(440a) Improving and Elucidating Isobutanol Tolerance in Escherichia Coli

Microbial conversion of lignocellulosic feedstocks to liquid biofuels represents a promising platform for sustainable production of transportation fuels.  However, a number of challenges exist, including the production of fuel molecules with desirable properties¹.  Recent advances in microbial engineering have led to development of metabolic pathways for producing higher molecular weight alcohols as next-generation biofuels.  In particular, Escherichia coli has been successfully engineered to produce isobutanol in high yield (86% of theoretical maximum)².  However, isobutanol is highly toxic to E. coli, which limits the maximum concentration that can be achieved during fermentation and reduces production.  The genetic basis of microbial solvent tolerance is poorly understood, hindering efforts to rationally engineer enhanced tolerance³.  In this work, we have sought to enhance the isobutanol tolerance in E. coli and elucidate the genetic basis of this tolerance at the genomic level.  Our aim is to advance the fundamental understanding of the genetic basis underlying microbial solvent tolerance, facilitating future efforts towards rationally engineering microbes with enhanced tolerance. 

We evolved isobutanol tolerant lines of E. coli by growing serial cultures on isobutanol spiked minimal media for several hundred generations.  Replicate populations were employed to explore the possibility of divergent evolution, and additionally, populations were evolved on different carbon sources (glucose and xylose, which are important constituents of cellulosic feedstocks) to investigate tolerance adaptations under different metabolic states.  Through our adaptive evolution approach, E. coli populations capable of growth at 2% isobutanol in glucose media and 1.75% isobutanol in xylose media have been obtained, representing 60% and 40% improvements in tolerance respectively, compared to the parent E. coli strain.

Advances in DNA sequencing technology have dramatically increased throughput and lowered costs⁴, but these new high-throughout sequencing technologies have not been fully exploited in the field of microbial engineering.  We are utilizing these new high-throughput technologies to investigate the genetic basis of isobutanol tolerance in our evolved E. coli lines.  Genome re-sequencing of isobutanol tolerant E. coli lines will be pursued, allowing for precise identification of mutations conferring enhanced tolerance.  Population samples from different evolutionary time points will be resequenced, particularly around points where major changes in isobutanol tolerance occur.  Given the multigenic nature of solvent tolerance, such temporal sequence information could provide insights into epistatic interactions involved in tolerance.

References

[1] United States. DOE. Genomics:GTL. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda. 2006.

[2] Atsumi, Shota, Taizo Hanai, and James C. Liao. "Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels." Nature 451 (2008): 86-91.

[3] Heipierper, Hermann J., Grit Neumann, Sjef Cornelissen, and Friedhelm Meinhardt. "Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems." Applied Microbiology and Biotechnology 74 (2007): 961-73.

[4] MacLean, Daniel, Johnathon D.G. Jones, and David Studholme. "Application of 'next-generation' sequencing technologies to microbial genetics." Nature Reviews Microbiology (2009).