title: Translation-dependent bioassay for amino acid quantification using auxotrophic microbes as biocatalysts of protein synthesis.
abstract: Bioassay for amino acid quantification is an important technology for a variety of fields, which allows for easy, inexpensive, and high-throughput analyses. Here, we describe a novel translation-dependent bioassay for the quantification of amino acids. For this, the gene encoding firefly luciferase was introduced into Lactococcus lactis auxotrophic to Glu, His, Ile, Leu, Pro, Val, and Arg. After a preculture where luciferase expression was repressed, the cells were mixed with analytes, synthetic medium, and an inducer for luciferase expression. Luminescence response to the target amino acid appeared just after mixing, and linear standard curves for these amino acids were obtained during 15-60-min incubation periods. The rapid quantification of amino acids has neither been reported in previous works on bioassays nor is it theoretically feasible with conventional methods, which require incubation times of more than 4 h to allow for the growth of the microbe used. In contrast, our assay was shown to depend on protein translation, rather than on cell growth. Furthermore, replacement of the luciferase gene with that of the green fluorescent protein (GFP) or β-galactosidase allowed for fluorescent and colorimetric detection of the amino acids, respectively. Significantly, when a Gln-auxotrophic Escherichia coli mutant was created and transformed by a luciferase expression plasmid, a linear standard curve for Gln was observed in 15 min. These results demonstrate that this methodology can provide versatile bioassays by adopting various combinations of marker genes and host strains according to the analytes and experimental circumstances.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Kurokawa, Imizu, Toyama, Japan.|Asano Active Enzyme Molecule Project, ERATO, Japan Science and Technology Agency, Kurokawa, Imizu, Toyama, Japan.|Earth-Life Science Institute, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Kameya
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Kurokawa, Imizu, Toyama, Japan. asano@pu-toyama.ac.jp.|Asano Active Enzyme Molecule Project, ERATO, Japan Science and Technology Agency, Kurokawa, Imizu, Toyama, Japan. asano@pu-toyama.ac.jp.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D001681:Biological Assay / Q000295:instrumentation*; D002138:Calibration; D003124:Colorimetry / Q000379:methods*; D017930:Genes, Reporter; D018698:Glutamic Acid / Q000172:deficiency; D005973:Glutamine / Q000172:deficiency; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000172:deficiency; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014633:Valine / Q000172:deficiency; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D005973:Glutamine; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D006639:Histidine; D008156:Luciferases; D001616:beta-Galactosidase; D014633:Valine
keywords: Amino acid quantification; Auxotroph; Bioassay; Lactic acid bacteria