Efficient cloning system for construction of gene silencing vectors in Aspergillus niger

Oliveira, José Miguel; Veen, Douwe van der; Graaff, L.H. de

doi:10.1007/s00253-008-1640-x

Cited by 20 publications

(13 citation statements)

References 44 publications

(61 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ongoing optimization of existing RNAi systems demonstrates the importance of such tools in deciphering gene functions, particularly when gene deletions by homologous recombination occur only at very low frequencies (Caracuel-Rios & Talbot, 2008;Nguyen et al, 2008;Oliveira et al, 2008;Shafran et al, 2008). Several RNAi approaches have been successfully performed using vectors that contain intron or spacer sequences between two inversely oriented target gene fragments to express dsRNA with a hairpin structure (e.g.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, it was shown that RNAi can be used effectively to manipulate a number of biotechnologically relevant ascomycetes (Janus et al, 2007;Oliveira et al, 2008;Takeno et al, 2005;Ullán et al, 2008;Yamada et al, 2007;Zhang et al, 2007). For example, RNAi was used to silence regulators of biosynthetic pathways as well as to downregulate the expression of genes involved in the synthesis of secondary metabolites such as fatty acids or b-lactam antibiotics.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

2009

View full text Add to dashboard Cite

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing system that downregulates target gene expression. Here, we provide several lines of evidence for RNA silencing in the industrial β-lactam antibiotic producer Penicillium chrysogenum using the DsRed reporter gene under the control of the constitutive trpC promoter or the inducible xylP promoter. The functional RNAi system was verified by detection of siRNAs that hybridized exclusively with gene-specific 32P-labelled RNA probes. Moreover, when RNAi was used to silence the endogenous PcbrlA morphogene that controls conidiophore development, a dramatic reduction in the formation of conidiospores was observed in 47 % of the corresponding transformants. Evidence that RNAi in P. chrysogenum is dependent on a Dicer peptide was provided with a strain lacking Pcdcl2. In the ΔPcdcl2 background, silencing of the PcbrlA gene was tested. None of the transformants analysed showed a developmental defect. The applicability of the RNAi system in P. chrysogenum was finally demonstrated by silencing the Pcku70 gene to increase homologous recombination frequency. This led to the generation of single and double knockout mutants.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

2009

View full text Add to dashboard Cite

show abstract

“…QPCR. RNA isolation, cDNA synthesis, quantitative real-time PCR (QPCR), and data analysis were performed as described previously (37). In brief, total RNA was extracted, quantified, and mixed with an exogenous RNA reference transcript.…”

mentioning

confidence: 99%

Shotgun Proteomics of Aspergillus niger Microsomes upon d -Xylose Induction

Oliveira

Passel

Schaap

et al. 2010

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the D-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. D-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.Aspergillus niger is a soil-dwelling filamentous fungus with a high capacity for decomposing plant materials. Many of the secreted depolymerizing enzymes have important biotechnological applications, e.g., to modify plant-derived food products. Homologous protein secretion in filamentous fungi may yield up to 20 g per liter of extracellular medium (14). For this reason, A. niger has been used intensively as a cell factory for enzyme production (3,14,32). In contrast to this, yields are much lower for heterologous protein secretion, with exceptions (11,18,35,59).The secretory potential of A. niger is not well understood, and only a limited number of functional studies have been performed to investigate the major components of the fungal secretion pathway. These studies have addressed overall transcriptional and translational responses in A. niger by studying the impact of secretion stress-inducing chemicals, temperature shifts, protein overproduction, or growth on carbon sources that induce changes in secretory states (20,23,26).In recent years, high-throughput shotgun proteomics has been used to study cell organelle makeup and function. Through the combined use of liquid chromatography and tandem mass spectrometry (LC-MS/MS), various studies have identified many organelle-specific proteins, including proteins related to protein secretion (18,24,29,51,51). In the case of aspergilli, such studies have focused mainly on the secretome and not on the actual components of the secretory pathway (34,36,55).In a previous study, we...

show abstract

“…Fungal transformations were made using the pyrA deficient strain A. niger N593 for the homologous expression of the different McoG versions, using a similar protocol to the one described in Oliveira et al [18]. In brief, enzymatic degradation of fungal cell wall was used to generate Brought to you by | MIT Libraries Authenticated Download Date | 5/10/18 7:16 PM protoplasts, followed by purification by filtration and storage in isotonic sorbitol solution.…”

Section: Molecular Biology Techniques and Transformationmentioning

confidence: 99%

Structure and function of Aspergillus niger laccase McoG

Ferraroni¹,

Westphal²,

Borsani³

et al. 2017

Biocatalysis

View full text Add to dashboard Cite

Biocatalysis 2017; 3: 1-16 p-phenylenediamine sulphate. However, the activities of H253A and H253N with 2-amino-4-methylphenol and 2-amino-4-methoxyphenol are strongly reduced compared to that of wild type. The redox potentials and electron transfer rates (k s ) of wild type and variants were determined (McoG wt E°' is +453 mV), and especially the reduced k s values of H253A and H253N show strong correlation with their low activity on phenolic compounds. In summary, our results suggest that the His253 adaptation of McoG can be beneficial for the conversion of phenolic compounds.Keywords: Aspergillus niger; crystal structure; laccase; redox potential; metalloprotein; multicopper oxidase Data deposition: Coordinates have been deposited with the Protein Data Bank. Accession codes are 5LM8, 5LWW and 5LWX.Abbreviations: CV, cyclic voltammetry; DT, decane-1-thiol; MCO, multicopper oxidase; MaL, laccase from Melanocarpus albomyces; TaL, laccase from Thielavia arenaria; SAM, self-assembled monolayer; SCE, saturated calomel electrode; SHE, standard hydrogen electrode; DMPPDA, N,N-dimethyl-p-phenylenediamine; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). IntroductionMulticopper oxidases (MCOs) form a family of redox enzymes that catalyze the reduction of molecular oxygen into water by a four-electron transfer process. It includes laccases (EC 1.10.3.2), ascorbate oxidases (EC 1.10.3.3), bilirubin oxidases (EC 1.3.3.5) and ferroxidases (EC 1.16.3.1), which are key enzymes in many biological processes of prokaryotic and eukaryotic organisms [1,2]. Their ability to catalyze the oxidation of various aromatic substrates with the concomitant reduction of molecular oxygen to water as sole byproduct makes them interesting green biocatalysts. The MCO catalyzed redox process is mediated by two copper centers, which contain Abstract: The ascomycete Aspergillus niger produces several multicopper oxidases, but their biocatalytic properties remain largely unknown. Elucidation of the crystal structure of A. niger laccase McoG at 1.7 Å resolution revealed that the C-terminal tail of this glycoprotein blocks the T3 solvent channel and that a peroxide ion bridges the two T3 copper atoms. Remarkably, McoG contains a histidine (His253) instead of the common aspartate or glutamate expected to be involved in catalytic proton transfer with phenolic compounds. The crystal structure of H253D at 1.5 Å resolution resembles the wild type structure. McoG and the H253D, H253A and H253N variants have similar activities with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid or N,N-dimethyl-

show abstract

Efficient cloning system for construction of gene silencing vectors in Aspergillus niger

Cited by 20 publications

References 44 publications

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

Shotgun Proteomics of Aspergillus niger Microsomes upon d -Xylose Induction

Structure and function of Aspergillus niger laccase McoG

Contact Info

Product

Resources

About