I’ve been bad at updating the blog lately, as I have been busy writing on my thesis, taking care of our 1.5 year old son, and being involved in various smaller brewing research projects at work. We’ve had a number of results published during the autumn, including two papers on kveik (here and here) and one on adaptive evolution of a low-diacetyl lager strain (here). I’ll write a separate post about those if I have time. Anyways, the point of this post was to share an updated version of the brewing yeast family tree I’ve blogged about a couple times before. An interesting preprint, A polyploid admixed origin of beer yeasts derived from European and Asian wine populations by Fay et al. (link), was uploaded recently to bioRxiv. In it they propose that the Ale beer / Beer 1 strains are derived from admixture between strains of the Sake/Asian and European Wine populations. In the study they sequenced a number of commercial Wyeast, Fermentis and Lallemand strains, which I retrieved the sequence data for (Bioproject PRJNA504476) and added to the previous version of the tree consisting mainly of White Labs strains (here and here). Below you’ll find the tree in PDF format (click on the image below) together with some observations by ‘qq’ and me. For clarity I decided to keep out the non-brewing strains from the 1011 yeast genomes.

Here are some comments from ‘qq’ (with minor modification by me):

Safbrew WB-06 and Wyeast 1388 Belgian Strong (“Duvel”) – With both of them STA1+, it is no great surprise to see them both up in Beer2 near WLP570 which supposedly came to Duvel from McEwans.

Lallemand BRY-97 – Surprisingly, this strain doesn’t group with the Beer 1 US strains, but rather in the Mixed group. As supposedly one of the key strains in the story of US yeast going from East to West, what is this doing here and not in the main US group?
Muntons English – Presumably not Munton’s Gold but the “ordinary” Munton’s dry yeast, which shows up in a lot of kits. The story goes that this was the old EDME yeast related to Windsor/S-33 which is consistent with what we see here.

Brewferm Lager – not on the chart but according to Table S2 this falls in the Mixed group.

Lallemand Munich – with the other German hefeweizen strains as you’d expect
Wyeast 3068 – supposedly Weihenstephan 68, the classic German wheat (and supposedly the origin of Danstar Munich Classic?)

Wyeast 1007 German – the internet had thought this could be close to K-97 and WLP036, but WLP003 German II makes sense
Wyeast 2565 Kolsch – Makes sense that it’s close to our old friend WLP800 Pilsner.
Wyeast 3463 Forbidden Fruit – assumed to be from Hoegaarden Verboden Vrucht (link), plausible that it’s in that WLP410/510 Belgian Wit II group although actually WLP400 Belgian Wit is meant to be Hoegaarden
Wyeast 3787 Trappist HG – supposedly from Westmalle, close to that WLP400 “Hoegaarden” and WLP530 “Abbey Ale”
Wyeast 3942 Belgian Wheat – supposedly from De Dolle, a Belgian brewery not known for its wits but the yeast falls in that wit group.

Wyeast 1764 Pacman, Wyeast seem to have stopped offering Rogue’s yeast from their Private Collection but Imperial A18 Joystick is meant to be the same. Supposed to be a better-behaved derivative of Chico and this seems to confirm that ancestry.

Wyeast 1275 Thames Valley (“Brakspear”) – close to WLP023 Burton Ale which despite the name is also meant to come from Brakspear.
Wyeast 1332 Northwest – not surprising that it’s close to WLP041 Pacific (“Redhook”) in the WLP002/007 group as it’s meant to come from Hales of Seattle. Mike Hale spent a year in England at Gale’s and brought the yeast back with him (link) and a former brewer at Gale’s has specifically said Gale’s used Whitbread B (link). Supposedly Gale’s got their yeast from Brickwoods, the main brewery in Portsmouth who were bought by Whitbread in 1971. This supports the idea that the WLP002/007 group represents the Whitbread B family, perhaps the most important group of British industrial yeasts. The Gale’s yeast is now used by Marble among others.
Wyeast 1968 London ESB – bit surprising that it’s not closer to WLP002 English since the internet reckons they both come from Fuller’s. But neither of them seem to quite have the “marmalade-iness” of real Fuller’s beer, either they’ve mutated or weren’t actually from Fuller’s in the first place.
Escarpment Labs Vermont Ale – The ‘classic’ NEIPA strain is closely related to Wyeast 1968 in the Whitbread B group.
Coopers Australian Ale Yeast – presumably the dry yeast from their kits? Seems to be an outlier of the main UK Beer1 group which makes sense for an Australian yeast if somewhat distant from WLP009 Australian Ale also supposedly from Coopers.
Wyeast 1098 British Ale – Wyeast 1098 and 1099 are both meant to come from Whitbread, and you will see tables on the internet saying that 1098 is equivalent to WLP007 Dry English. It’s clearly not, it’s close to WLP017 Whitbread II  (an elusive Vault strain) and 1318 London Ale III. It’s a shame that we don’t have sequence for 1099 but its brewing numbers suggest that it’s not much like WLP007 either.
Wyeast 1318 London Ale III – Seems to be another member of that little Whitbread II subfamily.  Traditionally it’s linked to Boddington’s which I never quite believed but Boddies had all sorts of yeast problems in the 1980s and were bought by Whitbread in 1989 so it’s plausible that the original yeast was ultimately replaced by one from the yeast bank at head office (perhaps after they’d tried others?). 1318 is a super-fashionable strain that everyone seems to be using for NEIPAs and is known for hop biotransformation, so it might be interesting to test its relatives for that.
Wyeast 1945 NeoBritannia – An exclusive that Wyeast used to do for Northern Brewer before the ABInBev takeover. Close relative of 1318 in the Whitbread II group.
Wyeast 1469 West Yorkshire – Was fully expecting this to be a Beer2 strain! 1469 is meant to come from Timothy Taylor, who got their yeast from Oldham, who got their yeast from John Smith’s. The John Smith yeast also went to Harvey’s (the source of VTT-A81062, a Beer2 strain). So it’s a bit of a surprise that 1469 is in the heart of the UK Beer1 strains, closest to WLP022 Essex (“Ridleys”). So either the traditional stories aren’t true, there’s been contamination/mixups, or we’re looking at John Smith being some kind of multistrain with both Beer 1’s and Beer 2’s in it.
Wyeast 1028 London Ale (“Worthington White Shield”) and Wyeast 1728 Scottish Ale “McEwans” – wasn’t expecting them to be so close, and for 1728 to be so far from WLP028 Edinburgh (also “McEwans” but at other end of main UK group). Also interesting to see them close to the WLP011 European and WLP072 French pairing, and some way from WLP013 which is also meant to be from White Shield.
Wyeast 1187 Ringwood – as expected close to WLP005 British and NCYC1187. In general Wyeast strains seem to have diverged more than White Labs, and this is a good example.
Safale S-04 – Closely related to WLP006 Bedford (“Charles Wells”) and WLP013 London, even though internet tradition always called it a dry version of Whitbread B. It’s nowhere close to the Whitbread strains.

Hopefully this should be useful both for finding yeast substitutes and elucidating the history of these strains. As genome sequencing becomes cheaper and more accessible all the time, we will certainly be able to update the tree with more strains in the future.

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.