Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica
Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. I...
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Veröffentlicht in: | Science China. Life sciences 2021-12, Vol.64 (12), p.2114-2128 |
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Sprache: | eng |
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Zusammenfassung: | Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in
Yarrowia lipolytica
through regulation of NHEJ repair process, employment of defective
Ura
marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.67, 22.74 and 1.87 times, respectively. We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of
CrtE, CrtB
and
CrtI
, with 23.8 times higher production than rDNA integration through homologous recombination (HR). The NHEJ-mediated genomic library technology also achieved random and scattered integration of
loxP
and
vox
sites, with the copy number up to 65 and 53, respectively, creating potential for further application of recombinase mediated genome rearrangement in
Y. lipolytica
. This work provides a high-efficient NHEJ-mediated genomic library technology, which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h. This novel technology also lays an excellent foundation for the development of other genetic technologies in
Y. lipolytica
. |
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ISSN: | 1674-7305 1869-1889 |
DOI: | 10.1007/s11427-020-1885-x |