Biotransformation of Daidzein, Genistein, and Naringenin by Streptomyces Species Isolated from High-Altitude Soil of Nepal

Flavonoids have achieved widespread importance in pharmaceutical, food, and cosmetics industries. Furthermore, modification of these naturally occurring flavonoids to structurally diverse compounds through whole cell biotransformation with enhanced biological activities has numerous biotechnological applications. The present study investigated the biotransformation potential of Streptomyces species isolated from a high-altitude-soil sample towards selected flavonoid molecules. The biotransformed metabolites were confirmed by comparing the HPLC chromatogram with authentic compounds and LC-MS/MS analysis. Of these isolates, Streptomyces species G-18 (Accession number: MW663767.1) catalyzed isoflavone molecules daidzein and genistein to produce hydroxylated products at 24 h of reaction condition in a whole cell system. The hydroxylation of daidzein (4′,7-dihydroxyisoflavone) was confirmed at 3′-position of the B ring to produce 3′,4′,7-trihydroxyisoflavone. In addition, Streptomyces species G-14 (Accession number: MW663770.1) and Streptomyces species S4L (Accession number: MW663769.1) also revealed the transformation of daidzein (4′,7-dihydroxyisoflavone) to hydroxy daidzein at a distinct position than that of G-18 isolates, whereas thee Streptomyces species S4L reaction mixture with naringenin as a substrate also revealed the hydroxylated product. Our results demonstrated that microorganisms isolated from different ecological niches have broad application.


Introduction
Streptomyces are Gram-positive bacteria known for their production of diverse groups of secondary metabolites; many of which are now commercially used as antibiotics [1]. Over the years, these microorganisms evolved ecological roles by involving in changing soil dynamics, pathogenesis, nutrients exhaustions, and various abiotic and biotic stresses as well [2]. e secondary metabolites produced by Streptomyces provide the organism with competitive advantages to cope with the changing environment [3]. Genome analysis revealed that genes involved in biosynthesis of secondary metabolites are mostly clustered and their expression is highly regulated by different regulatory enzymes. Streptomyces are not only known for their production of diverse secondary metabolites but also used as a host for the production of foreign molecules through various biochemical engineering processes [4]. e unique metabolic diversity and enzymatic capabilities of Streptomyces make them a robust host for the production of diverse chemical constituents. Streptomyces griseus catalyzes a wide variety of biotransformation reactions towards flavonoids [5]. e majority of S. griseus catalytic reactions include O-and N-dealkylations, hydroxylation, epoxidation, and carbon bond fission [6,7]. Microbial biotransformations provide the necessary precursors for the production of new compounds [8]. In addition, microorganisms have tendencies to modify functional groups of secondary metabolites to enhance their biological activities. Daidzein, a soy isoflavone, was hydroxylated by Aspergillus oryaze to form 6-hydroxydadizein and 8-hydroxydaidzein [9]. Moreover, this hydroxylated daidzein has ten-fold increase in antityrosinase activity compared with the precursor compounds of daidzein [10]. Hence, identification of a new microorganism with robust potential to convert foreign molecules is of scientific interest.
Flavonoids are a diverse group of phytochemicals that are produced by various plants species and play an important role in plant growth and development, as well as to defend against microorganisms and pests serving as means of plant-animal warfare [11]. Although plants are the potential source of flavonoids, many these flavonoids can be produced by the microorganisms through the whole cell biotransformation systems or through metabolic pathway engineering [12]. Interestingly, flavonoids that are not produced by plants can be produced by microbes through specific enzymatic modification [13]. Many microorganisms can perform several biochemical reactions such as hydroxylation, methylation, and glycosylation through the specific set of enzymes [14]. In addition, the advantage of using the whole cell biotransformation systems is that the microbes produce the necessary cofactors needed for the biochemical reactions. Furthermore, microorganisms isolated from different climatic and geographic conditions are proven to be effective for the production of diverse enzymes with different catalytic properties, which in turn play important roles in the production of diverse secondary metabolites. Hence, this study was designed in such a way to evaluate the biotransformation of flavonoid compounds daidzein, genistein, and naringenin by Streptomyces species isolated from a high-altitude-soil sample.

Soil Sample Collection and Species Isolation.
e soil samples for the isolation of Streptomyces species were collected from Gosaikunda, Langtang National Park, Nepal, 4380 masl (28.0820°N, 85.4150°E). e Streptomyces species were isolated in an ISP2 medium with the serial dilution method using a sterile spreader. All strains were characterized by their Gram staining, growth patterns, and colony morphology.

Growth Analysis.
e growth curve of isolated Streptomyces species (G-10, G-14, G-18, and S4L) was investigated by growing them for 96 h in 50 mL of the R2YE medium, inoculated individually using 1 mL of preculture inoculums of each species. e isolates were incubated on a rotary shaker (250 rpm) at 28°C for four days. An aliquot of 1 mL of the culture broth was withdrawn at 12 h interval, from 0 h to 93 h. e cell mass was harvested at 12,000 rpm for 5 min, and the supernatant was discarded. e pellets were washed with distilled water and dried in oven at 70°C. Dry weight of cell mass was measured using a weighing machine, and graphs were plotted as dry cell weight vs. time.

Whole Cell Biotransformation.
Whole cell biotransformation of flavonoids using Streptomyces species was carried out in the R2YE medium. Flavonoid substrate molecules were individually dissolved in dimethyl sulfoxide, and 100 mM of stock solution of each substrate was prepared. Streptomyces species were grown in the R2YE medium using the preculture inoculums. 100 μM of substrate molecules daidzein, genistein, naringenin, and quercetin were fed into 72 h culture broth of Streptomyces species. e progresses of the biotransformation reactions were monitored after 24 h of reaction. Metabolites were extracted using ethylacetate as an extracting solvent. e organic layer was dried using a vacuum evaporator. e metabolites were analyzed by High-Performance Liquid Chromatography (LC-2030 Shimadzu, Kyoto, Japan) equipped with a UV detector and C18 column (4.6 × 150 mm, 5 μm particle size). e flow rate of the solvent was 1.0 mL/min, and the injection volume was 10 μL. e mobile phase used for the analysis was acetonitrile (CH 3 CN): water (0.1% TFA) � 3 : 7 (v/v). e UV detection was carried out at 254 nm. e mass spectrometry was performed with an Agilent Jet Stream Electrospray Ionization source (AJS ESI) in MS2 scan and positive polarity mode. Acetonitrile and 0.1% formic acid in water in the ratio of 3 : 7 were used throughout the run. e flow rate was 0.5 mL/min. e column temperature was 30°C, and injection volume was 2 μL.

Growth Curve Analysis of Streptomyces Species.
Prior to the biotransformation of flavonoids, the growth pattern of isolated Streptomyces species was evaluated. ese isolates G-10, G-14, G-18, and S4L were grown in the R2YE medium for 96 h at 28°C. e Streptomyces species grew exponentially until 68 hours in the R2YE medium ( Figure 1). e highest dry weight biomass (mg/mL) was recorded by the incubation of 20 h to 48 h of period, which revealed the exponential growth phase. However, from 48 h onward, the growth was stagnant, and no significant increase in biomass was observed. Among the analyzed Streptomyces species, the highest growth was observed in Streptomyces species G-18, followed by Streptomyces species G-10.

Biotransformation of Flavonoids.
e whole cell biotransformation reaction with isolated Streptomyces species was carried out with flavonoids as substrate molecules. Analysis of the reaction products was carried out using HPLC equipped with a UV detector. Preliminary analysis revealed that, among several flavonoid molecules screened for the biotransformation potential by Streptomyces species, G-18 and G14 showed the biotransformed products toward daidzein as a substrate. Furthermore, G-18 also revealed the biotransformation product towards genistein, whereas S4L showed the biotransformation potential towards naringenin as a substrate. However, all other evaluated substrates did not reveal any product formation in our preliminary investigation (Table 1).
Further confirmation of the reaction metabolites was carried out by comparison of the HPLC chromatogram with authentic compounds. e substrate and reaction products were clearly separated. e reaction product of daidzein was matched completely with the authentic 7,3′,4′-trihydroxyisoflavone. Hence, daidzein biotransformation by Streptomyces species G-18 was at the 3′-position (Figure 2(a))), whereas Streptomyces species S4L and G-14 had a slight shift in the product peak, and this could be due to the hydroxylation of daidzein at 6-and 8-position (see supplementary data (available here)), whereas with genistein, the product peak was distinct from genistein, as observed by comparision of the HPLC chromatogram of authentic genistein, reaction product of Streptomyces species G-18, and controlonly Streptomyces species G-18 without substrate (Figure 2(b)). Moreover, reaction products of Streptomyces species S4L show distinct product peaks with naringenin as a substrate in comparison of the HPLC chromatogram with the control reactions (Figure 2(c)). e product identification and characterization of biotransformation products were performed by mass spectrometry analysis. A schematic representation of the biotransformation by the isolated species is shown in Figure 3.

Mass Spectrometry Analysis of Metabolites.
Further confirmation of the biotransformation products was carried out by mass spectrometry analysis. e reaction mixture of Streptomyces sp. G18 with daidzein showed two distinct MS peaks [M + H] + � 254.9 corresponding to daidzein and [M + H] + � 270.9 product. e increase of molecular mass by 16 units is due to the addition of one hydroxy group in daidzein. Based on the comparative HPLC chromatogram with authentic 3′,7,4′-trihydroxyisoflavone and further analysis by mass spectrometry, we confirm that the daidzein was biotransformed by Streptomyces species G-18 to the 3′position ( Figure 4).  Table 1: Screening of flavone, isoflavone, and flavanone against Streptomyces species for biotransformation of diverse substrate molecules. Analysis was based on the HPLC chromatogram of unknown peaks, where (+) means biotransformation positive results and means no biotransformation based on the unknown peak in the HPLC chromatogram.

Substrate
Streptomyces sp. S4L Streptomyces sp. G10 which is hydroxygenistein ( Figure 5). Based on the MS spectra, we speculate that the genistein must be hydroxylated at the 3′-position, whereas Streptomyces species S4L metabolized the naringenin (m/z � 272) to hydroxylated naringenin (m/z � 288), evident by the increase in the product peak by 16 units compared to naringenin ( Figure 6).

Discussion
Soy isoflavones are naturally occurring phytochemicals that are associated with diverse health benefits [15]. In addition, they are biotransformed through oxidative and reductive reaction in diverse microorganism, significantly increasing the biological activities compared to original substrates [16].
Recently, food, cosmetics, and nutraceutical industries have shown interest in the functional derivatives of the isoflavones and flavones. erefore, scientists all around the world are trying to understand the biosynthetic pathways for the production of valuable bioactive compounds using whole cell or enzymatic biotransformation. Esaki [19]. Although plants are the source of the flavonoids, extraction and purification do not meet the industrial demands. Moreover, many nonnatural flavonoid molecules can be produced using a microbial host. Choi et al. have reported that O-methyltransferase from Streptomyces avermitilis involved in the production of 4′,7-dihydroxy-3′methoxyisoflavone, a nonnatural compound [20]. Hence, microbial production of biotransformed molecules using whole cell or enzymes is alternative. Recent advancement in genetic engineering has made it possible to manipulate genes and engineer the host microorganism as well for efficient production of nonnatural isoflavonoids in a microbial host. Among the known microbial hosts, Streptomyces has been proven to be an effective host for the production of diverse chemical compounds. e biosynthetic gene cluster of secondary metabolites producing Streptomyces sp. involves several enzymes, which might be effective for the biotransformation of diverse molecules. Furthermore, cofactors such as NADH and/or NADPH required for the enzymatic  modification of the flavonoid molecules are produced endogenously with in the microbial host [21]. It has been reported that the overexpression of cytochrome p450 enzyme CYP105D7 with its redox partners FdxH and FprD in S. avermitilis host could produce 112.5 mg of 7,3′,4′-trihydroxyisoflavone [22,23].
Despite the significant effort in the past for the production of ortho-hydroxylated daidzein using whole microbial cell or enzymatic reaction, industrial-scale production of ortho-dihyroxyisoflavone (ODI) is still a big challenge. e bioconversion of flavonoids and isoflavonoids by microorganisms is very low and also hindered by solubility of substrate molecules in an aqueous solution. Moreover, ortho-dihydroxylated metabolites undergoe rapid degradation by the dioxygenase enzymes. Hence, screening of robust microorganisms from different environments as well as a protein engineering approach can overcome such limitations. In our investigation, Streptomyces species G-18 revealed hydroxylated products of daidzein and genistein. Daidzein was hydroxylated at the 3′position, which is confirmed by comparing the HPLC chromatogram of authentic compounds and mass spectrometry analysis. Furthermore, S4L and G-14 also hydroxylated daidzein at different positions than that of G-18 as confirmed by comparing the HPLC chromatogram. is clearly indicates that enzymes involved in daidzein biotransformation by these strains are different and highly regioselective. However, the bioconversion of daidzein to hydroxydaidzein by the stain S4L and G-14 is very low to purify the products for structure elucidation. Based on the position in the HPLC chromatogram, we speculate that hydroxylation took place at the 6-and 8-position of daidzein by strain S4L and G-14. On the other hand, strain S4L also revealed the biotransformation potential toward naringenin. Mass spectrometry analysis revealed the increase in mass by 16 units; hence, the product is hydroxylated naringenin. e hydroxylated product of naringenin might be eriodictyol; as reported in cases of microbial biotransformation, the product of naringenin is eriodictyol [24]. Further optimization of the reaction products might increase the production of these hydroxylated metabolites, which enable for structure identification using NMR.

Conclusions
Streptomyces species are well known for production of diverse secondary metabolites and proven to be the effective host for the production of diverse compounds. However, Streptomyces species isolated from the highaltitude-soil sample have not been explored for biotransformation potential towards flavonoids. High altitude with a cold environment could be the source of unique biodiversity. In the present study, we have successfully demonstrated that Streptomyces species isolated from high-altitude-soil samples are effective for the biotransformation of daidzein (4′,7-dihydroxyisoflavone) to 3′,4′,7-trihydroxyisoflavone and genistein (4′,5,7-trihydroxyisoflavone) to hydroxygenistein. Furthermore, isolated species also revealed the biotransformation potential towards naringenin to hydroxynaringenin. Furthermore, identification of the specific enzymes from the isolated species as well as host engineering for the efficient production of the hydroxylated isoflavonoid and flavonoid molecules has tremendous industrial importance. Moreover, our results open up the possibilities in future for the search of a novel biocatalyst from diverse ecological niches.
Data Availability e datasets used and/or analyzed during the current research are available from the corresponding author on reasonable request.  (b) Figure 6: Mass spectra of naringenin and its products by Streptomyces species S4L.

Conflicts of Interest
International Journal of Microbiology