Ethanol tolerance. Large concentrations of ethanol can be toxic to yeast, which has limited the production capacity of many yeast strains used in industry. Gregory Stephanopoulos, the Willard Henry Dow Professor of Chemical Engineering at MIT and colleagues at MIT and the Whitehead Institute for Biomedical Research have identified a new way to boost yeast tolerance to ethanol by simply altering the composition of the medium in which the yeast are grown.

Toxicity is probably the single most important problem in cost-effective biofuels production.

—Gregory Stephanopoulos

Ethanol and other alcohols can disrupt yeast cell membranes, eventually killing the cells. The MIT team found that adding potassium and hydroxide ions to the medium in which yeast grow can help cells compensate for that membrane damage. By making these changes, the researchers were able to boost yeast’s ethanol production by about 80%. They also showed that this approach works with commercial yeast strains and other types of alcohols, including propanol and butanol, which are even more toxic to yeast.

The more we understand about why a molecule is toxic, and methods that will make these organisms more tolerant, the more people will get ideas about how to attack other, more severe problems of toxicity.

—Gregory Stephanopoulos

The research team began this project searching for a gene or group of genes that could be manipulated to make yeast more tolerant to ethanol, but this approach did not yield much success. However, when the researchers began to experiment with altering the medium in which yeast grow, they found some significant results.

By augmenting the yeast’s environment with potassium chloride, and increasing the pH with potassium hydroxide, the researchers were able to boost ethanol production significantly. They also found that these changes did not affect the biochemical pathway used by the yeast to produce ethanol—the yeast continued to produce ethanol at the same per-cell rate as long as they remained viable.

Instead, the changes influenced their electrochemical membrane gradients—differences in ion concentrations inside and outside the membrane, which produce energy that the cell can harness to control the flow of various molecules into and out of the cell.

Ethanol increases the porosity of the cell membrane, making it very difficult for cells to maintain their electrochemical gradients. Increasing the potassium concentration and pH outside the cells helps them to strengthen the gradients and survive longer; the longer they survive, the more ethanol they produce.

By reinforcing these gradients, we’re energizing yeast to allow them to withstand harsher conditions and continue production. What’s also exciting to us is that this could apply beyond ethanol to more advanced biofuel alcohols that upset cell membranes in the same way.

—Felix Lam, lead author

The researchers found that they could also prolong survival, but not as much, by engineering the yeast cells to express more potassium and proton pumps, which are located in the cell membrane and pump potassium in and protons out.

Before yeast begin their work producing ethanol, the starting material, usually corn, must be broken down into glucose. A significant feature of the new MIT study is that the researchers did their experiments at very high concentrations of glucose. While many studies have examined ways to boost ethanol tolerance at low glucose levels, the MIT team used concentrations of about 300 grams per liter, similar to what would be found in an industrial biofuel fermenter.

If you really want to be relevant, you’ve got to go to these levels. Otherwise, what you learn at low ethanol levels is not likely to translate to industrial production.

—Stephanopoulos

Lonnie Ingram, director of the Florida Center for Renewable Chemicals and Fuels at the University of Florida, who was not involved in the study, describes the MIT team’s discovery as “remarkable and unexpected.”

Few would have anticipated these results, which show that increasing electrochemical gradients across membranes provide a dramatic increase in alcohol tolerance. This discovery will have direct applications in commercial processes for alcohol production from high concentrations of sugar.

—Lonnie Ingram

In more recent experiments, the MIT researchers have used this method to bump ethanol productivity even higher than reported in the Science paper. They are also working on using this approach to boost the ethanol yield from various industrial feedstocks that, because of starting compounds inherently toxic to yeast, now have low yields.

The research was funded by the MIT Energy Initiative and the Department of Energy.

Thermotolerance. Researchers at Chalmers University of Technology have demonstrated that, with a simple mutation, yeast can become more thermo tolerant, enabling them to grow in higher than normal temperatures. The findings may result in ethanol being more effectively manufactured for vehicle fuel, as well as increase the possibility of using residual waste as a raw material.

Currently, if industrial yeast cultivation is not cooled, the yeast cells die from the heat they themselves produce. Yeast cultivation is currently cooled to 30 ˚C, the temperature at which the yeast cells can best do their job of producing ethanol.

However, the production of bioethanol could be both less expensive and more effective if the temperature could be maintained at 40 ˚C. A great deal of money could be saved on the cooling costs, and the risk of bacterial growth would decrease. In addition, the raw material, for example starch, must be broken down into sugars that the yeast can use, a process that functions best at high temperatures.

As it turns out, a simple mutation is sufficient [to make yeast thermotolerant]. Yeast has a molecule in its cell membrane called ergosterol, instead of cholesterol which humans have. The mutation exchanges ergosterol for a more bent molecule called fecosterol. This has several different effects on the cells, which enables the yeast to grow at 40 degrees.

—Jens Nielsen, professor of systems biology and head of the research team

The yeast has not been genetically engineered by the researchers; rather, they have used adaptive laboratory evolution to produce it. The method allows new characteristics to be produced without knowing which mutations are required to achieve them.

Three yeast cultivations were subjected to a temperature of about 40 ˚C. After just over three months, when more than 300 generations had passed, the yeast suddenly started to grow effectively in all three cultivations. The researchers analysed the genetic structure and metabolism in three yeast strains from each cultivation. They concluded that while several different mutations had occurred in the strains, all the strains had the mutation that produced fecosterol.

Since that mutation took place in three independent cultivations, it appears to be the most important factor in terms of the yeast becoming thermotolerant. This shows how rapidly evolution can change an organism. It is interesting that the structure in fecosterol is the same as in sterol-like molecules, which protect some bacteria and plants against high temperatures.

—Jens Nielsen

One important characteristic of the yeast strains is that they are stable—they pass on their thermotolerancy to future generations.

This characteristic, and the fact that the researchers now know exactly what makes yeast thermotolerant, could have a major impact on bioethanol production, including current bioethanol production which is based on sugar beets or corn.

I believe that our results have very great potential for this type of development. In order to use residual waste of this kind, the substance lignocellulose must be broken down, which is difficult. The enzymes needed for decomposition work best at high temperatures. From a long-term perspective, our results may also increase the possibility of using yeast to produce more advanced biofuel that more closely resembles oil-based fuel.

—Jens Nielsen

The research was funded by the Novo Nordisk Foundation, the European Research Council and the Swedish Research Council. It was conducted at Chalmers University of Technology, and the Science for Life Laboratory did the DNA sequencing and bioinformatic analyses.

Resources

• Felix H. Lam, Adel Ghaderi, Gerald R. Fink, and Gregory Stephanopoulos (2014) “Engineering alcohol tolerance in yeast” Science 346 (6205), 71-75 doi: 10.1126/science.1257859

• Luis Caspeta, Yun Chen, Payam Ghiaci, Amir Feizi, Steen Buskov, Björn M. Hallström, Dina Petranovic, and Jens Nielsen (2014) “Altered sterol composition renders yeast thermotolerant” Science 346 (6205), 75-78 doi: 10.1126/science.1258137

• Clint Cheng and Katy C. Kao (2014) “How to survive being hot and inebriated” Science 346 (6205), 35 doi: 10.1126/science.1260127