Expanding the Genetic Toolbox to Improve Metabolic Engineering in the Industrial Oleaginous Yeast, Yarrowia lipolytic

The oleaginous yeast, Y. lipolytica, is becoming a popular host for industrial biotechnology because of its ability to grow on non-conventional feedstocks and naturally accumulate significant amounts of lipids. With new genome editing technologies, engineering novel pathways to produce lipid-derived oleochemicals has become easier. The goal, however, is to expand the genetic toolbox to improve the efficiency of metabolic engineering such that production capacities could expand from proof-of-concept shake flasks to an industrial scale.;Building efficient metabolic circuits require controlling strength and timing of several enzymes in a metabolic pathway. One method to do this is through transcription---using suitable promoters to control expression of genes that code for enzymes. Native promoters have limited application because of complex regulation and non-tunable expression. Engineering hybrid promoters alleviates these issues to obtain predictable and tunable gene expression. In Y. lipolytica, how to design these promoters is not fully understood, resulting in only a handful of engineered promoters to date.;In this work, we aim to develop tools for gene expression by investigating promoter architecture and designing tunable systems. In addition to Upstream Activating Sequences (UAS), tuning promoter strength can be achieved by varying sequence in the core promoter, TATA motif, and adjacent proximal sequences.;UASs can modulate transcription strength and inducibility, enabling controlled timing of expression. A promoter of the acyl-CoA oxidase 2 (POX2) from the beta-oxidation pathway was truncated heuristically to identify oleic acid (OA) UAS sequences. By fusing tandem repeats of the OA UAS elements, tunable yet inducible fatty acid hybrid promoters were engineered.;The current approaches to identify novel UAS elements in Y. lipolytica are laborious. Therefore, we investigated DNA accessibility through nucleosome positioning to determine if a relationship between POX2 UASs and DNA accessibility can be inferred. The goal is to eventually apply this approach develop newer hybrid promoters efficiently.;Finally, the hybrid fatty acid inducible promoter we developed was used to rationally engineering a Y. lipolytica strain capable of producing high amounts of free fatty acids. By localizing the fatty acyl / fatty aldehyde reductase in the peroxisome, we compartmentalized fatty alcohol production. This strategy led to upwards of 500 mg/L of fatty alcohols produced. It is a promising route to eventually make short to medium chain fatty alcohols in Y. lipolytica by utilizing the native ?-oxidation machinery.