Presentation Information
[3Plant-03]Heterologous production of corosolic acid, a "phyto-insulin", in agroinfiltrated Nicotiana benthamiana leaves
○Hikaru Seki1,2, Jutapat Romsuk1, Much Zaenal Fanani1, Kenji Miura3,4, Toshiya Muranaka2 (1. Department of Biotechnology, Graduate School of Engineering, The University of Osaka (Japan), 2. Institute for Open and Transdisciplinary Research Initiatives, The University of Osaka (Japan), 3. College of Biological Sciences, University of Tsukuba (Japan), 4. Tsukuba-Plant Innovation Research Center (Japan))
Keywords:
Plant specialized metabolites,Triterpenoids,Nicotiana benthamiana,Transient expression,Agroinfiltration
[Purpose]
Corosolic acid (2α-hydroxy-ursolic acid), a pentacyclic triterpenoid found in the mature leaves of Lagerstroemia speciosa (commonly called Banaba), is well known as a “phyto-insulin” and has recently gained attention for its insulin-like effects, but without triggering anti-insulin antibody production. In this study, we aimed to develop a biotechnological platform for producing corosolic acid by transient co-expression of biosynthetic enzymes in agroinfiltrated Nicotiana benthamiana leaves.
[Method]
We used the Tsukuba system, an agroinfiltration-based method for transient protein expression in plants. This system was developed for high-level transient expression of foreign proteins using a binary vector with a geminiviral replication system and a double terminator. We employed this system to produce corosolic acid in N. benthamiana leaves by co-expressing five enzymes. An Agrobacterium suspension containing the desired constructs was combined and infiltrated into the leaves of 5-week-old N. benthamiana plants using a needleless syringe. To prevent tissue necrosis, ascorbic acid solution was sprayed onto the leaves on days 1, 3, and 5 after infiltration. The leaves from three plants were collected for triterpenoid analysis seven days post-infiltration.
[Results]
We carried out simultaneous transient expression of an oxidosqualene cyclase from the medicinal legume Bauhinia forficata (BfOSC3), which mainly synthesizes α-amyrin from 2,3-oxidosqualene—a C30 acyclic precursor produced via the mevalonate pathway—along with Medicago truncatula CYP716A12 to catalyze the oxidation of α-amyrin, resulting in ursolic acid as a direct precursor for corosolic acid, and Avicennia marina CYP716C53 to catalyze the C-2α-hydroxylation of ursolic acid to form corosolic acid. We also increased the yield by co-expressing a feedback-insensitive form of Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the mevalonate pathway, along with a cytochrome P450 reductase as an electron transfer partner for CYPs. This approach yielded a corosolic acid content of 18.4 ± 3.6 mg/g dw. This amount is 4.3 times higher than the average level reported in mature banaba leaves. It also noted that the highest corosolic acid concentration is found in mature banaba leaves. Therefore, it is important to highlight that the yield of 18.4 ± 3.6 mg/g dw in N. benthamiana leaves was obtained 7 days after agroinfiltration.
[Consideration]
One advantage of this method is that it is very easy to introduce multiple genes at once by mixing and inoculating several Agrobacterium strains, each carrying a binary vector with the desired foreign gene. This feature is highly beneficial for flexible metabolic pathway design and on-demand compound production by combining enzyme genes. Moving forward, it is crucial to develop optimized "chassis N. benthamiana strains" as platforms to enhance production yield.
[Conclusion]
This study achieved high-yield heterologous production of corosolic acid, known as a “phyto-insulin,” in N. benthamiana leaves by combining the Tsukuba system and protein engineering. Our results offer an alternative method for producing health-beneficial triterpenoids beyond extraction from natural plant sources, which will also promote innovation in drug discovery.
[Acknowledgment]
This work was supported by JST GteX Program Japan Grant Number JPMJGX23B0.
Corosolic acid (2α-hydroxy-ursolic acid), a pentacyclic triterpenoid found in the mature leaves of Lagerstroemia speciosa (commonly called Banaba), is well known as a “phyto-insulin” and has recently gained attention for its insulin-like effects, but without triggering anti-insulin antibody production. In this study, we aimed to develop a biotechnological platform for producing corosolic acid by transient co-expression of biosynthetic enzymes in agroinfiltrated Nicotiana benthamiana leaves.
[Method]
We used the Tsukuba system, an agroinfiltration-based method for transient protein expression in plants. This system was developed for high-level transient expression of foreign proteins using a binary vector with a geminiviral replication system and a double terminator. We employed this system to produce corosolic acid in N. benthamiana leaves by co-expressing five enzymes. An Agrobacterium suspension containing the desired constructs was combined and infiltrated into the leaves of 5-week-old N. benthamiana plants using a needleless syringe. To prevent tissue necrosis, ascorbic acid solution was sprayed onto the leaves on days 1, 3, and 5 after infiltration. The leaves from three plants were collected for triterpenoid analysis seven days post-infiltration.
[Results]
We carried out simultaneous transient expression of an oxidosqualene cyclase from the medicinal legume Bauhinia forficata (BfOSC3), which mainly synthesizes α-amyrin from 2,3-oxidosqualene—a C30 acyclic precursor produced via the mevalonate pathway—along with Medicago truncatula CYP716A12 to catalyze the oxidation of α-amyrin, resulting in ursolic acid as a direct precursor for corosolic acid, and Avicennia marina CYP716C53 to catalyze the C-2α-hydroxylation of ursolic acid to form corosolic acid. We also increased the yield by co-expressing a feedback-insensitive form of Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the mevalonate pathway, along with a cytochrome P450 reductase as an electron transfer partner for CYPs. This approach yielded a corosolic acid content of 18.4 ± 3.6 mg/g dw. This amount is 4.3 times higher than the average level reported in mature banaba leaves. It also noted that the highest corosolic acid concentration is found in mature banaba leaves. Therefore, it is important to highlight that the yield of 18.4 ± 3.6 mg/g dw in N. benthamiana leaves was obtained 7 days after agroinfiltration.
[Consideration]
One advantage of this method is that it is very easy to introduce multiple genes at once by mixing and inoculating several Agrobacterium strains, each carrying a binary vector with the desired foreign gene. This feature is highly beneficial for flexible metabolic pathway design and on-demand compound production by combining enzyme genes. Moving forward, it is crucial to develop optimized "chassis N. benthamiana strains" as platforms to enhance production yield.
[Conclusion]
This study achieved high-yield heterologous production of corosolic acid, known as a “phyto-insulin,” in N. benthamiana leaves by combining the Tsukuba system and protein engineering. Our results offer an alternative method for producing health-beneficial triterpenoids beyond extraction from natural plant sources, which will also promote innovation in drug discovery.
[Acknowledgment]
This work was supported by JST GteX Program Japan Grant Number JPMJGX23B0.
Comment
To browse or post comments, you must log in.Log in