海洋链霉菌Streptomyces lohii的巴弗洛霉素生物合成后修饰途径解析和高产巴弗洛霉素A1工程菌构建

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	海洋链霉菌Streptomyces lohii的巴弗洛霉素生物合成后修饰途径解析和高产巴弗洛霉素A1工程菌构建
其他题名	生物化学与分子生物学
	李众
导师	链霉菌，巴弗洛霉素，生物合成，延胡索酸腺苷酰转移酶，调控基因
	2017-05
学位授予单位	中国科学院大学
学位授予地点	青岛
学位专业	巴弗洛霉素（bafilomycins）是一类核心骨架经I型聚酮合成酶装配合成的属于B类Plecomacrolide家族的十六元大环内酯。迄今为止，已有近30种巴弗洛霉素被发现，该类化合物的生物活性多样，一直受到药物学家的关注，但是对哺乳动物的高毒性阻碍了它们在临床上的应用。巴弗洛霉素A1作为所有巴弗洛霉素的母核结构具有很强的囊泡型H+-ATPase抑制活性，有望用于骨质疏松的治疗。分离自巴布亚新几内亚皇冠岛的海洋链霉菌Streptomyces lohii ATCC BAA-1276在常规培养条件下可以产生巴弗洛霉素A1，C1，B1。在前期的研究中，我们已经完成了S. lohii的巴弗洛霉素生物合成基因簇的鉴定，但是其后修饰途径一直未得到阐明。本研究通过体内基因敲除和体外酶反应首次完成对S. lohii的巴弗洛霉素的后修饰途径（巴弗洛霉素A1→巴弗洛霉素C1→巴弗洛霉素B1）解析：新颖的延胡索酸腺苷酰转移酶Orf3负责延胡索酸的激活，催化形成延胡索酰磷酸腺苷（延胡索酰AMP）；延胡索酸转移酶Orf2将延胡索酰AMP转移到巴弗洛霉素A1的21位羟基形成巴弗洛霉素C1；酰胺合成酶BafY催化巴弗洛霉素C1的羧基与由酰基辅酶A连接酶BafX和5-氨基乙酰丙酸（5-ALA）合酶BafZ催化形成的五碳结构单元C5N的氨基形成酰胺键得到巴弗洛霉素B1。由于巴弗洛霉素的结构复杂，通过基因工程的手段构建巴弗洛霉素A1的高产菌株，具有化学全合成难以比拟的低成本，绿色环保等诸多优势。本研究通过基因敲除，回补和过表达实验初步确定了S. lohii巴弗洛霉素生物合成基因簇中AfsR家族调控基因（bafG）和LuxR家族调控基因（orf1）在巴弗洛霉素生物合成中起正调控作用。我们通过在只产巴弗洛霉素A1的突变株（S. lohii ∆orf2&orf3）中过表达Orf1，最终得到巴弗洛霉素A1的产量达535.1 ± 25.0 mg/L的高产菌株。延胡索酸作为三羧酸循环中的重要中间产物，存在诸多天然产物的骨架中，但其引入机制一直是未解之谜。本研究首次发现并鉴定了延胡索酸腺苷酰转移酶Orf3和延胡索酸转移酶Orf2，为延胡索酸的引入机制提供了范例。整个巴弗洛霉素生物合成后修饰途径的阐明，体现了微生物初级代谢产物与次级代谢产物之间的“沟通”，为高效低毒的巴弗洛霉素或新型药物分子的理性设计提供了新思路。
关键词	理学
摘要	中文
其他摘要	Bafilomycins, belongs to class B Plecomacrolides, are a group of 16-membered macrolide biosynthetized by type I polyketide synthase. From now on, nearly thirty bafilomycin derivatives have been isolated and identified. Despite bafilomycins have attracted significant attention from pharmaceutists due to their diverse bioactivities, the toxicity prevents them from clinical application. As the core structure of all bafilomycins, bafilomycin A1 is a potent and specific vacuolar H+-ATPase (V-ATPase) which might be applied in the therapy of osteoporosis in the future. Streptomyces lohii ATCC BAA-1276, isolated from the Crown Island, Papua New Guinea can produce bafilomycin A1, C1, B1. Although the bafilomycin biosynthetic gene cluster have been elucidated in our previous study, but the post- polyketide synthase (PKS) tailoring steps for structural diversification and bioactivity improvement remain largely unknown. In this study, the tailoring steps of bafilomycin biosynthetic pathway has been elucidated for the first time through in vivo gene inactivation and in vitro reconstitution of enzyme activities: Orf3 is characterized as a novel fumarate adenylyltransferase activing the fumarate by forming the fumaryl-AMP; Orf2 with high substrate specificity is responsible for transferring the fumarate moiety from fumaryl-AMP to bafilomycin A1, giving rise to bafilomycin C1; acyl-CoA ligase BafX and 5-aminolevulinic acid (5-ALA) synthase BafZ cooperatively synthesize the C5N unit, and ATP-dependent amino synthetase BafY catalyzes the amide bond formation between bafilomycin C1 and C5N. Due to the complex structure of bafilomycins, to generate the bafilomycin A1 high-yielding strains through genetic engineering is more eco-friendly and low-cost compared with total synthesis. In this study, BafG and Orf1 have been identified as AfsR family and LuxR family activator involved in bafilomycins biosynthesis through in vivo gene inactivation, complementation and overexpression, respectively. Finally, the Orf1 was overexpressed in the Streptomyces lohii Δorf2&orf3 to give a maximum bafilomycin A1 production titer of 535.1 ± 25.0 mg/L to meet the industry application. Fumarate, as an important intermediate in the tricarboxylic acid cycle, exist in several structures of natural products. But the incorporation mechanism of fumarate into the natural product have not been elucidated so far. The biotransformation of bafilomycin A1 to bafilomycin C1 by Orf2 and Orf3 will give a paradigm. The bafilomycin biosynthetic pathway show us an interesting crosstalk between primary metabolites and secondary metabolites, providing a new horizon for the rational design of efficient and low-toxicity bafilomycin derivatives or new drugs.
作者部门	酶工程团队
公开日期	2020-12-01
学位类型	硕士 ; 学位论文
语种	中文
文献类型	学位论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/9976
专题	酶工程研究组
作者单位	中国科学院大学
推荐引用方式 GB/T 7714	李众. 海洋链霉菌Streptomyces lohii的巴弗洛霉素生物合成后修饰途径解析和高产巴弗洛霉素A1工程菌构建[D]. 青岛. 中国科学院大学,2017.