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

[Image: see text] 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...

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Autores principales: Liu, Juan, Xiao, Zhiqiang, Zhang, Siqi, Wang, Zhen, Chen, Yun, Shan, Yang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10311525/
https://www.ncbi.nlm.nih.gov/pubmed/37310069
http://dx.doi.org/10.1021/acs.jafc.3c02071
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author Liu, Juan
Xiao, Zhiqiang
Zhang, Siqi
Wang, Zhen
Chen, Yun
Shan, Yang
author_facet Liu, Juan
Xiao, Zhiqiang
Zhang, Siqi
Wang, Zhen
Chen, Yun
Shan, Yang
author_sort Liu, Juan
collection PubMed
description [Image: see text] 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 × 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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spelling pubmed-103115252023-07-01 Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli Liu, Juan Xiao, Zhiqiang Zhang, Siqi Wang, Zhen Chen, Yun Shan, Yang J Agric Food Chem [Image: see text] 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 × 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. American Chemical Society 2023-06-13 /pmc/articles/PMC10311525/ /pubmed/37310069 http://dx.doi.org/10.1021/acs.jafc.3c02071 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Liu, Juan
Xiao, Zhiqiang
Zhang, Siqi
Wang, Zhen
Chen, Yun
Shan, Yang
Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
title Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
title_full Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
title_fullStr Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
title_full_unstemmed Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
title_short Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli
title_sort restricting promiscuity of plant flavonoid 3′-hydroxylase and 4′-o-methyltransferase improves the biosynthesis of (2s)-hesperetin in e. coli
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10311525/
https://www.ncbi.nlm.nih.gov/pubmed/37310069
http://dx.doi.org/10.1021/acs.jafc.3c02071
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