Mapping of Nonhomologous End Joining-Mediated Integration Facilitates Genome-Scale Trackable Mutagenesis in Yarrowia lipolytica

Genome-scale mutagenesis, phenotypic screening, and tracking the causal mutations is a powerful approach for genetic analysis. However, classic mutagenesis approaches require extensive effort to identify causal mutations. It is desirable to demonstrate a powerful approach for rapid trackable mutagenesis. Here, we mapped the distribution of nonhomologous end joining (NHEJ)mediated integration for the first time and demonstrated that it can be used for constructing the genome-scale trackable mutagenesis library in Yarrowia lipolytica. The sequencing of 9.15 x 10(5) insertions showed that NHEJ-mediated integration inserted DNA randomly across the chromosomes, and the transcriptional regulatory regions exhibited integration preference. The insertions were located in both nucleosome-occupancy regions and nucleosome-free regions. Using NHEJ-mediated integration to construct the genome-scale mutagenesis library, the new targets that improved beta-carotene biosynthesis and acetic acid tolerance were identified rapidly. This mutagenesis approach is readily applicable to other organisms with strong NHEJ preference and will contribute to cell factory construction.