美登木素生物合成的几个问题初探
其他题名A Preliminary Study on the Biosynthesis of Maytansinoids
康前进
学位类型博士
导师沈月毛
2008-05-26
学位授予单位中国科学院昆明植物研究所
学位授予地点昆明植物研究所
学位专业植物学
关键词美登木素 滑桃树 内生菌 生物合成 “glycolate” 特殊延伸单元
摘要本论文在美登木素化合物生物合成研究以及我们课题组前期对于高等植物来源的该类化合物生源探讨基础之上,从以下方面展开了研究。 第一部分主要是化合物分离工作,从滑桃树茎皮的 85 % 乙醇提取物中分离得到23个化合物,其中 5-sarmentogenin-3-O--L-rhamnopyranoside (1), 5-sarmentogenin (2), 11-oxo-uzarigenin-3-O--L-rhomnopyranoside (3), 5-gitoxigenine-3--L-rhamnopyranoside (4), 5-oleandrigenin-3-O-- rhamnopyranoside (5), 5-oleandrigenin-3-O--glucopyranosyl-(1→4)-- rhamnopyranoside (6), -anhydroepiditoxigenin-3-O--glucopyranosyl-(1→4)- -L-rhamnopyranoside (7), (+)-dihydrodehydrodiconiferyl alcohol 4-O-β-(6″-O-galloyl)-glucopyranoside (14), 4,4′-O-dimethylellagic acid 3-(2″-O-acetyl)--rhamnopyranoside (19), ethyl O-β-D-(6′-galloyl)-glucopyrano side (21) 为新化合物。对分离到的鞣花酸类和部分苯丙素类化合物进行了抗氧化活性测定,显示新化合物14的IC50为22.8 μM,抗氧化活性较好;通过MTT分析实验得出,新化合物14、19和21在浓度为0.4 M时对H2O2诱导的PC12凋亡也具有一定的保护作用。所分离到的化合物类型涵盖了强心苷类、苯丙素类、萜类和甾体类、鞣花酸类以及其它类型,其中强心苷类化合物为首次从大戟科植物分离得到,苯丙素类、鞣花酸类化合物在大戟科植物中也具有一定的化学分类学意义。根据AHBA(3-氨基-5-羟基苯甲酸,美登木素类化合物的保守起始单元)合成酶基因和CT (氨甲酰基转移酶)基因保守区域设计引物并进行PCR扩增筛选,获得阳性滑桃树内生链霉菌 Strepomyces sp. M27m3。从它的10升YMG固体发酵物中分离得到了5个化合物,其中化合物cis-cyclo(11-hydroxyprolylleucyl) (1),cis-cyclo(11-ene-prolylleucyl) (2)是2个新的环二肽类化合物。从另一株AHBA合成酶基因阳性的滑桃树内生菌 Streptomyces sp. 5B的10升YMG固体培养基的发酵产物中分离得到了7个化合物,其中endophyticterpene KA (1),endophyticterpene KB (2),(2E,10E)-13-(3-hydroxy-4-methoxyphenyl)-2,6,10-trimethyl-7-one-5,9-dodecadienal (3),为新化合物;但是两株内生链霉菌均没有得到预期的美登木素类化合物。 第二部分对美登木素生物合成过程中特殊延伸单元 “glycolate” 的酰基载体蛋白功能进行了研究,对负责合成该单元的 asm13-17 五个单基因,利用 Redirect 技术分别进行了突变株的构建。对所有突变株的发酵产物进行了 HPLC 以及 LC-MS 检测和分析,结果发现突变株 asm13、asm15、asm16、asm17 的产物均为 10-demethoxy-AP-3,但在 asm14 突变株没有检测到任何安莎类的化合物,不过改变 HPLC 的条件后检测到了一个 N-acetyl-triketide 的产物。表明了 Asm14 不仅具有合成 “glycolate” 功能,而且是聚酮链延伸过程所必需的因子,从而具有双重功能。为了进一步验证基因 asm14 的功能和特异性,选择了基因 asm14 以及来源于格尔登素产生菌的同源基因 gdmJ,对 asm14 和 asm14-17 两个突变株进行了回补,结果使得这两个突变株分别恢复了产生安丝菌素和 10-demethoxy-AP-3 的能力,表明 asm14 和 gdmJ 具有一定的功能互补性。 论文的第三部分还对其它微生物的化学成分进行了研究。从美登木内生链霉菌 Streptomyces sp. 05B分离得到了以下7个化合物,其中 decahydro-4,8a-dimethylnaphthalene-1,4a,5-triol (1), (1S,4S,5R,6S,7S,10S) -4,5,10,11-cadinanetriol (2), cis-cyclo(11-hydroxyprolylleucyl) (3) 为新化合物。从滑桃树内生真菌Fusarium sp. 0219A20中分离到了5 个已知化合物。 本论文的最后一部分主要是对I 型PKS生物合成途径中的“glycolate” 特殊延伸单元的研究进行了综述。
其他摘要On the basis of maytansinoids biosynthesis and previous research in our laboratory on biological origin of these compounds from higher plants, this dissertation mainly composed of four parts. In the first part, the research on chemical components from the stem bark of Trewia nudiflora which is a rich source of maytansinoid compounds was carried out. 23 compounds including cardenolides, terpenes, steroids, phenyl propanoids, ellagic acids and miscellaneous were purified from the 85 % ethanol extract of the stem bark of T. nudiflora, and 5-sarmentogenin-3-O--L-rhamnopyranoside (1), 5-sarmentogenin (2), 11-oxo-uzarigenin-3-O--L-rhomnopyranoside (3), 5-gitoxigenine-3--L- rhamnopyranoside (4), 5-oleandrigenin-3-O--rhamnopyranoside (5), 5-oleandrigenin-3-O--glucopyranosyl-(1→4)--rhamnopyranoside (6),- anhydroepiditoxigenin-3-O--glucopyranosyl-(1→4)--L-rhamnopyranoside (7), (+)-dihydrodehydrodiconiferyl alcohol 4-O-β-(6″-O-galloyl)-gluco- pyranoside (14), 4,4′-O-dimethylellagic acid 3-(2″-O-acetyl)--rhamnopyrano- side (19), ethyl O-β-D-(6′-galloyl)-glucopyranoside (21) were designated new in structures. The antioxidant activities of ellagic acids and some of the phenyl propanoids were evaluated by the DPPH radical-scavenging assay. Compound 14 showed significant antioxidant activity and the IC50 was 22.8 μM. Furthermore, the compounds 14, 19 and 21 could also rescue the H2O2-induced PC12 cell death at 0.4 μM in MTT assay. Besides, cardenolides were first isolated from Euphorbiaceae species and the phenyl propanoids and ellagic acid possessed significance on chemotaxonomy of Euphorbiaceae. Among the endophytic actinomycetes isolated from T. nudiflora., Strepomyces sp. M27m3 which was positive in PCR screening with both primers of 3-amino-5-hydroxybenzoic acid (AHBA, starter unit of maytansinoids) synthase gene and carbamoyltransferase (CT) gene involved in the biosynthesis of maytansinoids was studied by chemical components isolation. 5 compounds were purified from 10 L YMG fermentation product among which cis-cyclo(11-hydroxyprolylleucyl) (1) and cis-cyclo(11-ene-prolylleucyl) (2) were two new cyclodipeptides. The other endophytic Streptomyces sp. 5B isolated from T. nudiflora which was also positive in AHBA PCR reaction was fermented with 10 L solid YMG and 7 compounds were obtained, among which endophyticterpene KA (1), endophyticterpene KB (2) and (2E,10E)-13-(3-hydroxy-4-methoxyphenyl)-2,6,10-trimethyl-7-one-5,9-dodecadi-enal (3) were designated as new compounds. However, no compounds chemically related to maytansinoids was discovered probably because of the influence on microbe metabolites by fermentation conditions, especially to endophytes from plant. In the second part, the function of acyl carrier protein Asm14 was investigated, involved in the biosynthesis of the unusual “Glycolate” chain extension unit. asm13-17 was individually inactivated, and a segment covering asm14-17 was deleted by Redirect Technology. Metabolites of the six mutants were analyzed by HPLC and LC-MS, and the mutants of asm13, asm15, asm16, asm17 all lost the ansamitocin productivity but instead accumulated 10-demethoxyl-AP-3. However, not any kind of ansamitocin was isolated from asm14 mutant. By adjusting the mobile phase of HPLC, a product of N-acetyl-triketide derivative was found. The above results indicated that asm14 was not only essential for the formation of “Glycolate”, but also required for the downstream polyketide chain extension. Further experiment on function and specificity of asm14 was carried out with asm14 and gdmJ from geldanamycin-producing strain. These two genes were introduced into asm14 and asm14-17 mutants, which led to the regained productivity of ansamitocins and 10-demethoxy-ansamitocins respectively. The third part was on research of chemical components of other endophyte microbes. 7 compounds were isolated from endohphytic Strptomyces sp. MH05B of Maytenus hookeri Loes, among which decahydro-4,8a-dimethylnaphthalene-1,4a,5-triol (1), (1S,4S,5R,6S,7S,10S)- 4,5,10,11-cadinanetriol (2), cis-cyclo(11-hydroxyprolyl- leucyl) (3) were new in structure. In addition, 5 known compounds were isolated from the endophytic fungi Fusarium sp. 0219A20 of T. nudiflora. In the last part, the author reviewed on the previous research of unsual extention unit of “glycolate” in biosynthesis of type I polyketide.
页数194
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/370
专题昆明植物所硕博研究生毕业学位论文
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康前进. 美登木素生物合成的几个问题初探[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2008.
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