title: Screening of an Escherichia coli promoter library for a phenylalanine biosensor.
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.
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Mahr
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany.
authors.forename: Raphael Freiherr
authors.initials: RF
authors.lastname: von Boeselager
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Wiechert
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany. j.frunzke@fz-juelich.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Frunzke
mesh_terms: D026905:Amino Acid Transport Systems / Q000235:genetics*; D015374:Biosensing Techniques; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods; D005690:Galactose / Q000378:metabolism*; D015723:Gene Library; D057166:High-Throughput Screening Assays; D010649:Phenylalanine / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D026905:Amino Acid Transport Systems; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C069255:mtr protein, E coli; D010649:Phenylalanine; D005690:Galactose
keywords: Aromatic amino acids; Biosensor; GFP library; Phenylalanine; Population heterogeneity; Random mutagenesis