Meyerozyma guilliermondii醇脱氢酶理性改造及其催化生物基呋喃醛还原的研究

由生物质为原料合成生物基化学品是当前的一个研究热点。生物基呋喃醛如5-羟甲基糠醛（HMF）及糠醛是重要的生物基平台化合物，可转化为各种高附加值中间体。生物催化生物基呋喃醛高值化转化具有选择性高、条件温和、环境友好等优点。获得高底物耐受性、高活性、稳定的生物催化剂是实现高毒性呋喃醛高值化生物转化的关键。本项目以自行筛选所得、高HMF耐受性的Meyerozyma guilliermondii SC1103为研究对象，挖掘催化HMF选择性还原的关键醇脱氢酶；解析关键酶及其与底物复合物分子的三维空间结构，揭示酶与底物键合及催化关键氨基酸位点，阐明其催化机制；进一步对酶分子中与底物相互作用的关键氨基酸残基进行理性设计和定点突变，获得高效目标酶，揭示该酶结构与功能关系；借助分子克隆技术等构建催化性能优异的重组菌；辅以固定化等手段，建立高效合成生物基呋喃醇的生物催化体系。

Recently, the production of biobased chemicals from biomass has attracted growing interest. Biobased furan aldehydes such as 5-hydroxymethylfurfural (HMF) and furfural, important biobased platform chemicals, could be transformed into a variety of value-added intermediates. Biocatalytic valorization of biobased furans has many advantages such as exquisite selectivity, mild reaction conditions and environmental friendliness. Highly substrate-tolerant, active and stable biocatalysts are key to implement efficient biotransformation of toxic furans. Recently, we have isolated a new and highly HMF-tolerant yeast strain - Meyerozyma guilliermondii SC1103 capable of selectively reducing HMF. In this project, the key alcohol dehydrogenase responsible for the HMF reduction will be mined from the genome of this yeast. The spatial structures of the key enzyme and enzyme-substrate complex will be resolved to reveal the key amino acid residues in the substrate-binding and catalytic sites of this enzyme, thus uncovering the reaction mechanism. Then, rational design and site-directed mutagenesis of the key amino acid residues in the enzyme active center will be conducted to afford an efficient and stable HMF reductase and unveil the structure-function relationship of the enzyme. By molecular cloning, the recombinant strain with good catalytic performances will be constructed. The combination of this new biocatalyst and some strategies such as cell immobilization allows to establish an efficient biocatalytic approach toward furan alcohols.