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 PnisZ-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.

中文翻译：

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通过新型高效筛选系统构建的具有新兴特性的重组乳酸乳球菌菌株

在乳酸乳球菌NZ9000中基因组减少2.83％之后，在我们以前的工作中获得了蛋白质生产的良好候选宿主。但是，基因删除过程既费时又费力。在这里，我们提出了一种方便的基因缺失方法，适用于乳酸乳球菌NZ9000的大规模基因组还原。构建了包含视觉选择标记PnisZ-lacZ的质粒pNZ5417，其允许更有效和方便地筛选基因缺失突变体。使用该质粒，在乳酸乳球菌9k-3中逐步删除了两个大的非必需DNA区域L-4A和L-5A，占染色体的1.25％。与亲本菌株相比，突变乳球菌9k-5A显示出更好的生长特性，可转化性，碳代谢能力和氨基酸生物合成。因此，