Chrysoeriol is a 3′-O
-methoxy flavone, chemically a derivative of luteolin, which belongs to the flavone group of compounds and is abundantly present in many types of plants, vegetables, and fruits, including medicinal herbs [1
]. It has been studied for various biological functions, such as neuroprotective [2
], cardio protection [3
], anti-inflammation, anti-cancer [4
], and antimicrobial effects [5
]. Chrysoeriol is well known for its anti-CK2
activity, which inhibits different molecular forms of CK2
at a lower concentration than other flavonoids [6
]. Structurally, luteolin has a catechol group in its B ring, and these catechol groups tend to be modified by catechol-O
-methyltransferases, which are widely distributed in human organs, such as the liver, intestines, lungs, and brain [7
]. One of the derivatives, diosmetin, which has a 4′-methoxy group (para-position) in luteolin, is considered to be more common than chrysoeriol, which has 3′-methoxy (meta-position) luteolin. However, both of these compounds are reported to be natural prodrugs in cancer prevention [8
In particular, chrysoeriol has been naturally isolated from Artemisia
, which is a large genus of plants, and contains an artemisinin derivative, which is now used worldwide to treat malaria, which is caused by Plasmodium falciparum
]. Hence, it might be possible that chrysoeriol could have anti-microbial activities. Although flavonoids from natural products have been showing antibacterial actions against various pathogenic organisms, including a well-known methicillin-resistant Staphylococcus aureus
(MRSA), and synergistic antimicrobial effects [10
]. However, a core flavonoid compound, luteolin, has anti-MRSA activity that was previously reported to act on the bacterial cell wall, as was further proved mechanistically by disrupting the MRSA cytoplasmic membrane [12
]. Chrysoeriol has also been found to have anti-microbial effects against different bacteria [14
]. These are interesting experimental facts that suggest that such flavonoids and their derivatives might have pharmaceutical importance and industrial applications. Besides Artemisia
, chrysoeriol was also isolated from other plant sources for various reasons: Digitalis purpurea
, for the induction of nitric-acid synthase by blocking nuclear factor activator protein (κB AP-1) [15
]; Artemisia copa
, vasorelaxing and hypotensive effects study through Ca2+
inhibition influx [16
]; Capsicum frutescens
, to study and compare the antioxidant and antimicrobial activities of phenolic compounds [14
]; and Tecoma stans
, lipase inhibitory activity for type 2 diabetes mellitus [17
Most of these important compounds are either extracted from plant biomass or chemically synthesized. Neither of these techniques is environmentally friendly or economical. Recently, the application of a genetic engineering tool to design a microbial platform has been widely used to synthesize such compounds by expression of a specific plant or microbial genetic materials. Microbes, especially actinomycetes, are a diverse source of simple and complex natural products that cover most of the antibiotics in use today [18
]. The biosynthetic pathway of these natural products involves post-secondary metabolite modification enzymes, which have recently been mimicked by researchers to use for flavonoid modifications in microbial hosts, such as Escherichia coli
Luteolin is one of the abundant flavones found in plants. It occurs as both O
- and C
-glycosides. A recent review shows that the abundance of apigenin and luteolin in free and glycosides form in different citrus, teas, fruits, vegetables, olive oil, honey, and dry herbal plants [20
]. However, the occurrence of chrysoeriol is limited in plants, and it is not easily available from commercial vendors. In contrast, luteolin is easily obtainable from diverse suppliers at a relatively cheaper price. In this context, the biosynthesis of chrysoeriol using luteolin as starting precursor would be a more economical, eco-friendly, and sustainable approach.
This study highlights the microbial production of an important antimicrobial agent, chrysoeriol, by the use of a post-modifying enzyme, sugar O
-methyltransferase (SpnK) from Saccharopolyspora spinose
). This is the first report of using microbial sugar (rhamnose) O
-methyltransferase from a spinosyn biosynthesis gene cluster to catalyze plant metabolite luteolin methylation at the 3′-OH position. Recently, we observed SpnK as a promiscuous enzyme with broad substrate flexibility towards plant polyphenols [21
]. Thus, here, we employed SpnK to catalyze an O
-methylation reaction over luteolin to synthesize chysoeriol. The microbially produced chrysoeriol was compared to luteolin and diosmetin for anti-bacterial activity against various Gram-positive and Gram-negative bacteria. Chrysoeriol exhibited strong anti-microbial activity in a smaller dose than that needed for luteolin or diosmetin.
Numerous pathogens including both Gram-negative and Gram-positive bacteria are resistant to multiple antibiotics. Pathogens always tend to find ways to resist the treatment. According to the list of threatening pathogens given by the World Health Organization (WHO), Proteus
, E. coli, Klebsiella
, and Pseudomonas
fall under the deadly infectious pathogens, and methicillin-resistant S. aureus
falls under high risk, thus suggesting a need to develop new antibiotics for their control (www.who.int
). Thus, there is an urgent need to develop antibiotics that are effective against multiple drug-resistant pathogens. Recent reports show increasing infections by the aforementioned pathogens in hospitals. In this context, the development and discovery of new antibacterial agents effective against a broad spectrum of multi-drug-resistant Gram-positive and Gram-negative pathogens is of prime importance.
The search for new antibiotics takes a significantly long time and requires several approval and clinical trial steps. Thus, many researchers develop compounds based on structure-activity relationships (SAR) and optimize them for better activities against particular target pathogen based on the previously known structure and their activities [25
]. Yet, the development of molecules by post-modifications and other derivatization is another widely accepted approach for developing novel molecules with potent activities [27
]. Recently, diverse plant and microbial secondary metabolites have been modified by enzymatic and microbial biotransformation with the aim of developing novel agents with stronger activities against diverse pathogens, cancers, and other communicable and non-communicable diseases. When compared with chemical synthesis, enzymatic synthesis is the most advantageous. Chemical methods are long, health hazardous, and result in more toxic intermediates. However, whole-cell biotransformation uses the fully grown cells, which contain all the needed enzymes and cofactors for the chemical reaction to occur within the cell. The adequate concentration of co-factors and enzymes provides the favorable conditions that modulate the activity of multi-enzymatic complexes, resulting in the increase of reaction conversion rates [30
]. The production of antibiotics via microbial biotransformation not only involves supply of purified precursor molecules but may also encompass complex substrates that can be biotransformed into compounds rich in active substances [28
In this study, a simple approach of regiospecific modification of luteolin, a plant metabolite, was applied to modify it into a methyl-group-conjugated chrysoeriol derivative. A sugar O-methyltransferase gene spnK from actinomycetes S. spinosa was cloned into an E. coli BL21 (DE3) host for selective biotransformation of luteolin. As a result, we successfully achieved chrysoeriol, a 3′-O-methylated derivative in the culture broth. A similar derivative, but methyl-group conjugated at the 4′-O-position of luteolin, diosmetin, is abundantly present in plants as diosmin. However, the occurrence of chrysoeriol is limited. Thus, biosynthesis of this molecule is of significant interest among biochemists. Moreover, the molecule is active against a wide range of Gram-negative and Gram-positive pathogens, including WHO-listed critical and high-risk pathogens such as MRSA and Proteus.
A study by Teffo et. al. [31
] showed four methyl-ether derivatives of kaempferol, such as 3, 5, 7-trihydroxy-4′-methoxyflavone, 5, 7, 4′-trihydroxy-3, 6-dimethoxyflavone, 5, 7-dihydroxy-3, 6, 4′-trimethoxyflavone (santin), and 5-hydroxy -3, 7, 4′-trimethoxyflavone, from the leaf powder of Dodonaea viscosa Jacq
. var. angustifolia
. These molecules showed MIC values ranging from 16 µg/mL to above 250 µg/mL against S. aureus
, Enterococcus faecalis
, E. coli
, and Pseudomonas aeruginosa.
Similarly, Choi et. al. [32
] recently developed a bio-renovation technique to produce diverse derivatives of genistein using whole cells of Bacillus amyloliquefaciens
KCTC 13588. Among the isolated four derivatives, 4′-O
-isopropyl genistein exhibited antibacterial activity against MRSA and MSSA strains with better activities (MIC 8 µg/mL or above) than genistein. However, the 3′-O
-methylated luteolin derivative, also called chrysoeriol, exhibited a significantly lower MIC value of 1.25 µg/mL against S. aureus
CCARM 3640 (MRSA), which is 16 times lower than that of its parent compound and only 2.5 times higher than ampicillin [33
]. It is not only active against MRSA, but it is also active against seven other Gram-positive strains (MRSA and MSSA) and a Gram-negative P. hauseri
strain (Table 2
). Among all the tested organisms, chrysoeriol exhibited better activity than luteolin in the disc diffusion assay when a 40 µg/disc compound dissolved in DMSO was loaded over a sterile disc. We also compared the activity using diosmetin under identical conditions and concentrations. However, it did not show any activity against the tested pathogens. The evidence revealed a significance of modification of luteolin at the 3′-OH, particularly by the alkyl group. However, a previous report showed the significance of free 3′, 4′, 5′-trihydroxy groups in the B-ring and of the free 3-OH group for antibacterial activity [31
]. Flavonoids inhibited DNA synthesis in Gram-negative P. vulgaris
and RNA synthesis in Gram-positive S. aureus
]. Other potential targets of polyphenols have long been studied [34
]. The reports also provide evidence that the lowest MIC value reported against S. aureus
is (0.06–2.0) µg/mL by panduratin A [36
]. Similarly, other bioactive polyphenols include epigallocatechin gallate, which killed 56 clinical isolates of Helicobacter pylori
, causative agents of ulcers and gastric cancers, at MIC values of 100 µg/mL [37
All such evidence shows the anti-microbial potential of diverse polyphenols isolated from plants. However, selective production of these molecules from native plants suffers from several difficulties, such as the high consumption of both time and plant biomass, low yield, and the difficulty of extraction and purification. Thus, microbial production of such bioactive molecules by simple biotransformation using engineered microbial cells is always superlative. In this study, we produced bioactive chrysoeriol from luteolin by a simple biotransformation approach using an E. coli BL21 (DE3) host harboring the spnK gene. The metabolite showed significantly improved antibacterial activity against a wide range of Gram-positive MRSA and MSSA and Gram-negative P. hauseri pathogens. Therefore, chrysoeriol has great potential as an antibiotic against superbugs.
4. Materials and Methods
4.1. General Procedures
Luteolin and diosmetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropyl β
-D-1-thiogalactopyranoside (IPTG) was purchased from GeneChem, Inc. (Daejeon, Korea). All the other chemicals and reagents used in this study were of the highest chemical grade. E. coli
cells were grown in Luria-Bertani (LB) broth or an agar medium supplemented with ampicillin 100 μg/mL. Previously constructed plasmid pET32a+ SpnK [19
] was used for transformation into E. coli
BL21 (DE) for biotransformation in LB medium. Pathogenic strains such as Staphylococcus. aureus
CCARM 3640 (MRSA), S. aureus
CCARM 3089 (MRSA), S. aureus
CCARM 33591 (MRSA), S. aureus
CCARM 0205 (MSSA), S. aureus
CCARM 0204 (MSSA), S. aureus
CCARM 0027 (MSSA), S. aureus
CCARM 3090 (MRSA), S. aureus
CCARM 3634 (MRSA), and S. aureus
CCARM 3635 (MRSA), Salmonella enterica
ATCC 14028, Kocuria rhizophilla
NBRC 12708, Bacillus subtilis
ATCC 6633, Proteus hauseri
NBRC 3851, Enterococcus faecalis
19433, E. faecalis
19434, Klebsiella pneumonia
ATCC 10031, and E. coli
ATCC 25922 were obtained from Professor Seung-Young Kim (Sun Moon University, Korea) [32
4.2. Whole-Cell Biotransformation and Validation
A recombinant strain of E. coli BL21 (DE3) harboring pET32a+ SpnK was inoculated in 5 mL of LB broth medium and incubated at 37 °C supplementing ampicillin antibiotic (100 mg/mL) for the selection and maintenance of the recombinant strain. Out of the 5 mL culture, 500 μL as seed culture was transferred to the same medium (50 mL culture volume) in two different shake flasks and incubated at 37 °C. One flask culture was induced with 0.5 mM IPTG after the optical density at 600 nm (OD600nm) reached 0.7; the other was not induced, but they were both incubated at 20 °C for 20 h in a similar condition. The culture without IPTG induction was a control for the experiment. Both cultures were supplemented with 200 μM of luteolin as substrate dissolved in dimethylsulfoxide (DMSO) and were incubated at 20 °C for 48 h at 200 rpm for the bioconversion experiment.
Later, a culture broth of 500 μL from each flask was transferred to micro-centrifuge tubes, and an equal volume of chilled high-grade methanol was added, mixed vigorously by vortex, and centrifuged at 13,475× g for 15 min. The supernatant separated from the cell debris was analyzed by a high-performance liquid chromatography coupled with a photodiode array (HPLC-PDA) detector, followed by confirmation via high-resolution quantitative time-of-flight electrospray-ionization mass spectrometry (HRQTOF-ESI-MS).
For collecting a sample to characterize structurally, the biotransformation experiment was done using two 2-L shake flasks, each containing 500 mL of culture. The pure fraction of the compound was collected via preparative HPLC.
4.3. Analytical Methods
The reverse-phase high-performance liquid-chromatography photo-diode array (HPLC-PDA) analysis was performed with a C18 column (Mightysil RP-18 GP (4.6 mm × 250 mm, 5 μm) connected to a PDA (290 nm) using binary conditions of H2O (0.1% trifluoroacetic acid buffer) and 100% acetonitrile (ACN) at a flow rate of 1 mL/min for 35 min. At this condition, the ACN concentration began from 20% (0–5 min), 70% (5–20 min), 100% (20–30 min), 50% (30–33 min), and 20% (33–35 min). The purification of the compounds was carried out by preparative HPLC with a C18 column (YMC-Pack ODS-AQ, 250 mm × 20 mm ID., 10 μm) connected to a UV detector (290 nm) using a 36-min binary program with ACN of 20% (0–5 min), 50% (5–12 min), 70% (12–20 min), 90% (20–28 min), 50% (28–33 min), and 20% (33–36 min) at a flow rate of 10 mL/min.
The HRQTOF-ESI-MS was performed in positive ion mode using an Acquity mass spectrometer (Waters, Milford, MA, USA), which was coupled with a Synapt G2-S system (Waters). However, for nuclear magnetic resonance (NMR) studies, a purified molecule was dissolved in hexadeuterodimethyl-sulfoxide (DMSO-d6) from Sigma-Aldrich. The compound was characterized by a 900-MHz NMR spectrometer equipped with the TCI CryoProbe (5 mm). In addition to 1H and 13C, two-dimensional NMRs, such as heteronuclear single-quantum correlation [HSQC] and heteronuclear multiple-bond correlation [HMBC], were performed for structural confirmation.
4.4. Biological Activities
4.4.1. Disc Diffusion Assay
The antibacterial activity of the newly synthesized compound was first examined by disc diffusion assay using the aforementioned microbial strains cultured in a Mueller Hinton agar (MHA) plate (Difco, Baltimore, MD, USA). Paper discs of 7-mm diameter were prepared and autoclaved. The discs were placed in each plate in a marked region after spreading the pathogen uniformly. Then, 4 microliters of 10 µg/mL of luteolin, diosmetin, and chrysoeriol were added to each disc, and the plates were kept for bacterial growth in a 37 °C incubator. The zone of inhibition was observed after the pathogen growth was visible. The diameter of the zone of inhibition was noted for each pathogen. Dimethyl sulfoxide (DMSO) was used as a control for the zone of inhibition, as all the compounds were dissolved in DMSO.
4.4.2. Measurement of the Colony-Forming Unit (CFU)
The colony-forming unit was observed for two pathogens, S. aureus 3640 (MRSA) and Proteus hauseri NBRC 3851. The pathogens were cultured until the O.D600 reached around 0.5. The cells were taken by centrifugation at 3000 rpm for 10 min, and the aliquot was removed. The cells were suspended in 5 mL of phosphate buffer (pH 7.0). Then, 1 mL of cells in phosphate buffer was taken into separate polypropylene tubes, in which 40 µg/mL of each compound was added separately. The result was compared with standard luteolin and the control (no compound added), which were also prepared under the identical conditions. The experimental cells were incubated at 37 °C in an incubator under shaking condition. Samples were taken at 0 min, 2 h, 4 h, and 6 h for each of the luteolin, chrysoeriol, and control. The sample was appropriately diluted and plated on MHA and incubated at 37 °C in a plate incubator for overnight growth. The colony-forming units (CFU) were counted the following day.
4.4.3. Measurement of Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration (MIC) was also calculated for two strains, S. aureus 3640 (MRSA), which Gram-positive, and Proteus hauseri NBRC 3851, which is a Gram-negative pathogen. The pathogens were cultured until they reached O.D600 of 0.6. LB broth (1 mL/tube) was dispensed into the 15-mL polypropylene tube. Different concentrations (80 µg/mL, 60 µg/mL, 50 µg/mL, 40 µg/mL, 30 µg/mL, 20 µg/mL, 10 µg/mL, 5 µg/mL, 2.5 µg/mL, 1.25 µg/mL, 625 ng/mL, 312.5 ng/mL, 156 ng/mL, and 78 ng/mL) of the compounds (chrysoeriol and luteolin) were added into each tube, in which 20 µL of inoculum were inoculated from the culture of O.D600 of 0.6. The tubes were kept for growth at 37 °C. After 16 h of incubation, growth (turbidity) was monitored by the unaided eye, and the MIC was noted. The growth was compared to sterile MH broth and culture without antibacterial compounds as controls. The test was repeated three times to confirm the MIC value.