METHOD FOR SCREENING AND QUANTIFYING ISOPRENE BIOSYNTHESIS ENZYME ACTIVITY

摘要

A method of performing high-throughput screening of various enzymatic activities with high sensitivity using artificial genetic circuits is provided. Particularly, the invention is screening and quantifying the activity of isoprene biosynthesis enzymes using an artificial genetic circuit capable of sensing isoprene. The artificial genetic circuit comprises an isoprene-sensing transcriptional regulator which recognizes isoprene, at least one reporter gene, a isoprene-sensing transcriptional regulator binding region and promoters for genes encoding isoprene-sensing transcriptional regulators and reporter proteins.;The artificial genetic circuit detects isoprene liberated from many enzymatic reactions and measures the activity of reporter genes. This system is widely applicable for high throughput and quantitative screening of isoprene biosynthesis enzymes and MEP/MVA pathway enzymes.;Therefore, the invention can be advantageously used in the protein engineering technology for enzyme modification. Particularly, it can provide a quantitative investigation of enzymatic activity, and thus can be applied to molecular evolution technology.

主权项

1. A method of detecting, screening or quantifying the activity of one or more isoprene biosynthesis enzymes using an artificial genetic circuit, the method comprising the steps of:
(a) providing an artificial genetic circuit for detecting isoprene or microorganisms containing in their chromosomal DNA or cytoplasm an artificial genetic circuit for detecting isoprene, the artificial genetic circuit comprising:
(i) a gene encoding an isoprene-sensing transcriptional regulator which recognizes isoprene,(ii) at least one reporter gene selected from the group consisting of fluorescence protein-encoding genes and antibiotic resistance genes, and(iii) a gene expression regulatory region consisting of a promoter regulating the expression of the isoprene-sensing transcriptional regulator, a region to which the isoprene-sensing transcriptional regulator binds to induce the expression of a downstream reporter gene, and a promoter regulating the expression of the reporter gene; (b) providing a clone or gene library containing one or more of a gene encoding an isoprene biosynthesis enzyme; (c) introducing the clone or gene library and the artificial gene circuit for detecting isoprene into host microorganisms to prepare recombinant microorganisms or introducing the clone or gene library into the microorganisms containing the artificial gene circuit or detecting isoprene to prepare recombinant microorganisms; (d) treating the recombinant microorganisms with a compound capable of liberating isoprene by an enzymatic reaction; and (e) detecting or quantifying the activity of the reporter protein whose expression is induced by sensing isoprene liberated by the enzymatic reaction.