阴沟肠杆菌中乙酸代谢溢流消除机制研究

During aerobic, high glucose consumption and rapid growth stage, the bacteria will partially oxidize glucose into acetate, not fully oxidize it through the TCA cycle. This phenomenon was known as acetate overflow. Acetate overflow can balance the proteins required for bacterial energy synthesis and biomass synthesis to support the rapid growth. However, the accumulation of acetate will promote superoxide generation, reduce intracellular pH, eventually lead to bacterial death. Researchers have tried many methods such as knocking out the acetate synthesis pathway, activating TCA cycle to inhibit the acetate overflow, but did not achieve the desired effect. In our previous study, we found that there is no negative effect caused by acetate overflow during the culture of Enterobacter cloacae SDM. Further studies showed that 2,3-butanediol (BD) synthesis might cooperate with alcohol production to eliminate the acetate overflow in strain SDM. In this project, we would use E. cloacae SDM as the research object for the study of the acetate overflow elimination mechanism. The project would determine the flow direction of acetate metabolic process and the function of alcohol production. Then, the specific mechanisms of BD synthesis pathway involved in the elimination of acetate overflow and the coordinated expression mechanism of key enzymes in BD biosynthesis and alcohol production would be clarified. Finally, the global influence of acetate overflow elimination in E. cloacae would be analyzed. The result of the project would lay the theoretical foundation to improve the application potential of industrial microorganisms by acetate overflow elimination.

好氧、高葡萄糖消耗及快速生长条件下，细菌会将葡萄糖部分氧化生成乙酸，而非通过TCA循环充分氧化，该现象称为乙酸代谢溢流。乙酸代谢溢流可以平衡细菌能量合成及生物量合成所需蛋白量以支持快速生长，但乙酸积累会促进超氧化物产生，降低胞内pH，最终导致细菌死亡。研究者尝试通过敲除乙酸合成途径，激活TCA循环等多种手段抑制乙酸代谢溢流的发生，但均未取得理想效果。申请者前期研究发现阴沟肠杆菌快速生长过程中并不存在乙酸代谢溢流造成的负面影响，该菌株可能通过2,3-丁二醇（BD）合成与乙醇生成的协同作用实现乙酸代谢溢流的消除。本项目拟确定阴沟肠杆菌乙酸代谢溢流消除中乙酸的流向及乙醇生成的功能；揭示BD合成参与乙酸代谢溢流消除的具体作用；阐明BD合成与乙醇生成关键酶协同表达的调控机制；分析乙酸代谢溢流消除对阴沟肠杆菌的全局影响；为通过消除乙酸代谢溢流提高工业微生物的应用潜力提供理论依据。

乙酸代谢溢流是微生物在好氧、高葡萄糖消耗及快速生长情况下呈现出的与厌氧状态相似的表型，将葡萄糖部分氧化生成乙酸而非通过TCA完全氧化来提供ATP。乙酸代谢溢流会抑制工业微生物的生长、降低目的产物产率及增加产物纯化的成本。课题组研究发现，2,3-丁二醇（2,3-BD）生产菌株阴沟肠杆菌SDM中存在乙酸先合成后消除的现象，推测2,3-BD合成可能参与乙酸代谢溢流的消除。本项目研究确认乙酸代谢溢流的消除过程伴随2,3-BD的合成；后续通过基因敲除及过表达、基因转录水平分析、同位素示踪、还原力扰动等一系列手段，确定了阴沟肠杆菌SDM中乙酸代谢溢流消除的机制，阐明了2,3-BD合成途径在乙酸消除过程中的作用与具体生理意义：2,3-BD合成通过提供NADH支持由乙酸向乙醇的还原转化，推动乙酸的消除。此外，本项目研究揭示了参与乙酸代谢溢流消除的2,3-BD及乙醇合成途径在转录及调控上的独立关系，验证了乙酸代谢溢流消除机制在细菌中具有存在的广泛性与功能的普适性，并最终使用代谢工程改造的阴沟肠杆菌SDM，通过调控还原力平衡，实现了以几丁质为底物的乙醇与乙偶姻的联产，证明了乙酸代谢溢流消除机制的实际应用价值。