Naturally Occurring Flavonoids and Isoflavonoids and Their Microbial Transformation: A Review

Flavonoids and isoflavonoids are polyphenolic secondary metabolites usually produced by plants adapting to changing ecological environments over a long period of time. Therefore, their biosynthesis pathways are considered as the most distinctive natural product pathway in plants. Seemingly, the flavonoids and isoflavones from fungi and actinomycetes have been relatively overlooked. In this review, we summarized and classified the isoflavones and flavonoids derived from fungi and actinomycetes and described their biological activities. Increasing attention has been paid to bioactive substances derived from microorganism whole-cell biotransformation. Additionally, we described the utilization of isoflavones and flavonoids as substrates by fungi and actinomycetes for biotransformation through hydroxylation, methylation, halogenation, glycosylation, dehydrogenation, cyclisation, and hydrogenation reactions to obtain rare and highly active biofunctional derivatives. Overall, among all microorganisms, actinomycetes are the main producers of flavonoids. In our review, we also summarized the functional genes involved in flavonoid biosynthesis.


Introduction
Flavonoids and isoflavonoids are versatile natural compounds and subdivisions of polyphenols that represent a large proportion of secondary metabolites produced by higher plants and are a rich part of the human diet [1]. They are playing multiple roles in the physiology and ecology of individual plant species. Flavonoids are a type of yellow pigment derived from 2-phenyl chromogenone as the parent nucleus and a series of compounds with C6-C3-C6 as the basic skeleton. Isoflavonoids are a subclass of flavonoids characterized by possessing a benzene-ring connected to C-3 instead of C-2 [2]. Phenylpropanoid and polyketone compounds are normally catalyzed by chalcone synthase to produce chalcones, and then cyclization of chalcones leads to generate flavonoids. Isoflavonoids, originating from the same biochemical pathway as flavonoids, are derived by aryl migration in a 2-phenylchroman skeleton under the catalysis of 2-hydroxyisoflavanone synthase [3][4][5][6]. Flavonoids and isoflavonoids are well-known natural products with extensive pharmacological activities and extremely low toxicity and therefore have become the focus and hotspots of drug discovery and development [7][8][9]. Most plants contain isoflavonoids and flavonoids that play important roles in the growth and development of plants as well as in antibacterial and disease-preventing aspects [10,11]. More importantly, they possess OH-1049. These compounds showed antioxidant activity in vitro [36]. Compounds 9 and 10 were acetylated by adding pyridine and Ac2O to afford 3′,4′,7-triacetoxyisoflavone and 8-chloro-3′,4′,5,7tetraacetoxyisoflavone, respectively [37].

Flavonoids
Flavonoid derivatives are natural products commonly found in medicinal plants and are synthesised from phenylpropanoid and acetate-derived precursors [11]. Some flavonoids have also been discovered from fungi and actinomycetes. We also classified these flavonoids into three
The strain Streptomyces sp. ERINLG-4 was isolated from the soil samples collected from a depth of 5-15 cm in the Doddabetta forest. The EtOAc extract of the strain showed potent cytotoxic activity in vitro against the A549 lung adenocarcinoma cancer cell line. The following work led to the isolation of an active component, quercetin-3-O-β-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (72), showing prominent cytotoxic activity against the A549 lung cancer cell line with an IC50 value of 82 μg/mL. However, it showed no toxicity against the Vero normal cell line, up to 2000 μg/mL [59]. Three flavonoids, named myricetin (50), myricitrin (73), and quercitrin (74) were obtained from the endophytic fungus X. papulis BCRC 09F0222 isolated from hairy woody plants [48].

Microbiological Transformation of Isoflavonoids and Flavonoids
Biotransformation is a well-known process of effectively obtaining isoflavones and flavonoids. The main reactions in the conversion process include hydroxylation, methylation, glycosylation, and cyclisation, through which some of the rare or expensive isoflavones and flavonoids can be obtained [63,64]. In this section, we review the biotransformation of isoflavones and flavonoids by fungi and actinomycetes, and the compounds involved in the microbiological transformation are shown in Figures 7 and 8, respectively.
The endophytic fungus Xylaria papulis BCRC 09F0222 was isolated from hairy woody plants. Three flavonoids, named myricetin (50), myricitrin (73), and quercitrin (74), were obtained from this strain [48]. Since the fungus was cultivated on rice, which is a feeding plant known to produce myricetin and other flavonoids [49], these metabolites might be derived from rice or synthesized by modifying the existing flavonoid precursors in culture medium. Dechlorochlorflavonin (56), a known flavonoid derivative, was isolated from the fungus Aspergillus candidus Bdf-2 derived from insects. Compound 56 was assessed for antibacterial activities against Ralstonia solanacearum and Staphylococcus aureus ATCC29213 with MIC values of 64 µg/mL. However, no antioxidant activity was detected [50].
In addition, chlorflavonin (58), 3 -bromo-2 ,5-dihydroxy-3,7,8-trimethoxyflavone (61), and dechlorochlorflavonin (62) were isolated from the fermentation broth of the fungus Acanthostigmella sp. CL12082. Compound 61 showed strong antifungal activity against Aspergillus fumigatus and Candida albicans with IC 50 values of 0.54 and 0.11 µg/mL. Compound 58 displayed significant activity against the growth of pathogenic fungi, C. albicans and A. fusigatus with IC 50 values of 0.035 and 0.10 µg/mL, as well as against the growth of HeLa cells IC 50 values of 20 µg/mL. At the same time, these three compounds showed weak inhibition of growth of pathogenic fungi, Cryptococus neoformans with IC 50 values of 20, 12, and 16 µg/mL, respectively [54]. Quercetin (63) was produced by an endophytic fungi Psathyrella candolleana from the seed of Ginkgo biloba. It displayed antibacterial activity against S. aureus with MIC values of 0.3906 mg/mL [55].
A study reported the isolation and determination of WS7528 (64), produced by Streptomyces sp. No. 7528 derived from a soil sample obtained at Nara Prefecture, Japan. It was tested orally and subcutaneously in immature rats to verify its effect on the growth of the uterus, which had also weak anti-inflammatory activity and could induce growth of the cell line MCF-7 [56]. Two flavonoids, rhamnazin (65) and cirsimaritin (66), obtained from microbial sources for the first time, were obtained from protoplast fusion between Streptomyces strains Merv 1996 and Merv 7409. Compound 65 showed remarkable activities against filamentous fungi B. fabae and A. niger with concentrations of 2.5 and 1.0 µg/mL, respectively. Compound 66 showed a strong antifungal activity in vitro against C. neoformans, C. albicans, Pichia angusta, and Rhodotorula minuta with MIC value of 1 µg/mL, while showed no inhibitory effect against filamentous fungi [57].
The strain Streptomyces sp. ERINLG-4 was isolated from the soil samples collected from a depth of 5-15 cm in the Doddabetta forest. The EtOAc extract of the strain showed potent cytotoxic activity in vitro against the A549 lung adenocarcinoma cancer cell line. The following work led to the isolation of an active component, quercetin-3-O-β-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (72), showing prominent cytotoxic activity against the A549 lung cancer cell line with an IC 50 value of 82 µg/mL. However, it showed no toxicity against the Vero normal cell line, up to 2000 µg/mL [59]. Three flavonoids, named myricetin (50), myricitrin (73), and quercitrin (74) were obtained from the endophytic fungus X. papulis BCRC 09F0222 isolated from hairy woody plants [48].

Microbiological Transformation of Isoflavonoids and Flavonoids
Biotransformation is a well-known process of effectively obtaining isoflavones and flavonoids. The main reactions in the conversion process include hydroxylation, methylation, glycosylation, and cyclisation, through which some of the rare or expensive isoflavones and flavonoids can be obtained [63,64]. In this section, we review the biotransformation of isoflavones and flavonoids by fungi and actinomycetes, and the compounds involved in the microbiological transformation are shown in Figures 7 and 8, respectively.

Development and Utilization of Flavonoids Synthesis Genes in Streptomyces
The availability of flavonoids in plants is limited by seasonal or regional variations, low abundance, and instability in the separation of single compounds from complex mixtures. Therefore, expressing plant biosynthesis genes in microorganisms is an effective method to produce these essential metabolites [97]. Streptomyces, one of the most important genera of actinomycetes, is diverse, and more than 50% of its species can produce various antibiotics and other active compounds of The study on the biotransformation of filamentous fungi found that Trichoderma harzianum NJ01 could convert puerarin (108) to 3 -hydroxypuerarin (109) with a conversion rate of up to 41% under the optimal conditions. In the DPPH free radical scavenging system, compound 109 was 20 times more active than 108. The solubility of 109 is 1.3 times higher than 108 [71]. Glabratephrin (110) was obtained from Tephrosia purpurea and transformed into pseudosemiglabrin (111) by the culture of A. niger. The transformation process is realized by using the open loop and the closed loop of the five-member ring [72].
Prenylated flavonoids are usually sourced from medicinal plants, and because of restrictions on bioavailability and the high extraction costs, it is essential to study their biological origins. Studies on the biotransformation showed that naringenin 8-dimethylallyltransferase expressed by transgenic yeast could convert naringenin (116) into 8-dimethylallylnaringenin (117). This was an example to provide a method for the production of prenylated flavonoids that rarely occur in nature [73].
Biotransformation was performed using isoflavones and 4 -fluoroisoflavones as substrates by A. niger and Cunninghamella elegans. Both fungi rapidly convert isoflavones into several metabolites. They metabolize isoflavones (40 mg/L) with half-lives of 1.6 and 4.2 days, respectively. Twenty-three metabolites were preliminarily identified during the biotransformation of A. niger. In the early stage, the main metabolites were mono-hydroxyl and dihydroxyl isoflavones, and after 10 days, the main metabolites were dihydroxyl and trihydroxyl isoflavones. The hydroxylation of isoflavones usually occurs in the B ring. Among them, 3 ,4 -dihydroxyl analogues were the most abundant. Methoxy metabolites accumulate slowly during culture. In addition, some glycosides have been detected. However, 4 -fluoroisoflavones were not transformed during culture, indicating that there was regional selective hydroxylation in the initial metabolism of isoflavones [78].
The  (2) is present in soybean meal. Fermentation media containing soybean meal could be used to isolate two new isoflavonoids, 8-chlorogenistein (19) and 6,8-dichlorogenistein (252), from Streptomyces griseus. However, the strain could not produce compounds 19 and 252 without soybean meal, and this suggests that the strain could biotransform compound 2 into chlorinated metabolites 19 and 252. These new isoflavonoids were produced through microbial halogenation. When S. griseus was cultivated with radiologically labelled acetate or phenylalanine, no labelled isoflavones were obtained. The result illustrated that isoflavonoids isolated from streptomycetes may have originated from the medium containing plant-derived nutrient components rather than having a microbial biosynthetic origin [63].
Two flavanones, pinocembrin (54) and naringenin (116), two flavones, chrysin and apigenin, together with a flavonol, kaempferol, were catalyzed by CYP105D7, a cytochrome P450 from Streptomyces avermitilis [94]. Through experiments, CYP105D7 enabled the catalysis of the hydroxylation of 54 and 116. The 3 -position of 116 was hydroxylated by CYP105D7, while the hydroxylated position of 54 could not be determined. Nonetheless, the retention time detected in HPLC implied that 3-hydoxylation or 4 -hydoxylation did not occur.
Studies showed that ring A hydroxyflavones were transformed to the corresponding C-4 hydroxylated metabolites; the rate and yields of production were related to the distance between the C-4 carbonyl group and the hydroxyl group in the A ring. During the process of biotransformation in the culture of Streptomyces fulvissimus, 6-hydroxyflavone (102) and 7-hydroxyflavone (121) converted into 6,4 -dihydroxyflavone (119) and 7,4 -dihydroxyflavone (122) with a medium and slow reaction, respectively. 5-Hydroxyflavone (158) was completely transformed to 5,4 -dihydroxyflavone (283) and 5,3 ,4 -dihydroxyflavone (284) for two days with a rapid reaction. These results demonstrated that the highest rate of transformation was observed when the hydroxy group was closest to the carbonyl group [96].

Development and Utilization of Flavonoids Synthesis Genes in Streptomyces
The availability of flavonoids in plants is limited by seasonal or regional variations, low abundance, and instability in the separation of single compounds from complex mixtures. Therefore, expressing plant biosynthesis genes in microorganisms is an effective method to produce these essential metabolites [97]. Streptomyces, one of the most important genera of actinomycetes, is diverse, and more than 50% of its species can produce various antibiotics and other active compounds of different structural types [98,99]. Enzymes with various catalytic functions in Streptomyces are useful members of an artificial gene cluster constructed in E. coli for the production of plant-specific flavones, including isoflavones and unnatural compounds by fermentation [100,101]. Here, we summarize the genes involved in flavonoid synthesis.
The most common method of studying genes that produce flavonoids in Streptomyces is the introduction of gene fragments into E. coli for heterologous expression. In plants, chalcone is a precursor of flavonoid, which can be converted to flavanones by chalcone isomerase, and 4-coumarate: coenzyme A ligase (4CL) is an essential enzyme in the transformation process. The gene fragment that synthesises 4CL has been found in S. coelicolor A3(2) and can be introduced into E. coli for heterogenic expression, which greatly enhances flavonoid production [100,102]. In a study by Kim et al., the SaOMT-2 gene from S. avermitilis MA-4680 was inserted into E. coli Sa-2 for expression, and the enzyme encoded by the SaOMT-2 gene was found to methylate the 7-hydroxyl group of isoflavones and flavonoids. In particular, the biotransformation of E. coli Sa-2 resulted in the production of sakuranetin, an antifungal flavonoid [103]. A gene fragment from S. peucetius ATCC 27952 that encodes an O-methyltransferase (OMT), SpOMT2884, was expressed in E. coli, and the enzyme was found to methylate various flavonoids, with 7,8-dihydroxyflavone being the most favourable substrate. Bioconversion of 7, 8-dihydroxyflavone was as high as 96% under optimal conditions, and the resulting 7-hydroxy-8-methoxyflavone was purified for in vitro glycosylation to produce glucose. The results indicated that methylation enhances the stability of the substrate, whereas glycation was shown to increase the water solubility of the substrate [104]. The SpOMT7740 gene from S. peucetius ATCC27952 encoding an OMT was cloned into E. coli to methylate different types of flavonoid substrates. The enzyme catalyzes the formation of various natural and non-natural O-methoxides [95]. O-Methylated phenylpropanoids are usually found in small quantities in plants. However, studies have indicated that co-culture of E. coli and Streptomyces can activate the expression of the SaOMT2 gene and promote the production of O-methylated phenylpropanoids, thus providing a powerful pathway for the production of scarce and valuable O-methylated phenylpropanoids [24]. Studies have shown that methylase GerMIII, derived from Streptomyces sp. KCTC 0041BP, can methylate the 4-hydroxyl position of some flavonoids, and quercetin may be the most suitable substrate [105]. The gene encoding oleandomycin glycosyltransferase (OleD GT) from S. antibioticus was introduced into E. coli BL21(DE3) for expression, and the purified recombinant OleD GT catalyzed glycosylation of various flavonoids. Fixation of OleD GT in hybrid nanoparticles of Fe 3 O 4 /silica/NiO is an effective technique for maintaining its high activity [106]. Not only E. coli but also Streptomyces can be used as engineered strains for heterologous expression of flavonoid-producing genes. In a study, flavonoid and stilbene biosynthesis genes were introduced for heterologous expression in S. venezuelae DHS2001, with the deletion of the native pikromycin polyketide synthesis gene, and the resulting strain produced racemic naringenin and pinocembrin from 4-coumaric acid and cinnamic acid, respectively. The yield of these two flavonoids was considerably increased by codon optimisation, and this is the first report of the phenylpropanoid biosynthesis pathway in the Streptomyces genus [107]. Marin et al. used the heterologous biosynthesis of industrial actinomyces S. coelicolor and Streptomyces albus to express the novo biosynthesis genes of three important flavonols, namely myricetin, kaempferol, and quercetin [108]. The shuffled biphenyl dioxygenase holoenzyme encoded by the bphA1 (2072) A2A3A4 gene cluster of S. lividans exhibits a wide range of substrate specificity and hydroxylates several flavonoids and isoflavones; some of the obtained products possess free radical scavenging activity [89]. Prenyltransferase expressed by the prenyltransferase gene from Streptomyces prenyltransferase HypSc (SCO7190) was shown to exhibit extensive substrate specificity in the host plant, tomato, and therefore, tomatoes accumulate prenylated flavonoids. This is the first report of prenylated flavonoid accumulation in transgenic plants [109]. Through gene exploration of S. clavuligerus, three naringenin-producing genes, namely ncs, ncyP, and tal, were found, all of which are indispensable for the process. This is the first report on the natural production of naringenin in actinomycetes [110]. The queD gene from S. eurythermus T encodes quercetinase (QueDHis6), which can convert several flavonols. The biological function of this enzyme was studied and was found to mainly play a role in detoxification rather than in catabolism [111]. The discovery of genes involved in the production of flavonoids by Streptomyces combined with newly developed techniques and increased genetic knowledge to manipulate the biosynthesis pathways is an effective way to achieve large-scale production of the essential flavonoids.

Conclusions
Flavonoids and isoflavonoids have attracted considerable attention due to their low toxicity and remarkable biological activity. Flavonoid and isoflavone pathways are considered the most characteristic natural product pathways in plants. However, microbial sources of isoflavones and flavonoids have received little attention. This is the first review of the fungal and actinomycete sources of isoflavones and flavonoids. Only 24 new compounds of isoflavones and flavonoids from fungi and actinomycetes have been identified; however, some of these are highly potent bioactive compounds that might be potential drug candidates. Compounds 61 and 58 from Acanthostigmella sp. CL12082 demonstrated potent antifungal activity. Compound 61 displayed strong antifungal activity against C. albicans and A. fumigatus, with IC 50 values of 0.11 and 0.54 µg/mL, respectively. Similarly, compound 58 displayed extremely strong activity against the fungi, C. albicans and A. fumigatus, with IC 50 values of 0.035 and 0.10 µg/mL, respectively [54]. Compound 76 from Streptomyces sp. (strain G246) showed broad-spectrum antimicrobial activity, and its antimicrobial activity was far superior than that of its positive controls, streptomycin and cycloheximide. Compound 76 displayed excellent inhibitory activity against P. aeruginosa, S. enterica, E. faecalis, S. aureus, B. cereus, and C. albicans with IC 50 values of 16, 32, 8, 1, 4, and 8 µg/mL, respectively [60]. The new compounds 77-79, derived from marine Streptomyces sp. G248, also demonstrated significant broad-spectrum antimicrobial activity against six pathogens, with IC 50 values in the range of 1-16 µg/mL, and their activities were much higher than those of the positive controls, streptomycin and cycloheximide [61]. To ascertain the therapeutic potential of these compounds, particularly of lavandulylated and halogen atom-bearing flavonoids, further pharmacological, chemical, and toxicological studies are required. Nearly all the isoflavones and flavones from actinomycetes are reported to be produced by Streptomyces sp.; however, two studies have reported the isolation of isoflavones and flavones from actinomycetes, M. aurantiaca 110B and Amycolatopsis sp. [33,43]. The culture medium of M. aurantiaca 110B for producing compounds 20, 21, and 71 was investigated, and the results indicated that the compounds could be detected in the culture medium only when soybean cake is added to the medium. The results also indicated that M. aurantiaca 110B could transform plant daidzein into fucosylated derivatives. Notably, the fermentation medium in which Amycolatopsis sp. produces isoflavones also comprises of soybean meal. Whether these two strains have similar transformation pathways to produce isoflavonoid glycosides requires further investigation.
Because of the low bioavailability of isoflavones and flavonoids, the bioconversion of isoflavones and flavonoids has gradually gained research attention [80]. In this review, we summarised the biotransformation of isoflavones and flavonoids by fungi and actinomycetes. Understanding the biotransformation process helps us elucidate not only the metabolic pathways of these compounds but also their mechanisms of action, toxicity, and pharmacological activity [112][113][114]. A. niger and Streptomyces sp. are the most adaptable fungi and actinomycetes, respectively, and are the most commonly used biotransformation strains because of their ability to use various isoflavones and flavonoids as substrates for biosynthesis and biotransformation. They usually undergo hydroxylation, methylation, glycosylation, cyclisation, halogenation, and double bond reduction to produce isoflavones and flavonoids, which are more beneficial to human health; for instance, methylation enhances substrate stability, hydroxylation enhances antioxidant activity, and glycosylation enhances water solubility. The biotransformation processes in fungi and actinomycetes for producing isoflavone-and flavonoid-derived drugs have gained considerable attention.
Use of bacteria to produce plant-derived compounds is a new metabolic engineering technology that can help us synthesise important drugs and produce bioactive substances. Multiple flavonoid biosynthesis genes of Streptomyces sp. can be heterogeneously expressed in E. coli or optimisation of the gene expression strategy can be employed to obtain heterogenous expression strains. Moreover, transfer of the biosynthesis pathway from the original microorganism to a more compliant alien host may provide an effective platform for the production of desired levels of flavonoids or other novel compounds. To date, only flavonoid synthesis genes have been studied in Streptomyces sp., and no isoflavone synthesis gene has been discovered yet. Future research should focus on elucidating the generation mechanism of isoflavones and flavonoids in microorganisms to promote the utilisation of these compounds in the pharmaceutical field.