Manufacturing technologies
Medical & healthcare, Functional foods, Industrial materials

High-yield bioactive triterpenoid production by heterologous
expression of biosynthetic genes in Nicotiana benthamiana
Department of Biotechnology, Graduate School of Engineering
Associate Professor Hikaru Seki

主要図表
(事事 務 局 にてhttps://researchmap.jp/read0152753?lang=en

Abstract
Maslinic acid is a natural triterpenoid contained in olive fruit. It
β-amyrin
CYP716C53
CYP716A12
synthase (bAS)
has been reported that maslinic acid is very effective in improving
locomotive ability of old subjects in a clinical test. Transient protein
expression in Nicotiana benthamiana leaves via agroinfiltration isa simpler and flexible method for producing high levels of recombinant proteins. In this study, maslinic acid was produced in N.
C-28
C-2α
benthamiana leaves by transiently introducing five biosynthetic
maslinic acid
2,3-oxidosqualene β-amyrin
oleanolic acidenzyme genes for maslinic acid, yielding 20.7 times higher conFigure 1 Engineered biosynthetic pathway of maslinic acid constructed incentration than that in olive fruit (cv. Kalamata), which have been
Nicotiana benthamiana leaves
主要図表2reported to contain high maslinic acid concentration. This study
図1．ベンサミアナタバコ葉に構築したマスリン酸生合成経路
established a general strategy for rapidly producing substantial
Figure 1 Engineered biosynthetic pathway of maslinic acid constructed in Nicotiana benthamiana leaves
quantities of useful triterpenoids.

Manufacturing technologies▍ Background & Results
Triterpenoids are a class of structurally diverse plant specialized(secondary) metabolites commonly derived from 2,3-oxidosqualene(the precursor of sterols, steroids, and triterpenoids). These compounds have been shown to have various health beneficial properties. Maslinic acid contained in olive fruits is very effective in improving locomotive ability of elderly people. Supplement products
containing maslinic acid were accepted by the Consumer Affairs
Agency as “Foods with Functional Claims” in Japan.
The plant N. benthamiana is used as a heterologous expression host for rapid and high-level recombinant protein production
by agroinfiltration-based transient transformation. The Tsukuba
system is one of the most powerful transient protein expression
systems utilizing agroinfiltration, based on the use of a geminiviral
replication vector for boosting expression. In this study, we used
Tsukuba system to produce maslinic acid by transiently expressing
five biosynthetic enzymes of maslinic acid. Using this system, wedemonstrate that maslinic acid was produced in N. benthamianaleaves, yielding 27.2 ± 3.0 mg/g dw at 7 days after agroinfiltration.
This concentration is 20.7 times higher than that in olive fruit (cv.
Kalamata), which have been reported to contain high maslinic acidconcentration. In conclusion, this study established a frameworkfor optimizing triterpenoids production via agrobacterium-mediatedtransient expression in N. benthamiana using the Tsukuba system.

tHMGRbAS

Maslinic acid(maintaining skeletal muscle)

CPR

CYP716A12 CYP716C53

Engineered pathway
ABIJKC DEF

Figure 2 Schematic representation of the agroinfiltration process to construct
engineered pathway of maslinic acid in N. benthamiana leaves
図2．アグロ浸潤法による⼀過的なマスリン酸⽣合成経路構築のスキーム

Figure 2 Schematic representation of the agroinfiltration process to construct engineered pathway
of maslinic acid in N. benthamiana leaves

Maslinic acidproduction

27.2 ± 3.0mg/g dw▍ Significance of the research and Future perspective
The accumulation levels of triterpenoids in raw plant materials
are sometimes very low and may also depend on the age of theplant. In this study, we produced substantial amounts of maslinicacid, as an example of a health-beneficial plant-derived triterpenoid, by transiently transformed N. benthamiana leaves in a week.
This research will contribute to the development of a more efficientplatform for synthesizing useful triterpenoids and sustainable useof plant resources.

補足図表+: With- : Without
ND: Not detectedAtHMGR-ND ND ND++ND

ND+bAS+CPR+++

CYP716A12-++CYP716C53--+ND+

Supplementary Figure Schematic representation of the agroinfiltration process
to construct
engineered pathway of maslinic acid in N. benthamiana leaves
補⾜図．ベンサミアナタバコ葉におけるマスリン酸合成量
Supplementary Figure Schematic representation of the agroinfiltration process to construct
engineered pathway of maslinic acid in N. benthamiana leaves

P a t e n t
Romsuk, Jutapat; Yasumoto, Shuhei; Fukushima, Ery Odette et al. High-yield bioactive triterpenoid production by heterologous expression in Nicotiana
benthamiana using the Tsukuba system. Frontiers in Plant Science. 2022, 13, 991909. doi: 10.3389/fpls.2022.991909
Chung, Soo Yeon; Seki, Hikaru; Fujisawa Yukiko et al. A cellulose synthase-derived enzyme catalyses 3-O-glucuronosylation in saponin biosynthesis.
Tr e a t i s e
Nature Communications. 2020, 11(1): 5664. doi: 10.1038/s41467-020-19399-0
Seki, Hikaru; Tamura, Keita; Muranaka, Toshiya. Plant-derived isoprenoid sweeteners: recent progress in biosynthetic gene discovery and perspectives on
microbial production. Bioscience, biotechnology, and biochemistry. 2018, 82(6) 927-934. doi: 10.1080/09168451.2017.1387514
URLK e y w o r d phytochemical, functional ingredients, dietary supplement, medicinal plants