LAUSR

The engineering of Yarrowia lipolytica to accumulate lipids with high titers and productivities has been enabled with a handful of constitutive promoters for pathway engineering. However, the development of promoters that are both strong and lipid responsive could greatly benefit the bioproduction efficiency of lipid‐derived oleochemicals in oleaginous yeast. In this study, a fatty acid regulated hybrid promoter for use in Y. lipolytica is engineered. A 200 bp upstream regulatory sequence in the peroxisomal acyl CoA oxidase 2 (POX2) promoter is identified. Further analysis of the promoter sequence reveal a regulatory sequence, that when used in tandem repeats, lead to a 48‐fold induction of gene expression relative to glucose and fourfold higher than the native POX2 promoter. To date, this is the strongest inducible promoter reported in Y. lipolytica. Taken together, the results show that it is possible to engineer strong promoters that retain strong inducibility. These types of promoters will be useful in controlling metabolism and as fatty acid sensors.