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1.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478725

RESUMO

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.


Assuntos
Aspergillus oryzae/genética , Bioquímica/métodos , Proteínas Fúngicas/biossíntese , Mutação/genética , Aspergillus oryzae/metabolismo , DNA/metabolismo , Polietilenoglicóis/química , Protoplastos/metabolismo , Esporos Fúngicos/metabolismo
2.
J Biosci Bioeng ; 130(3): 227-232, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32487497

RESUMO

Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.


Assuntos
Aspergillus oryzae/efeitos dos fármacos , Aspergillus oryzae/genética , Farmacorresistência Fúngica/genética , Edição de Genes , Genes Fúngicos/genética , Marcadores Genéticos/genética , Piritiamina/farmacologia , Sistemas CRISPR-Cas/genética
3.
Nat Commun ; 11(1): 1106, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107379

RESUMO

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.


Assuntos
Aspergillus flavus/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genômica , Aspergillus flavus/classificação , Aspergillus flavus/enzimologia , Aspergillus oryzae/classificação , Aspergillus oryzae/enzimologia , Reatores Biológicos , Metabolismo dos Carboidratos/genética , Produtos Agrícolas/microbiologia , DNA Fúngico/genética , Fermentação , Alimentos e Bebidas Fermentados , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Redes e Vias Metabólicas/genética , Família Multigênica , Fenótipo , Filogenia , Doenças das Plantas/prevenção & controle , Metabolismo Secundário/genética
4.
J Agric Food Chem ; 68(9): 2757-2764, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32026695

RESUMO

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Cloreto de Sódio/metabolismo , Aspergillus oryzae/química , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/química , Mutagênese , Peptídeo Hidrolases/química , Alimentos de Soja/análise , Alimentos de Soja/microbiologia
5.
J Agric Food Chem ; 68(5): 1419-1426, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31888328

RESUMO

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.


Assuntos
Bebidas Alcoólicas/microbiologia , Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeos/metabolismo , alfa-Glucosidases/metabolismo , Bebidas Alcoólicas/análise , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Etanol/metabolismo , Fermentação , Proteínas Fúngicas/genética , Glicosídeos/química , Glicosilação , Maltose/metabolismo , Oryza/metabolismo , Oryza/microbiologia , alfa-Glucosidases/genética
6.
J Biosci Bioeng ; 129(5): 573-580, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31919019

RESUMO

The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-ß-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A. oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.


Assuntos
Aspergillus oryzae/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Aspergillus oryzae/química , Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Glicoproteínas/genética , Polissacarídeos/química , alfa-Amilases/genética , alfa-Amilases/metabolismo
7.
J Biosci Bioeng ; 129(2): 150-154, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31492608

RESUMO

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80-100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-ß-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30-35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2-8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.


Assuntos
Aspergillus oryzae/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Glucana 1,4-alfa-Glucosidase/genética , Regiões Promotoras Genéticas , Aspergillus oryzae/genética , Biocatálise , Hidrolases de Éster Carboxílico/genética , Eletroforese em Gel de Poliacrilamida , Cinética , Peso Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(2): 1174-1180, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882449

RESUMO

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.


Assuntos
Alcaloides/biossíntese , Ascomicetos/enzimologia , Ascomicetos/metabolismo , Indolizidinas/metabolismo , Policetídeo Sintases/metabolismo , Fosfato de Piridoxal/metabolismo , Alcaloides/genética , Alcaloides/isolamento & purificação , Antibacterianos/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Catálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Hidroxilação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Família Multigênica , Filogenia , Policetídeo Sintases/classificação , Policetídeo Sintases/genética , Policetídeos , Fosfato de Piridoxal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
DNA Res ; 26(6): 465-472, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31755931

RESUMO

Aspergillus oryzae is an industrially useful species, of which various strains have been identified; however, their genetic relationships remain unclear. A. oryzae was previously thought to be asexual and unable to undergo crossbreeding. However, recent studies revealed the sexual reproduction of Aspergillus flavus, a species closely related to A. oryzae. To investigate potential sexual reproduction in A. oryzae and evolutionary history among A. oryzae and A. flavus strains, we assembled 82 draft genomes of A. oryzae strains used practically. The phylogenetic tree of concatenated genes confirmed that A. oryzae was monophyletic and nested in one of the clades of A. flavus but formed several clades with different genomic structures. Our results suggest that A. oryzae strains have undergone multiple inter-genomic recombination events between A. oryzae ancestors, although sexual recombination among domesticated species did not appear to have occurred during the domestication process, at least in the past few decades. Through inter- and intra-cladal comparative analysis, we found that evolutionary pressure induced by the domestication of A. oryzae appears to selectively cause non-synonymous and gap mutations in genes involved in fermentation characteristics, as well as intra-genomic rearrangements, with the conservation of industrially useful catalytic enzyme-encoding genes.


Assuntos
Adaptação Fisiológica/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genômica/métodos , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Aspergillus oryzae/classificação , Aspergillus oryzae/metabolismo , Evolução Molecular , Fermentação/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiologia Industrial/métodos , Mutação , Filogenia , Recombinação Genética , Reprodução/genética , Especificidade da Espécie
10.
BMC Biotechnol ; 19(1): 70, 2019 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-31655589

RESUMO

BACKGROUND: Aspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid. Thus, for the production of valuable secondary metabolites by genetic engineering, the species is considered a clean host, enabling easy purification from cultured cells. A. oryzae has been evaluated for secondary metabolite production utilizing strong constitutive promoters of genes responsible for primary metabolism. However, secondary metabolites are typically produced by residual nutrition after microbial cells grow to the stationary phase and primary metabolism slows. We focused on a promoter of the secondary metabolism gene kojA, a component of the kojic acid biosynthetic gene cluster, for the production of other secondary metabolites by A. oryzae. RESULTS: A kojA disruptant that does not produce kojic acid was utilized as a host strain for production. Using this host strain, a mutant that expressed a polyketide synthase gene involved in polyketide secondary metabolite production under the kojA gene promoter was constructed. Then, polyketide production and polyketide synthase gene expression were observed every 24 h in liquid culture. From days 0 to 10 of culture, the polyketide was continuously produced, and the synthase gene expression was maintained. Therefore, the kojA promoter was activated, and it enabled the continuous production of polyketide for 10 days. CONCLUSIONS: The combined use of the kojA gene promoter and a kojA disruptant proved useful for the continuous production of a polyketide secondary metabolite in A. oryzae. These findings suggest that this combination can be applied to other secondary metabolites for long-term production.


Assuntos
Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Policetídeos/metabolismo , Regiões Promotoras Genéticas/genética
11.
Curr Microbiol ; 76(12): 1443-1451, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31541261

RESUMO

U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5'-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.


Assuntos
Aspergillus oryzae/genética , Sistemas CRISPR-Cas , Proteínas Fúngicas/genética , Edição de Genes , Regiões Promotoras Genéticas , RNA Polimerase III/genética , RNA Fúngico/genética , RNA Guia/genética , Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Polimerase III/metabolismo , RNA Fúngico/metabolismo , RNA Guia/metabolismo
12.
J Ind Microbiol Biotechnol ; 46(12): 1769-1780, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31541399

RESUMO

The ZRT, IRT-like protein (ZIP) family exists in many species and plays an important role in many biological processes, but little is known about ZIP genes in Aspergillus oryzae. Here, 10 ZIP genes in A. oryzae were identified and these were classified into four groups based on phylogenetic analysis. The structures of these AoZip genes were determined, which indicated a great divergence of AoZip members from different groups. Synteny analysis revealed that AoZip7, AoZip8, and AoZip10 are conserved among Aspergillus species. We also found that the promoter regions of AoZip2, AoZip7, AoZip8, and AoZip10 contain multiple conserved response elements. Expression analysis revealed that AoZips exhibited different expression patterns in response to different metal treatments. Moreover, overexpression and RNA-interference (RNAi) of AoZip2 led to a decrease in mycelium growth diameter and inhibited conidia formation. AoZip2 overexpression and RNAi strains showed distinct sensitivity to severely Zn/Mn-depleted stress. In addition, kojic acid production was markedly lower in AoZip2 overexpression and RNAi strains than in the control strains, and the expression of kojA, kojR, and kojT was down-regulated in AoZip2 overexpression and RNAi strains. This study provides new insights into our understanding of ZIP genes and lays a foundation for further investigation of their roles in Aspergillus oryzae.


Assuntos
Aspergillus oryzae/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas Fúngicas/metabolismo , Pironas/metabolismo , Aspergillus oryzae/genética , Proteínas de Transporte de Cátions/genética , Proteínas Fúngicas/genética , Filogenia
13.
J Appl Microbiol ; 127(5): 1511-1520, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31429498

RESUMO

AIMS: To use genome-wide single nucleotide polymorphisms (total SNPs) to develop a molecular method for distinguishing Aspergillus flavus and Aspergillus oryzae. METHODS AND RESULTS: Thirteen A. flavus and eleven A. oryzae genome sequences were obtained from the National Center for Biotechnology Information. These sequences were analysed by Mauve, a multiple-genome alignment program, to extract total SNPs between isolates of A. flavus, A. oryzae, or the two species. Averages of total SNPs of A. flavus isolates belonging to the same sclerotial morphotype (L-type = 178 952 ± 14 033; S-type = 133 188 ± 16 430) and A. oryzae isolates (152 336 ± 49 124) were consistently lower than those between the morphotypes and between the two species. Averages of total SNPs for L-type vs S-type (300 116 ± 1562) and S-type A. flavus vs A. oryzae (301 797 ± 4123) were similar but were 36% greater than that of L-type A. flavus vs A. oryzae (226 240 ± 10 779). Based on the devised criterion, ATCC 12892, Aspergillus oryzae (Ahlburg) Cohn, which had an averaged total SNPs 10-fold greater than that of other A. oryzae isolates, was determined to be close to Aspergillus parasiticus. Atoxigenic A. flavus field isolates, WRRL1519 and NRRL35739, were shown to more closely resemble A. oryzae than toxigenic L-type A. flavus. Biocontrol strains AF36 and K49 were genetically close to toxigenic L-type A. flavus. NRRL21882, the active agent of the commercialized biocontrol product Afla-Guard® GR, was genetically distant from all other A. flavus isolates. CONCLUSIONS: The close genetic relatedness between A. flavus and A. oryzae was confirmed and the evolutionary origins of atoxigenic A. flavus biocontrol strains were revealed. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a greater understanding of genome similarity and dissimilarity between A. flavus and A. oryzae. The method can be an auxiliary technique for identifying A. flavus, A. oryzae.


Assuntos
Aspergillus flavus/genética , Aspergillus oryzae/genética , Genoma Fúngico , Aflatoxinas/metabolismo , Aspergillus flavus/isolamento & purificação , Aspergillus flavus/metabolismo , Aspergillus oryzae/isolamento & purificação , Aspergillus oryzae/metabolismo , Sequência de Bases , Polimorfismo de Nucleotídeo Único
14.
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
15.
Mol Biotechnol ; 61(10): 754-762, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392585

RESUMO

Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5ß,6α,8ß-trihydroxycariolan, 5ß,8ß-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.


Assuntos
Agaricales/genética , Vias Biossintéticas , Sesquiterpenos Policíclicos/metabolismo , Sequenciamento Completo do Genoma/métodos , Agaricales/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/crescimento & desenvolvimento , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Família Multigênica
16.
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
17.
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
18.
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
19.
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
20.
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
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