Yarrowia lipolytica is a yeast species that utilizes non-homologous end joining as the main mechanism for repairing double-strand DNA breaks. During this process, DNA fragments integrate into non-target loci, which can have unexpected effects on the resulting recombinant strain. By designing integration into the selected genomic loci, we can obtain strains with stably inserted DNA fragments at the desired locus. Bioinformatically, we identified 12 genomic loci for targeted integration and compared them with random integration at zeta locus, which appears multiple times in the genome. To evaluate integration sites, we used a reporter gene for green fluorescent protein (GFP), which can be easily measured with a spectrophotometer. Using Golden Gate assembly and Gibson cloning methods we assembled recombinant plasmid DNA molecules containing an expression cassette with the gene for the heterologous GFP. Plasmids differed in the homologous DNA sequence for each integration site. We used two different strains of Y. lipolytica: CH-164 and PO1f. We obtained transformants with integration at all target sites. Production of GFP protein varied among the transformants, with the highest observed at the integration site B1, which is located on the B chromosome, at a locus between two genes whose function is unknown. Through spectrophotometric measurement of fluorescence, SDS-polyacrylamide gel electrophoresis and Western blot protein detection, we successfully detected expressed GFP.
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