UC Riverside

In this work, I investigated strategies to improve recombinant protein production in yeast by targeting both transcriptional regulation and protein secretion. First, I characterized how mutations within the promoter region of Saccharomyces cerevisiae influence gene expression. Targeted mutagenesis revealed that increasing GC content within the TATA box and pre-initiation complex consistently reduced expression, identifying these regions as highly sensitive to sequence composition. I then expanded this approach using CRISPR-enabled trackable genome engineering (CREATE) to introduce a broader range of mutation types across an extended promoter region. This analysis confirmed that GC-rich substitutions were strongly associated with decreased expression, while certain AT- or mixed-base substitutions in the scanning region and transcriptional start site contributed to increased promoter activity. Finally, I adapted a mid-western (colony blot) secretion assay for use in Kluyveromyces marxianus. Using HiBiT-tagged constructs, I established a range of low, medium and high secretion outputs across multiple promoter–signal peptide–gene combinations. Together, these results demonstrate that combining promoter engineering with secretion screening provides a systematic framework for optimizing protein production in yeast.