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.

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.

A low-temperature polygalacturonase from P. occitanis: characterization and application in juice clarification.

An extracellular endo-polygalacturonase (PGase) was purified, after a single purification step, from the constitutive and hyperpectinolytic CT1 mutant of Penicillium occitanis. This enzyme named PG2 has a molecular weight of 42kDa. It was optimally active at 35°C and pH6 with more than 85% of activity at pH7 in contrast to the majority of fungal PGase, generally acting at 50°C and pH5. The specific activity obtained was among the highest ones, 31397.26U/mg. The PGase activity increased with the decrease of the degree of methylation (DM) of pectin, but it was also able to degrade the highly methyl-esterified substrates, 70% (DM) and 90% (DM), with almost 80% and 40% of residual activity respectively. Interestingly, PG2 is completely inhibited by DEPC, suggesting the implication of a Histidine residue in the active site. The sequencing of P. occitanis whole genome allowed us to identify the pga2 gene encoding PG2 and to localize the His residue, target of DEPC, while it was absent in the PG1 that resisted to DEPC. Besides that, the potentialities of PG2 have been put in use in juice clarification of pear, banana and citrus juice.

Single cell oil production from a newly isolated Candida viswanathii Y-E4 and agro-industrial by-products valorization.

Microbial lipids have drawn increasing attention in recent years as promising raw materials for biodiesel and added-value compounds production. To this end, new oleaginous yeast, Candida viswanathii Y-E4 was isolated, characterized and used for single cell oil (SCO) production. Physiologic and nutritional parameters optimization was carried out for improved biomass and lipid production. Y-E4 strain was able to use a wide range of substrates, especially C5 and C6 sugars as well as glycerol and hydrophobic substrates. The fatty acid profile analysis showed that oleic acid was the main component produced using different substrates. Batch and fed-bath fermentation were conducted using glucose as carbon source. Lipid production rate is twice higher in fed-batch culture providing a lipid content of 50 % (w/w). To minimize the SCO production cost, C. viswanathii Y-E4 was evaluated for its capacity to use different agro-industrial by-products for microbial oil production and changes in the fatty acid profile were monitored.

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.

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.

Constitutive over-expression of pectinases in Penicillium occitanis CT1 mutant is transcriptionally regulated.

The CT1 mutant of Penicillium occitanis hyperproduces extracellular pectinases constitutively since it secretes pectinases even on glucose-containing medium. We show here that all other hydrolytic enzymes remain at low activities in CT1, confirming the specificity of the regulatory mutation towards pectinases. We isolated, by RT-PCR and through the construction of a cDNA library, three fragments coding for: a pectin lyase (pnl1), a polygalacturonase (pga1) and a pectate lyase (pal1). These fragments were used as probes in Northern blots analysis of the wild type strain CL100 and the CT1 mutant of P. occitanis grown in three culture conditions. The CT1 mutant showed a very high amount of pnl1, pga1 and pal1 mRNA either in pectin, glucose or glycerol grown cells while in the wild type CL100 strain, all transcripts were undetectable even on pectin. These results suggest that the CT1 mutation affects a trans-regulatory transcriptional factor regulating pectinase expression.

Genomic organization of a polygalacturonase gene from a hyperpectinolytic mutant strain of Penicillium occitanis.

The pga1 gene encoding an endopolygalacturonase was isolated from a hyperpectinolytic mutant strain of Penicillium occitanis. It consists of an ORF of 1.155 kbp encoding a putative protein of 346 amino acids with a predicted molecular mass of 39 kDa, belonging to the family 28 of glycosyl hydrolases. The deduced amino acid sequence comprises a putative 38 N terminal amino acids of the prepropeptide. The nature and position of amino acids comprising the active site as well as the overall three-dimensional structure were well conserved between the P. occitanis pga1 and all polygalacturonases. The coding region of the pga1 gene is interrupted by three short introns of 57, 53 and 65 bp in length. In addition to the determination of the transcription start site, the promoter sequence from the pga1 gene was analysed. It showed the conservation of known response elements for CreA and Hap2-3-4 factors. Southern blot analysis at high stringency shows that the isolated polygalacturonase gene exists as a single copy in the fungus genome. Northern blot analysis confirmed the constitutive hyperpectinolytic nature of the hyperpectinolytic CT1 mutation as high levels of pga1 mRNA were observed either on pectin or on glucose-grown cells.

Cloning, genomic organisation and mRNA expression of a pectin lyase gene from a mutant strain of Penicillium occitanis.

The regulatory cis elements of fungal pectinases are well studied in Aspergillus genera but little is known in other fungal species. A genomic bank from Penicillium occitanis fungus is constructed and screened by previously isolated cDNA probe of a pectin lyase. From several isolated clones, the nucleotide sequence of the pectin lyase gene was completed and led to the identification of introns and promoter-terminator regions. A streaking future was found in pnl gene of P. occitanis: it exhibits the highest nucleotide homology with the pnlA of Aspergillus niger but the positions of its 4 introns is completely identical to that of A. niger pnlB gene. In addition to the determination of transcription start site, the promoter sequence from the pnl gene was analysed. It showed the conservation of known consensus sequences -CreA, Hap2-3-4, PacC ...-, and the existence of a particular sequence -CCTGA- which is similar to that already found to be specific of pectinolytic gene in Aspergillus, CCCTGA. This result suggests that the corresponding regulatory trans-acting factor should be the same as in Aspergillus.