New Hydroxylactones and Chloro-Hydroxylactones Obtained by Biotransformation of Bicyclic Halolactones and Their Antibacterial Activity.

The aim of this study was to obtain new halolactones with a gem-dimethyl group in the cyclohexane ring (at the C-3 or C-5 carbon) and a methyl group in the lactone ring and then subject them to biotransformations using filamentous fungi. Halolactones in the form of mixtures of two diasteroisomers were subjected to screening biotransformations, which showed that only compounds with a gem-dimethyl group located at the C-5 carbon were transformed. Strains from the genus Fusarium carried out hydrolytic dehalogenation, while strains from the genus Absidia carried out hydroxylation of the C-7 carbon. Both substrates and biotransformation products were then tested for antimicrobial activity against multidrug-resistant strains of both bacteria and yeast-like fungi. The highest antifungal activity against C. dubliniensis and C. albicans strains was obtained for compound 5b, while antimicrobial activity against S. aureus MRSA was obtained for compound 4a.

Biotransformation of (-)-α-Bisabolol by Absidia coerulea.

(-)-α-Bisabolol, a bioactive monocyclic sesquiterpene alcohol, has been used in pharmaceutical and cosmetic products with anti-inflammatory, antibacterial and skin-caring properties. However, the poor water solubility of (-)-α-bisabolol limits its pharmaceutical applications. It has been recognized that microbial transformation is a very useful approach to generate more polar metabolites. Fifteen microorganisms were screened for their ability to metabolize (-)-α-bisabolol in order to obtain its more polar derivatives, and the filamentous fungus Absidia coerulea was selected for scale-up fermentation. Seven new and four known metabolites were obtained from biotransformation of (-)-α-bisabolol (1), and all the metabolites exhibited higher aqueous solubility than that of the parent compound 1. The structures of newly formed metabolites were established as (1R,5R,7S)- and (1R,5S,7S)-5-hydroxy-α-bisabolol (2 and 3), (1R,5R,7S,10S)-5-hydroxybisabolol oxide B (4), (1R,7S,10S)-1-hydroxybisabolol oxide B (5), 12-hydroxy-α-bisabolol (7), (1S,3R,4S,7S)- and (1S,3S,4S,7S)-3,4-dihydroxy-α-bisabolol (8 and 10) on the basis of spectroscopic analyses. These compounds could also be used as reference standards for the detection and identification of the metabolic products of 1 in the mammalian system.

Optimization of (-)-cubebin biotransformation to (-)-hinokinin by the marine fungus Absidia coerulea 3A9.

The genus Absidia is widely used in the biotransformation of different classes of natural products. This study evaluates the ability of the Absidia coerulea 3A9 marine derived strain isolated from the ascidian Distaplia stilyfera to perform biotransformations by conducting assays with (-)-cubebin, as substrate. The experiment was optimized using the experimental design proposed by Plackett-Burman for seven factors and eight experiments, to establish the biotransformation conditions that would allow maximum production of biotransformed dibenzylbutyrolactone (-)-hinokinin. An analytical method based on Reverse-Phase-High Performance Liquid Chromatography (RP-HPLC) was developed to quantify the fungal biotransformation product. The factor that influenced the (-)-hinokinin peak area the most positively was the percentage of seawater (%seawater) given that its %relative standard deviation (%RSD) showed a 32.92% deviation from the real value.

The impact of COVID-19 outbreak on the incidence of acute invasive fungal rhinosinusitis.

BACKGROUND: Acute invasive fungal rhinosinusitis (AIFRS) is aggressive morbidity affecting immunocompromised patients. Coronavirus disease 2019 (COVID-19) may allow secondary fungal disease through a propensity to cause respiratory infection by affecting the immune system leading to dysregulation and reduced numbers of T lymphocytes, CD4+T, and CD8+T cells, altering the innate immunity. The aim of this study is to evaluate the incidence of acute invasive fungal rhinosinusitis (AIFRS) in COVID-19 patients. METHODOLOGY: Data for acute invasive rhinosinusitis was obtained from the Otorhinolaryngology departments at our tertiary hospital at the period from January 2017 to December 2020. Then the risk factors of comorbid diseases and fungal types between post-COVID-19 and non-COVID-19 groups regarding the incidence of AIFRS are compared. RESULTS: Consequently, the incidence of AIFRS showed a more significant difference (P < 0.05) in post-COVID-19 patients than in non-COVID-19 especially in immunocompromised patients, diabetic, renal, and liver dysfunction patients as well as patients with risk factors of AIFRS. The most common organisms affecting patients with AIFRS are Rhizopus oryzae, Aspergillus fumigatus, and Absidia mucor. CONCLUSIONS: The incidence of AIFRS is markedly prominent in post-COVID-19 patients than in those of non-COVID-19, especially in immunocompromised, diabetic, renal, and liver dysfunction patients and patients with risk factors for rhinosinusitis.

Microbial Conjugation Studies of Licochalcones and Xanthohumol.

Microbial conjugation studies of licochalcones (1-4) and xanthohumol (5) were performed by using the fungi Mucor hiemalis and Absidia coerulea. As a result, one new glucosylated metabolite was produced by M. hiemalis whereas four new and three known sulfated metabolites were obtained by transformation with A. coerulea. Chemical structures of all the metabolites were elucidated on the basis of 1D-, 2D-NMR and mass spectroscopic data analyses. These results could contribute to a better understanding of the metabolic fates of licochalcones and xanthohumol in mammalian systems. Although licochalcone A 4'-sulfate (7) showed less cytotoxic activity against human cancer cell lines compared to its substrate licochalcone A, its activity was fairly retained with the IC50 values in the range of 27.35-43.07 µM.

Identification of Absidia orchidis steroid 11ß-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone.

Hydrocortisone is an effective anti-inflammatory drug and also an important intermediate for synthesis of other steroid drugs. The filamentous fungus Absidia orchidis is renowned for biotransformation of acetylated cortexolone through 11ß-hydroxylation to produce hydrocortisone. However, due to the presence of 11α-hydroxylase in A. orchidis, the 11α-OH by-product epi-hydrocortisone is always produced in a 1:1 M ratio with hydrocortisone. In order to decrease epi-hydrocortisone production, Saccharomyces cerevisiae was engineered in this work as an alternative way to produce hydrocortisone through biotransformation. Through transcriptomic analysis coupled with genetic verification in S. cerevisiae, the A. orchidis steroid 11ß-hydroxylation system was characterized, including a cytochrome P450 enzyme CYP5311B2 and its associated redox partners cytochrome P450 reductase and cytochrome b5. CYP5311B2 produces a mix of stereoisomers containing 11ß- and 11α-hydroxylation derivatives in a 4:1 M ratio. This fungal steroid 11ß-hydroxylation system was reconstituted in S. cerevisiae for hydrocortisone production, resulting in a productivity of 22 mg/L·d. Protein engineering of CYP5311B2 generated a R126D/Y398F variant, which had 3 times higher hydrocortisone productivity compared to the wild type. Elimination of C20-hydroxylation by-products and optimization of the expression of A. orchidis 11ß-hydroxylation system genes further increased hydrocortisone productivity by 238% to 223 mg/L·d. In addition, a novel steroid transporter ClCDR4 gene was identified from Cochliobolus lunatus, overexpression of which further increased hydrocortisone productivity to 268 mg/L·d in S. cerevisiae. Through increasing cell mass, 1060 mg/L hydrocortisone was obtained in 48 h and the highest productivity reached 667 mg/L·d. This is the highest hydrocortisone titer reported for yeast biotransformation system so far.

Regioselective O-glycosylation of flavonoids by fungi Beauveria bassiana, Absidia coerulea and Absidia glauca.

In the present study, the species: Beauveria bassiana, Absidia coerulea and Absidia glauca were used in biotransformation of flavones (chrysin, apigenin, luteolin, diosmetin) and flavanones (pinocembrin, naringenin, eriodictyol, hesperetin). The Beauveria bassiana AM 278 strain catalyzed the methylglucose attachment reactions to the flavonoid molecule at positions C7 and C3'. The application of the Absidia genus (A. coerulea AM 93, A. glauca AM 177) as the biocatalyst resulted in the formation of glucosides with a sugar molecule present at C7 and C3' positions of flavonoids skeleton. Nine of obtained products have not been previously reported in the literature.

Absidia panacisoli sp. nov., isolated from rhizosphere of Panax notoginseng.

A strain (SYPF 7183T) was isolated from rhizosphere soil of Panax notoginseng in southwest China. Phylogenetic analyses indicated that strain SYPF 7183T was distinct from the other Absidia species with well-supported values. Strain SYPF 7183T produced spherical or subpyriform sporangia and short cylindrical sporangiospores. The azygospores were globose to oval. Based on morphological and phylogenetic evidence, the novel strain Absidia panacisoli sp. nov. is proposed.

Application of α- and ß-naphthoflavones as monooxygenase inhibitors of Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651 in transformation of 17α-methyltestosterone.

In this work, 17α-methyltestosterone was effectively hydroxylated by Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651. A. coerulea KCh 93 afforded 6ß-, 12ß-, 7α-, 11α-, 15α-hydroxy derivatives with 44%, 29%, 6%, 5% and 9% yields, respectively. S. racemosum KCh 105 afforded 7α-, 15α- and 11α-hydroxy derivatives with yields of 45%, 19% and 17%, respectively. Chaetomium sp. KCh 6651 afforded 15α-, 11α-, 7α-, 6ß-, 9α-, 14α-hydroxy and 6ß,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6ß,14α-dihydroxy derivatives were determined as new compounds. Effect of various sources of nitrogen and carbon in the media on biotransformations were tested, however did not affect the degree of substrate conversion or the composition of the products formed. The addition of α- or ß-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.

(R)-panaxadiol by whole cells of filamentous fungus Absidia coerulea AS 3.3382.

The microbial transformation of 20(R)-panaxadiol (PD) by the fungus Absidia coerulea AS 3.3382 afforded three new and three known metabolites. The structures of the metabolites were characterized as 3-oxo-20(R)-panaxadiol (1), 3-oxo-7ß- hydroxyl-20(R)-panaxadiol (2), 3-oxo-22ß-hydroxyl-20(R)-panaxadiol (3), 3-oxo- 7ß,22ß-dihydroxyl-20(R)-panaxadiol (4), 3-oxo-7ß,24ß-dihydroxyl-20(R)-panaxadiol (5), and 3-oxo-7ß,24α-dihydroxyl-20(R)-panaxadiol (6). Among them, 2-4 were new compounds. In addition, compounds 3 and 4 exhibited significant anti-hepatic fibrosis activity.

Biotransformation of a major beer prenylflavonoid - isoxanthohumol.

Microbial transformations of isoxanthohumol (1), a beer prenylated flavonoid, by 51 fungi were investigated. Many of the tested fungi cultures were capable of effective transformation of 1. Mucor hiemalis and Fusarium oxysporum converted isoxanthohumol (1) into isoxanthohumol 7-O-ß-d-glucopyranoside (3) and (2R)-2″-(2″'-hydroxyisopropyl)-dihydrofurano[2″,3″:7,8]-4″,5-hydroxy-5-methoxyflavanone (4), respectively. No product was obtained in the transformation of 1 by Absidia glauca conducted in a phosphate buffer. In the same medium, Beauveria bassiana converted isoxanthohumol (1) to isoxanthohumol 7-O-ß-d-4″'-O-methylglucopyranoside (2).

Two Novel Fungal Phenolic UDP Glycosyltransferases from Absidia coerulea and Rhizopus japonicus.

In the present study, two novel phenolic UDP glycosyltransferases (P-UGTs), UGT58A1 and UGT59A1, which can transfer sugar moieties from active donors to phenolic acceptors to generate corresponding glycosides, were identified in the fungal kingdom. UGT58A1 (from Absidia coerulea) and UGT59A1 (from Rhizopus japonicas) share a low degree of homology with known UGTs from animals, plants, bacteria, and viruses. These two P-UGTs are membrane-bound proteins with an N-terminal signal peptide and a transmembrane domain at the C terminus. Recombinant UGT58A1 and UGT59A1 are able to regioselectively and stereoselectively glycosylate a variety of phenolic aglycones to generate the corresponding glycosides. Phylogenetic analysis revealed the novelty of UGT58A1 and UGT59A1 in primary sequences in that they are distantly related to other UGTs and form a totally new evolutionary branch. Moreover, UGT58A1 and UGT59A1 represent the first members of the UGT58 and UGT59 families, respectively. Homology modeling and mutational analysis implied the sugar donor binding sites and key catalytic sites, which provided insights into the catalytic mechanism of UGT58A1. These results not only provide an efficient enzymatic tool for the synthesis of bioactive glycosides but also create a starting point for the identification of P-UGTs from fungi at the molecular level.IMPORTANCE Thus far, there have been many reports on the glycosylation of phenolics by fungal cells. However, no P-UGTs have ever been identified in fungi. Our study identified fungal P-UGTs at the molecular level and confirmed the existence of the UGT58 and UGT59 families. The novel sequence information on UGT58A1 and UGT59A1 shed light on the exciting and new P-UGTs hiding in the fungal kingdom, which would lead to the characterization of novel P-UGTs from fungi. Molecular identification of fungal P-UGTs not only is theoretically significant for a better understanding of the evolution of UGT families but also can be applied as a powerful tool in the glycodiversification of bioactive natural products for drug discovery.

Synthesis and Biotransformation of Bicyclic Unsaturated Lactones with Three or Four Methyl Groups.

The aim of this study was to obtain new unsaturated lactones by chemical synthesis and their microbial transformations using fungal strains. Some of these strains were able to transform unsaturated lactones into different hydroxy or epoxy derivatives. Strains of Syncephalastrum racemosum and Absidia cylindrospora gave products with a hydroxy group introduced into a tertiary carbon, while the Penicillium vermiculatum strain hydroxylated primary carbons. The Syncephalastrum racemosum strain hydroxylated both substrates in an allylic position. Using the Absidia cylindrospora and Penicillium vermiculatum strains led to the obtained epoxylactones. The structures of all lactones were established on the basis of spectroscopic data.

Microbial Glycosylation of Daidzein, Genistein and Biochanin A: Two New Glucosides of Biochanin A.

Biotransformation of daidzein, genistein and biochanin A by three selected filamentous fungi was investigated. As a result of biotransformations, six glycosylation products were obtained. Fungus Beauveria bassiana converted all tested isoflavones to 4″-O-methyl-7-O-glucosyl derivatives, whereas Absidia coerulea and Absidia glauca were able to transform genistein and biochanin A to genistin and sissotrin, respectively. In the culture of Absidia coerulea, in addition to the sissotrin, the product of glucosylation at position 5 was formed. Two of the obtained compounds have not been published so far: 4″-O-methyl-7-O-glucosyl biochanin A and 5-O-glucosyl biochanin A (isosissotrin). Biotransformation products were obtained with 22%-40% isolated yield.

Biotransformation studies of cresol red by Absidia spinosa M15.

Cresol Red, a commercial dye that used widely to color nylon, wool, cotton, and polyacrylonitrile-modified nylon in the massive textile manufacture is toxic recalcitrant. Absidia spinosa M15, a novel fungal strain isolated from a tropical rain forest, was found to decolorize Cresol Red 65% within 30 d under agitation condition. UV-Vis spectroscopy, TLC analysis and mass spectra of samples after decolorization process in culture medium confirmed final decolorization of Cresol Red. Two metabolites were identified in the treated medium: benzeneacetic acid (tR 9.6 min and m/z 136) and benzoic acid (tR 5.7 min and m/z 122). Laccase showed the significant activity (133.8 U/L) in biomass obtained at the end of experiment demonstrates role of the enzyme in the decolorization process.

Hydroxylation of DHEA and its analogues by Absidia coerulea AM93. Can an inducible microbial hydroxylase catalyze 7α- and 7ß-hydroxylation of 5-ene and 5α-dihydro C19-steroids?

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and 5α-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis of the hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to the mixture of allyl 7-hydroxy derivatives, while EpiA and 5α-androstan-3,17-dione were converted mainly to 7α- and 7ß-alcohols accompanied by 9α- and 11α-hydroxy derivatives. On the basis of (i) time course analysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells at different pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between the C-H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, it is postulated that the same enzyme can catalyze the oxidation of C7-Hα as well as C7-Hß bonds in 5-ene and 5α-dihydro C19-steroids. Correlations observed between the structure of the substrate and the regioselectivity of hydroxylation suggest that 7ß-hydroxylation may occur in the normal binding enzyme-substrate complex, while 7α-hydroxylation-in the reverse inverted binding complex.

Facial and systemic mucormycosis caused by Lichtheimia corymbifera in a goat: case report and literature review of fungal infections in goats.

An 8-y-old Pygora doe was presented to the University of California-Davis, Veterinary Medical Teaching Hospital because of non-healing facial swelling of 2-wk duration. The lesion grew despite medical treatment, causing discomfort masticating, little-to-no airflow from the right nasal passage, and led to euthanasia. On gross examination, a large facial mass with a draining tract through the skin and hard palate was identified. On section, the mass was brown-pink, homogeneous, and friable. Abscess-like masses were identified in the lungs and kidney. Histopathology of the face, including oral and nasal cavities, salivary glands, and lymph nodes, as well as the lung and kidney lesions, revealed large areas of necrosis with numerous wide ribbon-like, mostly aseptate, fungal hyphae consistent with zygomycetes. PCR for fungal organisms performed on formalin-fixed, paraffin-embedded tissue from the face identified Lichtheimia corymbifera (formerly Absidia corymbifera) of the order Mucorales and an Aspergillus sp. The lesion was suspected to have started either as a fungal rhinitis or dental feed impaction, subsequently spreading to the face and systemically to the lungs and kidney. We describe here the lesions associated with facial mucormycosis in a goat and present a literature review of L. corymbifera infection in veterinary species and fungal infections in goats.

Mucormycosis as a rare cause of severe gastrointestinal bleeding after multivisceral transplantation.

Mucormycosis, an emerging fungal infection in solid organ transplant patients, is mostly located in rhino-orbito-cerebral, pulmonary, and cutaneous areas, or disseminated with poor prognosis. A 4-year-old girl with chronic intestinal pseudo-obstruction syndrome underwent a modified multivisceral transplantation, including half of the stomach, the duodeno-pancreas, the small bowel, and the right colon. On postoperative day 5, a digestive perforation was suspected. Surgical exploration found a small necrotic area on the native stomach, which was externally drained. The next day, massive gastric bleeding occurred. During the emergency laparotomy, 2 hemorrhagic ulcers were found and resected from the transplanted stomach. Pathology and fungal culture showed mucormycosis caused by Lichtheimia (formerly Absidia) ramosa in both the transplanted and native stomach. High-dose intravenous liposomal amphotericin B was immediately started. No other site of fungal infection was found. The child recovered, and 3 years after transplantation, is alive and well, off parenteral nutrition. The originality of this case is the very early presentation after transplantation, the unusual site, and the complete recovery after rapid medico-surgical management. The origin of the fungus and treatment are discussed.

Microbial transformation of 20(S)-protopanaxatriol by Absidia corymbifera and their cytotoxic activities against two human prostate cancer cell lines.

Seven hydroxylates of 20(S)-protopanaxatriol (1) transformed by Absidia corymbifera AS 3.3387 were isolated and identified by spectral methods including 2D-NMR. Among them, 7ß-hydroxyl-20(S)-protopanaxatriol (2), 7α-hydroxyl-20(S)-protopanaxatriol (3), and 7ß, 15α-dihydroxyl-20(S)-protopanaxatriol (7) are new compounds. The metabolites 2, 6, 7, and 8 showed the more potent inhibitory effects against DU-145 and PC-3 cell lines than the substrate.

Disseminated zygomycosis due to Mycocladus corymbifera with cutaneous and cerebral involvement.

Fungal infections caused by zygomycetes are important and potentially life threatening infections. These opportunistic moulds have been increasingly implicated in human disease and are most frequently seen in immune compromised patients. We report a case of disseminated infection with Mycocladus corymbifera involving the brain, lungs, kidneys and skin in a 16-year-old patient with acute lymphoblastic leukaemia. The skin lesions played a significant role in the diagnosis of mucormycosis. These infections have an exceedingly high mortality rate and early recognition of cutaneous lesions is essential to successful management.