Broadly applicable genetic tools for fungi

Engineering of microbial hosts for industrial use has became significantly easier and faster in the recent years. This has been enabled by novel molecular biology tools and genome editing methods. However, the gene expression tools required for optimization of the engineered organisms are often inefficient, or completely lacking especially in less-established production hosts. This hinders the rapid development of novel biotechnological applications. This PhD work focused on developing synthetic biology tools that allow accurate control of gene expression in a broad spectrum of fungal species. The outcomes of this thesis represent an important advance in the field of biological engineering because fungi comprise an industrially important group of organisms. The developed tools provide high utility especially in applications in which precise balancing or strong gene expression is required. In the first part of this work (Publication I and II), a library of modular genetic parts, including a set of core promoters and synthetic transcription factors was established in Saccharomyces cerevisiae. These tools enable construction of complex genetic devices and they provided a broad gene expression range. Next part of this work focuses on development of universal gene expression system for fungi (Publication III). The functionality of the system was demonstrated in eight diverse fungal hosts: in six yeasts and two filamentous fungi. Importantly, due to universal performance of the system, these tools also makes it possible to introduce novel eukaryotic microbes for biotechnological use. In the final part, the developed synthetic tools were applied in a protein production application (Publication IV). The selected example protein product was lipase B of Candida antarctica (calB), and the production was carried out in filamentous fungus Trichoderma reesei. The obtained protein levels were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter. In addition, due to highly constitutive expression provided by synthetic expression system, it was possible to produce highly pure protein in cellulase-repressing glucose medium eliminating the secretion of unwanted background enzymes.