Evolution of alcohol synthesis

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yeast anaerobic metabolism

We need to appreciate beer more. Alcohol has a long history in human affairs, and has been important in purifying and preserving food and drink, and in making our parties livelier. We owe it all to a tiny little microorganism, Saccharomyces cerevisiae, which converts complex plant sugars into smaller, simpler, more socially potent molecules of ethanol. This is a remarkable process that seems to be entirely to our benefit (it has even been argued that beer is proof of the existence of God*), but recent research has shown that the little buggers do it all entirely for their own selfish reasons, and they've been busily making alcohol that has gone undrunk by humankind for tens of millions of years.

In order to explain how we know this, forgive me, but I must explain some very basic biochemistry, and summarize what cells do to extract energy from sugar. We start with a 6-carbon sugar molecule. As a first step, called glycolysis, enzymes in the cell snap the molecule in half, liberating a little bit of energy and producing two 3-carbon molecules, called pyruvate.

Pyruvate gets passed on to the next step, called the citric acid cycle. This is a series of reactions that breaks the 3-carbon chain down carbon by carbon, liberating yet more energy at each step. It's all the steps after glycolysis that extract the bulk of the energy from the sugar molecule, but there's a catch: these steps require oxygen to run (this is also called the aerobic pathway). No oxygen, no citric acid cycle. Glycolysis can run, but some of the reaction products (especially a compound called NADH) accumulate, and soon enough that reaction would get choked off, too.

In the absence of oxygen, cells can continue to get that little bit of energy from glycolysis if only they can get rid of the accumulating reaction products somehow. In us, our cells do that by carrying out an additional reaction to convert excess pyruvate and NADH to another 3-carbon molecule, lactate, and NAD+. Lactate diffuses out of the cells and into the blood stream, forming lactic acid. When you are exercising anaerobically, that is, making your cells work harder than you can deliver oxygen to them, they limp along by dumping 3-carbon molecules in the form of lactic acid so they can keep burning sugar inefficiently. Once you're done working out, and your oxygen intake catches up, the lactate is converted back to pyruvate and can be burned completely and efficiently in the citric acid cycle.

Yeast do something different. If they are under anaerobic conditions, say, deep in the flesh of some decaying fruit, or in a wine bottle, they have the same problem: they want to keep their metabolism going by carrying out glycolysis, but to do that they have to get rid of accumulating products, somehow. They don't do it by making lactic acid, though (thank goodness—if they did, fermentation would produce a vinegary acid). Instead, they take the 3-carbon pyruvate and split off one carbon, producing CO2, which is given off as a gas. Any homebrew beer makers out there will be familiar with the idea of monitoring fermentation by observing the gas being produced.

The 2-carbon molecule left behind is called acetaldehyde. Acetaldehyde is further processed by an enzyme called alcohol dehydrogenase, Adh for short, which also recycles NADH. Adh converts the 2-carbon acetaldehyde into another 2-carbon molecule, ethanol. Alcohol. Booze.

Just like us, yeast produce this byproduct to keep going under anaerobic conditions, and when oxygen is available, they try to recover the energy in the alcohol. Familiar brewers' yeast has two forms of alcohol dehydrogenase: Adh1, which favors the production of alcohol from acetaldehyde, and Adh2, which more effectively runs the reaction in reverse, producing acetaldehyde from alcohol, and allowing the 2-carbon molecule to be fed back into the citric acid cycle.

If you'd rather see this in a simple biochemical diagram of the yeast pathways, click on the picture below: it says the same thing I just wrote up there.

yeast anaerobic metabolism
Enzymes in red are associated with gene duplications that, according to the transition redundant exchange clock, arose nearly contemporaneously. The make-accumulate-consume pathway is boxed. The shunting of the carbon atoms from pyruvate into (and then out of, blue arrows) ethanol is energy-expensive, consuming a molecule of ATP (green) for every molecule of ethanol generated. This ATP is not consumed if pyruvate is oxidatively decarboxylated directly to acetyl-coenzyme A to enter the citric acid cycle directly (dashed arrow to the right). If dioxygen is available, the recycling of NADH does not need the acetaldehyde-to-ethanol reduction.

The yeast method of handling anaerobic conditions is not particularly efficient. They have to burn a little extra energy to prepare acetaldehyde for the citric acid cycle (the steps in green in the diagram above), which wouldn't be necessary if they used a 3-carbon intermediate as we do. So, one question is why they use a relatively inefficient method to carry out anaerobic metabolism.

One explanation is that humans are responsible—we've been selecting for yeast that produce intoxicating byproducts. A prediction from that would be that alcohol production would be a relatively recent innovation. An alternative explanation is that yeast have been doing this as a clever strategy—flooding their environment with a poison that that they can tolerate but that other microorganisms cannot is a way to limit competition for resources. A prediction from this is that the yeast evolved first to produce ethanol, and only secondarily evolved the ability to recycle it. A recent study strongly supports the latter hypothesis.

First, molecular clock analysis of various yeasts suggests that the ethanol enzymes began to diversify about 80 million years ago…at about the time flowering plants started producing fleshy fruits (that meteor at the end of the Cretaceous may have had an abrupt impact on the lives of dinosaurs, but I wonder if the explosion of flowering plant species before that may have had an equally profound, if more drawn out, effect). Face it, people, the chemistry of beer is for the benefit of yeast, and didn't evolve for our enjoyment. Or if it were the result of domestication, it was by the undiscovered species Zymurgosaurus dipsomanius, not Homo sapiens.

The second line of evidence is very cool. It would be instructive to be able to directly examine the metabolism of yeast from 80 million years ago, and measure for ourselves the activity of their Adh enzyme. We don't have a time machine, unfortunately, but we do have the ability to reconstruct ancient genes.

The authors compared the sequences of Adh1 and Adh2 from S. cerevesiae and from 15 other Adh homologs in other yeast species. They then calculated the maximum likelihood gene sequence for the last common ancestor of these enzymes, the primordial alcohol enzyme, which they called AdhA. They then took modern yeast, removed their Adh1 and Adh2 genes, and replaced them with AdhA. Voilà, they have yeast from the Age of the Dinosaurs.

They then analyzed the chemical kinetics of this enzyme. The question was whether it was more like Adh1, the enzyme that primarily makes ethanol, or whether it was more like Adh2, the enzyme that primarily consumes alcohol. Did yeast evolve this enzyme to make a byproduct to inhibit its competitors, or did it evolve it to eat this byproduct?

The answer is that it was more like Adh1, and that early yeast were brewers, not drinkers.

Notably, the kinetic properties of the remaining ancestral AdhA candidates resembled those of Adh1 more than those of Adh2. From this, we inferred that the ancestral yeast did not have an Adh specialized for the consumption of ethanol, similar to modern Adh2, but rather had an Adh specialized for making ethanol, similar to modern Adh1. This suggests that before the Adh1-Adh2 duplication, the ancestral yeast did not consume ethanol. This implies that the ancestral yeast also did not accumulate ethanol under aerobic conditions for future consumption and that the make-accumulate-consume strategy emerged after Adh1 and Adh2 diverged. These interpretations are robust with respect to the ambiguities in the reconstructions.

We can assemble a history of yeast fermentation from this information now. The first step was the gradual evolution of efficient alcohol-producing enzymes that allowed the yeast to colonize and exploit rotting fruit exclusively. This occurred a very long time ago, in the Cretaceous. Next, there was a gene duplication event that produced two copies of Adh; initially, both would have done exactly the same thing, just allowing the lucky duplicators to pump out alcohol even faster. With two copies, though, one would have more freedom to drift and change its enzymatic properties without serious consequence to the owner. One fortuitous change would be a shift in enzyme kinetics in one copy to better promote conversion of alcohol back to acetaldehyde and enter back into the citric acid cycle. So, first they learned how to make an environmental poison to give them exclusive access to a food source, and then that same machinery was adapted to better allow them to eat that poison, permitting them recover some of the energy lost in secreting it.


*"Beer is proof that God loves us and wants us to be happy," Benjamin Franklin.


Thomson JM, Gaucher EA, Burgan MF, DeKee DW, Li T, Aris JP, Benner SA (2005) Resurrecting ancestral alcohol dehydrogenases from yeast. Nature Genetics 37:630-635.

Woolfit M, Wolfe K (2005) The gene duplication that greased society's wheels. Nature Genetics 37:566-567.

27 Comments

Very enjoyable. Thanks, I think I’ll have another!

Brewing preceded soap making by about 5000 years, making it the first biotechnology. This fact illuminates two principles: our species’ priorities (party forever, housework whenever) and just how long you can wait after a party before cleaning up the mess.

Those yeasties are fun little gi’s, aren’t they?

If God had intended me to drink beer, she’d have made it out of grape juice.

Further, the existence of beer proves that God loves us and wants us to be happy.

The brewing of beer from malted barley is a remarkable process. First, it required the develpment of barley. This can be attributed to neolithic genetic engineers who modified wild grasses into cultivated barley. The starch in barley or any other grain, however, cannot be fermented. Yeasts do not have the enzymes to break down starch (amylose) into more simple fermentable sugars. The sprouting barley seedlings can provide an enzyme called amylase that breaks down starch into maltose (a sugar). Maltose is a substance the yeast can turn into alcohol.

Some genius put together the whole process of partially sprouting the barley and then allowing the ground up sprouts to to produce sugar that could be fermented by yeasts.

I suppose that its use for over 5000 years is the reason the frankenfood fear people don’t object to beer. Nonetheless, it is a higher use of complex biotechnology.

Drink no longer water, but use a little wine for thy stomach’s sake and thine often infirmities.

Paul of Tarsus to Timothy

– Anti-spam: Replace “user” with “harlequin2”

Beer has been studied from many angles. Check out the Geography of Beer.

nidaros Wrote:

Some genius put together the whole process of partially sprouting the barley and then allowing the ground up sprouts to to produce sugar that could be fermented by yeasts.

Or.…the genius found the half-sprouted barley in the bottom of the grain bin, where it got wet and sprouted.…it then died due to lack of sun, and was fermented by the natural yeast that would be on the grain. The genius, while repelled by the smell, ate the grain, and drank the liquid anyway due to the fact that he was starving and out of better grain.….he got a buzz.…and the rest is history.

As an avid homebrewer (I make a porter beer that I call “Viking Piss” and a pale ale that I call “Ninkasi” after the Sumerian goddess of brewing), I’ve long been a fan of the humble little yeast. :>

Or . …the genius found the half-sprouted barley in the bottom of the grain bin, where it got wet and sprouted . …it then died due to lack of sun, and was fermented by the natural yeast that would be on the grain. The genius, while repelled by the smell, ate the grain, and drank the liquid anyway due to the fact that he was starving and out of better grain . ….he got a buzz . …and the rest is history.

Actually, the oldest known beer recipes, from Sumeria, used barley *bread* instead of the grains themselves. So more likely someone got a bunch of sprouted barley (malt) in his grain, ground it into bread anyway, soaked that bread in water (perhaps to soften it), forgot about it for a little while, some wild little yeasties landed, and voila. A new goddess is born.

Alas, the ancients also drank their beer flat – at the end of the fermenting process, the beer is naturally flat. Carbonating it requires the use of a fairly strong and secure bottle, which didn’t appear until much much later. Carbonated beer is produced by adding a small amount of sugar to the fermented beer while it is sealed in a container. The residual yeast in the beer use the added sugar to produce more alcohol and release more CO2, which, having no place to escape, dissolves into the beer to carbonate it. If your bottle isn’t strong enough, though, kapow. Beer bomb. Not funny, when it sends glass fragments out with the force of a hand grenade. So as you sip your brew, thank not only the ancient barley growers, but also the ancient dudes who learned how to turn sand into glass, and how to strengthen that glass by adding other ingredients to it. Without them, we’d still be drinking flat beer. Blecccchhhh. ;>

In many societies, moreover, beer/wine was drunk far far more often than water was. The reason is simple —– sanitation back then being what is was (or wasn’t), drinking alcoholic concoctions was far safer than drinking water. This was true as recently as colonial times in the US, where every village had its beer master and no one drank water if they could avoid it.

(Sings)

Beer is best best best best, Beer is best. It makes you fit and it makes you strong And it puts more muscles on yer old pom pom Beer makes bonny Britain. Beer has stood the test. What did Adam say to Eve? Beer is best!

’Twas on the good ship Victory Out in Trafalgar bay For miles and miles and miles around the gallant Frenchmen lay Nelson on his flagship Doling out tots of rum … When up the masthead there did run A message to all true …

(falls off chair)

two and a half minutes left in game 7, manu ginobli hits a three, spurs up by seven…

…I’m pretty happy those little yeasty beasties invented vodka oh so long ago. Now THAT’s intelligent design.

Well then how do we explain Jesus’s wine trick at the wedding?

“When you are exercising anaerobically, that is, making your cells work harder than you can deliver oxygen to them, they limp along by dumping 3-carbon molecules in the form of lactic acid so they can keep burning sugar inefficiently. Once you’re done working out, and your oxygen intake catches up, the lactate is converted back to pyruvate and can be burned completely and efficiently in the citric acid cycle”

Actually … Numerous studies have shown that a lot of lactate can be produced in the presence of adequate oxygen simply due to the inefficient control over glycolysis. ATP produced through oxidative phosphorylation remains high enough to support muscular activity, just too much pyruvate piles up so the muscle cells must produce and diffuse out lactate and a little pyruvate. Much of the lactate is then used immediately by muscle cells with a high oxidative potential and lower glycolytic potential (slow-twitch fibers) while much of the rest is used by the muscle with the highest aerobic potential (the heart). The Cori cycle described in the original post was worked out in frogs and works only minimally in mammals.

Don S Wrote:

Well then how do we explain Jesus’s wine trick at the wedding?

You are presupposing the story is true in the first place. Even pretending it were true, modern day magician tricksters have done similar things, eg Paul Daniels in the UK. If there happen to be several methods, then you are probably not going to be able to find sufficient evidence this long after the (alleged) event to determine which of them was actually used.

“Alas, the ancients also drank their beer flat “

I though all beer was flat. And warm.

I though all beer was flat. And warm.

Nah, only British.

:>

Lenny Frank Wrote:

In many societies, moreover, beer/wine was drunk far far more often than water was. The reason is simple ——- sanitation back then being what is was (or wasn’t), drinking alcoholic concoctions was far safer than drinking water. This was true as recently as colonial times in the US, where every village had its beer master and no one drank water if they could avoid it.

I’ve also thought that beer and wine were excellent ways to store surplus food. While they won’t last forever, they have a much longer shelf-life than either fruit or grain, which tend to get moldy and eaten by rats. A well brewed beer (i.e. not the mass-marketed swill we get these days) is very nutritious and good for you if consumed in moderation. Fermentation was a good way to keep a large part of the harvest from going to waste.

Steve Reuland wrote: “I’ve also thought that beer and wine were excellent ways to store surplus food. While they won’t last forever, they have a much longer shelf-life than either fruit or grain, which tend to get moldy and eaten by rats. A well brewed beer (i.e. not the mass-marketed swill we get these days) is very nutritious and good for you if consumed in moderation. Fermentation was a good way to keep a large part of the harvest from going to waste.”

Early American history bears this out, with an added level of efficiency. Grains were malted, then distilled in order to concentrate the alcohol. With a lack of reliable and rapid transport, it is much simpler to bring the excess grain produced on the early frontier to market as liquor rather than grain. And, it was cheap and plentiful. Historical records show that, by far, the time of greatest alcohol consumption in the US (per capita) was between 1800 and 1830.

It’s important to remember, though, that despite Benjamin Franklin’s love of beer, he advocated AGAINST drinking if for breakfast (a view that was considered rather eccentric at the time).

Also, ethanol does indeed make an excellent partial antiseptic, which will reduce levels of many bad microbes even at low concentrations. And since, to brew beer properly, one has to make conditions ideal for the yeast to outcompete whatever bacteria would love to jump in and rot the wort (with apologies to sticklers for suggesting that bacteria actually “jump” or “love”), the mere fact that it’s beer rather than rotten wort is a badge of relative purity.

But there’s the diuretic issue. It’s not much good to “hydrate” yourself if you’re just going to urinate out all the water you took in, plus more. This is why, back in the good ol’ days, a lot of the beer people drank was relatively “weak” in ethanol by today’s standards (this was especially true of the beer that was served to children).

No beer for breakfast? What am I supposed to chase the shots with? Orange juice?

natural cynic Wrote:

Historical records show that, by far, the time of greatest alcohol consumption in the US (per capita) was between 1800 and 1830.

Interesting, I just saw a show on PBS (can’t recall the name) and someone stated that during and after prohibition demand for alcohol went through the roof. He continued that more people wanted to drink due to prohibition. The numbers I examined ( Ethanol Consumption 1850-2002 ) indicate that you’re right about per capita alcohol consumption and PBS was either flat wrong or deceptive. The Cato Institute released this article here saying prohibition was a failure, yet their first figure is in accordance with the government’s figures.

Rev. Billy Sunday at the beginning of Prohibition Wrote:

The reign of tears is over. The slums will soon be a memory. We will turn our prisons into factories and our jails into storehouses and concribs. Men will walk upright now, women will smile and children will laugh. Hell will forever be for rent.

Juxtapose the above quote with any rhetoric about materialistic atheism, the more things change the more they stay the same.

I also found this

But there’s the diuretic issue. It’s not much good to “hydrate” yourself if you’re just going to urinate out all the water you took in, plus more. This is why, back in the good ol’ days, a lot of the beer people drank was relatively “weak” in ethanol by today’s standards (this was especially true of the beer that was served to children).

That is why there were several types of beer, usually divided into “small” beer (for ordinary day-to-day drinking) and “strong” beer (for getting snockered).

I’m thinking of making some more wine actually now it has been bought up. It’s very easy to make, so much so that we made some in my microbiology class in second year :)

Who says laboratory science has to be boring ;)

“Also, ethanol does indeed make an excellent partial antiseptic, which will reduce levels of many bad microbes even at low concentrations. And since, to brew beer properly, one has to make conditions ideal for the yeast to outcompete whatever bacteria would love to jump in and rot the wort (with apologies to sticklers for suggesting that bacteria actually “jump” or “love”), the mere fact that it’s beer rather than rotten wort is a badge of relative purity.”

Beer (alcohol in general actually) was much safer to drink for many years than a local populations general water supply. Good for avoiding Cholera and other microbes in any event ;)

‘m thinking of making some more wine actually now it has been bought up. It’s very easy to make, so much so that we made some in my microbiology class in second year :)

When I was a poor high school kid who, like all high school kids, wanted to get blitzed every weekend, me and my friends made our own mead. One of my friends had a father who kept beehives, so we always got as much free honey as we wanted. We’d fill a plastic garbage can with five gallons of water, put two or three pounds of honey in, drop a packet of baker’s yeast on top, cover it with some cloth to keep the bugs from falling in, wait a couple weeks, and voila.

Tasted bloody awful, but got the job done every weekend. :>

OH PEEZEE!

You had me at “We need to appreciate beer more.”

!!1oneone

=)

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This page contains a single entry by PZ Myers published on June 23, 2005 1:47 PM.

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