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

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posted on 27.12.2021, 19:38 by Xiaoqin Liu, Mengmeng Liu, Jin Zhang, Yizhao Chang, Zhiyong Cui, Boyang Ji, Jens Nielsen, Qingsheng Qi, Jin Hou
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 × 105 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 β-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.

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