Isolation of quercetin metabolizing bacteria
The soils from the tea planting area were diluted, plated on LB agar plates, and incubated for 48 h at 30 °C. As a result, 2.46 × 107 colony-forming units (CFU)/g soil of the total number of culturable bacteria were obtained on LB agar plates. This was slightly higher than the results from the soils collected from the city park reported as 1.96 × 107 CFU/g . However, it was similar to 2.5 × 108 CFU/g of culturable bacteria reported from the investigation of the soils in Chang Qing Graden (CQG), which consisted of hardwood trees aged more than 20 years . Although the bacterial species were not compared, it was inferred that soils with large amounts of organic matter were favored by microorganisms.
To elucidate the biotransformation performance of the isolated bacteria, quercetin was fed into the cells as a substrate. After the biotransformation processes, the samples prepared by ethyl acetate extraction were subjected to HPLC analysis . Approximately 200 colonies were tested, of which 11 colonies showed new peaks at different retention times of quercetin, therefore, 5.5% of bacteria isolated from tea planting soils were able to convert quercetin (Additional file 1). The biotransformation ability of the bacteria was isolated through subculture processes, and their biotransformation ability to generate products from quercetin was confirmed by repeated experiments. Among the bacteria possessing biotransformation activity, the most reactive strain was selected and investigated in the subsequent experiments.
Identification of quercetin metabolizing bacteria
16S rRNA sequencing was performed to identify isoquercitrin-producing bacteria isolated from tea-planting soils. After the amplified 16S rRNA from the genomic DNA of bacteria was introduced into a pGEMT-easy vector (Promega, USA), 16s RNA gene sequences were determined by nucleotide sequence analysis (Additional file 2). The nucleotide sequences were compared and analyzed using the 16S ribosomal RNA gene sequencing database with the blastn program of the Basic Local Alignment Search Tool (BLAST). The 16S rRNA sequence of bacteria showed high homology with Bacillus aryabhattai B8W22 (99.8%), Bacillus megaterium strain ATCC 14581 (98.67%), Bacillus flexus strain IFO15715 (98.67%), Bacillus zanthoxyli strain 1433 (99.65%), Bacillus iocasae strain S36 (96.72%), and Bacillus qingshengii strain G19 (97.97%). As shown in Fig. 1, the isolated Bacillus strain was closest to Bacillus iocasae and Bacillus horikoshii DSM8719 and belonged to the same group as Bacillus megaterium and Bacillus acidicola based on phylogenetic analysis. The isolated strain possessing the biotransformation activity of quercetin was named Bacillus sp. CSQ10.
Characterization of biotransformation performance of Bacillus sp. CSQ10
Since Bacillus sp. CSQ10 isolated from tea planting soils showed the ability to metabolize quercetin, and its biotransformation performance was investigated. The quercetin was added as substrate to cells resuspended in M9 medium supplemented with 2% glucose. After 12 h of incubation, 2 mL of cell culture was extracted with ethyl acetate to prepare the samples for HPLC analysis. As shown in Fig. 2, a new substance (P1) was observed at 11.09 min (Fig. 2C), except at 14.39 min (Fig. 2A), which is the retention time for quercetin. Although the UV absorbance of P1 was similar to that of quercetin, the maximum absorbance of band I shifted from 371 to 354 nm (boxes in Fig. 2A, C). It is known that the shift of band I maximum absorbance in flavonols is caused by a hypsochromic shift induced by structural changes at position 3 . Thus, it was assumed that P1 would undergo a conformational change at position 3 of quercetin. To verify this assumption, P1 was used for mass spectrometry (MS) to estimate its molecular weight. The results of MS analysis of P1 are shown in Fig. 2D. The ion peak at 487.1 [M + H]+ was estimated to be quercetin-hexose-Na+, 465.2 [M + H]+ was quercetin-hexose, and 302.8 [M + H]+ was quercetin. In addition, major ions such as 463.4 [M−H]−, 300.2 [M−H]−, 271.2 [M−H]−, 255 [M−H]−, 179.1 [M−H]−, and151.0 [M−H]− were observed in the ion negative MS analysis (Fig. 2E). The ionization pattern determined by MS analysis was the same as the MS fragmentation pattern of quercetin-3-O-glucoside in a previous study . This was further confirmed by analyzing commercial quercetin 3-O-glucoside. As shown in Fig. 2B, the retention time and UV absorbance of quercetin 3-O-glucoside are indistinguishable from those of P1. Therefore, it can be concluded that Bacillus sp. CSQ10 exhibits biotransformation activity to produce isoquercitrin through biotransformation using quercetin as a substrate.
Isoquercitrin had been first isolated from the seed pods of Cercis canadensis L. (eastern redbud)  and then from various plants [11,12,13,14,15,16,17,18, 34, 35]. Isoquercitrin is a biologically active substance of St. John’s wort (Hypericum perforatum L.) , a medicinal plant found in many plant-derived beverages such as fruits, vegetables, tea, and wine . Additionally, it is known that isoquercitrin exhibits various physiological activities such as anti-inflammatory and anti-obesity , antihyperglycemic , colon inflammation relief , skin cancer growth inhibition , and liver cancer growth inhibition . Despite these various physiological activities, it is challenging to supply isoquercitrin commercially because the isoquercitrin content in plants is insufficient. In this regard, it would be invaluable to isolate new bacterial strain to produce isoquercitrin by one-step of biotransformation.
Effects of media on biotransformation activity of Bacillus sp. CSQ10
The biotransformation efficiency of microorganisms has been reported to be modulated by the components of the media [24, 25]. The biotransformation activity of Bacillus sp. CSQ10 was investigated under different biotransformation media, including Luria Broth (LB) medium (BD-Difco, USA), Terrific Broth (TB) medium (BD-Difco, USA), yeast extract –peptone–dextrose (YPD) medium (BD-Difco, USA), Andrw’s Magic Media (AMM)-Glu medium , AMM-Gly medium, YM9 (10 g yeast extract and M9 salt)-Glu medium, and M9-Glu medium (Fig. 3). Following the same procedures described previously, Bacillus sp. CSQ10 cells adjusted to 0.6 of OD600 were fed 200 µM quercetin as a final concentration and incubated for 12 h at 30 °C. As a result, the highest amount of isoquercitrin was produced in YPD medium as 56.2 mg/L, followed by M9-glucose medium (36.3 mg/L), AMM-glucose medium (38.6 mg/L), and YM9-glucose medium (20.9 mg/L) (Fig. 3A). From the HPLC analysis, only one peak (11.09 min) was produced in the biotransformation medium of LB, TB, AMM-Glu, AMM-Gly, and M9-Glu, while two peaks were observed at 11.09 min and 12.07 min in the biotransformation medium of YPD and YM9-glucose (Fig. 3D). As verified above, the peak indicated as P1 was identified as isoquercitrin by comparing the elution time and UV absorbance with those of authentic isoquercitrin. Although the peak indicated as P2 had a different elution time, the UV absorbance spectrum was indistinguishable from that of isoquercitrin (Fig. 3D). Thus, it can be assumed to be a derivative of P1, isoquercitrin.
To characterize the new product of biotransformation, P2 was used for MS analysis. As shown in Fig. 3E, major ion peaks such as 528.7 [M + H]+, 506.7 [M + H]+, and 302.8 [M + H]+ were detected. It could be interpreted as that the molecular weight of 528.7 [M + H]+ was Na+ attached to quercetin-3-O-(6″-O-acetyl)-β-d-glucoside, 506.7 [M + H]+ was quercetin-3-O-(6″-O-acetyl)-β-d-glucoside, and 302.8 [M + H]+ was quercetin aglycone. In addition, major ion peaks, such as 301.5 [M−H]−, 463.5 [M−H]−, and 505.6 [M−H]− were observed in the ion-negative MS analysis (Fig. 3F). The molecular weights of 301.5 [M + H]−, 463.5 [M + H]−, and 505.6 [M + H]− were estimated to be quercetin, quercetin-3-O-glucoside, and quercetin-3-O-(6″-O-acetyl)-β-d-glucoside, respectively. Therefore, it was necessary to conduct nuclear magnetic resonance (NMR) analysis to verify exact structure of P2. For the preparation of NMR samples, 500 L of cells was reacted with quercetin and extracted with ethyl acetate. The extracts were then subjected to HPLC to purify P2 and 6 mg of P2 was obtained. The samples were subjected to 1H-NMR analysis following the procedures described by Kim et al. . The NMR data was analyzed as follows: 1H-NMR (400 MHz, Acetone-d6); δ 6.21 (H, d, J = 2.0 Hz), δ 6.41(H, d, J = 2.0 Hz), δ 7.59 (1H, d, J = 2.0 Hz), δ 6.84 (H, d, J = 2.0 Hz), δ 7.60 (H, dd, J = 2.0, 8.6 Hz), δ 5.11 (H, d, J = 7.7 Hz), δ 4.6 (H, m), and δ 4.17(H, m). The structure of P2 was determined by comparison with 1H-NMR spectrum published by Jeon et al. . Conclusively, the new product indicated as P2 was identified as quercetin-3-O-(6″-O-acetyl)-β-d-glucoside. The results revealed that the Bacillus sp. CSQ10 isolated from soil can convert quercetin to isoquercitrin and quercetin-3-O-(6″-O-acetyl)-β-d-glucoside. Moreover, it would be inferred that Bacillus sp. CSQ10 has genes that catalyze the biotransformation of quercetin, and the gene encoding an enzyme that attaches an acetyl group on the glucoside of isoquercitrin would be induced by components only in YPD and YM9-glucose.
Optimization of isoquercitrin production
To maximize the yield of isoquercitrin, biotransformation of quercetin by Bacillus sp. CSQ10 was examined under various experimental conditions, including temperature, cell density, and substrate concentration. First, the amount of isoquercitrin was determined at different temperatures, including 25, 30, and 37 °C. As shown in Fig. 4A, the biotransformation efficiency of isoquercitrin at 30 °C and 37 °C was similar (59 mg/L), whereas the efficiency decreased about threefold at 25 °C (20 mg/L). This is because the growth rate of Bacillus sp. CSQ10 was reduced at 25 °C compared to other culture temperatures, so the supply of UDP-glucose, a sugar donor, was limited; therefore, the biotransformation efficiency was lowered. To verify the effects of cell density on the biotransformation efficiency, the initial cell densities for feeding quercetin were adjusted to 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 of OD600. As a result, the biosynthesis of isoquercitrin increased as the cell density increased, and the highest biosynthesis (128 mg/L) was observed at a cell density of 3.0 of OD600 (Fig. 4B). However, no further increase in isoquercitrin biosynthesis was observed even when the cell density was increased to over 3.0 OD600 (Fig. 4B). Finally, the effects of quercetin concentration on isoquercitrin biosynthesis were investigated. The cell density of Bacillus sp. CSQ10 in M9-glucose medium was adjusted to 3.0 of OD600 and 10 different concentrations (100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM) were tested (Fig. 4C). The highest production of isoquercitrin was 128 mg/L with 600 μM of quercetin supply, and approximately 10% less biotransformation efficiency (approximately 118 mg/L) was observed with 400, 500, 700, and 800 μM of quercetin supply. Additionally, it was observed that the isoquercitrin production was slightly decreased at 900 μM of quercetin and rapidly decreased at 1000 μM of quercetin supply (Fig. 4C). This result seems to be caused by the antibacterial effect of quercetin on Bacillus sp. CSQ10, as well as by the metabolic load for isoquercitrin biosynthesis Based on these results, it can be concluded that the optimum conditions for biotransformation to produce isoquercitrin from quercetin are M9-glucose medium, 30 °C, and 600 μM quercetin.
Under optimized conditions, we monitored isoquercitrin production for 72 h (Fig. 5). The biotransformation rate to produce isoquercitrin was high at the initial stage until 4 h, and 123 mg/L of isoquercitrin was produced. Subsequently, the rate gradually decreases. The highest production of isoquercitrin was 193 mg/L after 24 h of incubation, while 11.3 mg/L of quercetin remained. However, no further increase in isoquercitrin biosynthesis was observed, even after 72 h of incubation. During this biotransformation process, the cell density gradually increased to 6.8 of OD600 value at 48 h, and then decreased slightly after 48 h.
Optimization of Quercetin-3-O-(6″-O-acetyl)-β-D-glucoside
Isolated Bacillus sp. CSQ10 possesses the ability to biotransform isoquercitrin to quercetin-3-O-(6″-O-acetyl)-β-D-glucoside, as well as quercetin to isoquercitrin with YM9-glu and YPD as transformation media. The first identification of quercetin-3-O-(6″-O-acetyl)-β-d-glucoside was from Petasites japonicus  and was recently reported in Pinus densiflora needles . It has been reported that quercetin-3-O-(6″-O-acetyl)-β-d-glucoside has biological effects such as protection of skin cells from UV radiation , aldose reductase inhibitory activity , and antioxidant activity. Although several types of biological activities have been reported, the process has been hampered to explore their biological activity and supply as a diet because of the difficulty in obtaining sufficient amounts. In this regard, the biotransformation of quercetin by Bacillus sp. CSQ10 is valuable for the production of quercetin-3-O-(6″-O-acetyl)-β-D-glucoside.
To achieve the maximum yield, the experimental conditions for quercetin-3-O-(6″-O-acetyl)-β-D-glucoside production were optimized by testing various temperatures, cell densities, and substrate supply concentrations in YPD medium (Fig. 6). The biosynthesis of quercetin-3-O-(6″-O-acetyl)-β-D-glucoside occurred at 37 °C during the biotransformation process (97 mg/L), and 200 mg/L of isoquercitrin was obtained (Fig. 6A). As the temperature decreased, the production of quercetin-3-O-(6″-O-acetyl)-β-d-glucoside also decreased. This could be attributed to the retarded growth rate of Bacillus sp. CSQ10 limits the supply of UDP-sugar donors and acetyl-CoA, thereby resulting in a decrease in biosynthesis. To investigate the effects of the initial cell density on the biotransformation of quercetin-3-O-(6″-O-acetyl)-β-D-glucoside production yield, quercetin was fed to Bacillus sp. CSQ10 at 1.0 to 10.0, OD600 in the YPD medium. The cells underwent the biotransformation process by incubating at 37 °C for 12 h, and the biotransformation yields were determined by HPLC analysis. As shown in Fig. 6B, the production of quercetin-3-O-(6″-O-acetyl)-β-D-glucoside was increased to the 6.0 of OD600 showing highest amount as 120 mg/L, and the biotransformation efficiency was gradually decreased. In contrast, the amount of isoquercitrin biosynthesized from quercetin was inversely proportional to the amount of quercetin-3-O-(6″-O-acetyl)-β-D-glucoside. Therefore, we inferred that Bacillus sp. CSQ10 can convert quercetin to isoquercitrin, and isoquercitrin to quercetin-3-O-(6″-O-acetyl)-β-D-glucoside in YPD media. To verify the effects of quercetin concentration, the range of 100 to 1,000 μM of quercetin was fed to Bacillus sp. CSQ10 was adjusted to 6.0 an OD600 values of YPD. As shown in Fig. 6C, the highest production of quercetin-3-O-(6″-O-acetyl)-β-D-glucoside as 118.9 mg/L was observed from 600 μM of quercetin supply and it was decreased with higher concentration of quercetin. Additionally, the production efficiency of isoquercitrin increased up to 900 μM in YPD media, while the highest efficiency was observed at 600 μM in M9-glu (Fig. 5C).
In summary, the optimal conditions for quercetin-3-O-(6″-O-acetyl)-β-d-glucoside biosynthesis from quercetin were YPD medium for biotransformation, 37 °C for temperature, and 600 μM of quercetin. As described in the previous section, we monitored the efficiency of quercetin-3-O-(6″-O-acetyl)-β-d-glucoside production for 72 h under the optimized experimental conditions (Fig. 7). Isoquercitrin biosynthesis was first observed during the initial stage, and then the biosynthesis of quercetin-3-O-(6″-O-acetyl)-β-d-glucoside was initiated. Only isoquercitrin (69.9 mg/L) was obtained after 4 h of incubation, whereas no quercetin-3-O-(6″-O-acetyl)-β-d-glucoside was detected. However, the biosynthesis of quercetin-3-O-(6″-O-acetyl)-β-d-glucoside accelerated from 6 h and reached a maximum biotransformation amount of 198 mg/L after 48 h of incubation. In the case of isoquercitrin, the maximum production was observed after 10 h incubation with 124 mg/L and tended to decrease gradually with increasing incubation time and production of quercetin-3-O-(6″-O-acetyl)-β-d-glucoside (Fig. 7). The density of cells was also monitored and reached an 8.0 of OD600 after 12 h of incubation.
WCBs based on microorganisms have been used to produce high value-added physiological substances from inexpensive substances. Unlike genetically engineered organisms, WCBs for biotransformation have advantages such as being free of antibiotics and inducers, including isopropyl β-D-1-thiogalactopyranoside (IPTG) and arabinose. In addition, there is no need to supply expensive cofactors, as required in enzymatic biosynthesis, because WCBs can utilize their own endogenous cofactors. Moreover, the processes to produce bioactive substances using WCBs were simple to expand to larger quantities. With the advantages of biosynthesis by WCBs, many microorganisms, including Bacillus species, owing to their versatile functions and biological safety, have been implanted in the process of synthesizing bioactive substances. In this study, we isolated microorganisms possessing metabolic mechanisms for isoquercitrin biosynthesis from the soils of tea plantations. Thus, quercetin-metabolizing Bacillus sp. CSQ10 was isolated and used as a WCB to produce isoquercitrin from quercetin. Additionally, Bacillus sp. CSQ10 can produce quercetin-3-O-(6″-O-acetyl)-β-D-glucoside when M9-glucose medium supplemented with yeast extract was used as the biotransformation medium. To achieve maximum biotransformation efficiency, the experimental conditions for the biosynthesis of isoquercitrin and quercetin-3-O-(6″-O-acetyl)-β-d-glucoside from quercetin were optimized. As a result, approximately 193 mg/L of isoquercitrin and 198.7 mg/L of quercetin-3-O-(6″-O-acetyl)-β-D-glucoside were produced from 181.2 mg/L of quercetin after 48 h of incubation. Conclusively, it would be invaluable to isolated new bacterial strain capable of producing a rare glycosylated flavonoind, isoquercitirn, by one-step biotransformation. Since the biotransformation using Bacillus sp. CSQ10 could be an efficient method to produce massive amount isoquercitrin compared to conventional methods, it would be appreciated by industrial fields. Moreover, it is noteworthy that Bacillus sp. CSQ10 contains genes encoding enzymes that metabolize quercetin, although they were uncharacterized. Although the genes were not identified at this stage of investigation, it would be next goals to be achieved. We believed to identify those genes was invaluable not only to enlarge the understanding of flavonoid metabolic pathway in newly isolate strain but also to provide clues for biosynthesizing biological active substances.
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