Restricting Promiscuity of Plant Flavonoid 3'-Hydroxylase and 4'-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
文献类型： 外文期刊
作者： Liu, Juan 1 ; Xiao, Zhiqiang 1 ; Zhang, Siqi 1 ; Wang, Zhen 1 ; Chen, Yun 4 ; Shan, Yang 1 ;
作者机构： 1.Hunan Univ, Coll Biol, Longping Branch, Changsha 410125, Peoples R China
2.Hunan Acad Agr Sci, Agr Prod Proc Inst, Changsha 410125, Peoples R China
3.Hunan Key Lab Fruits & Vegetables Storage Proc Qua, Hunan Agr Prod Proc Inst, Changsha 410125, Peoples R China
4.Chalmers Univ Technol, Dept Life Sci, SE-41296 Gothenburg, Sweden
关键词： Enzyme promiscuity; flavonoid; (2S)-hesperetin; directed evolution; flavonoid 3'-hydroxylase; flavonoid 4'-O-methyltransferase
期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.1； 五年影响因子：6.3 ）
ISSN： 0021-8561
年卷期： 2023 年 71 卷 25 期
页码：
收录情况： SCI
摘要： Enzyme promiscuity is evolutionarily advantageous to plants for gaining new enzyme functions when adapting to environmental challenges. However, this promiscuity can negatively affect the expression of genes encoding for plant enzymes in microorganisms. Here, we show that refining the promiscuity of flavonoid 3'-hydroxylase (F3'H) and 4'-O-methyltransferase (F4'OMT) improves (2S)-hesperetin production in Escherichia coli. First, we employed inverse molecular docking to screen a highly substrate-specific ThF3'H from Tricyrtis hirta, which could selectively convert 100 mg L-1 (2S)-naringenin to (2S)-eriodictyol but not (2S)-isosakuranetin, with a cytochrome P450 reductase from Arabidopsis thaliana. Second, we employed a directed evolution approach to restrict the promiscuity of MpOMT from Mentha x piperita. The strain harboring the MpOMT(S142V) mutant presented a remarkably increased preference for (2S)-eriodictyol. Finally, 27.5 mg L-1 (2S)-hesperetin was produced, while only minor amounts of (2S)-eriodictyol and (2S)-isosakuranetin accumulated as byproducts. This value represents a 14-fold increase in (2S)-hesperetin compared to the parental strain, along with a dramatic reduction in side products. Our work highlights the benefit of alleviating the promiscuity of plant enzymes when engineering production of natural products by microbial cell factories.
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