敲除腺苷化结构域(A-domain)生产新型的Pliapastatin脂肽类似物
毕业论文关键词：非核糖体肽合成酶；脂肽plipastatin；同源重组；枯草芽孢杆菌
Deletion of Aenylation Domain for Producing Novel Pliapastatin Derivatives
Abstract: Non-ribosomal peptides synthetases (NRPSs) are large enzymatic complexes, composed of several functional modules. It could catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering and combinatorial biosynthesis have recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence or newly biological activety. The aenylation (A) domains from NRPSs recognize and activate the substrate amino acids. Thus, in this study, the integrate plasmid pKS-6UD was constructed by using the lipopeptide plipastatin biosynthetic pathway as the research object. the mutant strain Bacillus subtilis pB2-L9 was obtained by knocking out the sixth adenylate (A) domain of plipastatin synthase in Bacillus subtilis pB2-L using homologous recombination. Three novel plipastatin derived lipopeptides were identified from the crude extract of Bacillus subtilis pB2-L9 by high resolution LC-ESI-MS/MS. Additionally, deletion of the sixth adenylate (A) domain in plipastatin synthase leads to premature hydrolysis of the products and module skipping in the process of catalytic product synthesis.
Key words: Non-ribosomal peptides synthetases; plipastatin; homologous recombination; Bacillus subtilis
摘要 1
Abstract 1
Key words 1
引言 1
1 材料与方法 3
1.1 菌株和质粒 3
1.2 培养基及溶液配制 3
1.3 主要生化及分子生物学试剂 3
1.4 主要仪器和设备 3
1.5枯草芽孢杆菌重组整合质粒的构建 4
1.6 B. subtilis 168菌株感受态的制备和转化 5
1.7 突变菌株的诱导重组和筛选 5
1.8 脂肽的生产和鉴定 5
2 结果与分析 6
2.1 重组整合质粒的构建 6
2.2突变菌株的获得 7
2.3新型脂肽类物质的结构鉴定 8
3 讨论 11
致谢 12
参考文献 12
引言
自然界中的微生物能够生产种类复杂具有生物活性的次级代谢产物，这些天然产物在生物体内主要是通过核糖体肽合酶（ribosomal peptide synthetase）和非核糖体肽合酶（non-ribosomal peptide synthetase, NRPS）合成1。而经NRPS合成的肽类具有结构复杂，后修饰途径多样化，生物活性广泛的特点，如抗菌、抗炎症、免疫抑制剂、抗病毒等。众所周知的有，表面活性剂如surfactin、抗真菌剂如fengycin、plipastatin和iturin，还有一些临床药物如cyclosporin A和daptomycin。鉴于这类化合物重要的生物活性，许多已经被应用于食品、农业、工业和医药等领域。人们对该类物质的NRPS进行了一系列生物合成的研究，对扩大微生物生产脂肽的多样性，增加天然活性化合物的数量，改善天然化合物的生物学活性和物理化学特性，提高天然化合物的产量，开发极具临床应用前景的药物前导分子，都具有重大的研究价值和意义。
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