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1.
J Biol Chem ; 300(5): 107274, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38588809

RESUMEN

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.


Asunto(s)
Proteínas R-SNARE , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacuolas , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Vacuolas/genética , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Fusión de Membrana , Exocitosis , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Mutación
2.
PLoS Genet ; 18(1): e1009965, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-35041649

RESUMEN

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.


Asunto(s)
Aspergillus/crecimiento & desarrollo , Gliotoxina/farmacología , Metiltransferasas/genética , Factores de Transcripción/genética , Aspergillus/efectos de los fármacos , Aspergillus/genética , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus oryzae/efectos de los fármacos , Aspergillus oryzae/genética , Aspergillus oryzae/crecimiento & desarrollo , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Gliotoxina/biosíntesis , RNA-Seq
3.
Fungal Genet Biol ; 173: 103909, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38885923

RESUMEN

In the filamentous fungus Aspergillus oryzae, large amounts of amylolytic enzymes are inducibly produced by isomaltose, which is converted from maltose incorporated via the maltose transporter MalP. In contrast, the preferred sugar glucose strongly represses the expression of both amylolytic and malP genes through carbon catabolite repression. Simultaneously, the addition of glucose triggers the endocytic degradation of MalP on the plasma membrane. In budding yeast, the signal-dependent ubiquitin modification of plasma membrane transporters leads to selective endocytosis into the vacuole for degradation. In addition, during glucose-induced MalP degradation, the homologous of E6AP C-terminus-type E3 ubiquitin ligase (HulA) is responsible for the ubiquitin modification of MalP, and the arrestin-like protein CreD is required for HulA targeting. Although CreD-mediated MalP internalization occurs in response to glucose, the mechanism by which CreD regulates HulA-dependent MalP ubiquitination remains unclear. In this study, we demonstrated that three (P/L)PxY motifs present in the CreD protein are essential for functioning as HulA adaptors so that HulA can recognize MalP in response to glucose stimulation, enabling MalP internalization. Furthermore, four lysine residues (three highly conserved among Aspergillus species and yeast and one conserved among Aspergillus species) of CreD were found to be necessary for its ubiquitination, resulting in efficient glucose-induced MalP endocytosis. The results of this study pave the way for elucidating the regulatory mechanism of MalP endocytic degradation through ubiquitination by the HulA-CreD complex at the molecular level.


Asunto(s)
Aspergillus oryzae , Endocitosis , Proteínas Fúngicas , Glucosa , Proteínas de Transporte de Monosacáridos , Ubiquitina-Proteína Ligasas , Ubiquitinación , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Aspergillus oryzae/enzimología , Glucosa/metabolismo , Endocitosis/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Maltosa/metabolismo , Proteolisis , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética
4.
J Sci Food Agric ; 103(8): 3939-3949, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-36352497

RESUMEN

BACKGROUND: The ester-synthesis enzymes influenced by environmental factors during Daqu-making process largely determine the flavor of Chinese liquor, but the main ester-synthesis enzyme and its key influencer remain unclear. Here, the volatile ester profiles over the whole Daqu-making process, under different treatments, for at least 90 days, were carefully analyzed, and the potential ester-synthesis enzymes, as well as their dependently environmental factors, were explored. RESULTS: In the detected 46 volatile esters, only the short-chain (C4-C8) and medium-chain (C9-C13) ester content obviously changed, as the primary contributor discriminating different samples. Their trends were both consistent with that of the alcohols and the primary metabolism, which included alcohol acyltransferases (AATs) reaction with alcohols and acyl-CoAs as the substrates. Among the potential ester-synthesis enzymes, the typical AAT activity also exhibited the highest correlation with the short- and medium-chain esters (r > 0.78, P < 0.05). The Mantel test between environmental factors and ester production showed that temperature of Daqu was directly correlated with the short-chain esters (r = 0.58, P < 0.01) and AAT activity (r = 0.56, P < 0.01). Further, the short- and medium-chain ester content in Daqu under the treatment nearer to the reported optimal temperature of 40-50 °C of AATs reaction was overall higher than that of the other treatment Daqu. CONCLUSION: This study revealed that the temperature-dependent AATs reaction was the main enzymatic method producing the short- and medium-chain esters over the whole Daqu-making process. The results could contribute to the flavor improvement of Baijiu. © 2022 Society of Chemical Industry.


Asunto(s)
Aciltransferasas , Ésteres , Ésteres/química , Temperatura , Aciltransferasas/metabolismo , Alcoholes , Fermentación
5.
Appl Microbiol Biotechnol ; 105(7): 2701-2711, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33760931

RESUMEN

Aspergillus species are closely associated with humanity through fermentation, infectious disease, and mycotoxin contamination of food. Members of this genus produce various enzymes to degrade plant polysaccharides, including starch, cellulose, xylan, and xyloglucan. This review focus on the machinery of the xyloglucan degradation using glycoside hydrolases, such as xyloglucanases, isoprimeverose-producing oligoxyloglucan hydrolases, and α-xylosidases, in Aspergillus species. Some xyloglucan degradation-related glycoside hydrolases are well conserved in this genus; however, other enzymes are not. Cooperative actions of these glycoside hydrolases are crucial for xyloglucan degradation in Aspergillus species. KEY POINTS: •Xyloglucan degradation-related enzymes of Aspergillus species are reviewed. •Each Aspergillus species possesses a different set of glycoside hydrolases. •The machinery of xyloglucan degradation of A. oryzae is overviewed.


Asunto(s)
Glucanos , Xilanos , Glicósido Hidrolasas/metabolismo , Especificidad por Sustrato
6.
Biosci Biotechnol Biochem ; 85(2): 452-463, 2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33604648

RESUMEN

The uptake of di/tripeptides is mediated by the proton-dependent oligopeptide transporter (POT) family. In this study, 3 POT family transporters, designated PotA, PotB, and PotC were identified in Aspergillus oryzae. Growth comparison of deletion mutants of these transporter genes suggested that PotB and PotC are responsible for di/tripeptide uptake. PotA, which had the highest sequence similarity to yeast POT (Ptr2), contributed little to the uptake. Nitrogen starvation induced potB and potC expression, but not potA expression. When 3 dipeptides were provided as nitrogen sources, the expression profiles of these genes were different. PrtR, a transcription factor that regulates proteolytic genes, was involved in regulation of potA and potB but not in potC expression. Only potC expression levels were dramatically reduced by disruption of ubrA, an orthologue of yeast ubiquitin ligase UBR1 responsible for PTR2 expression. Expression of individual POT genes is apparently controlled by different regulatory mechanisms.


Asunto(s)
Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Dipéptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulación Fúngica de la Expresión Génica , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/genética
7.
Biosci Biotechnol Biochem ; 85(9): 2076-2083, 2021 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-34245563

RESUMEN

We examined the role of the intracellular α-glucosidase gene malT, which is part of the maltose-utilizing cluster (MAL cluster) together with malR and malP, in amylolytic gene expression in Aspergillus oryzae. malT disruption severely affected fungal growth on medium containing maltose or starch. Furthermore, the transcription level of the α-amylase gene was significantly reduced by malT disruption. Given that the transcription factor AmyR responsible for amylolytic gene expression is activated by isomaltose converted from maltose incorporated into the cells, MalT may have transglycosylation activity that converts maltose to isomaltose. Indeed, transglycosylated products such as isomaltose/maltotriose and panose were generated from the substrate maltose by MalT purified from a malT-overexpressing strain. The results of this study, taken together, suggests that MalT plays a pivotal role in AmyR activation via its transglycosylation activity that converts maltose to the physiological inducer isomaltose.


Asunto(s)
Aspergillus oryzae/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Factores de Transcripción/metabolismo , alfa-Amilasas/metabolismo , alfa-Glucosidasas/metabolismo , Aspergillus oryzae/genética , Genes Fúngicos , Glicosilación , Maltosa/metabolismo , Proteolisis , alfa-Amilasas/genética
8.
Curr Genet ; 66(4): 729-747, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32072240

RESUMEN

Gene expression using alternative transcription start sites (TSSs) is an important transcriptional regulatory mechanism for environmental responses in eukaryotes. Here, we identify two alternative TSSs in the enolase-encoding gene (enoA) in Aspergillus oryzae, an industrially important filamentous fungus. TSS use in enoA is strictly dependent on the difference in glycolytic and gluconeogenic carbon sources. Transcription from the upstream TSS (uTSS) or downstream TSS (dTSS) predominantly occurs under gluconeogenic or glycolytic conditions, respectively. In addition to enoA, most glycolytic genes involved in reversible reactions possess alternative TSSs. The fbaA gene, which encodes fructose-bisphosphate aldolase, also shows stringent alternative TSS selection, similar to enoA. Alignment of promoter sequences of enolase-encoding genes in Aspergillus predicted two conserved regions that contain a putative cis-element required for enoA transcription from each TSS. However, uTSS-mediated transcription of the acuN gene, an enoA ortholog in Aspergillus nidulans, is not strictly dependent on carbon source, unlike enoA. Furthermore, enoA transcript levels in glycolytic conditions are higher than in gluconeogenic conditions. Conversely, acuN is more highly transcribed in gluconeogenic conditions. This suggests that the stringent usage of alternative TSSs and higher transcription in glycolytic conditions in enoA may reflect that the A. oryzae evolutionary genetic background was domesticated by exclusive growth in starch-rich environments. These findings provide novel insights into the complexity and diversity of transcriptional regulation of glycolytic/gluconeogenic genes among Aspergilli.


Asunto(s)
Aspergillus oryzae/genética , Fosfopiruvato Hidratasa/genética , Sitio de Iniciación de la Transcripción , Regiones no Traducidas 5' , Aspergillus nidulans/genética , Aspergillus nidulans/fisiología , Aspergillus oryzae/enzimología , Carbono/metabolismo , Elementos de Facilitación Genéticos , Regulación Fúngica de la Expresión Génica , Gluconeogénesis/genética , Glucólisis/fisiología , Intrones , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
9.
J Biol Chem ; 293(28): 10926-10936, 2018 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-29789424

RESUMEN

In response to changes in nutrient conditions, cells rearrange the composition of plasma membrane (PM) transporters to optimize their metabolic flux. Not only transcriptional gene regulation, but also inactivation of specific transporters is important for fast rearrangement of the PM. In eukaryotic cells, endocytosis plays a role in transporter inactivation, which is triggered by ubiquitination of these transporters. The Nedd4 family E3 ubiquitin ligase is responsible for ubiquitination of the PM transporters and requires that a series of α-arrestin proteins are targeted to these transporters. The mechanism by which an α-arrestin recognizes its cognate transporters in response to environmental signals is of intense scientific interest. Excess substrates or signal transduction pathways are known to initiate recognition of transporters by α-arrestins. Here, we identified an endocytic-sorting signal in the monocarboxylate transporter Jen1 from yeast (Saccharomyces cerevisiae), whose endocytic degradation depends on the Snf1-glucose signaling pathway. We found that the C-terminal 20-amino acid-long region of Jen1 contains an amino acid sequence required for association of Jen1 to the α-arrestin Rod1, as well as lysine residues important for glucose-induced Jen1 ubiquitination. Notably, fusion of this region to the methionine permease, Mup1, whose endocytosis is normally induced by excess methionine, was sufficient for Mup1 to undergo glucose-induced, Rod1-mediated endocytosis. Taken together, our results demonstrate that the Jen1 C-terminal region acts as a glucose-responding degron for α-arrestin-mediated endocytic degradation of Jen1.


Asunto(s)
Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Proteínas de la Membrana/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Simportadores/metabolismo , Ubiquitina/metabolismo , Membrana Celular/metabolismo , Endocitosis , Proteínas de la Membrana/genética , Transportadores de Ácidos Monocarboxílicos/genética , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Simportadores/genética , Ubiquitinación
10.
Mol Microbiol ; 110(2): 176-190, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29995996

RESUMEN

Carbon catabolite repression (CCR) is regulated by the C2 H2 -type transcription factor CreA/Cre1 in filamentous fungi including Aspergillus oryzae. We investigated the stability and subcellular localization of CreA in A. oryzae. The abundance of FLAG-tagged CreA (FLAG-CreA) was dramatically reduced after incubation in maltose and xylose, which stimulated the export of CreA from the nucleus to the cytoplasm. Mutation of a putative nuclear export signal resulted in nuclear retention and significant stabilization of CreA. These results suggest that CreA is rapidly degraded in the cytoplasm after export from the nucleus. The FLAG-CreA protein level was reduced by disruption of creB and creC, which encode the deubiquitinating enzyme complex involved in CCR. In contrast, FLAG-CreA stability was not affected by disruption of creD which encodes an arrestin-like protein required for CCR relief. Deletion of the last 40 C-terminal amino acids resulted in remarkable stabilization and increased abundance of FLAG-CreA, whereas deletion of the last 20 C-terminal amino acids had no apparent effect on CreA stability. This result suggests that the 20 amino acid region located between positions 390 and 409 of CreA is critical for the rapid degradation of CreA.


Asunto(s)
Secuencia de Aminoácidos , Aspergillus oryzae/metabolismo , Carbono/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Represoras/metabolismo , Eliminación de Secuencia , Aspergillus oryzae/genética , Represión Catabólica/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Maltosa/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Proteínas Represoras/genética , Xilosa/metabolismo
11.
PLoS Pathog ; 13(1): e1006096, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-28052140

RESUMEN

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).


Asunto(s)
Antifúngicos/farmacología , Aspergilosis/microbiología , Aspergillus fumigatus/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Aspergillus fumigatus/efectos de los fármacos , Azoles/farmacología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Farmacorresistencia Fúngica , Proteínas Fúngicas/metabolismo , Humanos , Itraconazol/farmacología , Mutación , Fenotipo , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
12.
Biosci Biotechnol Biochem ; 83(8): 1385-1401, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31159661

RESUMEN

The koji mold Aspergillus oryzae has been used in traditional Japanese food and beverage fermentation for over a thousand years. Amylolytic enzymes are important in sake fermentation, wherein production is induced by starch or malto-oligosaccharides. This inducible production requires at least two transcription activators, AmyR and MalR. Among amylolytic enzymes, glucoamylase GlaB is produced exclusively in solid-state culture and plays a critical role in sake fermentation owing to its contribution to glucose generation from starch. A recent study demonstrated that glaB gene expression is regulated by a novel transcription factor, FlbC, in addition to AmyR in solid-state culture. Amylolytic enzyme production is generally repressed by glucose due to carbon catabolite repression (CCR), which is mediated by the transcription factor CreA. Modifying CCR machinery, including CreA, can improve amylolytic enzyme production. This review focuses on the role of transcription factors in regulating A. oryzae amylolytic gene expression.


Asunto(s)
Aspergillus oryzae/genética , Regulación Fúngica de la Expresión Génica , Glucano 1,4-alfa-Glucosidasa/metabolismo , Proteínas Fúngicas/genética , Maltosa/metabolismo , Factores de Transcripción/metabolismo
13.
Chembiochem ; 19(14): 1492-1497, 2018 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-29718548

RESUMEN

Indole sesquiterpene sespendole, which has been isolated from the filamentous fungus Pseudobotrytis terrestris FKA-25, is a specific inhibitor of lipid droplet synthesis in mouse macrophages. The biosynthetic pathway that involves genes encoding six enzymes (spdEMBQHJ) was elucidated through heterologous expression of spd genes in Aspergillus oryzae, biotransformation experiments, and in vitro enzymatic reactions with a recombinant protein, thereby revealing the mechanism underlying the characteristic modification on the indole ring, catalyzed by a set of prenyltransferase (SpdE)/cytochrome P450 (SpdJ) enzymes. Functional analysis of the homologous genes encoding these enzymes involved in the biosynthesis of lolitrem allowed a biosynthetic pathway for the bicyclic ring skeleton fused to the indole ring to be proposed.

14.
Biosci Biotechnol Biochem ; 82(5): 827-835, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29517411

RESUMEN

The Zn2Cys6-type transcription factor MalR controls the expression of maltose-utilizing (MAL) cluster genes and the production of amylolytic enzymes in Aspergillus oryzae. In the present study, we demonstrated that MalR formed a complex with Hsp70 and Hsp90 chaperones under non-inducing conditions similar to the yeast counterpart Mal63 and that the complex was released from the chaperone complex after the addition of the inducer maltose. The MalR protein was constitutively localized in the nucleus and mutation in both the putative nuclear localization signals (NLSs) located in the zinc finger motif and the C-terminal region resulted in the loss of nuclear localization. This result indicated the involvement of NSLs in the MalR nuclear localization. However, mutation in both NLSs did not affect the dissociation mode of the MalR-Hsp70/Hsp90 complex, suggesting that MalR activation induced by maltose can occur regardless of its intracellular localization.

15.
Biosci Biotechnol Biochem ; 82(1): 139-147, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29191129

RESUMEN

The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.


Asunto(s)
Afidicolina/química , Aspergillus oryzae/genética , Retículo Endoplásmico/química , Farnesiltransferasa/química , Citoplasma/química , Citoplasma/enzimología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Farnesiltransferasa/genética , Fosfatos de Poliisoprenilo/química
16.
Biosci Biotechnol Biochem ; 82(10): 1840-1848, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30011258

RESUMEN

For strain improvement of Aspergillus oryzae, development of the transformation system is essential, wherein dominant selectable markers, including drug-resistant genes, are available. However, A. oryzae generally has a relatively high resistance to many antifungal drugs effective against yeasts and other filamentous fungi. In the course of the study, while investigating azole drug resistance in A. oryzae, we isolated a spontaneous mutant that exhibited high resistance to azole fungicides and found that pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporter genes were upregulated in the mutant; their overexpression in the wild-type strain increased azole drug resistance. While deletion of the gene designated atrG resulted in increased azole susceptibility, double deletion of atrG and another gene (atrA) resulted in further azole hypersensitivity. Overall, these results indicate that the ABC transporters AtrA and AtrG are involved in azole drug resistance in A. oryzae.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Aspergillus oryzae/efectos de los fármacos , Azoles/farmacología , Farmacorresistencia Fúngica/fisiología
17.
Biochem Biophys Res Commun ; 483(1): 522-527, 2017 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-28013049

RESUMEN

Upon nutrient starvation, eukaryotic cells exploit autophagy to reconstruct cellular components. Although autophagy is induced by depletion of various nutrients such as nitrogen, carbon, amino acids, and sulfur in yeast, it was previously ambiguous whether phosphate depletion could trigger the induction of autophagy. Here, we showed that phosphate depletion induced autophagy in Saccharomyces cerevisiae, albeit to a lesser extent than nitrogen starvation. It is known that rapid inactivation of the target of rapamycin complex 1 (TORC1) signaling pathway contributes to Atg13 dephosphorylation, which is one of the cues for autophagy induction. We found that phosphate starvation caused Atg13 dephosphorylation with slower kinetics than nitrogen starvation, suggesting that poor autophagic activity during phosphate starvation was associated with slower inactivation of the TORC1 pathway. Phosphate starvation-induced autophagy requires Atg11, an adaptor protein for selective autophagy, but not its cargo recognition domain. These results suggested that Atg11 plays important roles in low-level nonselective autophagy.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Fosfatos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Relacionadas con la Autofagia/genética , Cinética , Nitrógeno/química , Fosforilación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Transcripción/genética , Proteínas de Transporte Vesicular/genética
18.
Chembiochem ; 18(23): 2317-2322, 2017 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-28924980

RESUMEN

The diterpene pleuromutilin is a ribosome-targeting antibiotic isolated from basidiomycete fungi, such as Clitopilus pseudo-pinsitus. The functional characterization of all biosynthetic enzymes involved in pleuromutilin biosynthesis is reported and a biosynthetic pathway proposed. In vitro enzymatic reactions and mutational analysis revealed that a labdane-related diterpene synthase, Ple3, catalyzed two rounds of cyclization from geranylgeranyl diphosphate to premutilin possessing a characteristic 5-6-8-tricyclic carbon skeleton. Biotransformation experiments utilizing Aspergillus oryzae transformants possessing modification enzyme genes allowed the biosynthetic pathway from premutilin to pleuromutilin to be proposed. The present study sets the stage for the enzymatic synthesis of natural products isolated from basidiomycete fungi, which are a prolific source of structurally diverse and biologically active terpenoids.


Asunto(s)
Basidiomycota/genética , Diterpenos/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Aspergillus oryzae/metabolismo , Basidiomycota/enzimología , Vías Biosintéticas/genética , Ciclización , Diterpenos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Plásmidos/genética , Plásmidos/metabolismo , Compuestos Policíclicos , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Pleuromutilinas
19.
Appl Environ Microbiol ; 83(13)2017 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-28455339

RESUMEN

Aspergillusoryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzaeIMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillusoryzae Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB, which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae.


Asunto(s)
Arrestina/metabolismo , Aspergillus oryzae/metabolismo , Represión Catabólica , Endocitosis , Proteínas Fúngicas/genética , Proteínas de Transporte de Monosacáridos/genética , alfa-Amilasas/genética , Arrestina/genética , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Carbono/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Mutación , Fosforilación , Transporte de Proteínas , alfa-Amilasas/metabolismo
20.
Appl Microbiol Biotechnol ; 101(6): 2437-2446, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28064367

RESUMEN

Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.


Asunto(s)
Aspergillus oryzae/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Manosidasas/metabolismo , Respuesta de Proteína Desplegada , alfa-Amilasas/metabolismo , Aspergillus oryzae/enzimología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Glicosilación , Manosidasas/genética , Mutación , Plásmidos/química , Plásmidos/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , alfa-Amilasas/genética
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