Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University, No.94 Weijin Road, Nankai District, Tianjin, 300071, China.
Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland.
Microb Cell Fact. 2020 Dec 9;19(1):225. doi: 10.1186/s12934-020-01487-x.
In bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics.
The Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 ℃, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins.
PRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.
在生物工程中，由于发酵过程中的环境和工业压力，微生物的生长受到限制。本研究旨在构建具有基因组简化、低代谢负担和多应激耐受特性的产乳链菌肽底盘乳球菌菌株。
应用 Cre-loxP 重组系统来减少基因组并获得目标底盘菌株。删除乳球菌 N8 基因组中的一个噬菌体相关片段（PRF；19739bp），并选择突变株乳球菌 N8-1 进行多应激耐受研究。乳球菌 N8-1 的乳链菌肽免疫原性提高到 6500IU/mL，比野生型乳球菌 N8（4500IU/mL）高 44.44%。经溶菌酶处理 2 小时和乳酸处理 1 小时后，乳球菌 N8-1 的存活率分别比野生型菌株高 1000 倍和 10000 倍。在 39℃下，乳球菌 N8-1 仍能保持生长，而野生型菌株的生长则急剧下降。扫描电子显微镜显示，经溶菌酶处理后，乳球菌 N8-1 的细胞壁完整性得到很好的维持。串联质量标签标记定量蛋白质组学显示，乳球菌 N8-1 中有 33 个蛋白显著上调，9 个蛋白显著下调。这些差异蛋白参与碳水化合物和能量的运输/代谢、细胞壁和细胞表面蛋白的生物合成。
证明 PRF 缺失是实现乳球菌多应激耐受和乳链菌肽免疫原性的有效策略，从而为工业获得多应激耐受的工程菌株提供了新的视角，并扩展了乳酸菌在生物技术和合成生物学中的应用。此外，还确定了 PRF 的重要性，它可以赋予细菌重要的表型。
