运动发酵单胞菌和大肠杆菌间穿梭质粒的构建

Construction of Shuttle Plasmids between Zymomonas mobilis and Escherichia coli

运动发酵单胞菌(Zymomonas．mobilis)是目前发现的乙醇发酵能力最强的微生物之一,被认为是进行大规模乙醇生产的潜在菌种；但由于其可利用的碳源范围窄,所以对其进行基因工程改造首先需要有较好的载体系统．以Z．mobilis内源质粒pZM2和大肠杆菌(E．coli)质粒pBR328、pACYC184为出发质粒,构建了三个Z．mobilis-E．coli之间的克隆型穿梭质粒,即pZB1、pZB21和pZB31．对它们在Z．mobilis CP4中的稳定性、拷贝数的初步比较分析表明：pZB21最稳定、拷贝数最高,pZB31次之,pZB1最差．进一步以pZB21为载体,在Z．mo- bilis CP4中表达E．coli的talB基因,酶活水平为0．087 U/mg蛋白．证明pZB21是能用于基因表达的、较为理想的Z．moblis-E．coli之间的穿梭载体．

Zymomonas mobilis, one of the strains which have the highest ethanol yield and specific ethanol productivity, has been evaluated as a possible candidate for industrial ethanol production. However, its substrate range is limited. It is necessary to construct efficent shuttle vectors for its genetic manipulation. In this paper three new shuttle vectors between Z. mobilis -E. coli were constructed from the 2.7 kb cryptic plasmid pZM2 of Z. mobilis and pBR328 and pACYC184 of E. coli, that is, pZB1, pZB21 and pZB31. Their stability and copy number were examed in Z. mobilis CP4 and the results showed, pZB21＇s stability is best and it＇s copy number is highest, pZB1＇s stability is worst and it＇s copy number is lowerest. Moreover, E. coli K- 12 talB in Z. rnobilis CP4 could be expressed by using pZB21 as the vector. The enzyme activity of talB is 0. 087 U/mg total cell protein. These research results demonstrated that pZB21 is a good Z. rnobilis-E, coli shuttle vector.