利用双标记mTn5构建甲基杆菌MB200突变体库的研究

Use of Dual Marker Transposon to Construct Methylobacterium sp. MB200 Mutants

Methylobacterium sp. MB200是一种粉红色的、兼性甲基营养菌,它能以单碳化合物作为唯一碳源和能源,因此,可以作为氨基酸、工业酶等的生产菌株。以M. sp. MB200一碳代谢途径作为主要研究对象,利用mTn5-gusA-pgfp21转座子构建了甲醇利用菌的突变体库,在含有丁二酸的培养基上共获得了约12000株突变体,其中,甲醇营养缺陷的突变体119株,表现GUS活性的突变体1542株。然后,根据转座子已知序列,运用TAIL-PCR的方法克隆到了突变基因的部分序列,为进一步研究一碳代谢基因以及氨基酸和工业酶等的生产打下了基础。

Methylobacterium sp. MB200 is a pink and facuhatively methylotrophic bacteria, which can make a single carbon compounds as a single carbon cources energy and can be used as amino acids, epzymes industrial production strains. We mainly focus on one carbon metabolism of M. sp. MB200, using mTn5 -gusA -pgfp21 transposon mutagenesis to have construeted methylotrophic bacteria mutant pool. From the 12 000 transposon insertion mutants obtained on succinate, 119 methanol defective mutant strains were isolated, 1 542 performanced GUS activity. Then, according to the transposon known sequence, the use of TAIL-PCR method to have been cloned several mutant genes partial sequence.