posted on 2021-12-27, 19:38authored byXiaoqin 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.