基于细胞表面展示的乳酸链球菌素生物转化合成途径调控机制解析

Nisin is a bacteriocin approved for utilization as food additive, and playing vitally important roles in food industrial all over the world. The whole-cell biocatalyst process for producing nisin based on Escherichia coli cell surface-displaying system was carried out with an expectation of huge advantage compared with the traditional methods via Lactococcus lactis subsp. lactis. However, this biocatalyst process did not work as well as expected, the nisin yield was low and become much lower when the recollected E. coli cells were used. With a molecular interaction analyzer, this project intends to characterize the influence of cell surface display strategy on the affinity, stability and other enzymatic properties of recombinant enzyme, via the changes in the types as well as lengths of the linker peptides. Different promoters were used to shift the transcription levels of specific genes, and a multi-index analyzing method was used to track the nisin biocatalyst process, so as to explore the interaction mechanism between promoter strength, surface display abundance and nisin synthesis. Furthermore, the overall synergistic correlation mechanism between the enzyme surface presentation strategy, surface presentation abundance and nisin synthesis was further analyzed. This project will provide theoretical guidance for the potential of nisin synthesis by biotransformation, and provide methods and theoretical references for biotransformation and synthesis of other active peptides.

乳酸链球菌素(nisin)作为一种可抑制多种食源性病原菌的天然食品防腐剂，在国内外食品加工行业中起到重要作用。基于大肠杆菌表面展示技术的生物转化法合成nisin相比传统乳酸乳球菌发酵法具有独特的优势和潜力。然而，申请人通过前期工作发现，理论上的潜力并未体现为实际上的优势：nisin合成效价较低；菌体重复使用时nisin合成量下降明显。因此，本项目拟通过融合蛋白连接肽类型和长度变化，借助分子互作技术表征重组酶底物亲和力、稳定性等酶学性质的变化；利用不同强度启动子调整编码基因的转录水平，采用多层次检测体系追踪nisin生物转化合成过程，探究启动子强度、表面展示丰度、nisin合成之间的关联机制；并进一步解析酶的表面展示策略、表面展示丰度和nisin合成之间的全局性协同关联机制。本项目将为生物转化法合成nisin的潜力挖掘提供理论指导，并为生物转化法合成其他活性多肽提供方法和理论上的借鉴和参考。

Nisin是由乳酸乳球菌乳酸亚种(Lactococcus lactis subsp. lactis)产生的具有稳定的广谱抗菌作用小分子多肽，并且热稳定性好、安全无毒，多年来还没有引发大范围的抗性。本研究利用复合诱变技术积累Nisin正向调控突变，进而在表型影响因素研究的基础上通过多组学测序技术开展Nisin高强度合成内在影响机制的研究。.本研究以L. lactis subsp. lactis lxl为原始菌株，通过紫外线-ARTP复合诱变，结合遗传稳定性检测和发酵实验，选育出一株效价较高遗传稳定性较好的Nisin高产突变菌株，命名为L. lactis subsp. lactis A32。该菌株的Nisin效价为5085 IU/mL，是原始菌株效价的3.5倍。.在优化后的发酵条件下，突变株A32的发酵液效价提高4.8%，5339 IU/mL，但是提高幅度不大，说明与原始菌株lxl相比，A32对营养及发酵条件的需求变化不大。.结合表面活性剂在多种化合物发酵中起到的重要作用，结合Nisin合成过程中修饰阶段关键酶的细胞膜定位，因此研究表面活性剂对A32发酵过程的影响。结果表明，不同类型表面活性剂对Nisin发酵的影响规律迥异，对发酵液效价提高幅度最大的是曲拉通X-100，在发酵中期添加，能提高Nisin效价至5798 IU/mL。结合扫描电镜观察添加表面活性剂的发酵液中细胞形态变化，发现细胞形态的变化与Nisin合成能力的变化规律一致性较高，对发酵液效价有促进作用的表面活性剂对细胞形态的影响较小。.最后对A32和lxl进行全基因组测序和转录组分析，进一步探究基因突变和表型变化的关系。结果显示，A32基因组中有107个基因发生突变。在转录水平上，共有92个显著表达差异基因，通过GO富集分析，这些基因涉及ABC-ATPase代谢和转运、氨基酸的生物合成和运输、Nisin免疫等生理过程。同时与Nisin合成直接关联的nisK基因也突变，但在转录组学水平上为表现出显著性差异，而Nisin免疫基因nisEFG的表达量显著上调，这与A32对Nisin抗性的提高结果相一致。另外，从ABC转运子-能量代谢途径、DNA复制转录相关的差异基因以及与半胱氨酸-硫代谢相关途径方找到了强关联差异基因。为后续进一步探究调节Nisin合成途径或进一步提高Nisin产量提供了理论依据和组学参考。