可回用碱性中和剂-氧化镁多尺度强化L-乳酸生物合成机制的研究

This project plans to reveal the promotion mechanism of magnesium oxide in L-lactic acid biosynthesis using a multi-level study including the intracellular exploration, the extracellular characterization and the systematic intensification. Firstly, to reveal the influence of excess magnesium ions in gene transcription for L-lactic acid biosynthesis, a transcriptome analysis of Lactobacillus rhamnosus LA-04-1 under the stress of high magnesium ion concentration will be conducted, the transcription information will be further associated with the efficiency of L-lactic acid biosynthesis, and the target sites of excess magnesium ions will be identified. Secondly, to interprete the promotion effect of magnesium ion in bacteria metabolism, the flux analysis of the metabolic network model of L. rhamnosus LA-04-1 will be conducted based on the metabolomics, the relationship of substance and energy metabolism to L-lactic acid biosynthesis will be clarified, and the metabolic nodes significantly affected by excess magnesium ions will be located. In addition, to confirm the improvement effect, which provided by the fermentation microenvironment of recycled magnesium oxide, on L-lactic acid biosynthesis, the mechanisms (including interactions) of steric hindrance effect, distribution effect and diffusion effect among bacteria cells, substrates, product and the microenvironment will be studied, and the special composition of the recycled magnesium oxide will be investigated. Finally, in order to comprehensively reveal and systematically evaluate the enhancement mechanism of L-lactic acid biosynthesis, the building of kinetic and thermodynamic models will be conducted based on the intensification of the coupling system, and the interaction mechanisms of modules and the relationship between each module and L-lactic acid production efficiency will also be studied. Results obtained from this project will provide theoretical and technical basis for the efficient biosynthesis of L-lactic acid.

本课题拟从胞内、胞外和全局系统全面揭示氧化镁对L-乳酸生物合成的强化机理。首先，通过高浓度镁离子压力下Lactobacillus rhamnosus LA-04-1转录组学分析，探索转录与产酸效率的关联规律，确定镁离子影响的靶向位点，在转录水平揭示产物合成的强化机理；其次，基于代谢组学对高浓度镁离子胁迫下的菌体进行代谢通量分析，确定菌体代谢与L-乳酸合成的关联规律，定位关键代谢节点，阐释镁离子对产物代谢合成的强化规律；此外，研究回用氧化镁特殊微环境对L-乳酸合成的影响，揭示菌体、底物、产物与微环境之间的分配、扩散和空间位阻效应及其互作规律，并进行特异元素分析，确证回用氧化镁对L-乳酸合成的促进机理；最后，在系统集成基础上进行模型构建，研究模块互作机制及各模块与L-乳酸合成效率的关联规律，对L-乳酸合成强化机制进行全局认识和系统性评价。研究成果将为高效生物合成L-乳酸奠定理论和技术基础。

本项目在胞内转录、代谢，胞外微环境及工艺系统水平上多尺度深入揭示了可回用氧化镁对鼠李糖乳杆菌发酵合成L-乳酸的影响机制。在胞内水平进行了高浓度镁离子压力下乳酸菌转录组学及代谢分析；在胞外水平研究了可回用氧化镁提供的特殊发酵微环境对L-乳酸合成的影响；在系统水平开展了L-乳酸发酵分离耦合的工艺集成。基于转录组学分析，认知了相关代谢途径中的关键基因及其表达规律；利用TMT标记定量蛋白组学分析，鉴定了差异蛋白，进一步确定了差异蛋白所参与的主要生物过程；采用代谢轮廓变化分析，筛选了显著性差异代谢物，阐释了差异代谢物所富集的KEGG代谢通路；通过结构检测，揭示了回用氧化镁在发酵微环境上对乳酸菌产酸的促进效应；根据模型验证及发酵表征，确证了氧化镁回用的可行性；运用聚乙烯醇/四氧化三铁复合体系开展了乳酸菌固定化发酵，确证了发酵分离耦合系统中菌体回用和产物在线移除对产酸的强化作用。本项目在局部探索与全局认识相结合的模式下开展，为研究高浓度镁离子压力下基因表达及代谢规律的变化提供了数据支持，为菌体固定化修饰和系统强化规律的评价提供了依据。本项目的实施为开辟高效生物合成L-乳酸新途径奠定了理论和技术基础。