title: The development and application of a single-cell biosensor for the detection of l-methionine and branched-chain amino acids.
abstract: The detection and quantification of specific metabolites in single bacterial cells is a major goal for industrial biotechnology. We have developed a biosensor based on the transcriptional regulator Lrp that detects intracellular l-methionine and branched-chain amino acids in Corynebacterium glutamicum. In assays, fluorescence output showed a linear relationship with cytoplasmic concentrations of the effector amino acids. In increasing order, the affinity of Lrp for the amino acids is l-valine, l-isoleucine, l-leucine and l-methionine. The sensor was applied for online monitoring and analysis of cell-to-cell variability of l-valine production by the pyruvate dehydrogenase-deficient C. glutamicum strain ΔaceE. Finally, the sensor system was successfully used in a high-throughput (HT) FACS screen for the isolation of amino acid-producing mutants after random mutagenesis of a non-producing wild type strain. These applications illustrate how one of nature's sensor devices - transcriptional regulators - can be used for the analysis, directed evolution and HT screening for microbial strain development.
authors.affiliation: Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, Leo-Brandt-Straße, 52425 Jülich, Germany.
authors.forename: Nurije
authors.initials: N
authors.lastname: Mustafi
authors.forename: Alexander
authors.initials: A
authors.lastname: Grünberger
authors.forename: Dietrich
authors.initials: D
authors.lastname: Kohlheyer
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
authors.forename: Julia
authors.initials: J
authors.lastname: Frunzke
mesh_terms: D000597:Amino Acids, Branched-Chain / Q000032:analysis*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D048230:Corynebacterium glutamicum / Q000378:metabolism; D051756:Leucine-Responsive Regulatory Protein / Q000737:chemistry; D008715:Methionine / Q000032:analysis*; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D000597:Amino Acids, Branched-Chain; D051756:Leucine-Responsive Regulatory Protein; D008715:Methionine