Potential of Near-Infrared Spectroscopy for the Determination of Olive Oil Quality.

The analysis of the physico-chemical parameters of quality of olive oil is still carried out in laboratories using chemicals and generating waste, which is relatively costly and time-consuming. Among the various alternatives for the online or on-site measurement of these parameters, the available literature highlights the use of near-infrared spectroscopy (NIRS). This article intends to comprehensively review the state-of-the-art research and the actual potential of NIRS for the analysis of olive oil. A description of the features of the infrared spectrum of olive oil and a quick explanation of the fundamentals of NIRS and chemometrics are also included. From the results available in the literature, it can be concluded that the four most usual physico-chemical parameters that define the quality of olive oils, namely free acidity, peroxide value, K232, and K270, can be measured by NIRS with high precision. In addition, NIRS is suitable for the nutritional labeling of olive oil because of its great performance in predicting the contents in total fat, total saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids in olive oils. Other parameters of interest have the potential to be analyzed by NIRS, but the improvement of the mathematical models for their determination is required, since the errors of prediction reported so far are a bit high for practical application.

Molecular Mechanisms of Phosphate Sensing, Transport and Signalling in Streptomyces and Related Actinobacteria.

: Phosphorous, in the form of phosphate, is a key element in the nutrition of all living beings. In nature, it is present in the form of phosphate salts, organophosphates, and phosphonates. Bacteria transport inorganic phosphate by the high affinity phosphate transport system PstSCAB, and the low affinity PitH transporters. The PstSCAB system consists of four components. PstS is the phosphate binding protein and discriminates between arsenate and phosphate. In the Streptomyces species, the PstS protein, attached to the outer side of the cell membrane, is glycosylated and released as a soluble protein that lacks its phosphate binding ability. Transport of phosphate by the PstSCAB system is drastically regulated by the inorganic phosphate concentration and mediated by binding of phosphorylated PhoP to the promoter of the PstSCAB operon. In Mycobacterium smegmatis, an additional high affinity transport system, PhnCDE, is also under PhoP regulation. Additionally, Streptomyces have a duplicated low affinity phosphate transport system encoded by the pitH1-pitH2 genes. In this system phosphate is transported as a metal-phosphate complex in simport with protons. Expression of pitH2, but not that of pitH1 in Streptomyces coelicolor, is regulated by PhoP. Interestingly, in many Streptomyces species, three gene clusters pitH1-pstSCAB-ppk (for a polyphosphate kinase), are linked in a supercluster formed by nine genes related to phosphate metabolism. Glycerol-3-phosphate may be transported by the actinobacteria Corynebacterium glutamicum that contains a ugp gene cluster for glycerol-3-P uptake, but the ugp cluster is not present in Streptomyces genomes. Sugar phosphates and nucleotides are used as phosphate source by the Streptomyces species, but there is no evidence of the uhp gene involved in the transport of sugar phosphates. Sugar phosphates and nucleotides are dephosphorylated by extracellular phosphatases and nucleotidases. An isolated uhpT gene for a hexose phosphate antiporter is present in several pathogenic corynebacteria, such as Corynebacterium diphtheriae, but not in non-pathogenic ones. Phosphonates are molecules that contains phosphate linked covalently to a carbon atom through a very stable C-P bond. Their utilization requires the phnCDE genes for phosphonates/phosphate transport and genes for degradation, including those for the subunits of the C-P lyase. Strains of the Arthrobacter and Streptomyces genera were reported to degrade simple phosphonates, but bioinformatic analysis reveals that whole sets of genes for putative phosphonate degradation are present only in three Arthrobacter species and a few Streptomyces species. Genes encoding the C-P lyase subunits occur in several Streptomyces species associated with plant roots or with mangroves, but not in the laboratory model Streptomyces species; however, the phnCDE genes that encode phosphonates/phosphate transport systems are frequent in Streptomyces species, suggesting that these genes, in the absence of C-P lyase genes, might be used as surrogate phosphate transporters. In summary, Streptomyces and related actinobacteria seem to be less versatile in phosphate transport systems than Enterobacteria.

Thermal Analysis of a Magnetic Brake Using Infrared Techniques and 3D Cell Method with a New Convective Constitutive Matrix.

In this work we analyse the temperature distribution in a conductor disk in transitory regime. The disk is in motion in a stationary magnetic field generated by a permanent magnet and so, the electric currents induced inside it generate heat. The system acts as a magnetic brake and is analysed using infrared sensor techniques. In addition, for the simulation and analysis of the magnetic brake, a new thermal convective matrix for the 3D Cell Method (CM) is proposed. The results of the simulation have been verified by comparing the numerical results with those obtained by the Finite Element Method (FEM) and with experimental data obtained by infrared technology. The difference between the experimental results obtained by infrared sensors and those obtained in the simulations is less than 0.0459%.

Phytoremediation of highly contaminated mining soils by Jatropha curcas L. and production of catalytic carbons from the generated biomass.

This paper deals with the removal of heavy metals from marginal soil mixtures from the Cobre Las Cruces and Aznalcóllar mining areas containing high concentrations of metals (Cr, Fe, Ni, Cu, Zn, Cd, Hg, Pb and As) by means of phytoremediation using Jatropha curcas L., and the subsequent production of biocatalysts from the plant biomass. First, J. curcas L. was sowed in eight mixtures of these mining soils to study its adaption to these high-contaminated soils and its growth during 60 days in a greenhouse under conditions simulating the South of Spain's spring climate. Later, the most suitable soil mixtures for plant growth were used for 120-day phytoremediation under the same conditions. Heavy metal concentration in soils, roots, stems and leaves were measured by ICP-OES at the beginning, at the middle and at the end of the phytoremediation period, thus calculating the translocation and bioaccumulation factors. J. curcas L. was found to absorb great amounts of Fe (>3000 mg kg-1 plant) as well as notable amounts of Pb, Zn, Cu, Cr and Ni, and traces of As. Other metals with lower initial concentrations such as Cd, Hg and Sn were completely removed from soils. Finally, the plant biomass was subjected to pyrolysis to obtain catalytic biocarbons, assessing the optimal temperature for the pyrolytic process by means of thermogravimetric analysis and Raman spectroscopy.

Biosynthetic gene clusters for relevant secondary metabolites produced by Penicillium roqueforti in blue cheeses.

Ripening of blue-veined cheeses, such as the French Bleu and Roquefort, the Italian Gorgonzola, the English Stilton, the Danish Danablu or the Spanish Cabrales, Picón Bejes-Tresviso, and Valdeón, requires the growth and enzymatic activity of the mold Penicillium roqueforti, which is responsible for the characteristic texture, blue-green spots, and aroma of these types of cheeses. This filamentous fungus is able to synthesize different secondary metabolites, including andrastins, mycophenolic acid, and several mycotoxins, such as roquefortines C and D, PR-toxin and eremofortins, isofumigaclavines A and B, and festuclavine. This review provides a detailed description of the main secondary metabolites produced by P. roqueforti in blue cheese, giving a special emphasis to roquefortine, PR-toxin and mycophenolic acid, and their biosynthetic gene clusters and pathways. The knowledge of these clusters and secondary metabolism pathways, together with the ability of P. roqueforti to produce beneficial secondary metabolites, is of interest for commercial purposes.

Molecular characterization of the PR-toxin gene cluster in Penicillium roqueforti and Penicillium chrysogenum: cross talk of secondary metabolite pathways.

The PR-toxin is a potent mycotoxin produced by Penicillium roqueforti in moulded grains and grass silages and may contaminate blue-veined cheese. The PR-toxin derives from the 15 carbon atoms sesquiterpene aristolochene formed by the aristolochene synthase (encoded by ari1). We have cloned and sequenced a four gene cluster that includes the ari1 gene from P. roqueforti. Gene silencing of each of the four genes (named prx1 to prx4) resulted in a reduction of 65-75% in the production of PR-toxin indicating that the four genes encode enzymes involved in PR-toxin biosynthesis. Interestingly the four silenced mutants overproduce large amounts of mycophenolic acid, an antitumor compound formed by an unrelated pathway suggesting a cross-talk of PR-toxin and mycophenolic acid production. An eleven gene cluster that includes the above mentioned four prx genes and a 14-TMS drug/H(+) antiporter was found in the genome of Penicillium chrysogenum. This eleven gene cluster has been reported to be very poorly expressed in a transcriptomic study of P. chrysogenum genes under conditions of penicillin production (strongly aerated cultures). We found that this apparently silent gene cluster is able to produce PR-toxin in P. chrysogenum under static culture conditions on hydrated rice medium. Noteworthily, the production of PR-toxin was 2.6-fold higher in P. chrysogenum npe10, a strain deleted in the 56.8kb amplifiable region containing the pen gene cluster, than in the parental strain Wisconsin 54-1255 providing another example of cross-talk between secondary metabolite pathways in this fungus. A detailed PR-toxin biosynthesis pathway is proposed based on all available evidence.

Direct involvement of the CreA transcription factor in penicillin biosynthesis and expression of the pcbAB gene in Penicillium chrysogenum.

The transcription factor CreA is the main regulator responsible for carbon repression in filamentous fungi. CreA is a wide domain regulator that binds to regulatory elements in the promoters of target genes to repress their transcription. Penicillin biosynthesis and the expression of penicillin biosynthetic genes are subject to carbon repression. However, evidence of the participation of CreA in this regulation is still lacking, and previous studies on the promoter of the pcbC gene of Aspergillus nidulans indicated the lack of involvement of CreA in its regulation. Here we present clear evidence of the participation of CreA in carbon repression of penicillin biosynthesis and expression of the pcbAB gene, encoding the first enzyme of the pathway, in Penicillium chrysogenum. Mutations in cis of some of the putative CreA binding sites present in the pcbAB gene promoter fused to a reporter gene caused an important increase in the measured enzyme activity in glucose-containing medium, whereas activity in the medium with lactose was not affected. An RNAi strategy was used to attenuate the expression of the creA gene. Transformants expressing a small interfering RNA for creA showed higher penicillin production, and this increase was more evident when glucose was used as carbon source. These results confirm that CreA plays an important role in the regulation of penicillin biosynthesis in P. chrysogenum and opens the possibility of its utilization to improve the industrial production of this antibiotic.

Encapsulation of Benzaldehyde Produced by the Eco-Friendly Degradation of Amygdalin in the Apricot Kernel Debitterizing Wastewater.

In order to fully utilize the by-products of apricot kernel-debitterizing and address the chemical instability of benzaldehyde in the food industry, benzaldehyde was first prepared by adding the apricot kernel powder to degrade the amygdalin present in the apricot kernel-debitterizing water. Subsequently, ß-cyclodextrin was employed to encapsulate the benzaldehyde, and its encapsulation efficacy was evaluated through various techniques including Fourier transform infrared spectroscopy, thermogravimetric analysis, release kinetics fitting inhibitory effect and the effect on Botrytis cinerea. Finally, the encapsulation was explored via molecular docking and molecular dynamics simulations. The results indicate that the optimal preparation conditions for the benzaldehyde were 1.8 h, 53 °C and pH 5.8, and the encapsulation of benzaldehyde with ß-cyclodextrin (wall-core ratio of 5:1, mL/g) has been verified by the deceleration in the release rate, the enhanced thermal stability and the prolonged inhibition effect against Botrytis cinerea. The encapsulation proceeded spontaneously without steric hindrance in the simulation, which led to a reduction in the hydrophobic cavity of ß-cyclodextrin. In conclusion, the amygdalin in the debitterizing wastewater can be degraded in an eco-friendly way to produce benzaldehyde by adding apricot kernel powder, which contains ß-glucosidase; the encapsulation of benzaldehyde is stable, thus enhancing the utilization of amygdalin in the debitterizing wastewater of apricot kernels.

NRPSsp: non-ribosomal peptide synthase substrate predictor.

SUMMARY: Non-ribosomal peptide synthetases (NRPSs) are multi-modular enzymes, which biosynthesize many important peptide compounds produced by bacteria and fungi. Some studies have revealed that an individual domain within the NRPSs shows significant substrate selectivity. The discovery and characterization of non-ribosomal peptides are of great interest for the biotechnological industries. We have applied computational mining methods in order to build a database of NRPSs modules that bind to specific substrates. We have used this database to build a hidden Markov model predictor of substrates that bind to a given NRPS. AVAILABILITY: The database and the predictor are freely available on an easy-to-use website at www.nrpssp.com. CONTACT: carlos.prieto@unileon.es SUPPLEMENTARY INFORMATION: Supplementary data is available at Bioinformatics online.

Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer 'Streptomyces tsukubaensis'.

'Streptomyces tsukubaensis' was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain 'S. tsukubaensis' NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in 'S. tsukubaensis' necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to 'S. tsukubaensis' have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non-defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in 'S. tsukubaensis'.

Interconnected Set of Enzymes Provide Lysine Biosynthetic Intermediates and Ornithine Derivatives as Key Precursors for the Biosynthesis of Bioactive Secondary Metabolites.

Bacteria, filamentous fungi, and plants synthesize thousands of secondary metabolites with important biological and pharmacological activities. The biosynthesis of these metabolites is performed by networks of complex enzymes such as non-ribosomal peptide synthetases, polyketide synthases, and terpenoid biosynthetic enzymes. The efficient production of these metabolites is dependent upon the supply of precursors that arise from primary metabolism. In the last decades, an impressive array of biosynthetic enzymes that provide specific precursors and intermediates leading to secondary metabolites biosynthesis has been reported. Suitable knowledge of the elaborated pathways that synthesize these precursors or intermediates is essential for advancing chemical biology and the production of natural or semisynthetic biological products. Two of the more prolific routes that provide key precursors in the biosynthesis of antitumor, immunosuppressant, antifungal, or antibacterial compounds are the lysine and ornithine pathways, which are involved in the biosynthesis of ß-lactams and other non-ribosomal peptides, and bacterial and fungal siderophores. Detailed analysis of the molecular genetics and biochemistry of the enzyme system shows that they are formed by closely related components. Particularly the focus of this study is on molecular genetics and the enzymatic steps that lead to the formation of intermediates of the lysine pathway, such as α-aminoadipic acid, saccharopine, pipecolic acid, and related compounds, and of ornithine-derived molecules, such as N5-Acetyl-N5-Hydroxyornithine and N5-anhydromevalonyl-N5-hydroxyornithine, which are precursors of siderophores. We provide evidence that shows interesting functional relationships between the genes encoding the enzymes that synthesize these products. This information will contribute to a better understanding of the possibilities of advancing the industrial applications of synthetic biology.

The state-of-the-art research of the application of ultrasound to winemaking: A critical review.

As a promising non-thermal physical technology, ultrasound has attracted extensive attention in recent years, and has been applied to many food processing operation units, such as involving filtration, freezing, thawing, sterilization, cutting, extraction, aging, etc. It is also widely used in the processing of meat products, fruits and vegetables, and dairy products. With regard to its application in winemaking, most of the studies available in the literature are focused on the impact of ultrasound on a certain characteristic of wine, lacking of systematic sorting of these literatures. This review systematically summarizes and explores the current achievements and problems of the application of ultrasound to the different stages of winemaking, including extraction, fermentation, aging and sterilization. Summarizing the advantages and disadvantages of ultrasound application in winemaking and its development in future development.

The inducers 1,3-diaminopropane and spermidine produce a drastic increase in the expression of the penicillin biosynthetic genes for prolonged time, mediated by the laeA regulator.

We described previously that an autoinducer molecule, identified as 1,3-diaminopropane (1,3-DAP), is secreted by Penicillium chrysogenum and Acremonium chrysogenum. Using pH-controlled fermentor cultures we have observed in this work that 1,3-DAP and spermidine clearly stimulate the biosynthesis of benzylpenicillin in P. chrysogenum, both in defined and in complex penicillin production media. Both 1,3-DAP and spermidine, but not putrescine (1,4-diaminobutane), produce a drastic increase in the transcript levels of the penicillin biosynthetic genes pcbAB, pcbC and penDE. These polyamines do not affect the expression of the global pH-stress regulator pacC gene, thus excluding that the effect of 1,3-DAP and spermidine is due to a modification of the pH control mechanism. Expression of the three penicillin biosynthetic genes is drastically reduced in a laeA-knock-down mutant of P. chrysogenum, which produces very low levels of benzylpenicillin. Interestingly, 1,3-DAP and spermidine revert the effect of the laeA knock-down mutation, completely restoring the levels of penicillin production. Furthermore, 1,3-DAP and spermidine enhanced the expression of laeA in the parental strain and restored the levels of laeA transcripts in the laeA knock-down mutant. Taken together these results indicate that the stimulatory effect of the inducer molecules 1,3-DAP and spermidine is exerted, at least in part, through the stimulation of the expression of laeA, a global regulator that acts epigenetically on the expression of secondary metabolite genes by heterochromatin reorganization.

The regulatory factor PcRFX1 controls the expression of the three genes of ß-lactam biosynthesis in Penicillium chrysogenum.

Penicillin biosynthesis is subjected to a complex regulatory network of signalling molecules that may serve as model for other secondary metabolites. The information provided by the new "omics" era about Penicillium chrysogenum and the advances in the knowledge of molecular mechanisms responsible for improved productivity, make this fungus an excellent model to decipher the mechanisms controlling the penicillin biosynthetic pathway. In this work, we have characterized a novel transcription factor PcRFX1, which is an ortholog of the Acremonium chrysogenum CPCR1 and Penicillium marneffei RfxA regulatory proteins. PcRFX1 DNA binding sequences were found in the promoter region of the pcbAB, pcbC and penDE genes. We show in this article that these motifs control the expression of the ß-galactosidase lacZ reporter gene, indicating that they may direct the PcRFX1-mediated regulation of the penicillin biosynthetic genes. By means of Pcrfx1 gene knock-down and overexpression techniques we confirmed that PcRFX1 controls penicillin biosynthesis through the regulation of the pcbAB, pcbC and penDE transcription. Morphology and development seemed not to be controlled by this transcription factor under the conditions studied and only sporulation was slightly reduced after the silencing of the Pcrfx1 gene. A genome-wide analysis of processes putatively regulated by this transcription factor was carried out in P. chrysogenum. Results suggested that PcRFX1, in addition to regulate penicillin biosynthesis, is also involved in the control of several pathways of primary metabolism.

Proteome analysis of the penicillin producer Penicillium chrysogenum: characterization of protein changes during the industrial strain improvement.

Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by "blue silver" colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin biosynthesis in the high producer strains. These results provide useful information to improve the production of many other bioactive secondary metabolites.

The Penicillium chrysogenum extracellular proteome. Conversion from a food-rotting strain to a versatile cell factory for white biotechnology.

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize ß-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54-1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-ß-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54-1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.

Role of peroxisomes in the biosynthesis and secretion of ß-lactams and other secondary metabolites.

Peroxisomes are eukaryotic organelles surrounded by a single bilayer membrane, containing a variety of proteins depending on the organism; they mainly perform degradation reactions of toxic metabolites (detoxification), catabolism of linear and branched-chain fatty acids, and removal of H(2)O(2) (formed in some oxidative processes) by catalase. Proteins named peroxins are involved in recruiting, transporting, and introducing the peroxisomal matrix proteins into the peroxisomes. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. Initial evidence indicated that the penicillin biosynthetic enzyme isopenicillin N acyltransferase (IAT) of Penicillium chrysogenum is located inside peroxisomes. There is now solid evidence (based on electron microscopy and/or biochemical data) confirming that IAT and the phenylacetic acid- and fatty acid-activating enzymes are also located in peroxisomes. Similarly, the Acremonium chrysogenum CefD1 and CefD2 proteins that perform the central reactions (activation and epimerization of isopenicillin N) of the cephalosporin pathway are targeted to peroxisomes. Growing evidence supports the conclusion that some enzymes involved in the biosynthesis of mycotoxins (e.g., AK-toxin), and the biosynthesis of signaling molecules in plants (e.g., jasmonic acid or auxins) occur in peroxisomes. The high concentration of substrates (in many cases toxic to the cytoplasm) and enzymes inside the peroxisomes allows efficient synthesis of metabolites with interesting biological or pharmacological activities. This compartmentalization poses additional challenges to the cell due to the need to import the substrates into the peroxisomes and to export the final products; the transporters involved in these processes are still very poorly known. This article focuses on new aspects of the metabolic processes occurring in peroxisomes, namely the degradation and detoxification processes that lead to the biosynthesis and secretion of secondary metabolites.

Heterotrimeric Gα protein Pga1 from Penicillium chrysogenum triggers germination in response to carbon sources and affects negatively resistance to different stress conditions.

Heterotrimeric Gα protein Pga1 of Penicillium chrysogenum controls vegetative growth, conidiation and secondary metabolite production. In this work we studied the role of Pga1 in spore germination and resistance to different stress conditions. Strains G203R-T (expressing the dominant inactivating pga1(G203R) allele) and Δpga1 (deleted pga1) showed a delayed and asynchronic germination pattern, and a decrease in the percentage of germination, which occurred in only 70-80% of the total conidia. In contrast, in strains expressing the dominant activating pga1(G42R) allele, germination occurred at earlier times and in 100% of conidia. In addition, strains with the pga1(G42R) allele were able to bypass the carbon source (glucose or sucrose) requirement for germination in about 64% of conidia. Thus Pga1 plays an important, but not essential, role in germination, mediating carbon source sensing. Regulation of germination by Pga1 is probably mediated by cAMP, as intracellular levels of this secondary messenger undergo a peak before the onset of germination only in strains with an active Pga1. Pga1 activity is also a determinant factor in the resistance to different stress conditions. Absence or inactivation of Pga1 allow growth on SDS-containing minimal medium, increase resistance of conidia to thermal and oxidative stress, and increase resistance of vegetative mycelium to thermal and osmotic stress. In contrast, constitutive activation of Pga1 causes a decrease in the resistance of conidia to thermal stress and of vegetative mycelium to thermal and osmotic stress. Together with our previously reported results, we show in this work that Pga1 plays a central role in the regulation of the whole growth-developmental program of this biotechnologically important fungus.

Characterization of an autoinducer of penicillin biosynthesis in Penicillium chrysogenum.

Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of ß-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.

Production of Bio-Oils and Biochars from Olive Stones: Application of Biochars to the Esterification of Oleic Acid.

Olive stones are a by-product of the olive oil industry. In this work, the valorisation of olive stones through pyrolysis was attempted. Before pyrolysis, half of the samples were impregnated with sulphuric acid. Pyrolysis was carried out in a vertical tubular furnace with a ceramic support. The pyrolysis conditions assayed were: temperature between 400 and 600 °C, heating ramp between 5 and 20 °C∙min-1, and inert gas flow rate between 50 and 300 mL Ar∙min-1. Among them, temperature was the only parameter that influenced the pyrolysis product distribution. The most suitable temperature for obtaining biochar was 400 °C for both non-treated and pre-treated raw material, while for obtaining bio-oil, it was 600 °C for impregnated olive stones and 400 °C for the raw material. The impregnated olives stones led to bio-oils with much higher amounts of high-added-value products such as levoglucosenone and catechol. Finally, the biochars were impregnated with sulphuric acid and assayed as biocatalysts for the esterification of oleic acid with methanol in a stirred tank batch reactor at 60 °C for 30 min. Biochars from non-treated olive stones, which had lower specific surfaces, led to higher esterification yields (up to 96.2%).