Microbial hydroxylation of chiral bicyclic enones by Chaetomium sp.1 and Didymosphaeria igniaria cultures

The biotransformations of (R)-(?)-methyloctalone and (S)-(+)-methyloctalone were investigated using Chaetomium sp.1 KCH 6651 and Didymosphaeria igniaria KCH 6670 as biocatalysts, yielding mostly 6β- and 7β-hydroxy derivatives. During the incubation of (R)-methyloctalone with the Chaetomium sp.1 culture, three products were obtained: the trans-7β-hydroxy derivative in 50% yield, trans-6β-hydroxy derivative in 30% yield and cis-8α-hydroxy derivative in 6% yield. The (S)-enone was hydroxylated mainly in the 6α-position (with 60% yield). 6β- and 7β-hydroxy derivatives are new compounds, not previously described in the literature. The biotransformation of the two enantiomers of methyloctalone with D. igniaria also afforded monohydroxy derivatives. In both cases the main product of transformation was the allylic hydroxy derivative (27–35% yield). D. igniaria transformed the (R)-methyloctalone more rapidly whereas Chaetomium sp. 1 preferred the (S)-methyloctalone.     

Application of α- and β-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 6β-, 12β-, 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-, 6β-, 9α-, 14α-hydroxy and 6β,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6β,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 β-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.     

Biosynthesis of Carotenoids in Brevibacterium sp. KY-4313

The biosynthesis of 4-keto and 4,4′-diketo carotenoids in Brevibacterium sp. KY-4313 was studied. Echinenone and canthaxanthin were isolated from the cultures grown on a medium containing several n-alkanes. When glutathione was added to the bacterial cultures, the formation of canthaxanthin was inhibited while β-carotene and its hydroxy derivatives accumulated. It is suggested that these 4-hydroxy compounds, isocryptoxanthin, isozeaxanthin, and 4-hydroxy-4′-keto-β-carotene, are intermediates in the biosynthesis of canthaxanthin. In the presence of 2-(4-chlorophenylthio)-triethylamine hydrochloride or nicotine, lycopene and neurosporene accumulated. The β-carotene level decreased slightly but β-zeacarotene remained unchanged. β-carotene and its derivatives were resynthesized upon removal of the inhibitors. It was concluded that cyclization can take place at either the neurosporene or lycopene level in Brevibacterium sp. KY-4313.     

Microbial transformations of chalcones to produce food sweetener derivatives

Biotransformations of two substrates: chalcone (1) and 2′-hydroxychalcone (4) were carried out using four yeast strains and five filamentous fungi cultures. Substrate 1 was effectively hydrogenated in all of tested yeast cultures (80–99% of substrate conversion after 1 h of biotransformation) affording dihydrochalcone 2. In the cultures of filamentous fungi the reaction was much slower, however, Chaetomium sp. gave product 2 in 97% yield. After 12 h of incubation a reduction of dihydrochalcone 2 to alcohol 3 was additionally observed. After 3 days of biotransformation in the culture of Rhodotorula rubra product (S)-3 was obtained with 75% ee (enantiomeric excess) and 99% of conversion. Also after a 3-day biotransformation using the strain Fusarium culmorum product (R)-3 was obtained with 98% ee and 97% of conversion. In most of the tested strains a change in enantiomeric excess of compound 3 during the biotransformation process was noticed. In the culture of Rhodotorula glutinis after 3 h of transformation alcohol (R)-3 was formed with 47% ee and 31% of substrate conversion, whereas after 6 days the (S)-3 enantiomer was obtained with 99% ee and 91% of conversion. In the case of 2′-hydroxychalcone (4), the hydrogenation proceeded much slower and led to 2′-hydroxydihydrochalcone (5) – in the culture of Yarrowia lipolytica 97% of conversion was observed after 3 days. In all cultures of the tested strains no products of 2′-hydroxydihydrochalcone reduction were detected.     

Coincident isolation of a novel homoisoflavonoid from Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae)

We report on the first phytochemical investigation of a member of the African genus Resnova (Hyacinthoideae: Hyacinthaceae). From the dichloromethane extract of the bulbs of both Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae) a novel 3-benzyl-4-chromanone homoisoflavonoid, 5,6-dimethoxy-7-hydroxy-3-(4′-hydroxybenzyl)-4-chromanone, was isolated. A further 11 known homoisoflavonoids were also identified, the 12 in total presenting a clear biosynthetic sequence. Eight of the 12 compounds found were common to both species.     

The effect of a preparation from Chaetomium fungi on the growth of phytopathogenic fungi

We studied the fungicidal activity of a biological preparation from the fungi of the genus Chaetomium against soil phytopathogenic fungi Rhizoctonia solani and Fusarium oxysporum. The inhibitory effect of the preparation under study depended on its concentration, duration of storage, and growth characteristics of pure cultures of the phytopathogens. The highest (98.8%) inhibitory activity was observed on the third day of the interaction with Rhizoctonia solani. After a 2-year storage, this preparation was capable of inhibiting the growth of phytopathogens only at high doses. The preparation precluded the development of a bare patch but increased the productivity of potato plants. The preparation may serve as an alternative to chemical fungicides for plant protection.     

Efficiency of natural and engineered bacterial strains in the degradation of 4-chlorobenzoic acid in soil slurry

Two bacterial strains, the natural isolate Arthrobacter sp. FG1 and the engineered strain Pseudomonas putida PaW340/pDH5, were compared for their efficiency in the degradation of 4-chlorobenzoic acid in a slurry phase system. The recombinant strain was obtained by cloning the Arthrobacter sp. FG1 dehalogenase encoding genes in P. putida PaW340. In the slurry inoculated with pre-adapted cultures of Arthrobacter sp. FG1, the 4-chlorobenzoic acid degradation was found to be slower than that observed in the slurry inoculated with the recombinant strain P. putida PaW340/pDH5, regardless of the presence or absence of soil indigenous bacteria. Slurry inoculated with mixed cultures of Arthrobacter sp. FG1 and the 4-hyroxybenzoic acid degrader P. putida PaW340 did not show any improvement in 4-chlorobenzoic acid degradation.     

Steroid modification by filamentous fungus Drechslera sp.: Focus on 7-hydroxylase and 17β-hydroxysteroid dehydrogenase activities

Fungal strain Drechslera sp. Ph F-34 was shown to modify 3-oxo- and 3-hydroxy steroids of androstane series to form the corresponding allylic 7-alcohols and 17β-reduced derivatives thus evidencing the presence of 7α-, 7β-hydroxylase and 17β-hydroxysteroid dehydrogenase (17β-HSD) activities. The growing mycelium predominantly hydroxylated androsta-1,4-diene-3,17-dione (ADD) at the 7β-position, while much lower 7α-hydroxylation was observed. Along with 7β-hydroxy-ADD and its corresponding 7α-isomer, their respective 17β-alcohols were produced.In this study, transformation of ADD, androst-4-en-17β-ol-3-one (testosterone, TS) and 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone, DHEA) by resting mycelium of Drechslera sp. have been estimated in different conditions with regard to the inducibility and functionality of the 17β-HSD and 7-hydroxylase enzyme systems. Steroids of androstane, pregnane and cholane series were evaluated as inducers. The inhibitory analysis was provided using cycloheximide (CHX). Steroids were assayed using TLC and HPLC methods, and the structures were confirmed by mass-spectrometry, ¹H and ¹³C NMR spectroscopy data.17β-HSD of the mycelium constitutively reduced 17-carbonyl group of ADD and DHEA to form the corresponding 17β-alcohols, namely, androsta-1,4-diene-17β-ol-3-one (1-dehydro-TS), and androst-5-ene-3β,17β-diol. Production of the 7α- and 7β-hydroxylated derivatives depended on the induction conditions. The inducer effect relied on the steroid structure and decreased in the order: DHEA > pregnenolone > lithocholic acid. β-Sitosterol did not induce hydroxylase activity in Drechslera sp. CHX fully inhibited the synthesis of 7-hydroxylase in Drechslera mycelium thus providing selective 17-keto reduction.Results contribute to the diversity of steroid modifying enzymes in fungi and can be used at the development of novel biocatalysts for production of valuable steroid 7(α/β)- and 17β-alcohols.     

Unnatural enantiomer of chaetocin shows strong apoptosis-inducing activity through caspase-8/caspase-3 activation

Chaetocin, a natural product isolated from Chaetomium species fungi, was reported to have various biological activities, including antitumor and antifungal activities. Recently, we reported the first total synthesis of chaetocin and its derivatives. Here, we examined the cell-death-inducing activity of these compounds in human leukemia HL-60 cells. The unnatural enantiomer of chaetocin (ent-chaetocin) was more potent than chaetocin, and was found to induce apoptosis through the caspase-8/caspase-3 activation pathway.     

Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.     

Keratinophilic fungi from soil of Tarragona,Catalunya

97 soils samples of Tarragona city and outskirts were analysed. We were isolated the following keratinophilic fungi: Microsporum gypseum, Trichophyton ajelloi, Ctenomyces serratus, Malbranchea state of C. uncinatus, Chrysosporium tropicum, Chrysosporium evolceanui, Chrysosporium asperatum, Chrysosporium pruinosum (?), Auxarthron thaxteri, Gymnoascus petalosporus, Gymnoascus reesii, Chaetomium murorum, Chaetomium spirale and Chaetomium sp. On explaine the distribution of different isolated species.     

Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives

A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

Influence of light on phycobiliprotein production in three marine cyanobacterial cultures

Complementary chromatic adaptation, a photomorphogenetic response, known to occur in many cyanobacteria, enables them to efficiently absorb prevalent wavelengths of light in the environment. In the present study, we have described the influence of light on phycobiliprotein production in three marine phycoerythrin producing cyanobacterial cultures, namely, Lyngbya sp. A09DM, Phormidium sp. A27DM and Halomicronema sp. A32DM. A comparative study (UV-visible overlay spectra and SDS-PAGE analyses) of phycobiliproteins purified from all the three cultures grown in white, yellow, red and green lights has been confirmed. White light was taken as control. Red and green lights were taken to check their effect on phycocyanin and phycoerythrin production, respectively. Yellow light was studied as its wavelength falls in between green and red light. Lyngbya sp. A09DM was found to be the best chromatically adapting cyanobacterium followed by Halomicronema sp. A32DM. These two cultures can be placed in group III chromatic adaptors. Phormidium sp. A27DM was the least chromatically adapting culture and can be placed in group II chromatic adaptors. The study signifies that even light plays an important role along with nutrient availability in adapting cultures to changing environmental conditions.     

Stress resistance and C1 metabolism involved in plant colonization of a methanotroph Methylosinus sp. B4S

Methanotrophs are widespread and have been isolated from various environments including the phyllosphere. In this study, we characterized the plant colonization by Methylosinus sp. B4S, an α-proteobacterial methanotroph isolated from plant leaf. The gfp-tagged Methylosinus sp. B4S cells were observed to colonize Arabidopsis leaf surfaces by forming aggregates. We cloned and sequenced the general stress response genes, phyR, nepR and ecfG, from Methylosinus sp. B4S. In vitro analysis showed that the phyR expression level was increased after heat shock challenge, and phyR was shown to be involved in resistance to heat shock and UV light. In the phyllospheric condition, the gene expression level of phyR as well as mmoX and mxaF was found to be relatively high, compared with methane-grown liquid cultures. The phyR-deletion strain as well as the wild-type strain inoculated on Arabidopsis leaves proliferated at the initial phase and then gradually decreased during plant colonization. These results have shed light firstly on the importance of general stress resistance and C1 metabolism in methanotroph living in the phyllosphere.     

Effects of organic solvents on activity and stability of lipases produced by thermotolerant fungi in solid-state fermentation

Dried solid-state fermented solids (biocatalysts) produced by seven thermotolerant fungal strains were tested for lipase activity and stability in organic solvents. Two strains of Rhizopus sp. (19 and 43a) produced biocatalysts (L-19 and L-43a) that showed high lipase activities (74 and 72 U/g of dry matter, respectively) comparable to Lipozyme® RM IM (118 U/g DM). The use of the dipole moment of the organic solvents along with their classification based on the functional groups (non-polar, protic polar, aprotic polar) allowed the establishment of four different relative activity profiles for the seven biocatalysts evaluated. Compared to a biocatalyst not exposed to the organic solvent (100% relative activity), all biocatalysts showed a high relative activity (greater than 90%) in aprotic polar solvents (acetonitrile, acetone and ethyl acetate), whereas in protic polar solvents (ethanol and i-propanol) activity was reduced (lower than 40%). In addition, the incubation of biocatalysts L-19 and L-43a in i-amyl alcohol increased lipase activity in the synthesis of ethyl oleate 3.36 and 1.46 times, respectively. L-19 activity also increased after incubation in toluene (2.0 times), i-propanol (1.5 times) and acetonitrile (1.3 times) at temperatures from 30 to 50 °C. The results suggest that these biocatalysts can be used for a broad range of lipase reactions.     

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.     

Relationship between the Unicellular Red Alga Porphyridium sp. and Its Predator, the Dinoflagellate Gymnodinium sp

Contamination of algae cultivated outdoors by various microorganisms, such as bacteria, fungi, algae, and protozoa, can affect growth and product quality, sometimes causing fast collapse of the cultures. The main contaminant of Porphyridium cultures grown outdoors in Israel is a Gymnodinium sp., a dinoflagellate that feeds on the alga. Comparison of the effects of various environmental conditions, i.e., pH, salinity, and temperature, on Gymnodinium and Porphyridium species revealed that the Gymnodinium sp. has sharp optimum curves, whereas the Porphyridium sp. has a wider range of optimum conditions and is also more resistant to extreme environmental variables. The mode of preying on the alga was observed, and the specificity of the Gymnodinium sp. for the Porphyridium sp. was shown. In addition, Gymnodinium extract was shown to contain enzymatic degrading activity specific to the Porphyridium sp. cell wall polysaccharide.     

Degradation of Carbazole by Microbial Cells Immobilized in Magnetic Gellan Gum Gel Beads

Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized Sphingomonas sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for Sphingomonas sp. strain XLDN2-5. After comparison with agar, alginate, and κ-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe₃O₄ nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g⁻¹ saturation magnetization. When the mixture of gellan gel and the Fe₃O₄ nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe₃O₄ nanoparticles was 9 mg ml⁻¹ and the saturation magnetization of magnetically immobilized cells was 11.08 emu g⁻¹. Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.     

Ecology of Thermophilic Fungi in Mushroom Compost, with Emphasis on Scytalidium thermophilum and Growth Stimulation of Agaricus bisporus Mycelium

Twenty-two species of thermophilic fungi were isolated from mushroom compost. Scytalidium thermophilum was present in the compost ingredients, fresh straw, horse droppings, and drainage from compost and dominated the fungal biota of compost after preparation. Of 34 species of thermophilic fungi tested, 9 promoted mycelial growth of Agaricus bisporus on sterilized compost: Chaetomium thermophilum, an unidentified Chaetomium sp., Malbranchea sulfurea, Myriococcum thermophilum, S. thermophilum, Stilbella thermophila, Thielavia terrestris, and two unidentified basidiomycetes. These species will be considered for future experiments on inoculation and more controlled preparation of compost.