As I mentioned a couple of posts ago, the research project I’ve been dedicating most of my time to the past 18 months is one based on the adaptive evolution of newly created lager hybrids. Our manuscript based on the work was accepted in late November 2017, and today the article was published in the latest issue of Applied and Environmental Microbiology. Please have a look if you are interested!

I thought I’d write up a short summary of our results. It is known that the genomes of newly created hybrids (particularly interspecies ones) tend to be quite unstable. This means that their genomes, along with their phenotypic properties, can change as the hybrid is grown for several generations. This is not very desirable in a brewing environment, where yeast is reused for multiple consecutive fermentations. We decided to exploit this instability, by growing (and adapting) some of our hybrids in media containing high ethanol concentrations for 130+ generations.

We started with four yeast strains, one a S. cerevisiae strain, while the other three were S. cerevisiae × S. eubayanus hybrids. The S. cerevisiae strain was A-81062, an ale strain that was a common parent strain for all three hybrids (this is Y1 in the study). The hybrids were all produced in our previous studies, and they included an allotetraploid one (Y2), an allotriploid one (Y3), and a POF- meiotic segregant, which was approximately diploid (Y4). The allotetraploid hybrid had two copies of the chromosomes from both parent strains, while the allotriploid hybrid had two copies of the S. cerevisiae chromosomes, but only one copy of the S. eubayanus chromosomes. We were interested in seeing if higher ploidy levels resulted in increased adaptation. This is because higher ploidy hybrids tend to be more unstable, and in layman’s terms ‘have more starting material’ available for adaptation.

What we then did with these four yeast strains, was grow them in 30 consecutive small-scale fermentations in two different growth media containing 10% ethanol (see (A) in image above). We took samples after 10, 20 and 30 fermentations, and isolated single cell isolates on agar (B). During the small-scale fermentations, we monitored optical density to see if there was an improvement in growth over time. This there was for all four yeast strains as seen in the image below:

We then performed high-throughput screening of all our isolates using a liquid handling robot and cultivations in 96-well plates (C). The growth media contained malt extract and was again supplemented with ethanol. We monitored sugar concentrations using HPLC and selected isolates which fermented the fast. We saw an improvement in fermentation speed compared to the original strains for the majority of the adapted isolates. We then selected a couple of the best-performing isolates from each yeast strain, and performed small-scale wort fermentations with them (D). Again, we saw that most adapted isolates performed better than the original (wild-type) strains:

After this, we selected two isolates from each strain (one from each growth media used during the adaptation process) and performed 2L-scale wort fermentations with them (E). We monitored these fermentations more carefully, and analysed the resulting beers afterwards for aroma compounds. We not only saw that the isolates again appeared to ferment faster (Y1-Y4: A-D):

The adapted isolates also appeared to produce lower concentrations of undesirable aroma compounds such as higher alcohols and diacetyl, while producing higher concentrations of many desirable aroma compounds such as esters:

So, to sum up the results up until this point: by growing the yeast strains in an high ethanol environment, we were able to isolate adapted strains that not only appeared to ferment faster in wort, but also produced beer with a more desirable aroma profile. Interestingly, we saw this improvement with all four strains, not only the hybrids!

What we wanted to do next was try to elucidate what genomic changes had occured, and try to see if we could link any of them to the phenotype change that we observed. We sequenced the adapted strains (Illumina 150bp paired-end) and compared them with the original strains. First we looked at overall DNA content (measured with flow cytometry and SYTOX Green staining), and saw quite large changes in many of the adapted strains. I won’t copy the numbers in here (have a look in the article if you are interested), but we saw quite large decreases in DNA content in the isolates obtained from the tetraploid hybrid, while the isolates remained more similar (one exception though). We then looked at copy number changes of individual chromosomes. We saw, for example, that the hybrids on average tended to lose more of their S. eubayanus chromosomes, and that S. cerevisiae chromosomes VII and XIV were amplified in multiple isolates:

Interestingly, chromosome VII of A-81062 contains multiple maltose/maltotriose transporters, and we did actually see a positive correlation between the sugar utilization rate and estimated copy numbers of these maltose transporters:

We also looked at single nucleotide polymorphisms occuring in the coding sequence of genes that resulted in amino acid changes. We found several mutations that hit common genes across the adapted isolates, such as IRA2, HSP150 and MNN4. In addition, some genes known to be linked with ethanol tolerance, such as UTH1, were affected.

So while there were no very obvious genomic changes that would explain the improved phenotype in the adapted isolates, we saw several factors that appeared to contribute to it. We unfortunately weren’t able to test them through reverse engineering as this was outside the scope of the current project, but it would definitely be interesting to do so in the future!

Our study demonstrates the possibility of improving newly created lager yeast hybrids (and also ale strains!) through adaptive evolution by generating stable and superior variants that possess traits that are desirable in beer fermentation (fast fermentation rate and desirable aroma profile). Thanks for reading!

Abstract:

Interspecific hybridization is a valuable tool for developing and improving brewing yeast in a number of industry-relevant aspects. However, the genomes of newly formed hybrids can be unstable. Here, we exploited this trait by adapting four brewing yeast strains, three of which were de novo interspecific lager hybrids with different ploidy levels, to high ethanol concentrations in an attempt to generate variant strains with improved fermentation performance in high-gravity wort. Through a batch fermentation-based adaptation process and selection based on a two-step screening process, we obtained eight variant strains which we compared to the wild-type strains in 2-liter-scale wort fermentations replicating industrial conditions. The results revealed that the adapted variants outperformed the strains from which they were derived, and the majority also possessed several desirable brewing-relevant traits, such as increased ester formation and ethanol tolerance, as well as decreased diacetyl formation. The variants obtained from the polyploid hybrids appeared to show greater improvements in fermentation performance than those derived from diploid strains. Interestingly, it was not only the hybrid strains, but also the Saccharomyces cerevisiae parent strain, that appeared to adapt and showed considerable changes in genome size. Genome sequencing and ploidy analysis revealed that changes had occurred at both the chromosome and single nucleotide levels in all variants. Our study demonstrates the possibility of improving de novo lager yeast hybrids through adaptive evolution by generating stable and superior variants that possess traits relevant to industrial lager beer fermentation.

IMPORTANCE Recent studies have shown that hybridization is a valuable tool for creating new and diverse strains of lager yeast. Adaptive evolution is another strain development tool that can be applied in order to improve upon desirable traits. Here, we apply adaptive evolution to newly created lager yeast hybrids by subjecting them to environments containing high ethanol levels. We isolated and characterized a number of adapted variants which possess improved fermentation properties and ethanol tolerance. Genome analysis revealed substantial changes in the variants compared to the original strains. These improved variant strains were produced without any genetic modification and are suitable for industrial lager beer fermentations.

 

Citation: Krogerus K, Holmström S, Gibson B. 2018. Enhanced wort fermentation with de novo lager hybrids adapted to high-ethanol environments. Appl Environ Microbiol 84:e02302-17. https://doi.org/10.1128/AEM.02302-17.