Understanding the Role of Epigenetics in Fatty Acid Regulated Promoters in Yarrowia Lipolytica

Authors: 
Blenner, M., Clemson University
Shabbir Hussain, M., Clemson University
Edgecomb, S., Clemson University
The genetic toolbox for the industrial oleaginous yeast, Y. lipolytica, has been improved in recent years. These tools include CRISPR-Cas9 mediated genome editing to libraries of promoters for fine-tuned gene expression. Most recently, we have reported the development of a strong fatty acid inducible hybrid promoter built from activating and regulatory sequences derived from the POX2 promoter. This promoter is the strongest fatty acid inducible promoter reported for Y. lipolytica. having nearly 50-fold induction relative to glucose with expression ranging from 2 to 10-fold higher than the commonly used native inducible acyl CoA oxidase (POX2) promoter. Here, we describe our most recent work to characterize the transcription factor binding sites important for fatty acid responsiveness and how these sites interact with nucleosomes to lead strong repression in the absence of fatty acid, and induction in its presence. We have performed mutagenesis of predicted transcription factor binding sites in functionally important promoter segments as well as studies that knockout transcription factor genes. These studies are complemented with analysis of nucleosome mapping under repressed and inducing media condition. We also examine changes in nucleosome occupancy at different growth phases, and compare these changes to promoter and transcription factor binding at the same time points. Together, these data improve our understanding of how transcription factors and nucleosomes interact to control gene expression. We suggest that these principles could be broadly applied in the context of synthetic biology to engineer responsive promoters and more fine control of transcription.