An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader

Abstract

The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

Is It Time To Kill the Survival Curve? A Case for Disease Progression Factors in Microbial Pathogenesis and Host Defense Research

“…The generation of an inducible and robust Tn-seq in vivo following the initiation of infection would be ideal for the identification of DPFs. Although methods for inducible fungal transposon mutagenesis exist (34), they have yet to be optimized for use in vivo. In addition, inducible systems to drive CRIPSR/Cas9 or Cre-mediated gene editing to generate loss of function alleles at specific time points during disease progression can be used to characterize DPFs once the infection is established.…”