微生物全细胞催化黄酮类化合物酰化修饰及其影响机理研究

Flavonoids are kinds of natural polyhydroxyl compounds with special stereostructure and multiple bioactivity. Regioselective acylation of flavonoids can led to new compounds with significantly improvement in both physiochemical properties and anticancer activity, which made this field even more promising and have attracted much more attentions. Considering the urgent need of low side-effect anticancer agents from natural sources, basic researches on whole cell biocatalysis will be explored for regioselective modification of flavoids. The Relationship between the crucial environmental factors (including cultivation and the reaction conditions) and the biosyntheisis/catalytic performance of whole-cell biocatalyst will be originally investigated in acylation of flavonoids. By investigating influence of substrate structures on the catalytic performance of the whole-cells, the substrate recognition mechanism of whole cells and its enzyme molecular basis in acylation of flavonoids will be revealed for the first time. The researches will be helpful in achieving a breakthrough in the catalytic mechanism of whole-cell catalyzed modification of poly-hydroxyl compounds by regioselective acylation. Based on the above-mentioned research work, a novel, cost-attractive and highly efficient whole-cell biocatalysis system will be build for modification of flavonoids, instead of traditional chemical method or the enzymatic method for improving the process economy, reducing the material consumption and energy consumption in the modification process, and providing theoretical references to made a breakthrough in application of whole-cell biocatalysts in medicine industries, chemical industry and other industries in our country.

黄酮类化合物是一类具有独特化学结构和药理活性的天然多羟基化合物，其经选择性酰化修饰后可获得理化性质改善、抗癌活性增强且具有良好生物相容性的酯类衍生物，应用潜力极大。本项目基于天然产物来源的抗癌新药研发的迫切需求，针对目前黄酮结构修饰中化学法和酶法修饰技术存在的不足，拟以黄酮类酯类化合物合成为目标，采用微生物细胞为新型绿色催化剂，探讨黄酮类化合物的区域选择性酰化修饰，揭示培养要素对全细胞联接酶的诱导/阻遏表达调控及催化活性调控关系，阐明反应介质因素对全细胞催化黄酮类化合物区域选择性修饰、催化剂稳定性的影响机理及全细胞催化剂在反应中的底物识别机理，实现全细胞催化技术在黄酮类化合物结构修饰过程中关键科学问题的认识突破；在此基础上构建具有较高应用潜力的绿色全细胞催化体系，用于替代化学法和酶法、提高过程经济性，为推动全细胞催化技术在我国医药、化工、食品及相关新兴产业中的应用突破提供理论支持。

在本项目支持下，项目组以具有抗肿瘤活性且有应用局限性的黄酮类化合物为目标底物，从不同来源微生物中选育了高效、高选择性及底物谱广的微生物菌种，催化不同结构的黄酮类化合物的选择性酰化反应。①在理论层面上，阐明了培养条件对具有酰化活性的胞内酶合成表达调控的影响规律，揭示了反应介质对微生物催化反应特性及细胞稳定性的影响规律；阐明了不同种类的全细胞催化剂的底物结构识别特性及反应中底物/产物扩散-传质特性等，揭示微生物细胞经诱导培养后在催化黄酮类化合物区域选择性结构修饰中的反应机理及各相关因素对该反应的影响机理；实现了全细胞催化技术在黄酮类化合物结构修饰过程中关键科学问题的认识突破；相关成果在国内外刊物上发表高水平学术论文23篇。②在技术层面上，选育出了4种高选择性、高活性的全细胞催化剂；相应地构建起了具有较高应用潜力的黄酮酯类衍生物合成的绿色细胞催化体系，达到底物转化率>80%、区域选择性>90%；部分优选菌株操作稳定性>80%（4-5批次）；并已合成了一系列具有代表性的黄酮酯衍生物，这些衍生物具有良好的生物相容性，且较之原化合物具有更高的吸收特性，抗氧化活性及抗癌细胞增殖活性等均有增强；相关成果已申请国家发明专利1项、国际专利1项，授权国家发明专利2项。 ③培养了一批具有开阔的学术视野和扎实的生物催化与转化技术的高水平研究人才，项目负责人入选“广东省特支计划青年拔尖人才”，并培养博士研究生4名，硕士研究生7名。本项目的研究成果是对非水相生物催化理论和天然产物化学的新丰富，为推动全细胞催化技术在我国医药、化工及食品产业中的应用突破提供了理论和技术支持，同时也为天然产物来源的抗癌药物研发提供了新途径。