Abstract
Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that – upon deletion – should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used.
Highlights
The rapid and exact determination of amino acid concentrations is of fundamental importance for a wide range of applications including biological, medical, or food technological analyses
Abnormal levels of amino acids in human blood are diagnostic for amino acid disorder diseases [1], in animal nutrition concentrations of essential amino acids have a vital influence on animal weight gain [2,3], and in fermentation processes it is important to continuously monitor the consumption or production of certain amino acids [4,5]
So-called combinatorial approaches that are frequently used in metabolic engineering of microbial strains to overproduce certain amino acids of interest rely on the rapid screening of large mutant libraries [6]
Summary
The rapid and exact determination of amino acid concentrations is of fundamental importance for a wide range of applications including biological, medical, or food technological analyses. Complex mixtures of compounds are separated via liquid- or gas chromatography and subsequently the amount of amino acids is quantified using fluorescence-, UV-detection or mass spectrometry [7,8,9,10]. Drawbacks of these approaches are that the analyses are often very time-consuming, and that these techniques provide no information on the bioavailability and bioaccessability of the respective amino acids [11]. Depending on the analytical methodology used, technical difficulties like matrix interference, a low sensitivity, elaborate sample preparation procedures, multiplicity of peaks formed, or a bias of the derivatising agent used against certain amino acids can constrain the applicability of the technique as well as the validity of the results (see [12,13] and references therein)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
