title: Coexisting/Coexpressing Genomic Libraries (CoGeL) identify interactions among distantly located genetic loci for developing complex microbial phenotypes.
abstract: In engineering novel microbial strains for biotechnological applications, beyond a priori identifiable pathways to be engineered, it is becoming increasingly important to develop complex, ill-defined cellular phenotypes. One approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs. Such libraries are limited by their inability to identify interactions among distant genetic loci. To solve this problem, we constructed plasmid- and fosmid-based Escherichia coli Coexisting/Coexpressing Genomic Libraries (CoGeLs). As a proof of principle, four sets of two genes of the l-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes demonstrating the interaction between the two libraries. CoGeLs were also screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. CoGeL screening identified combination of genes known to enhance acid tolerance (gadBC operon and adiC), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold. arcZ is a small RNA that we show increases pH tolerance alone and together with recA.
authors: Department of Chemical & Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
authors: Sergios A
authors: SA
authors: Nicolaou
authors: Stefan M
authors: SM
authors: Gaida
authors: Eleftherios T
authors: ET
authors: Papoutsakis
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D056426:Genetic Loci; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000096:biosynthesis; D010641:Phenotype
chemical_list: D008239:Lysine