Creating a New Yeast Strain

After finishing the book differentiate or die, I thought what better way to differentiate my homebrew than with my own, self created, proprietary yeast strain. The problem is, i have no idea how to do this, short of using the same yeast over and over to create mutations and then manual select the mutated properties that I like.

Has anyone ever tried to do this and had any luck? Are there any books out there that you would recommend for this sort of thing?

I have heard that you can get yeast to spore, and exchange genetic information this way, but it is also very hard to do.

Let's note that some commercial breweries obtain a culture and brew with it over and over and over again. One I was associated with used the same strain from the day they opened to the day they closed 20 years later. Over time the strain develops "house character". This is, in effect, what you are talking about but perhaps not as dramatic as, say, selecting for POF gene expressive colonies from plates (not that one would want to do that anyway).

bb239605 said:

The problem is, i have no idea how to do this, short of using the same yeast over and over to create mutations and then manual select the mutated properties that I like.

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That's the ticket! It will take multiple years, though, and that's assuming you are brewing often. The yeast will gradually acclimatize to your brewery, but you should do some research into proper handling to make sure you aren't introducing undesirable selective pressures. Charlie Papazian did something like this (as have many commercial breweries). You should harvest in ways that encourage the properties you want.

There are lab techniques to introduce mutation, but it's very unlikely that you'll produce yeast with desirable qualities that way.

You could speed up the change to get

As the other posters have mentioned, you can achieve your goal "passively" by simply re-using the same yeast again and again. Inevitably, evolution will do its thing and you'll end up with something unique and well adapted to your brewing conditions. AFAIK, this is how the vast majority of commercial and retail yeast strains were developed.

However, you can take a more directed approach to produce something more specific to your wants, and/or drive the whole process more quickly. But its also a lot more work. The process is called "directed evolution", and the name pretty much sums up what you're going to do.

From the point of a biologist, this is both easy and hard. Getting things to evolve is easy (making them not evolve is the real trick). Getting them to evolve into something you want is the hard part.

To do this efficiently, you need two things, a source of mutations and a method of selecting those yeast which have the desired characteristics.

Mutation (the easy part):
For a technical description of mutations rates, you can follow this link:
http://www.genetics.org/content/148/4/1667.full

Mutation rates vary across the genome, but in yeast the average is 0.0027 mutations per genome per generation (i.e. every generation, one out of every 370 yeast will carry a new mutation). So mutation is easy - simply grow yeast!

You can enhance mutation rates easily as well - growing yeast in high salt (0.5M NaCl) increases the mutation rate. Even easier, put a flask of rapidly growing yeast in direct sun for a half hour or so - the UV will induce a whole whack of mutations. But these methods create new headaches, so you may want to avoid them.

Getting something good (the hard part)
While mutating is easy, getting a desirable product is much harder. Once things are mutated, two evolutionary forces will come into play - drift and selection. To achieve your desired goals you need to control both forms of evolution as much as you can. Ideally this would mean plating out each generation into single-cell colonies and selecting only those which have the characteristics you desire (this all but eliminates drift and gives you total control over selection). That is impossible, so instead we need to set up conditions that allow for us to select populations with desired characteristics, or at a minimum, bias conditions so that evolution will go the way we want it to.

Drift:
Contrary to popular belief, the vast majority of mutations will have no effect on the fitness of the yeast. That is not the same as saying these mutations will have no effect on your beer - it simply means they have no effect on the replication of the yeast. You could very well end up with a mutation that creates something wanted (more of a specific taste) without affecting the growth of the yeast itself. These types of mutations experience drift - i.e. the frequency of them in the yeast population varies randomly; mutations can disappear or become prevalent due to random chance. Unfortunately, there is little that can be done to control for this, aside from occasionally storing (freezing preferably) cultures so you can "rewind" if things go astray.

Selection:
Mutations that have an effect of the survival/replication of the yeast will experience selection; the removal of (or at least decrease in the frequency of) traits that lower the replication/survival of the yeast. This is the easiest factor we can manipulate - by growing yeast in conditions we desire (high gravity, or within a specific temperature range, etc) we can select for the yeast with characteristics we desire, as those with the characteristics we desire will outgrow those which lack those characteristics. You can also select by choosing which yeast get a second chance at growth; for example high flocculation can be selected for by growing the yeast which first drop out of fermentation (or if you're lazy, the yeast at the bottom of the yeast cake).

Selection can also be a problem - sometimes a mutation which causes one desired characteristic will create a secondary characteristic that is undesired. Which is why methods to enhance mutation rates can be problematic - for example, salt induction of mutation also selects for salt tolerance; something which tends to be associated with fusel alcohol production.

Actually Doing It:
There are two approaches you can take - using multiple small fermenters (1L or so) to run parallel batches, or keeping and re-using yeast from full-scale batches that have desired characteristics. The first option is faster, but suffers because characteristics prominent in small-scale fermentations do not always scale upto full-sized batches. The latter is slower, and gives you much less control over the whole process. I've attempted this a few times, the small-method gave the most noticeable changes, but my one attempt at the later method gave the best result. Either way:

1) When transferring yeast from one batch to the next, always transfer a minimal amount. This creates a founder effect; i.e. the small amount of yeast transfered means any mutation in that transfered yeast has a good chance at becoming a common trait.

2) Keep a frozen sample of each generation, so you can backtrack if needed.

3) Have strict selection criteria and stick to them. Even a one batch deviation can set you back months, and inconsistent selection will prevent desired traits from becoming predominant. My first attempt's selection was "tastes good"; meaning I didn't select much at all. Later attempts were much more specific (looking for specific flavors/odors under desired growth conditions) and were more successful.

I'd add here that in my experience the best way to do this is have a wort with minimal flavor - base malt at the desired OG and nothing else. This way you're not trying to select the yeast's characteristics against a background of malt and hop flavours/odors.

4) Start with something close. It is much easier to "fine tune" the characteristics of a strain close to what you want than it is to develop whole new characteristics in a strain.

5) Jump-start the whole process by crossing two strains. Yeast reproduce sexually, so by forcing two strains to interbreed you can generate hybrid that give you both more genetic material to work with, as well as allow you to select for desired traits from two separate strains. This is a complex process to get it to work, and requires access to equipment a little more advanced than what the average home brewer has, but it worked quite well for me. A detailed protocol is beyond what I've written here, but the coles notes version would be: sporulate, mix, select hybrids, grow. Simply mixing strains wont work - sexual reproduction doesn't occur much under the conditions found in beer, and instead you tend to end up with one strain out-competing the other.

6) Multi-culture method: You need several flasks. Inoculate a small amount of yeast into each one, and let it grow. Pick 1 or 2 of the flasks which are the "best" (based on your criteria), dump the rest, make up new flasks, and inoculate the new flasks with small amounts of yeast from the flasks you've selected. Keep repeating this until you get what you're looking for (don't forget to freeze samples so you can rewind if nessisary).

7) Large-culture method: Make beer, drink it, decide if the yeast did a good job. If it did, reuse the yeast for your next batch. If it didn't, chuck the yeast and brew using an older culture. Just make sure you're being consistent in judging whether a beer gets to pass its yeast onto the next "generation" of beer.

Hope that helps.

Bryan

This is a flabbergastingly correct post which somehow avoids all of the predominant misconceptions about evolution and genetics in the homebrewing community. I have only two minor objections:

Contrary to popular belief, the vast majority of mutations will have no effect on the fitness of the yeast.

This should probably read "very little effect". The population sizes involved are going to be huge, so drift won't be quite so powerful in decreasing variance in fitness.

Yeast reproduce sexually, so by forcing two strains to interbreed you can generate hybrid that give you both more genetic material to work with, as well as allow you to select for desired traits from two separate strains.

I don't think this is quite true for brewing strains. As far as I know, brewing strains (unlike lab strains) are all polyploid, and (perhaps as a result) are extremely reluctant to reproduce sexually. I would to very interested to learn otherwise.

ni* said:

This is a flabbergastingly correct post which somehow avoids all of the predominant misconceptions about evolution and genetics in the homebrewing community.

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Thanx, although I would say that I'm a biologist (and I work with yeast in the lab, on occasion) so I'd better damned well get it right.

ni* said:

I have only two minor objections:
Contrary to popular belief, the vast majority of mutations will have no effect on the fitness of the yeast.

This should probably read "very little effect". The population sizes involved are going to be huge, so drift won't be quite so powerful in decreasing variance in fitness.

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I'm not sure by what you mean with "very little effect". If you mean the effect of drift verses selection, than I partially agree. That said, the role of drift can be controlled, by controlling the amount of yeast transfered from culture to culture. The smaller the number of yeast transfered, the larger the founder effects, and thus the impact of drift will be accordingly larger. If you mean the impact of most mutations, than I disagree vehemently;the junk DNA content alone is enough to ensure that most mutations are truly neutral. I couldn't find the paper, but if I recall correctly about 1 in every 10,000 mutations in saccharomyces has a measurable effect on fitness.

ni* said:

Yeast reproduce sexually, so by forcing two strains to interbreed you can generate hybrid that give you both more genetic material to work with, as well as allow you to select for desired traits from two separate strains.

I don't think this is quite true for brewing strains. As far as I know, brewing strains (unlike lab strains) are all polyploid, and (perhaps as a result) are extremely reluctant to reproduce sexually. I would to very interested to learn otherwise.

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The genetics of brewing strains is somewhat complex, and varies greatly between lager and ale yeasts. Ale yeasts are generally the same as lab strains - i.e. they have a 2N chromosomal number. Lager yeasts are plain weird - some are truly polypolid (i.e. have duplicate copies of their entire genome), although most are aneuploid (have extra copies of a few chromosomes or parts of chromosomes). To make things even more complex, lager yeasts are also hybrids of two different species, although the exact degree of hybridization varies greatly strain-to-strain.

Their "desire" to engage in sexual reproduction pretty much follows their genomics - ale yeasts are far more likely to do it, and are far easier to coax into mating. Lager yeast can be made to sexually reproduce. In the lab we use chemical stressors to force sexual reproduction, which works well with all strains of yeast. The usual method applied by brewers (usually starvation, although super-high gravity brews works as well) only work for ale yeasts, and in that case, are fairly inefficient.

Bryan

Warthaug said:

I'm not sure by what you mean with "very little effect". If you mean the effect of drift verses selection, than I partially agree. That said, the role of drift can be controlled, by controlling the amount of yeast transfered from culture to culture. The smaller the number of yeast transfered, the larger the founder effects, and thus the impact of drift will be accordingly larger. If you mean the impact of most mutations, than I disagree vehemently;the junk DNA content alone is enough to ensure that most mutations are truly neutral. I couldn't find the paper, but if I recall correctly about 1 in every 10,000 mutations in saccharomyces has a measurable effect on fitness.

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Yes, I am referring to the effect of drift versus selection. I assumed this is what you meant because it was in the section you labeled "drift". Yes, I agree, it is easy to intentionally arrange things to increase the strength of drift, but unless you are doing this (intentionally or otherwise) drift will not entirely overwhelm selection.

As for the latter interpretation, yes, it's well established that most mutations are neutral, although it's worth pointing out that mutations in junk DNA won't have an impact on the beer, either.

The genetics of brewing strains is somewhat complex, and varies greatly between lager and ale yeasts. Ale yeasts are generally the same as lab strains - i.e. they have a 2N chromosomal number. Lager yeasts are plain weird - some are truly polypolid (i.e. have duplicate copies of their entire genome), although most are aneuploid (have extra copies of a few chromosomes or parts of chromosomes). To make things even more complex, lager yeasts are also hybrids of two different species, although the exact degree of hybridization varies greatly strain-to-strain.

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Thanks for this, I was unaware of the details. My yeast research experience is limited to lab strains, and I've never found the the time to look into brewing strains.

Very interesting posts. I am now more grateful than ever for Mr. Logdgson and Mr. White (actaully Dr. in both cases I think).

ni
Lab strains do have some brewery in their history, I recall a paper back in the '92-'95 range from the Lindegrens, IIRC, that showed S288C was a spore from a cross of Fleischman's yeast and a Carlsberg later strain. So, you're closer to beer in the lab than you thought.
Good karma

I've never really doubted that -- in a sense it's kind of obvious. Where else would researchers have gone to get their strains, after all, other than breweries and bakeries, who would be certain to have healthy strains with know growth parameters? They're still pretty different from anything in current use, though -- mostly obviously through the ho knockout, but I'm sure there are countless other differences.

Yeah, the differences are legion, but it could have been just beer, bread, or wine, rather than a mix. I can't remember the name of the early guy that argued against using heterothalic mutants, but that's certainly a good example If he'd have won out, it probably would have been pure brewery strains, because he was at Carlsberg, IIRC. FWIW, I know it's obvious; another point to consider is that industrial scale brewing and baking also had the money to float research 100 or so years ago; there wasn't federal grants like now.
Take 'er easy

I've had 2 PM's asking for more details of what I've done in the past, so here's another post on what I've done and how it worked.

My first attempt was in the mid-1990's; I was in undergrad, had just taken a course on microbrial organisms, and figured I could evolve a yeast for my home brewery (I'm ashamed to say my goal was to make a lager yeast I could use at room temp so I could brew a Molson Canadian clone). The method used was simple - I took about a dozen beer bottles, filled them with wort, inoculated them with yeast, and put an airlock on. When fermented, I tasted each one, decided which one was "best" (based on no consistent criteria), dumped the rest, and re-inoculated a bunch more bottles and repeated the whole process. I don't have my brewing notes with me at the moment, but I think I went through 20 or so brew "generations" before I quit. Needless to say, this didn't work. About all I did was remove the cold tolerance from a formerly cold-loving yeast. I don't recall my starting strain, but I think it was either wyeasts american lager or californian lager.

My next "custom" yeast was simply an accident. When I first started brewing I had a "house brew" that was basically a light ale that my lager-loving friends would drink. I brewed two (sometimes 3) batches of this stuff a month, and just kept re-pitching the same yeast over and over again. At some point the quality (I use the term loosely) started going down hill, so I bit the bullet and bought another packet of the same dried yeast I had started with. That batch wasn't what we remembered the good batches being, so I "rewound" my brewing process by re-culturing an older generation of yeast from a bottle. That worked, and I now had my own "house brand" of yeast. I started with muttons dried yeast, what I ended up with was crisper, with more balanced ester/fruitiness. I still have that yeast kicking around somewhere, but my pallet has improved and I'd now consider it an OK, rather than great, yeast.

Buoyed by that success I made more efforts at generating my own yeast, largely helped by entering grad school where I had access to proper lab equipment. I won't bore with too many details, other than to say I went back to my first method of using multiple small fermenters to run several "generations" in parallel. Having a better grasp of evolution, I was also inoculating these with extremely small quantities of yeast (1-5ul [1/1000 of a ml; about 0.00003 oz]) of an active ferment inorder to maximize founder effects. I had a lot of failures; the problem with using such small volumes is one freak cell can end up taking over the culture, producing some real nasty stuff.

In most cases I ended up quitting after a dozen or so generations, but there were a few notable successes. I produced something akin to turbo yeast, starting with champaign yeast, and the "final product" made from it was clean as could be. I evolved Wyeast 1098 (british ale) into a more cold-tolerant form that was otherwise identical to the original (I live in Canada, our basements can be very cold), I evolved another "house strain" of yeast; this time starting with the yeast cultured from a chimay and ended up with something optimized for the conditions in my basement and a little milder in taste. My pride and joy though was turning a wild yeast from my sour-dough culture into a passable beer yeast. It produces something like a cross between a trappist and a lambic, although the biggest victory was getting the damned thing to flocculate a little.

Both of the people who PM'd me also asked about crossing strains. I used the standard yeast-mating method (sporulation driven by potassium acetate, followed by breeding). An example of this method:
http://dunham.gs.washington.edu/sporulationdissection.htm
Note, that I would only follow this upto forming spores (culturing in SPO++); everything after that is for purifying spores from lab strains that carry specific markers; something you cannot do with brewing yeasts.

This is sufficient to induce sporulation in both ale and lager yeast (I've tried both, and saw sporulation under a microscope in both cases). Because these are "wild" yeasts there is not a convenient way to separate spores, so "purification" consisted of drying the sporulated mixture at ~50C (this'll kill yeast, but leave most of the spores viable). I would then mix the spores from the two strains under sex-favorable conditions, & let them grow for a while. Because there is no easy way to separate the parental strains from any hybrids, I would then spend a lot of time sub-culturing (i.e. taking really small volumes of growing yeast and inoculating a fresh vial of wort with it), looking for something that had the desired qualities.

Without my notes I'm not sure how many times I tried this, but I'd guess I tried it in about 1/3rd of my breeding attempts. The only good strain I produced where I tried breeding was my "turbo" yeast. Basically, as I was evolving this yeast I ended up with two "competing" strains; one which fermented fast, and a second which had better alcohol tolerance. I crossed them, in the hopes I could get something in the middle. I have zero evidence that I actually generated a hybrid in that case, but the yeast I ended up with had better alcohol tolerance than either of the starting strains, and appeared quite a bit different in the fermenter as well (it didn't flocculate as well, and formed an almost cobble-stone appearing layer on top of the fermenting wort).

I'm not too sure what else to add, but feel free to ask any questions...

Bryan

Wow that's awesome. Thanks for writing that all up.

As one of the people who PM'ed you, I am excited to hear that sporulating these guys is as simple as it is for standard lab strains. I had never looked into it but always assumed it would be quite difficult given the continuous asexual propagation in a brewery.

Sounds like you had to do a lot of work each time you tried one of those experiments. That's pretty impressive, as I am not sure I would be able to keep doing it for very long. Thanks again!

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dinertime said:

Wow that's awesome. Thanks for writing that all up.

As one of the people who PM'ed you, I am excited to hear that sporulating these guys is as simple as it is for standard lab strains.

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I didn't want to say who PM'ed me, but yep, you were one..

Sporulation is as simple, but it is not nearly as efficient, as with lab strains. I only did it once with lager strains and ended up with a handful of colonies on an YPD-agar plate after the heat-killing stage, plating a volume that would normally give you a lawn of yeast. Ale yeast do much better, but even with them you end up with only a tiny percent of the sporulation you would see with lab strains.

I think I forgot to mention it in my last post, but I have zero evidence that the spores of the different strains cross-bred - for all I know the yeast I got out of the procedure were either yeast that survived the heat-killing or spores that recombined to re-create the original parental strain. My "evidence" of recombination is that the new yeast behaved differently than either of the old yeast strains.

dinertime said:

I had never looked into it but always assumed it would be quite difficult given the continuous asexual propagation in a brewery.

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In the brewery I doubt you'd ever get sporulation (maybe in a really old yeast cake), but in my experience if you stress the yeast, they do sporulate.

dinertime said:

Sounds like you had to do a lot of work each time you tried one of those experiments. That's pretty impressive, as I am not sure I would be able to keep doing it for very long. Thanks again!

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Its not as much work as you'd think. So long as you make all your wort ahead of time (and sealing it in ready-to-go fermenters), its little more than inoculating wort 1-2X a week. Getting everything ready is a PITA, but after that its pretty easy.

Bryan

It is interesting to think of what one might use as a marker in ale yeast. I (strongly) suspect that most of the obvious unambiguously scorable traits (high alcohol tolerance, say, or strong clove flavor) are polygenic. If you had two easily scorable traits, it would be easy enough to sort out whether a true cross is occurring.

I wonder if, by chance (and lack of selection preserving it) some popular strain has lost the ability to metabolize some particular carbon source? Rather unlikely, I suppose, but convenient if so.

Don't all ale yeasts have trouble metabolizing maltotriose? Though I suppose what you're saying is to find a difference between two particular ale yeasts' metabolic profiles to use as one of the scorable phenotypes.

Surely growth rate on different carbon sources could be a useful factor in comparing different strains. You would need to make a variety of defined media (well, YP + different sugars) and follow by optical density, which one can't do at home. However, I think that for most of what we're talking about you would need a pretty well equipped lab anyway.

Reading optical density, or doing most of what has been described, doesn't seem at all beyond the scale of what can be accomplished at home for someone otherwise interested in these things. Only a tetrad dissection microscope would be particularly expensive (and it's the only piece of equipment involved that I do not know to be in the home of someone I know or another). Spectrophotometers are less than $100 on ebay, and you could get away with a hemocytometer if you were feeling especially cheap.

It definitely borders on slightly crazy, but it's not at all beyond the scale of what can be done at home.

As for maltotriose, brief googling leads me to understand that while lager and ale yeasts use different maltotriose transporters, which do different in efficiency, neither is severely hindered when growing on maltotriose, so that probably wouldn't work as a phenotype. A shame, as it would otherwise have been perfect.

If you're serious about trying to make a completely different yeast strain do what has been listed previously. If you're a crazy ass scientist and wanna make a strain that no one has gotten even close to, feel free to read further on an idea I have. FYI, this is a crazy ass idea and most likely will die unexplored on this page.

I would suggest looking into developing yeast with a completely altered phenotype. A recent FDA approved drug for cancer therapy, 5-aza-deoxyctyidine acts as a substitue for the normally DNA-incorporated base cytidine. The only difference is that the 5-aza form is unable to become methylated by specific enzymes. Methylation can cause a gene to become silenced, and is one of the primary forms of gene regulation in the cell. The reason this drug is used in cancer is because it can serve as a reset button for genes that may have been turned off or on during the transformation of the normal cell into the cancerogenic cell.

Nonetheless, if one used this drug on a yeast culture, to reset the genetic regulation in its current form, and then introduce it into the specific environment that you are hoping for it to proliferate in, you could directly alter the phenotype to that specific environment (fruits, high alcohol, etc) since epigenetic factors like DNA methylation form based on the organism's environment. With all of their genes accessible to activation, instead of being silenced by methylation, you have a good chance of allowing for survival. The process of allowing the yeast to slowly adapt over time is basically the same end-goal, just a slower process. You may end up with a lot of cells dying, but with variable environments you could eventually create a new strain.

So yah, crazy ass idea I know. But if you can get to Mexico and buy some of this stuff it may be worth a shot and a publication if it works =)

I need to get out of grad school...

Warthaug, holy crap, great posts! Thank you!
I wish I had an autoclave... though i do have a big pressure cooker...

One basic thing that I don't think has been mentioned (though I need to re-read this thread when I'm not so sleepy) something I remember from "how to brew" by Palmer. Flocculation vs attenuation. If you were to use the yeast cake from your primary and one from your secondary side by side; The one from your primary would be more apt to exhibit better flocculation and poor attenuation. And vice verse for the one from your secondary, and with successive generations this would get more dramatic.
In a fermenting beer there is a large diversity of genetics at any given time. The point at which a yeast falls out is one of the simplest ways pick a specific trait. If you had a yeast you liked that made a cloudy beer (not to your liking) you could take some of the first to fall for the next generation.
This is an important aspect to understand, as it could lead to complications; if say; you always used yeast from the bottom of bottles. Though there are more interesting things to bread yeast for... like say: yummy beer.

I attempted to collect wild yeast last fall. I had bad luck... mostly in the form of ethyl acetate. The wild rout is rather different as you will get many stains in any sample. It may be possible to weed out the weaker ones over time, but that's a major process in it's self.

To the Mods: thanks for not moving this to the yeast forum yet, there are far more nerds over here, which is keeping this thread quite interesting.