| In engineering novel microbial strains, one approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs.;A fundamental limitation in the application of such libraries in functional genomics or genome engineering is low expression of heterologous genes in hosts like Escherichia coli (low functional sample space). To overcome this limitation, a new strategy was developed by engineering hybrid organisms that possess transcriptional machinery capable of recognizing promoters from heterologous DNA. These hybrid organisms can then be employed for screening heterologous DNA libraries that express foreign genes, thus enlarging the sampling space that can be engaged in genome engineering and functional genomics. L. plantarum is a Gram+ prokaryote known to be solvent tolerant. Screening libraries of L. plantarum in an E. coli strain with a chromosomally integrated L. plantarum rpoD (coding for its major sigma factor) allowed identification of genetic determinants imparting ethanol tolerance in E. coli, whereby survival increased by up to 14-fold after 48 hours of exposure to 7% (v/v) ethanol.;Another limitation of single genomic libraries is their inability to identify interactions among distant genetic loci (limited genomic sample space). To solve this problem, plasmid- and fosmid-based E. coli Coexisting Genomic Libraries (CoGeLs) were constructed. These CoGeLs were screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. Screening identified a combination of genes known to enhance acid tolerance (e.g., gadBC ), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold.;Furthermore, simultaneous overexpression of non-coding sRNAs, DsrA, RprA and ArcZ, which are translational activators of RpoS, increased acid tolerance supra-additively up to 8500-fold during active cell growth and provided protection against carboxylic-acid and oxidative stress. The supra-additive effect results from the impact sRNA expression has on RpoS-protein levels, and the beneficial perturbation of the interconnected RpoS and H-NS networks to create superior tolerance during active cell growth. Synthetic acid tolerance could be engineered by overexpressing the sRNAs, which is a desirable phenotype for many biotechnological applications, especially during active cell growth. |
