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.

Thanks to some amazing contributions and discussions in the comment section of my previous post on this article (special thanks to ‘qq’), we’ve now managed to identify (its at least a good estimation) the majority of the White Labs strains from the set of brewing strains in the Gallone et al. 2016 paper.

In addition to the genotype data, the Gallone et al. 2016 paper contains a huge amount of phenotypic data on these strains, and this information could be very useful for (home)brewers as well now that we know which strains are which (at least most of them). I’ve compiled the following spreadsheet (with the help of ‘qq’) containing all the identifications and useful phenotypic data (e.g. POF, flocculation, sugar use, various tolerances, and ester concentrations). See the ‘Notes’ sheet for more information. Press the icon in the lower right corner to open the spreadsheet in a new window (recommended). Please feel free to leave feedback or suggestions for improvement if you find something unclear or missing!

[embeddoc url=”http://beer.suregork.com/wp-content/uploads/2017/12/White_Labs_Gallone_v1_1.xlsx” download=”none” viewer=”microsoft”]

Updated family trees

I also put together the following dendrograms displaying the White Labs codes for those interested in how they look in the family tree (click to enlarge). As an added bonus, if anyone is wondering where the lager yeasts fit in on the tree, I’ve expanded it with the S. cerevisiae sub-genomes of two Frohberg strains (‘A15’ (VTT-A63015) and W34/70; names in purple) and two Saaz strains (CBS1503 and CBS1538; names in blue). We’ve sequenced A15 ourselves, while the other sequence reads were pulled from Okuno et al. 2016.

In addition, the following dendrograms are also available:

• Higher resolution, including the bootstrap support values
• Dendrogram with the original strain codes

‘qq’ also put together the following dendrograms (Beer 1 and Beer 2 groups separately) from Figure 1 in the original study:

There are some really interesting observations that can be made, e.g.

• WLP540, the ‘Rochefort’ strain, appears to be of British origin (POF- as well), and not related to other Trappist strains.
• The group of ‘US’ strains appears to contain one English and one Belgian strain. The Belgian one could be WLP515.
• WLP800 and WLP320 appear closely related, and their phenotypes seem very similar. Anyone want to try making a lager with WLP320 American Hefeweizen?
• Not related to the White Labs strains at all, but two strains used for industrial lager production (Beer039 and Beer040) appear to be closely related to various Belgian Ale strains in the Beer2 group.
• As can be seen, the lager yeasts branch off in the beginning of the ‘Beer 1’ clade together with the Hefeweizen strains. This is similar to the result obtained by Goncalves et al. 2016.

Phenotypic diversity

I’ve also recreated the heatmap from Figure 3 in the original publication, now with the White Labs codes in place of the strain codes. Strains (rows) that are grouped closely together are phenotypically similar. You can see a nice division between the ‘Beer 1’ strains and the rest of the strains. Click to enlarge.

Furthermore, I performed Principal Component Analysis (PCA) on the phenotype dataset, and below you can find the scores and loadings plots for ‘PC1 vs PC2’ and ‘PC1 vs PC3’. These first three principal components explained about 40% of the variation in the dataset. In layman’s terms, you can interpret the plots in the following way:

• Strains close to each other on the ‘scores’ plot (top) are phenotypically similar.
• By looking at the corresponding location in the ‘loadings’ plot (bottom), you can find what phenotypic traits are strongly associated with those particular strains.
• The larger the magnitude of the phenotypic trait (i.e. the further from the origin), the more it contributes to these principal components.
• E.g. In the top of the ‘scores’ plot for ‘PC1 vs PC2’ we find ‘wine018’, ‘wine017’ and ‘WLP050’. Looking at the ‘loadings’ plot, we see that these strains are strongly associated with high production of ethyl, isoamyl, isobutyl and phenylethyl acetate, as well as isoamyl alcohol. The particular traits have a large effect on PC2.
• Use these plots e.g. to find similar strains (if you want to substitute one yeast for another for example), or strains associated with particular traits.

PC1 vs PC2

PC1 vs PC3

Presence of STA1 in the strains

Following the recent news about a potential S. cerevisiae var. diastaticus contamination in White Labs yeast, I also did a BLAST search for STA1 (GenBank: X02649.1), the gene encoding for an extracellular glucoamylase, in each of the assemblies. Interestingly, there were full hits in multiple strains (partial matches in many strains as well). The strains are:

• Beer002
• Beer013
• Beer059 (probably WLP026)
• Beer085 (very likely WLP570)
• Beer086 (probably WLP585)
• Beer091 (probably a WLP strain)
• Beer092 (probably a WLP strain)
• Wine019 (probably a WLP strain)

I also assembled the reads from the WLP570 sample in the 1002 yeast genomes project, and STA1 was present in the resulting assembly as well. While these strains appear to contain STA1, it is unclear if any of them are diastatic (this needs to be tested). Interestingly, all the STA1 sequences contain a ‘T’ insertion at position 2406, close to stop codon (position 2427-2429). With the insertion, the amino acid sequence is extended by 39 amino acids (and these amino acids are homologous to the C-terminus of the SGA1-encoded intracellular glucoamylase). All this data is in the Excel spreadsheet as well!

Other information

In addition to these identifications, the comment section of the previous post contains a lot of interesting discussion on topics such as the history and spread of different strains, and whether interspecies hybrids have been included in the study. Please have a look! This information would definitely be worth a post of its own, but I’m sure someone else would be better at putting together such a post than me.

Hopefully White Labs will now be willing to unblind their own strains. It would make this data much more valuable, and I’m not really sure what they have to gain from keeping it secret.

Briefly, for those wondering how the tree was constructed: the Illumina reads from the lager strains were aligned to a concatenated reference genome of S. cerevisiae and S. eubayanus. FreeBayes was used to call variants. A consensus sequence for each strain was created using BCFtools and the FreeBayes VCFs. Only the S. cerevisiae sub-genome was retained for subsequent analysis. In addition, regions with coverage < 5 were excluded. kSNP3 was used for SNP detection and phylogenetic analysis on the set of assemblies from Gallone et al. 2016 and the four consensus sequences. The resulting SNP matrix was fed into IQ-TREE to produce a maximum likelihood tree with 1000 ultrafast bootstrap approximations. While the tree is very similar to the one in the Gallone et al. 2016 paper, there are some minor differences resulting from the different methodology (they e.g. only looked at SNPs located in the coding sequence of genes, while this approach looks at the whole genome).

My previous post contains information and discussion about how the White Labs strain identification was done.

DISCLAIMER: These are only guesses based on a range of evidence.

NOTE: This post has been updated.

The paper on brewing yeast domestication authored by the Verstrepen lab and White Labs last year is a fantastic piece of work (i.e. Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts, Gallone et al. 2016, Cell). I’m sure most of you have seen it by now, so I won’t be going into any details about it. While the article and the work that has been done is amazing, there is one big negative about it: the strains used in the study have been coded, and there is no way of knowing what strain in the paper corresponds to what strain from White Labs or the Verstrepen lab. This means that the readers can’t really benefit from the huge amount of phenotypic data that the study generated. Wouldn’t be nice, for example, to know which White Labs strains are POF+, which ones can’t use maltotriose, which ones produce high concentrations of isoamyl acetate, or which ones sporulate easily?

There is one way of finding out what the coded strains are though, but unfortunately it isn’t completely straight-forward and at the moment it can only be done with a handful of strains. The authors have released the genome assemblies for each of the strains used in the study (available here: https://www.ncbi.nlm.nih.gov/bioproject/323691). The genomes of some White Labs strains have been sequenced in other studies, and performing a search for ‘WLP*’ in the NCBI database yields some hits (https://www.ncbi.nlm.nih.gov/biosample/?term=WLP*). Most of these Illumina reads are from a recent paper focusing on wine yeasts by Borneman et al. 2016. Luckily the strains weren’t coded in this study, and we know what White Labs strains these sequence reads are derived from.

What I then did, was download all of the raw sequence reads for any White Labs strains I could find. I then aligned them to a brewing yeast reference genome (VTT-A81062). After alignment, I looked for SNPs with FreeBayes, and used the resulting VCF files to create consensus sequences for each White Labs strain using BCFtools. I then used ParSNP to perform core genome alignment on these consensus sequences and all 157 of the assemblies from the Gallone et al. paper that I had obtained previously. ParSNP outputs a core genome phylogeny (generated with FastTree2) and a SNP matrix. I additionally produced a maximum likelihood phylogenetic tree using the SNP matrix and ExaML. To identify the White Labs strains from the coded strains in the Gallone et al. paper, I then looked for the closest hits in the phylogenetic tree (this was really obvious for most strains). In cases when there wasn’t one obvious hit, I also looked at Supplementary Table S1 in Gallone et al. 2016 for the reported origin and source of each strain.

Using this approach, I was able to identify eight White Labs strains from the set of coded strains. I know it is not much, but at least it is a good start, and it already gives some interesting and valuable information. First of all here are the results:

The question mark (?) after the ‘Code in paper’ means that there were two close hits, and I chose the match based on the reported origin and source as well. First of all, my suspicions regarding WLP099 were true (see this old post), with it being Beer033, a yeast grouping in the wine clade and unable to use maltotriose. Interestingly, WLP570 (Beer085) also seems to be unable to use maltotriose. This should be the ‘Duvel’ strain, and based on this information, it should only be able to produce bone-dry beers in worts supplemented with sugars (as Duvel is, if I’ve understood correctly). Another interesting observation we can make is that WLP705 appears to be Sake002. This is the only out of the seven Sake strains that wasn’t grouped in the Asian clade, but rather in the wine clade. Is this really a Sake yeast, or is it a mislabeled wine yeast? Anyways, there is a lot of interesting data to be extracted from these strains alone (esters, ethanol tolerance, etc.)!

While writing this blog post, I also noticed that the authors have released the Illumina reads to all the strains as well (in June 2017 apparently). This should allow me to confirm these results by looking at things such as chromosome copy numbers (through coverage) and SNP heterozygosity (the assemblies are haploid sequences). Unfortunately, I probably won’t have time to do that anytime soon though. But hopefully I can have a look at those later during the winter.

I will also be keeping an eye on the NCBI databases in case more White Labs strains are sequenced in the future!

I feel bad for again having to start off a post by apologizing for my inactivity here at the blog. It has been quite a busy summer for me, as my wife and I got our first child (his name is Ludwig) in the end of June. I’m currently on parental leave from my research work, so have also been busy with wrapping up my ongoing work, so I can continue with it when I return from my leave in March. Anyways, we have been working on lots of interesting hybrid-related projects at our lab, so I thought I’d briefly sum up some of the topics we’ve been working on:

Adaptive evolution of newly created lager hybrids

This is the project I’ve been dedicating most of my time to the past 18 months. While the results haven’t been published in a peer-reviewed journal yet, we’ve submitted a manuscript based on the results for review, and have uploaded a pre-print to bioRxiv for everyone to read. We and others before us, have noticed that many hybrids (particularly interspecies ones) tend to have quite unstable genomes. 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 isolated colonies of variant strains along the adaptation process, and through a screening step, we managed to select several variant strains that outperformed the original hybrids in wort fermentations. Many of these variants also had higher ethanol tolerance than the original hybrids, and produced a more desirable aroma profile (more esters, less higher alcohols, and less diacetyl). We sequenced the genomes of these variants, and noticed that all variants had undergone gains and losses of various chromosomes. The results seemed to suggest that the S. cerevisiae sub-genome in the hybrids was ‘preferred’, while the S. eubayanus sub-genome had undergone more losses. We also identified multiple single nucleotide polymorphisms and small indels which affected the coding sequence of many genes. Mutations in some of these genes had previously been reported to affect ethanol tolerance and general fitness. In addition, these variant strains appeared stable when grown for multiple generations. All in all, our study shows that interspecific hybridization, coupled with adaptive evolution, is a powerful tool for producing new strains with tailor-designed properties.

Here is a link to the pre-print: https://www.biorxiv.org/content/early/2017/10/16/204198

Alternative Saccharomyces interspecific hybrid combinations for lager fermentations

This is a project a MSc student, Jarkko Nikulin, worked on last year. We wanted to investigate whether S. eubayanus really is that important for lager (i.e. low-temperature) fermentations. Studies have shown that there are several other cold-tolerant Saccharomyces species (though not as cold-tolerant as S. eubayanus), and the questions was: can we replace S. eubayanus in a lager hybrid with one of these alternative Saccharomyces species? We generated a range of S. cerevisiae × (S. arboricola / S. eubayanus / S. mikatae / S. uvarum) hybrids and compared them in wort fermentations. We noticed that S. eubayanus seems to be dispensable, as many of the other alternative hybrids performed just as well in low-temperature wort fermentations. There is a quite a lot of potential to generate some really diverse and unique strains using these alternative Saccharomyces species.

Here is a link to the study: http://onlinelibrary.wiley.com/doi/10.1002/yea.3246/full/

The use of Saccharomyces eubayanus and its hybrids in wine-making

This is a project my colleague and fellow PhD student Frederico Magalhaes has been working on. In his PhD project he has been looking at the use of S. eubayanus for cider and wine-making. Many wine fermentations are carried out at lower temperatures, e.g. to increase aromatic complexity and decrease the growth of contaminants, and the use of S. eubayanus would allow for that. You can read more about Frederico’s work in the following blog post:

https://vttindustrialbiotechnology.com/2017/08/30/some-like-it-cold-vtts-cold-tolerant-yeast-strains-for-wine-and-cider-production/

Here is also a link to a published study: https://doi.org/10.1093/femsyr/fox049

Review on modern yeast development strategies

Finally, here is a review on modern brewing yeast design and development that I was involved in, and was written for the ISSY33 conference that took place this summer in Ireland. Some of the topics that are covered are non-conventional brewing yeast, hybridization and genome analysis.

Here is a link to the review: https://academic.oup.com/femsyr/article/17/4/fox038/3861261

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.