(798e) Expanding the Searchable Genomic Space: Heterologous Sigma Factor Expression for Screening of Metagenomic Libraries

Most bacteria found in nature cannot be cultured in a laboratory but are able to perform many interesting and valuable biocatalytic functions.  Discovery of the underlying genetics requires functional screening of heterologous and metagenomic libraries. A major limitation in the application of this screening is the low-level heterologous gene expression within the screening host, such as Escherichia coli. To overcome this limitation, we engineered E. coli strains that possess non-native transcription machinery capable of recognizing heterologous promoters. Such strains can be employed for screening heterologous DNA libraries, thus greatly enlarging the genomic space that can be functionally sampled. Specifically, we expressed in E. coli sigma factors from phylogenetically-distant bacteria (e.g. Lactobacillus plantarum, Bacillus subtilis). Strains expressing foreign sigma factors recognized a significantly greater fraction of promoters from multiple heterologous genomic libraries compared to the native E. coli transcription machinery alone (as quantified using a flow cytometry-based gfp-trap method). Importantly, high levels of cross-recognition were observed between foreign sigma factors and libraries of differing species. Effect of simultaneous expression of multiple heterologous sigma factors on mixed genomic libraries was explored and optimized. Screening fosmid-based genomic libraries of L. plantarum in an E. coli strain expressing the L. plantarum rpoD gene (coding for its major sigma factor) allowed the identification of L. plantarum genetic elements imparting ethanol tolerance in E. coli.