盐胁迫对植物乳杆菌细菌素合成的调控机制研究

Lactobacillus plantarum is important industrial strain, and used for the manufacture of fermented milk, meat and other products as a starter culture. Many strains from Lactobacillus plantarum can produce bacteriocins, and their potential as biopreservative starter culture in the preservation of fermented food has been recognized. However food matrix containing salt, has effect on the bacteriocin production. We have discovered that low level salt concentration (20g/L) in MRS can stimulate the bacteriocin MG production, and high level salt concentration (more than 20g/L) had negative effect on bacteriocin MG production in Lactobacillus plantarum KLDS1.0391. The mechanism involved is still unclear. Thus, this project aims to study the regulation mechanism of salt stress on bacteriocin biosynthesis in Lactobacillus plantarum KLDS1.0391. First of all, the genes which regulate the bacteriocin biosynthesis in the strain KLDS1.0391 under salt stress will be analyzed by transcriptomic analysis. Second, the binding ability of regulatory proteins to the regulation region of bacteriocin locus will be investigated by electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), and the regulation of salt stress on the biosynthesis of bacteriocin and the regulatory factors involved will be determined. Lastly, the bacteriocin amount will be determined after the construction of major regulatory gene deletion mutant and over-expression recombination, and the regulatory pathway of salt stress in bacteriocin biosynthesis will be clarified. The results will provide scientific basis for the improvement of bacteriocin production by optimum salt concentration in fermented foods, and will be helpful for the extend of the shelf-life of fermented foods.

植物乳杆菌是重要的工业菌株，作为发酵剂广泛用于乳、肉、腌制品等食品的生产加工中。很多植物乳杆菌能产细菌素，这样的菌株作为发酵剂在发酵食品防腐保鲜方面具有潜在应用价值，但食品基质成分如食盐常对菌株细菌素合成造成影响。我们前期研究发现，20g/L浓度的NaCI对植物乳杆菌KLDS1.0391细菌素MG合成具有促进作用，高浓度反而抑制其产生，其详细机制不清楚。本项目拟以植物乳杆菌KLDS1.0391为材料，通过转录组分析，分析盐胁迫调控该菌细菌素产生的相关基因；利用阻滞凝胶电泳（EMSA）和等温滴定量热（ITC）等技术，研究相关调控蛋白与细菌素MG合成调控区域的结合作用，确定调控细菌素合成的调控作用及调控因子；在此基础上，敲除或过表达调控因子编码基因，测定在盐胁迫条件下该菌株产细菌素的水平，阐明盐胁迫对细菌素产生的调控通路，为食品加工中合理使用盐浓度促进细菌素产生进而延长食品货架期提供理论依据。

作为生防菌种使用的植物乳杆菌KLDS1.0391，细菌素合成受食品基质中的食盐影响，目前还不清楚具体的调控机制。本项目以上述菌株为材料，从盐胁迫对植物乳杆菌细菌素合成的调控作用、重要调控蛋白与细菌素合成调控区域的相互作用、重要调控基因对植物乳杆菌细菌素合成的调控作用，以及外源赖氨酸调控盐胁迫时细菌素合成相关通路分析四个方面，阐述了盐胁迫对细菌素合成的调控通路。.结果发现，基因组信息和胁迫抗性实验都表明，该菌对NaCl胁迫具有良好的耐受能力。RNA-seq测序结果锚定了可能具有调节作用的purR以及purL基因，但体外实验表明，PurR与PurL蛋白与细菌素合成启动子无直接的结合作用，推测purR和purL基因可能并不直接参与细菌素合成的调控。而群体感应信号分子AI-2合成酶的编码基因luxS显著影响菌株的胁迫抗性，发现该基因缺失导致菌株胁迫抗性下降，细菌素生物合成量减少。.该菌处于NaCl胁迫时，核酸代谢、碳代谢、氨基酸代谢、膜运输系统和双组份系统等相关通路都很活跃，通过提高能量和物质的生成，进而保证菌体的生长和细菌素的合成。编码DNA聚合酶和RNA聚合酶的基因polA和rpoZ表达上调，新合成的DNA及RNA可作为细菌素合成的模板；编码甘氨酸-tRNA及酪氨酸-tRNA合成过程中的关键酶基因glyQ、glyS及tyrS的表达量显著上调，表明甘氨酸和酪氨酸的需求量增加，可能为细菌素的合成提供物质。实验也证实：能量合成及细菌素分泌途径也参与了该调控过程。.本项目的研究结果阐明了盐胁迫对细菌素产生的调控通路，发现：盐胁迫时，氨基酸代偿，有利于正向调节细菌素的生物合成。为在含盐食品中产细菌素植物乳杆菌的使用提供了重要的理论依据。