Presentation Information
[P01-079]Engineering Saccharomyces cerevisiae for Efficient β-Glucogallin Production via Sucrose Synthase-Mediated UDP-Glucose Regeneration
○Dawon Seo1, Ho Joon Kim1, Sang Woo Seo1,2,3,4,5 (1. School of Chemical and Biological Engineering, Seoul National University (Korea), 2. Interdisciplinary Program in Bioengineering, Seoul National University (Korea), 3. Institute of Chemical Processes, Engineering, Seoul National University (Korea), 4. Bio-MAX Institute, Seoul National University (Korea), 5. Institute of Engineering Research, Seoul National University (Korea))
Keywords:
β-Glucogallin,Saccharomyces cerevisiae,UDP-glucose regeneration,Metabolic engineering,Sucrose synthase
[Purpose] β-Glucogallin is a polyphenolic secondary metabolite found in plants and fungi with reported skin benefits, including barrier reinforcement, wound healing, and UV protection. However, its use is limited by low natural abundance and costly extraction. The global cosmeceuticals market, valued at USD 67–81 billion in 2024–2025, continues to expand, highlighting the demand for sustainable production of high-value bioactive compounds. In this study, we aimed to establish a sustainable microbial platform for β-glucogallin production in Saccharomyces cerevisiae.
[Method] To prevent product degradation, endogenous β-glucosidase genes were deleted. We then introduced UGT84A25a from Eucalyptus camaldulensis and evaluated its β-glucogallin-producing activity both in vitro and in vivo. For stable production, UGT84A25a was integrated and compared with plasmid-based expression. To enhance precursor supply, we introduced a sucrose synthase (SUS)-based UDP(G) regeneration strategy. SUS activity was first evaluated in vitro and then applied in vivo after disruption of sucrose-hydrolyzing genes. Based on growth and production balance, a sucrose:glucose ratio of 1:3 was selected and used for screening multiple SUS candidates. Finally, the selected SUS was combined with a gallic acid biosynthetic pathway together with UGT84A25a to enable β-glucogallin production.
[Results] Deletion of EXG1 and SPR1 prevented β-glucogallin degradation: the wild-type strain hydrolyzed β-glucogallin to gallic acid, whereas the knockout strain did not. In vitro assays confirmed β-glucogallin production by UGT84A25a. In vivo, Δbgl / UGT84A25a::YIR007W produced up to 21.03 mg/L β-glucogallin after 48 h in YPD, whereas plasmid-based expression produced 6.71 mg/L in YSC-His medium, both with 1 g/L gallic acid. SUS-based UDP-glucose regeneration increased β-glucogallin production in vitro, indicating precursor limitation. In vivo, GmSUS was first evaluated under mixed carbon-source conditions, and the best SUS candidate was selected at a sucrose:glucose ratio of 1:3. The final strain, carrying SUS, AroZ, PobA*, and UGT84A25a, produced β-glucogallin from sucrose and glucose, confirmed by LC-MS/MS in SRM mode with a precursor ion at m/z 331 (Q1).
[Consideration] These results indicate that both product stability and UDP-glucose availability are major determinants of β-glucogallin production in S. cerevisiae. Preventing β-glucogallin hydrolysis preserved product, while SUS-based UDP-glucose regeneration improved precursor supply. Balancing sucrose and glucose also supported both growth and production.
[Conclusion] We established an engineered S. cerevisiae platform for β-glucogallin production by combining β-glucosidase deletion, UGT84A25a expression, SUS-based UDP-glucose regeneration, and a gallic acid biosynthetic pathway. This strategy demonstrates the feasibility of sustainable microbial production of β-glucogallin and related glycosylated natural products.
[Method] To prevent product degradation, endogenous β-glucosidase genes were deleted. We then introduced UGT84A25a from Eucalyptus camaldulensis and evaluated its β-glucogallin-producing activity both in vitro and in vivo. For stable production, UGT84A25a was integrated and compared with plasmid-based expression. To enhance precursor supply, we introduced a sucrose synthase (SUS)-based UDP(G) regeneration strategy. SUS activity was first evaluated in vitro and then applied in vivo after disruption of sucrose-hydrolyzing genes. Based on growth and production balance, a sucrose:glucose ratio of 1:3 was selected and used for screening multiple SUS candidates. Finally, the selected SUS was combined with a gallic acid biosynthetic pathway together with UGT84A25a to enable β-glucogallin production.
[Results] Deletion of EXG1 and SPR1 prevented β-glucogallin degradation: the wild-type strain hydrolyzed β-glucogallin to gallic acid, whereas the knockout strain did not. In vitro assays confirmed β-glucogallin production by UGT84A25a. In vivo, Δbgl / UGT84A25a::YIR007W produced up to 21.03 mg/L β-glucogallin after 48 h in YPD, whereas plasmid-based expression produced 6.71 mg/L in YSC-His medium, both with 1 g/L gallic acid. SUS-based UDP-glucose regeneration increased β-glucogallin production in vitro, indicating precursor limitation. In vivo, GmSUS was first evaluated under mixed carbon-source conditions, and the best SUS candidate was selected at a sucrose:glucose ratio of 1:3. The final strain, carrying SUS, AroZ, PobA*, and UGT84A25a, produced β-glucogallin from sucrose and glucose, confirmed by LC-MS/MS in SRM mode with a precursor ion at m/z 331 (Q1).
[Consideration] These results indicate that both product stability and UDP-glucose availability are major determinants of β-glucogallin production in S. cerevisiae. Preventing β-glucogallin hydrolysis preserved product, while SUS-based UDP-glucose regeneration improved precursor supply. Balancing sucrose and glucose also supported both growth and production.
[Conclusion] We established an engineered S. cerevisiae platform for β-glucogallin production by combining β-glucosidase deletion, UGT84A25a expression, SUS-based UDP-glucose regeneration, and a gallic acid biosynthetic pathway. This strategy demonstrates the feasibility of sustainable microbial production of β-glucogallin and related glycosylated natural products.
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