Production and partial purification by PEG/citrate ATPS of a ß-galactosidase from the new promising isolate Cladosporium tenuissimum URM 7803.

ß-Galactosidase production, partial purification and characterization by a new fungal were investigated. Partial purification was performed by aqueous two-phase system (ATPS) using polyethylene glycol (PEG) molar mass, PEG concentration, citrate concentration and pH as the independent variables. Purification factor (PF), partition coefficient (K) and yield (Y) were the responses. After identification by rDNA sequencing and classification as Cladosporium tenuissimum URM 7803, this isolate achieved a maximum cell concentration and ß-galactosidase activity of 0.48 g/L and 462.1 U/mL, respectively. ß-Galactosidase partitioned preferentially for bottom salt-rich phase likely due to hydrophobicity and volume exclusion effect caused in the top phase by the high PEG concentration and molar mass. The highest value of PF (12.94) was obtained using 24% (w/w) PEG 8000 g/mol and 15% (w/w) citrate, while that of Y (79.76%) using 20% (w/w) PEG 400 g/mol and 25% (w/w) citrate, both at pH 6. The enzyme exhibited optimum temperature in crude and ATPS extracts in the ranges 35-50 °C and 40-55 °C, respectively, and optimum pH in the range 3.0-4.5, with a fall of enzyme activity under alkaline conditions. Some metal ions and detergents inhibited, while others stimulated enzyme activity. Finally, C. tenuissimum URM 7803 ß-galactosidase showed a profile suitable for prebiotics production.

Moringa straw as cellulase production inducer and cellulolytic fungi source / Residuo de moringa como inductor de la producción de celulasas y como fuente de hongos celulolíticos

Abstract Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and puri-fied 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606 U/l) and exoglucanase (205 U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of p-glucosidase activity (664 U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and p-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum. © 2019 Asociación Argentina de Microbiología. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Resumen Actualmente, la valorización de los residuos agroindustriales es de gran interés. En este trabajo se emplearon residuos de madera blanda de Moringa oleifera para la producción de celulasas de bajo costo. El objetivo fue aislar, identificar y caracterizar microorganismos con actividad celulolítica en diferentes fuentes de carbono. A partir de la biomasa de M. oleifera, se aislaron e identificaron 42 microorganismos productores de celulasas. Los hongos que presentaron los mayores halos de hidrólisis en carboximetilcelulosa como sustrato fueron identificados molecularmente como Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) y Cladosporium cladosporioides (FC2). Mediante fermentación sumergida, se evaluó la capacidad de estas cepas en la producción de celulasas utilizando celulosa cristalina (Avicel) y amorfa (CMC) como fuentes de carbono. P. funiculosum y C. cladosporioides presentaron las mayores actividades de endoglucanasa (606 U/l) y exoglucanasa (205 U/l) en medio Avicel, mientras que F. verticillioides mostró la mayor actividad de p-glucosidasa (664 U/l) en medio CMC. Además, se evaluó el efecto de tres medios de cultivo (A, B y C) sobre la producción de celulasas en P. funiculosum empleando residuos de moringa como fuente de carbono. Los resultados mostraron que en el medio C, la productividad volumétrica de celulasas se incrementó en 2,77; 8,26 y 2,30 veces para las actividades de endoglucanasa, exoglucanasa y p-glucosidasa, respectivamente. Los extractos enzimáticos producidos tienen gran potencial para su utilización biotecnológica, especialmente en la sacarificación de residuos de moringa y la producción de bioetanol de segunda generación. Este es el primer estudio del uso de la biomasa de M. oleifera para inducir la producción de diversas celulasas en P. funiculosum.

Moringa straw as cellulase production inducer and cellulolytic fungi source.

Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and purified 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606U/l) and exoglucanase (205U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of ß-glucosidase activity (664U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and ß-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum.

Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16.

Glucose oxidases are widely used in various industrial processes, including bread baking. In this study, a novel glucose oxidase gene, CngoxA, from Cladosporium neopsychrotolerans SL16, was cloned and expressed in Pichia pastoris. Recombinant CnGOXA exhibited maximal activity at 20 °C and pH 7.0, and was stable at 30 °C and pH 6.0-9.0 for 1 h, with a half-life of 15 min at 40 °C. Compared with CnGOXA, the half-life of its mutant CnGOXA-M1 (Y169C-A211C), at 40 °C increased approximately 48-fold, and was stable at 30 °C and pH 3.0-12.0 for 1 h. The kcat and catalytic efficiency of CnGOXA-M1 were enhanced 0.7- and 1.6-fold, respectively. Both enzymes were cold-adapted and highly resistant to SDS. Furthermore, CnGOXA-M1 had a more significant effect on bread volume than that of GOX from Aspergillus niger. These favorable enzymatic properties of CnGOXA-M1 make it a potentially useful enzyme for many industrial applications.

Molecular and morphological characterization of rice phylloplane fungi and determination of the antagonistic activity against rice pathogens.

Cladosporium spp. is a cosmopolitan fungal genus. In the literature, it has been reported as a biological agent for controlling several plant diseases, but its mechanism of action has never been clarified. The present study aims to identify Cladosporium spp. based on the DNA phylogeny of nine isolates obtained from the phylloplane of rice and their potential antagonistic activity against the main fungal pathogens that affect rice crop. Nine isolates of Cladosporium spp. were identified based on DNA phylogeny, molecular and morphological characterization, and their antagonistic effects with the rice pathogens C. miyabeanus, M. oryzae, M. albescens and S. oryzae. Four isolates were selected to study lytic enzymes such as ß-1,3-glucanase, chitinase and protease, and only one isolate was selected for a conidial germination and appressoria formation assay. The nine isolates were identified as C. cladosporioides, C. tenuissimum and C. subuliforme. Four isolates, identified as C. cladosporioides, inhibited the mycelial growth of rice pathogens such as C1H (68.59%) of S. oryzae, C5 G (74.32%) of C. miyabeanus, C11 G (75.97%) of M. oryzae and C24 G (77.39%) of M. albescens. C24 G showed a high activity of lytic enzymes, was tested against C. miyabeanus and M. oryzae, and inhibited conidial germination and appressorium formation by more than 59.36%. The characterization of C. cladosporioides suggested this species as a potential bioagent for the management of several rice diseases, especially rice blast. This is the first time that a potential biological agent from the genus Cladosporium identified at the species level was isolated from the rice phylloplane, and some of its mechanisms of action were demonstrated, such as increasing lytic enzyme activity against rice pathogens.

A conserved GH17 glycosyl hydrolase from plant pathogenic Dothideomycetes releases a DAMP causing cell death in tomato.

To facilitate infection, pathogens deploy a plethora of effectors to suppress basal host immunity induced by exogenous microbe-associated or endogenous damage-associated molecular patterns (DAMPs). In this study, we have characterized family 17 glycosyl hydrolases of the tomato pathogen Cladosporium fulvum (CfGH17) and studied their role in infection. Heterologous expression of CfGH17-1 to 5 by potato virus X in different tomato cultivars showed that CfGH17-1 and CfGH17-5 enzymes induce cell death in Cf-0, Cf-1 and Cf-5 but not in Cf-Ecp3 tomato cultivars or tobacco. Moreover, CfGH17-1 orthologues from other phytopathogens, including Dothistroma septosporum and Mycosphaerella fijiensis, also trigger cell death in tomato. CfGH17-1 and CfGH17-5 are predicted to be ß-1,3-glucanases and their enzymatic activity is required for the induction of cell death. CfGH17-1 hydrolyses laminarin, a linear 1,3-ß-glucan with 1,6-ß linkages. CfGH17-1 expression is down-regulated during the biotrophic phase of infection and up-regulated during the necrotrophic phase. Deletion of CfGH17-1 in C. fulvum did not reduce virulence on tomato, while constitutive expression of CfGH17-1 decreased virulence, suggesting that abundant presence of CfGH17-1 during biotrophic growth may release a DAMP that activates plant defence responses. Under natural conditions CfGH17-1 is suggested to play a role during saprophytic growth when the fungus thrives on dead host tissue, which is in line with its high levels of expression at late stages of infection when host tissues have become necrotic. We suggest that CfGH17-1 releases a DAMP from the host cell wall that is recognized by a yet unknown host plant receptor.

Heterologous expression, purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6-7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20-30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.

Insight into the cold adaptation and hemicellulose utilization of Cladosporium neopsychrotolerans from genome analysis and biochemical characterization.

The occurrence of Cladosporium in cold ecosystems has been evidenced long before, and most of the knowledge about nutrient utilization of this genus is sporadic. An alpine soil isolate C. neopsychrotolerans SL-16, showing great cold tolerance and significant lignocellulose-degrading capability, was sequenced to form a 35.9 Mb genome that contains 13,456 predicted genes. Functional annotation on predicted genes revealed a wide array of proteins involved in the transport and metabolism of carbohydrate, protein and lipid. Large numbers of transmembrane proteins (967) and CAZymes (571) were identified, and those related to hemicellulose degradation was the most abundant. To undermine the hemicellulose (xyaln as the main component) utilization mechanism of SL-16, the mRNA levels of 23 xylanolytic enzymes were quantified, and representatives of three glycoside hydrolase families were functionally characterized. The enzymes showed similar neutral, cold active and thermolabile properties and synergistic action on xylan degradation (the synergy degree up to 15.32). Kinetic analysis and sequence and structure comparison with mesophilic and thermophilic homologues indicated that these cold-active enzymes employed different cold adaptation strategies to function well in cold environment. These similar and complementary advantages in cold adaptation and catalysis might explain the high efficiency of lignocellulose conversion observed in SL-16 under low temperatures.

Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization.

Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the ß-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.

Production and Partial Characterization of an Alkaline Xylanase from a Novel Fungus Cladosporium oxysporum.

A new fungus Cladosporium oxysporum GQ-3 producing extracellular xylanase was isolated from decaying agricultural waste and identified based on the morphology and comparison of internal transcribed spacer (ITS) rDNA gene sequence. C. oxysporum produced maximum xylanase activity of 55.92 U/mL with wheat bran as a substrate and NH4Cl as a nitrogen source. Mg(2+) improved C. oxysporum xylanase production. Partially purified xylanase exhibited maximum activity at 50°C and pH 8.0, respectively, and showed the stable activity after 2-h treatment in pH 7.0-8.5 or below 55°C. Mg(2+) enhanced the xylanase activity by 2% while Cu(2+) had the highest inhibition ratio of 57.9%. Furthermore, C. oxysporum xylanase was resistant to most of tested neutral and alkaline proteases. Our findings indicated that Cladosporium oxysporum GQ-3 was a novel xylanase producer, which could be used in the textile processes or paper/feed industries.

Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

STABILITY OF NATIVE AND MODIFIED α-GALACTOSIDASE OF Cladosporium cladosporioides.

By modifying carbohydrate component of glycoproteins it is possible to elucidate its role in manifestation of structural and functional properties of the enzyme. The comparison of activity and stability of the native and modified by oxidation with sodium periodate α-galactosidase of Cladosporium cladosporioides was carried out. To determine α-galactosidase activity the authors used n-nitrophenyl synthetic substrate, as well as melibiose; raffinose and stachyose. Modification of the carbohydrate component had a significant effect on catalytic properties of the enzyme. Both the reduction of V and enzyme affinity for natural and synthetic substrates were observed The native enzyme retained more than 50% ofthe maximum activity in the range of 20-60 °C, while for the modified enzyme under the same conditions that temperature range was 30-50 °C. The modified α-galactosidase demonstrated a higher thermal stability under neutral pH conditions. The residual activity of the modified α-galactosidase was about 30% when treated with 70% (v/v) methanol, ethanol and propanol. About 50% of initial activity was observed when 40% ethanol and propanol, and 50% methanol were used. It was shown that the modification of C. cladosporioides α-galactosidase by sodium periodate is accompanied by a significant decrease in enzyme activity and stability, probably caused by topological changes in the tertiary and quaternary structure of the protein molecule.

Regulation of secondary metabolite production in the fungal tomato pathogen Cladosporium fulvum.

Cladosporium fulvum is a non-obligate biotrophic fungal tomato pathogen for which fifteen secondary metabolite (SM) gene clusters were previously identified in its genome. However, most of these SM biosynthetic pathways remain cryptic during growth in planta and in different in vitro conditions. The sole SM produced in vitro is the pigment cladofulvin. In this study, we attempted to activate cryptic pathways in order to identify new compounds produced by C. fulvum. For this purpose, we manipulated orthologues of the global regulators VeA, LaeA and HdaA known to regulate SM biosynthesis in other fungal species. In C. fulvum, deleting or over-expressing these regulators yielded no new detectable SMs. Yet, quantification of cladofulvin revealed that CfHdaA is an activator whilst CfVeA and CfLaeA seemed to act as repressors of cladofulvin production. In the wild type strain, cladofulvin biosynthesis was affected by the carbon source, with highest production under carbon limitation and traces only in presence of saccharose. Repression of cladofulvin production by saccharose was dependent on both CfVeA and CfLaeA. Deletion of CfVeA or CfLaeA caused production of sterile mycelia, whilst Δcfhdaa deletion mutants sporulated, suggesting that cladofulvin production is not linked to asexual reproduction. Profiling the transcription of these regulators showed that CfHdaA-mediated regulation of cladofulvin production is independent of both CfVeA and CfLaeA. Our data suggest CfLaeA directly affects cladofulvin production whilst the effect of CfVeA is indirect, suggesting a role for CfLaeA outside of the Velvet complex. In conclusion, our results showed that regulation of SM production in C. fulvum is different from other fungi and indicate that manipulation of global regulators is not a universal tool to discover new fungal natural products.

Biochemistry of lipolytic enzymes secreted by Penicillium solitum and Cladosporium cladosporioides.

Two distinct extracellular lipases were obtained from Penicillium solitum 194A, isolated from domestic compost, and Cladosporium cladosporioides 194B, isolated from dairy wastewater. These alkaline enzymes had molecular masses of 42 and 30 kDa, respectively. The P. solitum 194A lipase differed in mass from previously reported enzyme, indicating that it is a novel lipase, and indicating that penicillia can secrete lipase isoenzymes. The C. cladosporioides lipase was more active on esters of medium-chain acids, whereas the P. solitum lipase was more active on longer chained substrates. The C. cladosporioides enzyme displayed higher thermal stability than the P. solitum lipase, preserving full activity up to 48 °C and showing a T50 (10 min) of 60 °C. Their different catalytic properties and good protein stability should make these enzymes suitable for biotechnological applications. Furthermore, the combined use of these two fungal strains may prove to be valuable in lipid-rich waste management.

Secondary metabolism and biotrophic lifestyle in the tomato pathogen Cladosporium fulvum.

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.

Chemical modification of L-asparaginase from Cladosporium sp. for improved activity and thermal stability.

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (kd), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.

Role of glycosylation in secretion and stability of micromycetes α-galactosidase.

The effect of the glycosylation inhibitors (tunicamycin and 2-deoxy-D-glucose) on the activity, stability and production of fungal glycosidases has been studied. It was shown that inhibition of N-glycosylation sites did not affect the secretion of Aspergillus niger α-galactosidase, however reduced yield of Cladosporium cladosporioides and Penicillium canescens α-galactosidases. Changes in the level of O-glycosylation resulted in a significant reduction in the activity and stability of α-galactosidases of all three producers tested. Activity of the modified enzymes was significantly lower than that of the native ones, and was 2.6 and 0.33 U/mg for A. niger α-galactosidase, 3.3 and 32.5 U/mg for C. cladosporioides α-galactosidase, 11.66 and 31.1 U/mg for P. canescens α-galactosidase, respectively. A. niger α-galactosidase completely lost activity during purification and storage. The decrease of thermal stability at 55 °C by 20% was shown for C. cladosporioides and P. canescens α-galactosidases. It was also noted that O-deglycosylation led to a decrease in resistance of these enzymes to the action of proteases.

Detoxification of α-tomatine by Cladosporium fulvum is required for full virulence on tomato.

· α-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade α-tomatine into the less fungitoxic compounds ß-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine. · Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases. · We show that C. fulvum hydrolyzes α-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆cftom1 mutants of C. fulvum are more sensitive to α-tomatine and are less virulent than the wild-type fungus on tomato. · Although α-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously. Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.

Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp.

L-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 µmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, L-asparaginase has greater affinity towards L-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of L-glutamine. L-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, ß-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca(2+), Co(2+), Cu(2+), Mg(2+), Na(+), K(+) and Zn(2+) significantly affected enzyme activity whereas presence of Fe(3+), Pb(2+) and KI stimulated the activity. Detergents studied also enhanced L-asparaginase activity. In-vitro half-life of purified L-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei.

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including ß-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.