S149: Engineering E. coli for improved ethanol tolerance and ethanol production

Due to recent advances in DNA synthesis, sequencing, and strain engineering tools, we are able to rapidly engineer genomes, dissect complex phenotypes, and tailor recombinant strains for desired traits.  Here we will highlight the findings from our work on engineering ethanol tolerance and ethanol production in Escherichia coli. We have used genomic libraries comprised of over 10⁶overlapping clones to map the genome for ethanol tolerance, and using this approach, we identified 9 novel genetic targets involved in a variety of cellular processes that were confirmed to improve growth in ethanol (up to 6-fold). We further characterized the engineered ethanol stress response in these ethanol-tolerant clones by transcriptomic and proteomic analysis, which identified some similar cellular network changes in many of the ethanol-tolerant clones. 

As a platform to test our identified ethanol tolerance genetic targets and to enable further genome engineering for ethanol production, we constructed and optimized a heterologous E. coli ethanol production strain containing the Zymomonas mobilis pathway for ethanol production. Interestingly, we found that genes conferring ethanol tolerance do not necessarily improve ethanol production and that selections must be carefully designed in order to select for improved production of a desired compound. Through this work, several genes were newly identified and confirmed to improve ethanol tolerance and production. Additionally, our ongoing work focuses on developing new genome engineering tools (such as trackable recursive multiplex engineering (TRMR), trait-specific regulon engineering, and targeted multiplex protein engineering) and applying these tools to engineer tolerance and production of industrially-desirable compounds.