Silencing of Amylomyces rouxii aspartic II protease by siRNA to increase tyrosinase activity.

Amylomyces rouxii is a zygomycete that produces extracellular protease and tyrosinase. The tyrosinase activity is negatively regulated by the proteases and, which attempts to purify the tyrosinase (tyr) enzyme that has been hampered by the presence of a protease that co-purified with it. In this work we identified genes encoding aspartic protease II (aspII) and VI of A.

, a Vintage Model with a Cutting-Edge Profile in Biotechnology.

The discovery of penicillin entailed a decisive breakthrough in medicine. No other medical advance has ever had the same impact in the clinical practise. The fungus (reclassified as ) has been used for industrial production of penicillin ever since the forties of the past century; industrial biotechnology developed hand in hand with it, and currently is a thoroughly studied model for secondary metabolite production and regulation.

High-Efficiency Electroporation for Genetic Improvement of Fungal Strains.

Electroporation is a method for the introduction of molecules (usually nucleic acids) into a cell, consisting of submitting the cells to high-voltage and short electric pulses in the presence of the exogenous DNA/molecule. It is a versatile method, adaptable to different types of cells, from bacteria to cultured cells to higher eukaryotes, and thus has applications in many diverse fields, such as environmental biology, biotechnology, genetic engineering, and medicine. Electroporation has some advantages over other genetic transformation strategies, including the simplicity of the method, a wide range of adjustable parameters (possibility of optimization), high reproducibility and avoidance of the use of chemicals toxic to cells.

Hypoxia up-regulates VEGF ligand and downregulates VEGF soluble receptor mRNA expression in bovine granulosa cells in vitro.

Oxygen concentration (0) in antral ovarian follicles is below that found in most tissues, which is important for adequate granulosa cell function. The VEGF system is linked to angiogenesis and responds to changing 0 by stimulating neovascularization when levels are low. However, in the avascular granulosa cell layer of the follicle, VEGF action is directed to stimulating cell viability and steroidogenesis.

A method for the extraction of high quality fungal RNA suitable for RNA-seq.

Transcriptomic analysis is an OMICs technology that is becoming indispensable to understand and get a complete picture of cell functioning and adaptation to the environmental cues the cell is continuously receiving. Among the techniques available to perform transcriptomics, RNA-seq is becoming the method of choice. The quality of the RNA used for the generation of cDNA libraries and subsequent sequencing is crucial for the success of the process.

Genetic Transformation of the Filamentous Fungus of Antarctic Origin.

Cold-adapted fungi isolated from Antarctica, in particular those belonging to the genus , are producers of secondary metabolites with interesting bioactive properties as well as enzymes with potential biotechnological applications. However, at genetic level, the study of these fungi has been hindered by the lack of suitable genetic tools such as transformation systems. In fungi, the availability of transformation systems is a key to address the functional analysis of genes related with the production of a particular metabolite or enzyme.

NeVOmics: An Enrichment Tool for Gene Ontology and Functional Network Analysis and Visualization of Data from OMICs Technologies.

The increasing number of OMICs studies demands bioinformatic tools that aid in the analysis of large sets of genes or proteins to understand their roles in the cell and establish functional networks and pathways. In the last decade, over-representation or enrichment tools have played a successful role in the functional analysis of large gene/protein lists, which is evidenced by thousands of publications citing these tools. However, in most cases the results of these analyses are long lists of biological terms associated to proteins that are difficult to digest and interpret.

Co-ordinated expression of the VEGF system components in granulosa cells to develop a proangiogenic autocrine milieu during ovarian follicle development.

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected.

Electroporation of germinated conidia and young mycelium as an efficient transformation system for Acremonium chrysogenum.

Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant β-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated.

RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene.

The microaerophilic protozoan is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway.

[Role of G-protein alpha sub-units in the morphogenic processes of filamentous Ascomycota fungi].

The phylum Ascomycota comprises about 75% of all the fungal species described, and includes species of medical, phytosanitary, agricultural, and biotechnological importance. The ability to spread, explore, and colonise new substrates is a feature of critical importance for this group of organisms. In this regard, basic processes such as conidial germination, the extension of hyphae and sporulation, make up the backbone of development in most filamentous fungi.

Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia.

Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect-in a rapid, sensitive, specific, and reproducible way-the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples.

Proteomic analysis of the signaling pathway mediated by the heterotrimeric Gα protein Pga1 of Penicillium chrysogenum.

Background: The heterotrimeric Gα protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways.

Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia.

Filamentous fungi from extreme environments as a promising source of novel bioactive secondary metabolites.

Natural product search is undergoing resurgence upon the discovery of a huge previously unknown potential for secondary metabolite (SM) production hidden in microbial genomes. This is also the case for filamentous fungi, since their genomes contain a high number of "orphan" SM gene clusters. Recent estimates indicate that only 5% of existing fungal species have been described, thus the potential for the discovery of novel metabolites in fungi is huge.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum.

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5.

Reactive oxygen species regulate lovastatin biosynthesis in Aspergillus terreus during submerged and solid-state fermentations.

In a previous work we detected an important increase in reactive oxygen species (ROS) concentrations during idiophase in lovastatin fermentations. Hence, the objective of the present work was to determine if ROS contributes to the regulation of lovastatin biosynthesis. Exogenous antioxidants were used to reduce ROS accumulation.

Effect of a heterotrimeric G protein alpha subunit on conidia germination, stress response, and roquefortine C production in Penicillium roqueforti.

Heterotrimeric G protein signaling regulates many processes in fungi, such as development, pathogenicity, and secondary metabolite biosynthesis. For example, the Galpha subunit Pga1 from Penicillium chrysogenum regulates conidiation and secondary metabolite production in this fungus. The dominant activating allele, pga1G42R, encoding a constitutively active Pga1 Galpha subunit, was introduced in Penicillium roqueforti by transformation, resulting in a phenotype characterized by low sporulation and slow growth.

The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing.

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein.

Heterotrimeric Galpha protein Pga1 of Penicillium chrysogenum controls conidiation mainly by a cAMP-independent mechanism.

Fungal heterotrimeric G proteins regulate different processes related to development, such as colony growth and asexual sporulation, the main mechanism of propagation in filamentous fungi. To gain insight into the mechanisms controlling growth and differentiation in the industrial penicillin producer Penicillioum chrysogenum, we investigated the role of the heterotrimeric Galpha subunit Pga1 in conidiogenesis. A pga1 deleted strain (Deltapga1) and transformants with constitutively activated (pga1G42R) and inactivated (pga1G203R) Pga1 alpha subunits were obtained.

The pga1 gene of Penicillium chrysogenum NRRL 1951 encodes a heterotrimeric G protein alpha subunit that controls growth and development.

The pga1 gene of Penicillium chrysogenum NRRL 1951 has been cloned and shown to participate in the developmental program of this fungus. It encodes a protein showing a high degree of identity to group I alpha subunits of fungal heterotrimeric G proteins, presenting in its sequence all the distinctive characteristics of this group. Northern analysis revealed that pga1 is highly expressed in a constitutive manner in submerged cultures, while its expression changes during development on solid media cultures; it is higher during vegetative growth and decreases significantly at the time of conidiogenesis.

Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeats containing the penicillin gene cluster in Penicillium chrysogenum.

High penicillin-producing strains of Penicillium chrysogenum contain 6-14 copies of the three clustered structural biosynthetic genes, pcbAB, pcbC, and penDE [Barredo, J.L., Díez, B.