Microbial Biosynthesis of Chrysazin Derivatives in Recombinant Escherichia coli and Their Biological Activities

Anthraquinone and its derivatives show remarkable biological properties such as anticancer, antibacterial, antifungal, and antiviral activities. Hence, anthraquinones derivatives have been of prime interest in drug development. This study developed a recombinant Escherichia coli strain to modify chrysazin to chrysazin-8-O-α-l-rhamnoside (CR) and chrysazin-8-O-α-l-2′-O-methylrhamnoside (CRM) using rhamnosyl transferase and sugar-O-methyltransferase. Biosynthesized CR and CRM were structurally characterized using HPLC, high-resolution mass spectrometry, and various nuclear magnetic resonance analyses. Antimicrobial effects of chrysazin, CR, and CRM against 18 superbugs, including 14 Gram-positive and 4 Gram-negative pathogens, were investigated. CR and CRM exhibited antimicrobial activities against nine pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in a disk diffusion assay at a concentration of 40 µg per disk. There were MIC and MBC values of 7.81–31.25 µg/mL for CR and CRM against methicillin-sensitive S. aureus CCARM 0205 (MSSA) for which the parent chrysazin is more than >1000 µg/mL. Furthermore, the anti-proliferative properties of chrysazin, CR, and CRM were assayed using AGS, Huh7, HL60, and HaCaT cell lines. CR and CRM showed higher antibacterial and anticancer properties than chrysazin.


Introduction
Quinones are naturally occurring organic compounds found in higher plants, fungi, bacteria, and animals. They have a lot of structural varieties. Since they are found in different colors in nature, they are considered pigments [1]. Anthraquinones are the largest group of quinones, with various biological properties such as antioxidant [2], antifungal [3], antiviral [4], anti-diabetic [5], anti-inflammatory [6], and laxative [7] effects. They are also used as natural dyes in industries [8]. Several anthraquinones are widely used in the treatment of cancer [9][10][11]. They show cytotoxic activities through interaction with DNA, preferentially at cytosine/guanine-rich sites [12].
Synthesis of anthraquinone derivatives is of great interest recently. There are various methods for the synthesis of anthraquinones derivatives, including intramolecular condensation of aryl and o-aroylbenzoic acid using fuming sulfuric acid, benzoyl chloride, concentrated sulfuric acid, benzoyl chloride, zinc chloride, and POCl 3 /P 2 O 3 Cl 4 [13]. The chemical synthesis of anthraquinones derivatives is an expensive and difficult process [14].
Modification of anthraquinones can be performed by glycosylation, methylation, sulfation, prenylation, and so on. Glycosylation is an important process for increasing the solubility of hydrophobic compounds, improving stability, reducing toxicity, and modifying biological activities [15,16]. Methylation can alter the solubility, deactivate the reactive solubility of hydrophobic compounds, improving stability, reducing toxicity, and modifying biological activities [15,16]. Methylation can alter the solubility, deactivate the reactive hydroxyl group, increase the metabolic stability, increase membrane transport, and increase pharmaceutical properties [17][18][19].
Methyltransferases are important enzymes in the modification of different substrate. However, methylation of sugar is very rare [20,21]. Generally, transfer of the sugar group is catalyzed by glycosyltransferases (GTs) with activated NDP-sugars as sugar donors. Methylation reactions are catalyzed by O-methyltransferase (OMT) that catalyzes the transfer of the methyl group of S-adenosyl-L-methionine (SAM) to hydroxyl groups [15,22,23].
Chrysazin (Dantron; 1,8 dihydroxyanthraquinone) has been used as a medicine since ancient times. It can be found naturally. It is isolated from the root and rhizome of Rheum palmatum L. (Polygonaceae) [24]. It has a wide range of activities, such as antitumor activity, confirmed by different experiments [24,25].

General Procedures
Chrysazin/Dantron was purchased from Tokyo Chemical Industry (Tokyo, Japan). HPLC-grade acetonitrile and water were purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). All other chemicals used were of high analytical grade and commercially available. Isopropyl-β-D-1-thiogalactoside (IPTG) was purchased from GeneChem Inc. (Daejeon, Korea). SAM was purchased from Sigma-Aldrich (St. Louis, MO, USA). Escherichia coli BL21 (DE3) (Stratagene, La Jolla, CA, USA) was used as an expression and biotransformation host. Luria-Bertani (LB) broth medium and agar plates with appropriate antibiotics (ampicillin, kanamycin, chloramphenicol, and streptomycin, each at 50 µg/mL) were used for culture preparation, colony selection, and biotransformation. Pathogenic strains such as Staphylococcus. aureus

Culture Preparation and Whole-Cell Biotransformation
A seed culture of E. coli S2 was cultured in a 5 mL LB medium supplemented with ampicillin, kanamycin, chloramphenicol, and streptomycin (each at 50 µg/mL) and incubated at 37 • C for 3 h. From the 5 mL seed culture, 500 µL was transferred into a flask containing 50 mL LB broth with respective antibiotics and cultured at 37 • C for around 4 h until the optical density of cells at 600 nm (OD 600 ) reached 0.6-0.8. This culture had added to it 0.5 mm isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce protein expression, followed by incubation at 20 • C for 20 h. To determine optimal substrate concentration, glucose concentration, and time, different concentrations (1, 2, 4, 6, 8, 10 mm) of chrysazin and different concentrations (2%, 5%, 10%, 12%, and 15%) of sterile glucose solution were fed into the cell culture after induction. After 20 h of adding IPTG, chrysazin dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 400 µM was added along with 10% glucose. Samples (3 mL of culture broths) were withdrawn at 6, 12, 24, 36, 48, and 60 h for product analysis. After 48 h, compounds were extracted using a double volume of ethyl acetate (2:1, v/v) and a Soxhlet extractor. The Soxhlet extractor was kept still to separate the mixture for 4-6 h at room temperature after shaking. Ethyl acetate was removed under reduced pressure and dissolved in methanol. This sample was further analyzed by HPLC and mass spectrometry. To collect a sample to characterize structurally, the biotransformation experiment was performed using a fermenter (3 L of culture). The pure fraction of the compound was collected via preparatory-high-pressure liquid chromatography (prep-HPLC).
One-dimensional NMRs ( 1 H NMR and 13 C NMR) and two-dimensional NMRs (heteronuclear multiple quantum coherence (HMQC), rotating frame Overhauser enhancement spectroscopy (ROESY), and heteronuclear multiple bonded connectivity (HMBC)) were used as needed to elucidate the structure of the compound. . Human leukemia HL60 cells were cultured in RPMI1640 supplemented with 10% FBS, 1% penicillin-streptomycin-amphotericin B, and L-glutamine (2 mm) (Grand Island, NY, USA). HaCaT cell lines were grown in DMEM supplemented with 10% FBS, 100 µg/mL streptomycin, and 100 µg/mL benzylpenicillin. All cells were maintained at 37 • C in a humidified 5% CO 2 incubator. For cell growth assay, cells were seeded at 3 × 10 2 cells/well into white 96-well culture plates (SPL Life Sciences, Pochon, Korea), incubated at 37 • C in a humidified 5% CO 2 overnight, and then treated with each compound after serial dilution (200 µM, 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.16 µM, 1.56 µM, 0.78 µM) for 72 h. After that, 20 µL substrate solution (Promega) was added to each well. The plate was shaken for 5 min and kept in the dark for 10 min. Luminescence was measured using a multimode plate reader (BioTek, Inc., Winooski, VT, USA). IC 50 values were analyzed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). 12708, and Micrococcus luteus) and four Gram-negative bacteria (Escherichia coli ATCC 25922, Proteus hauseri NBRC 3851, Klebsiella pneumonia ATCC10031, and Salmonella enterica ATCC 14028) were used to test antibacterial activities of chrysazin and its derivatives. The paper disk diffusion assay on the Mueller-Hinton agar (MHA) plate was carried out according to Clinical Laboratory Standard Institute (CLSI) guidelines and the Kirby-Bauer method [32,33]. Inocula containing 10 8 colony forming units (CFU)/mL were spread onto MHA plates. Then, 40 µg/disk compounds were placed on the surface of inoculated agar plates using sterile paper disks of 6 mm (Advantec, Toyo Roshi Kaisha, Ltd., Japan). Samples were then incubated at 37 • C for 18-20 h. The zone of inhibition diameter was measured in millimeters for each pathogen. Dimethyl sulfoxide (DMSO) was used as a control for the zone of inhibition as all compounds were dissolved in DMSO. . The broth dilution method was used to determine MIC [34]. The MHB and sample were dispensed in a 96-well plate and serially diluted. The strain was inoculated into each well and cultured for 16-20 h at 37 • C. Each strain's suspension was adjusted to 0.5 McFarland standard (1 × 10 8 CFU/mL) and then diluted to 2.5 × 10 6 CFU/mL in MHB. After knowing the MIC, the MBC test was performed on a fresh MHB medium by inoculating cultured samples containing MIC compounds and experimental strains.

Biosynthesis of CR and CRM
The recombinant strain of E. coli strain S2 was generated by engineering E. coli BL21 (DE3), which contained a sugar transfer cassette and a sugar methylation cassette. It was cultured and prepared for biotransformation as mentioned in Section 2.2. For optimal production, different concentrations (1, 2, 4, 6, 8, and 10 mm) of chrysazin and different concentrations (2%, 5%, 10%, 12%, and 15%) of glucose were tested with different time intervals (6,12,24,36,48, and 60 h). It was observed that 400 µM, 10% glucose, and 48 h were suitable conditions ( Figure S1). The biotransformation system was induced with 0.5 mm of IPTG. After 20 h, 400 µM of chrysazin and 10% glucose were supplied into cell cultures. The extract from engineered E. coli strain S2 was analyzed by HPLC. The HPLC chromatogram of chrysazin was obtained with its standard retention time (tR) of 21.

Purification and Structural Elucidation of the Metabolite
Biotransformation was carried out through fermentation to collect CR and CRM for structure identification and further biological activity tests. The biotransformation reaction mixture was extracted with a double volume of ethyl acetate. The crude extract was subjected to preparatory-high-pressure liquid chromatography (prep-HPLC) for purification. After several rounds of prep-HPLC, purified compounds were obtained. The purified product was dried by lyophilization, dissolved in 400 µ L of deuterated dimethyl sulfoxide, and analyzed by nuclear magnetic resonance (NMR) spectroscopy (700 MHz) including 1D NMR ( 1 H-NMR and 13 C-NMR) and 2D NMR (HMBC, HSQC, COSY, and ROESY), as shown in Figures S2-S4, and Table 1 for structural elucidation.

Purification and Structural Elucidation of the Metabolite
Biotransformation was carried out through fermentation to collect CR and CRM for structure identification and further biological activity tests. The biotransformation reaction mixture was extracted with a double volume of ethyl acetate. The crude extract was subjected to preparatory-high-pressure liquid chromatography (prep-HPLC) for purification. After several rounds of prep-HPLC, purified compounds were obtained. The purified product was dried by lyophilization, dissolved in 400 µL of deuterated dimethyl sulfoxide, and analyzed by nuclear magnetic resonance (NMR) spectroscopy (700 MHz) including 1D NMR ( 1 H-NMR and 13 C-NMR) and 2D NMR (HMBC, HSQC, COSY, and ROESY), as shown in Figures S2-S4, and Table 1 for structural elucidation.
The 1 H-NMR of chrysin, CR, and CRM showed multiple peaks between 1.0 ppm and 13.0 ppm. In the case of CR, the rhamnose group was attached to the 8-OH group of chrysazin. The anomeric proton (1 -H) was consistent with δ 5.67 (d, J = 1.1 Hz, 1H), in which the anomeric proton coupling constant (J = 1.1 Hz) confirmed that the conjugation of rhamnose moiety had an α-configuration. In addition, with 13 C-NMR of CR, the anomeric carbon peak appeared at δ 99.08 ppm, with other peaks appearing between 70 and 80 ppm along with a CH 3 peak at 18.35 ppm. In the case of CRM, there was a methylation in the 2 -OH group of rhamnose in CR, where the OCH 3 spectrum was visible in both 1 H and 13 C NMR at 3.5 ppm and 59.44 ppm, respectively. Furthermore, to confirm the sugar and sugar-O-methylation conjugation, two-dimensional (2D)-NMR analyses such as 1 H-13 C HSQC, 1 H-13 C HMBC, 1 H-1 H COSY, and 1 H-1 H ROESY experiments were performed. Similarly, in CR, HSQC showed a cross peak illustrating a correlation between the anomeric C-1 proton (δ 5.67 ppm) and the anomeric carbon (δ 99.00 ppm). Moreover, the C-8 signal appearing at δ 157.18 ppm showed a direct correlation with the observed anomeric proton at δ 5.67 ppm in HMBC ( Figure S3). In the case of CRM, HSQC showed a cross peak illustrating a correlation between the C-2 protons (δ 3.71 ppm) and the carbon (δ 80.61 ppm), and HMBC showed a cross peak depicting the correlations between C-2 (δ 80.62 ppm) and the protons of the methoxy group (δ 3.50 ppm) ( Figure S4). Results shown above reveal that the glycosylated derivative of chrysazin (i.e., the chrysazin-8-O-α-L-rhamnoside) and methylated derivative of CR (i.e., chrysazin-8-O-α-L-2 -O-methylrhamnoside) produced by E. coli S2 whole-cell biotransformation were new compounds.

Anticancer Activities
The three compounds prepared were further analyzed for their in vitro cytotoxicities using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay against three different cancer cell lines ( Figure 4) and one normal cell line ( Figure S6). Two derivatives of chrysazin (i.e., CR and CRM) showed higher cytotoxicities than chrysazin. The 50% inhibitory concentration (IC 50 ) values of chrysazin for AGS, Huh7, and HL60 cells were 17.08, 30.53, and 22.24 (µM), respectively. Chrysazin-8-O-α-L-rhamnoside inhibited AGS, Huh7, and HL60 cells with 50% inhibitory concentration (IC 50 ) values of 28.58, 21.28, and 14.68 (µM), respectively. Here, CR showed better anticancer activities in Huh7 and HL60 than chrysazin. In the case of AGS cells, it showed slightly lower anticancer activity. In the case of chrysazin-8-O-α-L-2 -O-methylrhamnoside, it showed higher anticancer activities than chrysazin and CR. The 50% inhibitory concentration (IC 50 ) values of CRM for AGS, Huh7, and HL60 cells were 7.513, 4.467, and 4.540 (µM), respectively. In the case of the HaCaT normal cell line, the IC 50 values were >200 µM for chrysazin, CR, and CRM (Table S1 and Figure S6). These compounds had a lower inhibitory effect on normal cells. These results suggest that CR and CRM can remarkably reduce cell viabilities of AGS, Huh7, and Hl60 cells in a dose-dependent manner. This is the first report of the activity of these two compounds against AGS, Huh7, and HL60 cells.
normal cells. These results suggest that CR and CRM can remarkably reduce cell viabilities of AGS, Huh7, and Hl60 cells in a dose-dependent manner. This is the first report of the activity of these two compounds against AGS, Huh7, and HL60 cells.

Disk Diffusion Assay
Paper-disk diffusion assay was performed to determine the antimicrobial activity. All three compounds (chrysazin, CR, and CRM) were prepared at a concentration of 10 mg/mL. All compounds were added to each disk at a final concentration of 40 µ g/disk (4 µ L). Each disk was placed over Mueller-Hinton agar (MHA) plates spread with bacterial strains. The diameter of the zone of inhibition was measured after 18-20 h. Results of disk diffusion assays revealed that chrysazin did not show any antibacterial activity against 18 different human pathogens tested. However, CR and CRM exhibited antibacterial activities against Gram-positive bacteria S. aureus subsp. ( Figure S5 and Table S2). These results reveal that rhamnosylation and rhamnose methylation of chrysazin might be profitable for heightening its antibacterial activity against different Gram-positive bacteria.

Disk Diffusion Assay
Paper-disk diffusion assay was performed to determine the antimicrobial activity. All three compounds (chrysazin, CR, and CRM) were prepared at a concentration of 10 mg/mL. All compounds were added to each disk at a final concentration of 40 µg/disk (4 µL). Each disk was placed over Mueller-Hinton agar (MHA) plates spread with bacterial strains. The diameter of the zone of inhibition was measured after 18-20 h. Results of disk diffusion assays revealed that chrysazin did not show any antibacterial activity against 18 different human pathogens tested. However, CR and CRM exhibited antibacterial activities against Gram-positive bacteria S. aureus subsp. ( Figure S5 and Table S2). These results reveal that rhamnosylation and rhamnose methylation of chrysazin might be profitable for heightening its antibacterial activity against different Gram-positive bacteria.   (Table 3). MBC values were similar to or higher than MIC values in all tested strains. Chrysazin did not show antibacterial activity against different pathogenic bacteria but derivatives of it, i.e., CR and CRM, improve its antibacterial activity. The metabolite showed significantly improved antibacterial activity against a wide range of Gram-positive MRSA and MSSA pathogens. Therefore, CR and CRM have great potential as an antibiotic against superbugs.

Conclusions
In this study, we successfully engineered an E. coli strain for the sustainable production of different derivatives of chrysazin. Chrysazin-8-O-α-L-rhamnoside and chrysazin-8-Oα-L-2 -O-methylrhamnoside were novel compounds. We also evaluated their activities against three different cancer cell lines (AGS, Huh7, and HL60). CR and CRM exhibited higher cytotoxicities than their parental compound, chrysazin. More significantly, the evaluation of antibacterial activity revealed promising bioactivities of CR and CRM. This study establishes an engineered microbial platform that can be used to produce novel bioactive compounds. This microbial platform can be further fine-tuned for the production of novel derivatives of other anthraquinones or different compounds. Furthermore, the optimization of bioprocessing parameters and rational engineering of the host using various synthetic biological tools and metabolic engineering can be employed to enhance the production titer.  Table S1: Anticancer (IC50 (µM)) potential of chrysazin analogues against different cell lines.; Table S2: Antibacterial activities test against Gram-positive and Gram-negative bacteria via disc diffusion assay. The zone of inhibition (diameter) due to DMSO, chrysazin, chrysazin