While the earlier work by BESC researchers at DOE’s Oak Ridge National Laboratory and the University of California at Los Angeles was important from a proof-of-principle perspective, this new result represents a significant gain.

When we reported our initial finding four years ago, we were using Clostridium celluloyticium, which is a less complex organism from a metabolic engineering perspective. With this paper, we have successfully engineered similar traits in the much higher yielding Clostridium thermocellum, and that has taken us to new levels of consolidated bioprocessing efficiency.

—co-author James Liao, UCLA Henry Samueli School of Engineering and Applied Science

Consolidated bioprocessing refers to the bundling of several processes in a single microbe that can be used to extract sugar from a plant’s cellulose and convert those sugars into a biofuel. This approach can be used to combine several steps—pretreatment, enzyme treatment and fermentation—to produce biofuel at a lower cost. The process also helps overcome the challenges of recalcitrance, or a plant’s natural defenses to being chemically dismantled. Recalcitrance is one of the primary economic barriers to using lignocellulosic biomass such as corn stover and switchgrass as a feedstock for biofuels.

While the previous genetically engineered microbe achieved conversion results of 0.6 gram of isobutanol per liter, Clostridium thermocellum has produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50 ˚C within 75 h, corresponding to 41% of theoretical yield. Researchers accomplished this by inserting five genes into the microbe, enabling it to synthesize isobutanol.

Metabolic engineering for isobutanol production in C. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. In this work, we first cloned essential isobutanol pathway genes under different promoters to create various plasmid constructs in Escherichia coli. Then, these constructs were transformed and tested in C. thermocellum. Among these engineered strains, the best isobutanol producer was selected and the production conditions were optimized. We confirmed the expression of the overexpressed genes by their mRNA quantities. We also determined that both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (KIVD) expressed were responsible for isobutanol production. We further found that the plasmid was integrated into the chromosome by single crossover. The resulting strain was stable without antibiotic selection pressure.

—Lin et al.

The scientists view this as a clear next-generation advance over strategies that use yeast to create biofuels from cellulose.

In addition to this development, which moves the BESC team closer to the production goal of more than 20 grams per liter, the prospects of commercial realization of this approach are greatly enabled by the fact that the microbe works at temperatures high enough to keep competing bugs from contaminating the microbial fermentation tanks and interfering with the conversion process.

—Paul Gilna, director of BESC

The authors noted that microbial engineering challenges remain, but they are encouraged by this finding. Other authors of the paper are Beth Papanek, Lauren Riley and Adam Guss of ORNL and Paul Lin, Lou Mi, Amy Morioka, Kouki Yoshino, Sawako Konishi and Sharon Xu of UCLA.

BESC, led by ORNL, is one of three DOE Bioenergy Research Centers established by the DOE’s Office of Science in 2007. The centers support multidisciplinary, multi-institutional research teams pursuing the fundamental scientific breakthroughs needed to make production of cellulosic biofuels, or biofuels from nonfood plant fiber, cost-effective on a national scale. The three centers are coordinated at ORNL, Lawrence Berkeley National Laboratory and the University of Wisconsin-Madison in partnership with Michigan State University.

Resources

• Paul P. Lin, Luo Mi, Amy H. Morioka, Kouki M. Yoshino, Sawako Konishi, Sharon C. Xu, Beth A. Papanek, Lauren A. Riley, Adam M. Guss, James C. Liao (2015) “Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum,” Metabolic Engineering, Volume 31, Pages 44-52 doi: 10.1016/j.ymben.2015.07.001