土曲霉中Butyrolactone I异戊烯基转移酶基因的克隆与功能鉴定

Prenylbutyrolactone derivative Butyrolactone I, as an effective cyclin-dependent kinases (CDK) inhibitor, has been found to exhibit antiproliferative activity through selectively inhibiting CDK2 and CDK1. It is expected to be developed into a novel anticancer drug. However, the supply of Butyrolactone I was very limited because of the rare occurrence in nature, and difficulty in chemical synthesis due to the regio-selectivity. While its precursor without prenyl group, Butyrolactone II, could be efficiently synthesized by chemical method. In our previous studies, Butyrolactone I has been isolated from an Aspergillus terreus A8-4, and 14 kinds of possible prenyltransferase genes have been predicted and cloned successfully on the basis of bio-informatics analysis combined with PSI-BLAST. Meanwhile, Butyrolactone II has been successfully synthesized by chemical methods in 56% yield. Based on the previous results, this proposal is aimed to screen out and characterize the prenyltransferase gene, and construct the engineering strain for the chemo-enzymatic synthesis of Butyrolactone I. This project would exploit a potent biosynthetic approach of Butyrolactone I synthesis.

丁内酯类衍生物Butyrolactone I是有效的细胞周期蛋白激酶(CDK)抑制剂，有望开发成一种新型的抗肿瘤药物。但其在天然界中含量较低，化学合成不含异戊烯基取代的前体化合物Butyrolactone II容易且产率高，而进一步异戊烯基化则由于涉及位置选择性，使得合成困难，制约了其大量合成。在前期研究中，我们发现土曲霉A8-4能产生Butyrolactone I，进一步通过生物信息学手段进行预测,并从中成功克隆得到14种芳香类化合物异戊烯基转移酶基因。本项目拟在此基础上，对获得的基因进行外源表达及功能鉴定，以期筛选得到能够催化Butyrolactone II异戊烯基取代的异戊烯基转移酶基因，并利用基因工程技术对该化合物进行酶法合成，为大量制备Butyrolactone I提供新方法，具有理论及实际应用意义。

Butyrolactone I是有效的细胞周期蛋白激酶（CDK）抑制剂，有望开发成一种新型的抗肿瘤药物，但其在天然界中含量较低，化学合成不含异戊烯基取代的前体化合物butyrolactone II容易且产率高，而进一步异戊烯基化由于涉及位置选择性，使得合成困难，制约了butyrolactone I的大量合成。本项目研究旨在寻找butyrolactone I异戊烯基转移酶基因并进行外源表达，通过酶法高效合成该化合物。通过本项目实施，我们从土曲霉（Aspergillus terreus A8-4）中克隆得到新型芳香类化合物异戊烯基转移酶基因BuPT，利用重组BuPT能够催化化学合成得到的(+)-butyrolactone II和DMAPP发生异戊烯基化反应高效合成(+)-butyrolactone I，重组BuPT为首个发现的可以体外催化该反应的异戊烯基转移酶。