Efficient synthesis of crocins from crocetin by a microbial

Oct 17, 2018 - Crocins are the most important active ingredient found in Crocus sativus, a well-known “plant gold”. The glycosyltransferase-cataly...
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Cite This: J. Agric. Food Chem. 2018, 66, 11701−11708

Efficient Synthesis of Crocins from Crocetin by a Microbial Glycosyltransferase from Bacillus subtilis 168 Fangyu Ding,† Feng Liu,† Wenming Shao,† Jianlin Chu,‡,§ Bin Wu,† and Bingfang He*,†,‡ †

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College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing 211816, China ‡ School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing 211816, China § Jiangsu National Synergetic Innovation Center for Advanced Materials, 30 Puzhunan Road, Nanjing 211816, China S Supporting Information *

ABSTRACT: Crocins are the most important active ingredient found in Crocus sativus, a well-known “plant gold”. The glycosyltransferase-catalyzed glycosylation of crocetin is the last step of biosynthesizing crocins and contributes to their structural diversity. Crocin biosynthesis is now hampered by the lack of efficient glycosyltransferases with activity toward crocetin. In this study, two microbial glycosyltransferases (Bs-GT and Bc-GTA) were successfully mined based on the comprehensive analysis of the PSPG motif and the N-terminal motif of the target plant-derived UGT75L6 and Cs-GT2. Bs-GT from Bacillus subtilis 168, an enzyme with a higher activity of glycosylation toward crocetin than that of Bc-GTA, was characterized. The efficient synthesis of crocins from crocetin catalyzed by microbial GT (Bs-GT) was first reported with a high molecular conversion rate of 81.9%, resulting in the production of 476.8 mg/L of crocins. The glycosylation of crocetin on its carboxyl groups by Bs-GT specifically produced crocin-5 and crocin-3, the important rare crocins. KEYWORDS: glycosyltransferase, Bacillus subtilis, crocetin, crocins, biosynthesis

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INTRODUCTION Crocins (crocetin glycosyl esters), the main active ingredients identified from Crocus sativus,1 have historically been used as high-valued colorants in the food industry due to their excellent coloring properties.2 Crocins also exhibit a protective effect against atherosclerosis, the inhibition of tumor cell proliferation, and an antioxidant effect on free radicals.3−5 In Crocus sativus, glycosyltransferases (GTs) glycosylate crocetin and crocins on both the carboxyl- and glycosyl- groups, giving five kinds of crocins (crocin-1, crocin-2, crocin-3, crocin-4, and crocin-5) (Figure 1). Of these, crocin-1 is the main component, with four glycosyl groups connecting to crocetin.6 However, pharmaceutical study of crocins indicated that crocins were gradually hydrolyzed to crocetin monoglucoside or even crocetin before absorbing and exhibiting bioactivities. The diglucosylation on each side of the carboxyl group of crocetin like crocin-1 made it difficult to hydrolyze and thus resulted in lower bioavailability.7 Currently, crocins are mainly produced by extraction from Crocus sativus or Gardenia jasminoides, and the main product was crocin-1.8 Some rare glycosides in natural plants may exhibit distinct and important biological properties,9,10 while few studies on rare crocins exist due to the scarcity of Crocus sativus resources and the difficulties involved in isolating rare crocins from natural Crocus sativus.8,11 Although a chemical approach for crocetin and crocin synthesis has been reported, the process is fairly complicated and suffers from disadvantages, such as poor stereospecificity and low efficiency.12 Recently, crocetin, the precursor of crocins, has been biosynthesized in engineered Saccharomyces cerevisiae or Chlorella vulgaris.13,14 In this context, to achieve efficient crocin production, research should © 2018 American Chemical Society

be further focused on seeking more efficient glycosyltransferases for the glycosylation of crocetin.14,15 To date, only two plant glycosyltransferases (Cs-GT2 from Crocus sativus, and UGT75L6 from Gardenia jasminoides) have been identified for glycosylating the carboxyl groups of crocetin.6,16,17 Although various attempts have been made to produce crocins by enzymatic glycosylation or synthetic biology with Cs-GT2 or UGT75L6, the titers are low because of the poor heterologous expression of the plant-derived glycosyltransferases in engineered Escherichia coli. By far, no glycosyltransferases responsible for crocetin glycosylation have been purified, and there are no specific titer data for crocin production by enzymatic glycosylation.17 Currently, the rapid development of gene mining technology provides a practicable way to mine microbial glycosyltransferases with higher heterologous expression and catalytic efficiency for the efficient synthesis of valuable natural glycosides.10,18,19 For example, the enzymatic synthesis of salidroside was first achieved by a microbial glycosyltransferase from Bacillus licheniformis.20,21 Zhuang et al. screened and modified a microbial GT from Saccharomyces cerevisiae, and applied it to construct hybrid pathways for producing ginsenoside Rh2.19 Recent reports concerning the efficient syntheses of natural products by some microbial enzymes encouraged us to mine microbial glycosyltransferases for the enzymatic glycosylation of crocetin. Received: Revised: Accepted: Published: 11701

August 8, 2018 October 11, 2018 October 17, 2018 October 17, 2018 DOI: 10.1021/acs.jafc.8b04274 J. Agric. Food Chem. 2018, 66, 11701−11708

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Journal of Agricultural and Food Chemistry

Figure 1. Enzymatic synthesis of crocins from precursor crocetin by UGT75L6/Cs-GT2 and UGT94E5/Cs-GT1. jasminoides (UGT75L6) involved in the glycosylation of the carboxyl groups of crocetin were analyzed for key functional motifs. The homology model was built using similar crystal structures (PDB ID: 2pq6, 2vce, and 3hbf) as templates. All pairwise and multiple amino acid sequence alignments were carried out using ClustalX with standard parameters. Docking analysis was performed using Discovery Studio 4.0. The protein sequence logos of the conserved motifs were created by the online tool WebLogo (http://weblogo.berkeley.edu/ logo.cgi). On the basis of previous research and the docking analysis, two conserved motifs were deduced (PSPG motif: WCSQIEVLTHPSLGCFVTHCGWNSTLESLVCGVPVVAFPHWTDQ, and N-terminal motif: HVLLITYPAQGHINPALQF) and the complete protein sequence were input for a homologous sequence search using Standard Protein BLAST in the NCBI nonredundant protein sequences (nr) database.23 Glycosyltransferases with a higher similarity to both the PSPG motif (Plant Secondary Product Glycosyltransferase motif) and N-terminal motif were selected for further research. Cloning and Expressing of the Selected Glycosyltransferases. The coding regions of the seven mined microbial glycosyltransferases (Bs-GT from Bacillus subtilis 168, Bc-GTA and Bc-GTB from Bacillus cereus WQ9−2, Fg-GT from Fictibacillus gelatin, Bl-GT1 and Bl-GT5 from Bacillus licheniformis ZSP01, Bp-GT1 from Bacillus pumilus BF1; accession numbers can be found in Supporting Information, SI, Table S1) were amplified and inserted into pET-28a, resulting in pET-glycosyltransferases (primers can be found in SI Table S2). The pET-glycosyltransferase plasmids were transformed into E. coli-BL21 (DE3), and the recombinant strains of E. coli/pET-

In this study, two microbial glycosyltransferases (Bs-GT and Bc-GTA) from Bacillus subtilis 168 and Bacillus cereus WQ9−2 for crocetin glycosylation were successfully mined based on the key motifs of the plant glycosyltransferases (Cs-GT2 or UGT75L6). To the best of our knowledge, this is the first report on the synthesis of crocins from crocetin using microbial glycosyltransferases. Further, the glycosyltransferase Bs-GT with a higher glycosylating activity toward crocetin was purified and characterized. The enzymatic glycosylation of crocetin on the carboxyl groups to produce rare crocin-5 and crocin-3 was also optimized.

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MATERIALS AND METHODS

Strains and Chemicals. Crocetin and crocins were kindly provided by Professor Guangji Wang (China Pharmaceutical University).22 The plasmid pET-28 and host E. coli BL21 (DE3) were used for plasmid propagation and recombinant enzyme production. Bacillus cereus WQ9−2 (M2010010), Bacillus licheniformis ZSP01 (CCTCC M207021), Bacillus subtilis 168 (Bio-82277), Bacillus pumilus BF1 (MTCC B6033), and Fictibacillus gelatini (BNCC161713) strains were purchased from the China Center for Type Culture Collection. All the chemical reagents used were of analytical grade. Mining Glycosyltransferase Candidates from Bacteria for Crocins Synthesis. Both protein sequences and homology models of glycosyltransferases from Crocus sativus (Cs-GT2) and Gardenia 11702

DOI: 10.1021/acs.jafc.8b04274 J. Agric. Food Chem. 2018, 66, 11701−11708

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Journal of Agricultural and Food Chemistry glycosyltransferase were then cultivated at 37 °C in a Luria−Bertani (LB) medium containing 50 mg/L of kanamycin. When the OD600 of the culture reached 0.6, isopropyl β-D-thiogalactoside (IPTG) was added at a final concentration of 0.10 mM, and the culture was further induced at 20 °C for 24 h. Purification of the Selected Glycosyltransferases. The recombinant cells were harvested by centrifugation (12 000g, 10 min) and resuspended in Tris-HCl buffer (pH 8.0, 20 mM). After sonication, the cell suspension was centrifuged, and the supernatant was passed through a 0.22-μm filter before chromatography. The filtered cellular extract was applied to a 5 mL Ni-NTA resin column using an Ä KTA Purifier. The column was washed at a flow rate of 1 mL/min with four column volumes of Tris-HCl buffer (pH 8.0, 20 mM, containing 300 mM NaCl,), and eluted with Tris-HCl buffer (pH 8.0, 20 mM, containing 600 mM NaCl, 50 mM imidazole) at a flow rate of 2 mL/min. The eluent with the desired protein was then dialyzed for further analysis. The purified enzymes were quantified using a BCA Protein Assay Kit (TaRaKa, Dalian, China), and were further analyzed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). Functional Validation of the Mined Microbial Glycosyltransferases. The functional validation for the glycosylation of crocetin was carried out through whole cell transformation. The glycosylation was conducted by shaking flasks at 200 rpm and 30 °C containing glucose (20 g/L), crocetin (50 mg/L), and recombinant cells with cell concentrations at an OD600 of approximately 4 in NaH2PO4−Na2HPO4 buffer (pH 8.0, 50 mM). After 12 h of reaction, the reaction mixture was centrifuged and the supernatant was analyzed by high performance liquid chromatography (HPLC). The glycosylation activity of the seven candidates toward crocetin was also confirmed by enzymatic glycosylation with the lysate of recombinant cells. Besides, further confirmation with purified Bs-GT and Bc-GTA proteins was also conducted. The reaction mixture contained 20 μmol/L crocetin, 0.2 mmol/L uridine diphosphate glucose (UDP-Glc), and 3 mg/L glycosyltransferase protein in 50 mM Gly-NaOH buffer (pH 9.5). The substrate specificity of Bs-GT and Bc-GTA were studied with 0.1 mmol/L of docosahexaenoic acid, linoleic acid, lauric acid, cinnamic acid, vitamin A acid, ursolic acid, oleanolic acid, kaempferol, genistein, luteolin, and quercetin. The supernatants of the reactants were analyzed by HPLC and liquid chromatography−mass spectrometry (LC−MS). Characterization of Bs-GT. The enzymatic glycosylation of crocetin was carried out with the same conditions as described above. Reactions were terminated by adding an equal volume of methanol after 2 h. One unit of glycosyltransferase activity was defined as the activity that corresponds to the conversion of 1 μmol crocetin into crocin-5 per minute. The effects of temperature (20, 25, 30, 35, 40, and 45 °C) and pH (NaH2PO4−Na2HPO4: 6.0, 6.5, 7.0, 7.5, 8.0; Tris-HCl:7.5, 8.0, 8.5; and 8.9;Gly-NaOH: 8.5, 9.0, 9.5, 10.0, 10.5) on the activity and stability of Bs-GT were determined. Kinetic Analysis of Bs-GT. The kinetic parameters of Bs-GT for glycosylation of crocetin were determined with 5 mM UDP-Glc and varying concentrations of crocetin (0.05−1.0 mM) in 50 mM GlyNaOH buffer (pH 9.5). The kinetic parameters of Bs-GT toward UDP-Glc were determined with 1 mM crocetin and varying concentrations of UDP-Glc (0.05−0.5 mM). The reactions were carried out at 30 °C for 15 min, and then terminated by adding an equal volume of methanol. The kinetic parameters were calculated by nonlinear regression of the Michaelis−Menten equation using origin 8.0. The Kcat value was calculated using the predicted molecular mass of 4.5 × 104 g/mol for Bs-GT. Optimization for Enzymatic Synthesis of Crocins. The enzymatic glycosylation of crocetin was carried out at 30 °C in 50 mM Gly-NaOH buffer (pH 9.5) catalyzed by 50 mg/L purified BsGT for 12 h. The effect of crocetin concentration (0.1−1.6 mM) on glycosylation was determined with 10.0 mM UDP-Glc, and the effect of UDP-Glc concentration (1.0−10.0 mM) was analyzed with 1.0 mM crocetin. The time course for the biosynthesis of crocins was determined with 1.0 mM crocetin and 5.0 mM UDP-Glc. All these above experiments were performed in triplicate.

HPLC, LC−MS, and NMR Analysis of the Glycosylated Products. The reactants were analyzed by HPLC on Dionex P680 equipped with a C18 column (Kromasil 250 mm × 4.6 mm × 5 μm) at 420 nm at 30 °C. The following gradient elution program was used at a flow rate of 1.0 mL/min:1−17 min, water/methanol (50:50, v/v), 17−25 min, water/methanol (50:50, v/v to 75:25, v/v), 25−42 min, water/methanol (75:25, v/v), 42−45 min, water/methanol (75:25,v/ v to 50:50,v/v). LC−MS analysis was performed in negative ion mode on an Agilent 6520 Accurate-Mass Q-TOF LC−MS platform (Palo Alto, CA, U.S.A.). The ion spray was operated at 25 Arb N2/min, 3.5 kV, and 300 °C. Glycosylation products of crocetin were purified using an Agilent 1200 preparative HPLC system coupled with a reversephase Ultimate C18 column (21.2 × 250 mm2, 5 μm particles, Welch, Shanghai, China) and then analyzed using the proton and carbon nuclear magnetic resonance (1H NMR, 13C NMR) spectrum with samples dissolved in DMSO-d6 (Bruker Avance IIII 400).

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RESULTS AND DISCUSSION Mining of Microbial Glycosyltransferases for Crocetin Glycosylation. Biotransformation and synthetic biology are thought to be a promising method to produce crocins. Compared to plant-derived glycosyltransferases, microbial glycosyltransferases usually show higher heterologous expression level and catalytic proficiency, which facilitates them as potential biocatalysts for the biosynthesis of many valuable natural products.10,20 So far, only two plant-derived glycosyltransferases (Cs-GT2 from Crocus sativus and UGT75L6 from Gardenia jasminoides) have been identified for the glycosylation of crocetin to crocins (Figure 1), and their application is restricted due to the difficulty in heterologous expression.6,17 Microbial glycosyltransferases might be a promising alternative for crocin production; however, it is difficult to mine microbial glycosyltransferases for crocetin glycosylation based on the plant glycosyltransferases Cs-GT2 and UGT75L6, because of their extremely low sequence identity (usually 13.5% were picked out. Among these microbial glycosyltransferases, 23 candidates with higher similarities of both N-terminal motif and the PSPG motif were screened for further similarity analysis (Table S3). Glycosyltransferase candidates with higher similarity on the Nterminal motif (65−75% for Bc-GTA, Bs-GT, Bl-GT1, BlGT5, and Bp-GT1) and PSPG motif (59.1−63.6% for BcGTA, Bc-GTB, Bs-GT, and Fg-GT) were selected for further study, including for the expression and the glycosylation of crocetin (Table 1). Expression and Functional Validation of the Mined Microbial Glycosyltransferases. The seven selected microbial glycosyltransferases were cloned and successfully expressed in E. coli BL21 (Figure S4). Whole cell transformation experiments indicated that two glycosyltransferases (Bc-GTA and Bs-GT) were able to glycosylate the carboxyl group of crocetin. After 12 h reaction, 6.7% and 15.3% of crocetin were glycosylated by Bc-GTA and Bs-GT, respectively. Further enzymatic validation of the seven selected microbial glycosyltransferases was conducted using crude enzymes with UDPGlc as sugar donor. Data also showed that only Bc-GTA and Bs-GT showed glycosylation activity toward crocetin (Figure S5). Among the seven selected candidates, Bs-GT from Bacillus subtilis 168 and Bc-GTA from Bacillus cereus WQ9−2 with higher similarities, in both the N-terminal and PSPG motifs, to UGT75L6 and Cs-GT2 showed the ability to glycosylate crocetin. The phylogenetic analysis of Bs-GT and Bc-GTA with previously reported UGTs indicated that they clustered together with some glycosyltransferases with activities toward phenolic substrates (Figure S6). The presumably systematic names of Bs-GT and Bc-GTA were presumed as UDP-αglucose: flavones β-glucosyltransferase (EC 2.4.1.91) according to the phylogenetic analysis. Bs-GT shared a protein sequence identity of 90.8% to Bs-GT1, which was used for the glycosylation of morin and related polyphenols,26 while BcGTA showed 91.2% identity to Bc-GT1, which was reported as a multifunctional glycosyltransferase with activities on curcumin, p-Nitrothiophenol, and indirubin, generating Oglycoside, N-glycoside, and S-glycoside, respectively.27

Since the pronounced activity for glycosylating crocetin, the Bc-GTA and Bs-GT were purified by Ni-NTA resin column and analyzed by SDS-PAGE showing a single protein band with an apparent molecular mass of about 45 kDa (Figure 3).

Figure 3. SDS−PAGE of Bc-GTA and Bs-GT expressed in E. coliBL21(DE3). Lane1: protein marker; Lane2: lysate supernatant of E. coli-BL21(DE3)-28a; Lane 3: lysate supernatant of recombined E. coliBL21(DE3)-Bc-GTA cells; Lane4: purified protein of Bc-GTA; Lane5: lysate supernatant of recombined E. coli-BL21(DE3)-Bs-GT cells; and Lane6: purified protein of Bs-GT.

The glycosylation of crocetin catalyzed by the purified Bc-GTA and Bs-GT protein was further confirmed, and the glycosylation products were analyzed by HPLC (Figure 4), LC−MS, 1H NMR, and 13C NMR. A doublet centered at δ 5.03 ppm with a couple parameter J = 8.0 Hz indicates the βconfiguration of glucosides in crocin-3.28 Besides, the 1H NMR (Table S4)and 13C NMR data (Table S5) of crocin-3 was identical to the 1H NMR and 13C NMR data of crocetin-βdiglucosyl esters (crocin-3) that have been reported previously.29 The Bs-GT could convert 39% of crocetin (0.5 mM) to crocin-5 and crocin-3, which was higher than that catalyzed by Bc-GTA (13%). To our knowledge, this is the first time that microbial glycosyltransferases were used to glycosylate crocetin for the synthesis of crocins. Characterization of Bs-GT. In view of the higher glycosylation activity toward crocetin, Bs-GT was then further characterized. The purified Bs-GT exhibited the highest activity at 30 °C and remained stable at and below 25 °C (Figure S7). The Bs-GT showed the highest activity at a pH of 9.0−9.5, and was quite stable in the pH range of 9.0−10.0, indicating that Bs-GT was an alkaline glycosyltransferase. 11704

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were also synthesized by Bc-GTA (Figures S8 and S9) and the products were speculated on account of previously reported glycosylation products of quercetin.27 Although BlGT1 and Bp-GT1 also showed high glycosylation activity toward various flavonoids like quercetin, kaempferol, and genistein, they showed no glycosylation activity toward crocetin (Table S6).31 Thus, it was presumed that there was no direct correlation between the glycosylation activity toward crocetin and flavonoids. These results reflect the robust catalytic activity of microbial glycosyltransferases on flavonoids and other natural products. However, the substrate specificities of microbial glycosyltransferases are hard to predict. Meanwhile, compared to other flavonoids glycosyltransferases reported with conversion rate ranging from about 30% to 100%, Bs-GT and Bc-GTA showed relative high conversion of flavonoids tested (∼90% of Bs-GT and 40% to 80% of BcGTA, Table 2), indicating that Bs-GT and Bc-GTA might be potential biocatalysts for glucosylation of phenylpropanoid substrates.26,32−34 Optimization and Time Course for the Enzymatic Synthesis of Crocins. The enzymatic synthesis of crocins catalyzed by Bs-GT was primarily optimized. Due to the high activity and stability of Bs-GT under alkaline conditions, the glycosylation reaction was conducted in Gly-NaOH (pH 9.5) buffer, which is beneficial to increasing the solubility of crocetin.17,35 The effects of crocetin and UDP-glucose (UDPG) concentrations on the synthesis of crocins were estimated (Figure S10). The higher crocetin concentrations exceeding 1.0 mM reduced the glycosylation and conversion rate; the total conversion rate declined dramatically from 84.6% (1.0 mM) to 52.8% (1.6 mM). This might be caused by the limitation of crocetin solubility in the reaction system. The conversion rate increased markedly as the UDP-Glc concentration increased from 1.0−5.0 mM. Thus, 1.0 mM crocetin and 5.0 mM UDP-Glc was selected for the synthesis of crocins. The time course of crocetin glycosylation catalyzed by BsGT was determined. As Figure 5 shows, crocetin was mainly glycosylated at one side of the carboxyl group of crocetin, resulting in the production of crocin-5 during the first 4 h. As the reaction continued, the crocin-5 was further glycosylated, resulting in the production of crocin-3 with glycosyl in both sides of the carboxyl group of crocetin. After 12 h of reaction, 81.9% of crocetin (1.0 mM/L) was glycosylated to crocin-5 (0.353 mM, 172.97 mg/L) and crocin-3 (0.466 mM,303.83 mg/L). Crocin-3 could be efficiently synthesized as the main product through extending the reaction (data not shown). Notably, crocetin could not be fully glycosylated after extending the reaction to 24 h (data not shown). This might be caused by the inhabitation effect of the accumulated UDP in the reaction system. So, further study should be addressed in coupling Bs-GT with sucrose synthase for UDPG cyclic utilization.36 The current progress of crocin biosynthesis is summarized in Table 3. Dufresne et al. synthesized crocins from 300 mg/L crocetin through Crocus sativus cell culture, resulting in 9 mg/g crocins (mainly crocin-1), with a total conversion rate of 30%.35 The enzymatic synthesis of crocins with glycosyltransferases isolated from Crocus sativus (Cs-GT2) resulted in unnatural crocins with much longer glucosyl chains.6 Another plant glycosyltransferase (UGT75L6) identified from Gardenia jasminoides was heterologously expressed in E. coli and has been applied for glycosylation of crocetin, but no specific yield has yet been reported.6,17 The biosynthesis of crocins via

Figure 4. HPLC and MS analysis of the glycosylated products of crocetin. (A) HPLC chromatograms of crocins standards and Bs-GT, Bc-GTA catalyzed reactants. (B) MS spectra for products 1 and 2.

Considering the fact that Bs-GT showed higher stability at pH 9.0−10.5 and that higher pH was beneficial to dissolve of crocetin, pH 9.5 was selected as the optimized pH for glycosylation of crocetin. The kinetic study of Bs-GT toward crocetin glycosylation was also conducted at pH 9.5 and 30 °C, and the Km, Vmax, and Kcat values were determined to be 1.70 ± 0.094 mM, 37.67 ± 1.48 nkat/mg, and 1.69 ± 0.07 s−1, respectively. And the Km, Vmax, and Kcat values toward UDPGlc were determined to be 0.742 ± 0.114 mM, 44.68 ± 4.67 nkat/mg, and 2.01 ± 0.21 s−1, respectively. Recently, limited by the low expression of plant glycosyltransferase in engineered E. coli, the purification of UGT75L6 responsible for crocetin glycosylation was hardly achieved, and there is no literature on its characteristics.17 This is the first reported characterization of a glycosyltransferase with activity toward crocetin glycosylation. The substrate specificities of Bc-GTA and Bs-GT were also investigated (Table 2). Unexpectedly, both Bs-GT and BcGTA showed no activity toward the carboxyl groups of other tested natural products and physiologically active compounds except for that of crocetin. And unlike Cs-GT2 which showed little glycosylation activity toward flavonoids, both Bs-GT and Bc-GTA displayed high glycosylation activity toward the tested flavonoids.30 Take quercetin as an example, both Bs-GT and Bc-GTA could glycosylate quercetin, but they showed different regioselectivity (line 258−261). Bs-GT glucosylated quercetin on the 3-OH resulting quercetin 3-O-D-glucoside. In contrast, besides quercetin 3-O-D-glucoside, other monoglucosides including quercetin 7-O-D-glucoside and 4′-O-D-glucoside 11705

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Journal of Agricultural and Food Chemistry Table 2. Substrate Specificity of Bs-GT and Bc-GTA

a

ND = not detected.

mined Bs-GT from bacterium Bacillus subtilis 168 showed glycosylation activity toward crocetin. A total conversion rate of 81.9% from crocetin was achieved with a yield of 476.8 mg/ L crocins (crocin-3 and crocin-5). Both Bs-GT and Bc-GTA catalyzed the primary glucosylation of crocetin on the carboxyl groups, generating crocetin monoglucosyl (crocin-5) and diglucosyl esters (crocin-3),17,30 and no glucosylation activity toward the glucose groups of crocin-5 or crocin-3 was observed. From a practicality standpoint, the efficient microbial Bs-GT is a favorable candidate for the effective biosynthesis of rare crocin-5 and crocin-3. In summary, two microbial glycosyltransferases (Bs-GT and Bc-GTA) were successfully mined based on the comprehensive analysis of PSPG and N-terminal motifs of plant-derived UGT75L6 and Cs-GT2, which are responsible for the glycosylation of crocetin. Bs-GT with a higher glycosylation activity was characterized. Under the optimized conditions, the efficient glycosylation of crocetin was achieved and reached a high conversion rate of 81.9%, resulting in 476.8 mg/L of crocins. The glycosylation catalyzed by Bs-GT on the one or

Figure 5. Time course of the enzymatic glycosylation by Bs-GT under optimized conditions. Data represent averages of three biological replicates and the standard deviations of the average of results are denoted by error bars.

synthetic biology in engineered E. coli with plant glycosyltransferases was also successfully confirmed, but there are no reports for the specific titer data of crocins.15 In this study, the 11706

DOI: 10.1021/acs.jafc.8b04274 J. Agric. Food Chem. 2018, 66, 11701−11708

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Journal of Agricultural and Food Chemistry Table 3. Comparison of Crocins Biosynthesis production mode

products

yields 9 mg/g dry weight

enzymatic synthesis

non-natural crocins (mainly crocetin dineapolitanosyl-ester) non-natural crocins (crocetin eaters with 9, 11, 13, and 18 glucosyl groups) crocins(mainly crocin-1)

enzymatic synthesis

crocin-5 and crocin-3

476.8 mg/L

synthetic biology

crocins

detectable

cell culture enzymatic synthesis

catalysts

reference

Crocus sativus L. cells

Dufresne35

GTs from Crocus sativus, expressed in E.coli GTs from Gardenia jasminoides, expressed in E.coli Bs-GT from Bacillus subtilis, expressed in E.coli Cs-GT2 from Crocus sativus, expressed in E.coli

Moraga6 Nagatoshi17 this work Raghavan15

a

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both sides of the carboxyl groups of crocetin specifically produced crocin-5 and crocin-3, the important rare crocins.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04274.

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Sequence alignment (Figures S1 and S2) and docking analysis of plant glycosyltransferase (Figure S3), some experimental results (Figures S4, S5, S6, S7, S8, S9, S10, and S11), accession numbers (Table S1) and sequence analysis (Table S3) of microbial glycosyltransferases, primers (Table S2) for seven glycosyltransferase candidates expression, 1H NMR and 13C NMR data of crocin-3 (Table S4, S5), glycosylation activity of Bs-GT, Bc-GTA, Bl-GT1, and Bp-GT1 toward crocetin and flavonoids (Table S6) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: 86-25-58139902. Fax: 86-25-58139902. E-mail:bing [email protected]. ORCID

Bingfang He: 0000-0002-4502-9531 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (81673321, 21776135, 21506099). We also thank the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (NO. XTC1812).

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ABBREVIATIONS USED PSPG, Plant Secondary Product Glycosyltransferase; UDPGlc, Uridine diphosphate glucose

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REFERENCES

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