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1.
Arch Microbiol ; 202(1): 197-203, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31372664

RESUMO

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Transferases/genética , Transferases/metabolismo , Aspergillus fumigatus/classificação , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus nidulans/classificação , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Aspergillus niger/classificação , Parede Celular , Deleção de Genes , Mutação , Filogenia
2.
Appl Microbiol Biotechnol ; 103(19): 8105-8114, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392377

RESUMO

The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre-loxP-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter Pxln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of loxP site-specific recombination in the strain with cre driven by Pxln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase-encoding gene (oahA) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation (oahA deletion, pyc, mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre-loxP system is a powerful tool for genetic engineering in A. niger, which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.


Assuntos
Aspergillus niger/genética , Aspergillus niger/metabolismo , Ácidos Carboxílicos/metabolismo , Edição de Genes/métodos , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Recombinação Genética
3.
World J Microbiol Biotechnol ; 35(6): 93, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187335

RESUMO

Siderophores are extra-cellular inducible compounds produced by aerobic microorganisms and plants to overcome iron insolubility via its chelation and then uptake inside the cell. This work aims to study the characteristics of siderophore that is produced by a rhizosphere-inhabiting fungus. This fungus has been morphologically and molecularly identified as Aspergillus niger with the ability to produce 87% siderophore units. The obtained siderophore in PDB medium gave a positive result with tetrazolium test and a characteristic spectrum with a maximum absorbance at 450 nm in FeCl3 test that did not shift in response to different pH degrees (5-9). This indicates that the obtained siderophore is a trihydroxymate in nature. After purification, the FTIR and NMR analyses showed that the obtained siderophore is considered to be ferrichrome. The purified siderophore has been further evaluated as a tool to extract uranium, thorium and rare earth elements (REEs) from Egyptian phosphorites obtained from Abu Tartur Mine area. The inductively coupled plasma atomic emission spectroscopy analysis showed that the highest removal efficiency percentage was for uranium (69.5%), followed by samarium (66.7%), thorium (55%), lanthanum (51%), and cerium (50.1%). This result confirmed the ability of hydroxymate siderophores to chelate the aforementioned precious elements, a result that paves the way for bioleaching to replace abiotic techniques in order to save the cost of such elements in an environmentally friendly way.


Assuntos
Aspergillus niger/isolamento & purificação , Aspergillus niger/metabolismo , Sideróforos/isolamento & purificação , Sideróforos/metabolismo , Microbiologia do Solo , Aspergillus niger/classificação , Aspergillus niger/genética , Egito , Ácidos Graxos/análise , Ferricromo , Concentração de Íons de Hidrogênio , Ferro , Minerais , Fosfatos , Rizosfera
4.
Microbiol Res ; 223-225: 44-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178050

RESUMO

Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.


Assuntos
Aspergillus niger/genética , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Edição de Genes/métodos , Aspergillus niger/enzimologia , Sequência de Bases , Desoxirribonuclease I/genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Genes Fúngicos/genética , Mutagênese
5.
Mol Biotechnol ; 61(9): 663-673, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31228008

RESUMO

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.


Assuntos
Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Gossypium/genética , Larva/efeitos dos fármacos , Metanol/toxicidade , Mariposas/efeitos dos fármacos , Proteínas de Plantas/genética , Agrobacterium/genética , Agrobacterium/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Parede Celular/parasitologia , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Gossypium/parasitologia , Herbivoria/efeitos dos fármacos , Herbivoria/fisiologia , Inseticidas/química , Inseticidas/toxicidade , Larva/patogenicidade , Metanol/metabolismo , Mariposas/patogenicidade , Células Vegetais/metabolismo , Células Vegetais/parasitologia , Folhas de Planta/genética , Folhas de Planta/parasitologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transgenes
6.
J Ind Microbiol Biotechnol ; 46(11): 1517-1529, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31236777

RESUMO

Holocellulase production by Aspergillus niger using raw sugarcane bagasse (rSCB) as the enzyme-inducing substrate is hampered by the intrinsic recalcitrance of this material. Here we report that mild hydrothermal pretreatment of rSCB increases holocellulase secretion by A. niger. Quantitative proteomic analysis revealed that pretreated solids (PS) induced a pronounced up-regulation of endoglucanases and cellobiohydrolases compared to rSCB, which resulted in a 10.1-fold increase in glucose release during SCB saccharification. The combined use of PS and pretreatment liquor (PL), referred to as whole pretreated slurry (WPS), as carbon source induced a more balanced up-regulation of cellulases, hemicellulases and pectinases and resulted in the highest increase (4.8-fold) in the release of total reducing sugars from SCB. The use of PL as the sole carbon source induced the modulation of A. niger's secretome towards hemicellulose degradation. Mild pretreatment allowed the use of PL in downstream biological operations without the need for undesirable detoxification steps.


Assuntos
Aspergillus niger/enzimologia , Celulose/metabolismo , Glicosídeo Hidrolases/metabolismo , Saccharum/metabolismo , Aspergillus niger/genética , Celulase/metabolismo , Celulose 1,4-beta-Celobiosidase/metabolismo , Hidrólise , Proteômica
7.
Fungal Biol ; 123(6): 465-470, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31126423

RESUMO

Intron retention, one of the major types of alternative splicing in plants and animals, has also been reported existing in filamentous fungi's glycoside hydrolases. In this study, an intron-retained ß-glucosidase gene transcript (bgl1B) from A. niger B2 strain was obtained. Compared with the normally spliced transcript bgl1A, bgl1B had an extra 51bp insertion, which was confirmed to be the sixth (the last) intron of this ß-glucosidase gene. The bgl1A and bgl1B were expressed in Pichia pastoris and the purified enzymes were used to compare their catalytic properties. The results showed that the intron retention didn't impair the catalytic function. Instead, the intron-retained enzyme BGL1B had a better thermostability with a higher optimal temperature and a longer half-life under 50 °C. Also it exhibited a little higher kcat for 4-nitrophenyl-ß-d-glucopyranoside (PNPG) and a noticeable higher hydrolysis efficiency towards geniposide. This work suggested that the ß-glucosidase gene in A. niger most likely underwent an alternative splicing presented as intron retention type, and intron retention might be a source of enzyme diversity in fungi.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Íntrons , beta-Glucosidase/genética , Clonagem Molecular , Genes Fúngicos , Iridoides/metabolismo , Pichia/genética , beta-Glucosidase/metabolismo
8.
Acta Biochim Biophys Sin (Shanghai) ; 51(6): 638-644, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31081016

RESUMO

The purpose of this study was to obtain an engineered Aspergillus niger strain with high glucoamylase activity by overexpressing the glucoamylase gene glaA and α-amylase gene amyA in A. niger CICC2462. Three recombinant strains containing a single copy of amyA (1A), containing two copies of amyA (2A), and coexpressing amyA and glaA (AG), respectively, were constructed. The transcript levels of amyA in 1A and 2A were increased by 2.95 folds and 3.09 folds, respectively. The levels of amyA and glaA in AG were increased by 1.21 folds and 2.86 folds, but the maximum extracellular glucoamylase activities did not differ significantly. In addition, after 1% casein phosphopeptides (CPPs) was added to the fermentation medium, the maximum extracellular glucoamylase activities for strains 1A, 2A, and AG were 35,200, 37,300, and 40,710 U/ml, respectively, which were significantly higher than that of the parental strain CICC2462 (28,250 U/ml), while CPPs alone had no effect on the parental strain CICC2462. We demonstrate that overexpression of amyA and glaA substantially increases the expression and secretion of glucoamylase in A. niger, and CPPs effectively improves the yield of glucoamylase in recombinant A. niger strains overexpressing amyA and glaA. The newly developed strains and culture methods may have extensive industrial applications.


Assuntos
Aspergillus niger/genética , Proteínas Fúngicas/genética , Glucana 1,4-alfa-Glucosidase/genética , alfa-Amilases/genética , Aspergillus niger/enzimologia , Aspergillus niger/metabolismo , Caseínas/metabolismo , Caseínas/farmacologia , Fermentação/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Engenharia Genética/métodos , Glucana 1,4-alfa-Glucosidase/metabolismo , Fosfopeptídeos/metabolismo , Fosfopeptídeos/farmacologia , alfa-Amilases/metabolismo
9.
J Agric Food Chem ; 67(16): 4435-4443, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30945533

RESUMO

Aspergillus niger, which is a fungal pathogen, causes rot in a variety of fruits. In this study, the cystathionine ß-synthase cbsA gene was deleted by homologous recombination to study its role in sulfur metabolism and pathogenicity of A. niger. The results showed that Δ cbsA strain maintained normal mycelia growth and sporulation compared with the control strain A. niger MA 70.15, whereas the contents of cysteine and glutathione (GSH) increased significantly after cbsA deletion. However, Δ cbsA strain showed reduced endogenous H2S production. Further results showed that cbsA gene deletion induced higher resistance to cadmium stress and stronger infectivity to pears. It was also found that a stronger response of reactive oxygen species (ROS) production was induced in Δ cbsA mutant-infected pear compared with the control strain. In all, the present research suggested the important role of cbsA in sulfur metabolism and pathogenicity of A. niger in pear fruit.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/patogenicidade , Cistationina beta-Sintase/metabolismo , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Pyrus/microbiologia , Enxofre/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Cistationina beta-Sintase/genética , Cisteína/metabolismo , Frutas/microbiologia , Proteínas Fúngicas/genética , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Virulência
10.
Food Microbiol ; 82: 240-248, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027779

RESUMO

The Aspergillus niger aggregate contains 15 morphologically indistinguishable species which presence is related to ochratoxin A (OTA) and fumonisin B2 (FB2) contamination of foodstuffs. The taxonomy of this group was recently reevaluated and there is a need of new studies regarding the risk that these species might pose to food security. 258 isolates of A. niger aggregate obtained from a variety of products from Spain were classified by molecular methods being A. tubingensis the most frequently occurring (67.5%) followed by A. welwitschiae (19.4%) and A. niger (11.7%). Their potential ability to produce mycotoxins was evaluated by PCR protocols which allow a rapid detection of OTA and FB2 biosynthetic genes in their genomes. OTA production is not widespread in A. niger aggregate since only 17% of A. niger and 6% of A. welwitschiae isolates presented the complete biosynthetic cluster whereas the lack of the cluster was confirmed in all A. tubingensis isolates. On the other hand, A. niger and A. welwitschiae seem to be important FB2 producers with 97% and 29% of the isolates, respectively, presenting the complete cluster. The genes involved in OTA and FB2 were overexpressed in producing isolates and their expression was related to mycotoxin synthesis.


Assuntos
Aspergillus/isolamento & purificação , Aspergillus/metabolismo , Microbiologia de Alimentos , Micotoxinas/metabolismo , Aspergillus/classificação , Aspergillus/genética , Aspergillus niger/classificação , Aspergillus niger/genética , Aspergillus niger/isolamento & purificação , Aspergillus niger/metabolismo , DNA Fúngico/genética , Contaminação de Alimentos/análise , Fumonisinas/metabolismo , Expressão Gênica , Genoma Fúngico/genética , Família Multigênica , Micotoxinas/genética , Ocratoxinas/metabolismo , Filogenia , Análise de Sequência de DNA , Espanha
11.
Appl Microbiol Biotechnol ; 103(10): 4125-4136, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30963207

RESUMO

The AraR transcription factor of Aspergillus niger encodes a Zn(II)2Cys6 transcription factor required for the induction of genes encoding arabinolytic enzymes. One of the target genes of AraR is abfA, encoding an arabinofuranosidase. The expression of abfA as well as other L-arabinose-induced genes in A. niger requires the presence of L-arabinose or its derivative L-arabitol as an inducer to activate AraR-dependant gene expression. In this study, mutants were isolated that express L-arabinose-induced genes independently of the presence of an inducer under derepressing conditions. To obtain these mutants, a reporter strain was constructed in a ΔcreA background containing the L-arabinose-responsive promoter (PabfA) fused to the acetamidase (amdS) gene. Spores of the ΔcreA PabfA-amdS reporter strain were UV-mutagenized and mutants were obtained by their ability to grow on acetamide without the presence of inducer. From a total of 164 mutants, 15 mutants were identified to contain transacting mutations resulting in high arabinofuranosidase activity in the medium after growth under non-inducing conditions. Sequencing of the araR gene of the 15 constitutive mutants revealed that 14 mutants carried a mutation in AraR. Some mutations were found more than once and in total nine different point mutations were identified in AraR. The AraRN806I point mutation was reintroduced into a parental strain and confirmed that this point mutation leads to inducer-independent expression of AraR target genes. The inducer independent of L-arabinose-induced genes in the AraRN806I mutant was found to be sensitive to carbon catabolite repression, indicating that the CreA-mediated carbon catabolite repression is dominant over the AraRN806I mutant allele. These mutations in AraR provide new opportunities to improve arabinase production in industrial fungal strains.


Assuntos
Aspergillus niger/genética , Aspergillus niger/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabinose/metabolismo , Aspergillus niger/crescimento & desenvolvimento , Aspergillus niger/efeitos da radiação , Análise Mutacional de DNA , Mutagênese , Álcoois Açúcares/metabolismo , Raios Ultravioleta
12.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991752

RESUMO

: Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of PsLac1 and PsLac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4-6. Different substrate specificities were exhibited, and the lowest Km and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for PsLac1 and PsLac2, respectively. The industrially important dyes-Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5-were more efficiently decolorized by PsLac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and PsLac2 seemed more adapted to the presence of sea salt than PsLac1. The overall surface charges of the predicted PsLac three-dimensional models showed large negatively charged surfaces for PsLac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for PsLac1, as seen in proteins from terrestrial organisms.


Assuntos
Corantes/metabolismo , Fungos/enzimologia , Lacase/metabolismo , Sequência de Aminoácidos , Aspergillus niger/genética , Clonagem Molecular/métodos , Corantes/isolamento & purificação , Estabilidade Enzimática , Fungos/genética , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Lacase/química , Lacase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salinidade , Especificidade por Substrato , Temperatura
13.
Int J Biol Macromol ; 131: 1117-1124, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30910675

RESUMO

A preferable phytase for use in animal feeds for industrial applications should have high, optimal activity at low pH in the monogastric gut environment and high thermostability. To obtain enzymes with enhanced catalytic efficiency (pH 5.5) and excellent activity in acidic pH range, we performed structure-based rational design of a thermostable phytase (PhyAn). For this, six mutants based on different rational design strategies were constructed and heterologously expressed in Pichia pastoris. Particularly, the extracellular enzymatic activity was assessed to ensure that the produced enzymes met requirements of further analyses. Several positive mutants with enhanced catalytic efficiency or pH-profile shifts were carefully examined. Biochemical and kinetic investigations of purified mutants revealed that E79K, E80K, E79K + E80K and D68K had higher catalytic efficiency than the parent enzyme by approximately 49%, 67%, 86% and 15%, respectively. Moreover, the optimum pH of mutant Y65H was shifted from 5.0 to 3.0, and the peak of D68K shifted to pH 5.5. Analysis of the structural-functional relationships revealed that changes in amino acid charges, structural flexibility and space hindrance could significantly influence certain enzyme characteristics. Our results illustrate the feasibility and present a structural foundation for enhancing the phytase-catalytic efficiency and acid resistance by assembling mutations derived using rational design.


Assuntos
6-Fitase/química , 6-Fitase/metabolismo , Aspergillus niger/enzimologia , 6-Fitase/genética , 6-Fitase/isolamento & purificação , Aspergillus niger/genética , Catálise , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solventes
14.
FEMS Microbiol Lett ; 366(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30869784

RESUMO

In this study, production of fungal phytase in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 employing methanol-inducible OtAOX promoter and sucrose-inducible OtMal promoter was investigated in a high cell density fed-batch fermentation. Although a similar maximum cell concentration was obtained in both expression systems, the OtMal system gave ~2-fold higher phytase activity, specific yield, production yield, volumetric productivity and specific productivity rate compared with the OtAOX system. In addition to being more efficient, the OtMal system is more flexible because sucrose or sugarcane molasses can be utilized as less expensive carbon sources instead of glycerol in batch and fed-batch stages. Phytase yields from the OtMal system produced using sucrose or sugarcane molasses are comparable with those obtained with glycerol. We estimate the cost of phytase production by the OtMal system using sucrose or sugarcane molasses to be ~85% lower than the OtAOX system.


Assuntos
6-Fitase/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Microbiologia Industrial/métodos , Proteínas Recombinantes/genética , Saccharomycetales/genética , Sacarose/farmacologia , 6-Fitase/metabolismo , Aspergillus niger/enzimologia , Aspergillus niger/genética , Contagem de Células , Fermentação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Metanol/metabolismo , Melaço , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Sacarose/metabolismo , Termotolerância
15.
J Gen Appl Microbiol ; 65(5): 240-245, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-30905899

RESUMO

In this study, a mutant xylanase of high thermostability was obtained by site-directed mutagenesis. The homologous 3D structure of xylanase was successfully modeled and the mutation sites were predicted using bioinformatics software. Two amino acids of XynZF-2 were respectively substituted by cysteines (T205C and A52C) and a disulfide bridge was introduced into the C-terminal of XynZF-2. The mutant gene xynZFTA was cloned into pPIC9K and expressed in P. pastoris. The optimum temperature of the variant XynZFTA was improved from 45°C to 60°C, and XynZFTA retained greater than 90.0% activity (XynZF-2 retained only 50.0% activity) after treatment at 50°C for 5 min. The optimum pH of mutant xylanase was similar to XynZF-2 (pH = 5.0). The pH stability span (5.0~7.0) of the mutant xylanase was increased to 3.0~9.0. Overall, the results implied that the introduction of a disulfide bridge in the C-terminal structure improved the thermostability and pH stability of XynZF-2.


Assuntos
Aspergillus niger/enzimologia , Endo-1,4-beta-Xilanases/química , Proteínas Fúngicas/química , Aspergillus niger/genética , Domínio Catalítico , Cátions , Dissulfetos/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
16.
Int J Mol Sci ; 20(5)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841519

RESUMO

Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.


Assuntos
Aspergillus niger/enzimologia , Biocatálise , Dissacarídeos/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Quercetina/metabolismo , Aspergillus niger/genética , Dissacarídeos/química , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Microbiologia Industrial/métodos , Pichia/genética , Pichia/metabolismo , Quercetina/química , Rutina/química , Rutina/metabolismo
17.
Microb Cell Fact ; 18(1): 28, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717739

RESUMO

Citric acid is the world's largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates highly efficient genome-scale genetic perturbation in A. niger. In this review, we summarize the impact of systems biology on the citric acid molecular regulatory mechanisms, the advances in metabolic engineering strategies for enhancing citric acid production and discuss the development and application of CRISPR/Cas9 systems for genome editing in A. niger. We believe that future systems metabolic engineering efforts will redesign and engineer A. niger as a highly optimized cell factory for industrial citric acid production.


Assuntos
Aspergillus niger/genética , Aspergillus niger/metabolismo , Ácido Cítrico/metabolismo , Genoma Fúngico , Engenharia Metabólica , Sistemas CRISPR-Cas , Edição de Genes , Genômica , Microbiologia Industrial , Biologia de Sistemas
18.
Appl Microbiol Biotechnol ; 103(7): 2889-2902, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30758523

RESUMO

Citric acid production by Aspergillus niger and itaconic acid production by Aspergillus terreus are two major examples of technical scale fungal fermentations based on metabolic overflow of primary metabolism. Both organic acids are formed by the same metabolic pathway, but whereas citric acid is the end product in A. niger, A. terreus performs two additional enzymatic steps leading to itaconic acid. Despite of this high similarity, the optimization of the production process and the mechanism and regulation of overflow of these two acids has mostly been investigated independently, thereby ignoring respective knowledge from the other. In this review, we will highlight where the similarities and the real differences of these two processes occur, which involves various aspects of medium composition, metabolic regulation and compartmentation, transcriptional regulation, and gene evolution. These comparative data may facilitate further investigations of citric acid and itaconic acid accumulation and may contribute to improvements in their industrial production.


Assuntos
Aspergillus niger/metabolismo , Aspergillus/metabolismo , Ácido Cítrico/metabolismo , Succinatos/metabolismo , Aspergillus/genética , Aspergillus niger/genética , Fermentação , Redes e Vias Metabólicas
19.
Microbiology ; 165(4): 396-410, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30806615

RESUMO

Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving another ureohydrolase, 4-guanidinobutyrase (GBase), was reported. We present here a detailed characterization of A. niger GBase - the first fungal (and eukaryotic) enzyme to be studied in detail. A. niger GBase is a homohexamer with a native molecular weight of 336 kDa and an optimal pH of 7.5. Unlike arginase, the Mn2+ enzyme from the same fungus, purified GBase protein is associated with Zn2+ ions. A sensitive fluorescence assay was used to determine its kinetic parameters. GBase acted 25 times more efficiently on 4-guanidinobutyrate (GB) than 3-guanidinopropionic acid (GP). The Km for GB was 2.7±0.4 mM, whereas for GP it was 53.7±0.8 mM. While GB was an efficient nitrogen source, A. niger grew very poorly on GP. Constitutive expression of GBase favoured fungal growth on GP, indicating that GP catabolism is limited by intracellular GBase levels in A. niger. The absence of a specific GPase and the inability of GP to induce GBase expression confine the fungal growth on GP. That GP is a poor substrate for GBase and a very poor nitrogen source for A. niger offers an opportunity to select GBase specificity mutations. Further, it is now possible to compare two distinct ureohydrolases, namely arginase and GBase, from the same organism.


Assuntos
Aspergillus niger/enzimologia , Butiratos/metabolismo , Proteínas Fúngicas/metabolismo , Guanidinas/metabolismo , Ureo-Hidrolases/metabolismo , Agmatina/metabolismo , Arginase/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Cátions/química , Meios de Cultura/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Cinética , Peso Molecular , Mutação , Propionatos/metabolismo , Multimerização Proteica , Especificidade por Substrato , Ureo-Hidrolases/antagonistas & inibidores , Ureo-Hidrolases/química , Ureo-Hidrolases/genética
20.
Biosci Biotechnol Biochem ; 83(8): 1538-1546, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30720390

RESUMO

The transporter that exports citric acid (CA) generated in mitochondria to the cytosol is an important component of the CA production machinery of Aspergillus niger. In this report, we cloned and identified the gene cocA, encoding a 33.7-kDa putative mitochondrial citrate-oxoglutarate shuttle protein of the CA hyper-producer A. niger WU-2223L. The amount of CA produced by a representative cocA disruptant (35 g/L) was significantly lower than that produced by strain WU-2223L (63 g/L) after culture for 12 days under CA production conditions, and the phenotype of the cocA disruptant differed in part from that of strain WU-2223L. A cocA disruptant complemented with cocA exhibited the same phenotypes as those of strain WU-2223L. This report is the first to show that cocA and its protein product clearly contribute to substantial CA production by A. niger, and provides a significant insight into microbial organic acid production by fermentation. Abbreviations: CA: citric acid; CD medium: Czapek-Dox medium; CS: citrate synthase; CTP: citrate transport protein; HR: homologous recombination; MCF: mitochondrial carrier family; RT-PCR: reverse-transcription PCR; TCA: tricarboxylic acid.


Assuntos
Aspergillus niger/metabolismo , Proteínas de Transporte/genética , Ácido Cítrico/metabolismo , Genes Fúngicos , Ácidos Cetoglutáricos/metabolismo , Proteínas Mitocondriais/genética , Sequência de Aminoácidos , Aspergillus niger/genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Fermentação , Transporte Proteico , Transcrição Genética
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