Monitoring and control of protein production in fungi

The work presented in this Ph.D. thesis describes the utilization of fluorescent proteins to investigate, compare and optimize protein expression and secretion in three different hosts; Saccharomyces cerevisiae, Aspergillus nidulans and Aspergillus niger. The following questions were addressed:
• How is protein production affected on a single cell level due to environmental stress factors?
• How can we improve heterologous protein production in filamentous fungi, and how does production in Aspergillus nidulans compare to protein production in the industrially exploited Aspergillus niger?

In Chapter one a solid background to recombinant protein production and the eukaryotic secretory pathway is given. Industrial products from yeasts and Aspergilli are presented, demonstrating the importance of recombinant protein production in these hosts. Since secretion of recombinant proteins is often preferred, the secretory pathway is described in detail. Differences between yeasts and Aspergilli are highlighted when appropriate. In addition, genetic manipulations of the secretory pathway in yeasts and Aspergilli are discussed, with a focus on how to improve recombinant protein production in these hosts. This field has been under a lot of investigation during the last few decades, and results show that alterations of the secretory pathway may have different effects depending on the host and also depending on what product is being produced. The chapter provides a foundation for later chapters, where the secretory pathway and molecular mechanisms is discussed based on experimental work.

Chapter two describes different reporter systems utilized in order to facilitate investigations within yeasts and Aspergilli. Significant focus lies on reporter systems based on fluorescent proteins in order to be able to monitor or quantify different processes within a cell. The chapter will also give an insight to how the secretory pathway and recombinant protein production can be investigated by utilizing fluorescent proteins. S. cerevisiae is one of the most commonly used eukaryotic model organisms. Advantages include highly developed molecular tools as well as an ease of performing physiological characterizations with a high reproducibility. Furthermore, it can be used for production of pharmaceutical proteins as well as for bio-ethanol production.

In chapter three the impact of various environmental stress elements on the production of heterologous proteins in S. cerevisiae is investigated. A fluorescent reporter strain, producing an intracellular protein linked to tagRFP from the glycolytic PGK1 promoter is constructed. This strain is used to monitor the level of production in each cell when exposed to environmental stress. The cells are grown in shake flasks as well as bioreactors and protein levels are analyzed by flow cytometry. It is demonstrated that the fluorescent reporter can be used to study the effects on stress elements on a population basis. Production of the protein was affected when cells were exposed to lower pH values, ethanol stress, increased osmotic pressure and increased glucose availability. A shift in fluorescence distribution is seen when cells are exposed to 1%, 3% and 5% ethanol, and this demonstrates that the strain is sensitive to small changes in the environment. Furthermore, increased levels of NaCl might be a way to improve protein levels. This effect is either due to inductive effects on the promoter, or other physiological changes in the cell. As the glycolytic PGK1 promoter was used, it was not surprising that increasing levels of glucose led to higher production levels of the protein. In addition, it is shown that a binomial distribution occurs when cells are growing on ethanol after the diauxic shift. Two subpopulations occur, and this may be due to some cells having a higher sensitivity to ethanol, or some cells entering the stationary phase earlier than others. This study provides a more detailed knowledge of how protein production is affected due to various environmental stress factors. In the longer perspective, the approach could potentially be used for improving industrial processes where these stress factors are likely to occur.

In chapter four, a protein secretion reporter strain in A. nidulans is developed (Figure 1). The strain secretes mRFP through a carrier fusion to the well secreted glucoamylase from A. niger. Secretion of mRFP is verified through fluorescence measurements, microscopy and SDS-PAGE. The protein localizes primarily to the plasma membrane, septa and hyphal tips. The reporter strain is used to overexpress 14 genes within the secretory pathway, with the aim of investigating how protein secretion is affected. Genes were chosen based on other studies, and are from different compartments within the pathway. Several of the chosen genes have an impact on growth and protein secretion. An increase of protein secretion with 25% is seen when overexpressing the Rab GTPase RabD, a protein involved in transport between the Golgi and the plasma membrane. Other genes show substantial negative effects on protein secretion. Furthermore, single cell protein distribution is investigated by microscopy, and it is demonstrated that the secretory cargo localizes more to the plasma membrane when rabD is overexpressed. The study demonstrates the effect of secretory pathway engineering, and shows how a secretion reporter can be used in order to investigate the effect in the engineered strains. The study gives a deeper insight to recombinant protein secretion in Aspergilli, and the results are one step towards providing a detailed overview on the secretory pathway. The study can be used as a starting point for engineering Aspergilli cell factories in order to improve strains for recombinant protein secretion.