Presentation Information
[P03-448]Synthetic Biology–Driven Rewiring of Triterpenoid Biosynthesis toward Glycyrrhizin Production in Licorice Hairy Roots
○Zitai Wang1, Zhixian Song2, Shuhei Yasumoto2, Seki Hikaru1,2, Toshiya Muranaka1,2 (1. OTRI, Univ. Osaka (Japan), 2. Grad. Sch. Eng., Univ. Osaka (Japan))
Keywords:
Genome editing,Glycyrrhizin,Hairy root culture,Metabolic engineering
[Purpose]
Glycyrrhizin (GL), a triterpenoid saponin derived from the roots of Glycyrrhiza uralensis (Licorice), is highly valued for its various bioactive properties. To address the shortage of natural licorice resources, Agrobacterium rhizogenes–induced hairy roots have emerged as a promising alternative production platform. However, GL accumulation in hairy roots is extremely limited, as it shares the common precursor β-amyrin with the competing soyasaponin biosynthetic pathway. Our previous study demonstrated that CRISPR/Cas9-mediated knockout of two soyasaponin biosynthetic CYP genes (CYP93E3 and CYP72A566, DKO) induced GL accumulation, which was further enhanced by the combination of overexpression of CYP88D6 (DKO/88D6-OX), a key GL biosynthetic enzyme (Chiyo et al. 2024). In this study, we further engineered the metabolic pathway by co-overexpressing CYP88D6 and CYP72A154 on the DKO background (DKO/DOX) to enhance metabolic flux toward GL production.
[Method]
DKO/DOX hairy root lines were generated via A. rhizogenes-mediated transformation using a vector combining CRISPR/Cas9-mediated double knockout (CYP93E3 and CYP72A566) with co-overexpression of CYP88D6 and CYP72A154. Gene knockout and expression levels were confirmed by sequencing and qPCR, respectively. Metabolite profiles were analyzed and quantified using LC-MS with authentic standards.
[Results]
Thirty-one seeds were sown and infected with Agrobacterium harboring the DKO/DOX vector, resulting in the isolation of four independent DKO/DOX hairy root lines, as confirmed by sequencing and qPCR. LC-MS analysis revealed that GL was undetectable in the empty vector control hairy roots. In contrast, DKO/88D6-OX lines accumulated approximately 1.2 mg/g dry weight of GL. This level was further increased to approximately 5.4 mg/g DW in DKO/DOX lines with co-overexpression of CYP88D6 and CYP72A154.
[Consideration]
Further comparison of CYP72A154 overexpression with and without CYP88D6 on the same DKO background will help clarify their individual and synergistic contributions to metabolic flux through the GL pathway. In addition, optimization strategies should extend beyond P450 oxygenases to include redox partners such as cytochrome P450 reductase (CPR), which may improve catalytic efficiency.
[Conclusion]
These results demonstrate that a combinatorial metabolic engineering strategy, blocking competing pathways while reinforcing rate-limiting steps, can significantly enhance GL production in licorice hairy roots. This approach provides a strong foundation for future optimization toward industrial-scale production.
[Acknowledgement]
This work was partially supported by the Cabinet Office, Government of Japan, through programs for Bridging the gap between R&d and the IDeal society (society 5.0) and Generating Economic and social value (BRIDGE) on “Creation of industrial plants using simultaneously modified genome editing technology, Grant Number JPJ013009”.
Glycyrrhizin (GL), a triterpenoid saponin derived from the roots of Glycyrrhiza uralensis (Licorice), is highly valued for its various bioactive properties. To address the shortage of natural licorice resources, Agrobacterium rhizogenes–induced hairy roots have emerged as a promising alternative production platform. However, GL accumulation in hairy roots is extremely limited, as it shares the common precursor β-amyrin with the competing soyasaponin biosynthetic pathway. Our previous study demonstrated that CRISPR/Cas9-mediated knockout of two soyasaponin biosynthetic CYP genes (CYP93E3 and CYP72A566, DKO) induced GL accumulation, which was further enhanced by the combination of overexpression of CYP88D6 (DKO/88D6-OX), a key GL biosynthetic enzyme (Chiyo et al. 2024). In this study, we further engineered the metabolic pathway by co-overexpressing CYP88D6 and CYP72A154 on the DKO background (DKO/DOX) to enhance metabolic flux toward GL production.
[Method]
DKO/DOX hairy root lines were generated via A. rhizogenes-mediated transformation using a vector combining CRISPR/Cas9-mediated double knockout (CYP93E3 and CYP72A566) with co-overexpression of CYP88D6 and CYP72A154. Gene knockout and expression levels were confirmed by sequencing and qPCR, respectively. Metabolite profiles were analyzed and quantified using LC-MS with authentic standards.
[Results]
Thirty-one seeds were sown and infected with Agrobacterium harboring the DKO/DOX vector, resulting in the isolation of four independent DKO/DOX hairy root lines, as confirmed by sequencing and qPCR. LC-MS analysis revealed that GL was undetectable in the empty vector control hairy roots. In contrast, DKO/88D6-OX lines accumulated approximately 1.2 mg/g dry weight of GL. This level was further increased to approximately 5.4 mg/g DW in DKO/DOX lines with co-overexpression of CYP88D6 and CYP72A154.
[Consideration]
Further comparison of CYP72A154 overexpression with and without CYP88D6 on the same DKO background will help clarify their individual and synergistic contributions to metabolic flux through the GL pathway. In addition, optimization strategies should extend beyond P450 oxygenases to include redox partners such as cytochrome P450 reductase (CPR), which may improve catalytic efficiency.
[Conclusion]
These results demonstrate that a combinatorial metabolic engineering strategy, blocking competing pathways while reinforcing rate-limiting steps, can significantly enhance GL production in licorice hairy roots. This approach provides a strong foundation for future optimization toward industrial-scale production.
[Acknowledgement]
This work was partially supported by the Cabinet Office, Government of Japan, through programs for Bridging the gap between R&d and the IDeal society (society 5.0) and Generating Economic and social value (BRIDGE) on “Creation of industrial plants using simultaneously modified genome editing technology, Grant Number JPJ013009”.
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