木栓酮合酶TwOSC1的产物异构化研究  

Research on Isomerization of Product of Friedelin Synthase TwOSC1

作　　者：张凯月 邹旭 张晨烁 余源 王洋 ZHANG Kai-yue;ZOU Xu;ZHANG Chen-shuo;YU Yuan;WANG Yang(College of Life Sciences,North China University of Science and Technology,Tangshan Hebei 063210,China)

机构地区：[1]华北理工大学生命科学学院,河北唐山063210

出　　处：《华北理工大学学报（自然科学版）》2023年第3期110-117,124,共9页Journal of North China University of Science and Technology：Natural Science Edition

基　　金：国家自然科学基金(32171430);河北省自然科学基金(B2021209008);华北理工大学省属高校基本科研业务费项目(JQN2020003)。

摘　　要：来源于雷公藤的木栓酮合酶TwOSC1能够催化(3S)-2,3-氧化鲨烯生成木栓酮、α-香树脂醇和β-香树脂醇,三者比例为3:1:1。将TwOSC1引入酿酒酵母能够合成木栓酮,但产量较低,无法达到工业化生产的需求,TwOSC1催化活性低,产物专一性差是影响其产量的主要瓶颈。通过理性和半理性设计分析TwOSC1的关键氨基酸残基位点,利用蛋白质工程对关键氨基酸残基(N11、G280、I259、I548、M552)进行改造。研究结果表明,突变体N11I、N11A、N11W、N11R、N11F、I548W、I548L的木栓酮产量降低;突变体G280H、G280P、G280S、M552S的木栓酮产量提高,且TwOSC1G280H催化活性最好,是野生型TwOSC1产量的1.27倍;突变体I259A、I259E、I259K、I259M、I259R无催化活性,但突变体TwOSC1I259E合成α-香树脂醇的专一性较高,是野生型TwOSC1产量的5倍。研究初步探索了5个关键氨基酸残基位点对TwOSC1催化活性及产物专一性的影响,为后续深入探究TwOSC1关键氨基酸位点提供了有价值的参考。Friedelin is an important friedelane pentacyclic triterpenoid compound in plants.TwOSC1 as a friedelin synthase from Tripterygium wilfordii can catalyze the cyclization of(3S)-2,3-oxidosqualene to produce friedelin,α-amyrin andβ-amyrin,the ratio of the three is 3:1:1.The introduction of TwOSC1 into saccharomyces cerevisiae can synthesize friedelin,but the yield is low,which can not meet the demand of industrial production.The low catalytic activity of TwOSC1 and the poor product specificity are the main bottlenecks affecting its yield.In this study,the key amino acid residues of TwOSC1 were analyzed by rational and semi-rational design,and the key amino acid residues(N11,G280,I259,I548,M552)were modified by protein engineering.The effect of five key amino acid residues on the catalytic activity and product specificity of friedelin synthase TwOSC1 was preliminarily explored.The results show that the yield of friedelin of mutant N11I,N11A,N11W,N11R,N11F,I548W,I548L decreases.Mutants G280H,G280P,G280S and M552S increase the production of friedelin,and the catalytic activity of TwOSC1G280H is the best,its yield is about 1.27 times that of wild type TwOSC1.Mutants I259A,I259E,I259K,I259M and I259R have no catalytic activity,but the mutant TwOSC1I259E is synthesizedα-amyrin has high specificity,its yield is about 5 times that of wild type TwOSC1.It provides valuable reference data for further research on the key amino acid sites of TwOSC1.

关 键 词：蛋白质工程 木栓酮合酶 木栓酮 α-香树脂醇 β-香树脂醇 

分 类 号：Q814[生物学—生物工程]