Increasing expression level and copy number of aYarrowia lipolyticaplasmid through regulated centromere function

Manipulating gene expression using different types of plasmids (centromeric, 2-micron, and autonomously replicating sequence) can expand expression levels and enable a quick characterization of combinations, both of which are highly desired for metabolic engineering applications. However, for the powerful industrial workhorse Yarrowia lipolytica, only a low-copy CEN plasmid is available. In this study, we engineered the CEN plasmid from Y. lipolytica by fusing different promoters upstream of the centromeric region to regulate its function and expand its range. By doing this, we successfully improved the copy number and gene expression at plasmid level by 80% and enabled a dynamic range of nearly 2.7-fold. This improvement was seen to be independent from both the promoter and gene used in the expression cassette. These results present a better starting point for more potent plasmids in Y. lipolytica.