源于假单胞菌的硫醚单加氧酶工程菌的构建与改造及其在手性亚砜类药物合成中的应用

Asymmetric oxidation of thioethers catalyzed by monooxygenase is a novel pathway for green synthesis of chiral sulfoxide drugs. However, it is difficult to obtain monoxygenases with high catalytic efficiency. In our previous studies, a Pseudomonas bacterial strain expressing monooxygenase which can catalyze thioethers converts to chiral sulfoxide efficiently was obtained. The genome sequencing of this Pseudomonas was then performed. In this project, all genes encoding monooxygenase will be screened based on the genome information, followed by gene expression analysis. Meanwhile, protein purification will be performed to isolation monooxygenases from the total protein of our bacterial strain. Subsequently, candidate genes confirmed by these two steps will be cloned to expression vectors and transformed into E.coli. After optimization of recombinant protein expression, the whole cell or cell extract of genetically engineered bacterium will be used as catalyst to synthesize chiral sulfoxide for the screening of thioether monooxygenases. Afterwards, the coenzyme regeneration system of monooxygenases will be established and the DNA directed evolution of monooxygenase genes will be performed to improve the catalytic efficiency of monooxygenases selected. Thus, this project expects to obtain several novel efficient thioether monooxygenases with independent intellectual property rights. At last, our monoxygenases will be used to synthesize some widely uesd chiral sulfoxide druges to lay a fundation for their industrialized application in future. The process of this project is significant for the chiral sulfoxides asymmetric synthesis through biological catalysis and the green synthesis of chiral drug.

单加氧酶催化的硫醚底物的不对称氧化反应是手性亚砜类药物绿色合成的新途径之一，而寻求高催化效率和高底物耐受性的硫醚单加氧酶仍是其难点和瓶颈。课题组前期筛选发现了一株高效催化硫醚底物生成手性亚砜的产单加氧酶假单胞菌，并进行了基因组测序。本项目拟利用其基因组信息，系统发掘基因组中的单加氧酶相关基因，研究其表达情况；同时结合蛋白质分离纯化技术从菌株总蛋白中分离单加氧酶并进行质谱鉴定。结合前两步确定和克隆候选基因，构建基因工程菌作为催化剂，筛选获得能高效催化硫醚底物生成手性亚砜的单加氧酶及相应工程菌株。接着研究其辅酶再生机制和循环系统以提高酶的催化效率，并通过DNA定向进化技术改造和进化酶，力争获得高底物耐受性和高活性的硫醚单加氧酶，应用于一些常用手性亚砜类药物的直接生物催化合成，并初步探索相关催化机制。本项目的研究有助于实现手性亚砜类药物的绿色合成，对推动生物催化在手性药物合成中的应用有重要意义。

生物酶催化的不对称反应是手性亚砜类药物绿色合成的新途径之一，而寻求高催化效率和高底物耐受性的生物酶一直是该领域的难点和瓶颈。在本项目的资助下，课题组从前期获得的一株假单胞菌以及Genbank数据库中，筛选并克隆了11个氧化酶基因和15个还原酶基因。通过重组表达，获得了相应的重组酶蛋白和基因工程菌。以重组酶蛋白或者含重组酶蛋白的基因工程菌为催化剂，通过硫醚底物的不对称氧化或者消旋体亚砜底物的不对称拆分手段，可有效制备R构型或S构型手性亚砜。其中：项目组获得的氧化酶pmSty能有效催化2-10 mM浓度下苯甲硫醚的氧化，转化率为56-91%，获得ee值42-68%的（S）-亚砜产物；项目组获得的ppMsrB和arMsrB在底物浓度为2 mM时，能有效还原体系中的R构型苯甲亚砜，生成ee值分别为63.6%和71.9的S构型苯甲亚砜。更重要的是，本研究发现的还原酶pmMsrA和paMsrA可分别实现R构型手性亚砜在高达200 mM和320 mM（45 g/L）底物浓度下的生物催化合成，产物ee值均大于99%，且产率接近理论最大值。这是目前手性亚砜生物催化合成领域单一重组酶催化所能达到的最大底物浓度。本项目的实施有助于实现手性亚砜类药物的绿色合成，对推动生物催化在手性药物合成中的应用有重要价值。