PheAB基因在棒状杆菌染色体上的整合

Integration of PheAB Gene in the Chromosome of Corynebacteria

改造大肠杆菌质粒ｐＬＣＸ３１，切除其中的ｘｙｌＥ基因得到大肠杆菌质粒ｐＪＬ０１．将源 于大肠杆菌的分枝酸变位酶－预苯酸脱水酶基因（ｐｈｅＡＢ）２．３ ｋｂ Ｂａｍ ＨＩ片段克隆到大肠杆 菌质粒 ｐＪＬ０１中启动子Ｐ３２的下游，构建成质粒ｐＪＬ０２．再在ｐＪＬ０２的ＨｉｎｄⅢ位点接入棒状 杆菌染色体的ＨｉｎｄⅢ消化的随机片段，构建成带不同棒状杆菌染色体片段而不带棒状杆菌 自主复制顺序的棒状杆菌整合质粒ｐＪＬ０３．通过接合转移将这些质粒导入苯丙氨酸生产菌黄 色短杆菌３６２１中，获得一系列整合菌株．发酵试验筛选到几株整合菌株，它们的Ｌ－苯丙氨酸 产量较出发菌株显著提高．稳定性试验表明，各整合菌株在无选择压力的液体培养基中连续 传代１０次，整合质粒稳定性仍在９５％以上．以ｐｈｅＡＢ作探针，与整合菌株染色体ＤＮＡ的 Ｓｏｕｔｈｅｒｎ分子杂交试验证实ｐｈｅＡＢ基因整合在黄色短杆菌３６２１染色体上．

In order to improve the stability of recombinant plasmid in the engineered corynebacteria strains, several integrated strains had been constructed. plasmid pJL01 was constructed from E. colt plasmid pICX 31 which had no autonnomously replicating sequence in the cell of Corynebacteria. A Hind chromosomal DNA fragment of corynebacteria and a 2. 3 kb BamH I DNA fragment carring a E. colt bifunctional enzyme chorismate mutaseprephenate dehydratase gene(pheAB) from E. colt plasmid pPA1 were cloned into pJL01 to construct a integrative plasmid pJL 03. Several integrative plasmids were used to transform B. flavum 3621 by conjugation and a few integrated strains were collected. Fermentation tests showed that the yield of L-phenylalanine in several integrated strains increased significantly when compared to that of parent strain. The cells of integrated strains retained their plasmids more than 95 percent over 240 hours of growth in batch culture under nonselective conditions. The result of Southern hybridization of the chromosomal DNA of integrated strains using pheAB gene as a probe labeling with a-P32-dATP indicated that the pheAB gene has integrated into the chromosomes of integrated strains.