A New Triterpenoid Glucoside from a Novel Acidic Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis

Abstract

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-β-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-β-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-β-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min−1) was 3-fold higher than that at pH 7 (kcat = 3.8 min−1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.

Glycosylation of Ganoderic Acid G by Bacillus Glycosyltransferases

“…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.…”

Postharvest Drying Techniques Regulate Secondary Metabolites and Anti-Neuroinflammatory Activities of Ganoderma lucidum

“…In this study, we observed an increase in the monosaccharide and amino acid contents and levels of the enzymes responsible for the conversion of malate to pyruvate and acetyl-CoA to citrate and oxaloacetate, along with an increase in the ganoderic acid contents in some heat-dried samples. In previous studies, the ability of GL to produce ganoderic acid was affected by different culture conditions, and the effect of temperature on the ganoderic acid content was the highest among the various factors [ 59 , 60 ]. This suggests that, during heat-drying, the lack of oxygen activates the selective enzymes of the glyoxylate cycle, followed by gluconeogenesis, which results in the production of more carbohydrates and precursor molecules for the biosynthesis of ganoderic acids.…”

Biocatalytic Synthesis of a Novel Bioactive Ginsenoside Using UDP-Glycosyltransferase from Bacillus subtilis 168

“…Compared with plant-derived UGTs, microbial UGTs generally exhibit remarkable aglycone flexibility and poor regiospecificity [13][14][15][16][17][18]. These promiscuous UGTs could be exploited as robust biocatalysts for glycodiversification of natural and unnatural products for new drug discovery [19,20]. Bs-YjiC from Bacillus subtilis 168 is a flexible and effective UGT toward a numerous number of structurally diverse compounds [21,22].…”