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Transformation of progesterone by Isaria farinosa KCh KW 1.1 strain. Biotransformation conditions: 100 mL of cultivation medium (3% glucose, 1% bacteriological peptone) in 300 mL Erlenmeyer flasks, 24 °C, 150 r/min for 7 day
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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...
Citations
... These microorganisms are affiliated with the Department of Food Chemistry and Biocatalysis at the Wrocław University of Environmental and Life Sciences in Poland. The techniques for isolating entomopathogenic filamentous fungi, their reproduction, and genetic identification have been detailed in our earlier publications [42][43][44]. ...
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-β-D-(4″-O-methyl)-glucopyranoside and 6-bromo-8-nitroflavone 4′-O-β-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.
... B. bassiana exhibits a noteworthy capability for glycosylation of flavonoid compounds such as quercetin [37], warfarin [38], cannflavin B [39] and xanthohumol [40] as well as hydroxylation of steroids such as 1-dehydrotestosterone, testosterone, 19-nortestosterone, 17α-methyltestosterone and progesterone [32]. In addition, I. farinosa and I. fumosorosea strains show the ability to biotransform 2'-hydroxy-2-methylchalcone [41], 2'-methylflavone [30], methoxyflavones [42,43] and steroids [44,45]. Metarhizium strains can also be used as biocatalysts in the bioconversion of quercetin [46] and steroids [35,45]. ...
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.
... On this basis, the course of the transformations of the tested substrate in the culture of the B. bassiana KCh J1.5 strain can be determined as follows: the first stage is the hydroxylation leading to the 11α-hydroxyl derivative (3), which undergoes further hydroxylation leading to the 6β,11α-dihydroxy derivative, and the last step is the oxidation of the hydroxyl group at carbon C-11, leading to the formation of compound 5 ( Figure 2). An analogous cascade course was previously described during the biotransformation of progesterone leading to 6β-hydroxy-11-oxoprogesterone in cultures of Mucor M881 strains [51] and Isaria farinosa KCh KW1.1 [17,33]. On the other hand, the ability to dihydroxylate progesterone leading to the 6β,11α-dihydroxy derivative was previously observed in cultures of the following strains: Aspergillus nidulans VKPM F-1069 [45], A. niger N402 [43], A. ochraceus [49], Rhizomucor pusillus and Absidia griseolla var. ...
... In our previous manuscripts, we described the ability of the Isaria farinosa KCh KW 1.1 strain to hydroxylate progesterone and its derivatives effectively, leading to 6β,11αdihydroxy derivatives [17,33]. These metabolites were obtained during the transformation of progesterone, 11α-hydroxyprogesterone, and 16α,17α-epoxyprogesterone [17,33]. ...
... In our previous manuscripts, we described the ability of the Isaria farinosa KCh KW 1.1 strain to hydroxylate progesterone and its derivatives effectively, leading to 6β,11αdihydroxy derivatives [17,33]. These metabolites were obtained during the transformation of progesterone, 11α-hydroxyprogesterone, and 16α,17α-epoxyprogesterone [17,33]. However, during the transformation of 17α-hydroxyprogesterone, we isolated three hydroxy derivatives: 6β,17α-dihydroxyprogesterone, 12β,17α-dihydroxyprogesta-1,4-diene-3-one, and 6β,12β,17α-trihydroxyprogesterone [17]. ...
... Further, biocatalysts of hydroxylation were used including strains of entomopathogenic filamentous fungi. The Isaria farinosa KCh KW 1.1 strain possesses the ability to effectively dihydroxylate progesterone and its derivatives [17,33]. Isaria fumosorosea KCh J2 is known for its ability to carry out multienzymatic transformations of steroid compounds [34,35]. ...
... On this basis, the course of the transformations of the tested substrate in the culture of the B. bassiana KCh J1.5 strain can be determined as follows: the first stage is the hydroxylation leading to the 11α-hydroxyl derivative (3), which undergoes further hydroxylation leading to the 6β,11α-dihydroxy derivative, and the last step is the oxidation of the hydroxyl group at carbon C-11, leading to the formation of compound 5 ( Figure 2). An analogous cascade course was previously described during the biotransformation of progesterone leading to 6β-hydroxy-11-oxoprogesterone in cultures of Mucor M881 strains [51] and Isaria farinosa KCh KW1.1 [17,33]. On the other hand, the ability to dihydroxylate progesterone leading to the 6β,11α-dihydroxy derivative was previously observed in cultures of the following strains: Aspergillus nidulans VKPM F-1069 [45], A. niger N402 [43], A. ochraceus [49], Rhizomucor pusillus and Absidia griseolla var. ...
... In our previous manuscripts, we described the ability of the Isaria farinosa KCh KW 1.1 strain to hydroxylate progesterone and its derivatives effectively, leading to 6β,11αdihydroxy derivatives [17,33]. These metabolites were obtained during the transformation of progesterone, 11α-hydroxyprogesterone, and 16α,17α-epoxyprogesterone [17,33]. ...
This research aimed at obtaining new derivatives of pregn-1,4-diene-3,20-dione (Δ 1-pro-gesterone) (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 Δ 1-dehydrogenase (AcmB) from Ster-olibacterium 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), 6β,11α-dihydroxypregn-1,4-diene-3,20-dione (4), 6β-hydroxypregn-1,4-diene-3,11,20-trione (5), 6β,17α-dihydroxypregn-1,4-diene-3,20-dione (6), 6β,17β-dihydroxyandrost-1,4-diene-3-one (7), and 12β,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 17β-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.
... In the brain and other tissues, DHEA and some other steroids are prominently 7α-hydroxylated by CYP7B1, and the resulting derivatives can have serious effects on the brain and immune system [18]. In addition, 7α-OH-DHEA can affect human memory and cognitive processes and plays an important role in the treatment of autoimmune diseases [19]. ...
The hydroxylation of steroids in the C7β position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete Curvularia sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450cur) was identified as being heterologously expressed and functionally characterized in Pichia pastoris. Transcriptome data mining and differential expression analysis revealed that 12 putative genes in Curvularia sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450cur gene was increased more than 300-fold. A two-gene construct with a candidate P450cur gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant P. pastoris strains co-expressing fungal P450cur and CPR genes were obtained. The functional activity of the recombinant P450cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7β-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/β-hydroxylation of ADD and DHEA.
... 3β,7α-Dihydroxyandrost-5-en-17-one (7α-OH-DHEA) is a well-known biologically active steroid with immunomodulatory property which is widely used in medicine to treat rheumatoid arthritis and other autoimmune diseases [2][3][4]. The ability to catalyze 7α-hydroxylation of DHEA to form 7α-OH-DHEA was reported for the wild-type filamentous fungi representing Actinomucor, Gongronella, Fusarium, Cunninghamella, and some other genera [3,5,6]. ...
... The ability to catalyze 7α-hydroxylation of DHEA to form 7α-OH-DHEA was reported for the wild-type filamentous fungi representing Actinomucor, Gongronella, Fusarium, Cunninghamella, and some other genera [3,5,6]. However, when incubating with DHEA, most of the strains also produce, along with 7α-OH-DHEA, the epimeric 7β-OH-, as well as 11α-and 15α-hydroxylated metabolites [4,5,[7][8][9]. The soil-dwelling zygomycete Backusella lamprospora VKM F-944 selected in our lab is characterized by high regio-and stereospecific 7α-hydroxylase activity toward DHEA (Fig. 1) [10]. The 7α-hydroxylase activity of the fungus was shown to depend on the nutrient medium composition, mode, and time of the substrate addition [10]. ...
7α-Hydroxy dehydroepiandrosterone (7α-OH-prasterone, 7α-OH-DHEA) is a key steroid intermediate in the synthesis of valuable pharmaceuticals widely used in the treatment of autoimmune illness, rheumatoid arthritis, colitis, and other severe diseases. The steroid can be produced using a filamentous fungus, which is capable of regio- and stereospecific hydroxylation of the steroid 3β-alcohol (DHEA) in the allylic position C7. Here, we describe a method for highly selective microbial production of 7α-OH-DHEA from DHEA using the zygomycete Backusella lamprospora VKM F-944. The method ensures high yield of 7α-OH-DHEA (up to 89%, mol/mol) even at high concentration of the substrate DHEA (15 g/L).Key wordsFilamentous fungi
Backusella lamprospora
Biocatalysis Steroids Transformation DHEA 7α-Hydroxylation 7α-OH-DHEA
... The separation products were scraped out and extracted twice using ethyl acetate. The isolation and identification procedures were described in our previous papers [51,60,80]. The strains Lecanicillium lecanii NK3 and L. lecanii DSM 63098 were obtained from the collection of the Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences (Wrocław, Poland). ...
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-β-D-(4″-O-methylglucopyranosyl)-quercetin, (2) 3-O-β-D-(4″-O-methylglucopyranosyl)-quercetin and (3) 3-O-β-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.
... The microorganisms belong to the Department of Food Chemistry and Biocatalysis of the Wrocław University of Environmental and Life Sciences in Poland. The methods of isolation of entomopathogenic filamentous fungi, reproduction, and genetic identification were described in our previous papers [23,52,53]. ...
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-β-D-(4″-O-methyl)-glucopyranoside and 8-nitroflavan-4-ol 6-methylene-O-β-D-(4″-O-methyl)-glucopyranoside. 8-Bromo-6-chloroflavanone was transformed by this strain to 8-bromo-6-chloroflavan-4-ol 4′-O-β-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-β-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-β-D-(4″-O-methyl)-glucopyranoside, and 3′-bromo-5′-chloro-2′-hydroxychalcone to 8-bromo-6-chloroflavanone 3′-O-β-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.
... Anthracene was transformed into 2,3-dihydroxynaphtha-lene, 3-hydroy-2 -naphthoic acid, and muconic acid by Armillaria sp. F022 isolated from decayed wood in a tropical rain forest [39,40]. Dehydroepiandrosterone was transformed into hydroxy-dehydroepiandrosterone by Isaria farinosa isolated from the bodies of dead insects in an ore cave [41]. ...
... A white-rot fungus Armillaria sp. F022, which can convert anthracene, was isolated from the decayed wood in a tropical rain forest using an enrichment culture and with anthracene as the source of carbon and energy [39,40]. Using Lantana camara as the sole carbon source, a microorganism specific to LA was isolated from the soil. ...
Continuously growing demand for natural products with pharmacological activities has promoted the development of microbial transformation techniques, thereby facilitating the efficient production of natural products and the mining of new active compounds. Furthermore, due to the shortcomings and defects of microbial transformation, it is an important scientific issue of social and economic value to improve and optimize microbial transformation technology in increasing the yield and activity of transformed products. In this review, the aspects regarding the optimization of fermentation and the cross-disciplinary strategy, leading to the microbial transformation of increased levels of the high-efficiency process from natural products of a plant or microbial origin, were discussed. Additionally, due to the increasing craving for targeted and efficient methods for detecting transformed metabolites, analytical methods based on multiomics were also discussed. Such strategies can be well exploited and applied to the production of more efficient and more natural products from microbial resources.
... They belong to the large short chain dehydrogenase/reductase family, which includes numerous enzymes with a variety of functions (Grimm et al., 2000;Rasmussen et al., 2013). The activity of hydroxysteroid dehydrogenases during the reduction of steroid compounds and estrogens was detected before in C. fumosorosea (Kozłowska et al., 2017(Kozłowska et al., , 2019 as well as in C. farinosa (Kozłowska et al., 2018). In this study, it was proved that the above-mentioned enzymes are also involved in the reduction of the estrogen zearalenone in C. fumosorosea. ...
Zearalenone (ZEN) is a mycotoxin that poses risks to animals and humans. Its metabolism in humans is well understood, but its biotransformation by fungi that co-exist in soil with Fusarium is not yet elucidated. In this study, the ability of the commonly used biocontrol agent Cordyceps to eliminate ZEN was evaluated. Overall, 19 phase I and II derivatives of ZEN biotransformation by reduction, oxidation, sulfonation, glycosylation and glucuronidation were detected. Importantly, new metabolites of ZEN biotransformation by fungi were discovered. C. fumosorosea and C. farinosa bioconverted ZEN to its oxidized and sulfonated form. Additionally, C. fumosorosea biotransformed zearalenol, the reduced metabolite of ZEN, via oxidation and sulfonation. Mechanistically, cytochrome P450 of the Cordyceps spp. catalyzed the oxidation of ZEN and its metabolites. Sulfate adenylyltransferase and adenylyl-sulfate kinase were responsible for sulfonation in the ZEN biotransformation pathway in C. fumosorosea. Additionally, enzymes that may be involved in the glycosylation and reduction were determined by proteomic research. Moreover, ZEN was found to decrease the level of beauvericin in C. fumosorosea, which could lead to the reduction in environmental pollution with this compound.
