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

Abstract

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-β-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-β-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °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 (kcat/KM) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates.

Glycosylation of Ganoderic Acid G by Bacillus Glycosyltransferases

“…The four Bacillus GTs have been considered genetically distinct among the known triterpenoid-catalyzed GTs until now ( Figure 1 ). Previous studies revealed that BsUGT398, BsUGT489, and BtGT_16345 have catalytic specificity toward glycosylating the C-15 hydroxyl group of the GAA triterpenoid [ 16 , 17 ]. In addition, BsGT110 was the first GT that glycosylated the C-26 carboxyl group of the GAA triterpenoid.…”

“…In addition, BsUGT489 was found to glycosylate the C-3 hydroxyl group of GAG. Table 1 and Figure 5 summarize the comparative results of the biotransformation of GAA [ 15 , 16 , 17 ] and GAG with the four Bacillus GTs. The main structure of ganoderic acid contains five functional groups that could be modified: the C-3, C-7, C-12, and C-15 hydroxyl groups, and the C-26 carboxyl group.…”

“…Four Bacillus GTs were further identified, based on their genomes, as the major enzymes able to catalyze the glycosylation of GAA to GAA saponins. BsGT110 [ 15 ], BsUGT398, and BsUGT489 [ 16 ] were identified using the ATCC 6633 strain, and BtGT_16345 was identified using the GA A07 strain [ 17 ]. So far, only seven Bacillus GTs with triterpenoid glycosylation activity have been identified, including the four Bacillus GTs mentioned previously [ 14 ].…”

“…Phylogenetic analysis suggests that the four Bacillus GTs are genetically distinct from each other ( Figure 1 ) [ 14 ]. Although the four Bacillus GTs have been proven to have the ability to produce Ganoderma triterpenoid saponins, previous studies have only focused on GAA glycosylation [ 15 , 16 , 17 ]. Thus, it is worth investigating the catalytic activity of the four Bacillus GTs toward other triterpenoids.…”

Carbohydrate-Active Enzymes: Structure, Activity, and Reaction Products

“…The enzyme is also regioselective toward triterpenoid substrates. BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenin, quercetin, naringenin, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K) [11].…”