Cheng, L., Zhang, H., Cui, H. et al. (2020) Efficient production of the anti-aging drug
Cycloastragenol: insight from two Glycosidases by enzyme mining. Applied
Microbiology and Biotechnology: 104 9991-10004.
https://doi.org/10.1007/s00253-020-10966-5
Li, Q., Wu, T., Zhao, L et al. (2019). Highly Efficient Biotransformation of Astragaloside IV
to Cycloastragenol by Sugar-Stimulated β-Glucosidase and β-Xylosidase from
Dictyoglomus thermophilum. Journal of Microbiology and Biotechnology: 29 18821893.https://doi.org/10.4014/jmb.1807.07020
Wang, L., & Chen, Y. (2017). Efficient Biotransformation of Astragaloside IV to
Cycloastragenol by Bacillus sp. LG-502. Applied Biochemistry and Biotechnology:
183 1488-1502. https://doi.org/10.1007/s12010-017-2517-1
67
Conclusion
Looking at the results in chapter 1, multiple bacteria derived from human intestines
showed the ability to metabolize AIV. Initial screening efforts showed that the bacteria show
a preferential metabolism of AIV by either eliminating the C-3 xylose or C-6 glucose from the
AIV rather than both simultaneously. Also, two representative groups of gut microbes, LAB
and bifidobacteria showed mirroring metabolic pathways of AIV to CA production.
Bifidobacteria showed preferential elimination of C-3 xylose to produce the intermediate BraB.
Bifidobacteria also produced CA and showed no production of the intermediate CycB. LAB
on the other hand showed preferential elimination of the C-6 glucose to produce CycB. LAB
also showed preferential production of dehydrogenated product of CA, CA-2H rather than CA.
LAB showed no production of intermediate BraB production through multiple screening
efforts. These results suggest that various bacteria metabolize AIV in the intestinal system with
different pathways, and when combined mimic previous research of AIV metabolic profile
utilizing the consortium of intestinally derived bacteria.
Utilizing the information from chapter 1, a successful method to efficiently produce CA
by harnessing the ability of LAB and bifidobacteria was achieved in chapter 2. Traditional
single cell fermentation methods using either LAB or bifidobacteria resulted in long term
fermentation and low concentration production of CA. However, successfully combing the
AIV-metabolizing activity of LAB and bifidobacteria, specifically in that respective order
allowed for significantly higher production of CA. When timed right there was little to no
remaining intermediate of CycB and BraB produced in certain combinations of bacteria strains.
Also, the production of dehydrogenated product of CA, CA-2H, was kept to a minimum by
utilizing the washed cells of bifidobacteria to finish the biotransformation process. The dual
resting cell reaction with W. cibaria RD 12578 and B. pseudocatenulatum JCM 7041 in this
sequence showed the highest production of CA with 0.21 mM concentration with an 82% yield
to 0.25 mM AIV as the substrate.
In chapter 2, the results suggested that B. pseudocatenulatum JCM 7041 can not only
metabolize AIV to BraB but also convert the intermediate CycB to CA by eliminating the C-3
xylose. Through protein purification, the candidate protein band revealed a high score match
68
of the amino acid sequences of the peptides with those of 4-alpha-glucanotransferase derived
from Bifidobacterium pseudocatenulatum.
69
Acknowledgment
The studies presented here have been carried out from 2017-2023 at the laboratory of
Fermentation Physiology and Applied Microbiology, Division of Applied Life Science,
Graduate School of Agriculture, Kyoto University.
The author wishes to express his deepest gratitude to Professor Jun Ogawa for allowing me the
valuable experience to come to Japan and conduct experimentation and learn under his valuable
guidance, warm encouragement, and kind support.
The author greatly appreciates Associate Professor Shigenobu Kishino’s guidance, continued
advice, invaluable discussion, and constant support throughout this study.
The author also appreciates Professor S. Takahashi, Professor M. Ueda, Assistant Prof. A.
Ando, Assistant Prof. M. Takeuchi, Associate Prof. R. Hara, Dr. S. Park, Ms. N. Kitamura and
Mr. Y. Sugiyama for their kind suggestion and assistance throughout this work.
The author would also like to thank Ms. A. Saika, Mr. W. Shimada, Mr. D Toyama, Mr. T.
Morikawa, Mr. Yu-an Sui, Mr. S. Maruyama, Ms. M. Fujikawa, Mr. T. Shiraishi, Ms. A.
Yamamoto, Mr. K. Katsuyama, Mr. R. Kato for their valuable help and support in this work.
The author would also like to especially thank Ms. A. Kitamura for endless support and
assistance throughout this study.
The author greatly appreciates the other and former laboratory members of Fermentation
Physiology and Applied Microbiology, Division of Applied Life Science, Graduate School of
Agriculture, Kyoto University, along with the Central Research and Development Team 1G at
Kobayashi Pharmaceutical Co Ltd. for their assistance and cooperation with this work.
Finally, the author would like to acknowledge the unconditional support and love from the
author’s wife and his two children.
January 24, 2023
Daniel Makoto Takeuchi
70
Publications
1) Daniel M Takeuchi, Shigenobu Kishino, Yuuki Ozeki, Hiroyuki Fukami, and Jun Ogawa.
Analysis of astragaloside IV metabolism to cycloastragenol in human gut microorganism,
bifidobacteria, and lactic acid bacteria. Biosci Biotechnol Biochem, 86(10), 1467–1475
(2022).
2) Daniel M Takeuchi, Yuuki Ozeki, Hiroyuki Fukami, Shigenobu Kishino, and Jun Ogawa.
Efficient biotransformation of astragaloside IV to cycloastragenol through a two-step
reaction catalyzed by washed cells of lactic acid bacteria and bifidobacteria. In
preparation.
3) Daniel M Takeuchi, Yuuki Ozeki, Hiroyuki Fukami, Jun Ogawa, and Shigenobu Kishino.
Purification of astragaloside IV-hydrolyzing β-ᴅ-xylosidase from Bifidobacterium
pseudocatenulatum JCM 7041. In preparation.
71
...
論文の公開元へ
