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.

Bioreduction of 4'-Hydroxychalcone in Deep Eutectic Solvents: Optimization and Efficacy with Various Yeast Strains.

4'-dihydrochalcones are secondary metabolites isolated from many medicinal plants and from the resin known as 'dragon's blood'. Due to their biological potential, our research objective was to determine the possibilities of using biocatalysis processes carried out in deep eutectic solvents (DESs) to obtain 4'-dihydrochalcones as a model compound. The processes were carried out in a culture of the yeast Yarrowia lipolytica KCh 71 and also in cultures of strains of the genera Rhodotorula and Debaryomyces. Based on the experiments carried out, an optimum process temperature of 35 °C was chosen, and the most suitable DES contained glycerol as a hydrogen bond donor (HBD). For a medium with 30% water content (DES 11), the conversion observed after 24 h exceeded 70%, while increasing the amount of water to 50% resulted in a similar level of conversion after just 1 h. A fivefold increase in the amount of added substrate resulted in a reduction in conversion, which reached 30.3%. Of the other yeast strains tested, Rhodotorula marina KCh 77 and Rhodotorula rubra KCh 4 also proved to be good biocatalysts for the bioreduction process. For these strains, the conversion reached 95.4% and 95.1%, respectively. These findings highlight the potential of yeast as a biocatalyst for the selective reduction of α,ß-unsaturated ketones and the possibility of using a DESs as a reaction medium in this process.

Antimicrobial Properties of Flavonoid Derivatives with Bromine, Chlorine, and Nitro Group Obtained by Chemical Synthesis and Biotransformation Studies.

The search for new substances of natural origin, such as flavonoids, is necessary in the fight against the growing number of diseases and bacterial resistance to antibiotics. In our research, we wanted to check the influence of flavonoids with chlorine or bromine atoms and a nitro group on pathogenic and probiotic bacteria. We synthesized flavonoids using Claisen-Schmidt condensation and its modifications, and through biotransformation via entomopathogenic filamentous fungi, we obtained their glycoside derivatives. Biotransformation yielded two new flavonoid glycosides: 8-amino-6-chloroflavone 4'-O-ß-D-(4″-O-methyl)-glucopyranoside and 6-bromo-8-nitroflavone 4'-O-ß-D-(4″-O-methyl)-glucopyranoside. Subsequently, we checked the antimicrobial properties of the aforementioned aglycon flavonoid compounds against pathogenic and probiotic bacteria and yeast. Our studies revealed that flavones have superior inhibitory effects compared to chalcones and flavanones. Notably, 6-chloro-8-nitroflavone showed potent inhibitory activity against pathogenic bacteria. Conversely, flavanones 6-chloro-8-nitroflavanone and 6-bromo-8-nitroflavanone stimulated the growth of probiotic bacteria (Lactobacillus acidophilus and Pediococcus pentosaceus). Our research has shown that the presence of chlorine, bromine, and nitro groups has a significant effect on their antimicrobial properties.

Antimicrobial Activity of Chalcones with a Chlorine Atom and Their Glycosides.

Chalcones, secondary plant metabolites, exhibit various biological properties. The introduction of a chlorine and a glucosyl substituent to the chalcone could enhance its bioactivity and bioavailability. Such compounds can be obtained through a combination of chemical and biotechnological methods. Therefore, 4-chloro-2'-hydroxychalcone and 5'-chloro-2'-hydroxychalcone were obtained by synthesis and then glycosylated in two filamentous fungi strains cultures, i.e., Isaria fumosorosea KCH J2 and Beauveria bassiana KCH J1.5. The main site of the glycosylation of both compounds by I. fumosorosea KCH J2 was C-2' and C-3 when the second strain was utilized. The pharmacokinetics of these compounds were predicted using chemoinformatics tools. Furthermore, antimicrobial activity tests were performed. Compounds significantly inhibited the growth of the bacteria strains Escherichia coli 10536, Staphylococcus aureus DSM 799, and yeast Candida albicans DSM 1386. Nevertheless, the bacterial strain Pseudomonas aeruginosa DSM 939 exhibited significant resistance to their effects. The growth of lactic acid bacteria strain Lactococcus acidophilus KBiMZ 01 bacteria was moderately inhibited, but strains Lactococcus rhamnosus GG and Streptococcus thermophilus KBM-1 were completely inhibited. In summary, chalcones substituted with a chlorine demonstrated greater efficacy in inhibiting the microbial strains under examination compared to 2'-hydroxychalcone, while aglycones and their glycosides exhibited similar effectiveness.

Biotransformation of Flavonoids with -NO2, -CH3 Groups and -Br, -Cl Atoms by Entomopathogenic Filamentous Fungi.

Combining chemical and microbiological methods using entomopathogenic filamentous fungi makes obtaining flavonoid glycosides possible. In the presented study, biotransformations were carried out in cultures of Beauveria bassiana KCH J1.5, Isaria fumosorosea KCH J2, and Isaria farinosa KCH J2.6 strains on six flavonoid compounds obtained in chemical synthesis. As a result of the biotransformation of 6-methyl-8-nitroflavanone using the strain I. fumosorosea KCH J2, two products were obtained: 6-methyl-8-nitro-2-phenylchromane 4-O-ß-D-(4″-O-methyl)-glucopyranoside and 8-nitroflavan-4-ol 6-methylene-O-ß-D-(4″-O-methyl)-glucopyranoside. 8-Bromo-6-chloroflavanone was transformed by this strain to 8-bromo-6-chloroflavan-4-ol 4'-O-ß-D-(4″-O-methyl)-glucopyranoside. As a result of microbial transformation by I. farinosa KCH J2.6 effectively biotransformed only 8-bromo-6-chloroflavone into 8-bromo-6-chloroflavone 4'-O-ß-D-(4″-O-methyl)-glucopyranoside. B. bassiana KCH J1.5 was able to transform 6-methyl-8-nitroflavone to 6-methyl-8-nitroflavone 4'-O-ß-D-(4″-O-methyl)-glucopyranoside, and 3'-bromo-5'-chloro-2'-hydroxychalcone to 8-bromo-6-chloroflavanone 3'-O-ß-D-(4″-O-methyl)-glucopyranoside. None of the filamentous fungi used transformed 2'-hydroxy-5'-methyl-3'-nitrochalcone effectively. Obtained flavonoid derivatives could be used to fight against antibiotic-resistant bacteria. To the best of our knowledge, all the substrates and products presented in this work are new compounds and are described for the first time.

Biotransformation of Δ1-Progesterone Using Selected Entomopathogenic Filamentous Fungi and Prediction of Its Products' Bioactivity.

This research aimed at obtaining new derivatives of pregn-1,4-diene-3,20-dione (Δ1-progesterone) (2) through microbiological transformation. For the role of catalysts, we used six strains of entomopathogenic filamentous fungi (Beauveria bassiana KCh J1.5, Beauveria caledonica KCh J3.3, Isaria fumosorosea KCh J2, Isaria farinosa KCh KW1.1, Isaria tenuipes MU35, and Metarhizium robertsii MU4). The substrate (2) was obtained by carrying out an enzymatic 1,2-dehydrogenation on an increased scale (3.5 g/L) using a recombinant cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans. All selected strains were characterized by the high biotransformation capacity for the used substrate. As a result of the biotransformation, six steroid derivatives were obtained: 11α-hydroxypregn-1,4-diene-3,20-dione (3), 6ß,11α-dihydroxypregn-1,4-diene-3,20-dione (4), 6ß-hydroxypregn-1,4-diene-3,11,20-trione (5), 6ß,17α-dihydroxypregn-1,4-diene-3,20-dione (6), 6ß,17ß-dihydroxyandrost-1,4-diene-3-one (7), and 12ß,17α-dihydroxypregn-1,4-diene-3,20-dione (8). The results show evident variability of the biotransformation process between strains of the tested biocatalysts from different species described as entomopathogenic filamentous fungi. The obtained products were tested in silico using cheminformatics tools for their pharmacokinetic and pharmacodynamic properties, proving their potentially high biological activities. This study showed that the obtained compounds may have applications as effective inhibitors of testosterone 17ß-dehydrogenase. Most of the obtained products should, also with a high probability, find potential uses as androgen antagonists, a prostate as well as menopausal disorders treatment. They should also demonstrate immunosuppressive, erythropoiesis-stimulating, and anti-inflammatory properties.

Glycosylation of Quercetin by Selected Entomopathogenic Filamentous Fungi and Prediction of Its Products' Bioactivity.

Quercetin is the most abundant flavonoid in food products, including berries, apples, cauliflower, tea, cabbage, nuts, onions, red wine and fruit juices. It exhibits various biological activities and is used for medical applications, such as treating allergic, inflammatory and metabolic disorders, ophthalmic and cardiovascular diseases, and arthritis. However, its low water solubility may limit quercetin's therapeutic potential. One method of increasing the solubility of active compounds is their coupling to polar molecules, such as sugars. The attachment of a glucose unit impacts the stability and solubility of flavonoids and often determines their bioavailability and bioactivity. Entomopathogenic fungi are biocatalysts well known for their ability to attach glucose and its 4-O-methyl derivative to bioactive compounds, including flavonoids. We investigated the ability of cultures of entomopathogenic fungi belonging to Beauveria, Isaria, Metapochonia, Lecanicillium and Metarhizium genera to biotransform quercetin. Three major glycosylation products were detected: (1), 7-O-ß-D-(4″-O-methylglucopyranosyl)-quercetin, (2) 3-O-ß-D-(4″-O-methylglucopyranosyl)-quercetin and (3) 3-O-ß-D-(glucopyranosyl)-quercetin. The results show evident variability of the biotransformation process, both between strains of the tested biocatalysts from different species and between strains of the same species. Pharmacokinetic and pharmacodynamic properties of the obtained compounds were predicted with the use of cheminformatics tools. The study showed that the obtained compounds may have applications as effective modulators of intestinal flora and may be stronger hepato-, cardio- and vasoprotectants and free radical scavengers than quercetin.

Hydroxylation of Progesterone and Its Derivatives by the Entomopathogenic Strain Isaria farinosa KCh KW1.1.

Progesterone biotransformation is worth studying because of the high industrial value of its derivatives. This study investigated the catalytic ability of the entomopathogenic filamentous fungus strain Isaria farinosa KCh KW1.1 to transform progesterone derivatives: 11α-hydroxyprogesterone, 17α-hydroxyprogesterone, 16α,17α-epoxyprogesterone and pregnenolone. In the culture of Isaria farinosa KCh KW1.1, 11α-hydroxyprogesterone was effectively transformed into only one product: 6ß,11α-dihydroxyprogesterone. Transformation of 17α-hydroxyprogesterone gave three hydroxy derivatives: 6ß,17α-dihydroxyprogesterone, 12ß,17α-dihydroxyprogesterone and 6ß,12ß,17α-trihydroxyprogesterone. Two products: 6ß-hydroxy-16α,17α-epoxyprogesterone and 6ß,11α-dihydroxy-16α,17α-epoxyprogesterone, were obtained from the 16α,17α-epoxyprogesterone transformation. We isolated two compounds from the biotransformation medium with pregnenolone: 11α-hydroxy-7-oxopregnenolone and 5α,6α-epoxy-3ß,11α-dihydroxypregnan-7,20-dione. In this study, we observed only mono- and dihydroxy derivatives of the tested substrates, and the number of obtained products for each biotransformation did not exceed three.

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.

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.