Identifying and overcoming protein secretion bottlenecks in yeast and filamentous fungal cell factories

Yeasts and moulds are both types of fungi. While yeasts such as Saccharomyces cerevisiae are widely regarded as useful because of its roles in making beer and wine, and in making bread, most moulds are regarded as a nuisance because they cause visible spoilage of foods, even though some species have uses in making valuable chemicals (e.g. penicillin). In fact, both yeasts and moulds are very important and are used commercially to make proteins that are not naturally produced by that fungus. In those cases, the gene that is necessary to make the protein is introduced into the fungus. Examples are insulin (for diabetics), chymosin (for cheese production) and a variety of enzymes used in food processing. Also, basic information about how enzymes work requires a plentiful supply of protein which usually means that those proteins are produced by a 'cell factory' such as a fungus. A common difficulty in making proteins from cell factories is that the yields are lower than desired. That is because the cell senses that it is being asked to make, and secrete to the outside of the cell, a protein which it wouldn't ordinarily make. The cell becomes stressed and it initiates a series of events within the cell aimed at protecting itself which, in so-doing, reduces the yield of protein secreted to the outside of the cell. Some proteins give rise to a bigger stress than others but we do not know the rules that govern the stress. This project aims to identify the rules and, thereby, understand how to predict which proteins will cause most stress and then to take steps with the 'cell factory' organisms to counteract the stress. In that way, we hope to be able to improve the cell factories to make more of the desired proteins. In particular, we aim to improve the capacity of fungal cell factories to produce antibody molecules that could be useful clinically.

The yeasts Saccharomyces cerevisiae and Pichia pastoris are the main experimental organisms in this study. Both species are used as cell factories (in the laboratory and commercially) for secreted protein production. S. cerevisiae is also an excellent model organism for investigations into the basic events involved in protein secretion and stress responses. P. pastoris is less amenable to basic studies but is an outstanding cell factory. This project will create some tools for basic studies in P. pastoris as well as using both organisms for comparative studies with each other and with the principal filamentous fungal cell factory, Aspergillus niger. We will examine the secreted expression of lysozyme and derived variant molecules, as well as scFv antibody proteins. Many of the necessary strains are already available although some will be constructed within the project. Controlled and reproducible cell culture is a necessary part of the studies proposed. The main technical objectives and main methods to be used in the study are: 1. Use transcriptomics and proteomics to examine the stress responses due to expression of variant lysozymes in S. cerevisiae and P. pastoris. 2. Examine the fates of selected lysozyme variants, including the folded states, using imaging, conformational antibody approaches and protein turnover studies. 3. Express and examine resulting secreted protein yields and stress responses from scFv proteins, measure thermal stability of purified scFvs, and compare with lysozymes. 4. Use comparative genomics methods to compare stress responses from S. cerevisiae, P. pastoris and Aspergillus niger to find commonality and differences. 5. Define and test a strategy for rational strain improvement for optimized secretion of scFvs based on stress response and protein fate studies.