Promiscuous glycosyltransferase from Bacillus subtilis 168 for the

c Tianjin Key Laboratory on Technologies Enabling Development of Clinical. 10. Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical Univers...
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Article Cite This: J. Agric. Food Chem. 2018, 66, 943−949

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Use of a Promiscuous Glycosyltransferase from Bacillus subtilis 168 for the Enzymatic Synthesis of Novel Protopanaxatriol-Type Ginsenosides Longhai Dai,† Jiao Li,†,‡ Jiangang Yang,† Yueming Zhu,† Yan Men,† Yan Zeng,† Yi Cai,† Caixia Dong,§ Zhubo Dai,† Xueli Zhang,*,† and Yuanxia Sun*,† †

National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xiqi Road, Tianjin Airport Economic Area, Tianjin 300308, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China S Supporting Information *

ABSTRACT: Ginsenosides are the principal bioactive ingredients of Panax ginseng and possess diverse notable pharmacological activities. UDP-glycosyltransferase (UGT)-mediated glycosylation of the C6−OH and C20−OH of protopanaxatriol (PPT) is the prominent biological modification that contributes to the immense structural and functional diversity of PPT-type ginsenosides. In this study, the glycosylation of PPT and PPT-type ginsenosides was achieved using a promiscuous glycosyltransferase (Bs-YjiC) from Bacillus subtilis 168. PPT was selected as the probe for the in vitro glycodiversification of PPTtype ginsenosides using diverse UDP-sugars as sugar donors. Structural analysis of the newly biosynthesized products demonstrated that Bs-YjiC can transfer a glucosyl moiety to the free C3−OH, C6−OH, and C12−OH of PPT. Five PPT-type ginsenosides were biosynthesized, including ginsenoside Rh1 and four unnatural ginsenosides. The present study suggests flexible microbial UGTs play an important role in the enzymatic synthesis of novel ginsenosides. KEYWORDS: protopanaxatriol, protopanaxatriol-type ginsenosides, Bacillus UDP-glycosyltransferase, glycosylation, chemical diversification

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INTRODUCTION Ginseng (Panax ginseng C. A. Meyer), a herbaceous perennial herb of the Araliaceae family, is one of the most well-known and best-selling oriental medicine.1,2 This herb has been frequently used for more than 2000 years in traditional medicine in Eastern Asia to strengthen immunity, reduce fatigue, and provide nutrition.3 The major pharmacologically active components of ginseng are glycosylated dammarane-type tetracyclic triterpene saponins known as ginsenosides (ca. 2− 4% in the dried roots and rhizomes of ginseng), which exhibit diverse and notable pharmacological characteristics, such as antitumor, anticancer, antidiabetic, anti-inflammatory, antiaging, antifatigue, antihypertensive, and immune-system-enhancing activities.4−7 More than 140 naturally occurring ginsenosides have been isolated from different ginseng species.8 Most of these natural products can be classified into protopanaxadiol (PPD) and protopanaxatriol (PPT) groups according to the structures of their triterpene aglycons.9 Currently, most of the key genes involved in the PPD- and PPT-type-ginsenoside biosynthetic pathway have been functionally characterized. The initial biosynthetic step is the cyclization of 2,3-oxidosqualene to form dammarenediol-II (DM), which is catalyzed by DM synthase (PgDDS).4,10 DM is hydroxylated by PPD synthase (PgPPDS, CYP716A47) at its C12 position to form PPD,11 and PPD is converted further to PPT after hydroxylation is © 2018 American Chemical Society

catalyzed at its C6 position by PPT synthase (PgPPTS, CYP716A53v2).12 Additionally, several UDP-glycosyltransferases (UGTs) responsible for the glycosylation of the C3− OH and C20−OH of PPD and the C6−OH and C20−OH of PPT have been isolated from P. ginseng in the past 3 years, and a number of PPD-type ginsenosides (Rh2, CK, F2, Rg3, and Rd) and PPT-type ginsenosides (F1, Rh1, and Rg1) have been biosynthesized by introducing an engineered PPD- or PPTproducing pathway and ginseng UGT genes into yeast-cell factories.13−18 UGT-catalyzed glycosylation acts as the final biosynthetic step for ginsenosides, which present immense structural and functional diversity after the process. Some UGTs, especially those isolated from microbes, show high substrate flexibility and poor regiospecificity and thus can be exploited as effective biocatalysts for the glycorandomization of natural products with diverse structures.19 For example, UGT OleD from Streptomyces antibioticus, 20,21 MhGT1 from Mucor hiemalis,22 UGT58A1 from Absidia coerulea, UGT59A1 from Rhizopus japonicas,23 Bl-YjiC from Bacillus licheniformis,24,25 and BcGT1 from Bacillus cereus26,27 are frequently applied to glycodiversify Received: Revised: Accepted: Published: 943

August 22, 2017 January 17, 2018 January 17, 2018 January 17, 2018 DOI: 10.1021/acs.jafc.7b03907 J. Agric. Food Chem. 2018, 66, 943−949

Article

Journal of Agricultural and Food Chemistry

Figure 1. Glycosylation patterns of Bs-YjiC toward PPT and the intermediates. PgUGT1, PgUGT100, and PgUGT101, which are involved in the biosynthesis of the natural ginsenosides Rh1, F1, and Rg1, were isolated from ginseng in previous studies. Bold black arrows represent the major catalytic steps of the intermediates.

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a considerable number of important botanical natural products. Recently, UGT51, which exhibits broad acceptor tolerance, was isolated from Saccharomyces cerevisiae and demonstrated the ability to regiospecifically transfer a glucosyl moiety to the free C3−OH of PPD for the synthesis of the ginsenoside Rh2.28 Additionally, several UGTs responsible for the biosynthesis of natural and unnatural ginsenosides were isolated from several Bacillus strains, including UGT109A1, which is involved in the biosynthesis of such ginsenosides as Rh2 (3-O-β-D-glucopyranosyl-20(S)-protopanaxadiol), 12-O-β- D -glucopyranosyl20(S)-protopanaxadiol, 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, 3-O-β-D-glucopyranosyl20(S)-protopanaxatriol, 12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, and 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol;29 YjiC1, which is involved in the biosynthesis of 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol;30 and BSGT1, which is involved in the biosynthesis of the ginsenoside Ia (3-O-β-D-glucopyranosyl-20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol).31 On the basis of these studies, we investigated substrate-flexible UGTs from microbes for the biosynthesis of novel ginsenosides for drug discovery. Bs-YjiC from Bacillus subtilis 168 is a promiscuous and robust UGT toward a considerable number of structurally diverse types of natural and unnatural products, including several structurally diverse types of triterpenes.32 Furthermore, Bs-YjiC can glycosylate both the free C3−OH and C20−OH of PPD and PPD-type ginsenosides to synthesize a series of natural and unnatural ginsenosides (unpublished data). In this study, the potential of Bs-YjiC as a biocatalyst for the glycosylation of PPT and PPT-type ginsenosides was explored further. PPT was selected as the probe for the in vitro glycodiversification of PPT-type ginsenosides that use diverse UDP-sugars as sugar donors. Furthermore, the regiospecificity, stereospecificity, and glycosylation patterns of Bs-YjiC toward PPT were elucidated by analysis of the structures of the glycosylated products and the glycosylation process.

MATERIALS AND METHODS

Chemicals and Reagents. PPT and PPT-type ginsenosides (Rh1, F1, and Rg1) were obtained from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China). UDP-glucose (UDPG), UDPgalactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid (UDP-GlcA) were purchased from SigmaAldrich (St. Louis, MO). Glycosylation of PPT and PPT-Type Ginsenosides. Gene BsYjiC (NP_389104) was inserted into the BamHI and SalI restriction sites of a pET28a expression vector to generate an N-terminally His6 tagged gene. The subsequent expression and purification of Bs-YjiC were carried out as described previously.32 Briefly, Escherichia coli BL21 (DE3) cells harboring the recombinant pET28a-Bs-YjiC were precultured at 37 °C on LB medium containing 50 μg mL−1 kanamycin. After the OD600 reached 0.6−0.8, recombinant-Bs-YjiC production was induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and the cultures were incubated further at 37 °C and 200 rpm for 6−8 h. The recombinant E. coli cells containing the recombinant Bs-YjiC were collected by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 25 mM imidazole), and then disrupted with a French press. Cell debris was removed by centrifugation at 17 000g for 30 min. The supernatant containing the recombinant Bs-YjiC was purified with an AKTA Purifier system (GE Healthcare, Piscataway, NJ) coupled with a NiNTA agarose affinity column. Enzymatic assays (0.3 mL) containing 10 mM diverse UDP-sugars (UDPG, UDP-GlcA, UDP-Gal, or UDP-GlcNAc), 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 μg of purified Bs-YjiC, and 2 mM PPT or one of the PPT-type ginsenosides were carried out at 35 °C for 0.5 h. To determine the effect of UDPG concentrations (2, 4, 8, or 16 mM) on the glycosylation patterns of Bs-YjiC toward PPT, duplicate reactions were performed in the presence of 2 mM PPT. To analyze the glycosylated products with products 1−5 as aglycons, duplicate reactions were performed in the presence of 4 mM UDPG. The reactions were terminated by adding an equal volume of methanol and then analyzed by high-performance liquid chromatography (HPLC) and HPLC coupled with quantitative time-of-flight high-resolution electrospray-ionization mass spectrometry (HPLC-Q-TOF/ESI-MS). HPLC and HPLC-Q-TOF/ESI-MS Analyses of the Glycosylated Products. A total of 20 μL of reactants were examined by HPLC and HPLC-Q-TOF/ESI-MS, as described in our previous study.33 The XBC18 reverse-phase column (4.6 × 250 mm, 5 μm particles, Welch, Shanghai, China) connected to an Agilent 1260 HPLC system was eluted with solvent A (water and 0.1% formic acid) and solvent B 944

DOI: 10.1021/acs.jafc.7b03907 J. Agric. Food Chem. 2018, 66, 943−949

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Figure 2. HPLC-Q-TOF/ESI-MS analysis of the glycosylated products of PPT catalyzed by Bs-YjiC. (A) HPLC chromatograms of PPT and PPTtype ginsenoside standards, control reaction mixtures, and Bs-YjiC-catalyzed reactions. (B) MS spectra for products 1 (a), 2 (b), 3 (c), 4 (d), and 5 (e). (acetonitrile and 0.1% formic acid) by using a gradient program of 25−85% B over 25 min. The ESI probe was operated in the positiveion mode. Structural Analysis of the Glycosylated Products of PPT. For the structural analysis of products 1−5, a scale-up reaction (200 mL) was prepared as described above. The enzymatic reactions were terminated by adding an equal volume of methanol. Subsequently, the reactants were condensed under reduced-pressure distillation, and the residues were resuspended in methanol (10 mL). The glycosylated products were purified with an Agilent 1200 preparative HPLC system coupled with a reverse-phase preparative C18 column (21.2 × 250 mm, 5 μm particles, Welch, Shanghai, China). The preparative column was eluted with solvent A (water) and solvent B (methanol) using a gradient program of 50−85% B over 60 min. The flow rate was 10 mL/min, and the other HPLC conditions were as described above. After being vacuum freeze-dried, the purified products were dissolved in methanol-d4. 1D-NMR (1H NMR and 13C NMR) and 2D-NMR (heteronuclear multiple-bond correlation spectroscopy, HMBC; heteronuclear singular quantum correlation spectroscopy, HSQC; and homonuclear correlation spectroscopy, COSY) spectra were obtained using a Bruker DMX-600 NMR spectrometer. Kinetic Analysis of Bs-YjiC. For the kinetic analysis of Bs-YjiC toward PPT, Rh1, F1, and Rg1, the reaction mixtures (0.3 mL) containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM UDPG, 0.1 μg of purified Bs-YjiC, and varying concentrations of PPT (50− 600 μM), Rh1 (50−600 μM), F1(50−800 μM), and Rg1 (50−600 μM) were incubated at 35 °C for 20 min. All the subsequent steps were performed as described above. The kinetic parameters were calculated by nonlinear-regression analysis using GraphPad Prism 5.0 software. The turnover (kcat) values were calculated using the predicted molecular mass of 4.5 × 104 g mol−1 for Bs-YjiC.

was easily purified to homogeneity by one-step affinity chromatography on nickel−nitrilotriacetic acid (Ni−NTA)agarose (Figure S1). PPT is the common triterpene aglycon of PPT-type ginsenosides.15 Therefore, PPT was selected as the probe for the in vitro glycodiversification of PPT-type ginsenosides with UDPG as the sugar donor (Figure 1). Five new products (1−5) were identified from the Bs-YjiC-catalyzed reaction through HPLC analysis, whereas no new products were obtained from the control reaction mixtures catalyzed by total lysates from E. coli BL21 (DE3) expressing pET28a (Figure 2A). Further HPLC-Q-TOF/ESI-MS analysis confirmed that products 1 ([M + H]+ m/z+ ∼ 963.5477), 2 ([M + H]+ m/z+ ∼ 801.4994), 3 ([M + H]+ m/z+ ∼ 801.4974), 4 ([M + H]+ m/z+ ∼ 639.4437), and 5 ([M + H]+ m/z+ ∼ 639.4438) were the glycosylated derivatives of PPT (C30H52O4, calculated molecular weight, [M + H]+ m/z+ ∼ 477.3938) with 1−3 glucosyl moieties attached to the PPT skeleton (Figure 2B). For the elucidation of the regio- and stereospecificities of BsYjiC toward PPT, products 1−5 were purified by preparative HPLC, and their structures were elucidated on the basis of 1DNMR (1H NMR and 13C NMR) and 2D-NMR (HMBC, HSQC, and COSY) spectra (Figures S2−S26). For product 5, the observation of significant downfield shift (∼11 ppm) of C3 suggested that a glucosyl moiety was attached to the C3−OH of PPT (Table S1).21,33 Furthermore, the HMBC correlations of the sugar anomeric signal H-1′ (δH 4.34, d, J = 7.80 Hz) with C3 (δC 90.6) confirmed that product 5 was 3-O-β-Dglucopyranosyl-20(S)-protopanaxatriol. The 1H- and 13CNMR spectra of product 4 were consistent with those of the authentic ginsenoside Rh1 (Table S2).15 A notably significant 13 C downfield shift (∼12 ppm, “glycosylation shift”) at δ 80.9 (C6) indicated that a glucosyl moiety was attached to the C6−

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RESULTS AND DISCUSSION Glycodiversification of PPT with Bs-YjiC. N-terminal His6-tagged Bs-YjiC was expressed in E. coli BL21 (DE3) and 945

DOI: 10.1021/acs.jafc.7b03907 J. Agric. Food Chem. 2018, 66, 943−949

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Journal of Agricultural and Food Chemistry OH of PPT. In the HMBC data, long-range correlations between the sugar anomeric signal H-1′ (δH 4.35, d, J = 7.80 Hz) with C6 (δC 80.9) suggested that product 4 was 6-O-β-Dglucopyranosyl-20(S)-protopanaxatriol (ginsenoside Rh1). Product 3 exhibited spectroscopic data similar to those of products 4 and 5 (Table S3). The observation of significant downfield shifts for C3 (∼11 ppm) and C12 (∼7 ppm) suggested that glucosyl moieties were attached to the C3−OH and C12−OH of PPT. The HMBC correlations of sugar anomeric signal H-1′ (δH 4.35, d, J = 7.80 Hz) with C3 (δC 90.5) and sugar anomeric signal H-1′′ (δH 4.54, d, J = 7.80 Hz) with C12 (δC 79.3) suggested that product 3 was 3-O-β-Dglucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. For product 2, the 13C-NMR glycosylation shifts (∼11 ppm) of C3 (δC 91.0) and C6 (δC 80.8) indicated that glucosyl moieties were attached to the C3−OH and C6−OH of PPT (Table S4). In the HMBC data, the HMBC correlations of the sugar anomeric signals H-1′ (δH 4.37, d, J = 7.80 Hz) with C3 (δC 91.0) and H-2′′ (δH 4.35, d, J = 7.80 Hz) with C6 (δC 80.8) suggested β-glucosyl moieties were attached to the C3−OH and C6−OH of PPT. Thus, product 2 was determined to be 3O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. The 1H- and 13C-NMR spectra of product 1 were highly similar to those of products 2, 3, 4, and 5 (Table S5). The observation of significant 13C downfield shifts of C3 (∼12 ppm), C6 (∼12 ppm), and C12 (∼7 ppm) of the PPT skeleton indicated that glucosyl moieties were attached to the C3−OH, C6−OH, and C12−OH of PPT. Furthermore, the HMBC correlations of the sugar anomeric signals H-1′ (δH 4.36, d, J = 7.80 Hz) with C3 (δC 90.9), H-1′′ (δH 4.38, d, J = 7.80 Hz) with C6 (δC 80.7), and H-1′′′ (δH 4.54, d, J = 7.80 Hz) with C12 (δC 79.3) demonstrated that product 1 was 3-O-β-Dglucopyranosyl-6-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. The observation of large anomeric proton-coupling constants (J = 7.80) indicated that all the sugar moieties were attached to the PPT skeletons by β-glycosidic bonds via an inverting mechanism of Bs-YjiC. Thus, Bs-YjiC is the first reported UGT that can regiospecifically and stereospecifically glycosylate the free C3−OH, C6−OH, and C12− OH of PPT (Figure 1). Of these five glycosylated derivatives of PPT, products 1, 2, 3, and 5 were unnatural PPT-type ginsenosides, and products 1 and 3 were first synthesized in this study. Similar to other natural PPT-type ginsenosides, the newly biosynthesized ginsenosides in this study should possess novel biological and pharmacological activities.3 Notably, the deduced amino acid sequences of Bs-YjiC exhibited 94.39% identity with those of UGT109A1 from B. subtilis CTCC 63501. However, UGT109A1 can only catalyze a continuous two-step glycosylation of the free C3−OH and C12−OH of PPT to produce 3-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, 12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, and 3O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol.29 Thus, further studies based on homologous modeling and site-directed mutagenesis should be carried out to determine the key amino acids of Bs-YjiC that are involved in the regiospecific glycosylation of PPT. Compared with UGTs isolated from plants, some microbial UGTs are more flexible toward both the sugar donors and aglycon acceptors.34−37 Thus, reactions of PPT with UDPGlcA, UDP-Gal, and UDP-GlcNAc as sugar donors were performed under identical conditions as that of PPT and UDPG (Figure 3). HPLC analysis of the reactants confirmed that Bs-YjiC could glycosylate PPT with UDP-Gal and UDP-

Figure 3. HPLC analysis of the glycosylated products of PPT with diverse UDP-sugars as sugar donors.

GlcNAc as sugar donors, whereas no new products were observed in the reaction setup incubating PPT with UDP-GlcA or the control reaction without sugar donors. When UDP-Gal was used as the sugar donor, products a ([M + H]+ m/z+ ∼ 639.4434), b ([M + H]+ m/z+ ∼ 639.4443), and c ([M + H]+ m/z+ ∼ 639.4447) were identified as monogalactosides of PPT (Figure S27), as indicated by the HPLC-Q-TOF/ESI-MS analysis. Conversely, when UDP-GlcNAc was used as the sugar donor, products d ([M + H]+ m/z+ ∼ 680.4697) and e ([M + H] + m/z + ∼ 680.4709) were identified as mono-Nacetylglucosaminides of PPT (Figure S28). Furthermore, when UDP-Gal and UDP-GlcNAc were used as sugar donors, the number of newly formed products, number of attached sugar moieties, and conversion rates of PPT were considerably lower than those obtained when UDPG was used (Figures 2 and 3). These results suggested that the sugar moieties of UDPsugars played an important role in the glycosylation patterns and catalytic efficiencies of Bs-YjiC. Glycosylation Patterns of Bs-YjiC toward PPT. The concentration of UDPG plays an important role in the number and concentration ratio of UTG-catalyzed products.25 To determine the glycosylation patterns of Bs-YjiC toward PPT when UDPG was used as the glucosyl donor, duplicate reactions were carried out with various UDPG concentrations (2, 4, 8, and 16 mM) in the presence of 2 mM PPT (Figure 4A). At a low UDPG concentration (2 mM), products 3 (diglucoside) and 5 (monoglucoside) were the major products, indicating that Bs-YjiC favorably glycosylated the C3−OH and C12−OH of PPT to form products 5 and 3. When UDPG had a concentration twice as high of that of PPT, the concentration of product 5 decreased, and only a trace amount of product 1 was detected, whereas the concentrations of products 2 and 3 increased. When the concentration ratio of UDPG/PPT increased to 4 or 8, the concentrations of products 1, 2, and 3 increased, and product 4 was not detected. To confirm the glycosylation patterns of Bs-YjiC, we performed duplicate reactions using products 1, 2, 3, 4, and 5 as substrates (2 mM) in the presence of 4 mM UDPG (Figure 4B). When product 5 was used as the substrate, product 3 was the major product, and only trace amounts of products 1 and 2 were detected, indicating that Bs-YjiC favorably glycosylated the C12−OH of product 5 to form product 3 (Figure 1). Furthermore, the detection of products 1 and 2 suggested that Bs-YjiC can catalyze a continuous twostep glycosylation of the C6−OH and C12−OH of product 5 946

DOI: 10.1021/acs.jafc.7b03907 J. Agric. Food Chem. 2018, 66, 943−949

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Journal of Agricultural and Food Chemistry

Figure 5. HPLC analysis of the glycosylated products of Rh1, F1, and Rg1 catalyzed by Bs-YjiC.

min, [M + H]+ m/z+ ∼ 963.5504; product R3, RT = 6.4 min, [M + H]+ m/z+ ∼ 963.5497), two tetraglucosides (product R4, RT = 5.9 min, [M + H]+ m/z+ ∼ 1125.6016; product R5, RT = 5.6 min, [M + H]+ m/z+ ∼ 1125.6053), and one pentaglucoside (product R6, RT = 5.4 min, [M + H]+ m/z+ ∼ 1287.6802) were produced (Figure S31). Given the regio- and stereospecificity of Bs-YjiC toward PPT, most of the newly biosynthesized products were novel PPT-type ginsenosides. Kinetic Parameters of Bs-YjiC. The kinetic parameters of purified Bs-YjiC toward PPT and the ginsenosides Rh1, F1, and Rg1 were determined (Table 1 and Figure S32). The Km values

Figure 4. HPLC analysis of the glycosylated products with different concentrations of UDPG and different intermediates. (A) HPLC analysis of the glycosylated products with different concentrations of UDPG (2, 4, 8, and 16 mM) in the presence of 2 mM PPT. (B) HPLC analysis of the glycosylated products when products 1−5 are used as substrates.

(Figure 4B). With products 2 or 4 as the substrate, the identification of product 1 reconfirmed that it could be formed via a continuous two-step glycosylation reaction of product 4. Moreover, analysis of the glycosylated products from the use of products 2, 4, and 5 as substrates suggested that product 1 was mainly formed via a continuous two-step glycosylation of the C3−OH and C12−OH of product 4 (Figure 1). However, no new products were observed when products 1 and 3 were used as substrates. This result was consistent with the glycosylation patterns of Bs-YjiC, as shown in Figure 1. Glycosylation of the Ginsenosides Rh1, F1, and Rg1 with Bs-YjiC. We were interested in the glycosylation of other PPT-type ginsenosides with Bs-YjiC as a biocatalyst. Thus, reactions of ginsenosides Rh1, F1, and Rg1 with UDPG as the glucosyl donor were performed under conditions identical to those of PPT (Figure 5). With ginsenoside Rh1 (calculated molecular weight, [M + H]+ m/z+ ∼ 639.4471), one diglucoside (product R-1, retention time (RT) = 10.3 min, [M + H]+ m/z+ ∼ 801.4982) and one triglucoside (product R2, RT = 6.5 min, [M + H]+ m/z+ ∼ 963.5377) were confirmed by HPLC-Q-TOF/ESI-MS (Figure S29). In the case of ginsenoside F1 (calculated molecular weight, [M + H]+ m/z+ ∼ 639.4471), one diglucoside (product F-1, RT = 10.6 min, [M + H]+ m/z+ ∼ 801.4998), three triglucosides (product F-2, RT = 10.2 min, [M + H]+ m/z+ ∼ 963.5526; product F-3, RT = 9.0 min, [M + H]+ m/z+ ∼ 963.5520; product F-4, RT = 7.0 min, [M + H]+ m/z+ ∼ 963.5522), and two tetraglucosides (product F-5, RT = 6.4 min, [M + H]+ m/z+ ∼ 1125.6059; product F-6, RT = 5.5 min, [M + H]+ m/z+ ∼ 1125.6045) were detected (Figure S30). With Rg1, three triglucosides (product R1, RT = 7.0 min, [M + H]+ m/z+ ∼ 963.5491; product R2, RT = 6.8

Table 1. Kinetic Parameters of Bs-YjiC towards PPT and the Ginsenosides Rh1, F1, and Rg1 substrate

Km (μM)

PPT Rh1 F1 Rg1

103.60 ± 18.28 50.34 ± 11.25 211.80 ± 23.81 107.40 ± 11.48

kcat (s−1)

kcat/Km (s−1 μM−1)

± ± ± ±

0.22 0.30 0.11 0.21

23.16 14.95 22.45 23.09

1.22 0.76 0.97 0.74

of Bs-YjiC for PPT (103.60 μM), Rh1 (50.34 μM), F1 (211.80 μM), and Rg1 (107.40 μM) were comparable to those previously reported for ginseng UGTs and microbial UGTs involved in the biosynthesis of PPT-type ginsenosides.15,29,31 The turnover numbers (kcat) of Bs-YjiC for PPT, Rh1, F1, and Rg1 were 23.16 s−1, 14.95 s−1, 22.45 s−1, and 23.09 s−1, respectively. The kcat values of Bs-YjiC were much higher than those of previously reported ginseng UGTs and microbial UGTs15,29,31,37 and thus the catalytic efficiencies (kcat/Km) of Bs-YjiC toward PPT (0.22 μM−1 s−1), Rh1 (0.30 μM−1 s−1), F1 (0.11 μM−1 s−1), and Rg1 (0.21 μM−1 s−1) were considerably high. The high catalytic efficiencies of Bs-YjiC toward PPT and PPT-type ginsenosides and its broad tolerance of a considerable number of structurally diverse types of natural and unnatural products as acceptors were consistent with the previous notion that naturally occurring UGTs with high catalytic proficiencies are generally more flexible toward aglycons.20,32,37 In summary, Bs-YjiC from B. subtilis 168 was the first reported UGT that can transfer a glucosyl moiety to the free C3−OH, C6−OH, and C12−OH of PPT. Our findings provided significant insight into the important roles of 947

DOI: 10.1021/acs.jafc.7b03907 J. Agric. Food Chem. 2018, 66, 943−949

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Journal of Agricultural and Food Chemistry

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microbial UGTs in the enzymatic glycodiversification of ginsenosides. The pharmacological properties of these newly biosynthesized unnatural PPT-type ginsenosides should be studied further. Future structural studies of Bs-YjiC should be carried out to elucidate the structure−function relationship. Furthermore, it will be of particular interest to introduce BsYjiC or an engineered Bs-YjiC into PPT-producing chassis cells to synthesize these natural and unnatural ginsenosides or a specific ginsenoside via metabolic engineering.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03907. 1 H- and 13C-NMR spectral data for products 1−5 (Tables S1−S5), some experimental results (Figures S1 and S32), and HPLC-Q-TOF/ESI-MS and NMR analyses (Figures S2−S31) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], Tel.: +862284861983 (X.Z.). *E-mail: [email protected], Tel.: +862284861960 (Y.S.). ORCID

Longhai Dai: 0000-0001-5662-9252 Jiangang Yang: 0000-0001-9463-8862 Caixia Dong: 0000-0002-4749-9510 Funding

This work was supported by the National Natural Science Foundation of China (no. 21702226) and the Science and Technology Planning Project of Tianjin (no. 11ZCZDSY08900). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.7b03907 J. Agric. Food Chem. 2018, 66, 943−949