When it comes to making ethanol from things like sugarcane and corn, we've turned to the method that has been used for ethanol production for millennia: give yeast some sugar, and take away their oxygen. Just as they do when making beer or wine, the yeast take the sugar and partially metabolize it, releasing ethanol as a waste product.

While the basics are easy to do, it's turned out to be hard to get yeast to operate well in the sorts of environments that lead to efficient production of biofuels. At some level, the ethanol the yeast produce becomes toxic (as it is for us). And brewer's yeast tends to grow best at moderate temperatures (30 degrees Celsius), while biofuel production works best at temperatures of around 40 degrees Celsius.

So far, the approach used for getting yeast to be a better biofuel producer has not exactly been carefully planned: we've just continued to grow them in the harsh environment of a biofuel reactor and wait for evolution to take its course. But two papers that appear in today's Science describe targeted changes that greatly enhance the ability of yeast to survive in a biofuel reactor.

Insane in the membrane

The first study essentially repeated the evolution experiment that's given us existing commercial strains of yeast: a number of populations spent 300 generations growing at elevated temperatures. This produced a number of rearrangements of entire chromosomes, along with 30 single DNA base changes. The latter category affected a total of 18 genes, but every single strain tested picked up a mutation that wiped out the function of a gene called ERG3.

ERG3 is involved in the metabolism of sterols (such as cholesterol). These chemicals are involved in a variety of biological processes, but the one the authors suspect is involved here is their role in the cell's membrane, which separates its components from the environment. This membrane is anything but static; parts are pinching off as new material is inserted elsewhere, while clusters of proteins drift across the surface and interact. Altering the membrane's cholesterol composition can easily influence a wide variety of processes.

More specifically, high temperatures would be expected to increase a membrane's fluidity—the rate at which things flow around within the membrane. It's possible that, by changing the composition of the membrane's sterols, the mutation counteracts this effect.

The authors went back to a standard lab strain of yeast and knocked out the ERG3 gene there. When placed at elevated temperature, this single change caused the lab strain to grow at 86 percent of the rate of the strains that had spent 300 generations adapting to these conditions.

Avoiding an acid trip

Membranes also turned out to be essential for another bit of progress that focused on ethanol tolerance. At high enough levels, ethanol becomes toxic to humans. At somewhat higher levels, it actually becomes toxic to the yeast that make it. This limits the amount of ethanol you can produce from a single batch of yeast, lowering the efficiency of the whole process.

The authors reasoned that ethanol was probably disrupting the membranes slightly, making small holes that allow some of the internal contents of the cell to seep out into the environment and vice-versa. So, they figured it might be possible to partly reverse that by simply adding some of the normal cellular contents to the solution the yeast were growing in.

The test worked, in that it quickly identified potassium as a chemical that could help yeast keep producing ethanol even after it would normally have toxic effects. And, by supplying potassium in different forms, they were able to show that pH mattered as well. When potassium hydroxide, which increases pH, was added to the external solution, the yeast did even better than when they were given potassium in other forms. It increased the yield of ethanol by as much as 80 percent compared to cultures without it.

Normally in yeast, potassium is brought into the cell, while proteins (which lower pH) are expelled from it. So, the researchers added an extra copy of two genes to the yeast: one that pushes protons out of the cell, and another that pulls potassium inside. This combination increased ethanol yields by 27 percent—not as good as adding the chemicals, but still a significant boost.

Better biofuels?

Combined, the two show that simple genetic changes are enough to make tougher membranes and to let the cells tolerate a bit of membrane failure. The two research groups—one was in Boston, the other Scandinavia—worked separately, so we can't tell if the effects would be additive. Still, they give us a much better idea of how yeast can survive the harsh conditions of industrial ethanol production.

It's not clear whether this gives us a big advantage as things now stand, given that the best industrial strains of yeast have spent many, many more generations adapting to bioreactors; they're likely to have some of these changes already—along with many additional ones. Still, it's probably not a bad thing to have a better understanding of what's going on with the yeast strains we've become reliant upon.

And it's possible that these mutations will help with things other than ethanol. There's a lot of interest in developing biofuels with properties that are more similar to gasoline than ethanol, based on longer and possibly branched carbon backbones. These mutations are likely to help yeast tolerate high levels of those chemicals as well, and so could be introduced right from the start.

Science, 2014. DOI: 10.1126/science.1258137, 10.1126/science.1257859 (About DOIs).