Mutagenesis and mitotic recombination in Aspergillus niger, an expedition from gene to genome

Aspergillus niger is a cosmopolitan fungus and its spores can be found in air and soil worldwide. This saprophyte is used in food biotechnology for the production of proteins, mainly enzymes and for the production of organic acids. In the production of proteins, several problems are encountered such as repressed gene-expression, morphologic and genetic instability and undesired metabolite accumulation. To find solutions to these problems, often mutagenesis and mitotic recombination have been used, as described in this thesis. These mutations cannot simply be recombined by crosses, as Aspergillus niger is an asexual fungus. An alternative way to recombine mutations is via the parasexual cycle, also called mitotic recombination. To obtain more information of its possibilities we studied mitotic recombination of genetic markers on chromosome III. Recently the genome sequence of A. niger has been determined and this information has been linked to the genetic map. By improving the genetic map of chromosome III we were able to study crossing-over of linked markers and found that these cross-over events occurred predominantly in a small part of the chromosome. This mitotic recombination hotspot has some interesting physical features, but it remains uncertain if and which of them is responsible for the increase in crossing-over.LEI Universiteit LeidenMicrobiologi

Factors impacting upon Australian university student participation in educational exchange programs

With globalisation and technological innovation changing the global marketplace, research (e.g. Australian International Education Foundation, 1998; Kling, Alexander, McCorkle, & Martinez, 1999; Webb, Mayer, Pioche, & Allen, 1999) has shown that employers are seeking graduates with international competencies. Moreover, it is argued that it is the role of universities to prepare students to work in the new international context, thus meeting the needs of business and society (Fantini, Arias-Galicia, & Guay, 2001; Higher Education Council, 1990). International education opportunities such as student exchange programs have been identified as effective means through which university students may develop such skills and knowledge (Clyne & Rizvi, 1998; Fantini et al., 2001; Wallace, 1993). Currently, less than one percent of Australian undergraduate students complete part of their qualification in an overseas institution (IDP Education Australia, 1995). Much research to date has focused on issues such as psychological and social adjustment of international students. Yet, there is a paucity of literature relating to the effects of international education experiences for Australian domestic students. Hence, this paper will examine the issues affecting Australian students’ participation in academic mobility programs, with a particular focus on the factors influencing the decision to participate in a student exchange program

Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism

This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of L-arabinose. Expression of the three known L-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular activities Of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding D-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. L-Arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the L-arabinose catabolic pathway

Mutagenesis and mitotic recombination in Aspergillus niger, an expedition from gene to genome

Aspergillus niger is a cosmopolitan fungus and its spores can be found in air and soil worldwide. This saprophyte is used in food biotechnology for the production of proteins, mainly enzymes and for the production of organic acids. In the production of proteins, several problems are encountered such as repressed gene-expression, morphologic and genetic instability and undesired metabolite accumulation. To find solutions to these problems, often mutagenesis and mitotic recombination have been used, as described in this thesis. These mutations cannot simply be recombined by crosses, as Aspergillus niger is an asexual fungus. An alternative way to recombine mutations is via the parasexual cycle, also called mitotic recombination. To obtain more information of its possibilities we studied mitotic recombination of genetic markers on chromosome III. Recently the genome sequence of A. niger has been determined and this information has been linked to the genetic map. By improving the genetic map of chromosome III we were able to study crossing-over of linked markers and found that these cross-over events occurred predominantly in a small part of the chromosome. This mitotic recombination hotspot has some interesting physical features, but it remains uncertain if and which of them is responsible for the increase in crossing-over.</p

Disruption of the Aspergillus niger argB gene : a tool for transformation

We disrupted the Aspergillus niger gene argB, encoding ornithine transcarbamylase. Full characterisation of the argB deletion was performed by Southern blot analysis, growth tests and by means of mitotic recombination, complementation and transformation. The argB locus was found to be physically removed, thus creating an auxotrophic mutation. The latter can be supplemented by addition of arginine into the culture medium. The argB gene and its disruption do not correlate to the argI13 (formerly argB13) allele described. The (argB is on chromosome I whereas argI13 is on V. In addition, the argI13 mutation can only be complemented by the A. nidulans argB gene, whereas the new argB deletion can be complemented by both the A. niger and A. nidulans argB genes. The (argB strain has been used to generate several strains in a breeding programme and to study the expression of important genes, such as areA and kexB

Aspergillus as a host for heterologous protein production: the problem of proteases.

Homologous and heterologous protein production by filamentous fungi is often limited by the expression of proteases at high levels. By eliminating specific protease activities, protein production in Aspergillus niger can be improved considerably. Both classical mutagenesis and gene disruption techniques have yielded strains with reduced protease expression. Combinations of these mutations and disruptions result in a further reduction of protease activity. The coupling of efficient promoters to target genes allows their expression under conditions that repress the expression of several proteases, which further improves product yields. The strategies used have led to the development of a set of tester strains from which the appropriate genetic background for production can be selected