Improving Protein Production in Yeast with Targeted and Exploratory Approaches

Recombinant proteins have many applications, from enzymes and protein therapeutics to meat and materials alternatives, and there is significant interest in trying to maximize the productivity of protein expressing strains. In a recent webinar, Eric Abbate, Inscripta’s Director of Analytical Biochemistry and Applications Development, showed how the Onyx platform can be used to improve heterologous protein production in S. cerevisiae.

In the webinar, Abbate explained how he used the OnyxTM workflow to engineer S. cerevisiae to optimize production of cellobiohydrolase I (CBH1), an enzyme involved in cellulose degradation.

Abbate chose two kinds of libraries for his experiments: genome-wide libraries and targeted libraries. The genome-wide libraries were for gene knockouts, terminator insertions, and short deletions, while the targeted libraries focused on edits to the CBH1 promoter as well as targeted knockout libraries for genes previously shown to play a role in heterologous protein production.

Screening using both library types yielded several hits where the strains showed significantly improved CBH1 production compared to the base strain, up to a 1.65-fold improvement from various edit types.

In total, Abbate generated just under 15,000 edits and screened nearly 8,500 isolates for this experiment. This resulted in more than 70 unique hits. The greatest number of hits came from a shallowly screened library, which is why Inscripta recommends shallow screening of many libraries rather than deeply screening just a few when phenotyping capacity is constrained.

Abbate noted that the hits affect different genomic functions, from protein degradation and protein trafficking to transcription, translation, nuclear signaling and glycosylation pathways. Some of these hits were in genes that Abbate said would likely have been missed had he not cast a wide net with the untargeted, genome-wide edits.

These results demonstrate how a combination of informed and exploratory edits enable rapid improvement of strain productivity. What is more, these beneficial edits could be potentially combined for even greater increase in protein production. To learn about the different libraries we tested and gain insights into the mechanisms behind improved production, you can watch the highlights below and listen to the full webinar.