The impact of evolutionary dynamics and expression plasmid stability on long-term heterologous protein production by yeast

Abstract Background Baker’s yeast, Saccharomyces cerevisiae , is a widely used industrial host organism for heterologous protein production. Ensuring stable, high-level production over extended production campaigns is important for low-cost manufacturing. However, maintaining consistent yields over multiple generations is challenging as strain productivity may decline over time. Understanding potential evolutionary mechanisms underlying such decline is therefore important for optimising yeast-based expression systems in continuous manufacturing. Results In this study, diverse yeast libraries carrying high-copy-number whole-2-micron episomal expression plasmids producing a secreted SARS-CoV-2 spike protein fragment fused to mCherry were used to investigate fluorescence-based production stability during prolonged cultivation in yeast. We observed that while some strains maintained relatively stable mCherry fluorescence, most strains with high initial total fluorescence showed marked declines over 140 generations. Growth assays indicated that cultures with reduced mCherry fluorescence had a fitness advantage over high-fluorescence cultures. The decline was partly explained by phenotypic heterogeneity among genetically uniform cells; early-stage (10th generation) cultures contained distinct, non-fluorescent, low-fluorescence and high-fluorescence subpopulations (these subpopulation phenotypes were non-heritable), whereas evolved low-fluorescence cultures after 140 generations displayed loss of the high- and/or low-fluorescence subpopulations and reduced cell-to-cell heterogeneity. Despite this, the selected evolved low-fluorescence isolates had fluorescence restored to near-initial levels after being cured of their expression plasmids and retransformed with plasmids from early-stage, high-fluorescence cultures, indicating that, in the selected evolved low-fluorescence isolates analysed, plasmid-associated changes were sufficient to explain the stable reduction in mCherry fluorescence. Whole-plasmid sequencing of the episomal 2-micron expression plasmids isolated from low-fluorescence cultures at the 140th generation revealed mutations in key functional regions, including the methionine-regulated MET17 promoter driving SARS-CoV-2 protein expression, FRT recombination sites and the SARS-CoV-2 spike protein fragment coding sequence. Conclusion The results support a model in which prolonged expression of a burdensome secreted recombinant protein can be associated with early non-genetic heterogeneity in intracellular mCherry fluorescence, growth advantages of low-fluorescence cells and evolution of plasmid variants that reduce expression. The extent of fluorescence decline varied among genetically diverse strains, with some remaining comparatively stable, highlighting the potential of exploiting yeast strain diversity to obtain production strains with improved long-term stability.