Expanding the molecular toolbox and understanding of Komagataella phaffii for optimised heterologous protein production

Biomanufacturing of heterologous proteins from genetically engineered cell factories has been a growing industry since the first heterologous pharmaceutical protein, human insulin, was released to the market more than four decades ago. Biomanufacturing serves as an economically profitable industry with biopharmaceutical production representing a multi-billion-dollar market. Importantly, bioproduction processes have the potential to deliver more sustainable manufacturing strategies. Hence, engineering of cell factories constitutes an important research field to fully exploit the potential of biosynthetic production.

The non-conventional budding yeast, Komagataella phaffii, has gained increasing attention as an attractive host for the production of heterologous proteins and other valuable products. K. phaffii has proven valuable for bioengineers as a production platform thanks to several beneficial characteristics: ease of genetic manipulation and cultivation; possession of eukaryotic post-translational machinery; and strong secretory capacity. Additionally, K. phaffii has been designated as a generally recognized as safe (GRAS) organism by the US Food and Drug Administration, further strengthening its attractiveness as a production platform. However, K. phaffii is still relatively new on the market compared to other established microbial hosts, like the budding yeast Saccharomyces cerevisiae. As such, there is still much to learn about this organism to fully tap into its potential as a production host.

A challenge faced when using living organisms as workhorse for biomanufacturing is that such unnatural overproduction of a desired biomolecule is often associated with cellular burden. Strong expression, protein synthesis and processing required for heterologous product formation hijacks cellular resources, which limits normal cellular and physiological processes. Over time, this burden on the host cell metabolism can ultimately curb bioproduction. As such, these consequences of biomanufacturing impose a selective pressure on cells promoting evolutionary drift to evade burdensome production. Thus, design strategies that facilitate controlled bioproduction by alleviating these challenges and ensuring long-term robust biosynthetic production are required.

The dual focus of this thesis is to expand the molecular toolbox available for engineering of K. phaffii and evaluation of the molecular response to heterologous protein burden in K. phaffii in order to better understand and utilize this organism. The overall aim is to enhance the ability to engineer well-performing and robust K. phaffii cell factories.

First, I set out to summarize what is already reported on the topic of burden imposed by heterologous protein production in the two major budding yeast production hosts: K. phaffii and S. cerevisiae. This work was published as a literature review article and presented here in Chapter 2.

For the engineering of K. phaffii cell factories for heterologous protein production, expanding the available molecular toolbox became part of the journey for exploring K. phaffii as a host organism. This journey led me to generate a K. phaffii promoter library. By using a fluorescent reporter protein, we screened the promoter library for the identification of promoters with the potential to be used in bioengineering applications. For this, I collaborated with another research group to develop a new microplate reader-based high-throughput screening technique for fluorescent protein analysis of arrayed microbial colonies. The development and testing of this method is presented in a published research article in Chapter 3.

The results of the promoter library experiment itself were published in a research article and presented here in Chapter 4, where we also present an in silico approach to identify native promoters in genomes predicted to be powerful based on the codon usage of the downstream open reading frames. Specifically, the promoter driving expression of the Stationary phase induced 1 gene (P_SPI1) stood out as a strong constitutive promoter and was used in my following work in Chapter 5.

Finally, I engineered K. phaffii heterologous protein production strains for the characterization of burden. Production strains were screened in parallel microbioreactors and validated under industrially relevant conditions in chemostats. To unravel the molecular response of the production strains to varying levels of production, three ”omics” level responses were examined - transcriptome, proteome, and secretome (extracellular proteome). Thus, in the work presented in Chapter 5 (manuscript submitted to FEMS Yeast Research), cellular and molecular insight into the response of K. phaffii cell factories is presented.