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

Author

Listed:
  • Stefan M. Gaida

    (Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware)

  • Nicholas R. Sandoval

    (Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware)

  • Sergios A. Nicolaou

    (Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware)

  • Yili Chen

    (Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware)

  • Keerthi P. Venkataramanan

    (Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware)

  • Eleftherios T. Papoutsakis

    (Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware)

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.

Suggested Citation

  • Stefan M. Gaida & Nicholas R. Sandoval & Sergios A. Nicolaou & Yili Chen & Keerthi P. Venkataramanan & Eleftherios T. Papoutsakis, 2015. "Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries," Nature Communications, Nature, vol. 6(1), pages 1-10, November.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms8045
    DOI: 10.1038/ncomms8045
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    Cited by:

    1. David Forrest & Emily A. Warman & Amanda M. Erkelens & Remus T. Dame & David C. Grainger, 2022. "Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity," Nature Communications, Nature, vol. 13(1), pages 1-13, December.

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