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Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

Joosten, V. (author), Gouka, R.J. (author), van den Hondel, C.A.M.J.J. (author), Verrips, C.T. (author), Lokman, B.C. (author), TNO Kwaliteit van Leven TNO Voeding (author)

We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V HHs were...

article 2005

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The Aspergillus nidulans amdS gene as a marker for the identification of multicopy T-DNA integration events in Agrobacterium-mediated transformation of Aspergillus awamori

Michielse, C.B. (author), Ram, A.F.J. (author), van den Hondel, C.A.M.J.J. (author), TNO Voeding (author)

The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp. awamori. The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decrease in the transformation frequency, compared...

article 2004

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Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer

Michielse, C.B. (author), Salim, K. (author), Ragas, P. (author), Ram, A.F.J. (author), Kudla, B. (author), Jarry, B. (author), Punt, P.J. (author), van den Hondel, C.A.M.J.J. (author), TNO Voeding (author)

Two transformation systems, based on the use of CaCl2/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS+ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming...

article 2004

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Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

Michielse, C.B. (author), Ram, A.F.J. (author), Hooykaas, P.J.J. (author), van den Hondel, C.A.M.J.J. (author), TNO Voeding (author)

The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that...

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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Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application

Record, E. (author), Asther, M. (author), Sigoillot, C. (author), Pagès, S. (author), Punt, P.J. (author), Delattre, M. (author), Haon, M. (author), van den Hondel, C.A.M.J.J. (author), Sigoillot, J.C. (author), Lesage-Meessen, L. (author), Asther, M. (author), TNO Voeding (author)

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l...

article 2003

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Calnexin overexpression increases manganese peroxidase production in Aspergillus niger

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

Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required...

article 2002

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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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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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A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration

Maassen, C.B.M. (author), TNO Preventie en Gezondheid (author)

Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated from Lactobacillus casei to transform other...

article 1999

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Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: Evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system

Chaillou, S. (author), Pouwels, P.H. (author), Postma, P.W. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low- affinity facilitated-diffusion system with an apparent K(m)...

article 1999

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Expression and secretion of defined cutinase variants by Aspergillus awamori

van Gemeren, I.A. (author), Beijersbergen, A. (author), van den Hondel, C.A.M.J.J. (author), Verrips, C.T. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

Several cutinase variants derived by molecular modelling and site- directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae. The majority of these variants are successfully produced by the filamentous fungus Aspergillus awamori. However, the L51S and T179Y mutations caused reductions in...

article 1998

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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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Increased resistance to 14α-demethylase inhibitors (DMIs) in Aspergillus niger by coexpression of the Penicillium italicum eburicol 14α-demethylase (cyp51) and the A. Niger cytochrome P450 reductase (cprA) genes

van den Brink, H.J.M. (author), van Nistelrooy, H.J.G.M. (author), de Waard, M.A. (author), van den Hondel, C.A.M.J.J. (author), van Gorcom, R.F.M. (author), Prins Maurits Laboratorium TNO (author)

In this paper we describe the effects of over-expression of the Penicillium italicum gene encoding eburicol 14α-demethylase (cyp51), in Aspergillus niger strains with one or multiple copies of the gene encoding cytochrome P450 reductase (cpr A), on the eburicol 14α-demethylase activity. Eburicol 14α-demethylase activity was determined by...

article 1996

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A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium

Argnani, A. (author), Leer, R.J. (author), van Luijk, N. (author), Pouwels, P.H. (author), TNO Voeding (author)

A protocol was developed for the introduction of foreign plasmid DNA into various Bifidobacterium strains. The method, which is applicable to all Bifidobacterium species tested so far, is based on electroporation of bacteria made competent by preincubation in electroporation buffer for several hours at 4°C. Transformation of Bifidobacterium...

article 1996

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The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori

TNO Nutrition and Food Research (author), van Gemeren, I.A. (author), Beijersbergen, A. (author), Musters, W. (author), Gouka, R.J. (author), van den Hondel, C.A.M.J.J. (author), Verrips, C.T. (author)

A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analyzed in single-copy...

article 1996

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Expression of Bacillus subtilis levanase gene in Lactobacillus plantarum and Lactobacillus casei

Wanker, E. (author), Leer, R.J. (author), Pouwels, P.H. (author), Schwab, H. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

Two Lactobacillus-Escherichia coli shuttle vectors, harbouring the levanase gene from Bacillus subtilis under the control of its own promoter (pLPEW1) or behind the E. coli tac promoter (pE-SIEW2), were constructed. Lactobacillus plantarum showed the same growth characteristics on selective plates and in liquid media containing inulin, after...

article 1995

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A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori

Gouka, R.J. (author), Hessing, J.G.M. (author), Stam, H. (author), Musters, W. (author), van den Hondel, C.A.M.J.J. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awarnori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site...

article 1995

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Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger

van den Brink, J.M. (author), van Zeijl, C.M.J. (author), Brons, J.F. (author), van den Hondel, C.A.M.J.J. (author), van Gorcom, R.F.M. (author), Centraal Instituut voor Voedingsonderzoek TNO (author)

In this paper, we describe the cloning and molecular characterization of the Aspergillus niger cytochrome P450 reductase (CPR) gene, cprA. Attempts to clone the cprA gene by heterologous hybridization techniques were unsuccessful. Using the polymerase chain reaction (PCR) with degenerate primers based on conserved regions found in cpr genes from...

article 1995

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Glucoamylase overexpression in Aspergillus niger: Molecular genetic analysis of strains containing multiple copies of the glaA gene

Medisch Biologisch Laboratorium TNO (author), Verdoes, J.C. (author), Punt, P.J. (author), Schrickx, J.M. (author), van Verseveld, H.W. (author), Stouthamer, A.H. (author), van den Hondel, C.A.M.J.J. (author)

A strategy, based on the usage of the amdS selection marker and a cosmid vector containing four copies of the glucoamylase gene (glaA), was developed to obtain glucoamylase (GLA)-overproducing A. niger strains. With this strategy, fungal strains carrying up to 200 copies of the glaA gene could be isolated at a relatively high frequency. In each...

article 1993