Biotransformation of zearalenone to non-estrogenic compounds with two novel recombinant lactonases from Gliocladium.

BACKGROUND: The mycotoxin zearalenone (ZEA) produced by toxigenic fungi is widely present in cereals and its downstream products. The danger of ZEA linked to various human health issues has attracted increasing attention. Thus, powerful ZEA-degrading or detoxifying strategies are urgently needed. Biology-based detoxification methods are specific, efficient, and environmentally friendly and do not lead to negative effects during cereal decontamination. Among these, ZEA detoxification using degrading enzymes was documented to be a promising strategy in broad research. Here, two efficient ZEA-degrading lactonases from the genus Gliocladium, ZHDR52 and ZHDP83, were identified for the first time. This work studied the degradation capacity and properties of ZEA using purified recombinant ZHDR52 and ZHDP83. RESULTS: According to the ZEA degradation study, transformed Escherichia coli BL21(DE3) PLySs cells harboring the zhdr52 or zhdp83 gene could transform 20 µg/mL ZEA within 2 h and degrade > 90% of ZEA toxic derivatives, α/ß-zearalanol and α/ß-zearalenol, within 6 h. Biochemical analysis demonstrated that the optimal pH was 9.0 for ZHDR52 and ZHDP83, and the optimum temperature was 45 °C. The purified recombinant ZHDR52 and ZHDP83 retained > 90% activity over a wide range of pH values and temperatures (pH 7.0-10.0 and 35-50 °C). In addition, the specific activities of purified ZHDR52 and ZHDP83 against ZEA were 196.11 and 229.64 U/mg, respectively. The results of these two novel lactonases suggested that, compared with ZHD101, these two novel lactonases transformed ZEA into different products. The slight position variations in E126 and H242 in ZDHR52/ZEA and ZHDP83/ZEA obtained via structural modelling may explain the difference in degradation products. Moreover, the MCF-7 cell proliferation assay indicated that the products of ZEA degradation using ZHDR52 and ZHDP83 did not exhibit estrogenic activity. CONCLUSIONS: ZHDR52 and ZHDP83 are alkali ZEA-degrading enzymes that can efficiently and irreversibly degrade ZEA into non-estrogenic products, indicating that they are potential candidates for commercial application. This study identified two excellent lactonases for industrial ZEA detoxification.

Cyclic heptapeptides from the soil-derived fungus Clonostachys rosea.

Three new cyclic heptapeptides (1-3) together with three known compounds (4-6) were isolated from a solid rice culture of the soil-derived fungus Clonostachys rosea. Fermentation of the fungus on white beans instead of rice afforded a new γ-lactam (7) and a known γ-lactone (8) that were not detected in the former extracts. The structures of the new compounds were elucidated on the basis of 1D and 2D NMR spectra as well as by HRESIMS data. Compounds 1 and 4 exhibited significant cytotoxicity against the L5178Y mouse lymphoma cell line with IC50 values of 4.1 and 0.1 µM, respectively. Compound 4 also displayed cytotoxicity against the A2780 human ovarian cancer cell line with an IC50 value of 3.5 µM. The preliminary structure-activity relationships are discussed.

Two new echinocystic acid derivatives catalyzed by filamentous fungus Gliocladium roseum CGMCC 3.3657.

Biotransformation of Echinocystic acid (EA,1) using G. roseum CGMCC 3.3657 has been investigated, which leads to the isolation and identification of two novel Echinocystic acid derivatives, 4, 16α-dihydroxy-3,4-seco-olean-12-en-3,28-dioic acid (2) and 16α-hydroxy, A-homo-3α-oxa-olean-12-en-3-one-28-oic acid (3). Their structures have been elucidated by analysis of spectroscopic data. This biocatalysis could serve as an efficient tool complementary to classical chemical methods for the transformation of EA.

Media studies to enhance the production of verticillins facilitated by in situ chemical analysis.

Verticillins are a group of epipolythiodioxopiperazine alkaloids that have displayed potent cytotoxicity. To evaluate their potential further, a larger supply of these compounds was needed for both in vivo studies and analogue development via semisynthesis. To optimize the biosynthesis of these secondary metabolites, their production was analyzed in two different fungal strains (MSX59553 and MSX79542) under a suite of fermentation conditions. These studies were facilitated by the use of the droplet-liquid microjunction-surface sampling probe (droplet probe), which enables chemical analysis in situ directly from the surface of the cultures. These experiments showed that the production of verticillins was greatly affected by growth conditions; a significantly higher quantity of these alkaloids was noted when the fungal strains were grown on an oatmeal-based medium. Using these technologies to select the best among the tested growth conditions, the production of the verticillin analogues was increased while concomitantly decreasing the time required for fermentations from 5 weeks to about 11 days. Importantly, where we could previously supply 5-10 mg every 6 weeks, we are now able to supply 50-150 mg quantities of key analogues per month via laboratory scale fermentation.

Biotransformation of quercetin by Gliocladium deliquescens NRRL 1086.

With an attempt to synthesize high-value isoquercitrin (quercetin-3-O-ß-D-glucopyranoside), we carried out the biotransformation of quercetin (1) by Gliocladium deliquescens NRRL 1086. Along with the aimed product quercetin 3-O-ß-D-glycoside (2), three additional metabolites, 2-protocatechuoyl-phlorogucinol carboxylic acid (3), 2,4,6-trihydroxybenzoic acid (4), and protocatechuic acid (5), were also isolated. The time-course experiments revealed that there were two metabolic routes, regio-selectivity glycosylation and quercetin 2,3-dioxygenation, co-existing in the culture. Both glycosylation and oxidative cleavage rapidly took place after quercetin feeding; about 98% quercetin were consumed within the initial 8 h and the oxdized product (2-protocatechuoyl-phlorogucinol carboxylic acid) was hydrolyzed into two phenolic compounds (2,4,6-trihydroxybenzoic acid and protocatechuic acid). We also investigated the impact of glucose content and metal ions on the two reactions and found that high concentrations of glucose significantly inhibited the oxidative cleavage and improved the yield of isoquercitrin and that Ca2+, Fe2+, Mn2+, Mg2+, and Zn2+ inhibited glycosylation. To test the promiscuity of this culture, we selected other four flavonols as substrates; the results demonstrated its high regio-selectivity glycosylation ability towards flavonols at C-3 hydroxyl. In conclusion, our findings indicated that the versatile microbe of G. deliquescens NRRL 1086 maitained abundant enzymes, deserving further research.

Diversity synthesis of tetrahydroprotoberberines glycosides by combined chemical and microbial catalysis.

The present study was designed to construct the structurally diverse library of tetrahydroprotoberberines (THPBs) by combining the methods of chemical nonselective demethylation and microbial glycosylation. HPLC-MS/MS analyses tentatively identified 12 de-methylated and 9 glycosylated derivates of THPBs and 5 rarely oxidized glycosides of THPBs in the library. Through this effort, we achieved not only a variety of the THPBs and their glycosides but also tested the catalytic characteristics and capabilities of G. deliquescens NRRL 1086.

Glycosylation and sulfation of emodin by Gliocladium deliquescens NRRL 1086.

The present study was designed to explore the substrate scope and biocatalytic capability of Gliocladium deliquescens NRRL 1086 on phenolic natural products. Emodin was subjected to the fermentation culture of Gliocladium deliquescens NRRL 1086 according to the standard two-stage protocol. The biotransformation process was monitored by HPLC-DAD-MS, the main product was isolated by column chromatography, and the structure was elucidated on the basis of NMR spectroscopy. Emodin could be fully metabolized by Gliocladium deliquescens NRRL 1086, resulting in high yield of emodin 6-O-ß-D-glucopyranoside and small amount of sulfated product. In conclusion, our results may provide a convenient method to prepare emodin 6-O-ß-D-glucopyranoside and the microbe catalyzed glucosylation/sulfation will give an inspiration to pharmacokinetic model studies in vitro.

Chemical and microbial semi-synthesis of tetrahydroprotoberberines as inhibitors on tissue factor procoagulant activity.

To discover new inhibitors on tissue factor procoagulant activity, 21 tetrahydroprotoberberines were screened on the model of human THP-1 cells stimulated by lipopolysaccharide. Among these tetrahydroprotoberberines, several unique compounds were synthesized through microbial transformation: compound 6 (l-corydalmine) was obtained through regio-selective demethylation by Streptomyces griseus ATCC 13273, whereas compounds 4a, 4b, 5h, and 5i were microbial glycosylation products by Gliocladium deliquescens NRRL1086. The bioassay results showed that compounds 3 (tetrahydroberberine), 10 (tetrahydroberberrubine), and 5f (cinnamyl ester of 5) and 5i (glycosidic product of 5), exhibited the most potential effects, with IC(50) values of 8.35, 6.75, 3.75, and 8.79 nM, respectively. The preliminary structure and activity relationship analysis revealed that the 2,3-methylenedioxy group of the A ring was essential for the strong inhibitory effects, and the R configuration of the chiral center C-14 showed higher activity than S-form products. The formation of fatty acid or aromatic acid esters of compound 5, except the cinnamyl esters, would weaken its effects. It is also interesting to note that the glycosylation of tetrahydroprotoberberines will maintain and even enhance the inhibitory effects. Because of the importance of glycochemistry in new drug discovery and development, this deserves further exploration and may provide some guide on the semi-synthesis of tetrahydroprotoberberines as tissue factor pathway inhibitors. Our findings also provide some potential leading compounds for tissue factor-related diseases, such as cancer and cardiovascular diseases.

Studies on the metabolism of five model drugs by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS analysis.

It is well-known that cadavers may be colonized by microorganisms, but there is limited information if or to what extent these microbes are capable of metabolizing drugs or poisons, changing the concentrations and metabolic pattern of such compounds in postmortem samples. The aim of the present study was to develop a fungal biotransformation system as an in vitro model to investigate potential postmortem metabolism by fungi. Five model drugs (amitriptyline, metoprolol, mirtazapine, promethazine, and zolpidem) were each incubated with five model fungi known to colonize cadavers (Absidia repens, Aspergillus repens, Aspergillus terreus, Gliocladium viride, and Mortierella polycephala) and with Cunninghamella elegans (positive control). Incubations were performed in Sabouraud medium at 25 °C for 5 days. After centrifugation, a part of the supernatants was analyzed by liquid chromatography-tandem mass spectrometry with product ion scanning. Another part was analyzed by full scan gas chromatography-mass spectrometry after extraction and derivatization. All model drugs were metabolized by the control fungus resulting in two (metoprolol) to ten (amitriptyline) metabolites. Of the model fungi, only Abs. repens and M. polycephala metabolized the model drugs: amitriptyline was metabolized to six and five, metoprolol to two and two, mirtazapine to five and three, promethazine to six and nine, and zolpidem to three and four metabolites, respectively. The main metabolic reactions were demethylation, oxidation, and hydroxylation. The presented in vitro model is applicable to studying drug metabolism by fungi colonizing cadavers.

Unique biocatalytic resolution of racemic tetrahydroberberrubine via kinetic glycosylation and enantio-selective sulfation.

In this communication, we document a facile kinetic glycosylation resolution of racemic tetrahydroberberrubine. We also demonstrate that the enantiomeric excess of the resolved products is increased via a second resolution of the minor product of the first glycosylation resolution. This provides a rare example of tandem kinetic resolution of racemates.

Regio- and enantio-selective glycosylation of tetrahydroprotoberberines by Gliocladium deliquescens NRRL1086 resulting in unique alkaloidal glycosides.

The microbial transformation of a series of tetrahydroprotoberberines (THPBs, 1-5) by Gliocladium deliquescens NRRL1086 was investigated. In this research, the novel glycosylation of tetrahydroberberrubine (1) was observed with fast rate and high regio- and enantio-selectivity. One pair of unique enantiomorphic alkaloidal glycosides T-1 and T-2 was isolated and their structures were elucidated unambiguously by HR-MS, CD, 1D and 2D NMR spectrum. It is interesting that different amounts of glucose in the potato broth medium could influence the ratio of T-1 and T-2; in the 1.5% glucose medium, the ratio was about 15:1 and the yield of the S-form product T-1 may reach the theoretical maximum yield of about 50% which could provide one practical method to prepare the enantiomerically pure product and one alternative resolution method of tetrahydroberberrubine. The preliminary enzymatic research by using sodium dodecyl sulfate (SDS) and imidazole as glycosyltransferase and glycosidase inhibitors revealed that glycosyltransferase may contribute to glycosylation process. This is the first successful approach to glycosylation of tetrahydroprotoberberines.

Production of hydrocarbon compounds by endophytic fungi Gliocladium species grown on cellulose.

Endophytic fungi belonging to the genus Gliocladium are able to degrade plant cellulose and synthesize complex hydrocarbons under microaerophilic conditions. These fungi could thus be used to produce biofuels from cellulosics without the need for hydrolytic pretreatments. Gas chromatography-mass spectrometry-solid-phase micro-extraction (GC-MS-SPME) of head space gases from Gliocladium cultures demonstrated the production of C(6)-C(19) hydrocarbons including hexane, benzene, heptane, 3,4-dimethyl hexane, 1-octene, m-xylene, 3-methyl nonane, dodecane, tridecane, hexadecane and nonadecane directly from the cellulosic biomass. Hydrocarbon production was 100-fold higher in co-cultures of Gliocladium and Escherichia coli than in pure Gliocladium cultures. The dry mycelia weight is stable at stationary period in co-culture condition which may lead to synthesize more hydrocarbons. These fungi could potentially be developed into cost-effective biocatalysts for production of biofuels.

Cellulase and xylanase activity in relation to biofilm formation by two intertidal filamentous fungi in a novel polymethylmethacrylate conico-cylindrical flask.

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for fungal attachment was employed for production of cellulase by Chaetomium crispatum and xylanase by Gliocladium viride. The design allowed comparison of production between CCFs with hydrophobic surface (PMMA-CCF), hydrophilic glass surface (GS-CCF) and 500-ml Erlenmeyer flask (EF). Compared with the EF, endo-ß-1,4-glucanase and FPase (filter paper degradation) activities increased from 0.044 to 0.156 and from 0.008 to 0.021 IU/ml, respectively, in the PMMA-CCF, while growth of C. crispatum was higher by at most 1.38-fold compared with the other vessels. Xylanase production in the EF was at most 5.08-fold higher and growth of G. viride was at most 1.52-fold higher compared with the other vessels. Temporal pattern of biofilm development based on two-channel fluorescence detection of extracellular polymeric substances (EPSs) and whole cells in a confocal laser scanning microscope demonstrated increase by 100% in biovolume, 25% in thickness and 62.5% both in substratum coverage and total spreading of C. crispatum biofilm in PMMA-CCF over 6 days. Biovolume of G. viride biofilm in GS-CCF increased by 150% over 4 days while that in PMMA-CCF enhanced by 200% over 2 days. Biofilm thickness in PMMA-CCF was 44% higher compared with GS-CCF and increased by 175% over 2 days. Substratum coverage was 38% higher in GS-CCF compared with PMMA-CCF. Thus, reactor surface area and property, shear forces and biofilm formation influenced enzyme production.

Secalonic acid D as a novel DNA topoisomerase I inhibitor from marine lichen-derived fungus Gliocladium sp. T31.

CONTEXT: DNA topoisomerase I (topo I) is an essential enzyme which regulates the conformational changes in DNA topology by cleaving and rejoining DNA strands during normal cell growth. The inhibitors of topo I represent a major class of anticancer drugs. In our projects to isolate new anticancer agents from marine-derived fungi, secalonic acid D (SAD) with inhibitory activity on topo I was isolated from the fermentation broth of marine lichen-derived fungus Gliocladium sp. T31, which was collected from marine sediments in South Pole. OBJECTIVE: The inhibitory activity of SAD on topo I was investigated for the first time. MATERIALS AND METHODS: The inhibitory effect of SAD on topo I was determined via in vitro supercoil relaxation assays and electrophoretic mobility shift assay (EMSA) using plasmid substrate, pBR322. RESULTS: SAD displays a considerable inhibition on topo I in a dose-dependent manner with the minimum inhibitory concentration (MIC) of 0.4 µM. Unlike the prototypic DNA topo I poison camptothecin (CPT), SAD inhibits the binding of topo I to DNA but does not induce the formation of topo I-DNA covalent complexes. DISCUSSION AND CONCLUSION: SAD is an excellent topo I inhibitor and thus a significantly potential anticancer candidate.

Biological control of wood decay against fungal infection.

Wood (timber) is an important raw material for various purposes, and having biological composition it is susceptible to deterioration by various agents. The history of wood protection by impregnation with synthetic chemicals is almost two hundred years old. However, the ever-increasing public concern and the new environmental regulations on the use of chemicals have created the need for the development and the use of alternative methods for wood protection. Biological wood protection by antagonistic microbes alone or in combination with (bio)chemicals, is one of the most promising ways for the environmentally sound wood protection. The most effective biocontrol antagonists belong to genera Trichoderma, Gliocladium, Bacillus, Pseudomonas and Streptomyces. They compete for an ecological niche by consuming available nutrients as well as by secreting a spectrum of biochemicals effective against various fungal pathogens. The biochemicals include cell wall-degrading enzymes, siderophores, chelating iron and a wide variety of volatile and non-volatile antibiotics. In this review, the nature and the function of the antagonistic microbes in wood protection are discussed.

Microbial conversion of ruscogenin by Gliocladium deliquescens NRRL1086: glycosylation at C-1.

The glycosylation of ruscogenin (1) by Gliocladium deliquescens NRRL 1086 was observed and gave a regioselectively glycosylated product identified as ruscogenin 1-O-beta-D-glucopyranoside (2) by infrared, mass spectrometry, and nuclear magnetic resonance spectra. Time-course studies indicated that it appeared to be favorable to accumulate 2 when ruscogenin was added to the 24-h-old stage II culture, and the yield of 2 was about 20.1% during 120 approximately 168 h. It was noted that additional carbohydrates could significantly increase glycoside formation and the yield of 2 even reached as high as 68% compared with the control 20.1%. The primary investigation about the characteristics of the enzyme resulted that the reaction was blocked by beta-glycosidase inhibitor imidazole, however, was enhanced remarkably by glycosyltransferase inhibitor sodium dodecyl sulfate. To our knowledge, this is the first reported case of producing steroidal saponin by microbial transformation, and G. deliquescens NRRL1086 would be a practical and highly efficient tool in producing natural ruscogenin monoside.

Production, purification and characterization of taxol and 10DAB III from a new endophytic fungus Gliocladium sp. isolated from the Indian yew tree, Taxus baccata.

We have isolated endophytic fungi from Indian yew tree, Taxus baccata and then screened for taxol production. Out of the forty fungal cultures screened, one fungus Gliocladium sp. was found to produce taxol and 10DAB III (10 Deacetyl baccatin III). These compounds were purified by TLC, HPLC and characterized using UV-Spectroscopy, ESI-MS, MS/MS and proton NMR. One liter of Gliocladium sp. culture yielded 10 microg of taxol and 65 microg of 10 DAB III. The purified taxol from the fungus showed cytotoxicity towards cancer lines HL-60 (leukemia), A431 (epidermal carcinoma) and MCF-7 (breast cancer).

The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072).

An endophytic fungus, Gliocladium roseum (NRRL 50072), produced a series of volatile hydrocarbons and hydrocarbon derivatives on an oatmeal-based agar under microaerophilic conditions as analysed by solid-phase micro-extraction (SPME)-GC/MS. As an example, this organism produced an extensive series of the acetic acid esters of straight-chained alkanes including those of pentyl, hexyl, heptyl, octyl, sec-octyl and decyl alcohols. Other hydrocarbons were also produced by this organism, including undecane, 2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-trimethyl; and undecane, 4,4-dimethyl. Volatile hydrocarbons were also produced on a cellulose-based medium, including heptane, octane, benzene, and some branched hydrocarbons. An extract of the host plant, Eucryphia cordifolia (ulmo), supported the growth and hydrocarbon production of this fungus. Quantification of volatile organic compounds, as measured by proton transfer mass spectrometry (PTR-MS), indicated a level of organic substances in the order of 80 p.p.m.v. (parts per million by volume) in the air space above the oatmeal agar medium in an 18 day old culture. Scaling the PTR-MS profile the acetic acid heptyl ester was quantified (at 500 p.p.b.v.) and subsequently the amount of each compound in the GC/MS profile could be estimated; all yielded a total value of about 4.0 p.p.m.v. The hydrocarbon profile of G. roseum contains a number of compounds normally associated with diesel fuel and so the volatiles of this fungus have been dubbed 'myco-diesel'. Extraction of liquid cultures of the fungus revealed the presence of numerous fatty acids and other lipids. All of these findings have implications in energy production and utilization.

Hydroxylation of the triterpenoid nigranoic acid by the fungus Gliocladium roseum YMF1.00133.

The ability of the fungus Gliocladium roseum YMF1.00133 to transform the bioactive nigranoic acid (=(24Z)-9,19-cyclo-3,4-secolanosta-4(28),24-diene-3,26-dioic acid) was investigated. Three new products from the co-cultures of nigranoic acid and G. roseum YMF1.00133 were obtained by employing a combination of Sephadex LH-20 and silica-gel column chromatography. The major metabolite was identified as 15beta-hydroxynigranoic acid, and the minor metabolites as 6alpha,15beta-dihydroxynigranoic acid and 7beta,15beta-dihydroxynigranoic acid by mass spectrometry and NMR spectroscopy. This is the first report of the biotransformation of the A-ring-secocycloartene triterpenoid, nigranoic acid.