cc >> 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]
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&A session,
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]
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] 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] 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] 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] 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] 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]
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]
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]