Complete biosynthesis of the phenylethanoid glycoside verbascoside

Yang, Yijun; Xi, Daoyi; Liu, Tao

doi:10.1016/j.xplc.2023.100592

Cited by 12 publications

(13 citation statements)

References 63 publications

(91 reference statements)

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“…In conclusion, the highest hydroxytyrosol production to date has been achieved with the addition of glycerol through modifications of the carbon metabolic pathway, cofactor engineering, and fermentation optimization. In fact, in recent years, there have also been many studies related to the upstream and downstream products of hydroxytyrosol, such as tyrosol and hydroxytyrosol acetate. , The present study provides a strategy for the biosynthesis of other similar substances and lays the foundation for the biosynthesis of downstream substances such as verbascoside . However, there are still many strategies to be explored, and the supply of precursors needs to be further strengthened.…”

Section: Discussionmentioning

confidence: 88%

Promoting FADH₂ Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Wang,

Chen

et al. 2023

J. Agric. Food Chem.

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Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

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Section: Discussionmentioning

confidence: 88%

Promoting FADH₂ Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Wang,

Chen

et al. 2023

J. Agric. Food Chem.

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“…The recent elucidation of the biosynthetic pathway for verbascoside production in plants has even enabled its heterologous production in Escherichia coli . The in vitro production of verbascoside by plant cells, at a high level and without the need of extensive purification, offers a significant advantage.…”

Section: Resultsmentioning

confidence: 99%

Highly Efficient Verbascoside Production from Olive (Olea europea L. var. Cellina di Nardò) In Vitro Cell Cultures

Blando,

Mita,

Di Sansebastiano

et al. 2024

J. Agric. Food Chem.

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Olive (Olea europea L.) is one of the oldest and most important fruit tree species cultivated in the Mediterranean region. Various plant tissues, drupes, and olive oil contain several phenolics (including verbascoside, although it is present in the plant at a low level) that are well-known for their highly beneficial effects on human health. An in vitro olive cell suspension culture (cultivar Cellina di Nardo, "CdN") was established, characterized for its growth and morphological features. Furthermore, a vital and relatively uniform population of protoplasts was generated from the olive suspension culture to investigate their cellular characteristics during growth. The polyphenolic extract of the in vitro "CdN" olive cells contained almost exclusively verbascoside, as revealed by the UPLC-ESI-MS analysis. The content of verbascoside reached up to 100 mg/g DW, with an average production rate of approximately 50 mg/g DW over one year of culture. This level of production has not been previously reported in a limited number of previous studies. This remarkable production of verbascoside was associated with an exceptionally high antioxidant capacity. The high level of verbascoside production and purity of the extract make this system a promising tool for secondary metabolite production.

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“…The biosynthetic pathway for PhGs had been established in Ligustrum robustum and other Lamiaceae plants, where the hydroxylation of the Tyl and Cou units by CYP98 A20 [24] and its homologs was proposed as the final step. In contrast, PhGs in F. suspensa and C. macrophylla predominantly consist of di‐hydroxyl building blocks such as HyTyl and Caf, with only a few exceptions (Figure S1–2, S4) [31] .…”

Section: Resultsmentioning

confidence: 99%

“…Except for UGT85AF10/11, all other GTs favors for mono‐hydroxylated units such as Tyl and Cou. Additionally, a group of CYP98A enzymes [24] has been reported to hydroxylate Tyl or Cou units as the last step. However, these enzymes are insufficient to capture the full structural diversity of PhGs.…”

Section: Introductionmentioning

confidence: 99%

Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

Yao,

Wang,

Wang

et al. 2024

Angew Chem Int Ed

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Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'‐OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one‐pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6‐fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5‐100%, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhGs biosynthesis, but also serves as a proof‐of‐concept for cell‐free enzymatic construction of diverse natural products.

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Complete biosynthesis of the phenylethanoid glycoside verbascoside

Cited by 12 publications

References 63 publications

Promoting FADH₂ Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Promoting FADH₂ Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Highly Efficient Verbascoside Production from Olive (Olea europea L. var. Cellina di Nardò) In Vitro Cell Cultures

Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

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Complete biosynthesis of the phenylethanoid glycoside verbascoside

Cited by 12 publications

References 63 publications

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Highly Efficient Verbascoside Production from Olive (Olea europea L. var. Cellina di Nardò) In Vitro Cell Cultures

Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

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Promoting FADH₂ Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli

Promoting FADH₂ Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli