Presentation Information
[P03-361]In vitro Glycosylation with UDP-Glucose Regeneration Cascade
○Natsumi Miyoshi1, Takuma Suzuki2,3, Miyuki Sako1, Jonathan Ekaputra1, Kentaro Miyazaki2, Hiroya Tomita2,4, Kohsuke Honda2,4 (1. Graduate School of Engineering, The University of Osaka (Japan), 2. International Center for Biotechnology, The University of Osaka (Japan), 3. Research Fellow of Japan Society for the Promotion of Science (Japan), 4. Industrial Biotechnology Initiative Division, Institute for Open and Transdisciplinary Research Initiatives, The University of Osaka (Japan))
Keywords:
Enzyme cascade,Glycosylation,Thermophilic enzyme,Trehalose,UDP-glucose
In vitro enzyme cascades enable the artificial assembly of metabolic pathways, providing highly selective and stoichiometric production of target compounds. Because no living cells are involved, these systems allow flexible control of reaction conditions through simple parameters such as temperature and pH. However, their construction typically requires multiple purified enzymes, making the process time-consuming. To address this limitation, we employed thermostable enzymes as building blocks of the cascade. Thermostable enzymes, recombinantly produced in E. coli, can be easily semi-purified by heat treatment of the crude extracts at 70-80oC, significantly simplifying the enzyme preparation process.
In this study, we focused on glycosylation, a sugar-transferring reaction from a sugar donor to an acceptor (aglycone). Glycosylation enhances water solubility and modulates the biological activities of aglycones, improving their applicability in the pharmaceutical and food industries. Although UDP-glucose (UDPG) is a typical sugar donor, its high cost remains a major limitation for its practical application to glucoside production.
Here, we newly developed an in vitro UDPG-regeneration cascade with thermostable enzymes for high-yield glucoside production. We focused on UDP, generated as a by-product of glycosylation, and constructed a cascade that converts free UDP into UDPG, enabling glucoside production without the external addition of UDPG. The UDPG-regeneration cascade was integrated with an ATP-regeneration non-oxidative glycolysis (ArNOG) cascade, which was also reconstituted with thermophilic enzymes, as an ATP regeneration module (Suryatin Alim et al., 2022). Because most intermediates are recycled within the system, only starch and aglycones are consumed, while glucosides and acetate are generated as the final products. The cascade is potentially applicable to glycosylation of a wide variety of compounds by integrating different types of UDP-glycosyltransferase. More importantly, the reaction equilibrium in this cascade is more strongly biased towards UDPG formation than in conventional systems, such as those based on sucrose synthases.
To enhance UDPG production rates, enzyme concentrations were optimized using design of experiments (DOE). As a proof of concept, trehalose production via UDPG-dependent glycosylation of glucose was demonstrated. As a result, 98.1 mol% of trehalose was produced from 1 mM UDP and 10 mM glucose within 10 h.
In this study, we focused on glycosylation, a sugar-transferring reaction from a sugar donor to an acceptor (aglycone). Glycosylation enhances water solubility and modulates the biological activities of aglycones, improving their applicability in the pharmaceutical and food industries. Although UDP-glucose (UDPG) is a typical sugar donor, its high cost remains a major limitation for its practical application to glucoside production.
Here, we newly developed an in vitro UDPG-regeneration cascade with thermostable enzymes for high-yield glucoside production. We focused on UDP, generated as a by-product of glycosylation, and constructed a cascade that converts free UDP into UDPG, enabling glucoside production without the external addition of UDPG. The UDPG-regeneration cascade was integrated with an ATP-regeneration non-oxidative glycolysis (ArNOG) cascade, which was also reconstituted with thermophilic enzymes, as an ATP regeneration module (Suryatin Alim et al., 2022). Because most intermediates are recycled within the system, only starch and aglycones are consumed, while glucosides and acetate are generated as the final products. The cascade is potentially applicable to glycosylation of a wide variety of compounds by integrating different types of UDP-glycosyltransferase. More importantly, the reaction equilibrium in this cascade is more strongly biased towards UDPG formation than in conventional systems, such as those based on sucrose synthases.
To enhance UDPG production rates, enzyme concentrations were optimized using design of experiments (DOE). As a proof of concept, trehalose production via UDPG-dependent glycosylation of glucose was demonstrated. As a result, 98.1 mol% of trehalose was produced from 1 mM UDP and 10 mM glucose within 10 h.
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