title: Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.
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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, Delaware 19711, USA.
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Gaida
authors.affiliation: Department of Chemical and Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, Delaware 19711, USA.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Sandoval
authors.affiliation: Department of Chemical and Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, Delaware 19711, USA.
authors.forename: Sergios A
authors.initials: SA
authors.lastname: Nicolaou
authors.affiliation: Department of Chemical and Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, Delaware 19711, USA.
authors.forename: Yili
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemical and Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, Delaware 19711, USA.
authors.forename: Keerthi P
authors.initials: KP
authors.lastname: Venkataramanan
authors.affiliation: Department of Chemical and Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, Delaware 19711, USA.
authors.forename: Eleftherios T
authors.initials: ET
authors.lastname: Papoutsakis
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D000431:Ethanol / Q000633:toxicity; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D056426:Genetic Loci; D005820:Genetic Testing; D015698:Genomic Library; D049452:Green Fluorescent Proteins / Q000378:metabolism; D048191:Lactiplantibacillus plantarum / Q000187:drug effects / Q000235:genetics; D054892:Metagenome; D011401:Promoter Regions, Genetic; D017423:Sequence Analysis, RNA; D012808:Sigma Factor / Q000378:metabolism*; D013045:Species Specificity; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D012808:Sigma Factor; D049452:Green Fluorescent Proteins; D000431:Ethanol; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases