Key Laboratory of Molecular Microbiology and Technology, College of Life Sciences, Ministry of Education, Nankai University, Room 301, Tianjin, China.
Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
Microb Cell Fact. 2019 Nov 14;18(1):198. doi: 10.1186/s12934-019-1249-z.
After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000.
Plasmid pNZ5417 containing a visually selectable marker P-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis.
Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.
在乳球菌 NZ9000 经历了 2.83%的基因组减少后，我们在之前的工作中得到了一个用于蛋白质生产的优秀候选宿主。然而，基因删除过程既耗时又费力。在这里，我们提出了一种适用于乳球菌 NZ9000 大规模基因组减少的便捷基因删除方法。
构建了含有可视选择标记 P-lacZ 的质粒 pNZ5417，这使得基因缺失突变体的筛选更加高效和便捷。利用该质粒，我们逐步删除了乳球菌 9k-3 中占染色体 1.25%的两个大型非必需 DNA 区 L-4A 和 L-5A。与亲本菌株相比，突变株乳球菌 9k-5A 表现出更好的生长特性、转化能力、碳代谢能力和氨基酸生物合成能力。
因此，通过应用可视选择标记，本研究为乳球菌的大规模基因组删除提供了一种便捷高效的系统，这有助于快速进行基因组简化，并生成可作为细胞工厂使用的重组乳球菌菌株。
