Genomic hot spots for strong and stable expression sites in Yarrowia lipolytica

Integrative expression in Y. lipolytica has been accomplished by targeting the zeta site for multiple copy integration to promote strong expression and by site specific integration. While this provides for stable and strong integration, there is little control of copy number and/or expression strength. Therefore, being able to control expression strength and attain strong yet stable expression is essential for metabolic engineering applications. It is well known that in eukaryotes expression strength is genomic loci-dependent as there are several factors that contribute to controlling transcriptional activation and amplification at the genomic scale. Here, we sought to identify loci in Y. lipolytica that promote strong expression and study the physical environment that enables such expression levels. To identify these loci, we transformed linear DNA carrying a GFP reporter fluorescence gene under a strong hybrid promoter and allowed Y. lipolytica to randomly integrate the DNA into its genome. A library of fluorescent cells was sorted into bins of expression strength using Fluorescent Activated Cell Sorting (FACS). Our results indicate that expression strength varied based on integration locus. A combination of genome sequencing methods was used to identify GFP integration location in the genome. The highly fluorescent colonies were further characterized for expression strength under different carbon and nitrogen conditions to determine if local regulation influences heterologous gene expression. The information gained from these experiments will allow future metabolic engineering efforts to attain maximum expression from genetic cassettes when integrating onto the genome.