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ORNL Gene Discovery Can Lead to More Cost-Competitive Cellulosic Ethanol; Process Demonstrates Approach for Accelerated Industrial Strain Development

Researchers at the US Department of Energy’s Oak Ridge National Laboratory have identified a key gene in the bacterium Zymomomas mobilis—an anaerobic ethanologen—that, when overexpressed in a new Z. mobilis strain, delivers increased tolerance to acetic acid, a common inhibitor produced in biomass pretreatment. Increased tolerance can yield more cost-competitive cellulosic ethanol. An open access paper on their work was published online 19 May in the Proceedings of the National Academy of Sciences.

More broadly, the researchers say, their study shows that the application of systems biology tools holds promise for rational industrial microbial strain development. The combination of classical and systems biology tools used in their work is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies, they said.

Currently, biomass materials like corn stover and switchgrass must undergo a series of pretreatments to loosen the cellular structure enough to extract the sugar from cellulose. Steven Brown, staff microbiologist in the Biosciences Division and one of the inventors of the improved Z. mobilis strain, said these treatments add new challenges because, although they are necessary, they create a range of inhibitors that stall or stop microorganisms like Z. mobilis from performing the fermentation.

There are two ways to combat recalcitrance, or the difficulty created by the inhibitors. One way is to remove the inhibitors, but this method is very expensive and would not help biofuels become cost-competitive with gasoline. The second way is what we do, which is to develop microorganisms that are more tolerant of the inhibitors.

—Steven Brown

The non-mutated strain of Z. mobilis, for instance, cannot grow in the presence of one of the predominant inhibitors, acetate. However, when gene nhaA is over-expressed by inserting a slice of DNA containing the gene into the non-mutated strain, the bacterium can withstand acetate in its environment.

Brown and lead author Shihui Yang also looked at related genes in other microorganisms and found that the method translates in different organisms.

We took this gene and integrated it into a strain of yeast, and the improvements carried over into the yeast.

—Shihui Yang

Brown says this method of processing biomass for ethanol has the potential to become a “tool kit”—a combination of mutant genes that reduce the impact of specific inhibitors. The tool kit could expand quickly, too, as scientists now have more advanced DNA sequencing technology available to identify and resequence genes.

The present study shows the potential of systems biology tools and genetics for the rapid identification and characterization of process-relevant traits. The expression profiles generated in these studies are the most comprehensive for Z. mobilis to date and will likely serve as useful reference data for future systems biology studies. By demonstrating that Z. mobilis nhaA overexpression confers the AcR tolerance phenotype and that most of the advantage conferred is against the sodium ion, our data reinforce the idea that one obtains what one selects for during adaptive evolution experiments.

The present work also demonstrates S. cerevisiae Nat¨MHt antiporter gene overexpression enhances its tolerance to acetate with three different counter ions. Our study also provides a caveat in using reference genome sequences for SNP identification and insights into technological limitations. We have affirmed the notion that near-term pathway engineering approaches benefit from a combinatorial approach. The combined approach of employing the advantages of classical selection, which lack mechanistic a priori assumptions, with systems biology tools is a paradigm for industrial strain characterization and development.

—Yang et al.

ORNL microbiologists are currently sequencing other microorganisms used in biofuels production that could also be advantageous if genetically altered to resist different types of inhibitors.


  • Shihui Yang, Miriam L. Land, Dawn M. Klingeman, Dale A. Pelletier, Tse-Yuan S. Lu, Stanton L. Martin, Hao-Bo Guo, Jeremy C. Smith, and Steven D. Brown (2010) Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae. PNAS published ahead of print doi: 10.1073/pnas.0914506107



"There are two ways to combat recalcitrance, or the difficulty created by the inhibitors. One way is to remove the inhibitors, but this method is very expensive and would not help biofuels become cost-competitive with gasoline. The second way is what we do, which is to develop microorganisms that are more tolerant of the inhibitors."

Sounds like Steve has found a way to make this research valuable. Congrats Steve! Let us not forget however that for thousands of years man and nature have fermented everything from dandelions to hops. Making grain or wood alcohol is no secret. It has been done in backwoods stills and bathtubs for years. Converting biomass to cost competitive alcohol may be a larger challenge but we should not rely on proprietary genetic engineering to do so. It's good to know that ORNL is financed by the American taxpayer and these discoveries will benefit taxpayer investment.

Cellulosic alcohols can and must be produced on a mass scale for a relatively short period of time (30-40 years) until electrification replaces all but heavy lifting diesel and jet fuels.

Henry Gibson

Everybody neglects to do the arithmetic that can quickly show that all of the forests and farms and even lawns in the US can produce less than one tenth enough cellulose for the amount of energy used in the US.

Convert all electricity production to nuclear power plants, bought and even built in Canada, and use the coal saved to make diesel fuel, and, OH Yes!, throw a few logs and a few tons of sewage in with the coal to make you feel more organic. All coal is organic and is the remains of plants.

Corn kernels would be more efficiently converted to fuel if they were thrown into the coal as well. In fact it is likely that corn can be inserted into crude oil in small amounts and converted into gasoline with more energy efficiency and lower cost than by fermenting them.

It would be a great benefit to the US economy to build hundreds of factories all over the US that convert coal to diesel and gasoline, and all new cars should be built to burn pure methanol or ethanol as well as gasoline because methanol is easier to make and to store for long periods. Filling up every 200 miles instead of every 350 is not a great peoplem. How many times have automobile articles mentioned this limited range of gasoline cars. ..HG..

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