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Screening of an Escherichia coli promoter library for a phenylalanine biosensor

Applied Microbiology and Biotechnology volume 100, pages 6739–6753 (2016)Cite this article

Abstract

In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and l-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an l-tryptophan-specific transporter, was identified as suitable part for the construction of an l-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of l-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.

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  1. Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany

    Regina Mahr, Raphael Freiherr von Boeselager, Johanna Wiechert & Julia Frunzke

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This work was supported by the German Federal Ministry of Education and Research (BMBF OptoSys grant 031A167B) and the Helmholtz Association (Helmholtz grant VH-NG-716).

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Julia Frunzke declares that she has no conflict of interest. Regina Mahr declares that she has no conflict of interest. Raphael Freiherr von Boeselager declares that he has no conflict of interest. Johanna Wiechert declares that she has no conflict of interest.

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Mahr, R., von Boeselager, R.F., Wiechert, J. et al. Screening of an Escherichia coli promoter library for a phenylalanine biosensor. Appl Microbiol Biotechnol 100, 6739–6753 (2016). https://doi.org/10.1007/s00253-016-7575-8

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  • DOI: https://doi.org/10.1007/s00253-016-7575-8

Keywords

  • Biosensor
  • GFP library
  • Phenylalanine
  • Aromatic amino acids
  • Random mutagenesis
  • Population heterogeneity