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

• Published in: Nature communications 2015-05, Vol.6 (1), p.7045-7045, Article 7045
• Main Authors: Gaida, Stefan M., Sandoval, Nicholas R., Nicolaou, Sergios A., Chen, Yili, Venkataramanan, Keerthi P., Papoutsakis, Eleftherios T.
• Format: Article
• Language: English
• Subjects: 38/47; 42/40; 631/208/199; 631/326/41/2142; 631/61/191; Deoxyribonucleic acid; DNA; DNA-Directed RNA Polymerases - metabolism; E coli; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Ethanol; Ethanol - toxicity; Gene expression; Gene Expression Regulation, Bacterial - drug effects; Genes; Genes, Bacterial; Genetic Loci; Genetic Testing; Genome editing; Genomic Library; Genomics; Green Fluorescent Proteins - metabolism; Humanities and Social Sciences; Lactobacillus plantarum; Lactobacillus plantarum - drug effects; Lactobacillus plantarum - genetics; Metagenome; multidisciplinary; Promoter Regions, Genetic; Science; Science (multidisciplinary); Screening; Sequence Analysis, RNA; Sigma Factor - metabolism; Species Specificity; Transcription; Transcription, Genetic - drug effects
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms8045

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. Screening genomic or metagenomic libraries for interesting products or activities is often hampered by poor gene expression in a heterologous host. Here the authors show that the expression of a Lactobacillus sigma factor greatly enhances transcription of heterologous and environmental DNA in E. coli .