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1.
Int J Food Microbiol ; 322: 108585, 2020 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-32179333

RESUMO

A total of 20 dried date samples, chosen as representative among those available on the Perugia (Umbria, Central Italy) market, were analyzed for the possible occurrence of fungal species and related contamination by fungal secondary metabolites. Twenty-six isolates, representative of the total mycobiota, were obtained and morphologically identified as belonging to the genera Aspergillus, Penicillium and Cladosporium. Inside each genus, molecular characterization (by partial sequencing of ITS region and/or ß-tubulin and calmodulin regions for Aspergillus and Penicillium isolates or actin region for Cladosporium isolates) and in vitro mycotoxigenic profile characterization (by LC-MS/MS analysis) showed the presence of the following species: A. flavus, A. tubingensis, P. brevicompactum, P. chrysogenum, P. crustosum, P. glabrum, P. solitum, P. venetum, C. cladosporioides, C. limoniforme and C. halotolerans, with A. tubingensis as the prevalent species and P. crustosum, P. solitum, P. venetum and C. limoniforme first reported here on dates. Date packaging and format showed an effect on the incidence of isolated fungi, with the lowest incidence recovered from whole dates and in hermetic bag packaging. These findings can be useful both for dried dates producers and consumers, guiding them towards choices of packaging and format with a lower risk of mycotoxigenic species presence. However, no fungal metabolites were detected in the dried date samples analyzed, which were therefore regarded as safe for human consumption, underlining the absence of correspondence between fungal isolation and mycotoxin contaminations.


Assuntos
Microbiologia de Alimentos , Alimentos em Conserva/microbiologia , Fungos/isolamento & purificação , Phoeniceae/microbiologia , Aspergillus/classificação , Aspergillus/genética , Aspergillus/isolamento & purificação , Aspergillus/metabolismo , Cladosporium/classificação , Cladosporium/genética , Cladosporium/isolamento & purificação , Cladosporium/metabolismo , Embalagem de Alimentos/métodos , Frutas/microbiologia , Fungos/classificação , Fungos/genética , Fungos/metabolismo , Humanos , Itália , Micotoxinas/análise , Penicillium/classificação , Penicillium/genética , Penicillium/isolamento & purificação , Penicillium/metabolismo
2.
J Basic Microbiol ; 60(1): 82-88, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31650621

RESUMO

Involvement of LaeA in various biological processes of filamentous fungi has been demonstrated. However, its role in Penicillium digitatum, the causal agent of citrus postharvest green mold, remains unclear. In this study, a ΔPdLaeA mutant was constructed using homologous recombination. The production of conidia by the ΔPdLaeA mutant was reduced by half compared with that of the wild-type strain. The sensitivity of the ΔPdLaeA mutant increased under alkaline conditions. The virulence assay revealed that PdLaeA was dispensable for the virulence of P. digitatum. Comparative transcriptome analysis revealed that the function loss of PdLaeA resulted in the reduced expression of several secondary metabolite gene clusters. In addition, expression of several key regulators of conidiation (BrlA, FlbA, FlbC, FlbD, and FluG) was also downregulated in the ΔPdLaeA mutant. In summary, the present work demonstrated that PdLaeA was involved in the regulation of SM biosynthesis, as well as the development and environmental adaptation of P. digitatum.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Penicillium/genética , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Citrus/microbiologia , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Família Multigênica/genética , Penicillium/crescimento & desenvolvimento , Penicillium/metabolismo , Penicillium/fisiologia , Deleção de Sequência , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/fisiologia , Fatores de Transcrição/genética , Virulência/genética
3.
Int J Food Microbiol ; 314: 108389, 2020 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-31683087

RESUMO

Most of diseases of pomegranate fruit are caused by fungal pathogens, which provoke postharvest yield and economical losses. Aspergillus and Penicillium sensu lato (s.l.) are the main wound pathogens of pomegranate fruit. In the present investigation, the populations of Aspergillus and Penicillium s.l. isolated from pomegranate fruit in Southern Italy were characterized. Since the morphological identification of species belonging to these genera is laborious, molecular approaches, such as PCR and High-Resolution Melting (HRM), were used. Particularly, a specific primer pair was designed to discriminate, within the Penicillium s.l. population, Penicillium sensu stricto (s.s.) from Talaromyces strains. Then, a new HRM assay for species identification within Penicillium s.s. according to SNPs present in a portion of the beta-tubulin gene was set up. Similarly, Aspergillus sect. nigri population was characterized arranging a HRM assay, whose primer pair was designed on a portion of the calmodulin gene. According to these assays, 10% of the Penicillium s.l. population proved to be made up of Talaromyces biverticillius strains. Furthermore, six species of Penicillium s.s. (P. adametzioides, P. brevicompactum, P. citrinum, P. glabrum, P. pagulum, and P. johnkrugii) and four of black aspergilli (A. tubingensis, A. welwitschiae, A. japonicus, and A. uvarum) were identified; all species belonging to both genera disclosed different incidences in postharvest rotted pomegranate fruit. Moreover, since Aspergillus and Penicillium are potentially producers of mycotoxins, like ochratoxin A and fumonisins, the presence/absence of genes involved in mycotoxin biosynthetic pathways was tested. Some Aspergillus strains belonging to species A. welwitschiae proved to possess fumonisin genes. The setup of molecular tools to characterize Penicillium s.l. and Aspergillus sect. nigri species infecting pomegranate fruit after harvest is of paramount importance for their effective control, even more considering the ability of these fungal genera to produce mycotoxins, which are hazardous for human health and potentially present also in by-products.


Assuntos
Aspergillus/genética , Microbiologia de Alimentos , Penicillium/genética , /microbiologia , Aspergillus/classificação , Aspergillus/isolamento & purificação , Frutas/microbiologia , Genes Fúngicos/genética , Itália , Micotoxinas/genética , Técnicas de Amplificação de Ácido Nucleico , Penicillium/classificação , Penicillium/isolamento & purificação
4.
Enzyme Microb Technol ; 133: 109447, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874680

RESUMO

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.


Assuntos
Celulose , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Saccharum/metabolismo , Vapor , Celulose/metabolismo , Hidrólise , Penicillium/enzimologia , Penicillium/genética , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Enzyme Microb Technol ; 133: 109463, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874686

RESUMO

Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.


Assuntos
Sistemas CRISPR-Cas , Enzimas/biossíntese , Edição de Genes , Penicillium/enzimologia , Penicillium/genética , Genoma Fúngico , Microbiologia Industrial/métodos , Fenótipo
6.
Molecules ; 24(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569777

RESUMO

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0-9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s-1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.


Assuntos
Códon , Endo-1,4-beta-Xilanases/genética , Regulação da Expressão Gênica , Penicillium/enzimologia , Penicillium/genética , Pichia/genética , Proteínas Recombinantes , Sequência de Bases , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Ativação Enzimática , Vetores Genéticos/genética , Concentração de Íons de Hidrogênio , Temperatura , Termodinâmica
7.
Mar Drugs ; 17(9)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31480589

RESUMO

Identification and analysis of the whole genome of the marine-derived fungus Penicillium brasilianum HBU-136 revealed the presence of an interesting biosynthetic gene cluster (BGC) for non-ribosomal peptide synthetases (NRPS), highly homologous to the BGCs of indole-diketopiperazine derivatives. With the aid of genomic analysis, eight indole-diketopiperazines (1-8), including three new compounds, spirotryprostatin G (1), and cyclotryprostatins F and G (2 and 3), were obtained by large-scale cultivation of the fungal strain HBU-136 using rice medium with 1.0% MgCl2. The absolute configurations of 1-3 were determined by comparison of their experimental electronic circular dichroism (ECD) with calculated ECD spectra. Selective cytotoxicities were observed for compounds 1 and 4 against HL-60 cell line with the IC50 values of 6.0 and 7.9 µM, respectively, whereas 2, 3, and 5 against MCF-7 cell line with the IC50 values of 7.6, 10.8, and 5.1 µM, respectively.


Assuntos
Organismos Aquáticos/química , Dicetopiperazinas/química , Fungos/química , Fungos/genética , Indóis/química , Penicillium/química , Penicillium/genética , Organismos Aquáticos/genética , Linhagem Celular Tumoral , Dicroísmo Circular , Genoma/genética , Genômica , Células HL-60 , Humanos , Células MCF-7 , Família Multigênica/genética , Peptídeo Sintases/genética
8.
Int J Food Microbiol ; 309: 108312, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31499265

RESUMO

The characteristics and quality of home-made dry cured sausages can be recognized and associated with the region of origin. The characteristics of this type of sausages result from the superficial mycobiota that spontaneously colonizes the products. The aim of this study was to identify the house mycobiota associated with home-made dry cured sausages from different localities of Argentina and characterize the populations of Penicillium nalgiovense present by morphological and biochemical markers. To this end, 79 samples were collected from 10 localities of three main producing regions (Buenos Aires, Córdoba and La Pampa provinces). A total of 196 isolates belonging to six genera and 17 species were obtained. The predominant genus was Penicillium (134 of the isolates) and the predominant species was P. nalgiovense (108 isolates). The isoenzyme patterns of α-esterase (α-EST; EC 3.1.1.1) and Malate dehydrogenase NADP+(MDHP; EC 1.1.1.40) were characterized in 48 of these isolates (ten from Colonia Caroya, ten from Oncativo, ten from Tandil, nine from Mercedes and nine from La Pampa). A total of 26 bands were observed: 17 for α-EST and 9 for MDHP. α-EST was the most polymorphic isoenzyme, whereas MDPH presented no polymorphism. The results were subjected to numerical analysis. Cluster analysis revealed the formation of two groups: Group I formed by 24 isolates from Córdoba and Buenos Aires provinces and Group II with 24 isolates from La Pampa and Buenos Aires province. These data suggest the existence of morphological and biochemical variations among P. nalgiovense populations with different geographical origin.


Assuntos
Produtos da Carne/microbiologia , Penicillium/classificação , Penicillium/isolamento & purificação , Argentina , Esterases/genética , Fermentação , Contaminação de Alimentos/análise , Malato Desidrogenase/genética , Penicillium/genética
9.
Environ Sci Pollut Res Int ; 26(28): 29138-29156, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392610

RESUMO

The capacity of dispersion, persistence, and stability from biocontrol agents is essential before these organisms can be developed into a commercial product. Interactions that microorganisms establish with stone fruit trees may be beneficial in the exploitation of trees in agriculture as crop production. The natural background levels of Penicillium frequentans strain 909 dispersion, persistence, and stability were assessed after tree applications and postharvest conditions. A fingerprinting-based approach to trace genetic stability of P. frequentans along stored time and its release in the field was developed. P. frequentans was successfully traced and discriminated. This strain was dispersed well in treated trees, persisting in the ecosystem up to 2 weeks and staying genetically stable after 36 months of storage. However, the dispersal of P. frequentans was very limited on around untreated trees and soil. P. frequentans dispersed randomly into the air, and its presence reduced from the first day to disappear completely at 15-21 days after application. Great losses of P. frequentans and its increased dispersal in open field conditions probably resulted from rainfall. Biological control strategies with Pf909 were discussed.


Assuntos
Agentes de Controle Biológico , Produção Agrícola/métodos , Penicillium/fisiologia , Ecossistema , Frutas , Penicillium/genética , Espanha , Árvores
10.
BMC Biotechnol ; 19(1): 51, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345213

RESUMO

BACKGROUND: A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30 L), combined with an additional disulfide bridge in the N-terminal region. RESULTS: The Tm value of mutant PTxA-DB was improved from 21.3 °C to 76.6 °C, and its half-life was found to be 53.6 min at 60 °C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the ß-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30 L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values. CONCLUSIONS: These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.


Assuntos
Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Mutagênese , Penicillium/genética , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Dissulfetos/química , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutação , Penicillium/enzimologia , Conformação Proteica , Engenharia de Proteínas/métodos , Temperatura
11.
Fungal Biol ; 123(8): 584-593, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31345412

RESUMO

Penicillium digitatum is the major source of postharvest decay in citrus fruits worldwide. This fungus shows a limited host range, being able to infect mainly mature fruit belonging to the Rutaceae family. This highly specific host interaction has attracted the interest of the scientific community. Researchers have investigated the chemical interactions and specialized virulence strategies that facilitate this fungus's fruit colonization, thereby leading to a successful citrus infection. There are several factors that mediate and affect the interaction between P. digitatum and its host citrus, including hydrogen peroxide modulation, secretion of organic acids and consequently pH control, and other strategies described here. The recently achieved sequencing of the complete P. digitatum genome opened up new possibilities for exploration of the virulence factors related to the host-pathogen interaction. Through such techniques as RNAseq, RT-PCR and targeted gene knockout mediated by Agrobacterium tumefaciens, important genes involved in the fungal infection process in citrus have been reported, helping to elucidate the molecular mechanisms, metabolites and genetic components that are involved in the pathogenicity of P. digitatum. Understanding the infection process and fungal strategies represents an important step in developing ways to protect citrus from P. digitatum infection, possibly leading to more productive citriculture.


Assuntos
Citrus/microbiologia , Penicillium/fisiologia , Doenças das Plantas/microbiologia , Citrus/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Penicillium/genética
12.
Fungal Biol ; 123(8): 594-600, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31345413

RESUMO

Green mold, caused by Penicillium digitatum, is the most destructive post-harvest disease in citrus. Secondary metabolites produced by fungal phytopathogens have been associated with toxicity to their respective host through the interaction with a wide range of cell targets. Natural products have also been described as important molecules for biocontrol and competition in their respective environment. For P. digitatum, the production of indole alkaloids, tryptoquialanines A and B, have been reported. However, their biological role remains unknown. Mass Spectrometry Imaging (MSI) technique was applied here for the first time to monitor the secondary metabolites produced on the orange surface during infection in order to gain insights about the P. digitatum-citrus interaction mechanisms. Through the combination of MSI and molecular networking it was possible to report, for the first time, the production of tryptoquivalines and fumiquinazolines by P. digitatum and also the accumulation of tryptoquialanines on the fruit surface from 4 to 7 d post inoculation. P. digitatum was also evaluated concerning the ability to sinthesize indole alkaloids in vivo in the different citrus hosts. The biological role of tryptoquialanines was investigated and tryptoquialanine A was submitted to insecticidal bioassays that revealed its high toxicity against Aedes Aegypti, suggesting an important insecticidal action during orange decay.


Assuntos
Alcaloides/química , Alcaloides/metabolismo , Citrus/microbiologia , Indóis/química , Penicillium/química , Penicillium/metabolismo , Doenças das Plantas/microbiologia , Citrus/química , Citrus/classificação , Frutas/química , Frutas/microbiologia , Indóis/metabolismo , Espectrometria de Massas , Estrutura Molecular , Penicillium/genética , Metabolismo Secundário
13.
Carbohydr Res ; 482: 107738, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31280019

RESUMO

Xylan, a component of plant cell walls, is composed of a backbone of ß-1,4-linked xylopyranosyl units with a number of substituents. The complete degradation of xylan requires the action of several enzymes, among them ß-xylosidase. The fungus Penicillium purpurogenum secretes a number of enzymes participating in the degradation of xylan. In this study, a ß-xylosidase from this fungus was expressed in Pichia pastoris, and characterized. This enzyme (Xyl2) is a member of glycoside hydrolase family 3; it consists of a sequence of 792 residues including a signal peptide of 20 residues, with a theoretical molecular mass for the mature protein of 84.2 KDa and an isoelectric point of 5.07. The highest identity with a characterized fungal enzyme, is with a ß-xylosidase from Aspergillus oryzae (70%). The optimal activity of Xyl2 is found at pH 2.0 and 28 °C. The enzyme is most stable at pH 2.0 and conserves 40% of activity at 42 °C (after 1h incubation). The kinetic parameters for p-nitrophenyl-ß-d-xylopyranoside are: KM 0.53 mM, kcat 1*107 s-1 and kcat/KM 1.9*1010 M-1 s-1. The enzyme is about 10% active on p-nitrophenyl-α-l-arabinofuranoside. Xyl2 exhibits a high hydrolytic activity on xylooligosaccharides; it liberates xylose from beechwood and birchwood glucuronoxylan and it acts synergistically with endoxylanases in the degradation of xylan. Its low pH optimum make this enzyme particularly useful in potential applications requiring a low pH such as increasing the flavor of wine.


Assuntos
Penicillium/enzimologia , Penicillium/genética , Xilosidases/genética , Xilosidases/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Pichia/genética
14.
Mar Drugs ; 17(8)2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31357680

RESUMO

Overexpression of the global regulator LaeA in a marine-derived fungal strain of Penicillium dipodomyis YJ-11 induced obvious morphological changes and metabolic variations. Further chemical investigation of the mutant strain afforded a series of sorbicillinoids including two new ones named 10,11-dihydrobislongiquinolide (1) and 10,11,16,17-tetrahydrobislongiquinolide (2), as well as four known analogues, bislongiquinolide (3), 16,17-dihydrobislongiquinolide (4), sohirnone A (5), and 2',3'-dihydrosorbicillin (6). The results support that the global regulator LaeA is a useful tool in activating silent gene clusters in Penicillium strains to obtain previously undiscovered compounds.


Assuntos
Organismos Aquáticos/genética , Organismos Aquáticos/metabolismo , Produtos Biológicos/metabolismo , Fungos/genética , Fungos/metabolismo , Penicillium/genética , Penicillium/metabolismo , Genes Fúngicos/genética , Mutação/genética
15.
Int J Mol Sci ; 20(14)2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31337149

RESUMO

Penicillium italicum is the principal pathogen causing blue mold of citrus. Searching for novel antifungal agents is an important aspect of the post-harvest citrus industry because of the lack of higher effective and low toxic antifungal agents. Herein, the effects of 2-methoxy-1,4-naphthoquinone (MNQ) on P. italicum and its mechanism were carried out by a series of methods. MNQ had a significant anti-P. italicum effect with an MIC value of 5.0 µg/mL. The label-free protein profiling under different MNQ conditions identified a total of 3037 proteins in the control group and the treatment group. Among them, there were 129 differentially expressed proteins (DEPs, up-regulated > 2.0-fold or down-regulated < 0.5-fold, p < 0.05), 19 up-regulated proteins, 26 down-regulated proteins, and 67 proteins that were specific for the treatment group and another 17 proteins that were specific for the control group. Of these, 83 proteins were sub-categorized into 23 hierarchically-structured GO classifications. Most of the identified DEPs were involved in molecular function (47%), meanwhile 27% DEPs were involved in the cellular component and 26% DEPs were involved in the biological process. Twenty-eight proteins identified for differential metabolic pathways by KEGG were sub-categorized into 60 classifications. Functional characterization by GO and KEGG enrichment results suggests that the DEPs are mainly related to energy generation (mitochondrial carrier protein, glycoside hydrolase, acyl-CoA dehydrogenase, and ribulose-phosphate 3-epimerase), NADPH supply (enolase, pyruvate carboxylase), oxidative stress (catalase, glutathione synthetase), and pentose phosphate pathway (ribulose-phosphate 3-epimerase and xylulose 5-phosphate). Three of the down-regulated proteins selected randomly the nitro-reductase family protein, mono-oxygenase, and cytochrome P450 were verified using parallel reaction monitoring. These findings illustrated that MNQ may inhibit P. italicum by disrupting the metabolic processes, especially in energy metabolism and stimulus response that are both critical for the growth of the fungus. In conclusion, based on the molecular mechanisms, MNQ can be developed as a potential anti-fungi agent against P. italicum.


Assuntos
Proteínas Fúngicas/metabolismo , Naftoquinonas/farmacologia , Penicillium/efeitos dos fármacos , Penicillium/metabolismo , Proteoma , Proteômica , Biologia Computacional/métodos , Proteínas Fúngicas/genética , Ontologia Genética , Anotação de Sequência Molecular , Naftoquinonas/química , Penicillium/genética , Proteômica/métodos
16.
Int J Food Microbiol ; 305: 108243, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31200120

RESUMO

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.


Assuntos
Debaromyces/isolamento & purificação , Proteínas Fúngicas/genética , Produtos da Carne/microbiologia , Ocratoxinas/metabolismo , Penicillium chrysogenum/isolamento & purificação , Penicillium/metabolismo , Animais , Debaromyces/genética , Debaromyces/metabolismo , Microbiologia de Alimentos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Produtos da Carne/análise , Ocratoxinas/análise , Penicillium/genética , Penicillium/crescimento & desenvolvimento , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Proteômica , Metabolismo Secundário , Suínos
17.
PLoS One ; 14(5): e0216546, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31091286

RESUMO

Copper radical alcohol oxidases belonging to auxiliary activity family 5, subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides, as well as aliphatic alcohols. Despite their broad applied potential, so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A), which groups within an unmapped clade phylogenetically distant from those comprising AA5_2 members characterized to date. PruAA5_2 preferentially oxidized raffinose over galactose; however, its catalytic efficiency was 6.5 times higher on glycolaldehyde dimer compared to raffinose. Deep sequence analysis of characterized AA5_2 members highlighted amino acid pairs correlated to substrate range and conserved within the family. Moreover, PruAA5_2 activity spans substrate preferences previously reported for AA5 subfamily 1 and 2 members, identifying possible functional overlap across the AA5 family.


Assuntos
Clonagem Molecular/métodos , Oxirredutases/genética , Oxirredutases/metabolismo , Penicillium/enzimologia , Rafinose/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactose/química , Galactosídeos/química , Sequenciamento de Nucleotídeos em Larga Escala , Oxirredução , Penicillium/genética , Filogenia , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína
18.
J Microbiol Biotechnol ; 29(6): 984-988, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31091865

RESUMO

Blue mold in citrus is caused by Penicillium italicum. In this study, the P. italicum-specific primers were developed for rapid detection based on the conserved genes RPB1 and RPB2 among Penicillium genomes. The two primer pairs RPB1-a and RPB1-b proved to be specific to detect P. italicum. The PCR assay among 39 fungal isolates and the colonial, pathogenic morphologies and molecular methods validated the specificity and reliability of these two primer pairs. This report provided a method and P. italicum-specific primers, which might greatly contribute to citrus postharvest industry.


Assuntos
Citrus/microbiologia , Primers do DNA/normas , Microbiologia de Alimentos/métodos , Técnicas de Tipagem Micológica/métodos , Penicillium/genética , Doenças das Plantas/microbiologia , Proteínas de Bactérias/genética , Primers do DNA/genética , Penicillium/classificação , Reação em Cadeia da Polimerase , RNA Polimerase II/genética , Reprodutibilidade dos Testes , Especificidade da Espécie
19.
Genes (Basel) ; 10(5)2019 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035394

RESUMO

Despite the recent advancements in culturomics, isolation of the majority of environmental microbiota performing critical ecosystem services, such as bioremediation of contaminants, remains elusive. Towards this end, we conducted a metagenomics-guided comparative assessment of soil microbial diversity and functions present in uraniferous soils relative to those that grew in diffusion chambers (DC) or microbial traps (MT), followed by isolation of uranium (U) resistant microbiota. Shotgun metagenomic analysis performed on the soils used to establish the DC/MT chambers revealed Proteobacterial phyla and Burkholderia genus to be the most abundant among bacteria. The chamber-associated growth conditions further increased their abundances relative to the soils. Ascomycota was the most abundant fungal phylum in the chambers relative to the soils, with Penicillium as the most dominant genus. Metagenomics-based taxonomic findings completely mirrored the taxonomic composition of the retrieved isolates such that the U-resistant bacteria and fungi mainly belonged to Burkholderia and Penicillium species, thus confirming that the chambers facilitated proliferation and subsequent isolation of specific microbiota with environmentally relevant functions. Furthermore, shotgun metagenomic analysis also revealed that the gene classes for carbohydrate metabolism, virulence, and respiration predominated with functions related to stress response, membrane transport, and metabolism of aromatic compounds were also identified, albeit at lower levels. Of major note was the successful isolation of a potentially novel Penicillium species using the MT approach, as evidenced by whole genome sequence analysis and comparative genomic analysis, thus enhancing our overall understanding on the uranium cycling microbiota within the tested uraniferous soils.


Assuntos
Microbiota/genética , Microbiologia do Solo , Urânio/toxicidade , Ascomicetos/genética , Ascomicetos/efeitos da radiação , Biodegradação Ambiental , Burkholderia/genética , Burkholderia/efeitos da radiação , Ecossistema , Pradaria , Humanos , Metagenômica , Microbiota/efeitos da radiação , Penicillium/genética , Penicillium/efeitos da radiação , Rios , Estados Unidos
20.
ACS Chem Biol ; 14(6): 1227-1234, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31141338

RESUMO

Heterologous expression of secondary metabolite genes and gene clusters has been proven to be a successful strategy for identification of new natural products of cryptic or silent genes hidden in the genome sequences. It is also a useful tool to produce designed compounds by synthetic biology approaches. In this study, we demonstrate the potential usage of the gene locus pcr4401 in the fast-growing filamentous fungus Penicillium crustosum as an integration site for heterologous gene expression. The deduced polyketide synthase (PKS) Pcr4401 is involved in the dihydroxynaphthalene (DHN)-melanin pigment formation, and its deletion in P. crustosum PRB-2 led to an albino phenotype. Heterologous expression of pcr4401 in Aspergillus nidulans proved its function as the melanin precursor YWA1 synthase. To ensure gene expression after genomic integration and to easily identify the potential transformants by visualization, the gene locus of pcr4401 was chosen as an integration site. For heterologous expression in P. crustosum, the expression constructs were created by ligation-independent homologous recombination in Escherichia coli or Saccharomyces cerevisiae. A pyrG deficient strain was also created, so that both the pyrG and hph resistance gene can be used as selection markers. Successful expression in P. crustosum was demonstrated by using one uncharacterized PKS gene from Aspergillus and two from Penicillium strains. All three genes were successfully introduced, heterologously expressed, and their biosynthetic products elucidated. The results presented in this study demonstrated that P. crustosum can be used as a suitable host for heterologous expression of secondary metabolite genes.


Assuntos
Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Melaninas/genética , Penicillium/genética , Pigmentos Biológicos/genética , Aspergillus nidulans/genética , Escherichia coli/genética , Melaninas/metabolismo , Naftóis/metabolismo , Penicillium/metabolismo , Pigmentos Biológicos/metabolismo , Policetídeo Sintases/metabolismo , Saccharomyces cerevisiae/genética
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