基于细菌群感效应人工构建分子开关

Construct a molecular switch based on bacterial quorum sensing

摘要:

对生物体内已有的或者人工组装的生物合成途径进行优化操作涉及两个重要问题：代谢途径中关键酶的活性及蛋白表达水平。对于酶表达水平的研究，传统的做法是采用强启动子控制下的靶蛋白过量表达策略。靶蛋白的过量表达通常会导致细胞内积累大量的无活性包涵体，从而严重影响细胞的生理状态和相关生物途径的有效运转。针对这一问题，设计一种分子开关来精确调控生物合成过程中关键酶的表达水平，对于研究生物合成途径的代谢节律以及促进生物合成途径高效运转都具有重要的实用价值。基于细菌群落中普遍存在群感效应的基本原理并结合酶促催化的动力学特征，首先在大肠杆菌群落中建立信号分子高丝氨酸内酯 (AHL) 介导的细胞–细胞交流机制，将靶基因egfp置入到启动子PluxI的控制之下。在细胞生长过程中，产生的AHL累积到一定浓度启动靶基因表达。通过在细胞生长的不同阶段启动AHL降解酶AiiA的表达控制环境中信号分子AHL的浓度水平，从而控制靶基因egfp的转录效率，最终实现对靶蛋白EGFP表达水平的精确控制。通过检测细胞的生长状态、靶基因在mRNA水平、蛋白质水平的表达情况证明人工设计的分子开关可以便捷高效地控制靶基因表达水平，具有时空调节的严谨性。该分子开关有望广泛应用于代谢工程和合成生物学等研究领域中。

Abstract:

Engineering the existing or manual assembling biosynthetic pathways involves two important issues: the activity and expression level of key enzymes in the pathway. Concerning the enzyme expression study, the conventional approach is to use strong promoter to initiate the overexpression of the target protein. The excessive expression of the target protein usually result in the intracellular accumulation of a large number of inactive inclusion bodies, thereby seriously affect the physiological state of the cell and the effective functioning of the relevant biological pathways. To solve this problem, we would like to design a molecular switch to precisely manipulate the expression level of key enzymes in the biosynthetic process, which has important practical value for the study of metabolic rhythm of the biosynthetic pathway and to promote the efficiency of the biosynthetic pathway. Based on the basic principles of quorum sensing existing in the bacterial community and combining the dynamic characteristics of the enzymatic catalysis, we first established cell-cell communication mechanisms mediated by signal molecule homoserine lactone (AHL) in the E. coli community and target protein EGFP was expressed under the control of the promoter PluxI. In the process of cell growth, AHL accumulated to a certain concentration to start the expression of target gene egfp. At the different cell growth stages, AHL-degrading enzyme AiiA was induced and resulted in the degradation of AHL molecule in a controlled environment, thereby controlling the transcription efficiency of target gene egfp and ultimately achieve the precise control of the level of expression of the target protein EGFP. The detection of cell growth state, the mRNA level and protein expression level of the target gene showed the artificially designed molecular switch can control the level of expression of a target gene in a convenient and efficient manner with a spatial and temporal regulation of rigor. The molecular switch is expected to be widely used in the field of metabolic engineering and synthetic biology research areas.