Filamentous fungi for protein production and identification of superior cell factories - Genetic tools and expression systems

Proteins and enzymes are used in many practices of commercial and societal importance, and the efforts in genome sequencing have provided a plethora of novel enzymes with unknown catalytic potential. The majority of these enzymes are produced heterologously using fungal production hosts that have a long tradition of use in both academia and industry.

However, the heterologously produced enzyme may yield inadequately titers, have alternate enzymatic properties, or be catalytically inactive. While significant improvements can be made via optimizations, the inherent features that makes the native host a prominent producer, in contrast to the heterologous host, are largely unknown and unpredictable.

This necessitates the development of a broader repertoire of fungal hosts that can be employed in screening for production capabilities, in order to find the most suitable host. We ventured to facilitate the use of broader fungal host repertoires by developing gene expression platforms that simplify heterologous production endeavors in multiple fungal species.

To assist in this task, we established a novel CRISPR-based genetic tool for industrial research and development, characterized a new constitutive bidirectional promoter, and evaluated the use of automated microscale cultivation systems for filamentous fungi. Using our gene expression platform, we investigate the heterologous production of certain proteins in different filamentous fungal species of genus Aspergillus to identify the best producer organism for each protein.

Chapter 1 introduces essential topics to the field of filamentous fungi and heterologous enzyme production, and provides context by highlighting the importance of assaying multiple heterologous hosts for production of a given protein. Chapter 2 describes the synthetic biology tool-box for genetic engineering of filamentous fungi and how these approaches can be used to establish gene expression systems for heterologous production of enzymes and secondary metabolites.

This self-contained chapter also goes into depth with topics related to optimizing production of enzymes and secondary metabolites, including engineering of the secretory pathway and use of protease deficient strains in production. Chapter 3 describes the development of a novel bio-brick in genus Aspergillus, namely the bidirectional promoter of histone-genes h3 and h4.1, Ph3h4.

Within the scope of establishing expression platforms that are functional in a broader group of organisms, a goal of the study was to investigate this promoter in terms of its usability across genus Aspergillus. To accommodate this, we took the promoter from five species, belonging to five phylogenetic sections of genus Aspergillus, and applied them for heterologous expression of fluorescent proteins in Aspergillus nidulans.

Through expression analysis, we show that all five promoters provide strong expression from both directions in both solid-state and submerged cultivations. A second goal was to show that several of these promoters could be used for expression of a biosynthetic gene cluster. As proof-of concept, we used the Ph4h3 of A. niger and A. clavatus to express four genes from the putative malformin pathway within a single locus in A. nidulans.

Chapter 4 describes the usage of automated microscale submerged cultivations using two different methods; the Hamilton-robotics setup, and the stand-alone instrument BioLector II. These were used for physiological characterizations of different fungal strains and evaluation of productivity of a fluorescent protein.

Chapter 5 describes the usage of CRISPR endonuclease SpCas9 for the establishment of a uniform expression platform in four species of Aspergillus. This platform was used to express four different proteins; β-glucoronidase (uidA) from Escherichia coli, mRFP1, 6-MSA synthase (yanA) from A. niger, and a cellobiohydrolase.

From this, we observed differential production levels among the four species in a product-dependent manner. Chapter 6 describes the establishment of a novel genetic tool for genetic engineering of filamentous fungi, namely the CRISPR endonuclease MAD7™ from Inscripta™. The functionality of the established MAD7-CRISPR system was validated by mutagenesis of the albA pigment gene in A. niger.

Subsequently, we applied this CRISPR system in selected fungal species for generating basic working fungal strains (pyrGΔ, ku70Δ) that harbor a uniform expression platform, a modified version of the platform in Chapter 5, integrated in a defined integration site.