葡萄糖诱导的酪丁酸梭菌甘露醇代谢阻遏机制解析及解除研究

Industrial-scale production of butyric acid by Clostridium tyrobutyricum is limited by its low yield and selectivity. In our previous work, glucose and mannitol co-fermentation achieved high selectivity of butyric acid production, but carbon catabolite repression (CCR) was also observed, resulting in a decreased productivity of butyric acid . Now, the mechanism underlying this phenomenon is not clear. Bioinformatics analysis indicated that MtlR- and/or CcpA-dependent CCR may exist. To validate this hypothesis, RNA sequencing will be used for monitoring the gene expression level of mannitol metabolism-related genes (mainly focuses on mtlARFD, lacI, hprK/P and glucose phosphotransferase system genes) under various substrates and growth phases, and locating the key genes and transcriptional regulation factors which are responsible for the regulation of mannitol metabolism in C. tyrobutyricum. Based on this, the metabolic pathway and carbon catabolite repression of mannitol utilization in C. tyrobutyricum will be investigated. Furthermore, metabolic engineering strategies for relieving the CCR will be conducted with the aim to achieve efficient co-utilization of glucose and mannitol. These findings from our research is not only beneficial to achieve enhanced butyric acid production by C. tyrobutyricum, but also provide theoretical foundation for overcoming the carbon catabolite repression of mannitol utilization induced by glucose.

酪丁酸梭菌存在丁酸转化率和选择性低的问题，严重制约利用其发酵生产丁酸的产业化进程。前期研究发现，葡萄糖甘露醇共发酵可以实现高选择性丁酸生产（无副产物乙酸），但是存在严重的碳代谢阻遏效应（CCR），导致丁酸生产强度下降。目前，关于这一现象尚缺乏系统研究。根据生物信息学分析，我们推测转录调节蛋白mtlR和代谢物控制蛋白CcpA可能是参与该过程的关键调控因子。为证明此假说，本项目将以葡萄糖和葡萄糖/甘露醇混合物为底物，对不同培养阶段的样品进行转录组测序，获得在不同条件下甘露醇代谢及其调控相关基因（重点关注mtlARFD、lacI、hprK/P、葡萄糖PTS系统基因）表达水平的基础数据，定位关键的基因和转录调控因子，解析甘露醇代谢途径及其碳代谢阻遏机制，并通过代谢工程手段获得高效共利用葡萄糖和甘露醇的工程菌，从而全面提高丁酸发酵水平，为解除葡萄糖诱导的甘露醇代谢阻遏效应提供理论依据。

酪丁酸梭菌是丁酸和丁醇发酵的优势菌株。我们前期研究发现，葡萄糖甘露醇共发酵可以实现高选择性丁酸生产，但是存在严重的碳代谢阻遏效应，导致丁酸生产强度降低。根据转录组和生物信息学分析，一方面通过构建相关基因缺失和回补菌株，证实mtlARFD为酪丁酸梭菌中甘露醇转运和代谢的关键基因，其中mtlR为甘露醇转录激活因子；另一方面，对转录调节蛋白MtlR和代谢物控制蛋白CcpA的关键位点进行定点突变，证实CcpA在葡萄糖诱导的甘露醇代谢阻遏中起到了关键作用，为后续酪丁酸梭菌的碳代谢调控研究提供了很好的借鉴。此外，以酪丁酸梭菌作为底盘，通过代谢工程和过程工程手段对海带水解液（主要成分为甘露醇和葡萄糖）发酵产丁酸和丁醇的工艺进行了优化。在底物高效利用的前提下，实现了较高的丁酸选择性（100%）和丁醇浓度（12.15 g/L）。在本项目资助下，申报人还探究了电子载体诱导的高选择性丁酸发酵机制，并构建了可以直接利用未处理甘蔗糖蜜发酵产丁酸的工程菌。本项目发表SCI论文4篇，培养研究生4名。