sb1c00390_si_002.xlsx (67.3 MB)
Mapping of Nonhomologous End Joining-Mediated Integration Facilitates Genome-Scale Trackable Mutagenesis in Yarrowia lipolytica
dataset
posted on 2021-12-27, 19:38 authored by Xiaoqin Liu, Mengmeng Liu, Jin Zhang, Yizhao Chang, Zhiyong Cui, Boyang Ji, Jens Nielsen, Qingsheng Qi, Jin HouGenome-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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nonhomologous end joiningcell factory constructionacetic acid tolerance15 × 10rapid trackable mutagenesisscale trackable mutagenesisscale mutagenesis libraryyarrowia lipolytica strong nhej preferenceidentify causal mutations5 supscale mutagenesisyarrowia lipolyticacausal mutationsmutagenesis approachusing nhejreadily applicablepowerful approachphenotypic screeningoccupancy regionsnew targetsmediated integrationimproved βidentified rapidlygenetic analysisfree regionsfirst timecarotene biosynthesis