【摘要】 以庆大霉素生物合成基因簇为模版,构建了genB4基因阻断质粒pGB403.经接合转移导入绛红色小单孢菌G1008,得到一株genB4基因缺失工程菌GB4408.对菌株GB4408的发酵产物进行了TLC、HPLC和MS分析,结果表明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,阻断了从西索霉素到庆大霉素C1a的转化以及从威大霉素到庆大霉素C2的转化,这可能是genB4基因参与了庆大霉素绛红糖胺C4′和C5′的双脱氢作用.据此推论,在菌株GbK(△genK)基础上敲除了genB4基因,并成功获得一株主产西索霉素的工程菌GbKB4,进一步验证了genB4的功能.更多还原

【Abstract】 A recombinant plasmid pGB403 was constructed with gentamicin biosynthesis gene cluster as the template to study the function of genB4.Then,the plasmid pGB403 was transformed into Micromonospora purpurea G1008 by conjugation.A disrupt genB4 was obtained,i.e.,emqineering strain(GB4408).Its ferment extract was analysised by TLC, HPLC and MS.The GB4408 mainly produced accumulated intermediate G418,sisomicin and verdamycin instead of gentamicin C compounds.The metabolic flux from sisomicin to gentamicin C1 aand from verdamycin to getamicin C2 were blocked.This result indicated that genB4 might be responsible for the dehydrogenation at C4′-C5′of purpurosamine.Based on the speculation,genB4 was distrupted in the strain GbK(△genK),and the strain GbKB4 mainly produced sisomicin was successful constructed,which further verified the function of genB4.更多还原