Biology subjects

Wong, R. W. K.

Publications and source records attributed to Wong, R. W. K..

2 recordsLinked to original sources

bioRxiv2025

Recombinant Laccase Production Causes Alterations of the S. cerevisiae Proteome that are Dependent on the Strain Origins

Saccharomyces cerevisiae yeast is a widely used recombinant protein production host. Recombinant protein expression requires adaptation of the host cell proteome to accommodate recombinant expression. However, this adaptation has not been well characterized. A better understanding of the adaptation to recombinant protein expression may inform us of pathways important to the process of expression. The proteome of a laboratory yeast was measured each of the 4 days of recombinant laccase expression to determine the adaptations of the proteome. Whereas a sizeable portion of the proteome had altered levels in response to nutrient depletion in batch growth, a smaller portion of the changes was specific to the laccase expression. By comparing yeast strains of different origins and laccase production capacities, we found that each strain tends to display a distinct response to heterologous expression, regardless of the origin of the laccase. For example, the chaperones Hsp26 and Kar2 were specifically elevated in a whey-derived strain upon laccase expression. Nonetheless, the higher capacity to produce active recombinant laccase in some strains appears to be more strongly associated with small groups of proteins that are constitutively expressed at different levels. These results indicate that strains of different origins each provide a unique cellular milieu that, in some cases, is more favorable for the expression of a given recombinant protein. This study provides potential new targets for strain engineering to improve the yield of select recombinant proteins and provides the first insights into the dynamics of the yeast proteome during recombinant laccase expression. Key PointsO_LIProteomes of S. cerevisiae strains during recombinant laccase expression determined C_LIO_LIChanges to ribosomal & metabolic protein levels occur during recombinant expression C_LIO_LIUnique cellular milieu, rather than proteome shifts, is linked to higher yields C_LI

molecular biology↗

bioRxiv2024

Mining Yeast Diversity Unveils Novel Targets for Improved Heterologous Laccase Production in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a widely utilized host cell for recombinant protein production due to its well studied and annotated genome, its ability to secrete large and post-translationally modified proteins, fast growth and cost-effective culturing. However, recombinant protein yields from S. cerevisiae often fall behind that of other host systems. To address this, we developed a high throughput screen of wild, industrial and laboratory S. cerevisiae isolates to identify strains with a natural propensity for greater recombinant protein production, specifically focussing on laccase multicopper oxidases from the fungi Trametes trogii and Myceliophthora thermophila. Using this method, we identified 20 non-laboratory strains with higher capacity to produce active laccase. Interestingly, lower levels of laccase mRNA were measured in most cases, indicating that the drivers of elevated protein production capacity lie beyond the regulation of recombinant gene expression. We characterized the identified strains using complementary genomic and proteomic approaches to reveal several potential pathways driving the improved expression phenotype. Gene ontology analysis suggests broad changes in cellular metabolism, specifically in genes/proteins involved in carbohydrate catabolism, thiamine biosynthesis, transmembrane transport and vacuolar degradation. Targeted deletions of the hexose transporter HXT11 and the Coat protein complex II interacting paralogs PRM8 and 9, involved in ER to Golgi transport, resulted in significantly improved laccase production from the S288C laboratory strain. Whereas the deletion of the Hsp110 SSE1 gene, guided by our proteomic analysis, also led to higher laccase activity, we did not observe major changes of the protein homeostasis network within the strains with higher laccase activity. This study opens new avenues to leverage the vast diversity of Saccharomyces cerevisiae for recombinant protein production, as well as offers new strategies and insights to enhance recombinant protein yields of current strains.

molecular biology↗