Metabolic engineering requires precise control of gene expression to improve flux through target molecules. Oleaginous yeast Yarrowia lipolytica has been emerging as an industrial workhorse due to its high flux lipids biosynthetic pathways; however, fewer tools to control gene expression are available compared to model yeast S. cerevisiae. To date, most approaches to control gene expression use endogenous promoters or hybrid promoters. Both types of promoters have difficult to anticipate responses to different substrates and genetic contexts. Here, we develop a tunable expression system in Y. lipolytica by mutating the TATA Box region in different promoters. The 8bp TATA Box region was randomized through degenerate bases and promoter libraries created to express hrGFP. We used FACS to isolate TATA Box mutants resulting in different expression levels and validated individual mutants through RT-qPCR. The correlation between fluorescence intensity, relative transcriptional level and relative translational level is evaluated over time in the context of four commonly used yeast promoters TEF, TEF-Intron, EXP and POX3. To demonstrate the utility of finely tuned expression systems, we used the expression system to balance a multienzyme heterologous pathway for naringenin synthesis. This work generates a simple and reliable system for tuning the expression system in Y. lipolytica by modifying the TATA Box to balance a heterologous pathway

Engineering TATA Box to create a promoter library to balance heterologous pathways in Y. lipolytica

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.