BIOSYNTHESIS OF TAXOL

紫杉醇的生物合成

• 批准号: 6295002
• 负责人: RODNEY B CROTEAU
• 金额: $ 5.65万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6295002
• 关键词: Conifera; acylation; bioreactors; biosynthesis; cell free system; chemical kinetics; chemical structure; cyclization; cytochrome P450; diterpenes; enzyme mechanism; geranyl compound; mass spectrometry; medicinal plants; molecular cloning; paclitaxel; plant extracts; plant physiology; radiotracer; tissue /cell culture; transfection

Taxol, a highly functionalized diterpenoid, is an important anticancer drug isolated from yew (Taxus) species. The supply of this drug, and its precursors for semisynthesis, from natural sources is very limited, and total synthesis is not practical. Any attempt to improve the biological production of taxol and its congeners requires an understanding of the biosynthesis of this natural product and of the regulation of the pathway. A multi-step biogenetic scheme has been proposed based on the occurrence of defined taxoid metabolites and on analogy to biosynthetic transformations of simpler terpenoids; however, there is presently little experimentally-supported information on the biosynthesis of taxol, a process upon which future supply must depend. The long-term objective of this research is to increase the yields of this valuable drug and/or its semisynthetic precursors by engineering the overexpression of slow steps of the pathway in intact yew plants or derived cell cultures. This molecular approach offers a feasible solution to the taxol supply problem in that it involves the engineering of relatively few genes into an existing background for taxol production in which at least the latter steps of the pathway seem reasonably efficient. This goal will be reached by determining the number, types and sequence of enzymatic steps in the transformation of the ubiquitous isoprenoid branch-point intermediate, geranylgeranyl diphosphate, to the diterpenoid natural product, and by assessing the contribution of each step to pathway flux in order to evaluate importance as a cDNA cloning target. Defining this complex, multi-step pathway will be accomplished primarily through the use of cell-free enzyme systems from yew (Taxus) stem or cultured cells, combined with in vivo studies with labeled precursors, to determine the sequential progression from simple to complex metabolites. This systematic approach should identify the most appropriate target steps, and provide the necessary information and tools for cDNA isolation. The first two committed steps of the taxol pathway have been defined. The first, the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)- diene, is very slow if not rate limiting in the pathway, and the second, the cytochrome P450-catalyzed hydroxylation with allylic rearrangement of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol, is also very slow relative to subsequent pathway steps. PCR-based cDNA cloning strategies for both cyclase and P450 hydroxylase genes have been devised and these efforts constitute the first two specific aims. The third specific aim is a systematic approach to determining the sequence of oxygenation steps leading from taxa-4(20),11(12)-dien-5alpha-ol to the level of a pentaol and evaluating the contribution of each step to pathway flux and its importance as a cloning target. The forth aim focuses on the sequence of acylation steps in the progressive oxygenation of the taxane nucleus, the timing of C9-oxidation, and deciphering the enzymatic route to oxetane ring formation. In the final aim, transgenic Taxus systems will be engineered for overexpression of slow pathway steps using existing technologies, and the influence on the production yields of taxol, and related taxoids, will be determined.

紫杉醇是一种高度功能化的二萜，是一种重要的抗癌药物

项目成果

Preliminary assessment of the C13-side chain 2'-hydroxylase involved in taxol biosynthesis.

初步评估参与紫杉醇生物合成的 C13 侧链 2-羟化酶。

• DOI: 10.1016/j.bbrc.2005.08.119
• 发表时间: 2005
• 期刊: Biochemical and biophysical research communications.
• 作者: Long,RobertM;Croteau,Rodney
• 通讯作者: Croteau,Rodney

Molecular cloning of a taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.

• DOI: 10.1006/abbi.1999.1609
• 发表时间: 2000-02
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Kevin D. Walker;A. Schoendorf;R. Croteau

Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction.

• DOI: 10.1006/abbi.2000.1865
• 发表时间: 2000-07
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: David C. Williams;M. Wildung;A. Q. Jin;Dolan Dalal;John S. Oliver;R. Coates;R. Croteau

Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production.

紫杉醇生物合成：两种乙酰COA的识别和表征：将通路途径转移到紫杉醇产量的途径途径。

• DOI: 10.1016/j.abb.2009.05.018
• 发表时间: 2009-07-15
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Hampel D;Mau CJ;Croteau RB
• 通讯作者: Croteau RB

Synthesis and In Vitro Evaluation of Taxol oxetane ring D precursors.

• DOI: 10.1016/j.tetlet.2010.02.033
• 发表时间: 2010-04-14
• 期刊: Tetrahedron letters
• 影响因子: 1.8
• 作者: Kaspera R;Cape JL;Faraldos JA;Ketchum RE;Croteau RB
• 通讯作者: Croteau RB