Zhang Y F, Liu S Y, Du Y H, Feng W J, Liu J H, Qiao J J
Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
J Dairy Sci. 2014 May;97(5):2528-41. doi: 10.3168/jds.2013-7238. Epub 2014 Mar 5.
Nisin has been widely used in the food industry as a safe and natural preservative to increase the shelf time of many foods. In this study, genome shuffling was applied to improve nisin Z production of Lactococcus lactis ssp. lactis YF11 (YF11) via recursive protoplast fusion. Ultraviolet irradiation and diethyl sulfate mutagenesis were used to generate parental strains for genome shuffling. After 4 rounds of genome shuffling, the best-performing strain F44 was obtained, which showed dramatic improvements in tolerance to both glucose (ranging from 8 to 15% (wt/vol) and nisin (ranging from 5,000 to 14,000 IU/mL). Fed-batch fermentation showed that the nisin titer of F44 was up to 4,023 IU/mL, which was 2.4 times that of the starting strain YF11. Field emission scanning electron microscope micrographs of YF11 and F44 revealed the apparent differences in cell morphology. Whereas YF11 displayed long and thin cell morphology, F44 cells were short and thick and with a raised surface in the middle of the cell. With the increasing glucose and nisin content in the medium, cells of both YF11 and F44 tended to become shrunken; however, alterations in YF11 cells were more pronounced than those of F44 cells, especially when cultured in tolerance medium containing both nisin and glucose. Nuclear magnetic resonance analysis demonstrated that the structure of nisin from YF11 and F44 was the same. Expression profiling of nisin synthesis related genes by real-time quantitative PCR showed that the transcription level of nisin structural gene nisZ and immunity gene nisI of F44 was 48 and 130% higher than that of the starting strain YF11, respectively. These results could provide valuable insights into the molecular basis underlying the nisin overproduction mechanism in L. lactis, thus facilitating the future construction of industrial strains for nisin production.
乳酸链球菌素作为一种安全的天然防腐剂，已在食品工业中广泛应用，以延长多种食品的保质期。在本研究中，通过递归原生质体融合，应用基因组改组技术提高乳酸乳球菌乳亚种YF11（YF11）的乳酸链球菌素Z产量。利用紫外线照射和硫酸二乙酯诱变来产生用于基因组改组的亲本菌株。经过四轮基因组改组，获得了性能最佳的菌株F44，其对葡萄糖（范围从8%至15%（wt/vol））和乳酸链球菌素（范围从5000至14000 IU/mL）的耐受性都有显著提高。补料分批发酵表明，F44的乳酸链球菌素效价高达4023 IU/mL，是出发菌株YF11的2.4倍。YF11和F44的场发射扫描电子显微镜图像显示了细胞形态的明显差异。YF11呈现出细长的细胞形态，而F44细胞短而粗，且细胞中部表面凸起。随着培养基中葡萄糖和乳酸链球菌素含量的增加，YF11和F44的细胞都趋于收缩；然而，YF11细胞的变化比F44细胞更明显，尤其是在含有乳酸链球菌素和葡萄糖的耐受培养基中培养时。核磁共振分析表明，YF11和F44的乳酸链球菌素结构相同。通过实时定量PCR对乳酸链球菌素合成相关基因进行表达谱分析表明，F44的乳酸链球菌素结构基因nisZ和免疫基因nisI的转录水平分别比出发菌株YF11高48%和130%。这些结果可为乳酸乳球菌中乳酸链球菌素过量生产机制的分子基础提供有价值的见解，从而有助于未来构建用于生产乳酸链球菌素的工业菌株。
