【作者】 林强；

【导师】 洪文荣；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 庆大霉素拟三糖的生物合成代谢流已有较为详细的推测。理论推测庆大霉素生物合成基因簇上的许多基因参与庆大霉素中间体生物合成的修饰,是开发庆大霉素A,B和X族化合物的重要资源。本课题是在本实验室已建立的绛红色小单孢菌接合转移体系基础上,锁定genA,genF和genB4为目标基因,通过敲除这几个功能性基因,分析其代谢产物的变化,揭示氨基寡糖的生物合成与基因之间的关系。第一,genA基因功能的研究。以温敏型质粒pKC1139为载体,构建genA同源重组质粒pAB103。通过接合转移,将质粒导入绛红小单孢菌G1008,获得单交换菌株GA1。经安普霉素抗性筛选、PCR扩增检测与最终测序证明,得到genA缺失工程菌GA1048。其代谢产物采用MS分析,与出发菌G1008相比,工程菌GA1048不再合成庆大霉素C族组分,主要合成庆大霉素A2。genA基因失活导致庆大霉素生物合成代谢流中断,暗示genA基因参与加洛糖胺C-3"位的氨甲基化。第二,genF基因功能的研究。以质粒pJTU412为载体,构建genF同源重组质粒pFB104,并将其转化大肠杆菌ET12567。采用与研究genA相似的方法和出发菌,获得genF基因缺失工程菌GF208。提取其代谢产物,经HPLC和MS分析,结果显示GF208的庆大霉素生物合成代谢流未发生改变,仍是积累庆大霉素C族组分。表明genF基因不是编码甲基转移酶的基因,且不参与庆大霉素生物合成的修饰作用。第三,genB4基因功能的研究。采用与研究genA相似的方法和出发菌,构建同源重组质粒pGB403,获得genB4基因缺失工程菌GB4408(G1008△genB4)。其代谢产物经TLC、HPLC和MS分析结果表明,工程菌GB4408不再合成庆大霉素C族组分,而是积累中间代谢产物G418、西索霉素和威大霉素。genB4基因的失活,阻断了从西索霉素到庆大霉素Cla的转化以及从威大霉素到庆大霉素C2的转化,推测genB4基因参与庆大霉素绛红糖胺C4’和C5’的双脱氢作用。第四,主产西索霉素工程菌的构建。以大肠杆菌ET12567(pUZ8002/pGB403)为供体菌,与绛红色小单孢菌GbK进行接合转移。采用相同的研究方法,获得genK和genB4基因双缺失工程菌GbKB4。GbKB4发酵产物经TLC、HPLC和MS分析,结果表明工程菌GbKB4主要积累中间体西索霉素和庆大霉素X2。与工程菌GB4408相比,不再合成G418和威大霉素,成功构建了一株主产西索霉素的工程菌。更多还原

【Abstract】 The biosynthesis metabolic flux of gentamicin pseudotrisaccharide had been speculated in detail.Many genes in gentamicin biosynthesis gene cluster involved in the modification of gentamicin intermediate.They were important resource to exploit gentamicin A,B and X compounds.This study was based on the established Micromonospora purpurea conjugative transfer system and chose genA,genF,genB4 as the target genes.It revealed the relation between amino oligosaccharide biosynthesis and genes by knocking out these genes and analysising the change of metabolites.Firstly,research of the function of genA.Using the temperature-sensitive plasmid pKC1139 as vector,the recombinant plasmid pAB103 was constructed and introduced into the micromonospora purpurea G1008 by conjugation.Then the single crossover mutant GA1 was obtained.Apramycin resistance,PCR amplification and sequencing were used to confirm Micromonospora purpurea GA1048.MS was used to analysis the secondary metabolites.Gentamicin A2,instead of gentamicin C complex,was accumulated in Micromonospora purpurea GA1048,comparing with Micromonospora purpurea G1008.The inactivation of genA led to the changes of gentamicin biosynthesis flow,and genA might be responsible for the methylation at C-3" of garosamine.Secondly,research of the function of genF.Using the plasmid pJTU412 as vector,the recombinant plasmid pFB104 was constructed and introduced into the E.coli ET12567 by transformation.Taking the same method and parent strain as the genA research,the.genF in-frame deletion engineering strain GF208 was obtained.HPLC and MS were used to analysis the metabolites,and the results showed that the metabolic flow of gentamicin biosynthesis had no change and still accumulated getamicinC compounds.This indicated that the gene genF was not responsible for the methylation and didn’t participate in the biosynthesis modification of gentamicin.Thirdly,research of the function of genB4.Taking the same method and parent strain as the genA research,the plasmid pGB403 was constructed for the genB4 disruption.The engineering strain GB4408 was obtained.Its metabolites was analysised by TLC,HPLC and MS.The engineering strain GB4408 mainly produced intermediate G418,sisomicin and verdamycin instead of gentamicinC compounds.The metabolic flux from sisomicin to gentamicinCla and from verdamycin to getamicinC2 were blocked.This result indicated that genB4 might be responsible for the dehydrogenation at C-4’-C5’ of purpurosamine.Fourth,The construction of engineering strain mainly producing sisomicin.The E.coli ET12567(pUZ8002/pGB403)was as donor bacteria and then conjugated with the micromonospora purpurea GbK.Taking the same method,both the gene genK and the gene genB4 in-frame deletion engineering strain GbKB4 was obtained.TLC,HPLC and MS were used to analysis the metabolites.The result indicated that GbKB4 mainly accumulated intermediate sisomicin and getamicinX2 instead of G418 and verdamycin,comparing with the engineering strain GB4408.The strain mainly produced sisomicin was successful constructed.更多还原