基于尿酸胞内生物传感器定向改造黄嘌呤氧化酶底物抑制性的研究

Xanthine oxidase (XOD) is severely inhibited by its substrate of xanthine, even at low substrate concentration. Such inhibition limits the productivity and applications of industrial biocatalysis applying XOD, such as synthesis of ribavirin. However, up to date, there is still neither reports on the protein engineering of the substrate inhibition nor investigation of the underlying mechanism. In our previous studies, we constructed a novel Rhodobacter capsulatus XOD (RcXOD), which is of the best catalytic performance and typical substrate inhibition. Based on a transcription protein HucR of uric acid oxidase of Deinococcus radiodurans, we also constructed a uric acid biosensor, which quantitatively respond to the intracellular uric acid at the single cell level and then transform into green fluorescence. This biosensor could be potentially applied in the field of high-throughput screening of strains with high uric acid production. By using the RcXOD as model system, this project will engineer the uric acid biosensor into a novel one which will accurately respond to the XOD activity via the enzymatic product of uric acid, and then high-throughput screen the mutants with improved substrate inhibition by using fluorescence activated cell sorting (FACS). Firstly, we will finely tune the responsive properties of uric acid biosensor by engineering “ligand binding site” of HucR, optimizing the sequences of promoter, RBS and regulatory accessory regions of biosensor plasmid, and deleting the potential purine-degrading genes, xanthine dehydrogenase (xdh) and aldehyde oxidase (pao) and overexpressing the xanthine specific transport proteins (ygfO and yicE) of chassis host, Escherichia coli. Secondly, we will construct high quality mutant libraries with better performance based on substrate channel and catalytic mechanism by using site-directed saturation mutagenesis at the key amino acid residues responsible for the catalytic and electron transfer processes, substrate channel and their neighboring amino acid residues, and then screen and separate the mutation pools under different xanthine concentrations by using fluorescence activate cell sorting (FACS). Thirdly, the target mutant XODs will be purified to homogeneity to characterize their enzyme kinetics parameters under different substrate inhibitions. With the information of the mutated amino acid residue, structure, enzymatic properties, we could explore molecular mechanism underlying the substrate inhibition. This project could shed light on the high-throughput screening and directed evolution of other intracellular complex enzymes by using biosensor that recognizes the enzymatic substrate or product in the industrial biocatalysis field.

黄嘌呤氧化酶（XOD）的底物抑制问题限制了在工业生物催化领域的应用，但目前仍缺乏XOD底物抑制性的定向改造研究及其分子机理解析。申请人前期研究获取了一个催化性能优良的新型荚膜红细菌XOD，和一种能够将尿酸转变成绿色荧光信号的大肠杆菌生物传感器。本项目将以此XOD作为模式体系，以该尿酸传感器为起点构建响应XOD酶催化活力的新型尿酸胞内生物传感器，然后利用荧光流式细胞仪（FACS）分选底物抑制性改善的突变体。首先分别从尿酸酶转录蛋白、传感器质粒和底盘宿主三个层面，改造尿酸传感器响应尿酸的特异性、灵敏度和范围以及对底物的通透性；其次基于XOD底物通道和催化机制构建高质量突变体库，利用FACS分选不同底物浓度条件下酶活提高的突变体；最后表征各突变体酶的抑制动力学参数，从序列-功能关系角度深入解析XOD底物抑制分子机理。本项目的实施也将为基于小分子生物传感器定向改造其他复杂胞内酶类提供方法借鉴。

针对黄嘌呤氧化酶（XOD）的底物抑制问题，本项目以鲍氏不动杆菌XOD（AbXOD）为实验材料，系统开展了四个方面的研究：1）构建特异性高灵敏度胞内尿酸生物传感器；2）基于XOD底物通道和催化机制构建高底物耐受性突变体；3）突变体的抑制动力学表征及分子机理解析；4）底物耐受性突变体应用于高浓度嘌呤复杂食品体系的概念验证。重要结果有：1）敲除大肠杆菌基因组嘌呤代谢相关基因簇xdh和pao、组成型表达嘌呤转运蛋白UraA，建立了与外源尿酸代谢途径和生物传感器正交的底盘宿主菌，构建了适于高通量筛选XOD突变体的转录型尿酸诱导阻遏型胞内生物传感器。2）鲍氏不动杆菌与荚膜红细菌来源的黄嘌呤脱氢酶、藤黄节杆菌XOD、节杆菌XOD、牛乳XOD以及AbXOD均表现出明显的黄嘌呤底物抑制以及高浓度尿酸的产物抑制。其中，AbXOD随尿酸浓度增加Km值降低，表现为反竞争性抑制。3）以5 mM黄嘌呤为底物、314 nm波长下检测建立了高通量筛选体系，成功获取6个底物抑制效应降低、催化活性提高的、碱性突变体XOD，其中，Q201E、Q201C、P319K和P319Y完全解除底物抑制，更加适合于高浓度嘌呤底物。最优突变体Q201E和P319K的转化数分别为799.44 s-1和1474.70 s-1，为野生型的1.95倍和3.6倍。5）Q201位点突变造成活性中心两个柔环loop234-245和loop523-530的构象、与底物C2=O、N9、N7的氢键网络、π-π键以及钼中心稳定性的改变，影响了底物的稳定性和选择性，并使通道入口更加灵活，帮助底物更好地进入活性中心发生反应。P319K中对应于野生型的原底物通道消失，出现一条新的底物通道。新通道增长4 Å，与黄嘌呤与尿酸的最大结合能为野生型中原通道的5倍和1.25倍，结合能力下降与部分位点结合的概率降低，可能造成底物与酶亲和力降低，底物抑制解除。6）在鱼露这一代表性高嘌呤复杂食品体系中，P319K对黄嘌呤的降解效果为理论值的46.7%，而野生型完全丧失酶活。同时，P319K有效降低了鱼露中挥发性组分，特别是引起鱼露酸臭味的2-甲基丁醛等以及引起腐败味与鱼腥味的三甲胺等不良风味物质，提供了一个应用XOD改善食品风味的一个新思路。