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Cassie Immel

Good evening, everyone. Thank you for watching Wednesday Nite at the Lab. I'm Cassie Immel. Tonight, I will be presenting Dr. Chris Hittinger from the genetics department. He received his bachelor of science from Southeast Missouri State University and his doctor of philosophy from the UW Madison Laboratory of Genetics and the Howard Hughes Medical Institute. His research interests are diversity and evolution of yeast, carbon metabolism networks. His research fields include gene expression, genomics, microbial genetics, molecular genetics, and population or the evolution of yeast and fungi. Without further ado, I present Dr. Chris Hittinger.

APPLAUSE

Cassie Immel

>>

Chris Hittinger

Thank you very much for the opportunity to tell you this story. As you can tell, it's an exciting story, and I've been asked to stay still so that it films well. So I apologize if I go off script in that matter. But this is a story that involves a search for the missing progenitor of lager yeast on five continents and uses some of the great classic techniques of microbial ecology and isolated wild species out in the field with your hands with the latest cutting-edge techniques from genetics, and I think that's something we can get excited about. And I want to jump right in because, as Cassie told us, there are supplements to the educational experience this evening during the Q&Asession, and I certainly want to thank the generous folks at Capital Brewery for providing that, and I certainly don't want to be accused of delaying our ability to supplement our education as much as possible.

LAUGHTER

Chris Hittinger

So this is a talk about yeast, and that raises the question of what yeast are. Yeast are one-celled fungi. So what are fungi? Well, they're one of the three major groups of eukaryotic organisms that we encounter every day. And this is sort of a view of all life on Earth and how it's related. We have single-celled bacteria down here. 2.5 billion years ago, sorry, about two billion years ago, two bacteria came together and formed eukaryotic life which then gave rise to most of the big macroscopic large organisms that we encounter, and the includes organisms like plants and also organisms like you and I, animals, and of course fungi. Interestingly, fungi are actually more closely related to animals than they are to plants, and you can see that evolutionary biologists typically represent those relationships by the cursor, which isn't showing up, which you could actually see at the base of that triangle there where the fungi and the animals actually come together and share a common ancestor in the dawn of the Precambrian. So fungi are further divided into two major groups. They're the ascomycetes and basidiomycetes, and we probably have a lot more experience encountering basidiomycetes, at least their larger forms, and that's mushrooms, also corn smut, wheat rust are basidiomycetes. Ascomycetes are cup fungi and molds, notably penicillin and, of course, the bread mold neurospora, and yeasts. Now, you'll notice that yeasts are also present in basidiomycetes because when we ask what is yeast, the answer is a little bit complicated. There are actually many, many different forms of yeast. And this is something that's actually evolved multiple times. So the correct question is really, what are yeasts? They're several different lineages of fungi that have all evolved independently this single-celled lifestyle of being a one-celled fungus. So what are yeasts? They're single-celled fungi. They're microscopic organisms, and yet they're descended from this complex multicellular lineage. There are over 1500 recognized species of yeasts. So, if there's one take-home I hope people will bring back to their regular jobs or at home on television it's that there are many, many kinds of yeast, and they're incredibly diverse. In fact, genetically they're actually more diverse than all vertebrates. So there's an awful lot of diversity locked within this group of organisms. Also, in contrast with bacteria, they undergo both sexual and asexual reproduction. So they can produce sexually with a partner or they can reproduce more of themselves, as we see this budding yeast here is doing in the upper corner dividing and making a copy, a genetically identical copy of itself. Now, it turns out while yeasts have evolved multiple times from fungi, it turns out the usually people are talking about a particular group of yeasts that all share a common ancestor 500 million to a billion years ago, and those are the Saccharomycotina or the Hemiascomycete yeasts. By looking at these pictures, you can usually see why they're called Hemiascomycetes, and it's because a normal ascomycetes has eight spores in its sexual structure, and this is actually a whole collection of sexual structures; whereas, the Hemiascomycetes usually only form four spores. And so it's an half an ascus. Usually, yeast means one of these guys here, this little part of the tree that branches off from the rest of the fungi maybe 500 million years ago. Well, what do yeasts do? I'm embarrassed to say that we still don't know for most of them, and I mean that in the broadest sense. We don't know what they're doing out in nature on their own, and we don't know what they can do for us, so that's an interesting ecological question and also an interesting biotechnological question. We do know that they often have pretty strong associations with various insect species or plant species. And the ones that we know the most about, of course, are the ones that either make us sick, our pets sick, or our crops sick. And many of you have probably encountered yeasts going by the name of Candida. These cause yeast infections sometimes called thrush, in particular oral thrush or vaginal thrush, and these are usually a result of yeast overgrowing and causing an infection. Yeast also produce a number of the products that are near and dear to many of our hearts and make our lives more palatable or more entertaining. Soy sauce is a great example. They're probiotics. Perhaps less popular in some circles are Vegemite and Marmite. If you ever read the ingredients on the back of many of your processed foods you'll see that they add torula yeast and brewer's yeast to lots and lots of things. And, of course, why we're here today is because of these alcoholic beverages toward the end, particularly beer. And, of course, I should note that the same process that are used to make alcoholic beverages, or indeed to make bread rise, is also the process that we use in the biofuel industry today which is of increasing importance to this state and to the nation as a whole. Yeast can also spoil food and beverages, and that's another important consideration. So there's sort of a balance between these products that we regard as useful and desirable and the products that we regard as spoiled. And, in fact, there's some cultural differences in terms of what people considered spoiled versus a desirable product. One thing that yeast often seem to share is that many of them like simple sugars, but really only some of them ferment readily, and that's what we're going to talk about today because it's easy fermentation that makes for these products down here. So, in particular, when we're talking about the champion fermenters are Saccharomycetes yeast, and many of you are familiar with these products here that Saccharomyces likes to grow on. Saccharomyces likes to grow in these places because they are rich in the simple sugar glucose. Glucose is the most abundant simple sugar on the planet. It fuels life in many ways. It's the only source of carbon that our brain uses. And it's found at high levels in bread, grapes, and of course the wort that is used to make beer. Probably many of you don't realize but in nature Saccharomyces yeasts are actually found in association with oak trees in the northern hemisphere. And, in fact, when we go sample wild yeasts, we're actually usually too lazy to scrape off the bark, although we do sometimes, especially if we have the landowner's permission, but it's much easier and quicker just to go under the oak tree and grab soil back and bring it back to the lab. And so we often get yeasts this way. If you're immunocompromised, Saccharomyces yeast can actually make you sick. So the same unfiltered yeast you may consume one night, if you're immunocompromised can, in some cases, cause an infection. And, of course, as I mentioned earlier, yeasts are very important to the biofuel industry, principally right now making corn into ethanol, but increasingly making other products into ethanol as well. So Saccharomyces love glucose. It gets it in these places. What does it do with the glucose? Well, most of us, most organisms, when they encounter glucose, as we learned in high school biology and thanks to the work of 19th century microbiologist Louis Pasteur, they check to see if there's oxygen. And if there is oxygen, they respire it and break it down fully into carbon dioxide and ethanol. And they do this because they get lots and lots of ATPs, which are the energy currency of the cell. Now, most organisms do have a fermentation pathway, but, as you can see, they get very little ATP back from using this. So they really only like to use it when oxygen is not available. The products from that, of course, are carbon dioxide, which is what makes beer fizz, and also ethanol, which is the product that some people are consuming for that aren't interested in the fizzing in root beer. Now, if you don't believe that there is more energy captured here than here, I think all you would have to do is with parental guidance, of course, is to take a small amount of water and try to light that on fire versus a small amount of ethanol. And I think you'll see that there is still plenty of energy present in the ethanol and not many energy present in the water. Now, interestingly, Otto Warburg, in the early 20th century, actually observed that many tumor cells do something completely different. They don't check to see if oxygen is present, and instead they run glucose directly through glycolysis and ferment it. Interestingly, Saccharomyces yeast do exactly the same thing. If glucose levels are high, they ignore the presence of oxygen and make ethanol anyway. Interestingly, this is not because they've lost the ability to respire. This is actually a regulatory decision that they make. So they check to see if glucose is high enough that they can make a living off of these ATPs, and then they actually actively shut down respiration and crank up glycolysis and fermentation. Now, why would you do that? Well, this is faster because you don't need to get oxygen into the mitochondria and go through all the many, many, many steps of respiration. And so if you can wastefully use the resources quickly as possible, that can give you brief competitive advantage. Not only that, ethanol is toxic, especially to bacteria which are among the principal competitors of yeast. So you can claim as much of the resource as possible. As a bonus you poison your competition with a compound that you are able to tolerate, and then you can actually, after the resource has been cleaned with the ethanol, you can actually go back and respire it and get a few more ATP back later in the process. So, when we started this project looking for the missing ancestor of lager yeast, we knew that there were six species of Saccharomyces. And I've shown their evolutionary relationships here. And the genus Saccharomyces is a collection of these six species, originated probably about 10 to 20 million years ago. And that means all six of these species shared a common ancestor at this point in the past. Now, if we fast forward in the future, we can see that new species emerge, and that's what is occurring at each of these branch points. And so Saccharomyces cerevisiae and Saccharomyces paradoxus share a common ancestor at this point in the past. Now, of course, yeast evolve, their DNA evolves at a much faster rate than our DNA, and that's why when we actually look at the level of genetic divergence we see that these two closely related yeast species actually are about as related as human and mouse are at a genetic level. And so by the time we go out to the most distant member of this genus, Saccharomyces uvarum, it's actually at about human/chicken. And so that's how we end up with the comparison that yeast are more diverse than all vertebrates. There's a lot of diversity captured in yeasts. And I've drawn, perhaps the most relevant for this talk here, are the preferred temperatures that these organisms like to grow at. Now, historically, largely because of the temperature, Saccharomyces cerevisiae has been the historical yeast of choice, and it's been with us for thousands of years, and people have used it to make ales, wine, for distilling, to leaven bread, and of course in more recent times to make biofuels. Now, this is a dangerous situation to every disagree with your boss, but I was at Washington University in St. Louis and then University of Colorado School of Medicine with my immediate supervisor and mentor, Mark Johnston, and he is an ale man. We've had a rather longstanding disagreement about the relative merits of these two products. He is a traditionalist and prefers the older ale product, and as you can see, I'm endorsing a lager product right here. I don't think I consumed all of those myself. I'm sharing with my friends back there. But ales, as I mentioned, have been brewed for thousands of years. They've been with us a very long time. Whereas, lagers were invented in 15th century Bavaria, and that comes from the historical record. And, in fact, by the 16th century, Bavaria had actually passed very strict purity laws called the Reinheitsgebot, and, actually, by the middle 16th century it had actually banned brewing during the hottest months of summer. The lagering became a worldwide phenomenon by the 19th century as Germans went to many, many countries including our own country, and, in fact, many of my ancestors would have come over in the German -- to this country. So what is an ale and a lager for those of you who have not had this particular argument with your boss? Ales are things like pale ales, IPAs which are India pale ales, a cosh, ESB, scotch ale, porter, stout. Lagers are things like pilsners, helles, what we call Oktoberfest in this country, Vienna style lagers, Schwarzbiers, bock. And if all these names sound vaguely German and these mostly sound vaguely English, that's not a coincidence. That's another sort of short-hand. Most German beers that are popular in this country tend to be of German origin, and many of the English style beers in this country that are popular tend to be ales. So another classical way that people have distinguished ales from lagers is that ales are made by a top fermenting yeast and that it ferments vigorously and stays up in the water column until the end of fermentation. Whereas, traditionally lager yeasts fall, crash out of solution and ferment along the bottom. Sometimes that's called sedimentation or flocculation. Also, ales are often served minimally filtered, in some cases unfiltered. Whereas, lagers tend to be prized for their clarity and crisp taste. The other big difference, and I think you'll note why we were paying attention on the tree to the temperature preferences is that ales are brewed at a fairly warm temperature. And this, of course, makes sense because much of civilization grew up around the Mediterranean and refrigeration would not have been widely available. So it makes perfect sense that you would be brewing a product which was commensurate with the temperature around you. Whereas, lagers are brewed at a much colder temperature. And, in fact, they go through a long-term storage process which is the basis of the word lager. It actually comes from the German lagern, which means to store. And there it's stored near freezing for several weeks to help age and continue to ferment. So ales are brewed with Saccharomyces cerevisiae, and this is true 95-plus percent of the time. Whereas, lagers we have know at least since the mid-'80s are brewed for a hybrid of Saccharomyces cerevisiae and something else. And that something else is the topic of tonight's talk. So, lager yeast is sometimes scientifically called Saccharomyces pastorianus. It's alternate name is carlsbergensis, which is indeed named for the brewery in Denmark. And this is a sterile hybrid of Saccharomyces cerevisiae and something else. Now, when I say it's a sterile hybrid, I mean it's much like a mule, a cross between a horse and a donkey. It can produce a viable progeny, but that progeny is not longer able to reproduce. So a mule is not, excuse me, a donkey is not able of reproducing either with a horse or with a mule. Yeast can reproduce asexually. So, essentially, what we have are donkeys which can reproducing asexually repeatedly in the laboratory, and we call these lager yeast. So based on the cursory genetic information that we started to roll in in the mid-'80s, we knew that it was a hybrid of Saccharomyces cerevisiae, and it turns out we also knew that it was a hybrid of something down in this part of the tree. One of these early branching chickenish level strains. But it turned out that there was a lot of diversity within this part of the tree, and depending on which strain you compared to which, you could get DNA divergence, which we'll talk about in more detail later, ranging as high as 15% which is actually greater than human/mouse. So there was a lot going on here, and that really raises the question of how many wild species there were in this complex. So we get a little bit of information in 2009 when a Japanese research team associated with the Suntory Brewing Company published the genome sequence, and that's the complete list of genetic information for the lager yeast Saccharomyces pastorianus, and that made it really clear, actually, by looking at the genome sequences of all these other species that there was something missing because it was clear that every gene in the genome, it was 7% or so off from this species here which I'm not calling Saccharomyces uvarum. So, again, nothing was matching this other part of the lager yeast genome. So why couldn't we find it? Well, it turns out that if you look at the global funding for research and development and you scale each country by the amount of money that it spends on research and development, you get a world view that is somewhat different from the traditional geography.

LAUGHTER

Chris Hittinger

Now, a lot of this is research for defense purposes and research on biomedical research and other applications. So very little of it is the most important kind of research, research on yeast biodiversity.

LAUGHTER

Chris Hittinger

But it turns out that it's a pretty good proxy, actually, for the kind of research that goes on yeast biodiversity because if you look at the five big strain collections of yeast, they're all in the northern hemisphere and they're all pretty much in the countries you'd guess them to be in based on general research and development spending. So they're all in the northern hemisphere. The biggest one is actually in the Netherlands, right here at CBS. And we have a very nice one right down I39 in Peoria, Illinois, that one of our collaborators, Clet Kurtzman, runs at the USDA there. So this project began as a collaboration between a team in Portugal, -- more than five years ago looking for new species of Saccharomyces, and in particular they were interested in a particular kind of transporter that they new was present in lager yeast and they were looking for where they could find this species in the wild. And they were searching in Portugal, in other parts of Europe, they also had collaborators in Japan they were sampling with, and they've been to Oceana a couple of times. Nothing. No Saccharomyces eubayanus, no lager yeast. >> Is there any reason while Mongolia figures so prominently? >> That is South Korea, that is China, and that is Japan. >> That's Korea? >> Yep. And then there's North Korea.

LAUGHTER

Chris Hittinger

So I began collaborating with Professor Sampaio and Goncalves on another project actually, also related to Saccharomyces biodiversity, and I've isolated about 200 strains from the United States. But, again, at least in wild settings, nothing that could plausibly be Saccharomyces eubayanus. Now, again, I stress that people of course are recovering lager yeast all the time from industrial settings, but these are not wild progenitor, these are things that are controlled by people and have specific genetic marks that tell us that they're no longer wild, and we'll talk about what those are later. Where we really hit pay dirt was with our collaborator down in Argentina at the very tip of South America in northwestern Patagonia, and Diego Libkind is the one who, by spreading the search to a fifth continent, actually finally hit upon a wild reservoir for this new species of yeast. So why Patagonia? Well, Patagonia is a consistently cool environment. It's really pretty close to Antarctica. It's average temperature is not too dissimilar from what we experience here in Wisconsin. It doesn't have quite the Arctic extremes that we do because it's a little bit more buffered by the oceans, but it's pretty consistently cool with an average temperature of between six and eight degrees Celsius. And we can heat samples instead of on oak trees, they don't have oak trees in this part of the southern hemisphere. Instead what they have are southern beech trees which are actually only very distant relatives of the beech trees we have in the northern hemisphere. They're called Nothofagus trees. And much like we see on oak trees, though, when they're wounded they'll produce this sap, and Saccharomyces yeasts and other yeasts will actually ferment the sap, and so you can see the sap being fermented in these cases. But perhaps what's more interesting is they get infected by another kind of fungus, a parasitic fungus called sartoria, and this fungus actually ends up in a tug of war with the tree's immune system and ends up blowing up into these massive galls, they're called, and this infection actually becomes very high in sugar as the fungus tries to exploit to resources from the tree and the tree tries to keep things under control and keeps pumping more resources here. And it turns out that because they become rich in sugar and these are fairly soft structures, people in Argentina will actually chop these off and chop them up and put them on salads as a little bit of a sweetener. Apparently, in pre-Columbian times the Mapuche tribe would actually take these guys off, harvest them, take them back to their village, dipped them in water, and extract the sugars, and of course what we now know to be Saccharomyces yeasts and make a fermented beverage. It's no longer made very frequently from what I understand, so that may tell you about its relative quality to what we have today. So how do we actually isolate it? I've talked a couple times about how we get yeast or that we're getting yeast from the wild. So what we do is we take bark or soil, and usually soil is more convenient but we can also chop off bark, and in the case of the sartoria you can actually harvest the fungal parasite. Takes that back to the laboratory and basically you give it sugar and ethanol, ethanol in this case is basically a cheap antibiotic, and sometimes we add antibiotics to the mix too, and that's because bacteria as present hundreds or thousand of fold higher concentration than yeast are. So by giving them sugar and ethanol, we can wait for couple of weeks depending on the temperature of isolation, and then as the yeast start to produce carbon dioxide from fermentation and the media starts to get cloudy, that's a pretty good indication that we've isolated something from the wild. And then we can take it back and put it on petri plates and check to see if it's forming those tetrads we talked about, those himyasci, and if it is, then we can expose it to DNA fingerprinting. And based on sort of a preliminary DNA fingerprint, we can triage the strains and tell whether they are something that we might be interested in or something that we're not interested in. And it turns out, from the Patagonian studies in particular, we were getting predominately two species back. And so Diego preliminarily identified strains as either wild Saccharomyces uvarum from Patagonia, and, again, this is strains that looked kind of like the European Saccharomyces uvarum whose genome had already been sequenced, and also possibly this new species eubayanus which is the candidate for the contributor to lager yeast. And it turns out this actually seems to be a preference for which species of yeast is growing on which species of southern beech tree. And this is statistically significant, and it's a little easier to see if I highlight them for you. So you can see that uvarum really likes Nothofagus dombeyi, whereas eubayanus likes pumilio and Antarctica. And, again, this isn't an absolute distinction, but there's clearly some partition of resources going on, some niche partitioning going on as one species prefers one set of resources and the other prefers the other set of resources. And so because we're finding them in the same place, they're in sympatry in the same basic area, that sort of raises the question of whether they're really separate species, and, indeed, which one actually contributed to lager yeast. And so I've shown you evidence that they are ecological species in that they are partitioning out resources. We also crossed them in the lab and attempted to create the hybrid, and it turns out when you do that, they can only reproduce sexual spores 5% to 10% of the time, which suggests that there's a substantial barrier to gene flow. In other words, they're substantially sterile much like a mule is. Now, phylogenetic species, this is more a question of whether they are actually exchanging genes historically in the wild. And so this is a question where applying the latest sequencing technology is really the only way to get a look at every single gene. And so what we did is applied illumina sequencing which allows us to sample 36 letters of DNA at a time. And then we mapped all of those reads to the genome assembly that we already had for the European strain of uvarum, allowing a bunch of mismatches. And how this technology works is truly amazing and really could fill an entire Wednesday Nite at the Lab talk on its own. I'm not going to go into it too much. Suffice it to say that when the original Saccharomyces uvarum genome was sequenced 10 years ago, it cost more than $100,000, and, using this technology, we now can do it for $100. And this is also the kind of technology that is going to, as we'll talk about at the end, change the way you interact with your doctor in the future. But just to give you a quick rundown of what exactly I mean when I say the DNA differs. Here's an example of the Saccharomyces uvarum European strains right here. This just happens to be a random snippet from the genome, and if we look at the strains we're getting out of Patagonia, we can look at a piece of DNA that matches the existing sequence, and as we see if you look across at the guanines and the thymines and adenines and cytosines, there's only four possible letters but we see that at every position it's exactly the same. And geneticists sometimes get lazy and it also helps us to view the data if we just put dots where it's the same. And so this makes it really clear that for this stretch of DNA they have exactly the same sequence of DNA letters. Okay, what about our new strain, our Saccharomyces eubayanus? Well, if we look at this same stretch of DNA, it turns out that it's the same for a lot of it, but there are a few differences. And in this case there are actually five differences. And so it's clearly distinct from both the European and Patagonian strains of uvarum. But it's similar enough that we can still recognize it. So, of course, if we then look at the same piece of DNA from the lager yeast, it matches perfectly our Saccharomyces eubayanus strain. It's the same as all four of them in some places, but where the eubayanus differs, the lager yeast Saccharomyces pastorianus also differs and it differs in the same way. Now, just to show you that I didn't cherry pick things, I'm going to walk you across the chromosome. Now, of course, a chromosome goes on, in this case, for 500,000 letters so I can't show you all 500,000 letters because we don't have time, and I would lose count probably at number 124,000. That's usually where I lose count.

LAUGHTER

Chris Hittinger

But what we can do is we can average and sort of look at it mathematically. And if we do that, we can measure, going up here, how different each strain is from our European strain of Saccharomyces uvarum, and then how different each strain is from our new Patagonian species, Saccharomyces eubayanus. And if we do that, we can see that our new strain of eubayanus is, indeed, quite distinct from the uvarum that we see. On average, it's 7% divergent, so that means that 7% of those DNA letters it differs. Whereas, the other species that we're getting from Patagonia, uvarum, looks almost exactly like its European counterpart. There are few differences, but it's very, very similar. And, of course, if we compare it, again, to the eubayanus, it's about 7% divergent. Okay, so if we lay the lager yeast sequence across, we see that lager yeast is, of course, very distinct from uvarum, but when we compare it to eubayanus, it is very, very close to the zero line. And, indeed, the genomes are 99.56% identical, so at more than 99.5% of the letters they're exactly the same. So, we needed to look at a few other strains associated with brewing and that's partially because other groups had asserted these as potential candidates for having given rise to lager yeast. And it turns out what we see is that many of these strains are really a mish-mash of genetic variance that probably came together at some point after lagering began and are mixtures of these wild alleles that we're seeing. And this is a perfect example. This is a strain that was isolated from turbid beer in 1895, I believe in the Netherlands, and this was actually designated as the type strain of Saccharomyces bayanus, which means that it's the official taxonomic specimen that is forever associated with this particular name bayanus. But we see that it actually has uvarum alleles for hundreds of thousand of kilobases, exact matches, and that it looks very different from eubayanus. And, indeed, for a lot of its genome it actually is heterozygous in that it has both eubayanus and uvarum alleles. And here, basically, an entire half chromosome is a mixture. We have one copy of eubayanus and one copy of uvarum. So if we look at another strain, this is another brewing contaminate that was isolated in the '40s, and this organism is also a hybrid. However, it's a hybrid in slightly different places. So here it has entirely uvarum alleles. It's very close to uvarum. And over here, it's entirely eubayanus alleles. Now, an important feature of plotting things this way is you can see whenever the line goes up here, it also goes up here. Similarly, the orange line, whenever it goes down here, it also goes down here. And that tells us that there are really two alternatives here, much as I've drawn it, that is an uvarum alternative and a eubayanus alternative for this particular species complex and for this set of strains. Because if there were three alternatives or four alternatives, those lines would not necessarily have to move in parallel. So if we look at the whole genome, you can see that if we look at all 12 million base pairs, we see exactly the same set of patterns. We see that the orange and the green strains are very complex mixtures. They go up and down all over the place. Interestingly, we see that pattern that I just mentioned where whenever the green goes down on top, it also goes down on bottom indicating that there are probably only two alternatives. And the other thing we see is that the wild strains that we're getting out of Patagonia, the red and the blue strains, they basically hug the positions that they're supposed to be in. They're not picking up alternative alleles from the other species, and similarly, the purple lines basically track exactly what the blue line, and that tells us, again, that pastorianus is a hybrid of cerevisiae and this new species Saccharomyces eubayanus. Okay, so what else can we do with the genetic information other than just identify the ancestor? Well, we can ask if Saccharomyces pastorianus was domesticated or if it underwent genetic changes after that sterile hybrid form. And so we're going to be looking for radical changes, changes that really stick out. We've only had this genome data for really about a year and a half now, and so we haven't had a lot of a chance to work with it. We're going to look for places where it differs from the wild strains where we can look at those three brewing strains, the contaminants and the Saccharomyces pastorianus, and look for obvious cases as to what's changed in the genetic code. And, in particular, the ones we're going to focus on are cases where there might be extra or transferred genes from Saccharomyces cerevisiae because we know cerevisiae has been in use for brewing and winemaking and other processes for thousands of years. And we're also going to look for Saccharomyces eubayanus genes that have been inactivated very recently, possibly as a result of domestication. So we looked at the genomes of all of those strains on the previous slide for contributions from other species, in particular for contributions from cerevisiae. We actually looked at all possible candidates, but it turns out we found no foreign contributions in any of the uvarum strains or in our wild Patagonian strains of Saccharomyces eubayanus. We, of course, found that Saccharomyces pastorianus has lots of genes from Saccharomyces cerevisiae. It is, of course, a hybrid. But, interestingly, we found that pastorianus had not picked up genes from any other species, any of those other species on the graph. However, we find that the green strain that was a brewing contaminant and the bayanus type strain from turbid beer 115 years ago, it turns out that these strains have picked up multiple genes from Saccharomyces cerevisiae. Now, you remember they are mostly mixtures of uvarum and eubayanus, but they're also picking up strains from cerevisiae that are involved, sorry, genes from cerevisiae that are involved in maltose consumption, which are glucose-glucose disaccharides, glucose-glucose sugars. And this is, of course, at very high levels in wort, which you'll note that one of the main four ingredients in beer is, of course, malt and that is why it's called maltose. There are also genes that these strains picked up from cerevisiae that are involved in sucrose consumption. This is common table sugar that we all put in coffee in the morning. That breaks glucose-fructose bonds. Some brewers will actually add table sugar to their beers, so this, of course, makes sense, and it's also present at fairly high level naturally in wort. And, of course, in the brewing contaminant strain, we found a number of cerevisiae genes that are known to be involved in sedimentation and flocculation, which is another trait that's very important to the brewing process. This is the process of settling out and creating a bottom fermenting yeast. So this means that these two strains, in addition to being mixtures of uvarum and eubayanus, are actually triple hybrids in that they've gone on and also picked up genes from cerevisiae. And so these are really complex organisms. So if we look at the genes that are being picked up from cerevisiae, we can look and see if it's ever happening in the same way twice because that could indicate that a single genetic event happened and then it was advantageous and spread to other species. And here's probably the most striking example we found. And this is out on the end of a chromosome. This is a case where the fancy genetic statistics are actually harder to read than the actual sequence data itself. So I'll direct you to the sequence data. And you can see, much as before, I've split out read alleles and blue versions. And you can pretty readily see that all of the uvarum versions are in red, all of the eubayanus versions are in blue, and then Saccharomyces cerevisiae is different still. It's a third option. And what happens right here, right here, is all of the eubayanus alleles that are involved in brewing all the sudden switch over to the cerevisiae version. You can see they're all the same. Whereas, the wild Patagonian version is actually different still from uvarum but is much closer to uvarum than it is to cerevisiae because these are very closely related sister species. So this is an example of a single genetic event that was rare and fused a Saccharomyces cerevisiae chromosome onto a eubayanus chromosome, and this rapidly spread, through the population. Here's one more case and this is a case where a eubayanus gene actually gets broken by the domestication process. And so, here again, I'm showing you the red versions for uvarum and the blue versions for eubayanus. We see there are lots of dots meaning it's basically the same. What we see here is at that orange arrow there's actually a missing base. It's no longer a dot. It's actually a missing base, and because of the way the DNA code is read off, it actually throws the entire right half of the screen out of frame, and so the code is completely scrambled, and now, instead of having exactly the same protein sequences that are encoded for by the DNA, it's completely random and completely different and non-functional. And, again, it's, in all three cases, exactly the same lesion that is breaking the sequence. Now, this is an interesting gene because this is a low affinity sulfate transporter. And it turns out there's actually a patent on over-expressing, ie. making a lot of, the other sulfate transporter from Saccharomyces eubayanus. And this will actually increase the production of sulfites, which are made from sulfates by lager yeasts, and this particularly interesting because it turns out that lagers actually have considerably more sulfites than beers produced by ale yeast. And this is probably one of the reasons that lagers are able to age longer and retain their flavor better is because sulfites are natural flavor stabilizers. Okay, so I'm going to briefly summarize what we think happened genetically based on the data I've shown you. Of course, in Europe for thousands of years there have been ale type Saccharomyces cerevisiae that have been partially domesticated. Brewers have been passaging them from batch of beer to batch of beer. Eubayanus, at some point, fused with this ale type yeast and created a hybrid yeast, a sterile hybrid that is a mixture of cerevisiae and eubayanus. There were a number of genetic changes that happened, two of which I've talked about, the inactivation of this sulfate transporter and also the picking up maltose genes from Saccharomyces cerevisiae, genes for breaking down maltose, a common sugar. And, of course, in the brewing vats, the populations of yeast reach tremendous levels, into the trillions. Towards the end of the process, cells start to die and they release DNA. And it turns out we can actually do this process in the laboratory. You can get transformation as strains pick up DNA from other organisms. And, of course, brewing contaminate strains seem to include Saccharomyces eubayanus once it made its way into the brewery, and it can pretty readily pick up these partially domesticated genes from pastorianus because it has substantial portions of eubayanus that can recognize it as its own DNA. And because eubayanus is so closely related to uvarum, they share a common ancestor at about 7% genetic divergence, and we know that Saccharomyces uvarum is present at pretty high frequency in Europe, we think a lot of these complex triple hybrids informed as brewing contaminants crossed to wild European strains of uvarum, and that's how we end up with several of the triple hybrids. Okay, so to summarize in cartoon form, here's what we think happened. Pastorianus is a hybrid of cerevisiae and ale yeast and, of course, eubayanus. The lagering process itself was invented in the 15th century, but based on the date of the rise of trans-Atlantic trade, the modern lager yeast strains probably came into existence some time after the process became popularized but probably before the lager strains were spread around the world in the 19th century. And I've shown you genes involved in sugar consumption, sulfite production, and sedimentation all show the sort of hallmarks that we would expect for genes that were under the selection of humans, brewers specifically, or under the competitive selection going on between brewing strains. And, of course, I've also tried to explain how some of the more complex brewing contaminant strains and some of the more complex brewing strains seem to have formed, in some places, pretty complex triple hybrids. Hopefully I've also convince you that while the species complex of bayanus turns out to be pretty complicated, it really boils down to probably two naturally occurring species that have then been crossed to the traditional ale yeasts and remixed around a whole lot. Or to really, really boil things down, it's something like that.

LAUGHTER

Chris Hittinger

Okay, so I think I have a few minutes left here, so I want to mention that hopefully I've convinced you that it's not just cerevisiae, Saccharomyces are important in other processes. And now that we are starting to understand about the natural diversities out there, we're starting to look at these industrial processes in a new way and learn what is doing what process. We now know, for example, that lagers are made with hybrids of eubayanus and Saccharomyces cerevisiae. I've also shown you now that's it's pretty clear that a number of the brewing contaminant strains have substantial contributions from uvarum. There are also reports that champagne has contributions from uvarum, although I think with genome technologies this probably needs to be reexamined. There are also a number of reports using not quite genome scale data but close, at least it's solid enough that I believe it, and that Belgian beers are often actually hybrids involving this other species called Saccharomyces kudriavzevii, and there are also some wine strains and cider strains that appear to involve contributions from kudriavzevii. So this is another species that appears to be involved in some pretty interesting industrial processes, and we're very interested in following some of that work up. And, in particular, Belgian strains are famously diverse, for those of you who are beer aficionados, and we suspect that there may be a lot of interesting complex things going on in Belgian stains. As I mentioned, it's only some that are kudriavzevii hybrids, and so we're very interested in learning about other Belgian strains, and also Weiss strains are also another interesting mystery. And we actually have a number of strains that we've recently generated genome sequence data for from New Glarus, and we're very interested in taking a look at those to see which contributions are coming from where. And, again, that's because we're going to be able to do that because DNA contains this very powerful information, and the powerful information it contains can be used for that sort of ancestor determination. And this is from this month's National Geographic from researchers at the National Human Genome Research Institute, Heidi Parker and colleagues. And this is an example of how they've determined the genetic contributions to various dog breeds, and, as you can see, your pooches can be broken out into four major groups based on which fraction of which genes come from each of these groups. And, indeed, you can actually pay a company commercially to go out and look at your dog's DNA if you have a mutt and determine with some degree of probability which various dog breeds you mutt might have come from. So that could settle many arguments between spouses or start new ones.

LAUGHTER

Chris Hittinger

In all seriousness though, this can be done in humans also, and there are companies that will take your DNA and tell you something about your ancestry for as little as $200, or will tell you about your susceptibility to various diseases. And this is something I think we should all be thinking of, and, again, this is a topic that would go well beyond a Wednesday Nite at the Lab. We could be here until Thursday or Saturday morning discussing personalized medicine, but this is something that we're all going to have to deal with, is that as I mentioned the cost of sequencing a yeast genome in 10 years has gone from over $100,000 to less than $100, and human genome sequencing is rapidly getting to that point where all of us are going to be able to, if we want, to sequence our human genome and discuss the implication of those variations, our susceptibility to disease with our doctor. And of course this is also important because many tumors have particular genetic signatures and that can affect prognosis and treatment for particular kinds of cancer. So this is an area we should all be thinking of. But this same kind of technology that's creating this revolution, I hope will also create another revolution, and that is in what I might call personalized brewing. And, again, this could take two flavors I think. A, there are a lot of diverse strains that are in use by brewers that have a lot of interesting characteristics. We can do the sort of thing you can do with dogs and tell you where they came from, what they are, but, of course, stains can be manipulated and crossed in the laboratory, and if you want to mix variation I think there's a lot of opportunity to do so in a way that is now informed by the kinds of genetic tools that we have. And so to summarize a lot of data from a lot of different groups, I also want to stress that all these species of yeast have variations within species, much like humans are different from one another, except the variation is even greater. So within a species of yeast, we have variation as high as 3.5%. Now, the human/chimp level variation is about 1%. so these are extremely diverse strains of yeast. There are hundreds of strains in freezers around the world. All of these like to make ethanol from sugar, and they're all a little bit different. And, of course, this goes to our academic interests are in understanding their ecology and evolution, but I think brewers and biofuel producers are really going to be interested in how we can exploit this variation. And, in particular, for the biofuel industry, the problem is this. It's that our main workhorse is wired to work off glucose, and while this is great for producing beer, which is starting with maltose, a glucose-glucose dimer, this is a real problem because it creates a competition for food because corn is the principal way that people feed this chain. Whereas, we really need to be moving toward cellulosic products which use alternative sugars like xylose. The problem is the organism that is best at doing this aspect is really pretty hardwired to using glucose. So we need to understand how it is using glucose better so that we can understand how to get it to use other sugars and how to exploit the variation that exists in yeast for its preferences for xylose, for between glucose and xylose, and other potential sugars. So very briefly, I want to thank Jose Paulo Sampaio and Paula Goncalves who began searching for wild yeast as part of this project more than five years ago in Europe. And, of course, Diego Libkind who actually discovered Saccharomyces eubayanus down in the wilds of Patagonia deep in the Andes Mountains. Jim Dover for taming the bucking bronco of an illumina machine at Wash U and then at Colorado and helping make this cutting-edge technology something that we're all going to be able to take advantage of to do cutting-edge genetics for the next 5 to 10 years. And, of course, Mark Johnston for letting me work on lager when deep down he wanted me to be working on ale.

LAUGHTER

Chris Hittinger

And so I think we'll take maybe one or two questions in here and then perhaps go enjoy the full sensory array of the educational multimedia presentation involving taste and smell and other delights. So thank you.

APPLAUSE