(271a) Discovery of Novel Strong Yeast Promoters for Refactoring Fungal Natural Product Gene Clusters Conference: AIChE Annual MeetingYear: 2015Proceeding: 2015 AIChE Annual MeetingGroup: Food, Pharmaceutical & Bioengineering DivisionSession: Biocatalysis and Biosynthesis I: Techniques Time: Tuesday, November 10, 2015 - 8:30am-8:47am Authors: Ning, J., University of Illinois at Urbana-Champaign Zhao, H., University of Illinois at Urbana Champaign Refactoring fungal natural product gene clusters in Saccharomyces cerevisiae represents an attractive strategy for discovery of novel natural products. Robust function of these gene clusters involving large proteins like polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases especially depends on strong expression levels to drive production forward. Previous advances in yeast promoter engineering have focused on improving a limited set of well-characterized promoters, but few efforts have been applied to expand the diversity and range of promoters available. We have taken a two-pronged approach to identify new promoters for use in S. cerevisiae that extend the range of promoter strength and expression timing. Utilizing a yeast-optimized destabilized GFP reporter system, we leveraged preexisting expression data to identify several potent, previously unutilized native yeast promoters. Among them, the promoter for phosphoenolpyruvate carboxykinase (PCK1) displays expression 4-fold higher than the strongest reported constitutive native yeast promoter, TEF1p, and also 1.5-fold higher expression than the most commonly used inducible promoter, ADH2p. Hypothesizing that the vital role of PCK1 in catabolism being conserved among yeasts would lead to similar promoter strengths in related species, we cloned 17 promoters controlling the orthologous genes from various yeast species. Seven of them were found to be active in S. cerevisiae and maintained relatively similar expression profiles with varying strengths, resulting in a library of sequence-varied promoters for concerted expression of genes. We have also demonstrated the utility of these promoters by overexpressing multiple heterologous pathways to increase the titer of the corresponding natural products.