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《工程(英文)》 >> 2019年 第5卷 第2期 doi: 10.1016/j.eng.2018.11.029

利用异源酶组合构建酿酒酵母中咖啡酸的生物合成

a Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin
University, Tianjin 300072, China

b SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University,
Tianjin 300072, China

收稿日期: 2018-03-21 修回日期: 2018-09-25 录用日期: 2018-11-20 发布日期: 2019-03-02

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摘要

微生物中构建植物源天然产物的生物合成面临着许多挑战,尤其是当需要表达与激活植物源细胞色素P450酶的时候。通过从几种细菌中筛选HpaB和HpaC两种酶,本文在酿酒酵母中构建了具有活性的4-羟基苯乙酸3-羟化酶(4 HPA3H),它可以发挥植物源细胞色素P450酶的相似作用,用于生产咖啡酸。在与一个共同的酪氨酸氨裂解酶(TAL)协同作用下,不同的异源HpaB酶和HpaC酶组合在将底物L-酪氨酸转化为目标产物咖啡酸上展现出不同能力。以铜绿假单胞菌的HpaB酶和肠沙门氏菌的HpaC酶的异源酶组合可生产最高咖啡酸产量,摇瓶培养下可达到(289.4 ± 4.6)mg·L−1。酵母底盘细胞与异源酶的相容性得到了有效的改善,咖啡酸的产量比初始值提高了40倍。铜绿假单胞菌HpaB酶中黄素腺嘌呤二核苷酸(FAD)结合域周围的6个关键氨基酸残基与其他细菌来源的HpaB酶有明显区别,可能在影响酶活性方面起关键作用。综上,我们建立了一种有效的方法来构建高效的酵母系统用于合成非天然羟基苯丙烷类化合物。

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