Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.

Stefan M. Gaida,Keerthi P. Venkataramanan,Eleftherios T. Papoutsakis,Nicholas R. Sandoval,Yili Chen,Sergios A. Nicolaou

doi:10.1038/ncomms8045

Stefan M. Gaida, Keerthi P. Venkataramanan + Show 4 more

Open Access

https://doi.org/10.1038/ncomms8045

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Abstract

A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries 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. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain.

Highlights

A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli
We show that the expression of the Lactobacillus plantarum (Lpl) RpoD increases the functional genomic space in E. coli, as quantified by green fluorescence protein (GFP) expression using five heterologous genome-wide, promoter GFP-trap libraries
Lpl RpoD increases the GFP þ population in all the five libraries tested, which were constructed from phylogenetically diverse genomes, namely, those of Lpl, Bacillus subtilis (Bsu), Deinococcus radiodurans (Dra), Clostridium pasteurianum (Cpa) and C. acetobutylicum (Cac)

Summary

Introduction

A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. Using the LPL-trap library, we investigated whether expressing the major sigma factor of L. plantarum (RpoD) can increase the fraction of Lpl promoters recognized by E. coli (Fig. 1a).

Results

Conclusion