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1.
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
2.
Vet Parasitol ; 271: 57-63, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31303205

RESUMO

The poultry red mite, Dermanyssus gallinae, is a blood-feeding ectoparasite that affects egg-laying hens worldwide. Strategies to control this parasite have focused in the use of entomopathogenic fungi, such as Metarhizium anisopliae. However, only a few studies have evaluated the use of Aspergillus oryzae to control D. gallinae and none of them have employed native strains. In the work presented here, a novel entomopathogenic fungus was isolated from a dead D. gallinae. The results of phylogenetic analysis showed 100% similarity between the isolated strain and those of two species, A. oryzae and Aspergillus flavus, and 99.82% similarity with A. parvisclerotigenus, which were in the same branch of the Flavi section of the genus Aspergillus. This entomopathogenic fungus was a non-aflatoxin B1 producer, as shown by the presence of aflatoxin B1 in the conidial infection suspension. Morphological features of fungus in comparison with A. oryzae and A. flavus indicated that the isolated strain belonged to A. oryzae, and was named Aspergillus sp. Dg-1. The pathogenicity of Aspergillus sp. Dg-1 on D. gallinae at different life stages was then assessed under laboratory conditions. The experiments showed that the isolated strain significantly increased the mortality rate in adult mites, up to 24.83 ± 2.25, compared to the mortality rates in the control group, which were 15.17 ± 2.75 (P < 0.05). However, Aspergillus sp. Dg-1 did not have pathogenic effects on the second nymph stage of D. gallinae. Our findings demonstrate that Aspergillus sp. Dg-1 has pathogenic effects on D. gallinae in their adult stage, presenting biocontrol potential against D. gallinae.


Assuntos
Aspergillus oryzae/fisiologia , Infestações por Ácaros/microbiologia , Controle Biológico de Vetores , Doenças das Aves Domésticas/terapia , Trombiculidae/microbiologia , Animais , Aspergillus oryzae/classificação , Aspergillus oryzae/genética , Aspergillus oryzae/patogenicidade , Estágios do Ciclo de Vida , Filogenia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/parasitologia
3.
Biosci Biotechnol Biochem ; 83(8): 1385-1401, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31159661

RESUMO

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.


Assuntos
Aspergillus oryzae/genética , Regulação Fúngica da Expressão Gênica , Glucana 1,4-alfa-Glucosidase/metabolismo , Proteínas Fúngicas/genética , Maltose/metabolismo , Fatores de Transcrição/metabolismo
4.
Food Chem ; 292: 81-89, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054696

RESUMO

How to effectively increase or decrease the ability of A. oryzae to produce enzymes was the key to improve the quality of soy sauce. However, multi-core property of A. oryzae resulted in genetic instability of the new strain. Here, A. oryzae 3.042-3 which can stably produce mononuclear spores was constructed based on A. oryzae 3.042. A. oryzae 3.042-3-c obtained by transformation of the fragment of cis-CreA into A. oryzae 3.042-3 exhibited genetic stability. The fragment containing the cis-acting and the promoter CreA from A. oryzae was connected to chromosome VII in A. oryzae 3.042-3-c. Compared with A. oryzae 3.042-3, the cellulase activity of A. oryzae 3.042-3-c was reduced by 50.5% and the pectinase activity was decreased by 10.0%. At the end of the soy sauce fermentation, the salt-free solid content of A. oryzae 3.042-3-c was higher 58.9% than that of A. oryzae 3.042-3. The kinds and contents of the flavor components of the soy sauce from the fermentation by A. oryzae 3.042-3-c were higher than those of the A. oryzae 3.042 and A. oryzae 3.042-3, especially in alcohols and esters. HEMF was only found in the soy sauce from A. oryzae 3.042-3-c. The results indicated that the new strain A. oryzae 3.042-3-c could improve the quality of soy sauce from the low-salt solid fermentation by decreasing enzyme activity of cellulase and pectinase.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Alimentos de Soja/análise , Aspergillus oryzae/genética , Celulase/genética , Celulase/metabolismo , Cromossomos Fúngicos , Qualidade dos Alimentos , Proteínas Fúngicas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alimentos de Soja/microbiologia
5.
Gene ; 706: 106-114, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31039437

RESUMO

Biological significance of 18-carbon polyunsaturated fatty acids, γ-linolenic acid (GLA; C18:3 n-6) and dihomo-γ-linolenic acid (DGLA; C20:3 n-6) has gained much attention in the systematic development of optimized strains for industrial applications. In this work, a n-6 PUFAs-producing strain of Aspergillus oryzae was generated by manipulating metabolic reactions in fatty acid modification and triacylglycerol biosynthesis. The codon-optimized genes coding for Δ6-desaturase and Δ6-elongase of Pythium sp., and diacylglycerol acyltransferase 2 (mMaDGAT2) of Mortierella alpina were co-transformed in a single vector into A. oryzae BCC14614, yielding strain TD6E6-DGAT2. Comparative phenotypic analysis showed that a 70% increase of lipid titer was found in the engineered strain, which was a result of a significant increase in triacylglycerol (TAG) content (52.0 ±â€¯1.8% of total lipids), and corresponded to the increased size of lipid particles observed in the fungal cells. Interestingly, the proportions of GLA and DGLA in neutral lipids of the engineered strain were similar, with the highest titers obtained in the high C:N culture (29:0; 6% glucose) during the lipid-accumulating stage of growth. Time-course expression analysis of the engineered strain revealed transcriptional control of TAG biosynthesis through a co-operation between the native DGAT2 of A. oryzae and the transformed mMaDGAT2.


Assuntos
Aspergillus oryzae/metabolismo , Lipídeos/biossíntese , Engenharia Metabólica/métodos , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidônico/biossíntese , Aspergillus oryzae/genética , Aspergillus oryzae/fisiologia , Vias Biossintéticas , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Proteínas Fúngicas/genética , Mortierella/genética , Triglicerídeos/biossíntese , Ácido gama-Linolênico/biossíntese
6.
J Biosci Bioeng ; 128(2): 129-134, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30824179

RESUMO

Aspergillus luchuensis NBRC4314 recently underwent genome sequencing. We have not used the frequently used protoplast-polyethylene glycol (PEG) method but have used agrobacterium-mediated transformation (AMT) to genetically engineer this strain because it was difficult to generate protoplasts using commercial cell wall lytic enzymes. In this study, we initially investigated the various conditions for protoplast formation in A. luchuensis. We found that A. luchuensis protoplasts could be generated using a minimal medium for the preculture medium, a static culture for the preculture condition, and Yatalase and α-1,3-glucanase as cell-wall lytic enzymes. These protoplasts could then be transformed with the protoplast-PEG method. Because α-1,3-glucanase was needed to form protoplasts in A. luchuensis, we investigated the role of the α-1,3-glucan synthase gene agsE in protoplast formation, one of five α-1,3-glucan synthase genes in A. luchuensis and a homolog of the major α-1,3-glucan synthase agsB in Aspergillus nidulans. We disrupted agsE in A. luchuensis (ΔagsE) with AMT and found that protoplast formation in ΔagsE was comparable with protoplast formation in Aspergillus oryzae with Yatalase. The ΔagsE protoplasts were also competent for transformation with the protoplast-PEG method. Hence, agsE appears to inhibit protoplast formation in A. luchuensis.


Assuntos
Aspergillus oryzae/citologia , Aspergillus oryzae/genética , Glucosiltransferases/genética , Protoplastos/metabolismo , Transformação Genética , Aspergillus nidulans/genética
7.
Bioprocess Biosyst Eng ; 42(5): 753-761, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30805716

RESUMO

Kojic acid is a kind of secondary metabolites, whose biosynthesis pathway remains unclear to date. It is produced industrially by microbial fermentation, and thus, mutagenesis breeding still plays a vital role for obtaining strains with high kojic acid production. The starting strain KA-11 isolated from mildewed fruits was identified as Aspergillus oryzae and then subjected to a combined mutagenesis program including microwave mutagenesis, UV irradiation, heat-LiCl, atmospheric, and room temperature plasma (ARTP). The kojic acid production was increased by 47.0%, 87.1%, 126.2%, and 292.3% compared with the starting strain KA-11 after each mutagenesis stage. A mutant strain AR-47 with kojic acid production of 96.5 g/L in flask-shake fermentation was finally obtained. The fermentation time also decreased from 7 to 5 days. Real-time quantitative PCR was used to determine the transcriptional expression levels of genes that may be relevant to kojic acid biosynthesis, including kojA, kojR, kojT, AO090113000141, AO090113000143, AO090113000145, nrtA, and laeA. The results showed that the transcriptional expression levels of all these genes in high yield strain AR-47 had increased compared with the starting strain KA-11.


Assuntos
Aspergillus oryzae , Proteínas Fúngicas , Mutagênese , Pironas/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética
8.
Biosci Biotechnol Biochem ; 83(8): 1557-1569, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30776970

RESUMO

In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of Aspergillus oryzae RIB40 and RIB128 and performed inter-strain expression of 37 sequence-diverse genes for mimicking HI. Four HET domain genes were identified to cause severe growth inhibition in a strain- or sequence-specific manner. Furthermore, SNPs responsible for the inhibition of cell growth were identified. This study provides an important insight into the physiological significance of sequence diversity of HET domain genes and their potential functions in HI of A. oryzae.


Assuntos
Aspergillus oryzae/crescimento & desenvolvimento , Aspergillus oryzae/genética , Genes Fúngicos , Filogenia , Polimorfismo de Nucleotídeo Único , Especificidade da Espécie
9.
J Microbiol Biotechnol ; 29(4): 577-586, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30786701

RESUMO

The engineered Aspergillus oryzae has a high NADPH demand for the xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, which is the main enzyme responding for the NADPH regeneration. The open reading frame and cDNA of putative A. oryzae G6PDH (AoG6PDH) were obtained, which was followed by heterogeneous expression in Escherichia coli and was purified as a his6-tagged protein presently. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed the maximal activity at pH 7.5 and the optimal temperature was 50 °C. This enzyme had half-life time of 33.3 min at 40 °C. Kinetics assay showed that AoG6PDH was strictly dependent on NADP+ (Km = 6.3 µM, kcat = 1000.0 s-1, kcat/Km =158.7 s-1·µM-1) as cofactor. The Km and kcat/Km value of glucose-6-phosphate were 109.7 s-1·µM-1 and 9.1 -1·µM-1 respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate steady-state ordered BiBi mechanism, where NADP+ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae for improving the xylose utilization and yields of valued metabolites.


Assuntos
Aspergillus oryzae/metabolismo , Glucosefosfato Desidrogenase/biossíntese , Engenharia Metabólica/métodos , Via de Pentose Fosfato , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Clonagem Molecular , DNA Fúngico , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , NADP/metabolismo , Oxirredução , Via de Pentose Fosfato/genética , Temperatura Ambiente , Xilose/metabolismo
10.
J Ind Microbiol Biotechnol ; 46(2): 231-239, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30604237

RESUMO

Salt stress can trigger several physiological responses in microorganisms such as the increasing accumulation of unsaturated fatty acid, which was biosynthesized by delta-9 fatty acid desaturases (D9D) at the first step. In the present study, two D9D genes, designated AoD9D1 and AoD9D2, were isolated from Aspergillus oryzae. The expression analysis showed that AoD9D1 and AoD9D2 were upregulated under salt stress. To investigate the function of AoD9D, transgenic Saccharomyces cerevisiae strains that heterologously expressed AoD9D were exposed to salinity condition. These transgenic strains exhibited greater tolerance to salt stress than wild-type strains, and the heterologous expression of AoD9D increased the content in unsaturated fatty acids as compared to control cells. Moreover, AoD9D1 and AoD9D2 both contained fatty acid desaturase (FAD) and cytochrome b5-like Heme/Steroid-binding domains (Cyt-b5). S. cerevisiae separately transformed with the gene fragments coding for the FAD and Cyt-b5 domains in the AoD9D1 protein grew better and accumulated a higher concentration of unsaturated FAs than the control. Altogether, the heterologous expression of AoD9D enhanced the tolerance of transgenic S. cerevisiae to high salinity stress with increased accumulation of unsaturated fatty acid. The results provide some practical basis for the successful development of salt-tolerant fermentation microorganisms.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Saccharomyces cerevisiae/metabolismo , Tolerância ao Sal/genética , Estearoil-CoA Dessaturase/metabolismo , Sequência de Aminoácidos , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase/genética
11.
Braz J Microbiol ; 50(1): 43-52, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30637636

RESUMO

Ergosterol, a unique component of fungal cells, is not only important for fungal growth and stress responses but also holds great economic value. Limited studies have been performed on ergosterol biosynthesis in Aspergillus oryzae, a safe filamentous fungus that has been used for the manufacture of oriental fermented foods. This study revealed that the ergosterol biosynthesis pathway is conserved between Saccharomyces cerevisiae and A. oryzae 3.042 by treatment with ergosterol biosynthesis inhibitors and bioinformatics analysis. However, the ergosterol biosynthesis pathway in A. oryzae 3.042 is more complicated than that in S. cerevisiae as there are multiple paralogs encoding the same biosynthetic enzymes. Using RNA-seq, this study identified 138 and 104 differentially expressed genes (DEG) in response to the ergosterol biosynthesis inhibitors tebuconazole and terbinafine, respectively. The results showed that the most common DEGs were transport- and metabolism-related genes. There were only 17 DEGs regulated by both tebuconazole and terbinafine treatments and there were 256 DEGs between tebuconazole and terbinafine treatments. These results provide new information on A. oryzae ergosterol biosynthesis and regulation mechanisms, which may lay the foundation for genetic modification of the ergosterol biosynthesis pathway in A. oryzae.


Assuntos
Aspergillus oryzae/efeitos dos fármacos , Aspergillus oryzae/genética , Ergosterol/biossíntese , Fungicidas Industriais/farmacologia , Transcrição Genética/efeitos dos fármacos , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Terbinafina/farmacologia , Triazóis/farmacologia
12.
J Microbiol Biotechnol ; 29(2): 230-234, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30602269

RESUMO

Currently, the genetic modification of Aspergillus oryzae is mainly dependent on protoplastmediated transformation (PMT). In this study, we established a dual selection marker system in an industrial A. oryzae 3.042 strain by using Agrobacterium tumefaciens-mediated transformation (ATMT). We first constructed a uridine/uracil auxotrophic A. oryzae 3.042 strain and a pyrithiamine (PT)-resistance binary vector. Then, we established the ATMT system by using uridine/uracil auxotrophy and PT-resistance genes as selection markers. Finally, a dual selection marker ATMT system was developed. This study demonstrates a useful dual selection marker transformation system for genetic manipulations of A. oryzae 3.042.


Assuntos
Agrobacterium tumefaciens/genética , Aspergillus oryzae/genética , Genes Fúngicos/genética , Microbiologia Industrial/métodos , Transformação Genética , Antimetabólitos/farmacologia , Aspergillus oryzae/efeitos dos fármacos , Aspergillus oryzae/metabolismo , Biomarcadores , Resistência Microbiana a Medicamentos/genética , Vetores Genéticos , Piritiamina/farmacologia , Uracila/metabolismo , Uridina/metabolismo
13.
BMC Genomics ; 20(1): 16, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30621576

RESUMO

BACKGROUND: Transcription factors (TFs) specifically bind to DNA sequences and control the expression of target genes. AoXlnR is a key TF involved in the expression of xylanolytic and cellulolytic enzymes in the filamentous fungi, Aspergillus oryzae. Genomic SELEX-Seq (gSELEX-Seq) can reveal the in vitro binding sites of a TF in a genome. To date, the gene expression network controlled by AoXlnR in A. oryzae is not fully explored. In this study, the data from gSELEX-Seq analysis and data mining were applied toward a comprehensive investigation of the AoXlnR-regulated transcriptional network in A. oryzae. RESULTS: Around 2000 promoters were selected as AoXlnR-binding DNAs using gSELEX-Seq, consequently identifying the genes downstream of them. On the other hand, 72 differentially expressed genes (DEGs) related to AoXlnR had been determined by microarray analysis. The intersecting set of genes, that were found using the gSELEX-Seq and the microarray analysis, had 51 genes. Further, the canonical AoXlnR-binding motifs, 5'-GGCT(A/G) A-3', were successfully identified in gSELEX-Seq. The motif numbers in each promoter of the DEGs and differential expression levels were correlated by in silico analysis. The analysis showed that the presence of both 5'-GGCTAA-3' and 5'-GGCTGA-3' motif has significantly high correlation with the differential expression levels of the genes. CONCLUSIONS: Genes regulated directly by AoXlnR were identified by integrated mining of data obtained from gSELEX-Seq and microarray. The data mining of the promoters of differentially expressed genes revealed the close relation between the presence of the AoXlnR-binding motifs and the expression levels of the downstream genes. The knowledge obtained in this study can contribute greatly to the elucidation of AoXlnR-mediated cellulose and xylan metabolic network in A. oryzae. The pipeline, which is based on integrated mining of data consisting of both in vitro characterization of the DNA-binding sites and TF phenotype, can be a robust platform for comprehensive analysis of the gene expression network via the TFs.


Assuntos
Aspergillus oryzae/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genômica , Transativadores/genética , Sítios de Ligação , Celulose/genética , Regulação Fúngica da Expressão Gênica , Redes e Vias Metabólicas/genética , Análise em Microsséries , Regiões Promotoras Genéticas , Técnica de Seleção de Aptâmeros , Fatores de Transcrição/genética , Xilanos/genética
14.
BMC Genomics ; 20(1): 86, 2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678639

RESUMO

BACKGROUND: Nicotine-degrading microorganisms (NDMs) have recently received much attention since they can consume nicotine as carbon and nitrogen source for growth. In our previous work, we isolated an efficient nicotine-degrading fungus Aspergillus oryzae 112822 and first proposed a novel demethylation pathway of nicotine degradation in fungi. However, the underlying mechanisms of the demethylation pathway remain unresolved. In the present study, we performed a comparative transcriptome analysis to elucidate the molecular mechanisms of nicotine tolerance and degradation in A. oryzae 112822. RESULTS: We acquired a global view of the transcriptional regulation of A. oryzae 112822 exposed to nicotine and identified 4381 differentially expressed genes (DEGs) by nicotine treatment. Candidate genes encoding cytochrome P450 monooxygenases (CYPs), FAD-containing amine oxidase, molybdenum cofactor (Moco)-containing hydroxylase, and NADH-dependent and FAD-containing hydroxylase were proposed to participate in the demethylation pathway of nicotine degradation. Analysis of these data also revealed that increased energy was invested to drive nicotine detoxification. Nicotine treatment led to overproduction of reactive oxygen species (ROS), which formed intracellular oxidative stress that could induce the expression of several antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx). Thioredoxin system was induced to restore the intracellular redox homeostasis. Several glutathione S-transferases (GSTs) were induced, most likely to participate in phase II detoxification of nicotine by catalyzing the conjugation of glutathione (GSH) to active metabolites. The toxin efflux pumps, such as the ATP-Binding Cassette (ABC) transporters and the major facilitator superfamily (MFS) transporters, were overexpressed to overcome the intracellular toxin accumulation. By contrast, the metabolic pathways related to cellular growth and reproduction, such as ribosome biogenesis and DNA replication, were inhibited by nicotine treatment. CONCLUSION: These results revealed that complex regulation networks, involving detoxification, transport, and oxidative stress response accompanied by increased energy investment, were developed for nicotine tolerance and degradation in A. oryzae 112822. This work provided the first insight into the metabolic regulation of nicotine degradation and laid the foundation for further revealing the molecular mechanisms of the nicotine demethylation pathway in filamentous fungi.


Assuntos
Aspergillus oryzae/genética , Regulação Fúngica da Expressão Gênica , Nicotina/metabolismo , Aspergillus oryzae/metabolismo , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Redes e Vias Metabólicas/genética , Estresse Oxidativo/genética , Análise de Sequência de RNA
15.
Org Biomol Chem ; 17(2): 248-251, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30548032

RESUMO

A putative three-gene cluster for asperterpenoid A was identified. Step-wise reconstitution of this gene cluster in Aspergillus oryzae reveals that astC encodes a sesterterpene cyclase to synthesize preasperterpenoid A, which is dually oxidized by a P450 enzyme AstB to give asperterpenoid A along with a minor product asperterpenoid B, and asperterpenoid A is further oxidized by another P450 eznyme AstA to afford a new sesterterpenoid asperterpenoid C. Unexpectedly, asperterpenoids A and B, but not the final product asperterpenoid C, exhibit potent inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B with IC50 values of 3-6 µM.


Assuntos
Antituberculosos/metabolismo , Antituberculosos/farmacologia , Aspergillus oryzae/metabolismo , Mycobacterium tuberculosis/enzimologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Liases/metabolismo , Família Multigênica , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico
16.
Org Biomol Chem ; 17(2): 374-379, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30556556

RESUMO

Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M. theobromae identified an R-PKS gene, men1, and an NR-PKS gene, men2, which both exhibited highest expression levels during the menisporopsin A production phase. These were cloned into separate vectors for heterologous expression in Aspergillus oryzae NSAR1. Unexpectedly, coexpression of the two PKSs alone was sufficient to catalyze the formation of the macrocyclic polylactone, ascotrichalactone A, a structural derivative of menisporopsin A. The unanticipated esterification and cyclolactonization activities could reside in the unusual thioesterase domain of the NR-PKS, which is similar to that of the NRPS catalyzing elongation and cyclization of trilactone in enterobactin biosynthesis and that of modular PKSs catalyzing macrodiolide formation in elaiophylin and conglobatin biosyntheses.


Assuntos
Ascomicetos/metabolismo , Aspergillus oryzae/metabolismo , Vias Biossintéticas , Proteínas Fúngicas/metabolismo , Macrolídeos/metabolismo , Policetídeo Sintases/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Expressão Gênica , Genes Fúngicos , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/genética , Transcriptoma
17.
J Biotechnol ; 290: 44-52, 2019 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-30576682

RESUMO

Arabinogalactan proteins are proteoglycans located in the plant cell wall. Most arabinogalactan proteins are composed of carbohydrate moieties of ß-(1→3)-galactan main chains with ß-(1→6)-galactan side chains terminated by other glycans. In this study, three novel endo-ß-(1→3)-galactanases were identified and the substrate specificity was further studied using well-defined galactan oligomers. Linear and branched ß-(1→3)-linked galactans, which resemble the carbohydrate core of the arabinogalactan protein, were used for the characterization of endo-ß-(1→3)-galactanases. The identified enzymes required at least three consecutive galactose residues for activity. Non-substituted regions were preferred, but substituents in the -2 and +2 and in some cases also -1 and +1 subsites were tolerated to some extent, depending on the branching pattern, however at a significantly lower rate/frequency.


Assuntos
Galactose/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Plantas , beta-Galactosidase , Aspergillus oryzae/genética , Configuração de Carboidratos , Clonagem Molecular , Galactose/química , Oligossacarídeos/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , beta-Galactosidase/química , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
18.
J Sci Food Agric ; 99(7): 3359-3366, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30584796

RESUMO

BACKGROUND: The salt tolerance of proteases secreted by Aspergillus oryzae 3.042 closely relates to the utilization of raw materials and the quality of soy sauce. However, little is known about the salt-tolerant proteases and their salt-tolerant mechanisms. RESULTS: In this study, we isolated and identified a salt-tolerant alkaline protease (AP, approximately 29 kDa) produced by A. oryzae 3.042. It was considered as a metal-ion-independent serine protease. The optimum and stable pH values were both pH 9.0 and the optimum temperature was 40 °C. Over 20% relative activity of AP remained in the presence of 3.0 mol L-1 NaCl after 7 days, but its Km and Vmax were only mildly influenced by the presence of 3.0 mol L-1 NaCl, indicating its outstanding salt tolerance. Furthermore, AP was more stable than non-salt-tolerant protease at high salinity. The salt-tolerant mechanisms of AP could be due to more salt bridges, higher proportion of ordered secondary structures and stronger hydrophobic amino acid residues in the interior. CONCLUSIONS: The above results are vital for maintaining, activating and/or modulating the activity of AP in high-salt environments. They would also provide theoretical guidance for the modification of AP and the engineering of A. oryzae 3.042 so as to secrete more AP. © 2018 Society of Chemical Industry.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Aspergillus oryzae/química , Aspergillus oryzae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Cloreto de Sódio/química , Cloreto de Sódio/metabolismo , Temperatura Ambiente
19.
Appl Microbiol Biotechnol ; 102(20): 8739-8751, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30109399

RESUMO

Microbial L-malate production from renewable feedstock is a promising alternative to petroleum-based chemical synthesis. However, high L-malate production of Aspergillus oryzae was achieved to date using organic nitrogen, with inorganic nitrogen still unable to meet industrial applications. In the current study, we constructed a screening system and nitrogen supply strategy to improve L-malate production with ammonium sulphate [(NH4)2SO4] as the sole nitrogen source. First, we generated and identified a high-producing mutant FMME218-37, which stably boosted L-malate production from 30.73 to 78.12 g/L, using a combined screening system with morphological characteristics. Then, by analyzing the fermentation parameters and physiological characteristics, we further speculated the key factor was the unbalance of carbon and nitrogen absorption. Finally, the titer and productivity of L-malate was increased to 95.2 g/L and 0.57 g/(L h) by regulating the nitrogen supply module to balance carbon and nitrogen absorption, which represented the highest level in A. oryzae with (NH4)2SO4 as nitrogen source achieved to date. Moreover, our findings using a low-cost substrate may lead to building an economical cell factory of A. oryzae for L-malate production.


Assuntos
Sulfato de Amônio/metabolismo , Aspergillus oryzae/metabolismo , Malatos/metabolismo , Aspergillus oryzae/genética , Carbono/metabolismo , Fermentação
20.
World J Microbiol Biotechnol ; 34(2): 35, 2018 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-29427255

RESUMO

The High osmolarity glycerol (HOG) gene family plays crucial roles in various developmental and physiological processes in fungi, such as the permeability of cell membrane, chlamydospore formation and stress signaling. Although the function of HOG genes has been investigated in Saccharomyces cerevisiae and some filamentous fungi, a comprehensive analysis of HOG gene family has not been performed in Aspergillus oryzae, a fungi mainly used for the production of soy sauce. In this study, we identified and corrected a total of 90 HOG genes from the A. oryzae genome. According to the phylogenetic relationship, they were divided into four discrete groups (Group A-D) comprising of 16, 24, 30 and 20 proteins, respectively. Six conserved motifs and exon-intron structures were examined among all HOG proteins to reveal the diversity of AoHOG genes. Based on transcriptome technology, the expression patterns of AoHOG genes across all developmental stages was identified, suggesting that the AoHOG gene family mainly functions in the logarithmic phase of development. The expression profiles of AoHOG genes under different concentrations of salt stress indicated that AoHOG genes are extensively involved in salt stress response, with possibly different mechanisms. The genome-wide identification, evolutionary, gene structures and expression analyses of AoHOG genes provide a comprehensive overview of this gene family as well as their potential involvements in development and stress responses. Our results will facilitate further research on HOG gene family regarding their physiological and biochemical functions.


Assuntos
Aspergillus oryzae/genética , Perfilação da Expressão Gênica , Genes Fúngicos/genética , Estudo de Associação Genômica Ampla , Glicerol/metabolismo , Aspergillus oryzae/fisiologia , Evolução Molecular , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Concentração Osmolar , Pressão Osmótica , Filogenia , Tolerância ao Sal , Transdução de Sinais , Estresse Fisiológico , Transcriptoma
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