Evolution of yeast variation during continuous manufacturing processes

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. 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.
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.