基于双底物平行标记实验的13C代谢通量分析的新方法

13C metabolic flux analysis, labeling the substrate (carbon source) with stable isotopic tracer 13C, is a powerful tool on quantification of the microbial cell system. However, applying parallel labeling experiment to a medium containing two substrates has little been reported. The purpose of this research project is to construct a new method to quantify the cell metabolism and substrates utilization based on parallel labeling experiment, providing the foundation of releasing carbon catabolite repression and higher efficacy metabolism. This experiment has constructed a recombinant Escherichia coli that can simultaneous consume glycerol and glucose effectively. Based on this strain’s central metabolic network, this research project has explored a parallel labeling experiment, i.e. two labeling substrates ([1-13C] glucose and [1,3-13C] glycerol). Then metabolic profiling level and gene transcript level of central metabolic pathway and target product synthesis pathway will be measured to compare the parent strain and the target strain. According to the 13C abundance of intracellular metabolites, metabolic flux analysis will be done. The important gene transcript level, metabolic profiling level and fluxomics level will be integrated to construct a mathematic model for metabolic re-engineering. This research can do much help for expanding the application scope of 13C flux analysis from single substrate to multiple substrates. Meanwhile, this method can provide the quantification analysis to clarify the mechanism of simultaneous consumption of two substrates. This research has the important theoretical significance with practical application value.

13C代谢通量分析利用稳定同位素13C对碳源底物进行标记，是定量分析细胞系统强有力的工具。然而适用于双底物的平行标记实验鲜有报道。本项目旨在建立基于双底物平行标记实验的13C代谢通量分析的新方法，可定量分析双底物下细胞代谢与底物利用信息，为解除分解代谢阻遏、实现细胞的高效代谢打下基础。实验以一株高效共利用甘油和葡萄糖的大肠杆菌为研究对象，依据其代谢网络，合理设计[1-13C]葡萄糖和[1,3-13C]甘油的底物标记模式，建立平行标记实验方法。然后对照出发菌株和目标菌株，测定参与中心代谢以及目标产物合成途径的代谢谱水平和转录水平。依据各中间代谢物13C的丰度，计算代谢通量。关联重要的转录水平、代谢谱水平与代谢通量水平等数据，建立数学模型以指导代谢过程改造。本研究方法可将13C代谢通量分析扩展到双底物培养体系，并为双底物高效共利用机制阐明提供定量分析基础，具有重要的理论研究意义与应用价值。

13C代谢通量分析是利用13C稳定同位素对底物进行标记，通过标记实验分析胞内中间代谢物质量同位体的分布情况，从而准确定量胞内反应速率，在系统理解细胞代谢特性和指导代谢工程改造等方面起着重要作用。然而，单一底物标记的代谢通量分析不适用于培养基中存在两种底物混合培养的情况。随着组学技术与系统生物学的发展，建立基于双底物平行标记实验的13C代谢通量分析的新方法成为可能。本项目以一株高效共利用甘油和葡萄糖的大肠杆菌（ΔptsGglpK*）为研究对象，设计了[1-13C]葡萄糖和[1,3-13C]甘油的底物标记模式，确立了双底物代谢通量分析的计算方法；完成了目标菌株和出发菌株在基因转录水平和中间代谢物水平的差异分析。实验表明，ΔptsGglpK*以甘油和葡萄糖同时作为碳源时，碳源和能量代谢再分布：快速的甘油摄取影响了NADPH/NADH，降低ATP，激活ArcA调节系统，抑制TCA循环，引起乙酸溢流；葡萄糖从糖酵解途径流向磷酸戊糖途径，说明以甘油为主要碳源利用时需添加其他碳源以增加NADPH的供应。基于上述结果，确定了以ΔptsGglpK*为出发菌株合成丙酮醇的关键节点，并通过相应的基因工程改造，丙酮醇产量达到1.82 g/L。本项目的研究思路和技术方法为13C代谢通量分析扩展到双底物培养体系，及双底物高效共利用机制的阐明奠定了基础，并为以甘油为主要碳源生物转化为高价值化学品提供借鉴。