Yeasts and Alcohol
I asked myself a question a while ago; "why do some yeasts, alone of living organisms, produce ethanol"? Of all the animal and plant and fungi taxa only few yeasts do so. Despite this many animals and insects use ethanol. Humans, such as ourselves at TeePee Cider make cider with 8% ethanol. Others make bread, wine and beer. With the help of Saccharomycetes cerevisiae yeast. The earliest breads and beer made seem to have a common ancestor on a gruel circa 5000BCE in the Fertile Crescent of the Middle East using this yeast.
The fruit fly is another organism that uses ethanol, and other primates and many other animals consume ethanol to some degree.
In nature, ethanol is made primarily by S. cerevisiae yeast. To do this yeasts have the enzyme alcohol dehydrogenase enzyme, ADH1 and have been producing ethanol since angiosperms first began producing sugar-rich fruits, sap, and nectar in the Cretaceous age approximately 100 million years ago. This seems counter intuitive. Why when you can use oxygen to completely oxidise glucose to CO2 would you not? Ethanol still has energy left in the remaining C-C bond that could be utilised. The accepted wisdom or reason is that it creates an environment that suppresses other living organisms like bacteria. Bacteria in most circumstances, including those of ripe fruit reproduce much faster than yeast and would win the ecological competition for this resource if the reproduction rate was the major factor determining success. But is this the cause or a happy coincidence?
Yeasts like many living organisms produce adenosine triphosphate - ATP (the main energy molecule of cell metabolism). But unlike other organisms produce ethanol via fermentation of glucose instead of the usual aerobic respiration to CO2, when it’s in a sugar-rich food. Also S. cerevisiae can produce ethanol (by fermentation) in both aerobic as well as anaerobic conditions. Bread making is aerobic, cider and wine production is anaerobic. This observation that yeast use energy-wasteful ethanol fermentation even when complete oxidation certainly suggests that ethanol is definitely more than a byproduct or waste product.
To make sense of this an examination of the lineages of ADHs show that S. cerevisiae acquired the ability to make ethanol at the time angiosperms started to produce fruits, but there is an ancestral form far older. So perhaps there was another reason. Under aerobic conditions, organisms respire as oxygen is the final electron acceptor,, but S. cerevisiae preferentially exhibits alcoholic fermentation until the sugar reaches a low level. This phenomenon is called the Crabtree effect , and the yeasts expressing this trait called Crabtree-positive yeasts. In contrast, “Crabtree-negative” yeasts lack fermentative products, and under aerobic conditions, increased biomass ( more cells) and carbon dioxide are the sole products. ADH1 ( fermentation) and ADH2 (respiration) differ only by 24 out of 348 amino acids. Comparing both ADH1 and , ADH2 ( similar to the human form converts ethanol to acetaldehyde) the last common ancestor of both can be identified and is called AdhA The kinetic behaviour of AdhA suggests that it the ancestor was optimised to make, not consume ethanol under aerobic conditions
Normally cells have no problem metabolising sugar to CO2 via respiration so why does S. cervisiae close fermentation which is not as energy conserving? The answer appears to be the yeast also has multiple copies of hexose ( 5 C sugars) transporter genes, which brings glucose into the cell and floods the cells metabolic machinery, there is not enough ATP left to recycle back to ADP to use in the Krebs cycle (respiration route) to consume more sugar aerobically. Visualise this as a traffic jam after several lanes on a motorway merge. So by switching to fermentation the need for ATP decreases as fermentation uses NAD+/NADH for the final electron acceptor not oxygen ie adding another lane out of the traffic jam.
So in addition to the novel enzyme ADH the yeast has extra copies of hexose transporter proteins. Why? The answer lies in its evolutionary history and luckily that is best studied in a model like yeast. In 1996, the genome of the S. cerevisiae was the first eukaryotic genome sequenced. Since then S. cerevisiae has been frequently used to study evolutionary processes. It is easy to grow and manipulation in the laboratory in a multitude of environmental conditions, combined with its small genome size (around 6000 genes spread over 12 chromosomes) makes it possible to study evolution as it happens in populations growing for weeks, months by sequencing the genomes of evolved lineages, and track the genomic changes underlying phenotypic adaptations. Combining this data with the available information on biochemical pathways and regulatory circuits makes it possible to understand why specific genetic changes are adaptive under specific conditions. Especially gene duplication as drivers of evolutionary innovations in the natural world.
But one step back in this discussion. One of the most intriguing questions in evolution theory is how new phenotypes can emerge. Darwin's theory builds on gradual changes of existing features, and he admitted that the appearance of novel phenotypes was difficult to explain. Later, after the discovery of DNA as the genetic carrier and genes as the basic genetic unit, scientists argued that new features likely emerge from gene duplication. Almost 50 years ago, Susumo Ohno (1928 – 2000) a Japanese-American geneticist and evolutionary scientist and seminal researcher in the field of molecular evolution postulated a solution to a rare effect that is noted in evolution usually after major events- whole genome duplication of WGD ( already mention in the sections on apples).
If the species survives this, ( which is rare), the retained duplicated genes can suffer 4 fates. Firstly, one copy is lost. Secondly one copy can retain the ancestral function, so that the other copy is redundant and might be relieved from genetic recollection after random changes to the genetic code and so can acquire mutations that can create a novel function for the gene (neofunctionalization). Thirdly, if the ancestral gene displayed multiple activities, duplication can allow the different functions to be split over- and sometimes also optimised in- the different copies (subfunctionalisation). And fourthly, the two copies can keep performing the same ancestral function, thus introducing redundancy and/or increased activity of the gene. In the case of S. cerevisiae this WGD event was also followed by reciprocal translocation between chromosomes — resulting in extensive genome rearrangement. The most common outcome of this type of duplication is loss of the duplicated copy from the genome, around 92% of duplicated genes returned to single copy. Interestingly, the genome of present-day S. cerevisiae contains multiple duplicated regions and displays a high degree of redundancy, with around 20% of all genes being members of duplicated and in particular to this story the ADH enzyme genes.
This WGD happened in S cerevisiae about 100 million years ago in the Cretaceous period. Not only were the genes for hexose transport increased and ADH altered, several other genes controlling energy production were too. So the species we know now as S. Cervisiae was uniquely equipped to take a dominant position in microbiological fruit when angiosperm evolved and so prospered. They captured more of the sugar, they could overcome the rate limiting block of recycling ADP/ATP by using NAD+/NADH and fermentation and the production of ethanol as a result was bactericidal too. And even better these yeasts can covert to respiration when the glucose level drops and consume the ethanol they have excreted as long as it’s not too high. ( Humans have tweaked this and now some S. cerevisiae can tolerate alcohol levels to 13%. And so as the yeast biomass proliferates, the sugar concentration dwindles, and yeast switch to oxidatively metabolise sugars and the ethanol they have created, a strategy termed ‘make–accumulate–consume’.
And there you have it. A WGD occurred in the yeast, that allowed it not only to capture most glucose in the environment but also to ferment it, so outperforming bacteria and suppress them.
Evolutionary table of Saccharomycetes showing the dating of the WGD event. It occurred after Candida glabrata separation but before S. Uvarum branch