一株主产庆大霉素C2a工程菌的构建

Construction of Single Mainly Producing Gentamicin C2a Engineering Bacteria

为更好生产单组分庆大霉素C2a,拟构建一株主产庆大霉素C2a的工程菌.通过序列比对分析,锁定genB2为构建该工程菌的关键基因.以绛红小单孢菌G1008基因组为模板,PCR扩增genB2的上下游序列为同源交换臂,构建同源重组质粒pZB303.通过接合转移,将质粒pZB303导入绛红小单孢菌G1008,安普抗性及PCR扩增筛选得到一株genB2框内缺失工程菌GB102.发酵并提取代谢产物,再经TLC,HPLC,MS分析.结果表明,工程菌GB102主要积累庆大霉素C2a.有望用于生产单组分庆大霉素C2a.

A mainly producing gentamicin C2a engineering bacteria was planned to be built to produce monocomponent gentamicin C2a better.Through sequence comparative analysis,genB2 was presumed as key gene for constructing the engineering bacteria.The homologous recombinant plasmid pZB303 was built by taking micromonospora purpurea G1008 genome as the template,and the downstream sequence on PCR amplification genB2 as homologous exchangeable arm.Through conjugational transfer,the plasmid was guided into micromonospora purpurea G1008,and a strain of genB2 with deficiency of GB102 engineering bacteria in frame was screened out by Apragaz resistance and PCR amplification.Through fermentation and extracting metabolite,and TLC analysis,HPLC analysis,and MS analysis,we can conclude that engineering bacteria GB102 mainly accumulates gentamicin C2a and it is expected to be used for producing monocomponent gentamicin C2a.