Advancing the Genetic Engineering Landscape in Filamentous Fungi: Development of Novel Tools and High-Throughput Technologies

Filamentous fungi have been extensively used as a biotechnological resource for the production of enzymes and small molecules for many years. Their natural traits offer a vast repertoire of applications, ranging from healthcare and agriculture to manufacturing and new materials.

Their evolved lifestyle as natural decomposers of organic matter, makes them superior enzyme secreters, with one of the best secretory pathways in nature. As a result, they have established themselves as superior cell factories dominating the recombinant protein production market. Despite the many benefits they offer, their complex genetics and growth characteristics make them harder to genetically manipulate and handle than other well-established cell factories, such as yeasts and bacteria. For these simpler organisms, many high-throughput (HTP) tools are already in place, enabling quick and precise genetic engineering of large libraries of strains. These HTP technologies allow scientists to accelerate discoveries and facilitate the development of new bio-solutions for the challenges we face today.

However, the implementation of these HTP technologies in filamentous fungi is lagging behind, hindering the innovations and breakthroughs that the complex biology of these organisms offers. The work presented in this thesis aims to develop and advance the fungal genetic engineering space by establishing new technologies and methodologies and enhancing the research with these organisms.

First, we have expanded the genetic engineering toolkit of Aspergillus by establishing an alternative CRISPR/Cas system based on MAD7, an IP friendly nuclease. Moreover, we have developed a new method for single-step multi-copy integration of Gene Expression Cassettes (GECs), named RoCi. The simplicity of the system and the minimal pre-engineering work required renders this method very promising for superior cell factory construction.

Additionally, we have developed an automated pipeline for cell factory engineering of filamentous fungi, utilizing A. oryzae as a model organism. By creating specialized programs for different liquid handling equipment, we have been able to automate DNA amplification, purification, and fungal transformation, successfully utilizing the pipeline for two distinct projects. In one, we generated a library of 22 strains expressing different CAZymes, used to both develop the pipeline and screen unknown enzymes. With the workflows in place, we also constructed a larger library of over 100 strains, to investigate de novo generated Signal Peptides (SP) designed using machine learning. We have demonstrated how combining superior transformer models with larger throughput technologies in filamentous fungi holds the promise of unlocking new and more advanced bio-solutions.