Genome shuffling of Lactococcus lactis subspecies lactis YF11 for improving nisin Z production and comparative analysis.
Genome shuffling of Lactococcus lactis subspecies lactis YF11 for improving nisin Z production and comparative analysis.
复制标题
DOI：
10.3168/jds.2013-7238
复制
发表时间：
2014-05
影响因子：
3.5
通讯作者：
Y. F. Zhang;S. Y. Liu;Y. H. Du;Wenjing Feng;Jingui Liu;Jianjun Qiao
中科院分区：
文献类型：
--
作者：
Y. F. Zhang;S. Y. Liu;Y. H. Du;Wenjing Feng;Jingui Liu;Jianjun Qiao
Nisin作为一种安全、天然的防腐剂在食品工业中得到了广泛的应用，可以延长许多食品的保质期。本研究应用基因组改组技术提高乳酸乳球菌Nisin Z的产量。Lactis YF11(YF11)通过递归原生质体融合。采用紫外光照射和硫酸二乙酯诱变的方法获得基因组改组亲本菌株。经过4轮基因组改组后，获得了表现最好的菌株F44，该菌株对葡萄糖(8%至15%(wt/vol))和乳链菌素(5000至14000IU/mL)的耐受性均有显著提高。分批补料发酵结果表明，菌株F44的乳链菌素效价可达4023IU/mL，是出发菌株YF11的2.4倍。场发射扫描电子显微镜显示YF11和F44在细胞形态上有明显的差异。YF11细胞形态细长，F44细胞短而粗，细胞中部隆起。随着培养液中葡萄糖和Nisin含量的增加，YF11和F44细胞都有缩小的趋势，但YF11细胞的变化比F44细胞更明显，尤其是在同时含有Nisin和葡萄糖的耐受培养液中。核磁共振分析表明，YF11和F44的Nisin具有相同的结构。利用实时荧光定量聚合酶链式反应对乳链菌素合成相关基因的表达谱分析表明，F44菌株Nisin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%。这些结果可以为乳酸乳链菌素高产机制的分子基础提供有价值的见解，从而为未来乳链菌素工业菌株的构建提供帮助。
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.