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| 標題: | A Genome-Centric Approach Reveals a Novel Glycosyltransferase from the GA A07 Strain of Bacillus thuringiensis Responsible for Catalyzing 15-O-Glycosylation of Ganoderic Acid A |
| 作者: | Chang, Te-Sheng
Wang, Tzi-Yuan
Hsueh, Tzu-Yu
Lee, Yu-Wen
Chuang, Hsin-Mei
Cai, Wen-Xuan
Wu, Jiumn-Yih
Chien-Min Chiang(江建民)
Wu, Yu-Wei |
| 貢獻者: | Natl Univ Tainan, Dept Biol Sci & Technol
Acad Sinica, Biodivers Res Ctr
Natl Quemoy Univ, Dept Food Sci
Chia Nan Univ Pharm & Sci, Dept Biotechnol
Taipei Med Univ, Coll Med Sci & Technol, Grad Inst Biomed Informat
Taipei Med Univ Hosp, Clin Big Data Res Ctr |
| 關鍵字: | Nanopore sequencing
ganoderic acid
Bacillus thuringiensis
biotransformation
glycosyltransferase
whole genome sequencing |
| 日期: | 2019 |
| 上傳時間: | 2020-07-29 13:52:23 (UTC+8) |
| 出版者: | MDPI |
| 摘要: | Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15-O-beta-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis. Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes (FQZ25_16345, FQZ25_19840, and FQZ25_19010) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010, were successfully cloned and expressed in a soluble form in Escherichia coli, and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15-O-beta-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 degrees C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (k(cat)/K-M) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GAA07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates. |
| 關聯: | International Journal of Molecular Sciences, v.20, n.20, 5192 |
| 顯示於類別: | [生物科技系(所)] 期刊論文
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