Enzymatic Synthesis of Unnatural Ginsenosides Using a Promiscuous UDP-Glucosyltransferase from Bacillus subtilis

Abstract

Glycosylation, which is catalyzed by UDP-glycosyltransferases (UGTs), is an important biological modification for the structural and functional diversity of ginsenosides. In this study, the promiscuous UGT109A1 from Bacillus subtilis was used to synthesize unnatural ginsenosides from natural ginsenosides. UGT109A1 was heterologously expressed in Escherichia coli and then purified by Ni-NTA affinity chromatography. Ginsenosides Re, Rf, Rh1, and R1 were selected as the substrates to produce the corresponding derivatives by the recombinant UGT109A1. The results showed that UGT109A1 could transfer a glucosyl moiety to C3-OH of ginsenosides Re and R1, and C3-OH and C12-OH of ginsenosides Rf and Rh1, respectively, to produce unnatural ginsenosides 3,20-di-O-β-d-glucopyranosyl-6-O-[α-l-rhamnopyrano-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (1), 3,20-di-O-β-d-glucopyranosyl-6-O-[β-d-xylopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (6), 3-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (3), 3,12-di-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (2), 3,6-di-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (5), and 3,6,12-tri-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (4). Among the above products, 1, 2, 3, and 6 are new compounds. The maximal activity of UGT109A1 was achieved at the temperature of 40 °C, in the pH range of 8.0–10.0. The activity of UGT109A1 was considerably enhanced by Mg2+, Mn2+, and Ca2+, but was obviously reduced by Cu2+, Co2+, and Zn2+. The study demonstrated that UGT109A1 was effective in producing a series of unnatural ginsenosides through enzymatic reactions, which could pave a way to generate promising leads for new drug discovery.

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

“…According to the Carbohydrate-Active Enzymes (CAZy) database, GTs can be classified into 107 families, in which GTs that catalyze the glycosylation of small molecules, such as flavonoids and triterpenoids, are classified as GT1 [18]. Although over 500 thousands of GT have been discovered, there are only six bacterial GTs reported to catalyze glycosylation of triterpenoids, including BsYjiC from B. subtilis 168 [11,19,20,21,22], UGT109A1 from B. subtilis CTCG 63501 [23,24], BsGT1 from B. subtilis KCTC 1022 [25], BsUGT398 and BsUGT489 from B. subtilis ATCC 6633 [17], and BsGT110 from B. subtilis ATCC 6633 [present study]. Among them, the BsYjiC group (BsYjiC, BsUGT489, UGT109A1, and BsGT1) were highly similar, sharing more than 90% identity in their amino acid sequences [17], BsGT110 and BsUGT398, however, were not grouped with the BsYjiC group, and only had 31% and 33% identity, respectively, with the BsYjiC group (Figure 6).…”

“…For example, the well-known triterpenoid-catalyzing BsGT1 [25] had optimal activity at pH 7, BsYjiC [20], BsUGT398, and BsUGT489 [17] had optimal activity at pH 8, and UGT109A1 [23] had optimal activity at pH 9–10. These triterpenoid-catalyzing GTs had a broad neutral-alkaline range in their triterpenoid glycosylation activity [17,20,21,22,23,24,25]. According to the reaction mechanism of the inverting GTs, the side-chain of a key residue in the catalytic site of the enzyme should be deprotonated to serve as a base during the reaction.…”

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

“…NP_389104) from B. subtilis 168 [19,20,21,22,23], UGT109A1 (GenBank Protein accession no. ASY97769) from B. subtilis CTCG 63501 [24,28], BsGT1 (GenBank Protein accession no. ANP92054) from B. subtilis KCTC 1022 [29], and two GTs, BsUGT398 and BsUGT489, from B. subtilis ATCC 6633 (GenBank Protein accession nos.…”

“…Although over 500,000 GTs were identified in the CAZy database, only six microbial GTs were found to exhibit glycosylation activities toward triterpenoids, including BtGT_16345 (this study), BsYjiC [19,20,21,22,23], UGT109A1 [24,28], BsGT1 [29], BsUGT398, and BsUGT489 [4]. BtGT_16345, which was classified into the GT28 family, is the only exception out of the other five GTs that belong to the GT1 family [1].…”

“…BtGT_16345, which was classified into the GT28 family, is the only exception out of the other five GTs that belong to the GT1 family [1]. Previous studies showed that these GT1 enzymes catalyzed the glycosylation of several flavonoids at multiple positions to form mixture of flavonoid glycosides [19,20,21,22,23,24,28,29]. In the present study, BtGT_16345 exhibited similar catalytic activities toward seven flavonoids to form multiple products (Figure 7).…”