【作者】 王海娜；

【导师】 赵心清；

【作者基本信息】 大连理工大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 聚酮化合物是细菌、真菌或植物来源的、由低级羧酸通过连续缩合生成的一大类次级代谢产物,多种在临床和农牧业上有着重要用途的抗生素属于聚酮类化合物。FK506是一种具有免疫抑制活性的聚酮类化合物,因其高效低毒的特性被学者认为是临床上最具潜力的免疫抑制剂。由于FK506的原产菌产量比较低,使得通过发酵培养来提取次级代谢物的过程变得复杂而困难。因此利用生长速度较快,可实现规模发酵培养的大肠杆菌或遗传背景清晰、易于分子生物学操作的链霉菌进行FK506的异源合成是一条新的解决途径。FK506生物合成过程中较为特殊的一个延伸单位为甲氧基丙二酰ACP(MM-ACP),其生物合成基因簇包含fkbG,fkbH, fkbl, fkbJ和fkbK五个基因。本课题通过基因工程的方法将合成MM-ACP的五个基因进行单独克隆,构建了含有单个基因的表达载体pETG,pETH,pETI,pETJ和pETK,以及五个基因的串联表达载体pETGK,同时还构建了基于核糖体DNA同源整合的表达载体pZHGK。将构建好的重组载体转化进入大肠杆菌BAP1中进行蛋白的诱导表达,得到了大小正确的目标蛋白产物,并研究了提高蛋白可溶性表达的策略,利用带有链霉菌伴侣蛋白的表达载体pL1SL2获得了可溶目的蛋白。海洋放线菌是进行新颖活性物质生产的重要来源。本实验室前期分离鉴定了一株海洋链霉菌新种星海链霉菌,对其基因组测序结果分析表明,该菌种具有生产聚酮类化合物、氨基糖苷类化合物、非核糖体多肽类化合物等多种次生代谢物的潜力,可作为生产聚酮化合物的异源表达宿主。本课题对该菌株进行了遗传转化体系的建立研究,首先研究了合适的培养基,发现高氏一号培养基为合适的生孢子培养基；其次,对可以使用的遗传转化标记进行了研究,结果表明,安普霉素、硫链丝菌素等抗生素可以作为星海链霉菌的筛选标记。利用pSET152整合载体,成功进行了结合转移操作,获得了较高的结合转移效率。本论文的研究结果为实现MM-ACP在大肠杆菌中的异源表达奠定了基础,也为研究MM-ACP的拷贝数对FK506异源生产的影响提供了前提。同时,星海链霉菌遗传转化体系的建立,为进一步开发其作为异源表达宿主,进行FK506等重要的次生代谢产物的生产提供了良好的理论依据。更多还原

【Abstract】 Polyketides are a large family of natural products produced by bacteria, fungi and plants, and include many clinically and agriculturally important antibiotics. Polyketides are biosynthesized from many low-level fatty acids, such as acyl CoA, malonyl CoA, propionyl CoA and so on. The immunosuppressant FK506is a polyketide compound which is regarded as one of the most potential clinically immunosuppressant due to its high efficiency and low toxicity. However, the natural producer of FK506has slow growth rate and low yield, to achieve large-scale fermentation and production of FK506, choosing host strains with faster growth rate or clear genetic background for heterologous production is a new alternative strategy.The size of the biosynthetic gene cluster of FK506is about90kb, and approximately10different kinds of small molecules participate in the biosynthetic process. Among them, MM-ACP (methoxymalonyl-ACP) is the most special one, and the biosynthetic genes of MM-ACP include fkbG,fkbH, fkbI, fkbJ and fkbK with the respective size667bp,1088bp,1100bp,260bp and875bp, respectively. In this study, the biosynthetic genes of MM-ACP from Streptomyces tsukubaensis were cloned into the expression plasmid pET28a either separately or as a whole expression unit to achieve heterologous expression. Meanwhile an integrative vector based on the ribosome DNA sequence as homologous arms is constructed. The protein expression of the constructed strains was monitored by SDS-PAGE. All the five genes were heterologously expressed in E. coli BAP1with the right size. Further research was carried out to realize soluble expression through coexpression with plasmid pL1SL2which contains Streptomyces chaperonins, and soluable protein of FkbH was successfully obtained.Marine Streptomyces is the main source of novel natural products. Genomic sequencing of a novel species, S. xinghaiensis S187, which is one of the marine actinobacteria previously isolated in our lab, indicated that it has the potential to produce polyketides, and can be developed as alternative host for FK506production. Therefore, genetic transformation system of S187was attempted. S187was found to be sensitive to apramycin, kanamycin, thiostrepton and tetracycline, and was easily transformed with pSET152plasmid using Gaus No.1medium for conjugation.The results in this study indicated that modulation of MM-ACP biosynthetic genes to achieve soluble protein expression is important for the heterologous production of FK506. The establishment of genetic manipulation systems of S. xinghaiensis provided basis for heterologous production of FK506using this marine streptomycete as host strain.更多还原