Molecular characterization and modeling study of the Podr1 gene and genome-scale identification of whole ATP-binding cassette (ABC) transporters in Penicillium occitanis.

As a preliminary step to characterize genes encoding ATP-Binding-Cassette (ABC) proteins, we cloned a gene encoding an ABC transporter from P. occitanis using a PCR based approach followed by a genomic library screening and by additionally using whole genome sequencing results. The encoded protein has high similarity to the pleiotropic drug resistance protein subfamily members. Analysis of the cloned sequence revealed the presence of Walker A, Walker B and the ABC signature motifs at the nucleotide binding domains. Molecular docking resulted in predicting the most stable complex between the gene-encoding protein and cycloheximide. The southern blot results indicate that the gene is present as a single copy in the P. occitanis genome. The genome-scale identification of the PoABC superfamily members led to the characterization of 58 putative proteins divided into five subfamilies including: 12 ABCB, 24 ABCC, 1 ABCE, 5 ABCF, 15 ABCG, and of which 51 contain trans-membrane domains.

Large-scale analysis of the genome of the rare alkaline-halophilic Stachybotrys microspora reveals 46 cellulase genes.

Fungi are of great importance in biotechnology, for example in the production of enzymes and metabolites. The main goal of this study was to obtain a high-coverage draft of the Stachybotrys microspora genome and to annotate and analyze the genome sequence data. The rare fungus S. microspora N1 strain is distinguished by its ability to grow in an alkaline halophilic environment and to efficiently secrete cellulolytic enzymes. Here we report the draft genome sequence composed of 3715 contigs, a genome size of 35 343 854 bp, with a GC content of 53.31% and a coverage around 20.5×. The identification of cellulolytic genes and of their corresponding functions was carried out through analysis and annotation of the whole genome sequence. Forty-six cellulases were identified using the fungicompanion bioinformatic tool. Interestingly, an S. microspora endoglucanase selected from those with a low isoelectric point was predicted to have a halophilic profile and share significant homology with a well-known bacterial halophilic cellulase. These results confirm previous biochemical studies revealing a halophilic character, which is a very rare feature among fungal cellulases. All these properties suggest that cellulases of S. microspora may have potential for use in the biofuel, textile, and detergent industries.

Biocatalysts: beautiful creatures.

The chemical industry has come under increasing pressure to make chemical production more eco-friendly and independent to fossil resources. The development of industrial processes based on micro-organisms can especially help to eliminate the use or the generation of hazardous substances and can support the transition from dependence on fossil resources towards real sustainable and eco-safety industrial processes. The biocatalysts are the best solution given by nature that can be used to improve some biotechnological applications. In this research review, we report some peculiar properties of biocatalysts, implicated in a range of metabolic pathways and biotechnological tools.

Molecular and bioinformatics analyses reveal two differentially expressed intracellular GH1 ß-glucosidases from the rare alkalophilic fungus Stachybotrys microspora.

The present study reports the isolation and analysis of two novel GH1 ß-glucosidases from the alkalophilic fungus Stachybotrys microspora, using PCR and Nested-PCR. Three major gene fragments were obtained by PCR: the first two are very similar and constitute a novel gene, which was named Smbgl1A, and the third PCR fragment is part of a different gene, named Smbgl1B. The truncated gene sequences were completely filled using the recent partial whole genome sequencing data of S. microspora (data not yet published). Moreover, we investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects. In fact, RT-PCR analysis showed that while Smbgl1A was expressed when the fungus was grown in the presence of cellulose but not when grown with glucose, Smbgl1B was equally expressed under both conditions. The putative catalytic residues and the conserved glycone binding sites were identified. Zymogram analysis showed the intracellular production of ß-glucosidases in S. microspora. The predicted secondary structure exhibited a classical (ß/α)8 barrel fold, showing that both SmBGL1A and SmBGL1B belong to the GH1 family. Phylogenetic studies showed that SmBGL1A and SmBGL1B belong to the same branch as ß-glucosidases from Stachybotrys chlorohalonata and Stachybotrys chartarum. However, SmBGL1A and SmBGL1B form two distinct clades.

Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.

Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form.

Cloning and Genomic Organization of a Rhamnogalacturonase Gene from Locally Isolated Strain of Aspergillus niger.

The rhg gene encoding a rhamnogalacturonase was isolated from the novel strain A1 of Aspergillus niger. It consists of an ORF of 1.505 kb encoding a putative protein of 446 amino acids with a predicted molecular mass of 47 kDa, belonging to the family 28 of glycosyl hydrolases. The nature and position of amino acids comprising the active site as well as the three-dimensional structure were well conserved between the A. niger CTM10548 and fungal rhamnogalacturonases. The coding region of the rhg gene is interrupted by three short introns of 56 (introns 1 and 3) and 52 (intron 2) bp in length. The comparison of the peptide sequence with A. niger rhg sequences revealed that the A1 rhg should be an endo-rhamnogalacturonases, more homologous to rhg A than rhg B A. niger known enzymes. The comparison of rhg nucleotide sequence from A. niger A1 with rhg A from A. niger shows several base changes. Most of these changes (59 %) are located at the third base of codons suggesting maintaining the same enzyme function. We used the rhamnogalacturonase A from Aspergillus aculeatus as a template to build a structural model of rhg A1 that adopted a right-handed parallel ß-helix.

Medium initial pH and carbon source stimulate differential alkaline cellulase time course production in Stachybotrys microspora.

The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and ß-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong ß-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the ß-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and ß-glucosidases.

Improvement of cellulase and xylanase production by solid-state fermentation of Stachybotrys microspora.

The current study investigated the production of cellulases and xylanases from the rare fungus Stachybotrys microspora under solid-state fermentation (SSF) on wheat bran (WB). A comparison of both activities was first performed in submerged cultures using various concentrations of WB, glucose, and cellulose as substrates. The maximal activity of ß-glucosidases and xylanases was obtained with 2% and 4% WB, respectively, whereas cellulose yielded the highest endoglucanase production. The SSF conditions were therefore consequently optimized. A moisture content of 70% gave the most significant levels of enzyme production. Inoculation by spores led to better results than by preculture, with 10(5) spores per gram of dried matter as the best inoculum dose for all activities tested. Interestingly, the WB-based medium need not to be supplemented by an exogeneous nitrogen source. Considering the richness of S. microspora secreted proteins as lytic hydrolases, the crude extracellular enzyme extracts were successfully tested in two different biotechnological fields: protoplasting of fungi and subsequent extraction of their DNA, paper pulp hydrolysis to produce fermentable sugars.

Investigations on hydrolytic activities from Stachybotrys microspora and their use as an alternative in yeast DNA extraction.

Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple hydrolytic activities (cellulolytic and non-cellulolytic enzymes). The production of these biocatalysts was studied under submerged culture using glucose, cellulose, and wheat bran as carbon sources. Endoglucanases, pectinases, xylanases, ß-glucanases, chitinases, and proteases were induced on cellulose-based medium and repressed on glucose in both strains with higher amounts produced by the mutant. ß-glucosidases were roughly equally produced by both strains under glucose and cellulose conditions. The yield of chitinases, ß-glucanases, and proteases produced by Stachybotrys strains was as much higher than the commercialized lysing enzyme called "zymolyase," currently used in yeast DNA extraction. In this context, we showed that S. microspora hydrolases can be successfully applied in the extraction of yeast DNA.

Cloning, molecular characterization, and mRNA expression of the thermostable family 3 ß-glucosidase from the rare fungus Stachybotrys microspora.

The filamentous fungus Stachybotrys microspora possess a rich ß-glucosidase system composed of five ß-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the ß-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 ß-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of ß-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/ß)8 triose phosphate isomerase (TIM) barrel, (α/ß)5 sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel ß-glucosidase.

Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli.

The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with "eight right-handed parallel ß-helix" architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 °C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole.