Adding tools to the box: facilitating host strain engineering of Penicillium chrysogenum for the production of heterologous secondary metabolites

This thesis presents the adaption of the RNA-guided endonuclease Cas9 for engineering the genome of the ascomycete fungus P. chrysogenum, whereby the insertion of a donor DNA is greatly facilitated. The approach of delivering the Cas9 protein and the sgRNA as a preassembled ribonucleoprotein particle was further demonstrated in P. decumbens and led to verification of the Calbistrin BGC. This BGC was then selected as a proof-of-principle example for in vivo homologous recombination of a heterologous BGC into a BGC-reduced P. chrysogenum strain. Lastly, the Cas9-boosted homologous recombination frequency was utilized to demonstrate a time-saving approach for building and testing novel expression cassettes transcription factors such as an aldehyde-inducible promoter and protein degradation tags in P. chrysogenum. The genetic tools developed in this thesis speed up the strain construction approaches in this industrially relevant fungus. In the coming decade, precise, simultaneous-multi-loci-editing techniques of filamentous fungi will become routinely applied in research labs and researchers will increasingly consider fungi as a host organism for their purposes if the available toolbox is kept updated and reliable to apply.