(75d) Using Promoter Architecture to Guide Engineering of Strong Fatty Acid Inducible Hybrid Promoters in Yarrowia Lipolytica

Recently, there has been significant improvements in the genetic toolbox for the industrial oleaginous microbe, Y. lipolytica. These tools range from CRISPR-Cas9 mediated genome editing to libraries of hybrid promoters made from strong upstream activating sequences for tunable strength; however, strong inducible promoters remain undeveloped. In addition to tunable strength, having control of timing of gene expression can improve cellular efficiency by separating the growth and production phase. Inducible regulation is particularly important when certain products are toxic and inhibit growth, or when requiring the ability to switch on production at stationary phase. Here, we demonstrate the development of the only known and strongest fatty acid inducible promoter system in Y. lipolytica. The hybrid promoter has nearly 50-fold induction strength relative to glucose with expression ranging from 2 to 10-fold higher than the commonly used native inducible acyl CoA oxidase (POX2) promoter. Additional engineering is accomplished with tandem repeats of an approximately 200 bp fatty acid inducible element to create a tunable hybrid promoters with a significantly larger dynamic expression range than the native POX2 promoter. We explore the mechanism of regulation by investigating epigenetics associated with inducible regulation in the POX2 promoter. Libraries of promoter strengths are accomplished by manipulating the different core functional elements of the promoter system. While repressed in the absence of fatty acids, the hybrid promoter lacks catabolite repression in the presence of either glucose or glycerol in fatty acid containing media. This inducible genetic switch can be can be strongly activated at stationary phase with low concentrations of a wide range of fatty acids ranging from oleic acid to eicosapentaenoic acid. The activity of the inducible promoter correlates well with changes in intracellular fatty acid pools, showing strong promise in application as a tool for strain engineering.