METHOD FOR SCREENING AND QUANTIFYING ISOPRENE BIOSYNTHESIS ENZYME ACTIVITY

摘要

The present invention is a novel technology of performing high-throughput screening of various enzymatic activities with high sensitivity using artificial genetic circuits. More specifically, the invention is a novel screening and quantifying methods of isoprene biosynthesis enzymes activity using an artificial genetic circuit capable of sensing isoprene. The artificial genetic circuit (IspGESS) is comprising a gene encoding a isoprene-sensing transcriptional regulator which recognizes isoprene, at least one reporter gene such fluorescence protein-encoding genes, a isoprene-sensing transcriptional regulator binding region and promoters for genes encoding isoprene-sensing transcriptional regulators and reporter proteins. IspGESS detects isoprene liberated from many enzymatic reactions and allows to measure the activity of reporter genes, such as fluorescent reporter genes and antibiotic resistance genes. This system is shown that it is a widely applicable tool for high throughput and quantitative screening of isoprene biosynthesis enzymes and MEP/MVA pathway enzymes. Thus, the invention can be advantageously used in the protein engineering technology for enzyme modification. Particularly, it can quantitatively investigate enzymatic activity, and thus can be applied to molecular evolution technology.