Näkökulmia vapaan demokraattisen yhteiskunnan edellytyksiin

Lectio Praecursoria 8.8.201

A new vector for efficient gene targeting to the pyrG locus in Aspergillus niger.

Microbial Biotechnolog

Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses

Mutants with a defective non-homologous-end-joining (NHEJ) pathway have boosted functional genomics in filamentous fungi as they are very efficient recipient strains for gene-targeting approaches, achieving homologous recombination frequencies up to 100%. For example, deletion of the ku70 homologous gene kusA in Aspergillus niger resulted in a recipient strain in which deletions of essential or non-essential genes can efficiently be obtained. To verify that the mutant phenotype observed is the result of a gene deletion, a complementation approach has to be performed. Here, an intact copy of the gene is transformed back to the mutant, where it should integrate ectopically into the genome. However, ectopic complementation is difficult in NHEJ-deficient strains, and the gene will preferably integrate via homologous recombination at its endogenous locus. To circumvent that problem, we have constructed autonomously replicating vectors useful for many filamentous fungi which contain either the pyrG allele or a hygromycin resistance gene as selectable markers. Under selective conditions, the plasmids are maintained, allowing complementation analyses; once the selective pressure is removed, the plasmid becomes lost and the mutant phenotype prevails. Another disadvantage of NHEJ-defective strains is their increased sensitivity towards DNA damaging conditions such as radiation. Thus, mutant analyses in these genetic backgrounds are limited and can even be obscured by pleiotropic effects. The use of sexual crossings for the restoration of the NHEJ pathway is, however, impossible in imperfect filamentous fungi such as A. niger. We have therefore established a transiently disrupted kusA strain as recipient strain for gene-targeting approaches

Protoplast-mediated transformation of Madurella mycetomatis using hygromycin resistance as a selection marker

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies

Interrogation of the cell wall integrity pathway in Aspergillus niger identifies a putative negative regulator of transcription involved in chitin deposition

Microbial Biotechnolog

Natural variation and the role of Zn2Cys6 transcription factors SdrA, WarA and WarB in sorbic acid resistance of aspergillus niger

Weak acids, such as sorbic acid, are used as chemical food preservatives by the industry. Fungiovercome this weak-acid stress by inducing cellular responses mediated by transcription factors. In ourresearch, a large-scale sorbic acid resistance screening was performed on 100 A. niger sensu stricto strains isolated fromvarious sources to study strain variability in sorbic acid resistance. Theminimal inhibitory concentration of undissociated (MICu) sorbic acid at pH = 4 in the MEB of the A. niger strains varies between 4.0 mMand 7.0 mM, with the average out of 100 strains being 4.8 0.8 mM, when scored after 28 days. MICu valueswere roughly 1mMlowerwhen tested in commercial ice tea. Genome sequencingof the most sorbic-acid-sensitive strain among the isolates revealed a premature stop codon inside thesorbic acid response regulator encoding gene sdrA. Repairing this missense mutation increased thesorbic acid resistance, showing that the sorbic-acid-sensitive phenotype of this strain is caused by theloss of SdrA function. To identify additional transcription factors involved in weak-acid resistance,a transcription factor knock-out library consisting of 240 A. niger deletion strains was screened. Thescreen identified a novel transcription factor,WarB, which contributes to the resistance against a broadrange of weak acids, including sorbic acid. The roles of SdrA,WarA andWarB in weak-acid resistance,including sorbic acid, were compared by creating single, double and the triple knock-out strains. Allthree transcription factors were found to have an additive effect on the sorbic acid stress response.Microbial Biotechnolog

A set of isogenic auxotrophic strains for constructing multiple gene deletion mutants and parasexual crossings in Aspergillus niger

Microbial Biotechnolog

Structure elucidation and characterization of patulin synthase, insights into the formation of a fungal mycotoxin

Patulin synthase (PatE) from Penicillium expansum is a flavin-dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in fruit and fruit-derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. This confirmed that PatE is active not only on the proposed patulin precursor ascladiol but also on several aromatic alcohols including 5-hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl-alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin.</p