Cloning And Function Analysis Of New Hydroxylase In Taxus Chinensis

Taxol, arguably the most powerful anti-cancer drug, was extract from taxcus with low yields. This drug is now largely produced by semi- synthesis and natural extraction, which cannot satisfy the commercially demand. Under the condition of kowning the whole taxol metablisom pathway, the heterogeneous biosynthesis with synthetic biology concept maybe a potential approach for the large scale production of taxol. There are still some taxane hydroxyl step are unclear. In the current research, 4 new candidate taxane hydroxylase were isolated, and their activity were analyzed. The main results were as follows:(1) The cloning and analysis of new hydroxylase. Totally, 266 candidate taxane hydroxylase P450 EST were found from the taxcus cell transcriptome data, and 39 belong to CYP725 or CYP716, 4 hydroxylases(cyp1/cyp2/cyp3/cyp4) among them hold the whole ORF sequence were isolated from the Taxus chinesis c DNA. They has the similarity of 71%, 70%, 74 %, 45% with CYPA1, T10 H, T10 H and CYP716B1.(2) Four candidate hydroxylases expression by prokaryotic expression system. We connected four new hydroxylases to p ET-32 a with suitable enzymic site, and the effects of IPTG, time as well as temperature on protein expression were optimized. As a result, the optimal condition is IPTG 0.08 m M, 5 h and 20?. Also, the recombinant protein is inclusion body.(3) The function and acitivity of these new hydroxylases were investigated by by Carbon monoxide difference spectra Isolation and enzymatic reaction in vitro. The result showed that CYP2 maybe inactive because of no optimal absorption at 450 nm. In vitro system was composed of substrate, enzyme, electron transport chain, the regeneration system and buffer. Taxane mixture were extract by CH2Cl2/CH3 OH and identified by HPLC, and TC were obtained by using chromatography column filled with sephadex. The result showed that eluant with 70% C H3 CN contained more than 90% TC; this kind of taxane with no reaction with CYP2 proved recombinant protein is inactive, relevant reasons were discussed.