fertile allotetraploid intermediates (which were capable of efficient sporulation) using the following scheme:

Before I get into the properties of this set of eight strains, I’ll quickly go through the cause of the ‘POF phenotype’. Many strains of Saccharomyces produce vinyl phenols from hydroxycinnamic acids, and these phenolic compounds are considered undesirable in many beer types (especially lager beer). The most well-studied of these vinyl phenols is 4-vinyl guaiacol, which is formed from ferulic acid. We know, thanks to work by Mukai et al., that the ability of brewing yeast to produce volatile phenols is attributed to the adjacent PAD1 and FDC1 genes, both of which are needed for a POF+ phenotype. Wild yeast strains, such as S. eubayanus, tend to have functional PAD1 and FDC1 genes, while domesticated POF- brewing yeast have nonsense or frameshift mutations in these genes, making them non-functional (e.g. see the recent Gallone et al. paper in Cell). The biological function of this mechanism is to protect the cell from the toxic effects of hydroxycinnamic acids (mainly plants produce them as antibiotics), which is why one can expect to find only POF- strains among domesticated yeasts. So if any non-functional alleles of PAD1 or FDC1 are present in the hybrid genome, it should be possible to remove the POF+ phenotype through meiotic recombination (as we demonstrate).

For this set of eight strains, we began with three parent strains (P1-P3). Two of them are S. cerevisiae ale strains (P1: VTT-A81062, P2: WLP099) and one is the S. eubayanus type strain (P3: VTT-C12902). These strains were chosen for their varying properties. Of the three, P1 is the only strain that is able to use maltotriose during fermentation, P2 is the only strain that does not produce 4-vinyl guaiacol (i.e. it is POF-), while P3 is the cold-tolerant parent strain of lager yeast. We began by creating the three possible double hybrids (P1 × P2, P1 × P3, P2 × P3) through rare mating. Hybrid H1 (P1 × P3) was fertile (thanks to allotetraploidy), despite it being an interspecific hybrid, and it produced viable spores efficiently. So we sporulated Hybrid H1 and then mated a mixture of its spores with parent strain P2 to obtain the triple Hybrid T1 ((P1 × P3) × P2). Whole genome sequencing revealed that the sub-genome ratio in T1 was approximately 1:2:1 (P1:P2:P3). Hybrid T1 was also an allotetraploid and it was able to form viable spores. Knowing that the genome contained both functional and non-functional alleles of PAD1, we attempted to remove the POF+ phenotype from Hybrid T1 through sporulation according to the image below. What we did was sporulate Hybrid T1, isolate individual spore clones, and then screen them for the POF phenotype in media containing ferulic acid. POF+ spore clones would produce a strong clove-like aroma in the media, while POF- spore clones would not. Approximately 25% of the spore clones were POF− (as one would expect), and the best growing of these was given the name Hybrid T2, i.e. a POF− meiotic segregant of Hybrid T1.

We wanted to compare how these 8 brewing strains would perform in wort fermentations at lager brewing conditions (15 °P all-malt wort at 15 °C). There was considerable variation in fermentation performance between the eight strains, as you can see in the figure below (A). Of the 8 strains, Hybrid T1 and S. cerevisiae P2 had the highest overall fermentation rates, but these slowed down considerably after reaching 5.8% (v/v) alcohol. We looked at the sugars present in the beer, and saw that S. cerevisiae P2 was unable to ferment the maltotriose in the wort (D). Hybrid T1 had only consumed a small amount of the initial maltotriose present in the wort (D). Of the 8 strains, Hybrid T2 attenuated the best, followed closely by Hybrids H1 and H3 (A). These three hybrids had also used maltotriose efficiently (D). The beers produced with the 8 brewing strains also varied considerably in concentrations of aroma-active compounds (B). The most ester-rich (i.e. fruity) beers were produced with Hybrids H1, H3 and T1. When we compare the aroma profiles of the beers made with Hybrids T1 and T2, we see that the meiotic segregant T2 produced lower concentrations of most esters, while its beer contained higher concentrations of most higher alcohols. This could maybe be explained by it having lower activities or expression of alcohol acetyl transferases (e.g. ATF1 and ATF2). Of the 8 strains, the POF− S. cerevisiae P2 parent strain and Hybrid T2 were the only ones that did not produce any detectable amounts of 4-vinyl guaiacol (detection limit 0.2 mg L⁻¹), thus confirming their POF− phenotype (C). All other strains produced 4-vinyl guaiacol in concentrations above the flavour threshold of 0.3–0.5 mg L⁻¹. We finally compared the sequences of PAD1 and FDC1 in the 8strains, and found that Hybrid T2 only carried the PAD1 allele that was derived from S. cerevisiae P2 (E). This particular allele, contained a possible loss-of-function SNP at position 638 (A>G, resulting in an amino acid substitution of aspartate to glycine). The other strains, including Hybrid T1, which Hybrid T2 was derived from, carried either or both of the functional PAD1 alleles derived from S. cerevisiae P1 or S. eubayanus P3.

So there you have it, some cool things you can do with allotetraploid interspecific hybrids. We wanted to demonstrate that it is possible to construct complex yeast hybrids that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. If you are interested in some more info on the topic (e.g. some lipidomics analysis of these strains), I’m happy to announce that an article based on these results was recently published in Microbial Cell Factories:

Inheritance of brewing-relevant phenotypes in constructed Saccharomyces cerevisiae × Saccharomyces eubayanus hybrids

Kristoffer Krogerus, Tuulikki Seppänen-Laakso, Sandra Castillo, Brian Gibson

Microbial Cell Factories, 2017, 16:66. DOI:10.1186/s12934-017-0679-8

Please feel free to check it out (it is open access)! Here is the abstract:

Abstract

Background

Interspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity.

Results

Here we generated a set of five hybrids from three different parent strains, two of which contained DNA from all three parent strains. These hybrids were constructed with fertile allotetraploid intermediates, which were capable of efficient sporulation. We used these eight brewing strains to examine two brewing-relevant phenotypes: stress tolerance and phenolic off-flavour formation. Lipidomics and multivariate analysis revealed links between several lipid species and the ability to ferment in low temperatures and high ethanol concentrations. Unsaturated fatty acids, such as oleic acid, and ergosterol were shown to positively influence growth at high ethanol concentrations. The ability to produce phenolic off-flavours was also successfully removed from one of the hybrids, Hybrid T2, through meiotic segregation. The potential application of these strains in industrial fermentations was demonstrated in wort fermentations, which revealed that the meiotic segregant Hybrid T2 not only didn’t produce any phenolic off-flavours, but also reached the highest ethanol concentration and consumed the most maltotriose.

Conclusions

Our study demonstrates the possibility of constructing complex yeast hybrids that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. Yeast lipid composition was also shown to have a central role in determining ethanol and cold tolerance in brewing strains.