Genome-scale identification and characterization of ethanol tolerance genes in Escherichia coli
The identification of relevant gene targets for engineering a desired trait is a key step in combinatorial strain engineering. Here, we applied the multi-Scalar Analysis of Library Enrichments (SCALEs) approach to map ethanol tolerance onto 1,000,000 genomic-library clones in Escherichia coli. We assigned fitness scores to each of the ∼4,300 genes in E. coli, and through follow-up confirmatory studies identified 9 novel genetic targets (12 genes total) that increase E. coli ethanol tolerance (up to 6-fold improved growth). These genetic targets are involved in the processes related to cell membrane composition, translation, serine biosynthesis, and transcription regulation. Transcriptional profiling of the ethanol stress response in 5 of these ethanol-tolerant clones revealed a total of 700 genes with significantly altered expression (mapped to 615 significantly enriched gene ontology terms) across all five clones, with similar overall changes in global gene expression between two clone clusters. All ethanol-tolerant clones analyzed shared 6% of the overexpressed genes and showed enrichment for transcription regulation-related GO terms. iTRAQ-based proteomic analysis of ethanol-tolerant strains identified upregulation of proteins related to ROS mitigation, fatty acid biosynthesis, and vitamin biosynthesis as compared to the parent strain's ethanol response. The approach we outline here will be useful for engineering a variety of other traits and further improvements in alcohol tolerance.
► Genome-wide selections for ethanol tolerance genes using SCALEs. ► Identification of many novel genes for improved ethanol tolerance (up to 6-fold). ► Characterized altered transcriptomes and proteomes for ethanol-tolerant clones. ► Overexpression of transcription regulation-related genes linked to ethanol tolerance. ► Upregulated proteins for ROS mitigation, fatty acid, and vitamin biosynthesis.
Journal: Metabolic Engineering - Volume 15, January 2013, Pages 124–133