Evolution of yeast variation during continuous manufacturing processes

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Yeast is a proven cell factory for the production of therapeutic proteins, vaccines and other high-value biologics. The global market for these yeast-derived products is around $40Bn p.a. Yeast is attractive because it is fast-growing, eukaryotic (e.g., suitable for mammalian-protein expression), well characterised, safe and hardy. The biologics industry is increasingly shifting towards continuous manufacturing, with no interruptions to production. This is embraced by our non-academic partner, Phenotypeca, for lowcost vaccine manufacture; with obvious economic advantages. However, it also presents a problem that has so far been side-stepped, where the producing cell population becomes heterogeneous - showing mixed phenotypes - which can lower production yields over time. Theoretically, this can occur within 40-60 generations (a few days) after a production run has started. The metabolic burden of production could explain this decline, as it may create selection pressure for faster-growing low-yield variants. However, this significant problem has not yet been addressed, so this interdisciplinary project proposal is exceptionally timely. Yeast diversity in this context also presents intriguing academic questions. Experimental Plan and Methods The project will test the hypotheses that; (i) high-yield production drives diversification and selection in yeast cultures, and (ii) natural yeast diversity could be exploitable to address this problem. The specific objectives are: How rapidly do product-yields decline during continuous yeast culture? (Months 1-9). The project will focus on Saccharomyces cerevisiae, with which we have extensive experience and resources. The student will study key products of interest (e.g. albumin, amylases, VLP vaccines), corroborating key findings with other recombinant products. We have the relevant constructs and production yeast strains. Product measurement in this objective will be at the bulk population level. Yeasts will be analysed from batch-flask and fermenter culture. Is altered product-yield due to novel genotypes and/or novel phenotypic variants (i.e. nongenotypic heterogeneity, NGH) within cultures? (Months 10-20). Production by individual cells within populations will be compared by analysis of fluoro-tagged product with time-lapse microscopy and image-streaming flow cytometry. The student will use our microfluidics capability (CellASIC) for single-cell and NGH studies, with options for fermentation scale-up. Individual cells showing low or high production will be FACS-sorted and assayed for heritability of the production phenotype, to indicate genotypic or non-genotypic bases. One risk is semi-heritable epigenetic phenotypes, which we will mitigate with appropriate deletants and assay timescale. Genome re-sequencing of selected phenotypic variants will corroborate key conclusions. Can alternative yeast genetic-backgrounds or manipulation of NGH be harnessed to stabilise production over time? (Months 21-36). The supervisors have complementary expertise in genotypic and phenotypic heterogeneity. Using their available reagents, the student will select or refine natural strain backgrounds or NGH constructs (with results from Obj 2 steering a focus on genotypic or phenotypic heterogeneity, respectively) to develop novel production strains with decreased heterogeneity. These will be profiled for their production characteristics, hence the potential for improved manufacturing.