【作者】 曹孟婵；

【导师】 张部昌；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 红霉素（erythromycin）是一类广谱大环内酯类抗生素，主要由糖多孢红霉菌（Saccharopolyspora erythraea）合成。同其它抗生素一样，红霉素在临床上也出现了许多耐药病原菌，开发针对耐药性细菌的新药已非常迫切。现在推向市场的第三代红霉素—酮内酯类（ketolides）抗生素是使用化学方法对红霉素结构进行修饰完成。其结构上最大的特点将内酯环C-3位的L-克拉定糖（L-cladinose）替换成了羰基。因为羰基能避免诱导大环内酯-林可酰胺-链阳霉素B（macrolide-lincosamide-streptograminB，MLS_B）抗性，所以能大大提高对某些红霉素抗性菌的活性。随着分子生物学的发展和对抗生素生物合成过程分子机制的认识深入，组合生物合成方法渐渐成为探索合成新抗生素的方法之一。 红霉素生物合成分为两步：母核的合成及后修饰。母核的合成是以聚酮合成酶（polyketide sythase，PKS）来完成的。PKS是个复合酶系，由六套结构和功能相近的模块组成。红霉素母核中C-3位羟基是由模块6中KR6酶域决定的。用基因工程方法对KR6酶域的DNA序列进行敲除或替换可能会全部或部分地使KR6失活，使C-3位羟基变为酮基。位于KR6酶域催化活性中心的Tyr₂₆₉₉是个关键性氨基酸残基，突变Tyr₂₆₉₉可以使KR6完全使活。本文通过同源重组方法突变KR6酶域Tyr₂₆₉₉的密码子，构建可以合成DOEB而减少红霉素合成的糖多孢红霉菌A226突变体。 为了将KR6中催化三联体中Tyr₂₆₉₉（Y）密码子TAC换成Ala（A）密码子GCC，并保证有效的染色体整合和二次重组，突变位点两侧同源片段长度保持约500bp。以糖多孢红霉菌基因组DNA为模板，用PCR技术扩增出突变位点两侧DNA片段。然后再用重叠PCR技术将扩增出其合成基因KR6酶域及其附近酶域中的Tyr₂₆₉₉密码子TAC突变为Ala密码子GCC的大约1000bp DNA同源序列，克隆到载体pWHM3上，构建了同源重组质粒pWHM3-YA。将pWHM3-YA转化到糖多孢红霉菌A226原生质体中，然后筛选出pWHM3-YA整合到红霉素合成基因上的整合体A226-pWHM3-YA-A和A226-pWHM3-YA-B。将筛选得到的整合体A226-pWHM3-YA-A在无硫链丝菌肽（Thiostrepton，Thio）的R3M斜面更多还原

【Abstract】 Erythromycin was a kind of broad-spectrum macrolide antibiotics produced by Saccharopolyspora erythraea. Like other antibiotics, bacterial resistances to erythromycin also came out in clinic. It was urgent for us to develop new antibiotics, which could overcome some problems of bacterial resistance. The third generation erythromycin that had been gotten onto the market was one of ketolides that was synthesized by chemical modification of erythromycin. L-cladinose at position C3 of macrolactone ring had been replaced by carbonyl by chemical modification, this carbonyl enabled ketolides to bind to their targets without triggering the inducible resistance to macrolide-lincosamide-streptogramin B（MLSB）drugs, and so improve antimicrobial activity to a great extent. With the development of molecular biology and deep understanding of molecular mechanism of erythromycin biosynthesis process, combination biosynthesis became one way of exploring to synthesize antibiotics.Biosynthesis process of erythromycin had two steps: macrolactone ring synthesis and post modification. The macrolactone ring was synthesized by polyketide sythase（PKS）. PKS were multiplex enzymatic system and composed of six modules whose function and structure was close. Whether C3 hydroxyl of macrolactone ring would be reduced depended on the ketoreductase （KR6） domain of the sixth module of polyketide synthase, so we could make KR6 domain completely or partly inactivated and turn C3 hydroxyl into carbonyl by genetic engineering for the ketolides biosynthesis. Tyr₂₆₉₉ was a key amino acid in the catalytic site of KR6 domain. Mutant Tyr₂₆₉₉ could completely inactivate KR6. Saccharopolyspora erythraea A226-YA had been constructed with homologous recombination, and it could synthesize the compound 3-deoxy-3-oxo-erythronolide B without detecting erythromycin.In order to mutate the codon TAC of Tyr₂₆₉₉ to the codon GCC of Ala of catalytic triplet in KR6 and ensure effective chromosome integration and the second更多还原