Manufacturing technologies
Functional foods, Industrial materials, Medical / Healthcare

Development of sustainable production of useful secondary
metabolites by microorganisms and plants
Division of Biotechnology, Graduate School of Engineering
Associate Professor Hikaru Seki

Researchmap https://researchmap.jp/read0152753?lang=en

Professor Toshiya Muranaka

Researchmap https://researchmap.jp/bio?lang=en

Abstract
Glycyrrhizin, which is contained in the underground part of the
medicinal plant “licorice”, has many pharmacological properties,
such as anti-inflammatory, anti-ulcer and hepatoprotective activities. And also use as a natural sweetener because it is about 150
times sweeter than sugar. Almost all licorice is imported from overseas, but the decrease in the number of high-quality products andthe increase in prices have become a problem. Therefore, there
has been a demand to develop a sustainable alternative production method. We have been working on the isolation and functional
analysis of glycyrrhizin biosynthesis genes in licorice, including theglycosyltransferase of the second sugar in 2019, the glycosyltransferase of the first sugar in 2020. In 2021, we elucidated the molecular mechanism of glycyrrhizin-producing/non-producing licorice.
Furthermore, by introducing a total of seven genes into budding
yeast, we succeeded in production of glycyrrhizin in yeast.

Manufacturing technologies▍ Background & Results
Triterpene glycosides (generally called saponins) are a group ofcompounds in which multiple sugars are attached to the carbon
skeleton of a triterpene, and are known to be the main active ingredients in many herbal medicines. Glycyrrhizin, a saponin extracted
from licorice root, is used as a raw material for medicines such as
liver disease remedies, and also as a natural sweetener. The process of saponin synthesis in plant cells involves cytochrome P450
monooxygenase (CYP), which oxidizes and modifies the triterpene
carbon skeleton to produce aglycones with various structures,
followed by several UDP sugar-dependent glycosyltransferases(UGT), which transfer sugars to the aglycones (glycosylation). We
first isolated CYPs involved in glycyrrhizin biosynthesis, and thenUGT73P12 as the UGT that transfers the second sugar, glucuronic acid, in the second step of glycosylation (Nomura, Seki et al.
2019). However, the enzyme that catalyzes the glucuronic acidtransfer to the 3-position hydroxyl group, the first step of glycosylation, remained unknown. We used a gene co-expression analysis

β-amyrin11-oxo-β-amyrin

method to narrow down the list of genes to search for candidates
for the uncharacterized glucuronosyltransferase. As a result, wefound that a protein with unknown function (named CSyGT), unlike
UGT, which is highly similar to cellulose synthase, is an enzymethat catalyzes the glucuronyl transfer reaction to the hydroxyl groupat position 3 (Chung, Seki et al. 2020). We also elucidated the molecular mechanism of glycyrrhizin-producing/non-glycyrrhizin-producing licorice (Fanani et al 2021). Furthermore, by introducing a
total of seven plant enzyme genes, including CSyGT, into yeast, we
succeeded in production of glycyrrhizin in yeast for the first time in
the world.

▍ Significance of the research and Future perspectiveの関係で問題が生じた場合は，こちらでトリミング等の加工をさせていThis research is expected to pave the way for the industrialあります。
productionof glycyrrhizin using yeast and plants that have been
genetically modified with the genes discovered in this study, which
in turn will help prevent theoverharvesting of natural
resources and preserve theecosystem. Plants produce
a wide variety of specialized(secondary) metabolites, asmany as one million different types. By applying newtechnologies, such as synthetic biology and genome
editing, to many specialized
metabolites other than glycyrrhizin, we can expect
to build an environmentallyfriendly and sustainable
material production systemthat can be used as raw
materials for pharmaceuticals, functional foods, andindustrial materials.
Fig. 1 Licorice root
図 1. カンゾウの根（甘草）（主要図）

Glycyrrhetinicacid

Glycyrrhetinic acid
3-O-glucuronide

Glycyrrhizin

2,3-oxidosqualene
common precursorbAS

CYP88D6

CYP72A154
CYP72A63

CSyGT
UGT73P12
図 2. グリチルリチンの生合成経路および生合成酵素（主要図）

Fig. 2 Biosynthetic pathway of Glycyrrhizin

１2. ご自身の顔写真のご提供※ 印刷可能な解像度のものを電子ファイルにてご提供ください。
P a t e n t JP6344774, PCT/JP2020/39175
別途添付します。
Fanani, M.Z; Sawai, S; Seki, H. et al. Allylic hydroxylation activity is a source of saponin chemodiversity in the genus Glycyrrhiza . Plant Cell Physiol. 2021;
62(2): 262-271. doi: 10.1093/pcp/pcaa173
Chung, S.Y; Seki, H. et al. A cellulose synthase-derived enzyme catalyses 3-O -glucuronosylation in saponin biosynthesis. Nat. Commun. 2020; 11(1):Tr e a t i s e5664. doi: 10.1038/s41467-020-19399-0.
Nomura, Y; Seki, H. et al. Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin.
Plant J. 2019; 99(6): 1127-1143. doi: 10.1111/tpj.14409.
UR
L http://www.bio.eng.osaka-u.ac.jp/pl/index_e.html
K e y w o r d genome editing, medicinal plants, metabolic engineering, plant specialized metabolism, synthetic biology