利用抗性基因替换的方法将两个拷贝外源基因表达单元整合到枯草芽孢杆菌染色体基因组同一位点

本研究提供了一种新的整合多拷贝外源基因表达单元到枯草芽孢杆菌染色体同一位点的方法。以β-淀粉酶表达单元作为应用实例,本方法包括以下步骤：首先,构建含有一个拷贝β-淀粉酶表达单元的整合质粒pMLK83-CTBA,通过同源双交换获得单拷贝β-淀粉酶表达单元整合到枯草芽孢杆菌1A751染色体α-淀粉酶基因位点的菌株1A751[CTBA]Neo＋;然后,利用抗性基因替换质粒pVK71,将新霉素抗性基因替换为状观霉素抗性基因,得到1A751[CTBA]Neo-Spe＋;最后,整合质粒pMLK83-CTBA再以同源单交换方式整合到1A751[CTBA]Neo-Spe＋染色体,通过新霉素抗性和状观霉素抗性筛选出两个拷贝β-淀粉酶基因整合的重组菌1A751[CTBA2]Neo＋Spe＋。结果显示,利用此方法增加β-淀粉酶基因的拷贝数,能够显著和稳定地提高β-淀粉酶的表达量。

Integration of Two Copies of Exogenous Gene Expression Unit into Bacillus subtilis Genome by Substitution of Antibiotic Resistance Genes

A novel method for integrating multicopy exogenous gene expression unit into the same location of Bacillus subtilis genome was provided in this study. Using β-amylase expression unit as an example, the method includes the following steps： First of all, a integrating plasmid pMLK83-CTBA which contains one copy ofβ-amylase expression unit was constructed and subsequently transformed into Bacillus subtilis strain 1A751 to obtain the recombinant strain 1A751 [CTBA] Neo＋, in which one copy of β-amylase expression unit was integrated by double crossover. Then, using resistance gene replacement plasmid pVK71, 1A751 [CTBA] Neo-Spe＋ was obtained, in which neomycin resistance gene was substituted to spectinomycin resistance gene. Finally, the integrating plasmid pMLK83-CTBA was used again to integrate β-amylase expression unit into 1A751 [CTBA]Neo-Spe＋ by single crossover, and the recombinant strain 1A751 [CTBA2] Neo＋Spe＋ with two copies of β-amylase expression unit integrated in genome was selected by neomycin and spectinomycin resistance screening. The result shows that the β-amylase gene copies via this method can significantly and stably increase secretory expression of β-amylase enzyme activity.