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1.
Food Chem ; 305: 125382, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525590

RESUMO

During the mashing process for brewing beer, incomplete degradation of arabinoxylan in barley malt may cause an intense filterability problem. The present study cloned a putative arabinofuranosidase (AnAbf), one of the debranching enzymes, from Aspergillus niger, to explore its application for improving filterability. Recombinant AnAbf (rAnAbf) showed activity towards both synthetic and natural substrates, such as 4-nitrophenyl α-l-arabinofuranoside (pNPαAraf) and malt water extractable arabinoxylan (WEAX), which was maximized at a temperature of 50 °C and pH of 5.5. Metal ions did not increase the activity of rAnAbf, indicating a difference in its C-terminal domain from that of type II GH43 family members. rAnAbf also exhibited a synergistic effect with ß-xylanase against WEAX. The filtration rate of the wort increased by 12.8% after supplementing with rAnAbf during the initial stage of mashing. A slight decrease in viscosity and an unexpected increase in turbidity were observed.


Assuntos
Aspergillus niger/enzimologia , Cerveja/análise , Glicosídeo Hidrolases/metabolismo , Xilanos/metabolismo , Arabinose/análogos & derivados , Arabinose/metabolismo , Endo-1,4-beta-Xilanases , Proteínas Fúngicas/metabolismo , Temperatura Alta , Proteínas Recombinantes/metabolismo
2.
Pol J Microbiol ; 68(4): 493-504, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880893

RESUMO

Bird fecal matter is considered a potential source of pathogenic microbes such as yeast species that contaminate the environment. Therefore, it needs to be scrutinized to assess potential environmental health risks. The aim of this study was to investigate the diversity of the yeasts in pigeon fecal droppings, their antifungal susceptibility patterns, and virulence factors. We used culturing techniques to detect the yeasts in pigeon fecal droppings. The isolates were then characterized based on colony morphologies, microscopic examinations, and biochemical reactions. The molecular identification of all yeast isolates was performed by sequencing of the amplified ITS gene. Genes encoding virulence factors CAP1, CAP59, and PLB were also detected. Antifungal susceptibility patterns were examined by the disk diffusion method. A total of 46 yeast-like isolates were recovered, and they belonged to nine different genera, namely, Cryptococcus, Saccharomyces, Rhodotorula, Candida, Meyerozyma, Cyberlindnera, Rhodosporidium, Millerozyma, and Lodderomyces. The prevalence of two genera Cryptococcus and Rhodotorula was high. None of the yeast isolates exhibited any resistance to the antifungal drugs tested; however, all pathogenic Cryptococcus species were positive for virulence determinants like urease activity, growth at 37°C, melanin production, the PLB and CAP genes. This is the first report on the molecular diversity of yeast species, particularly, Cryptococcus species and their virulence attributes in pigeon fecal droppings in Saudi Arabia.Bird fecal matter is considered a potential source of pathogenic microbes such as yeast species that contaminate the environment. Therefore, it needs to be scrutinized to assess potential environmental health risks. The aim of this study was to investigate the diversity of the yeasts in pigeon fecal droppings, their antifungal susceptibility patterns, and virulence factors. We used culturing techniques to detect the yeasts in pigeon fecal droppings. The isolates were then characterized based on colony morphologies, microscopic examinations, and biochemical reactions. The molecular identification of all yeast isolates was performed by sequencing of the amplified ITS gene. Genes encoding virulence factors CAP1, CAP59, and PLB were also detected. Antifungal susceptibility patterns were examined by the disk diffusion method. A total of 46 yeast-like isolates were recovered, and they belonged to nine different genera, namely, Cryptococcus, Saccharomyces, Rhodotorula, Candida, Meyerozyma, Cyberlindnera, Rhodosporidium, Millerozyma, and Lodderomyces. The prevalence of two genera Cryptococcus and Rhodotorula was high. None of the yeast isolates exhibited any resistance to the antifungal drugs tested; however, all pathogenic Cryptococcus species were positive for virulence determinants like urease activity, growth at 37°C, melanin production, the PLB and CAP genes. This is the first report on the molecular diversity of yeast species, particularly, Cryptococcus species and their virulence attributes in pigeon fecal droppings in Saudi Arabia.


Assuntos
Columbidae/microbiologia , Fezes/microbiologia , Fatores de Virulência/metabolismo , Leveduras/efeitos dos fármacos , Leveduras/isolamento & purificação , Animais , Antifúngicos/farmacologia , Biodiversidade , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Testes de Sensibilidade Microbiana , Arábia Saudita , Fatores de Virulência/genética , Leveduras/genética , Leveduras/metabolismo
3.
Adv Exp Med Biol ; 1174: 161-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31713199

RESUMO

In recent years, much attention has focused on incorporating biological and bio-inspired nanomaterials into various applications that range from functionalising surfaces and enhancing biomolecule binding properties, to coating drugs for improved bioavailability and delivery. Hydrophobin proteins, which can spontaneously assemble into amphipathic layers at hydrophobic:hydrophilic interfaces, are exciting candidates for use as nanomaterials. These unique proteins, which are only expressed by filamentous fungi, have been the focus of increasing interest from the biotechnology industry, as evidenced by the sharply growing number of hydrophobin-associated publications and patents. Here, we explore the contribution of different hydrophobins to supporting fungal growth and development. We describe the key structural elements of hydrophobins and the molecular characteristics that underlie self-assembly of these proteins at interfaces. We outline the multiple roles that hydrophobins can play in supporting aerial growth of filamentous structures, facilitating spore dispersal and preventing an immune response in the infected host. The growing understanding of the hydrophobin protein structure and self-assembly process highlights the potential for hydrophobin proteins to be engineered for use in a variety of novel applications that require biocompatible coatings.


Assuntos
Proteínas Fúngicas , Nanoestruturas , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Nanoestruturas/química , Conformação Proteica
4.
J Agric Food Chem ; 67(41): 11444-11453, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31592644

RESUMO

Innovative approaches to develop flavors with high sensory appeal are critical in encouraging increased consumer preference and adoption of low sodium foods. Gas chromatography-olfactometry, coupled with stable isotope dilution assays and sensory experiments, led to the identification of the odorants responsible for an enhancement in saltiness perception of chicken broth prepared with thermally treated enzymatically hydrolyzed mushroom protein and cysteine, then reacted under kitchen-like cooking conditions. Comparative aroma extract dilution analysis revealed 36 odorants with flavor dilution factors between a range of 1 and 256. Sixteen odorants were quantitated and odor activity values (OAVs) calculated. Important odorants included 2-furfurylthiol (coffee, OAV 610), 1-(2-furyl)ethanethiol (meaty, OAV 78), 3-sulfanylpentan-2-one (catty, OAV 42), sotolon (maple, OAV 20), indole (animal, OAV 8), 2-methyl-3-(methyldithio)furan (meaty, OAV 3), and p-cresol (barnyard, OAV 1). An odor simulation model was evaluated in two consumer sensory studies. These studies confirmed that the addition of the aroma model increased the perceived saltiness of low sodium chicken broth (p < 0.05).


Assuntos
Agaricus/química , Cisteína/química , Proteínas Fúngicas/química , Odorantes/análise , Percepção Gustatória , Agaricus/metabolismo , Cloretos/análise , Cloretos/metabolismo , Cromatografia Gasosa , Culinária , Cisteína/metabolismo , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Temperatura Alta , Humanos , Hidrólise , Olfatometria , Olfato , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo
5.
J Med Microbiol ; 68(11): 1649-1654, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31609198

RESUMO

Myeloid C-type lectin receptors (CLRs) are innate immune recognition molecules that bind to microorganisms via their carbohydrate recognition domains. In this study, we utilized a library of CLRs that recognize fungal mannans. We used this library to screen against Pneumocystis carinii (Pc) homogenates or purified Pc major surface glycoprotein (Msg) present on Pneumocystis. The results demonstrated that all of the mammalian CLR hFc-fusions tested displayed significant interaction/binding with Pc organisms, and furthermore to isolated Msg. Highest Pc organism and Msg binding activities were with CLR members Mincle, Dectin-2, DC-SIGN and MCL. An immunofluorescence assay with the respective CLR hFc-fusions against whole Pc life forms corroborated these findings. Although some of these CLRs have been implicated previously as important for Pneumocystis pathogenesis (Dectin-1/Dectin-2/Mincle), this is the first analysis of head-to-head comparison of known fungal mannan binding CLR-hFc fusions with Pc. Lastly, heat treatment resulted in reducted CLR binding.


Assuntos
Proteínas Fúngicas/metabolismo , Lectinas Tipo C/metabolismo , Mananas/metabolismo , Glicoproteínas de Membrana/metabolismo , Infecções por Pneumocystis/metabolismo , Pneumocystis carinii/metabolismo , Humanos , Lectinas Tipo C/genética , Infecções por Pneumocystis/genética , Infecções por Pneumocystis/microbiologia , Pneumocystis carinii/genética , Ligação Proteica
6.
J Agric Food Chem ; 67(42): 11758-11768, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31577438

RESUMO

Patulin contamination not only is a menace to human health but also causes serious environmental problems worldwide due to the synthetic fungicides that are used to control it. This study focused on investigating the patulin degradation mechanism in Pichia caribbica at the molecular level. According to the results, P. caribbica (2 × 106 cells/mL) was able to degrade patulin from 20 µg/mL to an undetectable level in 72 h. The RNA-seq data showed patulin-induced oxidative stress and responses in P. caribbica. The deletion of PcCRG1 led to a significant decrease in patulin degradation by P. caribbica, whereas the overexpression of PcCRG1 accelerated the degradation of patulin. The study identified that PcCRG1 protein had the ability to degrade patulin in vitro. Overall, we demonstrated that the patulin degradation process in P. caribbica was more than one way; PcCRG1 was an S-adenosylmethionine-dependent methyltransferase and played an important role in the patulin degradation process in P. caribbica.


Assuntos
Proteínas Fúngicas/metabolismo , Fungicidas Industriais/metabolismo , Metiltransferases/metabolismo , Patulina/metabolismo , Pichia/metabolismo , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Metiltransferases/química , Metiltransferases/genética , Pichia/enzimologia , Pichia/genética , Alinhamento de Sequência
7.
J Agric Food Chem ; 67(40): 11025-11034, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31502841

RESUMO

Recently, we have observed a relationship between poor breadmaking quality and protease activities related to fungal infection. This study aims to identify potential gluten-degrading proteases secreted by fungi and to analyze effects of these proteases on rheological properties of dough and gluten. Fusarium graminearum-infected grain was used as a model system. Zymography showed that serine-type proteases secreted by F. graminearum degrade gluten proteins. Zymography followed by liquid chromatography-mass spectrometry (MS)/MS analysis predicted one serine carboxypeptidase and seven serine endo-peptidases to be candidate fungal proteases involved in gluten degradation. Effects of fungal proteases on the time-dependent rheological properties of dough and gluten were analyzed by small amplitude oscillatory shear rheology and large deformation extensional rheology. Our results indicate that fungal proteases degrade gluten proteins not only in the grain itself, but also during dough preparation and resting. Our study gives new insights into fungal proteases and their potential role in weakening of gluten.


Assuntos
Carboxipeptidases/metabolismo , Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Glutens/metabolismo , Doenças das Plantas/microbiologia , Triticum/microbiologia , Carboxipeptidases/química , Endopeptidases/química , Farinha/análise , Farinha/microbiologia , Proteínas Fúngicas/química , Fusarium/fisiologia , Glutens/análise , Espectrometria de Massas , Reologia , Triticum/química , Triticum/metabolismo
8.
J Agric Food Chem ; 67(39): 10984-10993, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31525294

RESUMO

The objective of the present study was to reveal the effects of four types of nitrogen sources (soymeal, yeast extract, KNO3, and ammonium tartrate) on the lipid metabolism of the oleaginous fungus Mortierella alpina using untargeted lipidomics, targeted fatty acid, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. Our results showed clear differences in the contents and compositions of lipids between four types of nitrogen sources. Soymeal and ammonium tartrate supplementation favored the accumulation of triglycerides with arachidonic acid (ARA) and C16-18 fatty acids, respectively. These results were further validated by our targeted fatty acid analysis. RT-qPCR analysis of related genes in M. alpina between the four nitrogen source conditions found that soymeal supplementation dramatically increased the expression of GPAT, ELOVL, and Δ12/Δ6 desaturase. Our findings provided new insights into the regulation of lipid biosynthesis in M. alpina and potential avenues for genetic manipulation and highlighted the importance of an optimal nitrogen source for ARA-rich oil production.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/biossíntese , Lipídeos/química , Espectrometria de Massas/métodos , Mortierella/metabolismo , Nitrogênio/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mortierella/química , Mortierella/enzimologia , Mortierella/genética
9.
J Agric Food Chem ; 67(38): 10744-10755, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31525900

RESUMO

We previously reported that ß-glucosidase BGL1 at low concentration (15 µg mL-1) from Coprinopsis cinerea exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 µg mL-1) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 µg mL-1) showed transglycosylation but BGL1 at high concentration (200 µg mL-1) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a ß(1 → 3) or ß(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-ß-d-gentiobiosyl-d-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.


Assuntos
Agaricales/enzimologia , Dissacarídeos/metabolismo , Proteínas Fúngicas/química , Oligossacarídeos/metabolismo , beta-Glucosidase/química , Agaricales/química , Agaricales/genética , Sequência de Aminoácidos , Dissacarídeos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Hidrólise , Cinética , Estrutura Molecular , Oligossacarídeos/química , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
10.
Microbiol Res ; 229: 126326, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31493702

RESUMO

Sclerotinia sclerotiorum (Lib.) de Bary produces a resistance structure called sclerotium, which guarantees its survival in soil for long periods. Morphological and melanization aspects during sclerotial development were evaluated by microscopy and qRT-PCR techniques. S. sclerotiorum produces sclerotia with different phases of maturation and melanization during growth in PDA medium. Using scanning electron microscopy we observed that there are no structural differences in the three stages of formation of melanized and non-melanized sclerotium. Through histochemical analysis we observed that the melanized sclerotium accumulates more glycogen and produces less protein than non-melanized sclerotia. Melanin was most commonly found in the rind of melanized sclerotia, and the highest concentration of lipofucsins was found in non-melanized sclerotia. These molecules are products of the lipid peroxidation pathway and are associated with oxidative stress during differentiation in fungi. The expression of histidine kinase (shk) and adenylate cyclase (sac) genes in melanized and non-melanized sclerotiawere also evaluated. The higher gene expression of shk and lesser expression of sac in non-melanized sclerotiais an indication of the participation of cell signaling in the development of these structures. The higher expression of polyketide synthase (pks), tyrosinase (tyr) and laccase (lac) in non-melanized sclerotia suggested that S. sclerotiorum can use the DHN and L-dopa pathways to produce melanin. Expression studies of the enzymes chitin synthase and glucan synthase suggest that this process occurs along with the formation of melanin. This is interesting since polysaccharides, such as chitin and ß-1,3-glucan, serve as a scaffold to which the melanin granules are cross-linked.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/fisiologia , Doenças das Plantas/microbiologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Melaninas/metabolismo , Estresse Oxidativo , Phaseolus/microbiologia
11.
Chemosphere ; 235: 995-1006, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561316

RESUMO

The influence of Cr(VI) on the degradation of tetrabromobisphenol A (TBBPA) by a typical species of white rot fungi, Pycnoporus sanguineus, was investigated in this study. The results showed that P. sanguineus together with its intracellular and extracellular enzyme could effectively degrade TBBPA. The degradation efficiency of TBBPA by both P. sanguineus and its enzymes decreased significantly when Cr(VI) concentration increased from 0 to 40 mg/L. The subsequent analysis about cellular distribution of TBBPA showed that the extracellular amount of TBBPA increased with the increment of Cr(VI) concentration, but the content of TBBPA inside fungal cells exhibited an opposite variation tendency. The inhibition of TBBPA degradation by P. sanguineus was partly attributed to the increase of cell membrane permeability and the decrease of cell membrane fluidity caused by Cr(VI). In addition, the decline of H+-ATPase and Mg2+-ATPase activities was also an important factor contributing to the suppression of TBBPA degradation in the system containing concomitant Cr(VI). Moreover, the activities of two typical extracellular lignin-degrading enzymes of P. sanguineus, MnP and Lac, were found to descend with ascended Cr(VI) level. Cr(VI) could also obviously suppress the gene expression of four intracellular enzymes implicated in TBBPA degradation, including two cytochrome P450s, glutathione S-transferases and pentachlorophenol 4-monooxygenase, which resulted in a decline of TBBPA degradation efficiency by fungal cells and intracellular enzyme in the presence of Cr(VI). Overall, this study provides new insights into the characteristics and mechanisms involved in TBBPA biodegradation by white rot fungi in an environment where heavy metals co-exist.


Assuntos
Biodegradação Ambiental , Cromo/toxicidade , Poluentes Ambientais/metabolismo , Bifenil Polibromatos/metabolismo , Pycnoporus/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Oxirredução , Pycnoporus/efeitos dos fármacos , Pycnoporus/crescimento & desenvolvimento
12.
World J Microbiol Biotechnol ; 35(10): 147, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31549247

RESUMO

The term mycoprotein refers to the protein-rich food made of filamentous fungal biomass that can be consumed as an alternative to meat. In this paper, the impact caused by the substitution of animal-origin meat in the human diet for mycoprotein on the health and the environment is reviewed. Presently, mycoprotein can be found in the supermarkets of developed countries in several forms (e.g. sausages and patties). Expansion to other markets depends on the reduction of the costs. Although scarce, the results of life cycle analyses of mycoprotein agree that this meat substitute causes an environmental impact similar to chicken and pork. In this context, the use of inexpensive agro-industrial residues as substrate for mycoprotein production has been investigated. This strategy is believed to reduce the costs involved in the fungal cultivation and lower the environmental impact of both the mycoprotein and the food industry. Moreover, several positive effects in health have been associated with the substitution of meat for mycoprotein, including improvements in blood cholesterol concentration and glycemic response. Mycoprotein has found a place in the market, but questions regarding the consumer's experience on the sensory and health aspects are still being investigated.


Assuntos
Aditivos Alimentares/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Animais , Meio Ambiente , Aditivos Alimentares/análise , Aditivos Alimentares/economia , Proteínas Fúngicas/análise , Proteínas Fúngicas/economia , Fungos/química , Fungos/crescimento & desenvolvimento , Saúde , Humanos
13.
Microb Cell Fact ; 18(1): 159, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542050

RESUMO

BACKGROUND: Xylanases randomly cleave the internal ß-1,4-glycosidic bonds in the xylan backbone and are grouped into different families in the carbohydrate-active enzyme (CAZy) database. Although multiple xylanases are detected in single strains of many filamentous fungi, no study has been reported on the composition, synergistic effect, and mode of action in a complete set of xylanases secreted by the same microorganism. RESULTS: All three xylanases secreted by Penicillium chrysogenum P33 were expressed and characterized. The enzymes Xyl1 and Xyl3 belong to the GH10 family and Xyl3 contains a CBM1 domain at its C-terminal, whereas Xyl2 belongs to the GH11 family. The optimal temperature/pH values were 35 °C/6.0, 50 °C/5.0 and 55 °C/6.0 for Xyl1, Xyl2, and Xyl3, respectively. The three xylanases exhibited synergistic effects, with the maximum synergy observed between Xyl3 and Xyl2, which are from different families. The synergy between xylanases could also improve the hydrolysis of cellulase (C), with the maximum amount of reducing sugars (5.68 mg/mL) observed using the combination of C + Xyl2 + Xyl3. Although the enzymatic activity of Xyl1 toward xylan was low, it was shown to be capable of hydrolyzing xylooligosaccharides into xylose. Xyl2 was shown to hydrolyze xylan to long-chain xylooligosaccharides, whereas Xyl3 hydrolyzed xylan to xylooligosaccharides with a lower degree of polymerization. CONCLUSIONS: Synergistic effect exists among different xylanases, and it was higher between xylanases from different families. The cooperation of hydrolysis modes comprised the primary mechanism for the observed synergy between different xylanases. This study demonstrated, for the first time, that the hydrolysates of GH11 xylanases can be further hydrolyzed by GH10 xylanases, but not vice versa.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimologia , Polissacarídeos/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucuronatos/metabolismo , Temperatura Alta , Hidrólise , Família Multigênica , Oligossacarídeos/metabolismo , Penicillium chrysogenum/química , Penicillium chrysogenum/genética , Domínios Proteicos , Xilanos/metabolismo
14.
World J Microbiol Biotechnol ; 35(9): 138, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451937

RESUMO

Monascus azaphilone pigments, including red, orange, and yellow, are world-famous food colorants. However, the pigments produced by different Monascus species vary in yields and compositions. The underlying mechanism is unclear. In this study, four wild-type Monascus strains, namely M. anka M7, M. purpureus M9, M. ruber C100, and M. aurantiacus M15, were selected as research objects according to the diversification of their pigments fermented in the same mediums and conditions. Twenty-three 3 kbp segments (300 bp overlap with adjacent segments) of the pigment gene cluster were amplified, sequenced, and assembled into the DNA sequences of the clusters. The DNA sequences of pigment biosynthetic gene clusters of the four strains showed 99.94% similarity according to the results of multiple alignment. The expression levels of 17 pigment biosynthetic genes of four strains were determined by using real-time quantitative PCR. The transcriptional regulation contributed more than the DNA sequence variation in Monascus pigments metabolism. Our result gives insight into the study of Monascus pigment biosynthesis.


Assuntos
Monascus/genética , Monascus/metabolismo , Pigmentos Biológicos/biossíntese , Transcrição Genética , Sequência de Aminoácidos , Sequência de Bases , Cor , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Variação Genética , Monascus/química , Monascus/classificação , Família Multigênica , Filogenia , Pigmentos Biológicos/química
15.
J Agric Food Chem ; 67(37): 10448-10457, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453693

RESUMO

Carabrone is isolated from Carpesium macrocephalum Franch. et Sav, which has good fungicidal activity, especially for Gaeumannomyces graminis (Get). According to previous studies, we speculated that carabrone targets the mitochondrial enzyme complex III of Get. To elucidate the mode of action, we used carabrone to induce oxidative stress and apoptosis in Get. Incubation with carabrone reduced the burst of reactive oxygen species (ROS) and mitochondrial membrane potential, as well as phosphatidylserine release. Carabrone caused ROS accumulation in mycelia by inhibiting the activity of antioxidase enzymes, among which inhibition of glutathione reductase (GR) activity was most obvious. The catalytic center of GR consists of l-cysteine residues that react with the α-methylene-γ-butyrolactone active site of carabrone. Additionally, a positive TUNEL reaction led to diffusion of the DNA electrophoresis band and upregulation of Ggmet1 and Ggmet2. We propose that carabrone inhibits antioxidant enzymes and promotes ROS overproduction, which causes membrane hyperpermeability, release of apoptotic factors, activation of the mitochondria-mediated apoptosis pathway, and fungal cell apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Ascomicetos/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ascomicetos/citologia , Ascomicetos/metabolismo , Asteraceae/química , Proteínas Fúngicas/metabolismo , Glutationa Redutase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
16.
Gene ; 718: 144073, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31446096

RESUMO

Cell morphology of the oleaginous fungus, Aspergillus oryzae BCC7051, was genetically engineered by disruption of non-essential genes involved in cell wall biosynthesis. Comparative phenotypic analysis of two disruptant strains defective either in α-1,3-glucan synthase 1 (ΔAoAgs1) or chitin synthase B (ΔAoChsB), and the wild type showed that the ΔAoAgs1 strain had no alterations in colonial growth and sporulation when grown on agar medium whereas the ΔAoChsB disruptant showed growth retardation and lower sporulation. However, tiny and loose pellets were found in the ΔAoAgs1 culture grown in liquid medium, where fungal pellet size was decreased by 35-50% of the wild type size. Further investigation of the ΔAoAgs1 mutant grown under stress-induced conditions, including high salt concentration, ionic strength and osmolarity, showed that its growth and development remained similar to that of the wild type. When cultivating the ΔAoAgs1 strain in a stirred-tank bioreactor, lipid production in terms of titer and productivity was significantly improved. As compared to the wild type, an increase of triacylglycerol and ergosterol contents with a proportional decrease in steryl ester content was observed in the ΔAoAgs1 strain. These results suggest that the morphologically engineered strain of A. oryzae is a robust cell chassis useful for exploitation in further production development of functional lipids with industrial significance.


Assuntos
Aspergillus oryzae/metabolismo , Ergosterol/biossíntese , Engenharia Metabólica , Microrganismos Geneticamente Modificados/metabolismo , Triglicerídeos/biossíntese , Aspergillus oryzae/genética , Quitina Sintase/genética , Quitina Sintase/metabolismo , Ergosterol/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Microrganismos Geneticamente Modificados/genética , Triglicerídeos/genética
17.
J Agric Food Chem ; 67(34): 9551-9559, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31379157

RESUMO

In oleaginous micro-organisms, nitrogen limitation activates adenosine monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of isocitrate dehydrogenase. We found that the overexpression of homologous AMPD in Mortierella alpina favored lipid synthesis over cell growth. Total fatty acid content in the recombinant strain was 15.0-34.3% higher than that in the control, even though their biomass was similar. During the early fermentation stage, the intracellular AMP level reduced by 40-60%, together with a 1.9-2.7-fold increase in citrate content compared with the control, therefore provided more precursors for fatty acid synthesis. Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by the blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid synthesis pathways. By coupling the energy balance with lipogenesis, this study provides new insights into cell metabolism under nitrogen-limited conditions and targets the regulation of fatty acid accumulation in oleaginous micro-organisms.


Assuntos
AMP Desaminase/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/metabolismo , Mortierella/enzimologia , AMP Desaminase/genética , Monofosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Proteínas Fúngicas/genética , Metabolismo dos Lipídeos , Mortierella/genética , Mortierella/crescimento & desenvolvimento , Mortierella/metabolismo
18.
J Med Microbiol ; 68(10): 1497-1506, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31460860

RESUMO

Aim. The aim of this work was to characterize the response of Candida albicans to atorvastatin, and to assess its in vivo antifungal capability.Methodology. The effect of atorvastatin on the growth and viability of C. albicans was assessed. The ability of the statin to alter cell permeability was quantified by measuring amino acid and protein leakage. The response of C. albicans to atorvastatin was assessed using label-free quantitative proteomics. The in vivo antifungal activity of atorvastatin was assessed using Galleria mellonella larvae infected with C. albicans.Results. Atorvastatin inhibited the growth of C. albicans. The atorvastatin-treated cells showed lower ergosterol levels than the controls, demonstrated increased calcofluor staining and released elevated quantities of amino acids and protein. Larvae infected with C. albicans showed a survival rate of 18.1±4.2 % at 144 h. In contrast, larvae administered atorvastatin (9.09 mg kg-1) displayed a survival rate of 60.2±6.4 % (P<0.05). Label-free quantitative proteomics identified 1575 proteins with 2 or more peptides and 465 proteins were differentially abundant (P<0.05). There was an increase in the abundance of enzymes with oxidoreductase and hydrolase activity in atorvastatin-treated cells, and squalene monooxygenase (4.52-fold increase) and lanosterol synthase (2.84-fold increase) were increased in abundance. Proteins such as small heat shock protein 21 (-6.33-fold) and glutathione peroxidase (-2.05-fold) were reduced in abundance.Conclusion. The results presented here indicate that atorvastatin inhibits the growth of C. albicans and is capable of increasing the survival of G. mellonella larvae infected with C. albicans.


Assuntos
Antifúngicos/farmacologia , Atorvastatina/farmacologia , Candida albicans/efeitos dos fármacos , Animais , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candidíase/microbiologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Larva/microbiologia , Mariposas/microbiologia
19.
J Agric Food Chem ; 67(37): 10505-10512, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31462045

RESUMO

An aspartic protease gene (Bsapa) was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris. The recombinant BsAPA showed maximal activity at pH 3.0 and 75 °C and remained stable at 70 °C and below, indicating the thermostable nature of BsAPA. However, heat inactivation still limits the application of BsAPA. To further improve its thermostability, an autocatalysis site (L205-F206) in BsAPA was identified and three mutants (F193W, K204P, and A371V) were generated based on the analysis of the structure neighboring the autocatalysis site. These mutants have improved thermostability, and their half-life at 75 °C increased by 0.5-, 0.2-, and 0.3-fold, respectively. A triple-site mutant (F193W/K204P/A371V) was generated, with 1.5-fold increased half-life at 80 and a 10.7 °C increased Tm, compared with those of the wild-type. These results indicate that autocatalysis of aspartic protease reduces enzyme thermostability. Furthermore, site-directed mutagenesis at regions near the autocatalysis site is an efficient approach to improve aspartic protease thermostability.


Assuntos
Ascomicetos/enzimologia , Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Ascomicetos/química , Ascomicetos/genética , Ácido Aspártico Proteases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Temperatura Alta , Cinética , Mutagênese Sítio-Dirigida , Mutação , Pichia/genética , Pichia/metabolismo
20.
J Microbiol ; 57(10): 893-899, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31463784

RESUMO

Fungal development is regulated by a variety of transcription factors in Aspergillus nidulans. Previous studies demonstrated that the NF-κB type velvet transcription factors regulate certain target genes that govern fungal differentiation and cellular metabolism. In this study, we characterize one of the VosA/VelB-inhibited developmental genes called vidA, which is predicted to encode a 581-amino acid protein with a C2H2 zinc finger domain at the C-terminus. Levels of vidA mRNA are high during the early and middle phases of asexual development and decrease during the late phase of asexual development and asexual spore (conidium) formation. Deletion of either vosA or velB results in increased vidA mRNA accumulation in conidia, suggesting that vidA transcript accumulation in conidia is repressed by VosA and VelB. Phenotypic analysis demonstrated that deletion of vidA causes decreased colony growth, reduced production of asexual spores, and abnormal formation of sexual fruiting bodies. In addition, the vidA deletion mutant conidia contain more trehalose and ß-glucan than wild type. Overall, these results suggest that VidA is a putative transcription factor that plays a key role in governing proper fungal growth, asexual and sexual development, and conidia formation in A. nidulans.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , beta-Glucanas/metabolismo
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