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.

Resources

• 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