1.The Cloning And Expression Of Dxr Gene Of MEP Pathway For Terpenoid Biosynthesis And Its Activity Test 2.Biosynthesis Of Iridoid Glucosides In Lamium Barbatum

The 2-methyl-D-erythritol-4-phosphate(MEP) pathway for isoprenoid biosynthesis has come under increased scrutiny as a target for novel antibacterial agents.1-Deoxy-D-xylulose 5-phosphate reductoisomerase(DXR) is a key enzyme of the pathway that catalyzes the rearrangement and nicotinamide adenine dinucleotide phosphate(NADPH)-dependent reduction of 1-deoxy-D-xylulose-5-phosphate(DXP) to MEP.The unique properties of DXR make it a remarkable and rational target for drug design.First,it is a vital enzyme for synthesis of isoprenoids in several eubacteria including the pathogenic bacteria.Second,there are no functional equivalents to DXR in humans,making it an attractive target for therapeutic intervention.In this study,we prepared this key enzyme of MEP pathway through genetic engineering and tested its activity by bioassay.In order to obtain purified DXR,full length DXR cDNA cloned by PCR from E.coli genomic DNA was inserted into pET-15b vector to construct pET15b-DXR plasmid.After confirmed by enzyme digest analysis and sequencing,the positive pET15b-DXR recombinant plasmid was transformed into E.coli BL21(DE3),and the protein induced by IPTG was examined by SDS-PAGE and Western-blotting analysis.The result of SDS-PAGE and Western-blotting showed that the relative molecular weight of the fusion protein was same as the expected value,namely about 42 kDa.The recombinant pET15b-DXR protein was purified by combination of(NH₄)₂SO₄ precipitation and Ni-NTA chromatography with a yield of about 10mg/L medium and more than 95%purity.The activity test indicated that this fusion protein can catalyze DXP to MEP in the presence of NADPH.Iridoid glucoside compounds are self-protecting substances of several plants,which have extensive biological activities.They are also the effective ingredients of many herbal medicines.Lamium barbatum belongs to Lamium family of Labiatae and contains abundant iridoid glucosides.C-13 labeled compound[3,4,5-¹³C₃]DXP,which is one of the key intermediates in the MEP pathway for the biosynthesis of terpenoids,was used as the precursor to feed the selected plant.By analysing the C-13 labeling pattern of iridoid glucoside compounds caryoptoside and lamalbid isolated from the plant after feeding and the incoorperation of the labeled DXP,we found that the iridoid glucosides in L.barbatum were biosynthesized through the newly established MEP biosynthetic pathway.