Flavonoids are plant secondary metabolites with a broad spectrum of biological activities. In nature, they occur mainly in the form of glycosides, but their extraction is often difficult and expensive, as is chemical synthesis. We have shown that biotransformations are an excellent method for obtaining flavonoid glycosides. We are the first team to describe the use of Isaria microorganisms in biotransformations of flavonoid compounds. In the present study as biocatalysts, we used one strain of Isaria fumosorosea KCH J2 isolated from a spider carcass in green areas of Wroclaw and two strains of I. farinosa (J1.4 and J1.6) isolated from insects found in already unused mines in Lower Silesia. The substrates were 3-hydroxyflavone, 3-methoxyflavone, quercetin (3,3′,4′,5,7-pentahydroxyflavone), and baicalein (5,6,7-trihydroxyflavone). For all the substrates that were used in this study, we obtained 4-O-methylglucopyranosides. In the case of substrates with a hydroxyl group in the third position, O-β-d-glucopyranosides were also formed. Isoquercetin that was obtained by biotransformation was used as a substrate to check the kinetics of the formation of flavonoid 4-O-methylglucopyranosides in I. fumosorosea KCH J2 culture. We did not observe the attachment of the methyl group to glucose unit in isoquercetin. Our finding suggest that the attachment of 4-O-methylglucopyranose occurs in one step.

Entomopathogenic fungi are known for their ability to carry out glycosylation of flavonoids, which usually results in the improvement of their stability and bioavailability. In this study we used a newly isolated strain of the entomopathogenic filamentous fungus Isaria fumosorosea KCH J2 as a biocatalyst. Our aim was to evaluate its ability to carry out the biotransformation of flavonoids and to obtain new flavonoid derivatives. The fungus was isolated from a spider’s carcass and molecularly identified using analysis of the ITS1-ITS2 rDNA sequence. As a result of biotransformation of 6-methylflavone two new products were obtained: 6-methylflavone 8-O-β-D-(4”-O-methyl)-glucopyranoside and 6-methylflavone 4’-O-β-D-(4”-O-methyl)-glucopyranoside. Chemical structures of the products were determined based on spectroscopic methods (¹H NMR, ¹³C NMR, COSY, HMBC, HSQC). Our research allowed us to discover a new species of filamentous fungus capable of carrying out glycosylation reactions and proved that I. fumosorosea KCH J2 is an effective biocatalyst for glycosylation of flavonoid compounds. For the first time we describe biotransformations of 6-methylflavone and the attachment of the sugar unit to the flavonoid substrate having no hydroxyl group. The possibility of using flavonoid aglycones is often limited by their low bioavailability due to poor solubility in water. The incorporation of a sugar unit improves the physical properties of tested compounds and thus increases the chance of using them as pharmaceuticals.

Entomopathogenic filamentous fungi of the genus Isaria are effective biocatalysts in the biotransformation of flavonoids as well as steroids. In the present study, the species Isaria fumosorosea and Isaria farinosa isolated from the environment were used. Their catalytic capacity to carry out biotransformations of flavones—unsubstituted, with hydroxy- and amino-substituents as well as a hydroxylated isoflavone—was investigated. Biotransformations of flavone, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, and daidzein resulted in the formation of O-methylglucosides, in the case of flavone and 5-hydroxyflavone with additional hydroxylations. 7-Aminoflavone was transformed into two acetamido derivatives. The following products were obtained: From flavone–flavone 2′-O-β-d-(4′′-O-methyl)-glucopyranoside, flavone 4′-O-β-d-(4′′-O-methyl)-glucopyranoside and 3′-hydroxyflavone 4′-O-β-d-(4′′-O-methyl)-glucopyranoside; from 5-hydroxyflavone–5-hydroxyflavone 4′-O-β-d-(4′′-O-methyl)-glucopyranoside; from 6-hydroxyflavone–flavone 6-O-β-d-(4′′-O-methyl)-glucopyranoside; from 7-hydroxyflavone–flavone 7-O-β-d-(4′′-O-methyl)-glucopyranoside; from daidzein–daidzein 7-O-β-d-(4′′-O-methyl)-glucopyranoside; and from 7-aminoflavone–7-acetamidoflavone and 7-acetamido-4′-hydroxyflavone. Seven of the products obtained by us have not been previously reported in the literature.

Steroid compounds are very interesting substrates for biotransformation due to their high biological activity and a high number of inactivated carbons which make chemical modification difficult. Microbial transformation can involve reactions which are complicated and uneconomical in chemical synthesis, and searching for a new effective biocatalyst is necessary. The best known entomopathogenic species used in steroid modification is Beauveria bassiana. In this study we tested the ability of Isaria farinosa, another entomopathogenic species, to transform several steroids. Twelve strains of the entomopathogenic filamentous fungus Isaria farinosa, collected in abandoned mines located in the area of the Lower Silesian Voivodeship, Poland, from insects’ bodies covered by fungus, were used as a biocatalyst. All the tested strains effectively transformed dehydroepiandrosterone (DHEA). We observed 7α- and 7β-hydroxy derivatives as well as changes in the percentage composition of the emerging products. Due to the similar metabolism of DHEA in all tested strains, one of them was selected for further investigation. In the culture of the selected strain, Isaria farinosa KCh KW1.1, transformations of androstenediol, androstenedione, adrenosterone, 17α-methyltestosterone, 17β-hydroxyandrost-1,4,6-triene-3-one and progesterone were performed. All the substrates were hydroxylated with high yield and stereoselectivity. We obtained 6β-hydroxyandrost-4-ene-3,11,17-trione, 15α,17β-dihydroxy-6β,7β-epoxyandrost-1,4-diene-3-one and 6β,11α-dihydroxyprogesterone. There is no evidence of either earlier microbial transformation of 17β-hydroxyandrost-1,4,6-triene-3-one or new epoxy derivatives. Isaria farinosa has a broad spectrum of highly effective steroid hydroxylases. The obtained 7-hydroxydehydroepiandrosterone has proven high biological activity and can be used in Alzheimer’s disease and as a key intermediate in the synthesis of aldosterone antagonists. Transformation of progesterone leads to high yield of 6β,11α-dihydroxyprogesterone and it is worth further study.

Beauveria bassiana is an entomopathogenic fungus used as a biological control agent. It is a well-known biocatalyst for the transformation of steroid compounds. Hydroxylations at the 7α or 11α position and oxidation to D-homo lactones are described in the literature. In our study, we examined the diversity of metabolism of five different B. bassiana strains and compared them to already known pathways. According to the literature, 7α and 11α-hydroxy derivatives as well as 3β,11α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one have been observed. Here we describe new DHEA metabolic pathways and two products not described before: 3β-hydroxy-17a-oxa-D-homo-androst-5-en-7,17-dione and 3β,11α-dihydroxyandrost-5-en-7,17-dione. We also used for the first time another species from this genus, Beauveria caledonica, for steroid transformation. DHEA was hydroxylated at the 7α, 7β and 11α positions and then reactions of oxidation and reduction leading to 3β,11α-dihydroxyandrost-5-en-7,17-dione were observed. All tested strains from the Beauveria genus effectively transformed the steroid substrate using several different enzymes, resulting in cascade transformation.

Flavonoids are widely described plant secondary metabolites with high and diverse pro-health properties. In nature, they occur mostly in the form of glycosides. Our research showed that an excellent way to obtain the sugar derivatives of flavonoids is through biotransformations with the use of entomopathogenic filamentous fungi as biocatalysts. In the current paper, we described the biotransformations of five methoxylated flavonoid compounds (2′-methoxyflavanone, 3′-methoxyflavanone, 4′-methoxyflavanone, 6-methoxyflavanone, and 6-methoxyflavone) in cultures of Isaria fumosorosea KCH J2. As a result, we obtained twelve new flavonoid 4-O-methylglucopyranosides. The products were purified with methods that enabled the reduction of the consumption of organic solvents (preparative TLC and flash chromatography). The structures of the products were confirmed with spectroscopic methods (NMR: ¹H, ¹³C, HSQC, HMBC, COSY). The compounds obtained by us expand the library of available flavonoid derivatives and can be used in biological research.