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 inEscherichia
coli. First, we employed inverse molecular docking to screen
a highly substrate-specific ThF3′H fromTricyrtis hirta, which could selectively convert 100 mg L–1(2S)-naringenin to (2S)-eriodictyol but not
(2S)-isosakuranetin, with a cytochrome P450 reductase
fromArabidopsis thaliana. Second, we employed a
directed evolution approach to restrict the promiscuity of MpOMT fromMentha × piperita. The strain harboring the MpOMTS142Vmutant 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.