Improving the Thermostability of Thermomyces lanuginosus Lipase by Restricting the Flexibility of N-Terminus and C-Terminus Simultaneously via the 25-Loop Substitutions.

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24-30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27…E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.

Studies on the morphology, phylogeny, and bioremediation potential of Penicillium citrinum and Paecilomyces variotii (Eurotiales) from oil-contaminated areas.

Crude oil pollution is one of the most arduous issues to address, as it is hazardous to both public health and the environment. The discovery of novel biosurfactants-producing fungi and bacteria is in high demand due to their excellent properties and wide range of applications. The aim of this research is to isolate a powerful biosurfactant-producing fungus from the crude oil site near Barauni oil refinery in Bihar, India. Standard protocols were used to collect samples from the site. An integrative taxonomic approach was used, which included morphological, molecular, and phylogenetic analysis. The use of plating samples on Bushnell-Hass (BH) media aided in the isolation of a fungal strain from an enrichment culture. Two fungal strains isolated from contaminated soils, Penicillium citrinum and Paecilomyces variotti, showed potent oil degrading activity in a single culture. For preliminary biosurfactants screening, drop collapse assays, oil spreading, and emulsification activity tests were used. The results showed that the cultures performed well in the screening test and were further evaluated for degradation capacity. Different treatment periods (0, 3, 6, 9, 12, and 15 days) were used to observe degradation in single cultures. A steady drop in pH, an alteration in optical density and an increase in carbon dioxide release showed the ability of fungal strain to degrade the crude oil in a single culture. Fungi mycelia provide a larger surface area for absorption and degradation of the pollutants in contaminated environment. They produce extracellular enzymes to degrade the oil, and at the same time absorb and utilise carbon, allowing them to remove toxic substances from the oil. Thus, they could be candidates for bioremediation of a hydrocarbon-contaminated site.

Employing Engineered Enolase Promoter for Efficient Expression of Thermomyces lanuginosus Lipase in Yarrowia lipolytica via a Self-Excisable Vector.

Yarrowia lipolytica is progressively being employed as a workhouse for recombinant protein expression. Here, we expanded the molecular toolbox by engineering the enolase promoter (pENO) and developed a new self-excisable vector, and based on this, a combined strategy was employed to enhance the expression of Thermomyces lanuginosus lipase (TLL) in Y. lipolytica. The strength of 11 truncated enolase promoters of different length was first identified using eGFP as a reporter. Seven of the truncated promoters were selected to examine their ability for driving TLL expression. Then, a series of enolase promoters with higher activities were developed by upstream fusing of different copies of UAS1B, and the recombinant strain Po1f/hp16e100-tll harboring the optimal promoter hp16e100 obtained a TLL activity of 447 U/mL. Additionally, a new self-excisable vector was developed based on a Cre/loxP recombination system, which achieved efficient markerless integration in Y. lipolytica. Subsequently, strains harboring one to four copies of the tll gene were constructed using this tool, with the three-copy strain Po1f/3tll showing the highest activity of 579 U/mL. The activity of Po1f/3tll was then increased to 720 U/mL by optimizing the shaking flask fermentation parameters. Moreover, the folding-related proteins Hac1, Pdi, and Kar2 were employed to further enhance TLL expression, and the TLL activity of the optimal recombinant strain Po1f/3tll-hac1-pdi-kar2 reached 1197 U/mL. By using this combined strategy, TLL activity was enhanced by approximately 39.9-fold compared to the initial strain. Thus, the new vector and the combined strategy could be a useful tool to engineer Y. lipolytica for high-level expression of heterologous protein.

Stepwise gating of the Sec61 protein-conducting channel by Sec63 and Sec62.

Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins, Sec62 and Sec63, but their functions are poorly defined. In the present study, we determined cryo-electron microscopy (cryo-EM) structures of several variants of Sec61-Sec62-Sec63 complexes from Saccharomyces cerevisiae and Thermomyces lanuginosus and show that Sec62 and Sec63 induce opening of the Sec61 channel. Without Sec62, the translocation pore of Sec61 remains closed by the plug domain, rendering the channel inactive. We further show that the lateral gate of Sec61 must first be partially opened by interactions between Sec61 and Sec63 in cytosolic and luminal domains, a simultaneous disruption of which completely closes the channel. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our study shows how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.

Impact of the disulfide bond on hydrolytic characteristics of a xylanase from Talaromyces thermophiles F1208.

A xylanase from Talaromyces thermophiles F1208 (T-Xyn) was used specifically to explore the effects of disulfide bond on hydrolytic activity. The T-Xyn-C122S-C166S mutant does not have the C122-C166 disulfide bond present in wild-type T-Xyn, whereas T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S mutants have an introduced disulfide bond, C38-C50, to T-Xyn and T-Xyn-C122S-C166S, respectively. The optimum pH of T-Xyn-T38C-S50C and T-Xyn-T38C-S50C-C122S-C166S was lower than that of T-Xyn and T-Xyn-C122S-C166S. The introduction of a disulfide bond caused a decrease in the optimum temperature and thermal stability of T-Xyn. The existence of a disulfide bond has a strong influence on the hydrolysis characteristics of T-Xyn, which caused changes in the composition and proportion of the hydrolysate products. T-Xyn-T38C-S50C produces the highest level of xylose when using beechwood xylan as the substrate, whereas T-Xyn produces the highest level of xylobiose and T-Xyn-T38C-S50C-C122S-C166S produces the largest amount of xylotriose. When birchwood xylan was used as the substrate, the introduction of a disulfide bond increased the content of xylose, decreased the content of xylotriose and a high degree of polymerization (DP ≥ 5) was observed. The hydrolysis of oat-spelt xylan is more complex with the introduction of the disulfide bond causing an increase in the degradation rate of xylotriose.

Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation.

Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents.IMPORTANCEThermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

Optimization of process parameters for improved chitinase activity from Thermomyces sp. by using artificial neural network and genetic algorithm.

Chitinase is responsible for the breaking down of chitin to N-acetyl-glucosamine units linked through (1-4)-glycosidic bond. The chitinases find several applications in waste management and pest control. The high yield with characteristics thermal stability of chitinase is the key to their industrial application. Therefore, the present work focuses on parameter optimization for chitinase production using fungus Thermomyces lanuginosus MTCC 9331. Three different optimization approaches, namely, response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) were used. The parameters under study were incubation time, pH and inoculum size. The central composite design with RSM was used for the optimization of the process parameters. Further, results were validated with GA and ANN. A multilayer feed-forward algorithm was performed for ANN, i.e., Levenberg-Marquardt, Bayesian Regularization, and Scaled Conjugate Gradient. The ANN predicted values gave higher chitinase activity, i.e., 102.24 U/L as compared to RSM-predicted values, i.e., 88.38 U/L. The predicted chitinase activity was also closer to the observed data at these levels. The validation study suggested that the highest activity of chitinase as predicted by ANN is in line with experimental analysis. The comparison of three different statistical approaches suggested that ANN gives better optimization results compared to the GA and RSM study.

Coimmobilization of different lipases: Simple layer by layer enzyme spatial ordering.

This paper shows the step by step coimmobilization of up to five different enzymes following two different orders in the coimmobilization to alter the effect of substrate diffusion limitations. The enzymes were the lipases A and B from Candida antarctica, the lipases from Rhizomocur miehei and, Themomyces lanuginosus and the phospholipase Lecitase Ultra. The utilized strategy was a layer by layer immobilization, coating the immobilized enzymes with polyethylenimine followed by the crosslinking of the enzyme and PEI with glutaraldehyde to prevent enzyme release, and them adding a new lipase layer. The use of previously inactivated biocatalysts (using diethyl p-nitrophenylphosphate) permitted to visualize the immobilization of each enzyme layer, which was later confirmed by SDS-PAGE. This also confirmed the successful and complete covalent crosslinking of the glutaraldehyde treated enzyme layers. Activity of the combibiocatalysts was followed using diverse substrates. The protocol was successful and permitted to immobilize in an ordered way the 5 different enzymes in a down-up distribution.

High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis.

Leucine aminopeptidase (LAP), an exopeptidase that releases amino acid residues, especially leucine, from the N-terminus of polypeptides, is often applied to debitter protein hydrolysate in the food industry. However, there are no thermostable and high activity enzymes that can be used in the food industry. In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum activity over a wide temperature range (30-80 °C). With regard to temperature stability, Thelap retained more than 90% activity when it was incubated at 65-75 °C for 2 h. K+ and Co2+ increased the enzyme activity of the recombinant Thelap, while Ba2+, Mn2+, Ni2+, Ca2+, Mg2+ and SDS inhibited its enzyme activity, and the inhibition capacity of Mg2+ was the weakest. Upon application in soy protein hydrolysis, Thelap could significantly increase the degree of hydrolysis and remove more hydrophobic amino acids from the N-terminal region of the polypeptide to decrease the bitterness.

Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences.

Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining.IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

Integrated Functional-Omics Analysis of Thermomyces lanuginosus Reveals its Potential for Simultaneous Production of Xylanase and Substituted Xylooligosaccharides.

Thermophiles have several beneficial properties for the conversion of biomass at high temperatures. Thermomyces lanuginosus is a thermophilic filamentous fungus that was shown to secrete 40 glycoside hydrolases and 25 proteases when grown on different carbon sources. Among the 13 identified glycoside hydrolases with high expression levels, 9 were reduced sugar glycosidases (RSGs) belonging to seven GH families, and 7 of the 10 identified proteases were exopeptidases belonging to six different protease families. High expression of RSGs and exopeptidases may allow the fungus to efficiently degrade oligosaccharides and oligopeptides in saprophytic habitats. There were no xylan side chain-degrading enzymes predicted in the genome of T. lanuginosus, and only one thermophilic GH11 xylanase (g4601.t1) and one GH43 xylosidase (g3706.t1) were detected by liquid chromatography-mass spectrometry/mass spectrometry when T. lanuginosus grown on xylan, which led to the accumulation of substituted xylooligosaccharides (SXOS) during corncob xylan degradation where SXOS output made up more than 8% of the total xylan. The SXOS are beneficial prebiotics and important inducers for enzymes secretion of microorganisms. Thus, T. lanuginosus exhibits distinct advantages in utilizing cheap raw materials producing one thermostable xylanase and the high value-added SXOS as well as microbial inoculants to compost by batch fermentation.

Microbial synthesis of mammalian metabolites of spironolactone by thermophilic fungus Thermomyces lanuginosus.

Mesophilic fungi are well recognized as models of mammalian drug metabolism. Thermophilic fungi remained unexplored despite having a unique mechanism of growing at higher temperatures and performing wide diverse reactions. The present investigation is directed to isolate a promising thermophilic fungal strain capable of biotransformation using spironolactone as a model drug. Two-stage fermentation protocol was followed for the process. The transformation of spironolactone was identified by HPLC and structure elucidation of the metabolites was done with the help of LC-MS/MS analysis and previous reports. A strain of Thermomyces lanuginosus isolated from decomposed banana peel waste was found to be most promising in transforming spironolactone to 4 metabolites viz.7α-thiospironolactone (M1) canrenone (M2), 7α-thiomethylspironolactone (M3) and 6ß-OH-7α-thiomethylspironolactone (M4), the major mammalian metabolites reported previously. The synthesis of metabolites of spironolactone by T. lanuginosus similar to mammals clearly states that this fungus possess enzyme system similar to mammals. Hence, this fungus has the potential to use as a model organism for studying drug metabolism.

Glycerol enhances fungal germination at the water-activity limit for life.

For the most-extreme fungal xerophiles, metabolic activity and cell division typically halts between 0.700 and 0.640 water activity (approximately 70.0-64.0% relative humidity). Here, we investigate whether glycerol can enhance xerophile germination under acute water-activity regimes, using an experimental system which represents the biophysical limit of Earth's biosphere. Spores from a variety of species, including Aspergillus penicillioides, Eurotium halophilicum, Xerochrysium xerophilum (formerly Chrysosporium xerophilum) and Xeromyces bisporus, were produced by cultures growing on media supplemented with glycerol (and contained up to 189 mg glycerol g dry spores-1 ). The ability of these spores to germinate, and the kinetics of germination, were then determined on a range of media designed to recreate stresses experienced in microbial habitats or anthropogenic systems (with water-activities from 0.765 to 0.575). For A. penicillioides, Eurotium amstelodami, E. halophilicum, X. xerophilum and X. bisporus, germination occurred at lower water-activities than previously recorded (0.640, 0.685, 0.651, 0.664 and 0.637 respectively). In addition, the kinetics of germination at low water-activities were substantially faster than those reported previously. Extrapolations indicated theoretical water-activity minima below these values; as low as 0.570 for A. penicillioides and X. bisporus. Glycerol is present at high concentrations (up to molar levels) in many types of microbial habitat. We discuss the likely role of glycerol in expanding the water-activity limit for microbial cell function in relation to temporal constraints and location of the microbial cell or habitat. The findings reported here have also critical implications for understanding the extremes of Earth's biosphere; for understanding the potency of disease-causing microorganisms; and in biotechnologies that operate at the limits of microbial function.

Structural and functional studies of the glycoside hydrolase family 3 ß-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. ß-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the ß-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the ß-glucosidase from H. jecorina (HjCel3A) with the ß-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 ß-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Diversification of ergot alkaloids in natural and modified fungi.

Several fungi in two different families--the Clavicipitaceae and the Trichocomaceae--produce different profiles of ergot alkaloids, many of which are important in agriculture and medicine. All ergot alkaloid producers share early steps before their pathways diverge to produce different end products. EasA, an oxidoreductase of the old yellow enzyme class, has alternate activities in different fungi resulting in branching of the pathway. Enzymes beyond the branch point differ among lineages. In the Clavicipitaceae, diversity is generated by the presence or absence and activities of lysergyl peptide synthetases, which interact to make lysergic acid amides and ergopeptines. The range of ergopeptines in a fungus may be controlled by the presence of multiple peptide synthetases as well as by the specificity of individual peptide synthetase domains. In the Trichocomaceae, diversity is generated by the presence or absence of the prenyl transferase encoded by easL (also called fgaPT1). Moreover, relaxed specificity of EasL appears to contribute to ergot alkaloid diversification. The profile of ergot alkaloids observed within a fungus also is affected by a delayed flux of intermediates through the pathway, which results in an accumulation of intermediates or early pathway byproducts to concentrations comparable to that of the pathway end product.

Purine utilization proteins in the Eurotiales: cellular compartmentalization, phylogenetic conservation and divergence.

The purine utilization pathway has been thoroughly characterized in Aspergillus nidulans. We establish here the subcellular distribution of seven key intracellular enzymes, xanthine dehydrogenase (HxA), urate oxidase (UaZ), 5-hydroxy-isourate hydrolase (UaX), 2-oxo-4-hydroxy-4-carboxy ureido imidazoline decarboxylase (UaW), allantoinase (AlX), allantoicase (AaX), ureidoglycolate lyase (UglA), and the fungal-specific α-ketoglutarate Fe(II)-dependent dioxygenase (XanA). HxA, AlX, AaX, UaW and XanA are cytosolic, while UaZ, UaX and UglA are peroxisomal. Peroxisomal localization was confirmed by using appropriate pex mutants. The pathway is largely, but not completely conserved in the Eurotiomycetes, noticeably in some species AaX is substituted by an alternative enzyme of probable bacterial origin. UaZ and the urate-xanthine UapA and UapC transporters, are also localized in specific cells of the conidiophore. We show that metabolic accumulation of uric acid occurring in uaZ null mutations is associated with an increased frequency of appearance of morphologically distinct colony sectors, diminished conidiospore production, UV resistance and an altered response to oxidation stress, which may provide a rationale for the conidiophore-specific localization. The pathway-specific transcription factor UaY is localized in both the cytoplasm and nuclei under non-inducing conditions, but it rapidly accumulates exclusively to the nuclei upon induction by uric acid.

Phylogeny and intraspecific variation of the extreme xerophile, Xeromyces bisporus.

The filamentous ascomycete Xeromyces bisporus is an extreme xerophile able to grow down to a water activity of 0.62. We have inferred the phylogenetic position of Xeromyces in relation to other xerophilic and xerotolerant fungi in the order Eurotiales. Using nrDNA and betatubulin sequences, we show that it is more closely related to the xerophilic foodborne species of the genus Chrysosporium, than to the genus Monascus. The taxonomy of X. bisporus and Monascus is discussed. Based on physiological, morphological, and phylogenetic distinctiveness, we suggest that Xeromyces should be retained as a separate genus.

Phosphate solubilization potential and stress tolerance of Eupenicillium parvum from tea soil.

Eupenicillium parvum was recorded for first time during isolation of phosphate-solubilizing microorganisms from the tea rhizosphere. The fungus developed a phosphate solubilization zone on modified Pikovskaya agar, supplemented with tricalcium phosphate. Quantitative estimation of phosphate solubilization in Pikovskaya broth showed high solubilization of tricalcium phosphate and aluminium phosphate. The fungus also solubilized North Carolina rock phosphate and Mussoorie rock phosphate, and exhibited high levels of tolerance against desiccation, acidity, salinity, aluminium, and iron. Solubilization of inorganic phosphates by the fungus was also observed under high stress levels of aluminium, iron, and desiccation, though the significant decline in phosphate solubilization was marked in the presence of aluminium than iron. The fungal isolate showed 100% identity with E. parvum strain NRRL 2095 ITS 1, 5.8S rRNA gene and ITS 2, complete sequence; and 28S rRNA gene, partial sequence.

The inability of Byssochlamys fulva to produce patulin is related to absence of 6-methylsalicylic acid synthase and isoepoxydon dehydrogenase genes.

Byssochlamys species are responsible for spoilage and degradation of fruits and silages. Under specific conditions they are able to produce mycotoxins. The aim of this study was to evaluate the potential of 19 different strains of Byssochlamys nivea and Byssochlamys fulva to produce patulin in relation with the presence of two genes involved in the patulin biosynthesis pathways in the genome of these fungal strains. The strains were characterized by macroscopic, microscopic examinations, internal transcribed spacer (ITS) rRNA and beta-tubulin fragment amplification and sequencing. All of the 8 B. nivea strains tested produced patulin. By contrast, none of the 11 strains of B. fulva produce this toxin. Two genes of the patulin biosynthetic pathway, a polyketide synthase (pks) and the isoepoxydon dehydrogenase (idh) were cloned from B. nivea. The deduced amino acid sequence of the polyketide synthase was 74% identical to the 6-methylsalicylic acid synthase gene of Penicillium griseofulvum and had the five functional domains characteristic of fungal type I polyketide synthases (beta-ketosynthase, acyltransferase, dehydratase, beta-ketoreductase and acyl carrier protein). The complete coding sequence of idh gene displayed after translation 88% of identity with P. griseofulvum IDH and 85% with P. expansum IDH, respectively. Both pks and idh messengers were strongly co-expressed during the production of 6-methylsalicylic acid and patulin. The presence of these genes was then investigated in the genome of B. nivea and B. fulva strains by PCR. All B. nivea strains possess the two genes, by contrast none of the B. fulva strains display these genes. The absence of 6-methylsalicylic acid and isoepoxydon dehydrogenase genes can explain the inability of B. fulva to produce patulin. In conclusion, B. fulva don't seem to be responsible for the occurrence of patulin by lack of genes.