(722a) Improved Functional Screening of Metagenomic Libraries Via Heterologous Sigma Factor Expression

The microbial diversity present in nature is an enormous stock of genetic material that remains largely unexplored. With fewer than 1% of organisms successfully cultured in the laboratory, this enormous genetic diversity can be harvested best in the form of metagenomic libraries. A major limitation in functional screening of heterologous genomic or metagenomic libraries is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we engineer E. coli strains that are capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among several sigma factors tested, the primary sigma factor RpoD from Lactobacillus plantarum (Lpl) is able to initiate transcription from many diverse sources of DNA. Using a promoter GFP-trap concept, we show greatly improved transcription initiation from DNA libraries with heterologous and metagenomic DNA (sourced from Lpl, Bacillus subtilis, Deinococcus radiodurans, Clostridium pasteurianum, C. acetobutylicum, and soil samples), thus enlarging the genomic space that can be functionally sampled in E. coli. We show that screening fosmid-based heterologous (Lpl) genomic library in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Strand-specific RNA-seq analysis confirms increased expression of heterologous genes in the engineered strain. Metagenomic and heterologous libraries were screened for functional traits in the wild type and engineered strains.