Design and characterization of auxotrophy-based amino acid biosensors

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.

Figure 1. Confirmation of the biosensors' auxotrophy.

The eleven biosensors were grown in minimal medium, which was supplemented with 3 mM of the required focal amino acid (+), devoid of any amino acid supplementation (0), or supplemented with 3 mM of each of the 19 other, proteinogenic amino acids (++). Growth of eight replicates was determined turbidometrically (OD_{600 nm}) after 18 h (histidine, arginine, tryptophan, isoleucine, methionine, phenylalanine, tyrosine, proline, and lysine) or 24 h (leucine and threonine) of cultivation. Boxplots: median (horizontal lines in boxes), interquartile range (boxes), 1.5-fold interquartile range (whiskers).

Figure 2. Amino acid-dependent growth of the eleven GFP-labeled biosensors.

Each biosensor was cultivated in minimal medium, to which increasing concentrations of the required focal amino acid have been added. Cell growth was measured either turbidometrically (OD_{600 nm}, ▪) or as relative fluorescence units (RFU, Δ) after 18 h (histidine, arginine, tryptophan, isoleucine, methionine, phenylalanine, tyrosine, proline and lysine) or 24 h (leucine and threonine) of cultivation. Means (±95% confidence interval) of four replicates are given.

Figure 3. Quantification of amino acid concentrations in the culture supernatant of two mutant E. coli strains.

Mean (±95% confidence interval) amount of (A) histidine produced by E. coli ΔhisL, and (B) tryptophan produced by E. coli ΔtrpcC as determined by conventional HPLC measurements (black bar) or the growth of the respective biosensors, which was quantified turbidometrically (OD_{600 nm}, grey bar) or GFP fluorescence emission (white bar). Different letters indicate significant differences (S-N-K test: P<0.05, n = 4).