酪丁酸梭菌代谢流迁移多尺度水平调控应用基础研究

A significant decrease in the production rates of various important metabolites, which is normally caused by the shift of the metabolic flux, has been frequently observed during the middle-late stages of most microbial fermentation processes. It has become a big issue in the application of industrial microbiology. Great research efforts have been made to alternate this natural process of products accumulation. Actually, a variety of strategies have been proposed to identify the fluctuations resulted from the metabolic flux shift and manipulate the dynamic process correspondingly, including an investigation in key parameters during the fermentation processes, a combination of quantitative analysis in the metabolic network and qualitative analysis in transcriptomics, and a correlation of the metabolic flux distribution between physiological regulation and biosynthesis system. It is clear that these strategies will play an important role in achieving high-yield and high-titer production of target metabolites, greatly limiting the accumulation of by-products, as well as facilitating product separation and purification. Based on the recent advances in innovative process developments, systems biology, and omics, including genomics, metabolomics, proteomics, transcriptomics, and enzynomics, this project will focus on the study of the metabolic regulation mechanism of Clostridium tyrobutyricum at multiple levels involving important metabolites, key enzymes, and related gene expressions, and the examination of the distribution and interaction of the intermediate metabolites in Clostridium tyrobutyricum at different conditions. Of special interests are the fermentation kinetics, molecular transfer and mixing, as well as the adaptation and regulation ability of the cells in response to the changes in their culture conditions. This work can help us better understand the multi-level mechanisms of the metabolic flux shift. We expect to reveal the biological nature of the multi-level metabolic regulation and demonstrate the relationship between metabolites accumulation and environmental disturbances, which is of great importance to promote the biorefinery industries based on artificial metabolic regulations and expand the production and application of various value-added microbial chemicals.

在工业微生物研究领域，许多微生物发酵过程存在代谢流迁移问题所致的中后期目的产物生成速率明显下降的现象。如何打破这种正常形成产物的动态平衡成为一大难题。因此，研究通过宏观反映出来的过程参数，将生理调控和生物合成过程中的代谢流分布相关联，无疑对过程调控具有至关重要的作用。本项目以酪丁酸梭菌发酵生产丁酸过程中代谢流迁移行为为研究对象，利用代谢组学技术，针对反应器水平的传递混合特性、微生物细胞自身对外界环境的适应和调节能力方面，开展代谢产物、关键酶及相关基因表达的多尺度酪丁酸梭菌产丁酸代谢调控机理研究，揭示酪丁酸梭菌在不同条件下代谢流迁移行为在多尺度水平上的实质，赋予多尺度代谢调控更清晰的生物学意义，为提出切实有效的过程控制方法和策略提供理论依据和丰富的数据基础。同时，探索强化目的代谢提高底物转化率，降低副产物生成的有效手段，为促进人工调控生物制备过程和源自微生物的重要化工原料生产应用奠定基础。

本课题通过底物水平、丁酸代谢途径中关键酶及基因水平及反应器水平的多尺度考察，探索了酪丁酸梭菌发酵产丁酸的新途径和新方法。.围绕底物利用范围及机制，研究了利用废弃生物质资源废纸和油菜秸秆发酵生产丁酸的可行性，解析了酪丁酸梭菌利用不同碳源为底物和不同环境条件下发酵产丁酸过程中代谢流迁移现象。采用优化实验考察了废纸和油菜秸秆的水解工艺。在重复批次发酵模式下以废纸水解液为碳源发酵产丁酸的最高浓度达到25.3 g/L，得率平均值0.41 g/g。在补料批次发酵模式下，丁酸浓度达到50.42 g/L，得率0.40 g/g，丁酸/乙酸比率43.43。以油菜秸秆为底物，重复发酵批次中最大丁酸产量达16.41 g/L，得率达0.37~0.55 g/g，产率达1.68~3.75 g/L/h。在补料批次发酵模式下，丁酸终产量达50.20 g/L，得率达0.38 g/g，产率达1.08 g/L/h。在丁酸代谢途径中的关键酶和基因水平上，结果表明，在发酵中后期，当乙酸代谢流发生迁移时，PTA、AK和BK的相对活力都有不同程度的降低，PTB的相对活力则持续增加。末期，在以葡萄糖为碳源生长的菌体中乙酸、丁酸形成关键酶均表现出较高的活力，而以水解液为碳源的关键酶活力最低。乙酸形成途径中编码关键酶基因的表达量有所下降，丁酸合成途径中编码关键酶基因的表达量先略有上升，然后又不同程度的下降，是由于基因的转录和表达不同步所造成的。编码乙酸激酶基因（ak）在以不同碳源为底物条件下，其表达量变化均无规律。同时解析了批次发酵模式下游离细胞与固定化细胞之间、不同pH条件下游离细胞之间以及固定化细胞之间的代谢流迁移情况。.在反应器水平上，开发了新型内置碟式纤维床反应器用于丁酸的发酵生产。借助海藻酸钙与纳米四氧化三铁混合物及羧基修饰磁性纳米颗对C. tyrobutyricum细胞进行固定化，通过对固定化细胞产酸能力、细胞相对活性、固定化率三方面的综合考察，与传统固定化方法相比较，磁性纳米颗粒的加入不仅使其具备了外加磁场条件下自主分离的特性，同时还提高了固定化细胞的产酸能力与重复使用次数。该工艺在工业应用中具有较好的适用性。