CRISPR-mediated expression platform for multi-species Aspergilli

Zofia Dorota Jarczynska Ferdinand Hans Kirchner Christina Spuur Nødvig Uffe Hasbro Mortensen
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark

The recent sequencing survey on many fungal species revealed a large repertoire of industrially or medically relevant enzymes and secondary metabolites. However, the industrial potential of these species is often hindered by the difficulties with the cultivation at laboratory conditions or in bioreactors. It is therefore beneficial to create heterologous expression platforms that facilitate the screening process of fungal genes for production of relevant enzymatic activities or secondary metabolites. Unfortunately, in many cases it is not possible to predict whether a given host possesses a physiology and metabolism compatible for formation of these products, resulting in an inefficient heterologous production. To increase the chance of successful heterologous production, we have created a flexible expression platform in various Aspergillus species. The system is based on the insertion of a reporter gene into defined locus in each of the different expression hosts. The reporter allows to assess the strength of expression from the various defined loci, as well as it can be replaced by a gene-expression cassette containing your favourite gene or pathway via marker-free homologous recombination mediated by CRISPR-Cas9 technology. Importantly, our setup allows different species to be transformed by the same gene-expression cassette to reduce DNA construction work. As a proof-of-concept, we chose three representatives of Aspergillus: the model fungus A. nidulans, and the fungal industrial workhorses A. niger and A. oryzae. We have used red fluorescent protein (RFP) as a reporter gene and inserted it into several defined integration sites in all three species. RFP production was confirmed through fluorescence microscopy and the three different strains constitute our versatile Aspergillus expression platform. We have tested the platform and replaced RFP in the different species with genes allowing for production of relevant enzymes and secondary metabolites.