The Study of a Novel Paeoniflorin-Converting Enzyme from Cunninghamella blakesleeana.

Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that is used in traditional herbal medicine and shows various protective effects on the cardio-cerebral vascular system. It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites. However, the bioavailability of paeoniflorin by oral administration is low, which greatly limits its clinical application. In this paper, a paeoniflorin-converting enzyme gene (G6046, GenBank accession numbers: OP856858) from Cunninghamella blakesleeana (AS 3.970) was identified by comparative analysis between MS analysis and transcriptomics. The expression, purification, enzyme activity, and structure of the conversion products produced by this paeoniflorin-converting enzyme were studied. The optimal conditions for the enzymatic activity were found to be pH 9, 45 °C, resulting in a specific enzyme activity of 14.56 U/mg. The products were separated and purified by high-performance counter-current chromatography (HPCCC). Two main components were isolated and identified, 2-amino-2-p-hydroxymethyl-methyl alcohol-benzoate (tirs-benzoate) and 1-benzoyloxy-2,3-propanediol (1-benzoyloxypropane-2,3-diol), via UPLC-Q-TOF-MS and NMR. Additionally, paeoniflorin demonstrated the ability to metabolize into benzoic acid via G6046 enzyme, which might exert antidepressant effects through the blood-brain barrier into the brain.

Production, immobilization and application of invertase from new wild strain Cunninghamella echinulata PA3S12MM.

AIMS: The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. METHODS AND RESULTS: The maximum production was verified in 8 days of cultivation at 28°C supplemented with 10 g L-1 apple peel, reaching 1054.85 U ml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15 ml of sucrose 500 g L-1 in a fixed bed reactor (total volume of 31 ml) produced 24.44 µmol min-1 of glucose and fructose at a residence time of 30 min and a conversion factor of 0.5. CONCLUSIONS: The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.

Oligochitosan Synthesized by Cunninghamella elegans, a Fungus from Caatinga (The Brazilian Savanna) Is a Better Antioxidant than Animal Chitosan.

Animal chitosan (Chit-A) is gaining more acceptance in daily activities. It is used in a range of products from food supplements for weight loss to even raw materials for producing nanoparticles and hydrogel drug carriers; however, it has low antioxidant activity. Fungal oligochitosan (OChit-F) was identified as a potential substitute for Chit-A. Cunninghamella elegans is a fungus found in the Brazilian savanna (Caatinga) that produces OligoChit-F, which is a relatively poorly studied compound. In this study, 4 kDa OChit-F with a 76% deacetylation degree was extracted from C. elegans. OChit-F showed antioxidant activity similar to that of Chit-A in only one in vitro test (copper chelation) but exhibited higher activity than that of Chit-A in three other tests (reducing power, hydroxyl radical scavenging, and iron chelation). These results indicate that OChit-F is a better antioxidant than Chit-A. In addition, Chit-A significantly increased the formation of calcium oxalate crystals in vitro, particularly those of the monohydrate (COM) type; however, OChit-F had no effect on this process in vitro. In summary, OChit-F had higher antioxidant activity than Chit-A and did not induce the formation of CaOx crystals. Thus, OChit-F can be used as a Chit-A substitute in applications affected by oxidative stress.

The oxygenated products of cryptotanshinone by biotransformation with Cunninghamella elegans exerting anti-neuroinflammatory effects by inhibiting TLR 4-mediated MAPK signaling pathway.

Cryptotanshinone (1), a major bioactive constituent in the traditional Chinese medicinal herb Dan-Shen Salvia miltiorrhiza Bunge, has been reported to possess remarkable pharmacological activities. To improve its bioactivities and physicochemical properties, in the present study, cryptotanshinone (1) was biotransformed with the fungus Cunninghamella elegans AS3.2028. Three oxygenated products (2-4) at C-3 of cryptotanshinone (1) were obtained, among them 2 was a new compound. Their structures were elucidated by comprehensive spectroscopic analysis including HRESIMS, NMR and ECD data. All of the biotransformation products (2-4) were found to inhibit significantly lipopolysaccharide-induced nitric oxide production in BV2 microglia cells with the IC50 values of 0.16-1.16 µM, approximately 2-20 folds stronger than the substrate (1). These biotransformation products also displayed remarkably improved inhibitory effects on the production of inflammatory cytokines (IL-1ß, IL-6, TNF-α, COX-2 and iNOS) in BV-2 cells via targeting TLR4 compared to substrate (1). The underlying mechanism of 2 was elucidated by comparative transcriptome analysis, which suggested that it reduced neuroinflammatory mainly through mitogen-activated protein kinase (MAPK) signaling pathway. Western blotting results revealed that 2 downregulated LPS-induced phosphorylation of JNK, ERK, and p38 in MAPK signaling pathway. These findings provide a basal material for the discovery of candidates in treating Alzheimer's disease.

Fungal transformation of norandrostenedione with Cunninghamella blakesleeana and anti-bacterial activity of the transformed products.

Although the discovery of antibiotics has decreased the spread and severity of infectious diseases, their uncontrolled use has lead to the emergence of bacterial resistance to existing chemotherapeutic agents. Bacterial disease thus remains a challenge for health authorities in worldwide and especially in sub-Saharan Africa. Despite their efficacy, the miss-use of medicinal plants for the treatment of infectious diseases couple to the farming and hunting activities has contribute enormously to the destruction of many medicinal plant species. In search of an alternative for new and effective agents against bacterial infection, norandrostenedion (19-nor-4-androsten-3,17-dione) (1), was biotransformed by Cunninghamella blakesleeana ATCC 8688A and yielded a new metabolite, 6α,10 ß -dihydroxy-19-nor-4-androsten-3-one (2), together with three known compounds, 10 ß -hydroxy-19-nor-4-androsten-3,17-dione (3), 6 ß,10 ß,17 ß -trihydroxy-19-nor-4-androsten-3-one (4) and 10 ß,17 ß -dihydroxy-19-nor-4-androsten-3-one (5). Their structures were elucidated on the basis ofspectroscopic techniques: NMR analysis (1D and 2D) and HRIE-MS and by comparison with previously reported data. In addition, the agar diffusion method was used to evaluate the diameter of the inhibition zone and INT colorimetric assay for MIC values. All metabolites obtained showed a potent and varied activity against tested bacteria. These results support the uses of biotransformation to develop new antimicrobial compounds for clinical application.

Microbial transformation of isocoronarin D by Cunninghamella echinulata NRRL 1386.

The diterpene isocoronarin D (1) is a bioactive major constituent of labdane diterpene from the aerial parts of Curcuma comosa Roxb. (Zingiberaceae), the Thai medicinal plant. Microbial transformation of 1 was performed by the fungus Cunninghamella echinulata NRRL 1386 to yield three new metabolites, 3ß-hydroxyisocoronarin D (2), 6α-hydroxyisocoronarin D (3) and 3ß,7α-dihydroxyisocoronarin D (4). The structures of the new compounds were elucidated by spectroscopic techniques.

A novel dihydroxylated derivative of artemisinin from microbial transformation.

Microbial transformation of artemisinin (1) by Cunninghamella elegans was investigated. Four isolated products were identified as 6ß-hydroxyartemisinin (2), 7α-hydroxyartemisinin (3), 7ß-hydroxyartemisinin (4), and 6ß,7α-dihydroxyartemisinin (5). The structures were elucidated by spectroscopic and X-ray crystallographic analysis. Product 5 is a novel compound and being reported here for the first time. It features two hydroxyl groups in its structure, and this is the first report on dihydroxylation of the artemisinin skeleton. Quantitative structure-activity relationship and molecular modeling studies indicate the modification of artemisinin skeleton will increase antimalarial activity and water solubility. The chemical syntheses of artemisinin derivatives at C6 or C7 position are impossible due to the lack of functional groups. 6ß,7α-Dihydroxyartemisinin is hydroxylated at both 6ß- and 7α-positions of artemisinin skeleton at the same time. Therefore, this new compound would be a good scaffold for further structural modification in the search for more potent antimalarial drugs.

Biotransfomation of cyperenoic acid by Cunninghamella elegans AS 3.2028 and the potent anti-angiogenic activities of its metabolites.

Cyperenoic acid (1) is one of the major sesquiterpenes isolated from Croton crassifolius, which exhibited potent anti-angiogenic activity. Traditional structural modification of 1 is difficult to perform by chemical technology due to the remarkable stability of the patchoulane skeleton. In order to overcome chemical synthesis difficulties and obtain structurally diverse derivations, microbial transformation of 1 by using Cunninghamella elegans AS 3.2028 was studied for the first time. Five new hydroxylated products 2-6 were obtained. Furthermore, cytotoxicity and anti-angiogenic activities of all the biotransformation products were evaluated by MTT assay and ELISA in HepG2 and MCF-7 cells. These results indicated that hydroxylated modification products 2-4 significantly inhibited VEGF release, which suggest the potential use of hydroxylated modification products for cancer therapy.

A microbial model of mammalian metabolism: biotransformation of 4,5-dimethoxyl-canthin-6-one using Cunninghamella blakesleeana CGMCC 3.970.

1. A filamentous fungus, Cunninghamella blakesleeana CGMCC 3.970, was applied as a microbial system to mimic mammalian metabolism of 4,5-dimethoxyl-canthin-6-one (1). Compound 1 belongs to canthin-6-one type alkaloids, which is a major bioactive constituent of a traditional Chinese medicine (the stems of Picrasma quassioides). 2. After 72 h of incubation in potato dextrose broth, 1 was metabolized to seven metabolites as follows: 4-methoxyl-5-hydroxyl-canthin-6-one (M1), 4-hydroxyl-5-methoxyl-canthin-6-one (M2), canthin-6-one (M3), canthin-6-one N-oxide (M4), 10-hydroxyl-4,5-dimethoxyl-canthin-6-one (M5), 1-methoxycarbonl-ß-carboline (M6), and 4-methoxyl-5-O-ß-D-glucopyranosyl-canthin-6-one (M7). 3. The structures of metabolites were determined using spectroscopic analyses, chemical methods, and comparison of NMR data with those of known compounds. Among them, M7 was a new compound. 4. The metabolic pathways of 1 were proposed, and the metabolic processes involved phase I (O-demethylation, dehydroxylation, demethoxylation, N-oxidation, hydroxylation, and oxidative ring cleavage) and phase II (glycosylation) reactions. 5. This was the first research on microbial transformation of canthin-6-one alkaloid, which could be a useful microbial model for producing the mammalian phase I and phase II metabolites of canthin-6-one alkaloids. 6. 1, M1-M5, and M7 are canthin-6-one alkaloids, whereas M6 belongs to ß-carboline type alkaloids. The strain of Cunninghamella blakesleeana can supply an approach to transform canthin-6-one type alkaloids into ß-carboline type alkaloids.

Biotransformation of patchoulol by Cunninghamella echinulata var. elegans.

Biocatalysis of patchoulol (PA) was performed by the fungus Cunninghamella echinulata var. elegans. Eight metabolites (1-8) including four new compounds were obtained, and their structures were elucidated as (5R,8S)-5,8 dihydroxypatchoulol (1), (5R*,9R*)-5,9 dihydroxypatchoulol (2), (6S*, 9S*)-6,9 dihydroxypatchoulol (3), and (4R*)-4 hydroxypatchoulol (4) by spectroscopic analysis. The absolute configuration of 1 was determined by single crystal X-ray diffraction.

[Microbial transformation of glycyrrhetinic acid by Cunninghamella blakesleeana].

A study on the microbial transformation of glycyrrhetinic acid (GA) was conducted by a fungus, Cunninghamella blakesleeana CGMCC 3.970 systematically. After incubation with the cell cultures of C. blakesleeana CGMCC 3.970 at 25 degrees C for 7 days on a rotary shaker operating at 135 r x min(-1), GA was converted into one major product and five minor products. The products were extracted and purified by solvent extraction, macroporous adsorbent resin, silica gel column chromatography, and semi-preparative RP-HPLC chromatography. Their structures were identified as 3-oxo-15α-hydroxy-18ß-glycyrrhetinic acid(1), 3-oxo-15ß-hydroxy-18ß-glycyrrhetinic acid (2), 7ß,15α-dihydroxy-18ß-glycyrrhetinic acid (3), 3-oxo-7ß, 15α-dihydroxy-18ß-glycyrrhetinic acid (4), 7ß-hydroxy-18ß-glycyrrhetinic acid(5) and 15α-hydroxy-18ß-glycyrrhetinic acid(6) by the analyses of MS, 1H-NMR and 13C-NMR spectroscopic data respectively. Among them, 2 was a new compound. These results suggest that C. blakesleeana CGMCC 3.970 has the capability of selective ketonization and hydroxylation for GA. [Key words] glycyrrhetinic acid; Cunninghamella blakesleeana CGMCC 3. 970; microbial transformation

Bioactive metabolites of Schisanlactone E transformed by Cunninghamella blakesleana AS 3.970.

Schisanlactone E (SE) is a major triterpene obtained from the plants of genus Kadsura. The aim of this research was to investigate the transformed metabolites of SE by fungi and evaluate the bioactivities of these products. After screening 10 strains of filamentous fungi, Cunninghamella blakesleana AS 3.970 was chosen as a potent organism to be used for the biotransformation of SE. 13 metabolites were obtained and determined to be new compounds through the use of spectroscopic data, including UV, 1D-, 2D-NMR, and HR-ESIMS. Furthermore, in an in vitro bioassay, metabolites 7 and 9 showed moderate inhibitory effects on the nitric oxide production in LPS-induced macrophages with IC50 values of 16.73, 5.91 µM, respectively; 9 could inhibit the proliferation of acetaldehyde-induced HSC-T6 cells, with the IC50 value of 21.4 µM. Preliminary findings on the structure-activity relationships for these metabolites were also discussed.

Stereo and regioselective microbial reduction of the clerodane diterpene 3,12-dioxo-15,16-epoxy-4-hydroxycleroda-13(16),14-diene.

The biotransformation of the clerodane diterpene, 3,12-dioxo-15,16-epoxy-4-hydroxy-cleroda-13(16),14-diene (1), obtained from Croton micans var. argyroglossum (Baill.) Mill., was investigated for the first time. Whole cells of Cunninghamella echinulata and Rhizopus stolonifer were used as enzymatic systems, and with both fungi the only biotransformation product obtained was the new ent-neo-clerodane diterpene (3R,4S,5S,8S,9R,10S)-3,4-dihydroxy-15,16-epoxy-12-oxo-cleroda-13(16),14-diene (2a). The absolute stereochemistry of 2a was inferred by comparison of its optical rotation with those of the chemical reduction product of 1 and its quasienantiomer 2c.

Harnessing indigenous plant seed oil for the production of bio-fuel by an oleaginous fungus, Cunninghamella blakesleeana- JSK2, isolated from tropical soil.

Cunninghamella blakesleeana- JSK2, a gamma-linolenic acid (GLA) producing tropical fungal isolate, was utilized as a tool to evaluate the influence of various plant seed oils on biomass, oleagenicity and bio-fuel production. The fungus accumulated 26 % total lipid of their dry biomass (2 g/l) and 13 % of GLA in its total fatty acid. Among the various plant seed oils tested as carbon sources for biotransformation studies, watermelon oil had an effect on biomass and total lipid increasing up to 9.24 g/l and 34 % respectively. Sunflower, pumpkin, and onion oil increased GLA content between 15-18 %. Interestingly, an indigenous biodiesel commodity, Pongamia pinnata oil showed tremendous effect on fatty acid profile in C. blakesleeana- JSK2, when used as a sole source of carbon. There was complete inhibition of GLA from 13 to 0 % and increase in oleic acid content, one of the key components of biodiesel to 70 % (from 20 % in control). Our results suggest the potential application of indigenous plant seed oils, particularly P. pinnata oil, for the production of economically valuable bio-fuel in oleaginous fungi in general, and C. blakesleeana- JSK2, in particular.

Characterization of in vitro and in vivo metabolites of carnosic acid, a natural antioxidant, by high performance liquid chromatography coupled with tandem mass spectrometry.

Carnosic acid (CA) is a widely employed antioxidant and the main active component in rosemary and sage, but its metabolism remains largely unknown. The present study investigated the metabolism of CA in vitro and in vivo for the first time, using high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (HPLC-Q-trap-MS). A couple of scan modes were adopted in mass spectrometer domain, including Q1 full scan, neutral loss scan-information dependent acquisition-enhanced product ion (NL-IDA-EPI) and precursor ion scan-information dependent acquisition-enhanced product ion (PI-IDA-EPI). In particular, a prediction was carried out on the basis of in vitro metabolism results, and gave birth to a multiple ion monitoring-information dependent acquisition-enhanced product ion (MIM-IDA-EPI) mode aiming to detect the trace metabolites in CA-treated biological samples. A total of ten metabolites (M4-13), along with three degradative products (M1-3), were identified for CA from in vitro metabolism models, including liver microsomes of human and rats (HLMs and RLMs), human intestinal microsomes (HIMs) and two species of Cunninghamella elegans. Twelve (U1-12) and six (F1-6) metabolites were detected from CA-treated urine and feces, respectively. In addition, five metabolites (SM2-6) in vivo were purified and definitely identified using NMR spectroscopy. The results of both in vitro and in vivo metabolism studies indicated poor metabolic stability for CA, and the glucuronidation and oxidation metabolisms extensively occurred for CA in vitro, while oxidation, glucuronidation and methylation were the main metabolic pathways observed in vivo.

Characterization of three Δ9-fatty acid desaturases with distinct substrate specificity from an oleaginous fungus Cunninghamella echinulata.

In oleaginous fungus Cunninghamella echinulata, Δ9-fatty acid desaturase introduces the first double bond into a saturated fatty acid. Three distinct genes, designated as d9dma, d9dmb and d9dmc, all encoding putative Δ9-fatty acid desaturases were isolated from this strain. The predicted proteins showed 79-87 % identity to other fungal Δ9-fatty acid desaturases. They all contain three conserved histidine boxes, C-terminal cytochrome b 5 fusion and four transmembrane domains characteristic of Δ9-desaturase. Each putative Δ9-desaturase gene from C. echinulata was able to complement the ole1 mutation in Saccharomyces cerevisiae L8-14C through heterologous expression. Analysis of the fatty acid composition of the transgenic yeast revealed that the conversion rates of 16:0 and 18:0 by D9DMA were obviously higher than those of D9DMB and D9DMC. In addition, D9DMA, D9DMB and D9DMC all had a substrate preference for 18:0 compared with 16:0. Of interest, D9DMA could saturate 12:0, 14:0, 16:0, 17:0, 18:0 and 20:0, while D9DMB saturated 14:0, 16:0, 17:0, 18:0 and 20:0. We also noticed that the transcriptional level of d9dma in C. echinulata was stimulated by cell growth but not by decline in temperature. In contrast, expression of d9dmb and d9dmc was regulated by neither cell growth nor decline in temperature in this strain.

Production of 8-prenylnaringenin from isoxanthohumol through biotransformation by fungi cells.

8-Prenylnaringenin (8PN), which presents in hop, enjoys fame as the most potential phytoestrogen. Although a number of health effects are attributed to 8PN, few reports are available about the production of it. In this work, screening of fungi to efficiently transform isoxanthohumol (IXN) into 8PN was designed. The biotransformation of IXN was significantly observed in Eupenicillium javanicum, Cunninghamella blakesleana, and Ceriporiopsis subvermispora under five kinds of transformation conditions. As a comparative result of IXN transformation, E. javanicum was the optimal biocatalyst to produce 8PN. Transformation caused by growing precultured fungal mycelia, a process designated as G2, was a favorable condition for IXN transformation in view of the yield of 8PN. The possible transformation pathway of 8PN bioproduction is postulated in this work. The construction of fungus and transformation mode derived from the current work is viable and an alternative procedure for 8PN formation.

Bioactive natural, biocatalytic, and semisynthetic tobacco cembranoids.

The two major Nicotiana tabacum tobacco cembranoids, (1 S,2 E,4 R,6 R,7 E,11 E)-2,7,11-cembratriene-4,6-diol (1) and its C-4 epimer, exhibit a wide range of interesting biological activities. Although the tumorigenesis inhibition activity of tobacco cembranoids have been known since the mid 1980's, only a limited number of investigations have targeted their optimization and structure-activity relationship. This study reports the isolation of the new (1 S,2 E,4 S,6 E,8 S,11 E)-2,6,11-cembratriene-8- O-methyl-4,8-diol (3) and the known (1 S,2 E,4 R,6 R,7 E,11 E)-2,7,11-cembratriene-4- O-methyl-4,6-diol (2) from fresh N. tabacum leaves. Cembranoid 2 showed good anti-migratory activity against prostate cancer cell lines, and was therefore subjected to microbial transformation and semisynthetic optimization studies. Biotransformation of 2 using the fungal strains Cunninghamella NRRL 5695 and Mucor ramannianus ATCC 9628 afforded new ( 4 and 5) and known ( 6 and 7) metabolites. Semisynthetic esterification, oxidation, epoxidation, and reaction with Lawesson's reagent afforded the new products 8- 14. Cembranoid 2 and its epoxidation product 9 showed potent anti-migratory activities against the highly metastatic human prostate cancer cell lines PC-3M-CT+ (spheroid disaggregation assay) and PC-3 (wound-healing assay).

Xenobiotic biotransformation of 4-methoxy-N-methyl-2-quinolone, isolated from Zanthoxylum monophyllum.

Phytochemical evaluation of Zanthoxylum monophyllum has led to the isolation of the alkaloid 4-methoxy-N-methyl-2-quinolone (1) with a significant activity against methicillin-resistant Staphylococcus aureus (MRSA), with an IC50 value of 1.5 microg/mL. Xenobiotic biotransformation of 1 has been conducted with the general goal of increasing the bioactivity of the compound and contributing new leads for further pharmacological research. Twenty-nine microorganisms were used for screening and two (Aspergillus flavus and Cunninghamella echinulata var. echinulata) were able to transform compound 1 to 4-methoxy-2-quinolone (2). Structural identification of the compounds was based on NMR, IR, and MS data.

[Microbiological transformation of paeoniflorin and albiflorin].

OBJECTIVE: To investigate the microbiological transformation of paeoniflorin and albiflorin. METHOD: The bacteria strains able to transform paeoniflorin and albiflorin were screened from 18 strains of microorganisms. The products were isolated by chromatography method and their structures were elucidated by spectral technology. RESULT: It was found that Cunninghamella blakesleana (AS 3.970) and Syncephalastrum racemosum (AS 3.264) could convert paeoniflorin and albiflorin efficiently, respectively. C. blakesleana could convert paeoniflorin to produce albiflorin, while S. racemosum could convert albiflorin to produce paeoniflorin. CONCLUSION: Paeoniflorin and albiflorin could be converted each other in definited condition.