Characterization of an efficient gene cloning strategy for Aspergillus niger based on an autonomously replicating plasmid: cloning of the nicB gene of A. niger

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger

“…However, gene expression by independent transformants of the identical integrative expression plasmid was highly different and varied up to 50-fold. Similar observations have been made with other integrative expression systems and have been attributed to different sites of integration and different copies of the integrated plasmids (Storms et al 2005;Verdoes et al 1994). We further plan to identify the integration sites of the plasmids which led to maximal expression of the model proteins, to use these chromosomal loci for the development of specific integrative expression plasmids to ensure high expression levels of the target proteins.…”

“…niger with selective markers have been established to clone and express heterologous genes (Punt and van den Hondel 1992;Storms et al 2005;Verdoes et al 1994). An expression system, that has been exploited for the production of most recombinant proteins in A. niger utilizes the maltose-inducible promoter of the glaA gene, encoding the highly expressed and efficiently secreted enzyme glycoamylase (Fowler et al 1990).…”

Efficient Generation of Aspergillus niger Knock Out Strains by Combining NHEJ Mutants and a Split Marker Approach

“…The A. niger nicB gene encodes a nicotinatenucleotide pyrophosphorylase. Identifi cation and the construction of a gene deletion cassette to disrupt nicB is based on a previous work by Verdoes et al 1994 , and will be described elsewhere in detail (Niu et al manuscript in preparation). The ΔnicB strain is auxotrophic for nicotinamide and needs supplementation of nicotinamide to be able to grow.…”

Genetic and Genomic Manipulations in Aspergillus niger

“…Protoplast-mediated transformation (Buxton et al 1985 ), modifi ed from a protocol originally developed for yeast, is the most widely used method. It provides good transformation effi ciency of up to 1,000 transformants per μg DNA for integrative vectors (Buxton et al 1985 ) to 10,000 transformants per μg DNA for episomal vectors (Verdoes et al 1994b ). Recently, this method has been adapted for high-throughput platform with transformation and regeneration conducted in microtiterplate (Gielesen and van den Berg 2013 ).…”

“…As in other fungal species, therefore, transformation results from genomic integration of transforming DNA at non-homologous sites as well as at the homologous region (Hynes 1996 ). However, with the isolation of the AMA1 sequence from A. nidulans (Gems et al 1991 ), autonomously replicating vectors for use in A. niger have been constructed (Storms et al 2005 ;Verdoes et al 1994b ). Advantages of such vectors are increased transformation effi ciencies (see previous section) and easy plasmid rescue.…”