Engineering improved ethanol production in Escherichia coli with a genome-wide approach

• Published in: Metabolic engineering Vol. 17; pp. 1 - 11
• Main Authors: Woodruff, Lauren B.A., Boyle, Nanette R., Gill, Ryan T.
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.05.2013
• Subjects: Biofuel; Chromosome Mapping - methods; Escherichia coli; Escherichia coli - physiology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Ethanol; Ethanol - metabolism; Fermentation; Gene Library; Genetic Enhancement - methods; Genome, Bacterial - genetics; Metabolic engineering; Production; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Tolerance; Tolerance; Metabolic engineering; Biofuel; Ethanol; Fermentation; Production
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2013.01.006

A key challenge to the commercial production of commodity chemical and fuels is the toxicity of such molecules to the microbial host. While a number of studies have attempted to engineer improved tolerance for such compounds, the majority of these studies have been performed in wild-type strains and culturing conditions that differ considerably from production conditions. Here we applied the multiscalar analysis of library enrichments (SCALEs) method and performed a growth selection in an ethanol production system to quantitatively map in parallel all genes in the genome onto ethanol tolerance and production. In order to perform the selection in an ethanol-producing system, we used a previously engineered Escherichia coli ethanol production strain (LW06; ATCC BAA-2466) (Woodruff et al., in press), as the host strain for the multiscalar genomic library analysis (>106 clones for each library of 1, 2, or 4kb overlapping genomic fragments). By testing individually selected clones, we confirmed that growth selections enriched for clones with both improved ethanol tolerance and production phenotypes. We performed combinatorial testing of the top genes identified (uspC, otsA, otsB) to investigate their ability to confer improved ethanol tolerance or ethanol production. We determined that overexpression of otsA was required for improved tolerance and productivity phenotypes, with the best performing strains showing up to 75% improvement relative to the parent production strain. ► Designed selections for improved ethanol production in a production platform. ► Genome mapping for ethanol production genetic targets using genomic libraries. ► Selected for genes conferring improved growth in ethanol and ethanol production. ► Greater than 50% improvement in ethanol productivity. ► Combinatorial analysis of identified genes for ethanol tolerance and production traits.