KIB OpenIR  > 昆明植物所硕博研究生毕业学位论文
Alternative TitlePost-PKS Modifications Study of Ansamitocin Biosynthesis
Thesis Advisor郝小江
Degree Grantor中国科学院昆明植物研究所
Place of Conferral昆明植物研究所
Degree Discipline植物学
Keyword安丝菌素酰胺n-苷 聚酮后修饰 酰胺n-糖基转移酶 Fadh2依赖型卤代酶
Abstract本文从基因失活、突变体次生代谢产物的分析、基因异源表达、重组蛋白催化特性几方面对安丝菌素后修饰酶Asm25(酰胺N-糖基转移酶)和Asm12(氯代酶)的功能进行了研究。同时还从云南美登木(Maytenus hookeri)的内生放线菌CS中克隆到了一个FADH2依赖型卤化酶并对其体外催化特性进行了研究。 通过基因敲除的方法得到糖基转移酶基因asm25的失活质粒,利用接合转移导入野生型菌株替换野生型的基因得到了突变体。Asm12是安丝菌素生物合成途径中编码卤代酶的基因,克隆asm12基因到pET32a+中,在大肠杆菌E. coli BL21(DE3)plysS成功表达了C端带有His标签的可溶性蛋白。通过MS 和 NMR来检测无细胞提取物及纯酶的体外催化活性,结果证明异源表达的Asm12 能转化proansmitocin生成19-chloroproansmitocin,但是不能催化L- tryptophan进行氯取代。 云南美登木的内生菌CS产生化合物naphthomycin A,其结构中含一个氯原子,根据FADH2-依赖型卤化酶的保守区设计简并引物从CS的基因组DNA中扩增到了卤代酶的基因片段cshaloA,以该基因片段作探针通过克隆杂交筛选,从CS基因组文库中得到了cshaloA的全长基因。本文的研究结果为选择性分离安丝菌素的衍生物提供了新的思路,另外酰胺N-糖基转移酶的初步研究也可以为进一步研究提供参考。
Other AbstractN-glycosyltransferase gene asm25 and halogenase gene asm12 were proved to be involved in the post-PKS modifications of ansamitocin through gene inactivation, chemical analysis, heterologous expression and in vitro enzymatic characterization. Enzymatic catalysis and gene inactivation had generated ansamitocin derivatives with peripheral structural modifications. In addition, a FADH2-dependent halogenase gene cshaloA was cloned and characterized in vitro from Streptomyces sp. CS, an endophytic bacterium of Maytenus hookeri. Gene asm25, the only glycosyltransferase homolog identified in ansamitocin biosynthetic gene cluster, was inactivated through in-frame deletion. Metabolite profile of the mutant was preliminarily surveyed through LC-ESI-MS and structures of the main accumulants were elucidated by MS and NMR. Inactivation of asm25 resulted in the accumulations of N-demethyl-AP-3 (NMDP-3) and N-demethyl-AP2 (NDMP-2), which was the same as the N-methyltransferase asm10 mutant. Therefore it could be concluded that N-methylation or N-glycosylation on the amide bond were branching steps in the biosynthesis of ansamitocins or N-demethyl-ansamitocinosides. Inactivation of halogenase gene asm12 established its catalytic activity as chloronation of C-19 on proansamitocin, which was the first modification on the PKS backbone. Analyzed through MS and NMR, Over-expressed His-tagged Asm12 from E. coli BL21(DE3)pLysS converted proansamitocin to 19-chloroproansamitocin but with no activity on L-tryptophan. In vitro characterization had proved Asm12 as FADH2-dependent halogenase and the Km value towards proansamitocin was 4.163 uM. Streptomyces sp. CS, endophytic bacterium of Maytenus hookeri, was found to produce naphthomycin A containing chlorine. Using degenerate primers designed based on the conserved regions of FADH2-dependent halogenases, a fragment of halogenase was amplified from the genome of S. sp. CS and named as cshaloA. Subsequent hybridization using the amplified fragment as probe had led to the cloning of the whole cshaloA, which had 95 % identity with Asm12 and contained two motifs conserved in FADH2-dependent halogenases. Over-expressed and purified CshaloA was analyzed as Asm12 and it was found they shared the same activity to convert proansamitocin to 19-chloroansamitocin with the presence of FADH2. All the above-mentioned results suggested new strategies for selective isolation of ansamitocin and its derivatives. And the pilot study of the Asm25 could be a good reference for next study.
Document Type学位论文
Recommended Citation
GB/T 7714
代焕琴. 安丝菌素生物合成的后修饰研究[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2006.
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