Location: Natural Products Utilization ResearchTitle: Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors) Author
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2014
Publication Date: 5/13/2014
Citation: Rocaboy-Faquet, E., Noquer, T., Romdhane, S., Bertrand, C., Dayan, F.E., Barthelmebs, L. 2014. Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors. Applied Microbiology and Biotechnology. 98:7243-7252. Interpretive Summary: 4-Hydroxyphenylpyruvate dioxygenase is a key enzyme in plants that is involved in the synthesis of plastoquinone and vitamin E. It is inhibited by certain herbicides. We have developed an assay that can be used to measure the presence of these herbicides at very low concentration. This assay does not require very advanced analytical instrumentation. It measures the formation of melanin-like compounds by E. coli overexpressing a plant HPPD with a spectrophotometer.
Technical Abstract: Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole cell colorimetric bioassay with a recombinant E. coli expressing a plant HPPD for the ß-triketones herbicide analysis. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. Addition of sulcotrione, a HPPD inhibitor, decreased the production of the pigment. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96 wells microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione and leptospermone were 0.069, 0.051, 0.038, and 20 µM, respectively, allowing to validate the whole cell colorimetric bioassay as a simple and cost effective alternative tool for a laboratory use. The bioassay results from spiked soil samples were confirmed with high-performance liquid chromatography.