基于光合辅酶再生的蓝藻生物催化不对称合成手性醇研究

Enantiopure alcohols are widely applied as the key building blocks in synthesis of enantiopure pharmaceuticals, agrochemicals and other fine chemicals due to their unique structure property. Asymmetric reduction of the corresponding prochiral ketones is an efficient and promising synthesis route for production of chiral alcohols. Whole cell biocatalysts, such as microbial and plant cells, are excellent catalysts for the asymmetric reduction reaction of prochiral ketones because of their intrinsic advantages, such as outstanding enantioselectivity, environment-friendly and mild reaction conditions, and in vivo regeneration of coenzyme (i.e. NADPH). . Based on our research experience in asymmetric synthesis and microalgal biotechnology, we proposed a technique for production of enantiopure alcohols through asymmetric reduction reaction depended upon photosynthetic regeneration of coenzyme with microalgae cells. The prochiral ketones are stereoselectively reduced to chiral alcohols catalyzed by the carbonyl reductase in microalgal cells. In this process, the coenzyme (i.e. NADPH) is directly regenerated through photosynthesis utilizing sunlight as energy. Excellent results were achieved in our preliminary research. Cyanobacteria and chlorophyta are eligible photo-biocatalysts for the asymmetric reduction reaction. Cyanobacteria are excellent candidate for the microalgal photo-biocatalysis because of its advantages, such as high growth rate and easy to cultivating, good ecological benefit, and outstanding catalytic activity. . In this proposal, Synechococcus sp. is chosen as the model cyanobacterium. We will devote our efforts to explore the key carbonyl reductase and discover the mechanism of photosynthetically regenerating NADPH, then to develop high efficient microalgal biocatalysis process. At first, the key carbonyl reductase responsible for the asymmetric reduction reaction will be retrieved and purified from the cyanobacterium cells, the structure and function of the reductase will be resolved. At the same time, the metabolic processes of NADPH regenerating through photosynthesis in microalgal cells will be discovered, and the methodology to improve the efficiency of NADPH regenerating will be developed based on promoting the plastoquinone (PQ) and ferrdoxin-NADP+ reductase activity or inhibiting the Calvin cycle.. This project may open up a new research field for biocatalysis and develop a novel kind of biocatalysts for asymmetric reduction reaction. Also, it will provide a green and sustainable development technique for production of chiral alcohols.

手性醇是手性医药及其它手性精细化学品合成的关键手性砌块与平台化合物。高效、绿色合成手性醇对手性技术的发展与应用至关重要。我们在之前的生物催化不对称合成研究基础上结合光合辅酶再生技术，开展了光合辅酶再生的微藻生物催化不对称合成手性醇的研究，取得了喜人结果。. 在此基础上，本项目以聚球藻为模式蓝藻，在酶及代谢层面上，深入揭示基于光合辅酶再生的蓝藻生物催化前手性羰基不对称合成手性醇的机理，以开发高效的微藻催化手性醇生产技术。项目将检索并分离目标藻中主要羰基还原酶，解析其结构与功能；分析光合辅酶再生代谢，通过强化光反应中的质体醌还原效率及铁氧还蛋白-NADP+还原酶活性同时抑制卡尔文循环以增强辅酶再生效率。通过这些研究为开发满足应用要求的工程化蓝藻生物催化技术提供技术基础与理论依据。本项目为生物催化不对称合成开创新领域、开拓新催化剂，为手性醇合成开辟绿色、可持续发展的新技术。

在国家自然科学基金的资助下我对“基于光合辅酶再生的蓝藻生物催化不对称合成手性醇研究”进行了研究，取得了如下结果：. 本项目首先对蓝细菌（Spirulina platensis）细胞内催化前手性芳香酮不对称还原的主要羰基还原酶利用高压匀浆破胞、盐析、层析等进行了分离，纯化出了一种高活性的芳香酮羰基还原酶，该酶为双亚基还原酶分子量大约为61.9 kDa，并获得该酶的酶学性质，为生物催化不对称合成提供新的酶及其基础数据。.分析了主要羰基还原酶结构与功能关系，表明羰基还原酶存在同一个保守的氨基酸结构PS00061，并在其中都存在Y（酪氨酸）酪氨酸（tyrosine, Y）和赖氨酸（lysine, K）活性催化位点。其二级结构均以无规卷曲为主，其次为β-片层和α-螺旋，无β-转角。这些基本结构信息为羰基还原酶检索，以及羰基还原酶的理性设计与进化改造提供理论指导。. 开发微藻光合辅酶再生代谢调控技术。从阻断微藻细胞内辅酶消耗与促进微藻光合作用两方面进行调控增强辅酶再生代谢。开发了抑制甘油醛-3-磷酸脱氢酶活性，直接减少Calvin循环对NADPH消耗，增加细胞内NADPH含量高达96.0%，促进不对称还原的产率达63.3%；通过添加光合作用促进剂促进NADPH再生，使细胞内NADPH量最高增加了73.1%，不对称还原的产率提高118.4%。. 进一步利用基因工程技术从克隆FNR基因，利用同源重组技术把FNR基因petH重组及微藻染色体增加FNR量以增强光合作用中FNR催化NADP+还原为NADPH的效率进而促进微藻不对称催化效率，NADPH量提高0.5倍，反应的催化提高0.2倍。当前还未见任何有关该方面的报道。.为了充分利用微藻生物催化后所产生大量的生物质，我们增加了利用微藻合成生物油脂进而转化为生物柴油的相关研究。制备出了相应的生物油脂，其转化为生物柴油的十六烷值为57.3，符合生物柴油的热值要求；获得了微藻油脂积累的最优工艺参数，在此条件下油脂含量的理论值为15.4%。以油脂合成的关键酶为切入点，通过柠檬酸和Mg2+两种乙酰辅酶A羧化酶促进剂强化微藻油脂合成过程。. 项目为微藻生物催化羰基不对称还原合成手性醇的生产过程开发奠定了理论基础。