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关键酶泛素化位点对柚皮素生物合成的影响
引用本文:李明佳,周景文,李江华.关键酶泛素化位点对柚皮素生物合成的影响[J].生物工程学报,2022,38(2):691-704.
作者姓名:李明佳  周景文  李江华
作者单位:江南大学 未来食品科学中心, 江苏 无锡 214122;江南大学 生物工程学院, 江苏 无锡 214122;江南大学 粮食发酵与食品生物制造国家工程研究中心, 江苏 无锡 214122
基金项目:国家自然科学基金创新研究群体项目(32021005)
摘    要:黄酮类化合物具有多种生物活性,在食品、药品、化妆品等领域都有重要应用.柚皮素是多种重要黄酮类化合物生物合成的平台化合物.泛素化是蛋白质翻译后修饰的重要一环,参与调控细胞的生命活动.泛素化的蛋白质通过泛素-蛋白酶体系统降解,对维持细胞正常生理活动具有重要意义,对外源蛋白的表达和积累也可能具有显著影响.文中利用荧光双分子互...

关 键 词:酿酒酵母  黄酮  对香豆酸  柚皮素  泛素化  蛋白质工程
收稿时间:2021/1/31 0:00:00

Effect of key enzymes ubiquitination sites on the biosynthesis of naringenin
LI Mingji,ZHOU Jingwen,LI Jianghua.Effect of key enzymes ubiquitination sites on the biosynthesis of naringenin[J].Chinese Journal of Biotechnology,2022,38(2):691-704.
Authors:LI Mingji  ZHOU Jingwen  LI Jianghua
Institution:Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China;School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;National Engineering Research Center for Cereal Fermantation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, Jiangsu, China
Abstract:Flavonoids have a variety of biological activities and have important applications in food, medicine, cosmetics, and many other fields. Naringenin is a platform chemical for the biosynthesis of many important flavonoids. Ubiquitination plays a pivotal role in the post-translational modification of proteins and participates in the regulation of cellular activities. Ubiquitinated proteins can be degraded by the ubiquitin-protease system, which is important for maintaining the physiological activities of cells, and may also exert a significant impact on the expression of exogenous proteins. In this study, a real-time in-situ detection system for ubiquitination modification has been established in Saccharomyces cerevisiae by using a fluorescence bimolecular complementation approach. The ubiquitination level of protein was characterized by fluorescence intensity. By using the approach, the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway have been obtained. The lysine residues of the relevant ubiquitination sites were mutated to arginine to reduce the ubiquitination level. The mutants of tyrosine ammonia-lyase (FjTAL) and chalcone synthase (SjCHS, SmCHS) showed decreased fluorescence, suggested that a decreased ubiquitination level. After fermentation verification, the S. cerevisiae expressing tyrosine ammonia-lyase FjTAL mutant FjTAL-K487R accumulated 74.2 mg/L p-coumaric acid at 72 h, which was 32.3% higher than that of the original FjTAL. The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin. The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.
Keywords:Saccharomyces cerevisiae  flavonoids  p-coumaric acid  naringenin  ubiquitination  protein engineering
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