【作者】 刘波；

【导师】 唐莉；

【作者基本信息】 大连理工大学 ， 生物化工， 2011， 硕士

【摘要】 天然产物在药物发现研究中具有重要的价值。来源于微生物、植物以及海洋生物的聚酮化合物以其多样性的结构和生物活性、独特的生物合成机制以及在组合生物合成中的应用价值,成为天然产物研究中的热点。Tautomycetin(TMC),最初于1989年在土壤放线菌Streptomyces griseochromogennes的发酵液中发现的具有抗真菌活性的聚酮化合物,含有独特的马来酸酐环结构。随后的研究发现其具有蛋白磷酸酶PP1选择性抑制活性和T细胞免疫抑制活性,使得TMC不但在抗肿瘤药物和免疫抑制药物研发中具有重要价值,而且在PP1的生化基础研究领域有着重要意义。目前,已克隆了TMC的生物合成基因簇tau,并在补料实验和序列分析推测的各基因功能的基础上提出了其生物合成的一个模型。本研究主要从Streptomyces griseochromogennes的两株基因重组突变株STQ1306和STQ1010的发酵液中提取分离TMC的新型衍生物,并通过生物转化实验探讨其生物合成机制。研究采用反相C18柱层析技术和HPLC半制备技术从菌株STQ1306和菌株STQ1010的发酵液中分别分离到3个(TMC-Ds,化合物7-9)和5个(TMC-Ks,化合物10-14)新型的TMC衍生物,并通过UV、MS、1D-NMR和2D-NMR确定了结构,其中化合物10-14等5个化合物是首次分离的。对TMC-K系列衍生物(化合物10-14)的结构分析表明在已鉴定的TMC生物合成基因簇之外可能存在非特异性的氧化酶负责C-7位和C-5位羟基的形成。聚酮合酶TauB的模块8的酰基转移酶结构域(AT8)的特异性不高,除了能以乙基丙二酰-CoA为底物外,也可以丙二酰-CoA和甲基丙二酰-CoA为底物。初步研究了培养基成分对菌株STQ1306各产物(TMC-Ds)生成的影响,发现CaCO3可能通过影响发酵液pH影响TMC-D系列产物的生成。对TMC-D系列衍生物(化合物7-9)的进行生物转化实验,由结果可推断脱羧酶TauD和P450氧化酶TauK的特异性不高,可催化多种底物。TMC-D2可由TauD的催化脱羧形成TMC, TMC-D3和TMC-D4在TauK和TauD的作用下形成TMC, TMC-D3和TMC-D4由TauD催化脱羧可分别形成TMC-K6和TMC-K5。因此TMC的生物合成在主要途径之外,存在多条支路途,这也是TMC中间体衍生物比较多的原因之一。研究结果一方面进一步了解了TMC的生物合成机制,另一方面为TMC构效关系研究提供结构特异的化合物,也为进一步的组合生物合成定向改造TMC打下基础。更多还原

【Abstract】 Natural products have provided considerable value to the pharmaceutical industry over the past half century, and continue to provide significant value in the discovery of novel chemical structures and bioactive lead molecules for clinical development. Polyketides mostly produced by microorganisms are a remarkable class of compounds, which exhibit a staggering range of functional and structural diversity, and boast a wealth of medicinally important activities. Besides, their unque biosynthesis mechanism makes them valuable in combinatorial biosynthesis.Tautomycetin(TMC), which was isolated from Streptomyces griseochromogennes in 1989, is a highly specific inhibitor of protein phosphatase I (PP1) and possesses anti-fungal and immunosuppressive activities. Thus, TMC is not only an interesting drug lead in antitumor drugs and immunosuppressive drugs development, but also a powerful biochemical tool in elucidation the roles of PP1 in various biological pathways. In addition, the unique structure of TMC featuring a rare dialkymalic anhydride moiety (DAM) and a polyketide chain draws attention to its biosynthetic chemistry. The tau biosynthetic gene cluster for TMC has been cloned and sequenced, and on the basis of functional assignments for each gene in the tau cluster from sequence analysis and feeding experiments, a model for TMC biosynthesis has been proposed.In this thesis, novel TMC analogues were isolated from the recombinant mutant strain STQ1306 and STQ1010, and their biosynthsis mechanism was studied by biotransformation experiment.Using RP-C18 column chromatography and semi-preparative HPLC,3 compounds (TMC-Ds, compounds 7-9) from strain STQ1306 and 5 compounds (TMC-Ks, compounds 10-14) from strain STQ1010 were obtained, and their structures were confirmed by UV, MS, 1D-NMR and 2D-NMR.On the basis of TMC-Ks (compounds 10-14), we proposed that there is non-specific oxidase involving the hydroxylation of C-5 and C-7 beyond the cloned gene cluster of TMC. And we proposed that the acyltransferase(AT) domains of module 8 in polyketide synthase TauB has relaxed substrate specificity, so it could accept either ethylmalonyl CoA, or malonyl CoA and methylmalonyl CoA as extender unit. The components of medium on the production of TMC-Ds by strain STQ1306 were studied, and it was found that CaCO3 affects production of TMC-Ds by affecting pH.Based on the results of the biotransformation experiments, we proposed that the decarboxylase TauD and P450 oxidase TauK have relaxed substrate specificity, and they could catalyze a variety of compounds. We proposed that TMC-D2 could be decarboxylated by TauD to produce TMC, TMC-D3 and TMC-D4 could be oxidated by TauK and decarboxylated by TauD to produce TMC. Besides, TMC-D3 and TMC-D4 also may only be decarboxylated by TauD to produce TMC-K6 and TMC-K5 respectively. Therefore, in addition to the main pathway of the biosynthesis of TMC, there are a number of shunt ways, which is also one of the reasons that there are more intermediate analogues of TMC.On the basis of the results in this thesis, the biosynthsis mechanism of TMC and its analogues was further studied, on the other side various compounds were provided for its structure activity relationships study.更多还原