Improving protein production in the yeast Saccharomyces cerevisiae: the role of non-coding RNAs

Abstract

Over the past few decades, significant benefits to humankind have been provided by advances in the production of recombinant proteins. However, an increased demand exists for more efficient and sustainable methods to enhance protein secretion in the industrially crucial expression system, yeast. This research introduces two innovative strategies for augmenting both endogenous and recombinant protein secretion in Saccharomyces cerevisiae, with an emphasis placed on the role of non-coding RNAs (ncRNAs). In the first strategy, the effect of ncRNAs on yeast protein secretion was explored through comprehensive screening for enhanced endogenous invertase secretion in ncRNA deletion strains. Emphasis was placed on the deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, and snRNAs. Three notable candidate ncRNAs - SUT418, SUT390, and SUT125 - were identified. When these ncRNAs were deleted, an improvement in endogenous invertase secretion was observed. An investigation revealed downregulation of the PIL1 gene in the SUT125 deletion strain. Given the established role of PIL1 as an eisosome component, critical for plasma membrane invaginations, it was found that PIL1 knockout resulted in enhanced secretion of endogenous invertase, heterologous GFP, and a single-chain Fv (scFv) fragment Pexelizumab (Pex). Interestingly, the signal peptide was found to dictate this enhanced secretion. This investigation led to the realization that PIL1 deletion substantially boosts protein secretion, potentially via the eisosome disruption and subsequently upregulation of sphingolipid biosynthesis, revealing a novel avenue for augmenting protein secretion in yeast. For the second strategy, the individual deletions of five protein-coding genes, known to enhance protein secretion, were produced. These genes - SNC2, HDA2, EMC1, OPI1, and VPS10 - are implicated in various processes, from membrane trafficking to protein sorting. These genes were deleted in diverse combinations to assess potential synergistic impacts on invertase secretion. It was found that the deletion of SNC2 alone yielded twice the invertase secretion. Greater enhancements in secretion were observed with the combined deletion of SNC2 & HDA2 and SNC2 & VPS10. Nonetheless, no further augmentation in secretion was discerned in combined deletions involving three gene deletions, suggesting an inherent limitation to protein secretion enhancement by multiple protein-coding gene deletions. In collaboration, an analysis of synthetic genetic interactions of ncRNAs in S. cerevisiae was conducted, with a focus on the subfunctionalisation of the U3 snoRNA genes SNR17A and SNR17B. A prior Synthetic Genetic Array (SGA) analysis had hinted at potential sub-functionalisation of SNR17A and SNR17B under stresses. The predominant function of U3 snoRNA was inferred to be primarily managed by SNR17A under stress conditions, while SNR17B appeared to serve alternative, yet-to-be-determined functions. In summary, invaluable insights into the regulation of protein secretion in yeast are provided by this research, unveiling two innovative strategies: ncRNA regulation and secretion related coding gene deletions. The pivotal role of ncRNAs and coding genes in enhancing protein secretion was elucidated. The constraints encountered with multiple protein-coding gene deletions was underscored, suggesting a limitation to secretion enhancement via this approach. Additionally, insights into the subfunctionalisation of U3 snoRNA SNR17A and SNR17B under stresses were deepened. Collectively, these discoveries set a foundation for subsequent research aimed at optimizing the efficiency and sustainability of protein secretion in yeast, further amplifying the horizons of recombinant protein production.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Raymond O'Keefe (Supervisor) & Catherine Millar (Supervisor)