In the first of three Features looking at aspects of alcohol, Siëlle Gramser discovers how yeast first opened the floodgates of intoxication.
Steven Benner jokingly calls himself a dilettante. A biochemist at the University of Florida in Gainesville, Benner dabbles in a wide range of disciplines, from bioinformatics to astrobiology. His aim is to gain insight into the basic chemical rules that govern how life works — both here and, ultimately, on other planets. But although science drew his gaze to the skies, it was alcohol that brought him back down to Earth. Or, to be more exact, the enzymes that can both make and consume it.
Alcohol dehydrogenase is best known as the enzyme that breaks down alcohol in the body, and as such it has been studied exhaustively. But Benner and other researchers in the field have now turned to its evolution, and their work is providing fresh insight into the puzzle of why some creatures, such as yeast, came to make alcohol and why so many others, including ourselves, can tolerate it.
Alcohol dehydrogenase — ADH for short — is a blanket term applied to a large and diverse group of enzymes. In many creatures, including ourselves, they help to convert alcohols, such as ethanol, into compounds that other enzymes can break down and extract energy from. But in a number of microorganisms, they can help the reverse reaction, making alcohols as part of the process of extracting energy from sugars.
The stars of these alcohol-producers are the yeasts. Not only do Saccharomyces species of yeast churn out oodles of ethanol, they can also tolerate far higher concentrations of it than other microorganisms. Brewer's yeast (S. cerevisiae) owes this ability to two alcohol dehydrogenases: ADH1, which makes ethanol, and ADH2, which breaks it down for use as an energy source. Yeast not only brews its own moonshine, it consumes it too — “to the last drop”, as Benner says.
At first sight, this makes no sense. Making ethanol from sugar and then consuming it is energetically far more wasteful than simply consuming the sugar. Researchers have long pondered why yeast goes to all that trouble. Although it might be nice to think that there is a creature out there whose raison d'être is to party, evolution doesn't work that way.
Make or break
Benner and his team came across the explanation when hunting for the origins of ADH in yeast. Benner is interested in combining the study of genes and proteins with geology and palaeontology to gain insight into the history of life on Earth and present-day protein function. “Every biomolecule is better understood if we know its history as well as its structure,” he says.
The ADH genes in yeast make an intriguing subject for this approach. When yeast gained its ability to make alcohol, it must have done so as a result of a selection pressure in its environment and, what is more, this would have had a knock-on effect on other creatures. So working out when and how the ADH enzymes came to be could open a small window onto what ecosystems were like back then.
ADH genes and the proteins they make are well studied and have been isolated from many different species of yeast, so Benner's team had plenty of useful material to work with. The goal was to reconstruct the original gene that was duplicated to give rise to ADH1 and ADH2, and to ask what its function was — did it make alcohol, or did it break it down?
From a database of the sequences of related ADH genes in various yeasts — combined with additional ADH genes specially sequenced for this study — Benner and his colleagues assembled an evolutionary tree of yeast ADH. This showed where the ancestral gene would have fitted in and helped the researchers work out its most likely amino-acid sequence. Inferring the past from the present isn't perfect, so they ended up with 12 slightly different candidate genes1.
The group then reconstructed all 12 genes and tested them in yeast to see how the enzymes they produced compared with today's ADH enzymes. The supposed ancestor turned out to be most similar to modern-day ADH1, the one that helps yeast make alcohol.
The same evolutionary tree helped the team to estimate when the ancestor gave rise to the two present ADH genes. This information offers some insight into what drove the strategy. Was it humans breeding yeasts and selecting them to accumulate alcohol? Or did the event take place long before that?
“Yeast ‘realized’ there was a lifestyle opportunity, which involved making large amounts of alcohol. Steven Benner, University of Florida”
The group found that duplication of the ancestral gene took place between 80 million and 60 million years ago, which means that humans could not have had anything to do with it. Rather, Benner thinks it was down to flowering plants. “The hypothesis is that it occurred near the time Earth first provided yeast with fleshy fruits,” he says. With their temptingly large amounts of sugar, the fruit called for a clever strategy. “Yeast ‘realized’ there was a lifestyle opportunity, which involved making large amounts of alcohol as a way of defending the resources against competing organisms,” Benner explains.
In other words, yeast came up with a way of ‘pickling’ the fruit by producing alcohol, which would have made the fruit toxic to its competitors. This had a knock-on effect on its wider ecosystem: as well as killing off its competitors, yeast had created a niche in fermenting fruit for any organism that could devise a way to cope with the alcohol.
It was around this time that the fruitflies emerged. Feeding on yeast and fruit juices in rotting fruit that can easily contain alcohol concentrations of 4% or more (about the same as beer), the fruitfly (Drosophila) and its larvae found themselves in need of a mechanism for breaking down alcohol. Drosophila came up with its own form of ADH, structurally unrelated to that of mammals and yeast. In fruitflies, ADH plays a role in alcohol tolerance but also in energy metabolism, allowing the fly to use alcohol — indeed many different alcohols — as energy sources.
Different species of Drosophila live on different fruits, which in turn produce different combinations of alcohols when they ferment. Given that the biology of ADH is well understood, and that fruitflies are ideal for doing genetics studies, scientists have turned to studying the enzyme to understand how natural selection shapes it to prefer different alcohols in different species. Such studies provide an elegant link between a creature's ecology and the molecular changes that allowed it to exploit its niche.
Luciano Matzkin, an evolutionary biologist at the University of Arizona in Tucson, recently looked at ADH in two species of Drosophila that feed on different plants. He compared the different versions of the Adh gene in each fly, and identified key changes to the enzymes' structures that could have helped the flies adapt to different alcohols2.
Although alcohol tolerance is clearly an important trait for fruitflies, it is not the only function ADH seems to have in Drosophila. “It has played various roles during the evolution of the fruitfly,” Matzkin points out. “It pops up in many different places.” One of these is related to how well flies can resist a hot environment. Different populations of flies living at different latitudes have different versions of the Adh gene. And these patterns can shift rapidly in response to climate change, giving scientists a ringside seat for watching evolution at work, as well as a way of seeing the effects of global warming on ecosystems.
Together with others, Ary Hoffmann, evolutionary geneticist at La Trobe University near Melbourne, Australia, found that a particular version of the Adh gene, called AdhS, in Australia has spread south by some 400 kilometres in only 20 years3. This version of the gene is associated with heat resistance. “Twenty years is rapid in evolutionary terms,” Hoffmann points out. The speed of change suggests that different versions of Adh can make a big difference to a fruitfly's survival.
ADH, it seems, is a versatile enzyme that has evolved in different times and settings. In fact, ADH activity is carried out by three families of enzymes that seem to have arisen independently. The families are spread among most major life forms — from bacteria to plants, yeast and animals. It seems as though the structure of ADH, which allows it to bind to alcohol as well as to several other chemicals, made it a useful enzyme under different circumstances.
The original purpose of the ADH now found in humans probably wasn't breaking down alcohol: the fact that the enzyme can do this simply came in handy later on. So, what was its original function? At the moment, nobody knows. But some are hazarding a guess. Ricard Albalat, an evolutionary geneticist at the University of Barcelona in Spain, believes it was used to break down other potentially harmful chemicals, such as formaldehyde4. “Formaldehyde can react with DNA and cause mutations,” notes Jan-Olov Höög, a medical biochemist at the Karolinska Institute in Stockholm, Sweden. “The ability to break it down is a crucial function of ADH.”
But whatever their true origins, there is clearly a lot more to these multitalented enzymes than just allowing us to get drunk. As researchers delve further into their history, these molecules are shedding light on the big questions of evolutionary biology. A surefire cause for celebration.
Thomson, J. M. et al. Nature Genet. 37, 630–635 (2005).
Matzkin, L. M. Mol. Ecol. 14, 2223–2231 (2005).
Umina, P. A., Weeks, A. R., Kearney, M. R., McKechnie, S. W. & Hoffmann, A. A. Science 308, 691–693 (2005).
Gonzàlez-Duarte, R. & Albalat, R. Heredity 95, 184–197 (2005).