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A fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae

Zune, Q. (author), Delepierre, A. (author), Gofflot, S. (author), Bauwens, J. (author), Twizere, J.C. (author), Punt, P.J. (author), Francis, F. (author), Toye, D. (author), Bawin, T. (author), Delvigne, F. (author)

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-staterelated physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilmreactor for the production of a Gla::green fluorescent protein (GFP) fusion protein by Aspergillus oryzae...

article 2015

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Quantitative iTRAQ secretome analysis of aspergillus niger reveals novel hydrolytic enzymes

Adav, S.S. (author), Li, A.A. (author), Manavalan, A. (author), Punt, P. (author), Sze, S.K. (author), TNO Kwaliteit van Leven (author)

The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics...

article 2010

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Aspergillus niger genome-wide analysis reveals a large number of novel alpha-glucan acting enzymes with unexpected expression profiles

Yuan, X.-L. (author), van der Kaaij, R.M. (author), van den Hondel, C.A.M.J.J. (author), Punt, P.J. (author), van der Maarel, M.J.E.C. (author), Dijkhuizen, L. (author), Ram, A.F.J. (author), TNO Kwaliteit van Leven (author)

The filamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of α-amylase, glucoamylase and α-glucosidase enzymes, members of the...

article 2008

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Substitution patterns in methylated potato starch as revealed from the structure and composition of fragments in enzymatic digests

Steeneken, P.A.M. (author), Tas, A.C. (author), Woortman, A.J.J. (author), Sanders, P. (author), Mijland, P.J.H.C. (author), de Weijs, L.G.R. (author), TNO Kwaliteit van Leven (author)

The effect of the granule structure on the methylation of starch was investigated by comparing the substitution patterns of potato starch methylated in granular suspension and in solution to DS 0.3. Substitution patterns were analyzed by successive digestion with α-amylase and amyloglucosidase, fractionation of the resulting malto...

article 2008

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Expression of human α1-proteinase inhibitor in Aspergillus niger

Karnaukhova, E. (author), Ophir, Y. (author), Trinh, L. (author), Dalal, N. (author), Punt, P.J. (author), Golding, B. (author), Shiloach, J. (author), TNO Kwaliteit van Leven (author)

Background: Human α1-proteinase inhibitor (α1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd...

article 2007

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A new method for screening and isolation of hypersecretion mutants in Aspergillus niger

Weenink, X.O. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), Ram, A.F.J. (author), TNO Kwaliteit van Leven (author)

Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants...

article 2006

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Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation

te Biesebeke, R. (author), van Biezen, N. (author), de Vos, W.M. (author), van den Hondel, C.A.M.J.J. (author), Punt, P.J. (author), TNO Kwaliteit van Leven (author)

Solid-state fermentation (SSF) with Aspergillus oryzae results in high levels of secreted protein. However, control mechanisms of gene expression in SSF have been only poorly studied. In this study we show that both glucoamylase (glaB) and protease (alpA, nptB) genes are highly expressed during surface cultivation on wheat-based solid medium,...

article 2005

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Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates

te Biesebeke, R. (author), Record, E. (author), van Biezen, N. (author), Heerikhuisen, M. (author), Franken, A. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), TNO Kwaliteit van Leven (author)

To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the...

article 2005

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Endoplasmic reticulum stress leads to the selective transcriptional downregulation of the glucoamylase gene in Aspergillus niger

Al-Sheikh, H. (author), Watson, A.J. (author), Lacey, G.A. (author), Punt, P.J. (author), MacKenzie, D.A. (author), Jeenes, D.J. (author), Pakula, T. (author), Penttilä, M. (author), Alcocer, M.J.C. (author), Archer, D.B. (author), TNO Voeding (author)

We describe a new endoplasmic reticulum (ER)-associated stress response in the filamentous fungus Aspergillus niger. The inhibition of protein folding within the ER leads to cellular responses known collectively as the unfolded protein response (UPR) and we show that the selective transcriptional downregulation of the gene encoding glucoamylase,...

article 2004

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Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain

TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO (author), Levasseur, A. (author), Pagès, S. (author), Fierobe, H.P. (author), Navarro, D. (author), Punt, P. (author), Belaïch, J.P. (author), Asther, M. (author), Record, E. (author)

A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergilhis niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was...

article 2004

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The role of the Aspergillus niger furin-type protease gene in processing of fungal proproteins and fusion proteins: Evidence for alternative processing of recombinant (fusion-) proteins

Punt, P.J. (author), Drint-Kuijvenhoven, A. (author), Lokman, B.C. (author), Spencer, J.A. (author), Jeenes, D. (author), Archer, D.A. (author), van den Hondel, C.A.M.J.J. (author)

We have characterized growth and protein processing characteristics of Aspergillus niger strains carrying a disrupted allele of the previously cloned and characterized kexB gene [Appl. Environ. Microbiol. 66 (2000) 363] encoding a furin-type endoprotease. Deletion of the single-copy gene confirms it to be non-essential but disruptant strains...

article 2003

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The effect of organic nitrogen sources on recombinant glucoamylase production by Aspergillus niger in chemostat culture

Swift, R.J. (author), Karandikar, A. (author), Griffen, A.M. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), Robson, G.D. (author), Trinci, A.P.J. (author), Wiebe, M.G. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 ± 0.01 h-1, pH 5.4). In cultures supplemented with L-alanine, L-methionine, casamino acids, or peptone, specific glucoamylase ...

article 2000

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Studies on the production of fungal peroxidases in Aspergillus niger

Conesa, A. (author), van den Hondel, C.A.M.J.J. (author), Punt, P.J. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and...

article 2000

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Expression of human lymphotoxin alpha in Aspergillus niger

Krasevec, N. (author), van de Hondel, C.A.M.J.J. (author), Komel, R. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

A gene-fusion expression strategy was applied for heterologous expression of human lymphotoxin alpha (LTα) in the Aspergillus niger AB1.13 protease-deficient strain. The LTα gene was fused with the A. niger glucoamylase GII-form as a carrier-gene, behind its transcription control and secretion signals. Special attention was paid to the influence...

article 2000

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Can hTNF-alpha be successfully produced and secreted in filamentous fungus Aspergillus niger?

Krasevec, N. (author), van den Hondel, C.A.M.J.J. (author), Komel, R. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

A gene-fusion expression strategy was applied for the heterologous expression of hTNF-α in A. niger AB1.13. The TNF-α gene was fused with the A. niger glucoamylase GII form as a carrier-gene, behind its transcription control and secretion signal. The protein was expressed in the cells in the form of a glucoamylase-fusion protein, but was not...

article 2000

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Glucoamylasegreen fluorescent protein fusions to monitor protein secretion in Aspergillus niger

TNO Voeding (author), Gordon, C.L. (author), Khalaj, V. (author), Ram, A.F.J. (author), Archer, D.B. (author), Brookman, J.L. (author), Trinci, A.P.J. (author), Jeenes, D.J. (author), Doonan, J.H. (author), Wells, B. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), Robson, G.D. (author)

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and...

article 2000

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Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger

Centraal Instituut voor Voedingsonderzoek TNO TNO Voeding (author), Ngiam, C. (author), Jeenes, D.J. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), Archer, D.B. (author)

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced...

article 2000

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Study of the glucoamylase promoter in Aspergillus niger using green fluorescent protein

Santerre Henriksen, A.L. (author), Even, S. (author), Müller, C. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), Nielsen, J. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

An Aspergillus niger strain expressing a red-shifted green fluorescent protein (GFP) in the cytoplasm under the control of the glucoamylase promoter (PglaA) was characterized with respect to its physiology and morphology. Although xylose acted as a repressor carbon source during batch cultivations, PglaA-driven GFP expression by the glucoamylase...

article 1999

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An improved colony-PCR method for filamentous fungi for amplification of pcr-fragments of several kilobases

van Zeijl, C.M.J. (author), van de Kamp, E.H.M. (author), Punt, P.J. (author), Selten, G.C.M. (author), Hauer, B. (author), van Gorcom, R.F.M. (author), van den Hondel, C.A.M.J.J. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

A method was developed to perform PCR directly on mycelial pellets or colonies treated with NOVOzym 234. The method allows rapid screening of large numbers of transformants of both sporulating and non-sporulating fungi for the presence of (co)transforming plasmid copies or for specific genetic modifications such as gene disruption and site...

article 1998

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Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

Withers, J.M. (author), Swift, R.J. (author), Wiebe, M.G. (author), Robson, G.D. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited chemostat culture at a dilution rate of 0.13 h-1....

article 1998