In this blog post I’m briefly going to summarize how I created and improved a hybrid yeast strain between Conan (isolated from a can of Heady Topper in 2013) and WLP644. I’ve used both strains in several homebrews previously, and I’ve really liked the fruity esters they produce during fermentation. While Conan produces a really nice aroma profile, it doesn’t seem to attenuate as well as say WLP001 (most probably as a result of incomplete maltotriose use). This means that it often leaves a slight sweetness in the beer, and is a bit unpredictable. With WLP644 I’m not that sure. I’m fairly sure it uses maltotriose and ferments the beer quite dry, but this observation is only based on a single homebrew I’ve done with it. It seems to grow and ferment slightly slower than most ale strains though. So, I had the idea to generate a hybrid between the strains, in order to create the ultimate ‘super fruity’ yeast strain:
- Lots and lots of fruity esters
- Complete maltotriose use for high attenuation
- Bonuses are fast fermentation and moderate flocculation
Spore-to-spore mating is the traditional method of generating yeast hybrids, but I myself am more of a fan of rare mating. Also, spore-to-spore mating couldn’t really be applied here in this case because of the poor sporulation ability of Conan. To begin, I needed to ‘tag’ the parent strains with selection markers. To do this, I selected spontaneous auxotrophs of Conan (ura-) and WLP644 (lys-) on 5-FOA and α-AA agar, respectively. This means that my Conan mutant strain isn’t able to synthesize its own uracil anymore, while the WLP644 mutant strain isn’t able to synthesize its own lysine anymore. Any hybrid between these two would inherit the functional genes from the other parent, meaning the hybrid is prototrophic again, and can again synthesize its own uracil and lysine. Hybrids can thus be selected on minimal media, which doesn’t contain uracil and lysine.
The actual hybridization process is easy, all I have to do is mix cultures of both parent strains in rich media (YP-Maltose in my case), incubate for 3 days, pellet, wash and starve the yeast, and finally spread it out on minimal media agar. Any colonies appearing on the minimal media agar are most likely hybrids. The other option is that the colonies are the parent strains, which have undergone spontaneous mutations to return to being prototrophic again. In order to ensure that we in fact have hybrids, the colonies are first purified by restreaking on fresh minimal media agar, and then transferring and streaking a single colony to YPM agar. DNA is then extracted from a single colony on the YPM plate, and interdelta-PCR is performed on the DNA.
Interdelta-PCR is a technique that is usable for differentiating Saccharomyces cerevisiae strains. Below you can see the gel and the profiles that our hybrids (H1-H4) and the parent strains produce. As we can see, the hybrids contain DNA (i.e. the bands) from both parent strains, so it is confirmed that we have successfully generated a hybrid. This is especially evident in the areas I’ve marked with red asterisks on the DNA Ladder. I won’t go into more details on the mating mechanisms, but the short answer is loss of heterozygosity at the MAT locus (for more details see here). Being a result of rare-mating, this hybrid will most likely be tetraploid (assuming that both parent strains are diploid), and contain approximately the whole genome of both parent strains (as can be seen from the gel below, hybrid H2 has already lost some DNA).
To stabilize the hybrids and also generate some potentially interesting phenotypes, I utilized meiotic recombination and segregation! During ascospore formation in yeast, meiosis and chromosomal crossover takes place, during which genes are shuffled between pairs of chromosomes. First we have to sporulate our hybrid (this can be done on 1% potassium acetate), then treat the forming ascospores with a lytic enzyme (e.g. Zymolyase), and finally dissect the ascospores with a micromanipulator. I choose to do this to hybrids H1 and H2. Both strains sporulated quite poorly and the spores had low viabilities as well (34% for H1 and 8% for H2). I selected the fastest growing colonies for further characterization, and this was four segregants from hybrid H1: H1/A4, H1/C3, H1/C4, H1/H1. The characterization was done in some small-scale (35 ml) wort fermentations. This was done in order to confirm that they still are able to grow and ferment well in wort and that they produce a lot of fruity esters.
The four segregants that were chosen for further characterization
The small-scale fermentations were carried out in 15 °P wort (specific gravity: 1.060), because I thought this would be representative of a typical IPA wort. 35 ml of wort was added to pre-weighed and airlock-capped plastic tubes (50 ml), after which 10 million cells / ml of wort of each yeast strain was added to start the fermentations. Fermentations were carried out at 18 °C (in retrospect, this might have been a little low), and they were monitored daily through mass loss. The alcohol content of the final beer was measured with an Anton Paar DMA5000M + Alcolyzer. Our HS-GC is in heavy use at the moment in other projects, so I wasn’t able to measure the individual ester concentrations. I did sniff the beers though to get a general idea of whether the hybrids are actually fruitier than the parent strains. The fermentations were done in wort that had only been hopped in the beginning of the boil, to make sure that the majority of the beer aroma was yeast- and not hop-derived.
The small-scale fermentation vessels
ABV% and pH of the beers
As you can see, there was considerable differences in fermentation rate between the strains. Of the parent strains, Conan started fast, but ended slow, while WLP644 was the other way around with a slow start and faster finish. In early stages of fermentation, the H1 hybrid was also doing well, but in the end it was two of the meiotic segregants, H1/A4 and H1/C4, that reached the lowest final gravities after two weeks of fermentation (1.013 and 1.009 respectively). The Conan and WLP644 parent strains reached final gravities of 1.015 and 1.017 (this would probably have dropped slightly with a couple of more days of fermentation). There was considerable variation in pH as well, as the lowest pH values were observed in the beers fermented with H1/A4 (4.39), while the highest pH values were observed in the beers fermented with H2, H1/C4 and H1/H1 (4.64, 4.61 and 4.69). The aromas of the beers were very similar, all featuring a similar fruity tone that reminded me of various stone fruits (peach and apricot). While there were some differences in intensity between the beers, I’m not sure how much I would trust just my nose. In my opinion the strongest fruity aroma was found in the beers fermented with Conan, F1/A4 and F1/H1. The weakest aroma was found in the fastest fermenting beer, i.e. F1/C4. However, as I mentioned the actual ester concentrations would need to be measured to actually draw any conclusions. All aromas were ‘clean’, with no signs of any phenolics (both parents are POF-). The WLP644 beer had a slight sulfuric note to it though. Another thing I observed during these fermentations, was that WLP644 flocculates poorly, while Conan and all the hybrids flocculated quite well.
The next step will be to actually brew and taste some beers brewed with these yeast strains, in order to see if there actually is any real world difference. For this I was planning on brewing up a 25-liter batch of APA wort (OG around 1.050, IBUs around 50, whirlpool hops, but no dry hopping), which I would then split into 5 fermentation vessels. To these I would then pitch: Conan, WLP644, H1, H1/A4 and H1/C4. I’ll return when it is time for the brewday and ultimately the tasting notes.
References and additional reading for those interested:
Krogerus K, Magalhães F, Vidgren V, Gibson B. 2015. New lager yeast strains generated by interspecific hybridization. Journal of Industrial Microbiology & Biotechnology 42: 769-778. DOI: 10.1007/s10295-015-1597-6
Steensels J, Snoek T, Meersman E, Nicolino M, Voordeckers K, Verstrepen K. 2014. Improving industrial yeast strains: exploiting natural and artificial diversity. FEMS Microbiology Reviews 38: 947-995.