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

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Biokatalys och enzymteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Biocatalysis and Enzyme Technology (hsv//eng)

Nyckelord

Enzyme promiscuity

flavonoid 3'-hydroxylase

directed evolution

flavonoid

flavonoid 4'-O-methyltransferase

(2S)-hesperetin

Publikations- och innehållstyp

art (ämneskategori)

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