植物乳杆菌苯乳酸生物合成途径的分子调控机理

Phenyllactic acid (PLA) in lactic acid bacteria (LAB), generally recognized as safe, is one of food grade biological preservatives and the research to improve the yield of LAB PLA has became the hot spot of current food preservation area. In previous studies, 14 strains of Lactobacillus plantarum with different PLA yield levels were isolated and 3 of their whole genomes were sequenced by the project group. Further more, the fact that yield of PLA was influenced by the concentration of glucose in media was discovered. In this project, a high PLA yield strain (YM-5-2) and two low PLA yield strains (YM-4-3 and AY01) will be selected as study material. The incubation time of the biggest difference of the PLA yield between YM-5-2 and YM-4-3 or YM-5-2 and AY01 was determined. The culture environment of YM-5-2 was optimized. In these situations, the differences in gene transcription and metabolite profilings between YM-5-2 and YM-4-3, YM-5-2 and AY01 or between before and after optimization of YM-5-2 were investigated. The molecular regulation model of L. plantarum PLA biosynthesis was established, which was validated by fermentation experiment. At last, partial crucial candidate genes were chosen, and their specific functions in the process of PLA production were determined by heterogeneous expression and gene knock-out technology. This project will help us to understand deeply the molecular regulation mechanism of LAB PLA biosynthesis pathway and the role of environmental factors plays on, and provide theoretical basis for screening high yield strains efficiently and improve fermentation strains systematicity.

乳酸菌（LAB）产生的苯乳酸（PLA）是一种公认安全的食品级生物防腐剂，提高LAB PLA产量成为当前食品保藏领域研究热点。前期研究中，项目组分离到14株产不同水平PLA植物乳杆菌，测定了其中3株全基因组序列，并发现PLA产量受培养基中葡萄糖浓度影响。本项目拟以PLA高产菌株（YM-5-2）和低产菌株（YM-4-3和AY01）为材料，确定YM-5-2对YM-4-3和AY01 PLA产量差别最大培养时间点，优化YM-5-2培养环境，在此基础上，考察高产与低产菌株、YM-5-2优化前后的基因转录和代谢物谱差异情况，构建植物乳杆菌PLA生物合成分子调控模型，并利用发酵实验验证。最后选取部分关键候选基因，利用异源表达和基因敲除技术，确定它们在PLA产生过程中的具体功能。本项目有助于深入了解LAB PLA生物合成分子调控机理及环境因子在其中的作用，为高效筛选高产菌株和系统性改造发酵菌株提供理论依据。

本项目以植物乳杆菌（Lactobacillus plantarum）苯乳酸（Phenyllactic acid，PLA）中产菌株YM-4-3和低产菌株S1为研究材料，时间—产量动力学分析发现24 h是YM-4-3对S1的PLA产量差别最大培养时间点。在此基础上，我们优化了YM-4-3的培养条件以及培养基配方，得到样品（YM-4-3y）的PLA产量高达400.74 mg/L，是未优化时的7.61倍。优化试验还发现苯丙氨酸和葡萄糖是影响植物乳杆菌PLA产量的两种关键环境因子。S1全基因组测序以及与PLA中产菌株（YM-4-3、AY01和YM-5-2n）比较基因组分析发现，4个菌株具有2185个共有基因，S1具有完整的PLA核心合成路径和从头合成路径，而中产菌株由于缺少预苯酸脱水酶编码基因，不具完整从头合成路径；至于辅助路径，S1比中产菌株多3个NADH氧化酶基因。另外，3条路径已有基因具有很高同源性（84.36～100%）。为了系统阐述YM-4-3y和YM-4-3高产PLA的分子机制，我们测定并分析了S1、YM-4-3和YM-4-3y的转录组和代谢组。结果发现，相比于S1，YM-4-3y和/或YM-4-3的PLA核心合成路径，糖酵解途径、磷酸戊糖途径、三羧酸循环等中心碳代谢较强，而从头合成途径和氨基酸及其衍生物生物合成途径较弱。三组学联合分析表明，PLA中高产菌株通过增强核心合成途径、中心碳代谢途径和辅助途径，弱化从头合成途径和氨基酸及其衍生物合成途径，为细胞提供足够前体、补偿或平衡细胞能量，使能量和代谢流向核心合成路径倾斜，从而促进PLA在细胞内大量积累。利用基因敲除和异源表达技术，我们分别研究了8个和5个PLA可能相关基因的生物学功能。结果表明，Omt1、Pdh1、CitC、CysE、Pst1、Hpp1、Hpp3、Hpp4和Hpp5等9个基因通过正、负性调控方式参与植物乳杆菌PLA和/或p-OH-PLA的生物合成过程。此外，Omt1基因敲除突变株，Pdh1基因异源表达重组菌发酵上清液对食源性病原菌的抑制效果分别减少和增加，这跟预期结果完全一致，进一步佐证了其生物学功能与有机酸产生相关。总之，我们的研究初步阐明了植物乳杆菌PLA生物合成途径的分子调控机理，为研究芳香族氨基酸衍生物生物合成以及后续系统性改造发酵菌株提供理论依据。