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Strawn, Garrett

University of British Columbia

The yeast Saccharomyces cerevisiae is an attractive host for the recombinant production of proteins. Despite widespread, the typically low protein yield is a severe disadvantage compared to other host systems. To address this limitation, the objective of this research is to identify gene deletions that influence recombinant protein production in S. cerevisiae. Specifically, we have used an industrially relevant fungal laccase enzyme, Lcc1, isolated from Trametes trogii as our model recombinant protein. We utilized synthetic genetic array (SGA) methodology to produce a library of 4,790 laccase expressing gene deletion strains and a novel soft agarose overlay assay to assess quantities of active secreted laccase. We identified 66 gene deletions that resulted in increased secreted laccase. Gene ontology (GO) analysis revealed an enrichment for processes such as vacuolar targeting, proteolysis, autophagy, and vesicle trafficking. Further validation of the levels of secreted laccase from the identified mutant strains was done in liquid cultures resulting in a set of 17 gene deletions with significantly increased secreted laccase activity from that of a reference strain. Notably, deletions of the endoplasmic reticulum (ER) membrane localized flippase, ARV1, a component of the SKI complex, SKI3, and PMT2, an ER membrane protein-O-mannosyltransferase all resulted in an over 4-fold increase in secreted laccase activity. Through complementation experiments and independent gene deletions, we were able to verify the role of six gene deletions in increasing secreted laccase activity. We have also identified a set of 207 gene deletions which decrease secreted laccase activity that could serve as potential targets for overexpression. When assessed in liquid cultures, deletion of the ER co-chaperone SCJ1, resulted in almost no secreted laccase activity. However, upon overexpression of the co-chaperone, secreted laccase activity was also decreased compared to that of a reference strain suggesting the basal expression level is optimal to produce the recombinant laccase. Our results firstly demonstrate the application of high throughput screening techniques for investigating effects on recombinant protein production. Additionally, we have identified a set of gene deletions with uninvestigated roles in recombinant laccase production thus elaborating upon previously identified bottlenecks limiting recombinant protein production in S. cerevisiae.

Text

2023-10-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83099

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/