乳链菌肽细胞分子传感器的构建与应用

【背景】乳链菌肽主要是由乳酸乳球菌生产的一类多肽，对革兰氏阳性菌有抑菌作用，是目前联合国粮食及农业组织/世界卫生组织唯一批准使用的天然食品防腐剂。但是其产量低、缺乏简便高效的检测方法，限制了其研究和应用。【目的】构建一种可输出肉眼可见红色荧光的细胞分子传感器，以期能简单方便地检测样品中的乳链菌肽，同时应用该传感器筛选乳链菌肽生产菌株。【方法】用Golden-Gate克隆方法构建含乳链菌肽诱导启动子和下游红色荧光蛋白基因(两种)的载体，转入Lactococcus lactis中。用细胞传感器筛选可能的乳链菌肽生产菌株。【结果】构建的两种乳链菌肽细胞分子传感器都能对2?200 ng/mL乳链菌肽有灵敏的响应，可用于定量测定。两种传感器的最大荧光强度和表型也有所不同。利用细胞传感器确定了Lactococcus lactis ATCC 11454乳链菌肽的产生，同时排除了一个能产其他抗菌化合物的菌株。【结论】构建的细胞分子传感器能特异性地响应乳链菌肽，并能简单快速地筛选乳链菌肽菌株。

Construction of nisin whole-cell biosensors

Background] Nisin is a small antibacterial peptide mainly produced by Lactococcus lactis. It is the only natural food preservative approved for wide applications. However, its low biosynthesis yield, and lack of simple and efficient detection methods limit its research and applications. Objective] To develop a simple and efficient method to detect nisin, the nisin controlled gene expression (NICE) system and a visible red fluorescent protein were adopted for construction of a whole-cell nisin biosensor. The biosensor was used to quickly screen for nisin-producing candidate strains. Methods] A Golden-Gate cloning method was used to construct a vector which contains a nisin-inducible promoter (Pnis) for regulating a red fluorescent protein gene (rfp). Two rfp genes, identical in protein sequences but different in DNA sequences, were tested. The constructed biosensor plasmids were transferred into Lactococcus lactis, generating the whole-cell nisin biosensors and further used for the screening. Results] Both of the two whole-cell nisin biosensors could specifically and quantitatively respond to nisin varied from 2 to 200 ng/mL. Moreover, the biosensor was applied to screen candidate strains and identified a nisin-producing strain Lactococcus lactis ATCC 11454. Conclusion] The developed whole-cell nisin biosensor can specifically respond to nisin, and can be used to simply and quickly identify nisin-producing strains.