Abstract
The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology is presented. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.
Highlights
The oleaginous yeast Yarrowia lipolytica is an attractive host for industrial production of fatty-acid derived chemicals, organic acids, and enzymes [1]–[3]
The most widely applied CRISPR/Cas9 system for genome editing combines three parts: (1) a sgRNA [10], composed of a site-specific crRNA fused to a tracrRNA, which binds Cas9 endonuclease, (2) the Cas9 endonuclease, capable of creating dsDNA breaks, and (3) a dsDNA repair template, which is used by the homologous recombination (HR) pathway to repair the dsDNA break
In pCfB4906, a Y. lipolytica codon-optimized Cas9 gene from Streptococcus pyogenes is under control of the Tef promoter and terminator and has been integrated into the IntB integration site [23] with the help of a hygromycin resistance marker
Summary
The oleaginous yeast Yarrowia lipolytica is an attractive host for industrial production of fatty-acid derived chemicals, organic acids, and enzymes [1]–[3]. The engineering efforts so far have been hampered by limited genome targeting efficiencies in Y. lipolytica and by the requirement for selection markers, which need to be recycled. The low genome targeting efficiencies are due to a high rate of non-homologous end joining (NHEJ) for repair of DNA double-strand (ds) breaks in contrast to Saccharomyces cerevisiae, where the homologous recombination (HR) mechanism is the dominating repair pathway [7][8]. To overcome the above-mentioned limitations of marker-based genome editing, the CRISPR/Cas system has been successfully implemented in several yeast species [9]. The most widely applied CRISPR/Cas system for genome editing combines three parts: (1) a sgRNA [10], composed of a site-specific crRNA fused to a tracrRNA, which binds Cas endonuclease, (2) the Cas endonuclease, capable of creating dsDNA breaks, and (3) a dsDNA repair template, which is used by the HR pathway to repair the dsDNA break
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