Ionic liquids—neutral salts that attain a liquid state at temperatures mostly below 100 ˚C—are very effective in the pretreatment of biomass to make its cellulose highly accessible to cellulase enzymes that hydrolyze it into glucose for fermentation by microbes. However, ionic liquids are toxic to biofuel-producing microbes such as the yeast Saccharomyces cerevisiae. This toxicity is enough to make the yeast as much as 70% less efficient at turning sugar into biofuel.

Now, researchers from the University of Wisconsin-Madison and several Department of Energy laboratories have identified two changes to a single gene in S. cerevisiae that enable the yeast to tolerate ionic liquids used as biomass pretreatment chemicals. They published their findings recently in the journal Genetics.

The researchers evaluated 136 yeast isolates and found one strain with outstanding tolerance to ionic liquids. They screened DNA sequences from this strain and identified a pair of genes key to surviving the otherwise toxic pretreatment chemicals. One of the genes, called SGE1, makes a protein that settles in the yeast cell membrane and works as a pump to remove toxins.

A change of just two individual nucleotides among more than 12 million that make up the yeast genome are enough to increase the production of those cellular pumps and protect yeast from ionic liquids.

The researchers used the gene-editing tool CRISPR to alter a strain of an ionic liquid-susceptible yeast, introducing the two single-nucleotide changes and successfully producing a yeast that can survive—and ferment—alongside amounts of ionic liquid that are normally toxic.

Now anyone using this yeast can look at a specific gene in their own strain and tell whether it’s compatible and useful with an ionic liquid process or not. It’s a simple engineering procedure, which doesn’t take long and isn’t expensive. And it can be fixed with CRISPR in a matter of a week or two.

—Trey Sato, UW–Madison-based Great Lakes Bioenergy Research Center and co-corresponding author

Sato says the next step is to try out the modified yeast outside the lab, incorporating the real-world plant material used as biofuel feedstock.

Collaborators on the study included Michael Thelen of the Joint BioEnergy Institute and UW-Madison genetics professors Audrey Gasch and Chris Hittinger and biochemistry and bacteriology professor Robert Landick.

This research was supported by grants from the Department of Energy (DE-SC0018409, DE-FC02-07ER64494 and DE-AC02-05CH11231).

Resources

• Douglas A. Higgins, Megan K. Young, Mary Tremaine, Maria Sardi, Jenna M. Fletcher, Margaret Agnew, Lisa Liu, Quinn Dickinson, David Peris, Russell L. Wrobel, Chris Todd Hittinger, Audrey P. Gasch, Steven W. Singer, Blake A. Simmons, Robert Landick, Michael P. Thelen and Trey K. Sato (2018) “Natural Variation in the Multidrug Efflux Pump SGE1 Underlies Ionic Liquid Tolerance in Yeast” Genetics doi: 10.1534/genetics.118.301161