南欧大戟巨大戟醇酯生物合成关键基因的鉴定与功能分析

	南欧大戟巨大戟醇酯生物合成关键基因的鉴定与功能分析
	李冬梅
导师	方欣
关键词	南欧大戟 Euphorbia, Ingenol ester, Acyltransferase 巨大戟醇酯乙酰基转移酶
摘要	南欧大戟（Euphorbia peplus）是一种药用植物，以其良好生物活性的二萜类化合物而闻名。2012 年，从中分离到的巨大戟醇-3-O-当归酸酯（ingenol mebutate, PEP005, Picato）的乳膏制剂被 FDA 批准作为治疗日光性角化病的一类药物进入市场[1]。本课题组长期从事大戟科二萜类化合物的结构和活性研究[2-7]，并在与中科院遗传发育所杨崇林课题组合作中，发现了巨大戟烷类化合物 20-脱氧巨大戟醇及其类似物如 HEP14 可促进溶酶体生成。这类化合物优异的生物活性吸引了对其生物合成的研究，但迄今为止，只报道了从 GGPP 到续随子烷的生物合成途径[8]。巨大戟醇-3-O-当归酸酯和 HEP14 分子中 3 位和 5 位的当归酰基被认为是主要的活性基团，在这些位置还存在其他类型的酰基修饰，然而催化这些酰基化反应的酶尚未报道。为了分离鉴定巨大戟醇合成途径中催化酰基化的酶基因，本论文基于本课题组前期对南欧大戟进行基因组和转录组测序数据，根据 BAHD 保守结构域从南欧大戟基因组中发现了 100 多个 BAHD 家族基因。通过对这 100 多个 BAHD 基因的进化分析，挑选出属于第五亚家族的 BAHD 基因，该亚家族包含了已报道的催化二萜类化合物酰基化的基因。通过与已知的南欧大戟巨大戟烷合成途径基因的共表达分析，最后获得了 10 个候选基因。本论文通过体外酶活实验检测了 10 个候选基因的功能。课题组前期利用植物化学手段，从南欧大戟中分离得到巨大戟醇及假白榄烷类化合物作为可能的酰基受体。本论文分别利用原核表达体系和烟草瞬时转化体系，获得了目的基因蛋白。然后利用化学合成方法合成了乙酰辅酶 A、苯甲酰辅酶 A、当归酰辅酶 A 等作为酰基供体。然后分别以不同的酶与不同的酰基受体和酰基供体的组合进行体外酶活实验，反应产物通过高效液相色谱质谱联用检测。经多轮酶活反应，最终发现其中一个酶能够乙酰化 20-脱氧巨大戟醇，该结果通过与化学合成的 20-脱氧巨大戟醇乙酰化产物比对确认无误。为了鉴定产物乙酰化位置，我们分别利用酶的混杂催化活性，发现该酶还能够乙酰化 20-脱氧巨大戟醇 3 位苯甲酰酯，从而确定该酶的功能为乙酰化 20-脱氧巨大戟醇的 5 位羟基。本论文首次报道了对催化巨大戟醇类二萜的乙酰化酶的克隆鉴定，该项工作为植物代谢工程提高南欧大戟中活性巨大戟烷化合物的含量奠定了基础。; Euphorbia peplus is a medicinal herb known for its bioactive diterpenoids including ingenol mebutate. In 2012, the cream preparation of ingenol mebutate isolated from Euphorbia peplus was approved by the FDA as a first-in-class drug for treatment of actinic keratosis[1]. Our research group has been engaged in the study of the chemistry and activity of E. peplus diterpenoids for a long time[2-7] . Recently, in cooperation with Yang Chonglin's group (Institute of Genetics and Development, Chinese Academy of Sciences), we have discovered that the ingenol compounds 20-deoxyingenol and its analogs such as HEP14 could enhance lysosomal biogenesis. The excellent biological activity of these compounds has attracted research on their biosynthesis. However, so far, only the biosynthetic pathway from GGPP to lathyrane has been reported[8] . The angelic acyl group at positions 3 and 5 in the ingenol mebutate and HEP14 molecules are considered to be the main active groups. There are other types of acyl modifications at these positions, however the enzymes catalyzing these acylation reactions have not been reported. To isolate and identify the gene of the enzyme that catalyzes acylation in the ingenol synthesis pathway, we found more than 100 acyl-CoA-dependent BAHD family genes in the genome of E. peplus according to BAHD conserved domains, based on the previous genome and transcriptome sequencing data of E. peplus.Through phylogenetic analysis of thses 100 BAHD genes, the BAHD genes belonging to the fifth subfamily, contains genes that have been reported to catalyze the acylation of diterpenoids, were selected for further analysis. Finally, 10 candidate genes were obtained by co-expression analysis with the known E. peplus ingenane synthesis pathway genes. The function of selected 10 BAHD enzymes was charactrized by in vitro enzymatic assay. Previously, ingenol and jatrophane compounds from E. peplus was isolated by phytochemical methods as possible acyl acceptors. In this paper, the target gene protein was obtained by using prokaryotic expression system and Nicotiana benthamiana transiently transformation system respectively. Then, acetyl CoA, benzoyl CoA and angeloyl CoA were synthesized as acyl donors by chemical synthesis. Then, in vitroenzyme activity experiments were carried out with different combinations of enzymes and different acyl acceptors and acyl donors, The products were detected by liquid chromatography-mass spectrometry. After several rounds of enzymatic assaies, one of the enzymes was finally found to be able to acetylate 20-deoxyingenol, which was confirmed by comparison with the chemically synthesized 20-deoxyingenol acetylated product. To identify the location of the acetyl group, we exploited the promiscuous catalytic activities of the enzymes, and found that the enzyme could also acetylate the 3- O-acetyl-20-deoxyingenol, thus confirming that the function of the enzyme was to acetylate 20-deoxyingenol at 5-O
语种	中文
	2022-06
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75187
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	李冬梅. 南欧大戟巨大戟醇酯生物合成关键基因的鉴定与功能分析[D]. 中国科学院大学,2022.

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