Penicillium chrysogenum: Beyond the penicillin.

Almost one century after the Sir Alexander Fleming's fortuitous discovery of penicillin and the identification of the fungal producer as Penicillium notatum, later Penicillium chrysogenum (currently reidentified as Penicillium rubens), the molecular mechanisms behind the massive production of penicillin titers by industrial strains could be considered almost fully characterized. However, this filamentous fungus is not only circumscribed to penicillin, and instead, it seems to be full of surprises, thereby producing important metabolites and providing expanded biotechnological applications. This review, in addition to summarizing the classical role of P. chrysogenum as penicillin producer, highlights its ability to generate an array of additional bioactive secondary metabolites and enzymes, together with the use of this microorganism in relevant biotechnological processes, such as bioremediation, biocontrol, production of bioactive nanoparticles and compounds with pharmaceutical interest, revalorization of agricultural and food-derived wastes or the enhancement of food industrial processes and the agricultural production.

Targeting Trypanothione Metabolism in Trypanosomatids.

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Miltefosine and Nifuratel Combination: A Promising Therapy for the Treatment of Leishmania donovani Visceral Leishmaniasis.

Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel-a nitrofurantoin used against vaginal infections-as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine-the only oral drug currently used against leishmaniasis-with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.

Biosynthesis and beneficial effects of microbial gibberellins on crops for sustainable agriculture.

Soil microbes promote plant growth through several mechanisms such as secretion of chemical compounds including plant growth hormones. Among the phytohormones, auxins, ethylene, cytokinins, abscisic acid and gibberellins are the best understood compounds. Gibberellins were first isolated in 1935 from the fungus Gibberella fujikuroi and are synthesized by several soil microbes. The effect of gibberellins on plant growth and development has been studied, as has the biosynthesis pathways, enzymes, genes and their regulation. This review revisits the history of gibberellin research highlighting microbial gibberellins and their effects on plant health with an emphasis on the early discoveries and current advances that can find vital applications in agricultural practices.

Auxins of microbial origin and their use in agriculture.

To maintain the world population demand, a sustainable agriculture is needed. Since current global vision is more friendly with the environment, eco-friendly alternatives are desirable. In this sense, plant growth-promoting rhizobacteria could be the choice for the management of soil-borne diseases of crop plants. These rhizobacteria secrete chemical compounds which act as phytohormones. Indole-3-acetic acid (IAA) is the most common plant hormone of the auxin class which regulates various processes of plant growth. IAA compound, in which structure can be found a carboxylic acid attached through a methylene group to the C-3 position of an indole ring, is produced both by plants and microorganisms. Plant growth-promoting rhizobacteria and fungi secrete IAA to promote the plant growth. In this review, IAA production and mechanisms of action by bacteria and fungi along with the metabolic pathways evolved in the IAA secretion and commercial prospects are revised.Key points• Many microorganisms produce auxins which help the plant growth promotion.• These auxins improve the plant growth by several mechanisms.• The auxins are produced through different mechanisms.

Antimicrobial secondary metabolites from agriculturally important bacteria as next-generation pesticides.

The whole organisms can be packaged as biopesticides, but secondary metabolites secreted by microorganisms can also have a wide range of biological activities that either protect the plant against pests and pathogens or act as plant growth promotors which can be beneficial for the agricultural crops. In this review, we have compiled information about the most important secondary metabolites of three important bacterial genera currently used in agriculture pest and disease management.

Screening Marine Natural Products for New Drug Leads against Trypanosomatids and Malaria.

Neglected Tropical Diseases (NTD) represent a serious threat to humans, especially for those living in poor or developing countries. Almost one-sixth of the world population is at risk of suffering from these diseases and many thousands die because of NTDs, to which we should add the sanitary, labor and social issues that hinder the economic development of these countries. Protozoan-borne diseases are responsible for more than one million deaths every year. Visceral leishmaniasis, Chagas disease or sleeping sickness are among the most lethal NTDs. Despite not being considered an NTD by the World Health Organization (WHO), malaria must be added to this sinister group. Malaria, caused by the apicomplexan parasite Plasmodium falciparum, is responsible for thousands of deaths each year. The treatment of this disease has been losing effectiveness year after year. Many of the medicines currently in use are obsolete due to their gradual loss of efficacy, their intrinsic toxicity and the emergence of drug resistance or a lack of adherence to treatment. Therefore, there is an urgent and global need for new drugs. Despite this, the scant interest shown by most of the stakeholders involved in the pharmaceutical industry makes our present therapeutic arsenal scarce, and until recently, the search for new drugs has not been seriously addressed. The sources of new drugs for these and other pathologies include natural products, synthetic molecules or repurposing drugs. The most frequent sources of natural products are microorganisms, e.g., bacteria, fungi, yeasts, algae and plants, which are able to synthesize many drugs that are currently in use (e.g. antimicrobials, antitumor, immunosuppressants, etc.). The marine environment is another well-established source of bioactive natural products, with recent applications against parasites, bacteria and other pathogens which affect humans and animals. Drug discovery techniques have rapidly advanced since the beginning of the millennium. The combination of novel techniques that include the genetic modification of pathogens, bioimaging and robotics has given rise to the standardization of High-Performance Screening platforms in the discovery of drugs. These advancements have accelerated the discovery of new chemical entities with antiparasitic effects. This review presents critical updates regarding the use of High-Throughput Screening (HTS) in the discovery of drugs for NTDs transmitted by protozoa, including malaria, and its application in the discovery of new drugs of marine origin.

Antimicrobial secondary metabolites from agriculturally important fungi as next biocontrol agents.

Synthetic chemical pesticides have been used for many years to increase the yield of agricultural crops. However, in the future, this approach is likely to be limited due to negative impacts on human health and the environment. Therefore, studies of the secondary metabolites produced by agriculturally important microorganisms have an important role in improving the quality of the crops entering the human food chain. In this review, we have compiled information about the most important secondary metabolites of fungal species currently used in agriculture pest and disease management.

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.

Richness, infestation and specificity of spinturnicid mites (Acari: Spinturnicidae) on bats in southern Oaxaca, Mexico.

Studies of mites on bats in the Mexican state Oaxaca are scarce. Our objective was therefore to evaluate the richness, infestation, and specificity of spinturnicid mites on bats in southern Oaxaca, Mexico. Bats were monthly captured from April 2010 to February 2011, in four sites using four mist-nets; also, we visited natural (crevices) and artificial roosts (tunnel). Of each bat we account the number of spinturnicid mites, considering the area of the body where they were collected. Mites were preserved in 70 % ethanol and later they were mounted on microscope slides in Hoyer's medium. We captured bats of 15 species, of which eight species were infested. We recorded seven spinturnicid mites: five of the genus Periglischrus, one of the genus Cameronieta, and one of the genus Mesoperiglischrus. Periglischrus caligus, P. iheringi, and Periglischrus sp. are new records on Artibeus lituratus, Glossophaga soricina, and G. commissarisi, respectively. More infested bat species were Artibeus jamaicensis (93.8 %), A. lituratus (88.9 %), G. commissarisi and Sturnira parvidens (both 66.7 %). Prevalence of A. jamaicensis and A. lituratus was significantly higher than most other bat species. Although prevalence percentage was high, mean and median intensity were low. Spinturnicid mites were recorded in particular areas of a bat's body; therefore, they could be an additional tool for the taxonomic identification of bats.

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.

Polyamine Metabolism for Drug Intervention in Trypanosomatids.

Neglected tropical diseases transmitted by trypanosomatids include three major human scourges that globally affect the world's poorest people: African trypanosomiasis or sleeping sickness, American trypanosomiasis or Chagas disease and different types of leishmaniasis. Different metabolic pathways have been targeted to find antitrypanosomatid drugs, including polyamine metabolism. Since their discovery, the naturally occurring polyamines, putrescine, spermidine and spermine, have been considered important metabolites involved in cell growth. With a complex metabolism involving biosynthesis, catabolism and interconversion, the synthesis of putrescine and spermidine was targeted by thousands of compounds in an effort to produce cell growth blockade in tumor and infectious processes with limited success. However, the discovery of eflornithine (DFMO) as a curative drug against sleeping sickness encouraged researchers to develop new molecules against these diseases. Polyamine synthesis inhibitors have also provided insight into the peculiarities of this pathway between the host and the parasite, and also among different trypanosomatid species, thus allowing the search for new specific chemical entities aimed to treat these diseases and leading to the investigation of target-based scaffolds. The main molecular targets include the enzymes involved in polyamine biosynthesis (ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine synthase), enzymes participating in their uptake from the environment, and the enzymes involved in the redox balance of the parasite. In this review, we summarize the research behind polyamine-based treatments, the current trends, and the main challenges in this field.

In Vitro and Ex Vivo Synergistic Effect of Pyrvinium Pamoate Combined with Miltefosine and Paromomycin against Leishmania.

One of the major drawbacks of current treatments for neglected tropical diseases is the low safety of the drugs used and the emergence of resistance. Leishmaniasis is a group of neglected diseases caused by protozoa of the trypanosomatidae family that lacks preventive vaccines and whose pharmacological treatments are scarce and unsafe. Combination therapy is a strategy that could solve the above-mentioned problems, due to the participation of several mechanisms of action and the reduction in the amount of drug necessary to obtain the therapeutic effect. In addition, this approach also increases the odds of finding an effective drug following the repurposing strategy. From the previous screening of two collections of repositioning drugs, we found that pyrvinium pamoate had a potent leishmanicidal effect. For this reason, we decided to combine it separately with two clinically used leishmanicidal drugs, miltefosine and paromomycin. These combinations were tested in axenic amastigotes of Leishmania infantum obtained from bone marrow cells and in intramacrophagic amastigotes obtained from primary cultures of splenic cells, both cell types coming from experimentally infected mice. Some of the combinations showed synergistic behavior, especially in the case of the combination of pyrvinium pamoate with paromomycin, and exhibited low cytotoxicity and good tolerability on intestinal murine organoids, which reveal the potential of these combinations for the treatment of leishmaniasis.

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.

Secondary Metabolites Produced by the Blue-Cheese Ripening Mold Penicillium roqueforti; Biosynthesis and Regulation Mechanisms.

Filamentous fungi are an important source of natural products. The mold Penicillium roqueforti, which is well-known for being responsible for the characteristic texture, blue-green spots, and aroma of the so-called blue-veined cheeses (French Bleu, Roquefort, Gorgonzola, Stilton, Cabrales, and Valdeón, among others), is able to synthesize different secondary metabolites, including andrastins and mycophenolic acid, as well as several mycotoxins, such as Roquefortines C and D, PR-toxin and eremofortins, Isofumigaclavines A and B, festuclavine, and Annullatins D and F. This review provides a detailed description of the biosynthetic gene clusters and pathways of the main secondary metabolites produced by P. roqueforti, as well as an overview of the regulatory mechanisms controlling secondary metabolism in this filamentous fungus.

Biotechnological Fungal Platforms for the Production of Biosynthetic Cannabinoids.

Cannabinoids are bioactive meroterpenoids comprising prenylated polyketide molecules that can modulate a wide range of physiological processes. Cannabinoids have been shown to possess various medical/therapeutic effects, such as anti-convulsive, anti-anxiety, anti-psychotic, antinausea, and anti-microbial properties. The increasing interest in their beneficial effects and application as clinically useful drugs has promoted the development of heterologous biosynthetic platforms for the industrial production of these compounds. This approach can help circumvent the drawbacks associated with extraction from naturally occurring plants or chemical synthesis. In this review, we provide an overview of the fungal platforms developed by genetic engineering for the biosynthetic production of cannabinoids. Different yeast species, such as Komagataella phaffii (formerly P. pastoris) and Saccharomyces cerevisiae, have been genetically modified to include the cannabinoid biosynthetic pathway and to improve metabolic fluxes in order to increase cannabinoid titers. In addition, we engineered the filamentous fungus Penicillium chrysogenum for the first time as a host microorganism for the production of Δ9-tetrahydrocannabinolic acid from intermediates (cannabigerolic acid and olivetolic acid), thereby showing the potential of filamentous fungi as alternative platforms for cannabinoid biosynthesis upon optimization.

Further Investigations of Nitroheterocyclic Compounds as Potential Antikinetoplastid Drug Candidates.

Due to the lack of specific vaccines, management of the trypanosomatid-caused neglected tropical diseases (sleeping sickness, Chagas disease and leishmaniasis) relies exclusively on pharmacological treatments. Current drugs against them are scarce, old and exhibit disadvantages, such as adverse effects, parenteral administration, chemical instability and high costs which are often unaffordable for endemic low-income countries. Discoveries of new pharmacological entities for the treatment of these diseases are scarce, since most of the big pharmaceutical companies find this market unattractive. In order to fill the pipeline of compounds and replace existing ones, highly translatable drug screening platforms have been developed in the last two decades. Thousands of molecules have been tested, including nitroheterocyclic compounds, such as benznidazole and nifurtimox, which had already provided potent and effective effects against Chagas disease. More recently, fexinidazole has been added as a new drug against African trypanosomiasis. Despite the success of nitroheterocycles, they had been discarded from drug discovery campaigns due to their mutagenic potential, but now they represent a promising source of inspiration for oral drugs that can replace those currently on the market. The examples provided by the trypanocidal activity of fexinidazole and the promising efficacy of the derivative DNDi-0690 against leishmaniasis seem to open a new window of opportunity for these compounds that were discovered in the 1960s. In this review, we show the current uses of nitroheterocycles and the novel derived molecules that are being synthesized against these neglected diseases.

Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506).

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.

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.