Glucose is a six-carbon sugar that is relatively easy to ferment; xylose is a five-carbon sugar that has been much more difficult to utilize in ethanol production. The new strain, made by combining, optimizing and adding to earlier advances, reduces or eliminates several major inefficiencies associated with current biofuel production methods.

To overcome these bottlenecks, we engineered yeasts to co-ferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular ß-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing co-consumption of cellobiose and xylose.

The resulting yeast strains, co-fermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from co-fermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising co-fermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production.

—Ha et al.

The new yeast strain is at least 20% more efficient at converting xylose to ethanol than other strains, making it the best xylose-fermenting strain reported in any study, according to Jin.

The Energy Biosciences Institute, a BP-funded initiative, supported the research. A paper on their work was published in the Proceedings of the National Academy of Sciences (PNAS).

S. cerevisiae has been used for centuries in baking and brewing because it efficiently ferments sugars and in the process produces ethanol and carbon dioxide. The biofuel industry uses this yeast to convert plant sugars to bioethanol. While S. cerevisiae is very good at utilizing glucose, a building block of cellulose and the primary sugar in plants, it cannot use xylose, a secondary but significant component of the lignocellulose that makes up plant stems and leaves. Most yeast strains that are engineered to metabolize xylose do so very slowly.

Xylose is a wood sugar, a five-carbon sugar that is very abundant in lignocellulosic biomass but not in our food. Most yeast cannot ferment xylose.

—Yong-Su Jin

Yeasts altered to take up xylose is that they will suck up all the glucose in a mixture before they will touch the xylose, Jin said. A glucose transporter on the surface of the yeast prefers to bind to glucose. The yeast’s extremely slow metabolism of xylose also adds significantly to the cost of biofuels production.

If you do the fermentation by using only cellobiose or xylose, it takes 48 hours. But if you do the co-fermentation with the cellobiose and xylose, double the amount of sugar is consumed in the same amount of time and produces more than double the amount of ethanol. It’s a huge synergistic effect of co-fermentation.

—Suk-Jin Ha, U of I postdoc and lead author

The team first gave the yeast a cellobiose transporter. Cellobiose, a part of plant cell walls, consists of two glucose sugars linked together. Cellobiose is traditionally converted to glucose outside the yeast cell before entering the cell through glucose transporters for conversion to ethanol. Having a cellobiose transporter means that the engineered yeast can bring cellobiose directly into the cell. Only after the cellobiose is inside the cell is it converted to glucose.

This approach, initially developed by co-corresponding author Jamie Cate at the Lawrence Berkeley National Laboratory and the University of California at Berkeley, eliminates the costly step of adding a cellobiose-degrading enzyme to the lignocellulose mixture before the yeast consumes it.

It has the added advantage of circumventing the yeast’s own preference for glucose. Because the glucose can now “sneak” into the yeast in the form of cellobiose, the glucose transporters can focus on drawing xylose into the cell instead. Cate worked with Jonathan Galazka, of UC Berkeley, to clone the transporter and enzyme used in the new strain.

The team then tackled the problems associated with xylose metabolism. The researchers inserted three genes into S. cerevisiae from a xylose-consuming yeast, Picchia stipitis.

Graduate student Soo Rin Kim at the University of Illinois identified a bottleneck in this metabolic pathway, however. By adjusting the relative production of these enzymes, the researchers eliminated the bottleneck and boosted the speed and efficiency of xylose metabolism in the new strain.

They also engineered an artificial isoenzyme that balanced the proportion of two important cofactors so that the accumulation of xylitol, a byproduct in the xylose assimilitary pathway, could be minimized. Finally, the team used evolutionary engineering to optimize the new strain’s ability to utilize xylose.

The cost benefits of this advance in co-fermentation are very significant, Jin said.

We don’t have to do two separate fermentations. We can do it all in one pot. And the yield is even higher than the industry standard. We are pretty sure that this research can be commercialized very soon.

—Yong-Su Jin

Jin noted that the research was the result of a successful collaboration among principal investigators in the Energy Biosciences Institute and a BP scientist, Xiaomin Yang, who played a key role in developing the co-fermentation concept and coordinating the collaboration.

Resources

• Suk-Jin Ha, Jonathan M. Galazka, Soo Rin Kim, Jin-Ho Choi, Xiaomin Yang, Jin-Ho Seo, N. Louise Glass, Jamie H. D. Cate, and Yong-Su Jin (2010) Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation PNAS doi: 10.1073/pnas.1010456108