Restricting Promiscuity of Plant Flavonoid 3'-Hydroxylase and 4'-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
Journal article, 2023

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.

Enzyme promiscuity

flavonoid 3'-hydroxylase

directed evolution

flavonoid

flavonoid 4'-O-methyltransferase

(2S)-hesperetin

Author

Juan Liu

Chalmers, Life Sciences, Systems and Synthetic Biology

Zhiqiang Xiao

Hunan Academy of Agricultural Sciences

Hunan University

Hunan Key Lab Fruits & Vegetables Storage Proc Qua

Siqi Zhang

Hunan Academy of Agricultural Sciences

Hunan University

Hunan Key Lab Fruits & Vegetables Storage Proc Qua

Zhen Wang

Hunan University

Hunan Academy of Agricultural Sciences

Hunan Key Lab Fruits & Vegetables Storage Proc Qua

Yun Chen

Chalmers, Life Sciences, Systems and Synthetic Biology

Yang Shan

Hunan University

Hunan Academy of Agricultural Sciences

Hunan Key Lab Fruits & Vegetables Storage Proc Qua

Journal of Agricultural and Food Chemistry

0021-8561 (ISSN) 1520-5118 (eISSN)

Vol. 71 25 9826-9835

Subject Categories

Biochemistry and Molecular Biology

Biocatalysis and Enzyme Technology

DOI

10.1021/acs.jafc.3c02071

PubMed

37310069

More information

Latest update

9/6/2023 9