Establishment Of Genomic Library Technology Based On Non-Homologous End Joining Mechanism In Yarrowia Lipolytica

Genomic variant libraries are conducive to obtaining dominant strains with different traits,which is convenient for exploring the biotechnological applications of microorganisms and the bioinformatics of unknown functional genes.In order to efficiently achieve genome-scale alterations and create various mutants with distinct phenotypes,we developed a genomic library technology based on the mechanism of non-homologous end joining in Yarrowia lipolytica.In this study,we found that heterologous genes were randomly integrated into different locations of the genome through non-homologous end joining(NHEJ)in strains with fatty alcohol overproduction and validated the positive effect of two targeted locations disruption with unknown functions on fatty alcohol overproduction was verified in Y.lipolytica.In order to further explore the potential of NHEJ repair mechanism of Y.lipolytica in obtaining diverse strains and tapping genomic biological information,we utilized attempts on the overexpression of DNA ligase IV(DL4)and the core NHEJ factors PAXX and XRCC4 involved in NHEJ pathway,the optimization of marker with truncated promoter,and the multiple rounds of iterative transformation,in which the fluorescence intensity was increased by 4.67 times,22.74 times and 1.87 times,respectively.The genome recombination technology activated by NHEJ to diverse organism was successfully established in Y.lipolytica,achieving multiple heterologous genes randomly integrating into the genome and numerous strains with diverse phenotypes obtained within 96 hours.The integration of three foreign genes of CrtE,CrtB and CrtI into the genome via NHEJ library technology was achieved,obtaining a series of strains with different lycopene production.Compared with the strains with lycopene expression module integrated to r DNA site by homologous recombination,the lycopene production of the strains obtained by NHEJ library technology increased by 23.88 times.At the same time,this study used NHEJ library technology to efficiently and randomly integrate the recombinase recognition sites lox P and vox into the genome of Y.lipolytica,and the copy number reached up to 65 and 53,respectively,paving an avenue for the application of recombinase system-mediated genome rearrangement technology in Y.lipolytica.This efficient and convenient genomic operation technology can be applied to facilitate the development of genome editing technology.