人工辅酶偏好性甲酸脱氢酶创制及选择性代谢调控研究

In this project, we will modify the coenzyme binding domain of the formate dehydrogenase by combining rational design and iterative saturation mutation method, then, screen nicotinamide cytosine dinucleotide (NCD) dependent mutants and characterize their biochemistry and enzymological properties. Using crystal structure data and dynamic simulation virtual screening, we will acquire high-performance NCD-dependent formate dehydrogenase mutants, then establish a universal directed evolution method for NCD-dependent enzymes, and elucidate the chemical bond basis of NCD-mutant recognition. In model microorganism hosts, energy conversion efficiency and matching rules betweeen NCD-dependent energy input module, formate dehydrogenase mutants, and energy utilization modules will be studied. Proteins in modules and different fluorescent proteins will be fusionally expressed, respectively, and their quantitative relationship will be determined. We will regulate the protein expression levels of the two modules by genetic engineering means and obtain positive clones containing quantitative energy transfer chains using high-throughput cell sorting technology. By analyzing intracellular and extracellular small molecules and optimizing related biological elements, we will use formic acid as a switch to selectively regulate NCD mediated intracellular metabolic reactions and reveal adaptive mechanism of biological elements associated with artificial coenzyme NCD. It will provide elements, methods and ideas for reconstruction the artificial redox metabolic network and selective regulation of intracellular metabolism and other chemical biology researches.

本项目拟采用理性设计与迭代饱和突变相结合的方法，改造甲酸脱氢酶的辅酶结合域，筛选可识别人工辅酶烟酰胺胞嘧啶二核苷酸(NCD)的突变体，并进行生化和酶学表征，通过晶体结构数据和动力学模拟虚拟筛选，获得高性能NCD偏好性甲酸脱氢酶突变体，建立NCD依赖型酶的普适定向进化方法，阐明其识别NCD的化学键基础；在模式微生物宿主中，以NCD偏好性甲酸脱氢酶为能量输入模块，研究其与能量利用模块间转化效率及其匹配规律；将模块蛋白分别与不同荧光蛋白融合表达并确定定量关系，通过基因工程手段调控两模块蛋白表达水平，利用高通量细胞分选技术，获得由双模块构成定量能量传递链的阳性克隆；通过分析胞内外小分子，优化相关生物元件，实现以甲酸为开关，NCD介导选择性调控胞内代谢反应，揭示人工辅酶NCD关联的生物学元件适配性机制，为重构氧化还原代谢网络，利用外源能量选择性调控胞内代谢等化学生物学研究提供元件、方法和思路。

本项目以甲酸脱氢酶(FDH)晶体结构数据和动力学模拟虚拟筛选为基础，通过理性设计与迭代饱和突变相结合改造FDH辅酶结合域，经高通量筛选获得可识别人工辅酶烟酰胺胞嘧啶二核苷酸(NCD)的高活性突变体，其中突变体FDH* (V198I/C256I/P260S/E261P/S381N/S383F)对NCD的偏好性比野生型提高了3700倍；通过解析突变体晶体结构，阐明了NCD依赖型酶定向进化辅酶识别的化学键基础；搭建了以甲酸为能量源，NCD介导选择性调控丙酮酸到苹果酸的能量定向输入管线，构建了甲酸驱动的丙酮酸还原羧化纯酶体系和胞内合成体系，甲酸可为苹果酸的合成同时提供碳和还原力；在NCD自给型菌株中构建甲酸驱动的D-乳酸合成菌株，显著提高D-乳酸含量；通过NCD偏好性FDH*能量源的原核细胞模型，揭示能量输入与能量利用间转化匹配规律；分析胞内相关代谢小分子，阐明人工辅酶NCD依赖生物学元件匹配机制，为利用外源能量源选择性调控胞内代谢和重构氧化还原代谢网络提供了新方法和工具。在此基础上，整合NCD依赖性甲醇脱氢酶(MDH*)和甲醛脱氢酶(FalDH*)，为甲醇能量源的原子经济C1生物转化反应器构建生产高附加值化学品或药物及代谢通路选择性调控奠定生物技术基础。