Multienzymatic biotransformation of flavokawain B by entomopathogenic filamentous fungi: structural modifications and pharmacological predictions.

BACKGROUND: Flavokawain B is one of the naturally occurring chalcones in the kava plant (Piper methysticum). It exhibits anticancer, anti-inflammatory and antimalarial properties. Due to its therapeutic potential, flavokawain B holds promise for the treatment of many diseases. However, due to its poor bioavailability and low aqueous solubility, its application remains limited. The attachment of a sugar unit impacts the stability and solubility of flavonoids and often determines their bioavailability and bioactivity. Biotransformation is an environmentally friendly way to improve the properties of compounds, for example, to increase their hydrophilicity and thus affect their bioavailability. Recent studies proved that entomopathogenic filamentous fungi from the genera Isaria and Beauveria can perform O-methylglycosylation of hydroxyflavonoids or O-demethylation and hydroxylation of selected chalcones and flavones. RESULTS: In the present study, we examined the ability of entomopathogenic filamentous fungal strains of Beauveria bassiana, Beauveria caledonica, Isaria farinosa, Isaria fumosorosea, and Isaria tenuipes to transform flavokawain B into its glycosylated derivatives. The main process occurring during the reaction is O-demethylation and/or hydroxylation followed by 4-O-methylglycosylation. The substrate used was characterized by low susceptibility to transformations compared to our previously described transformations of flavones and chalcones in the cultures of the tested strains. However, in the culture of the B. bassiana KCh J1.5 and BBT, Metarhizium robertsii MU4, and I. tenuipes MU35, the expected methylglycosides were obtained with high yields. Cheminformatic analyses indicated altered physicochemical and pharmacokinetic properties in the derivatives compared to flavokawain B. Pharmacological predictions suggested potential anticarcinogenic activity, caspase 3 stimulation, and antileishmanial effects. CONCLUSIONS: In summary, the study provided valuable insights into the enzymatic transformations of flavokawain B by entomopathogenic filamentous fungi, elucidating the structural modifications and predicting potential pharmacological activities of the obtained derivatives. The findings contribute to the understanding of the biocatalytic capabilities of these microbial cultures and the potential therapeutic applications of the modified flavokawain B derivatives.

Conversion of Phytochemicals by Lactobacilli: (Phospho)-ß-glucosidases Are Specific for Glucosylated Phytochemicals Rather than Disaccharides.

Food-fermenting lactobacilli convert glycosylated phytochemicals to glycosyl hydrolases and thereby alter their biological activity. This study aimed to investigate the microbial transformation of ß-glucosides of phytochemicals in comparison with utilization of cellobiose. Four homofermentative and four heterofermentative lactobacilli were selected to represent the metabolic diversity of Lactobacillaceae. The genomes of Lactobacillus crispatus, Companilactobacillus paralimentarius, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum encoded for 8 to 22 enzymes, predominantly phospho-ß-glucosidases, with predicted activity on ß-glucosides. Levilactobacillus hammesii and Furfurilactobacillus milii encoded for 3 ß-glucosidases, Furfurilactobacillus rossiae for one, and Fructilactobacillus sanfranciscensis for none. The hydrolysis of amygdalin, esculin, salicin, glucosides of quercetin and genistein, and ginsenosides demonstrated that several strains hydrolyzed ß-glucosides of phytochemicals but not cellobiose. Taken together, several of the carbohydrate-active enzymes of food-fermenting lactobacilli are specific for glycosides of phytochemicals.

Glycosylation of Methylflavonoids in the Cultures of Entomopathogenic Filamentous Fungi as a Tool for Obtaining New Biologically Active Compounds.

Flavonoid compounds are secondary plant metabolites with numerous biological activities; they naturally occur mainly in the form of glycosides. The glucosyl moiety attached to the flavonoid core makes them more stable and water-soluble. The methyl derivatives of flavonoids also show increased stability and intestinal absorption. Our study showed that such flavonoids can be obtained by combined chemical and biotechnological methods with entomopathogenic filamentous fungi as glycosylation biocatalysts. In the current paper, two flavonoids, i.e., 2'-hydroxy-4-methylchalcone and 4'-methylflavone, have been synthesized and biotransformed in the cultures of two strains of entomopathogenic filamentous fungi Isaria fumosorosea KCH J2 and Beauveria bassiana KCH J1.5. Biotransformation of 2'-hydroxy-4-methylchalcone resulted in the formation of two dihydrochalcone glucopyranoside derivatives in the culture of I. fumosorosea KCH J2 and chalcone glucopyranoside derivative in the case of B. bassiana KCH J1.5. 4'-Methylflavone was transformed in the culture of I. fumosorosea KCH J2 into four products, i.e., 4'-hydroxymethylflavone, flavone 4'-methylene-O-ß-d-(4″-O-methyl)-glucopyranoside, flavone 4'-carboxylic acid, and 4'-methylflavone 3-O-ß-d-(4″-O-methyl)-glucopyranoside. 4'-Methylflavone was not efficiently biotransformed in the culture of B. bassiana KCH J1.5. The computer-aided simulations based on the chemical structures of the obtained compounds showed their improved physicochemical properties and antimicrobial, anticarcinogenic, hepatoprotective, and cardioprotective potential.

4'-Methylflavanone Glycosides Obtained Using Biotransformation in the Entomopathogenic Filamentous Fungi Cultures as Potential Anticarcinogenic, Antimicrobial, and Hepatoprotective Agents.

Flavonoid compounds exhibit numerous biological activities and significantly impact human health. The presence of methyl or glucosyl moieties attached to the flavonoid core remarkably modifies their physicochemical properties and improves intestinal absorption. Combined chemical and biotechnological methods can be applied to obtain such derivatives. In the presented study, 4'-methylflavanone was synthesized and biotransformed in the cultures of three strains of entomopathogenic filamentous fungi, i.e., Isaria fumosorosea KCH J2, Beauveria bassiana KCH J1.5, and Isaria farinosa KCH J2.1. The microbial transformation products in the culture of I. fumosorosea KCH J2, flavanone 4'-methylene-O-ß-D-(4″-O-methyl)-glucopyranoside, 2-phenyl-(4'-hydroxymethyl)-4-hydroxychromane, and flavanone 4'-carboxylic acid were obtained. Biotransformation of 4'-methylflavanone in the culture of B. bassiana KCH J1.5 resulted in the formation of one main product, i.e., flavanone 4'-methylene-O-ß-D-(4″-O-methyl)-glucopyranoside. In the case of I. farinosa KCH J2.6 as a biocatalyst, three products, i.e., flavanone 4'-methylene-O-ß-D-(4″-O-methyl)-glucopyranoside, flavanone 4'-carboxylic acid, and 4'-hydroxymethylflavanone 4-O-ß-D-(4″-O-methyl)-glucopyranoside were obtained. The Swiss-ADME online simulations confirmed the increase in water solubility of 4'-methylflavanone glycosides and analyses performed using the Way2Drug Pass Online prediction tool indicated that flavanone 4'-methylene-O-ß-D-(4″-O-methyl)-glucopyranoside and 4'-hydroxymethylflavanone 4-O-ß-D-(4″-O-methyl)-glucopyranoside, which had not been previously reported in the literature, are promising anticarcinogenic, antimicrobial, and hepatoprotective agents.

Cascade biotransformation of estrogens by Isaria fumosorosea KCh J2.

Estrone, estradiol, ethynylestradiol and estrone 3-methyl ether underwent a biotransformation process in the submerged culture of Isaria fumosorosea KCh J2. Estrone was transformed into seven metabolites, four of which were glycosylated. Estradiol was selectively glycosylated at C-3 and then transformed to D-ring lactone. Ethynylestradiol was coupled with methylglucoside and 6ß-hydroxyderivative was obtained. Estrone 3-methyl ether was not transformed indicating that a free hydroxyl group at C-3 is necessary for glycosylation. Baeyer-Villiger oxidation combined with hydroxylation and glycosylation was observed. All glycosides obtained in this study are 3-O-ß-methylglucosides.

Biotransformation of steroids by entomopathogenic strains of Isaria farinosa.

BACKGROUND: 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. RESULTS: 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 7ß-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, 17ß-hydroxyandrost-1,4,6-triene-3-one and progesterone were performed. All the substrates were hydroxylated with high yield and stereoselectivity. We obtained 6ß-hydroxyandrost-4-ene-3,11,17-trione, 15α,17ß-dihydroxy-6ß,7ß-epoxyandrost-1,4-diene-3-one and 6ß,11α-dihydroxyprogesterone. There is no evidence of either earlier microbial transformation of 17ß-hydroxyandrost-1,4,6-triene-3-one or new epoxy derivatives. CONCLUSIONS: 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 6ß,11α-dihydroxyprogesterone and it is worth further study.

Isaria fumosorosea KCh J2 Entomopathogenic Strain as an Effective Biocatalyst for Steroid Compound Transformations.

The catalytic activity of enzymes produced by an entomopathogenic filamentous fungus (Isaria fumosorosea KCh J2) towards selected steroid compounds (androstenedione, adrenosterone, progesterone, 17α-methyltestosterone and dehydroepiandrosterone) was investigated. All tested substrates were efficiently transformed. The structure of the substrate has a crucial impact on regio- and stereoselectivity of hydroxylation since it affects binding to the active site of the enzyme. Androstenedione was hydroxylated in the 7α-position to give a key intermediate in the synthesis of the diuretic-7α-hydroxyandrost-4-ene-3,17-dione with 82% conversion. Adrenosterone and 17α-methyltestosterone were hydroxylated in the 6ß-position. Hydroxylated derivatives such as 15ß-hydroxy-17α-methyltestosterone and 6ß,12ß-dihydroxy-17α-methyltestosterone were also observed. In the culture of Isaria fumosorosea KCh J2, DHEA was effectively hydroxylated in the C-7 position and then oxidized to give 7-oxo-DHEA, 3ß,7α- and 3ß,7ß-dihydroxy-17a-oxa-d-homo-androst-5-ene-17-one. We obtained 7ß-OH-DHEA lactone with 82% yield during 3 days transformation of highly concentrated (5 g/L) DHEA.