Tn5转座诱变选育冠菌素耐高温生产菌株及其发酵条件研究

利用双亲接合的方法将含有Tn5转座子的质粒pRL1063a导入丁香假单胞菌大豆致病变种（Pseudomonas syingae pv. glycinea）PG4180中,在含有卡那霉素和链霉素的MG平板上筛选抗性接合子。通过诱变,从874个突变株中筛选得到2株高温下产生冠菌素的突变株MFB132和MFB141。进而对菌株MFB141的发酵条件进行了优化,其最佳发酵条件为：2%葡萄糖为碳源,0.1%氯化铵为氮源,培养基初始pH为6.8,装液量为50 mL / 500mL,接种量为2%,发酵温度由原来的18℃上升到28℃,而发酵时间由原来的168 h缩短到108 h。在最优化条件下,出发菌株28℃下冠菌素产量仅为1.8 mg/L,而突变株MFB141在28℃下的产量达到37.66 mg/L,大约是出发菌株的21倍。

Screening of High Temperature Tolerant Coronatine Producing Strains by Tn5 Transposon Mutation and Studies on its Fermentation Conditions

The plasmid pRL1063a containing Tn5 transposon was transferred into Pseudomonas syingae pv. glycinea PG4180 by conjunction and associators were screened on MG plates with kanamycin and strepmycin. Out of 874 insertion mutants, two mutants which could produce coronatine at high temperature were obtained. They were named as MFB132 and MFB141, respectively. The optimal fermentation conditions for MFB141 were as follows: 2% glucose as carbon source, 0.1% NH4Cl as nitrogen source, the initial pH was 6.8, medium cubage was 50 mL/500mL, and inoculation size was 2%. The fermentation temperature rose from original 18℃ to 28℃ and fermentation time was shortened from 168 hours to 108 hours. The coronatine yield of MFB141 under optimized condition reached to 37.66 mg/L at 28℃, which was 21 times of that of wild type strain (1.8 mg/mL).