中国红豆杉紫杉烷10β-羟化酶的分子克隆及在酿酒酵母中的表达

Taxol Biosynthesis: Molecular Cloning of a Cytochrome P450 Taxane 10 β-hydroxylase cDNA from Taxus chinensis and Expression in Saccharomyces cerevisiae

天然产物紫杉醇是一种重要的抗癌药。其天然来源的匮乏和缺少商业上可行的化学全合成促进了对紫杉醇生物来源的深入研究。紫杉醇的生物合成是一个复杂的多步过程,主要包括四环骨架的构建和加入各种羟基和酰基基团,其中羟基的加入由细胞色素P450氧化酶催化。我们从中国红豆杉愈伤组织细胞mRNA构建的cDNA文库中克隆得到几个P450 cDNA片段。其中一个长1 494 bp的cDNA片段,编码497个氨基酸,推断蛋白分子量为56470 Da,等电点为9.42。序列比较显示,这个推断蛋白包含多个细胞色素P450氧化酶的保守区,具有典型的细胞色素P450氧化酶的特征,进一步的比较发现其与已鉴定的东北红豆杉紫杉烷-10 β-羟化酶有92%的同源性。将这个cDNA片段连接在质粒pYeDP60上构建表达载体,导入相应的酵母表达菌株WHT和WVS进行表达,获得相应大小的蛋白表达条带,功能鉴定的工作正在进行中。

The natural product Taxol,produced by Taxus species, is an important anticancer drug. The low yield of Taxol from natural resources and the lack of a commerically viable total synthesis have rendered that Taxol is produced primarily by semisynthesis methods. The biosynthesis of Taxol from plant primary metabolism is a very complex process, including near 20-step Taxol biosynthetic pathways to construct its tetracyclic skeleton and the addition of the various oxygen and acyl functional groups. A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. Several full-length cytochrome P450-dependent monooxygenase sequences were obtained from a cDNA library constructed from mRNA isolated from Taxus chinensis callus culture cells. All Taxus species are known to be very closely related, so we may designed several pairs of primers to clone full-length taxane 10 β - hydroxylase gene from T. chinensis according to the both ends of taxane 10 β - hydroxylase sequences from T. cuspidata. A distinctive PCR product with expected size of 1. 5 kb was amplified by PCR screening T. chinensis cDNA library. The PCR product was cloned into T-ended vector pMD18-T (TaKaRa) for DNA sequencing. This determination has been performed on the strands to confirm the accurracy of sequence. The full-length cDNA contains a 1494 bp open reading frame, which encodes 497 amino acids deduced protein of molecular weight 56470 Da with theoretical pI of 9. 42. The deduced ami-no acid sequences includes an N-terminal hydrophobic helix required for anchoring in endoplasmic reticu-lum (ER) membrane, a prolich-rich hinge region modulating folding and substrate access to the catalytic pocket, preceded by a cluster of basic residues (the halt-transfer signal) between the hydrophobic amino-terminal membrane anchoring segement and the globular part of the protein. The deduced amino acid sequence comprises several of signature features of other cytochrome P450-dependent monooxygenasesof plant origin in eluding. The PFGGGLRTCPG domain of this putative protein contains the most consensus sequence (F-X-X-G-X-R-X-C-X-G) with the absolutely conserved cysteine that serves as fifth ligand to heme iron; the ESLR domain, has the absolutely conserved E-X-X-R motif in helix K, and the PSRF domain form an E-R-R triad that is thought to form a salt bridge that locks the Cys pocket in position and assures heme association with the protein; and finally, the ASYDTT domain contains a highly conserved Thr that is thought to be involved in binding and activation, which corresponds to the proton transfer groove on the distal side of the heme. Comparison with that of cytochrome P450 taxane 10 β-hydroxylase cDNA from Taxus cuspidata revealed significant homology (92% identity) and indicated they have an very closer relationship. The Sactharomyces cerevisiae WHT employed as the expression host, when grown on galactose, expresses the HTR gene encoding an Helianthus tuberosus NADPH-cytochrome P450 reductase which is essential for functional catalysis by the coexpressed cytochrome P450 monooxygenase. When suitable substrates for test of function are available, function of expression of this target enzymes P450 clone will follow confirmation. Cloning and expression of new Taxol biosyn-thetic hydroxylase genes not only improve detail understanding of biosynthetic pathway of Taxol but also improve the production yields and obtain novel taxane metabolites and deriatives by bioengineering manipulations.