【作者】 张琳；

【导师】 林娟；

【作者基本信息】 福州大学 ， 生物工程（专业学位）， 2022， 硕士

【摘要】 邻氨基苯甲酸合酶（ANTA合酶）是色氨酸等下游代谢产物合成过程的关键限速酶,本文以ANTA合酶TrpE亚基作为研究对象,基于生物信息学分析,采用定点突变与半理性设计相结合的方法对TrpE进行分子改造,以降低L-Trp的反馈抑制并提高其催化效率,为L-Trp的发酵生产提供高产稳定的工程菌株。主要研究内容如下:1、采用生物信息学方法对TrpE亚基进行结构预测与分析。预测结果表明,ANTA合酶TrpE亚基具有PRK13564结构域,不含信号肽和无跨膜区域,为亲水不稳定蛋白;其二级结构主要以折叠和螺旋为主。2、使用SWISS MODEL对TrpE进行同源建模,通过SAVES v5.0对构建的模型结构进行合理性评估,使用Auto Dock Tools将TrpE分别与小分子配体L-Trp、CHA进行分子对接,选取符合能量最低且最符合催化机制的结构作为目标对接结果。3、TrpE催化结构域存在2个结合腔:抑制剂（L-Trp）结合腔和底物（CHA）结合腔。以重组质粒p ET-28a-P_lac-trp ED为模板,根据结合腔附近氨基酸的性质和结合腔入口的空间位阻,采用定点突变以及关键位点的饱和突变等方法,筛选到L-Trp产量提高2.26倍的两点突变体S40F/L365A。4、TrpE-Trp和TrpE^S40F/L365A-Trp的分子动力学模拟结果表明,突变导致酶整体结构柔性降低,阻碍抑制剂进入结合腔,突变体与L-Trp的结合能力更弱,减弱了氨基酸残基总的能量贡献,TrpE^S40F/L365A的L-Trp结合中心处的相互作用力明显比TrpE少,不利于L-Trp与TrpE的结合,有效的解除了反馈抑制。TrpE-CHA和TrpE^S40F/L365A-CHA的分子动力学模拟结果表明,突变提高了酶Loop环2（T425-R431）氨基酸残基及结合腔入口处组成α-螺旋的氨基酸残基的柔性,优化了突变位点附近的能量分布,减小了降解底物的空间位阻,增加了活性中心同底物的相互作用力,导致亲合能提高,底物更易结合在催化中心,有利于质子的传递和裂解的发生,从而提高催化效率。更多还原

【Abstract】 Anthranilate synthase（ANTA synthase）is a key rate-limiting enzyme in the synthesis of downstream metabolites such as tryptophan.In this study,the TrpE subunit of ANTA synthase was used as the research object.Based on bioinformatics analysis,site-directed mutation combined with semi-rational design was used to carry out molecular modification of TrpE.In order to reduce the feedback inhibition of L-Trpand improve its catalytic efficiency,to provide high yield and stable engineering strain for L-Trpfermentation production.The main research contents are as follows:1.The TrpE subunit structure was predicted and analyzed by bioinformatics method.The results showed that the TrpE subunit of ANTA synthase was a hydrophilic unstable protein with PRK13564 domain and no signal peptide or transmembrane region.Its secondary structure mainly consists of folding and spiral.2.SWISS MODEL was used for homologous modeling of TrpE,and The rationality of the MODEL structure was evaluated with SAVES V5.0.Auto Dock Tools was used to dock TrpE with small molecular ligand L-Trpand CHA.The structure with the lowest energy and most consistent with the catalytic mechanism is selected as the target docking result.3.There are two binding cavities in the TrpE catalytic domain:the inhibitor（L-Trp）binding cavity and the substrate（CHA）binding cavity.Using recombinant plasmid p ET-28a-P_lac-trp ED as template,S40F/L365A with 2.26 times higher yield of L-Trpwas screened by site-directed mutation and saturation mutation at key sites according to the properties of amino acids near the binding cavity and steric hindrment at the entrance of catalytic cavity.4.Molecular dynamics simulation results of TrpE-Trpand TrpE^S40F/L365A-Trpshowed that the mutation reduced the flexibility of the overall structure of the enzyme,hindered the entry of inhibitors into the binding cavity,weakened the binding ability of the mutant to L-Trp,and weakened the total energy contribution of amino acid residues.The interaction force at the L-Trpbinding center of TrpE^S40F/L365Ais significantly less than that of TrpE,which is not conducive to the combination of L-Trpand TrpE and effectively removes the feedback inhibition.Molecular dynamics simulation results of TrpE-CHA and TrpE^S40F/L365A-CHA showed that mutations improved the flexibility of amino acid residues in enzyme Loop 2（T425-R431）andα-helical amino acid residues at the entrance of catalytic cavity,optimized the energy distribution near the mutation site,and reduced the steric hindrance of degradation substrates.The interaction between the active center and the substrate is increased,resulting in improved affinity energy,and the substrate is more easily bound to the catalytic center,which is conducive to proton transfer and cleavage,thus improving the catalytic efficiency.更多还原