Leenhouts K, Bolhuis A, Venema G, Kok J
Department of Genetics, University of Groningen, Haren, The Netherlands.
Appl Microbiol Biotechnol. 1998 Apr;49(4):417-23. doi: 10.1007/s002530051192.
A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.
开发了一种食品级载体系统，该系统可使多个质粒拷贝稳定整合到乳酸乳球菌的染色体中。该载体由乳球菌质粒pWV01的正向复制起点（Ori+）、作为选择标记的戊糖片球菌PPE1.0的蔗糖基因、多克隆位点以及一个特征明确的染色体区域的乳球菌DNA片段组成。该系统包括两株乳酸乳球菌，LL108和LL302，它们产生Ori+载体复制所必需的pWV01 RepA蛋白。这些辅助菌株允许从同源背景构建和分离整合质粒的复制形式。在含有蔗糖作为唯一可发酵糖的培养基上选择转化体后，质粒在乳酸乳球菌MG1363中的单交换整合导致扩增至约20拷贝/染色体的水平。在选择性生长条件下，扩增是稳定的。在含葡萄糖的培养基中，检测到整合质粒拷贝有有限的丢失，丢失率为每代（7.5 - 15）×10⁻²拷贝。分离出一株菌株MG124，在经过120代非选择性生长后，该菌株保留了11个整合拷贝。这些结果表明，这里描述的单交换整合系统代表了一种简单的方法，用于构建携带多个感兴趣基因拷贝的稳定食品级菌株。
