药学毕业产红霉素c的糖多孢红霉菌a226g突变体的构建.docVIP

XX大学 毕业论文 产红霉素C的糖多抱红霉菌A226G.突变体的构建 姓 名： 2014年6月25日 产红霉素C的糖多范红霉菌A226G-突变体的构建 作者：袁华黄训端张部昌刘道琴赵皓孔小卫张书 祥 【摘要】 糖多抱红霉菌eryG基因编码产物为红霉素3- 0 甲基转移酶 (EryG),失活eryG基因能阻断红霉素A的生物合成，积累其中间产物红霉素Co 以糖多砲红霉菌A226基因组DNA为模版，用重叠PCR方法扩增岀eryG基因 两侧约1600bp DNA片段(AG),其中去除了 EryG上S腺苜甲硫氨酸结合区域 对应的290bp DNA片段。将该片段克隆于质粒pWHM3,构建了同源重组质粒 pWHMAGo PEG介导原生质体转化法将pWHMAG转入糖多泡红霉菌A226屮, 通过染色休同源重组突变染色体上eryG基因。利用薄层层析、PCR和质谱方法 筛选出一株eryG基因突变的糖多抱红霉菌A226G■突变体，此突变体主耍积累屮 间产物红霉素C而不再合成红霉素A。通过eryG基因的功能补偿，糖多抱红霉 菌基因工程菌A226G?G+口J以恢复红霉索A的合成能力。 【关键词】 糖多他红霉菌；红霉素；3- 0甲基转移酶；同源重组；功 能补偿 ABSTRACT Saccharopolyspora erythraea erythromycin 3 0 methyltransferase (EryG), encoded by the eryG gene, could play an important role in erythromycin biosynthesis, and the inactivation of eryG gene would lead to erythromycin C accumulation in the eryG mutant fermentation culture. Taking Sac. erythraea genomic DNA as a template, about 1,600bp DNA fragment with deletion of a 290bp fragment which contained S adenosylmethionine binding domain in eryG gene was amplified by overlap PCR and cloned into the vector pWHM3 to construct homologous recombination plasmid pWHMAG. It was then introduced into Sac. erythraea by PEG mediation, and two integrants (I and II) were selected. After integrant I had grown for three generations on R3M media without thiostrepton (Thio), they were protoplasted and plated on R3M media without Thio. One mutant, A226G-, was screened and identified by TLC, PCR and MS analyzes. The A226G- mutant accumulated erythromycin C instead of erythromycin A in the culture. By introducing cryG gene expression vector into A226G? mutant, erythromycin A production was restored, but the yield was somewhat lower than that of its parent strain A226. KEY WORDS Saccharopolyspora erythraea; Erythromycin; 3 O mcthyltransfcrasc; Homologous recombination; Function compensation 红霉素(erythromycin , Er)是由革兰阳性细菌糖多抱红霉菌 (Saccharopolyspora erythraea)合成的次生代谢产物，为一类人环内酯类抗生索， 包括红霉素A(ErA)、红霉素B(ErB)、红霉素C(ErC)和红霉素D(ErD)等。这四种 红霉素均有抑菌活性，但在临床上抑菌活性最高，用途最广泛的是ErA [1, 2]。 红霉素的生