烟酸脱氢酶的高效异源表达及其底物识别的分子基础

Biocatalysis has become the frontier due to its advantages of high efficiency and green nature. But the low activity and poor selectivity of the existing enzymes limit their applications in industrial process with non-natural substrate and environment. The key problem is that the molecular recognition mechanism of the enzyme catalysis process has not been resolved. It is very difficult to reform the performance of enzyme, thus the industrial enzyme source is limited. Nicotinate dehydrogenase from Comamonas testosterone JA1（CT-NDH） was selected as the target biocatalyst and the hydroxylation of 3-cyano-pyridine was selected as the model reaction. Base on the genome-editing technique of CRISPR/Cas9, the high efficiency hetroexpression of CT-NDH will be studied to improve its activity expression level in Pseudomonas putida KT2440. The crystal structure of CT-NDH will be resolved through protein crystallization technology. Based on the crystal structure, the interaction between CT-NDH and the pyridine substrates and their dynamic structure changes will be studied by using the methods of molecular dynamics/quantum mechanics. The molecular recognition mechanism of CT-NDH towards the pyridine substrates will be resolved at the molecular level. The construction of highly selective CT-NDH towards 3-cyano-pyridine will be realized via rational design. Based on the kinetics studies, the process will be intensified and the reaction efficiency will be improved to realize the high efficiency bioproduction of 3-cyano-6-hydroxy-pyridine. This project is not only a very important supplement to the existing theory of nicotinate dehydrogenase with significant theoretical value, but also has very clear and broad application prospects.

生物催化过程高效绿色，是当前热点研究领域。生物催化剂-酶是生物催化的核心，但在工业过程中，酶面对非天然底物和非天然环境，存在酶活低和选择性不高等问题。关键在于缺乏对酶催化过程分子识别机制的深入研究和解析，性能改造困难，来源有限。本项目拟选择C. testosterone JA1烟酸脱氢酶（CT-NDH），以3-氰基吡啶羟基化反应为模型。基于基因组编辑技术CRISPR/Cas9开展其在恶臭假单胞菌KT2440中的高效表达研究。采用蛋白质结晶技术获得晶体并解析其空间结构。采用分子动力学/量子力学研究酶和底物间相互作用和动态结构变化，在分子水平上解析酶对吡啶类底物识别的分子机制。通过理性设计，构建针对3-氰基吡啶的高选择性CT-NDH；深入研究催化过程动力学，实现过程强化，改善反应效率，实现3-氰基-6-羟基吡啶的高效生物制造。本项目研究不仅具有重要的的理论价值，其应用前景也非常明确和广阔。

烟酸脱氢酶是一类能够区域特异性催化吡啶衍生物C6羟基化的酶类，在用于制备6-羟基烟酸、6-羟基3-羧基吡啶、6-羟基-3-氰基吡啶等具有重要应用价值的化学品上显得尤为关键。目前，国内外烟酸脱氢酶的相关研究较少，高效异源表达体系鲜见报道。. 本课题主要从两方面出发开展研究，一方面构建高效的基因组编辑系统并进行底盘细胞的改造，另一方面构建烟酸脱氢酶高效表达体系并对其催化机制进行探索。. 在底盘细胞改造方面，首先在恶臭假单胞菌KT2440中建立了高效的CRISPR/Cas9系统，利用该系统，基因敲除、基因替换、基因插入等操作可以在5天内完成，编辑效率超过了70%。同时还开发了宿主更为广、目标靶点更多的pSEVA6BE-NG编辑器，成功地在双位点与三位点，以90%与40%的效率实现了碱基同时编辑。通过运用CRISPR/Cas9基因编辑工具成功的对KT2440没食子酸途径实现改造，没食子酸产量达3589.02 mg/L，相较于出发菌株提高了44.76倍。同样的对虫媒假单胞菌L48原儿茶酸途径进行编辑，将原儿茶酸产量提高到了5.05 g/L。. 在烟酸脱氢酶的高效表达研究和催化机制探索方面：首先在恶臭假单胞菌KT2440及其基因删减型菌株EM42中构建基因组整合类T7表达模块。选取非必需基因作为整合位点，并通过优化整合位点与拷贝数获得了可以提高异源蛋白表达水平的底盘细胞KTCM与EMCM。并在底盘细胞KTCM与EMCM中优化了类T7表达系统的诱导表达的条件。用此底盘细胞成功提高了烟酸脱氢酶的表达水平，达82 U/L。其次在非模式菌睾酮丛毛单胞菌 CNB-2中构建重组表达体系，实现了烟酸脱氢酶的异源功能表达，随后利用多层次表达强化策略，改善基因转录，翻译及蛋白质折叠组装效率，包括类T7系统整合，分子伴侣辅助表达，启动子优化，RBS优化，亚基平衡表达等，显著提高了烟酸脱氢酶表达水平，达192 U/L。通过多序列比对策略成功确定了烟酸脱氢酶中催化3-氰基吡啶的关键氨基酸片段，研究其催化机制，并对该片段中氨基酸进行定点突变提高催化活性。通过发酵过程和催化工艺优化，实现了6-羟基-3-氰基吡啶的高效制备，产率达0.6 g/L/h。.