Whether triple-distilled, aged in oak, or bootlegged, beverages containing ethanol have been prized by nearly all human societies. Ethanol, however, has uses beyond “liquid courage”; it is considered a possible substitute fuel for gasoline with many benefits. It has a higher octane than gas, emits fewer pollutants, and has the potential to be a sustainable, carbon-neutral fuel source. Ideally, ethanol would be produced from cellulosic biomass rather than from starches, such as corn. Cellulosic ethanol feedstocks would include wood and cardboard waste, or dedicated biofuel crops like switchgrass. An obvious advantage of these sources is that they include waste products, rather than food. Additionally, switchgrass can be grown using less resources than corn, and cellulosic biomass has the potential to produce more ethanol per volume of plant than starch. However, producing ethanol from cellulose is expensive, partly because fuel ethanol is currently produced through fermentation by the same yeast strains as those used to make our alcoholic drinks.
Like humans, yeast process starches rather than cellulose. Both starch and cellulose are polymers—long chains—of the simple sugar glucose. The main difference between the two materials is in the shape of the chemical linkage between glucose units. Because of this difference in link shape, “there are differences in the active sites of the enzymes used to break down these polymers,” explains professor of molecular and cell biology Jamie Cate. “Normal yeast just doesn’t have the right enzymes to break down cellulose.”
Professor Cate’s research group at UC Berkeley is a member laboratory of the Energy Biosciences Institute (EBI), an interdisciplinary, collaborative research organization that takes a full systems approach to developing sustainable biofuels (see “Natural Inspiration“). Toward this goal, Cate postulated that yeast could be engineered to digest cellulose; you just have to identify and insert the right genes. This was recently achieved by Dr. Jonathan Galazka, a graduate of Cate’s research group. He created a yeast strain with the ability to grow on short chains of cellulose by inserting genes taken from the fungus Neurospora crassa, which naturally grows on cellulosic biomass in grasslands.
Galazka’s work builds on a previous collaboration with Professor Louise Glass here at Berkeley that took a global look at the N. crassa genome and identified which of its genes were necessary for cellulose digestion. For this experiment, the team began by growing the fungus on pure sugar, and noting the genes it expressed. Then they switched the diet to cellulose. Within hours, they could monitor changes in the genes. “We know all of the genes in Neurospora,” says Cate. “We don’t necessarily know what they’re doing. By using this switch from sucrose to cellulose we’re able to have Neurospora tell us what genes are important, by which genes it turns on and turns off.” On comparing the “important” genes to a bioinformatics database, the researchers found that these genes are quite common in cellulose-eating fungi. Based on comparisons to other known organisms, they predicted that the genes prompt N. crassa to produce two major types of proteins: one that transports short chains of cellulose from the outside environment into the cell, and one that could break down the short chains to glucose inside the cell. Galazka’s project tested this hypothesis. “He showed that these putative transporters, when put into yeast, made the yeast all of a sudden able to take in short chains of glucose and actually use them for growth to make ethanol.”
This is an important achievement for the cellulosic ethanol community. The current process for making ethanol from cellulose is long and expensive, involving treatments with chemicals and enzyme cocktails to break the cellulose down into single sugars, followed by purification treatments, fermentation with yeast, and finally separation of the ethanol from waste products. Using cellulose-eating yeast eliminates the need to break down cellulose all the way to single sugars; instead, converting cellulose to shorter glucose chains known as cellodextrins is sufficient. This removes a few of the processing steps, making ethanol production quicker and cheaper. It also leads to less contamination. “If you have a big vat of glucose, everything likes to grow on glucose. If you only break down the cellulose to cellodextrins, not as many things can grow on that, so you eliminate contaminant organisms,” Cate explains.
The next steps for this project will include incorporating the cellodextrin transport systems into the less-studied, but more robust strains of yeast that are used in industry. But the work won’t stop there, as Cate explains. “In the end it has to be a whole process. What plants do you grow, how do you harvest them, break them down, and treat them so you can extract the sugars, how do you ferment them, and how do you isolate the ethanol?” The Cate laboratory, together with other member labs of EBI, is addressing these broad-spectrum questions of the global energy challenge.
Hannah Ray is a graduate student in materials science.