Regulation Of DNA Break Repair Mechanism Of Rhodotorula Toruloides

Rhodotorula toruloides has great potential in industrial application and can accumulate lipids and carotenoids in cells.So far,the multi-omics information of R.toruloides has been obtained,and the genetic manipulation methods of R.toruloides based on Agrobacterium tumefaciens-mediated transformation(ATMT)and electroporation transformation has been established.These methods provide a basis for improving production traits,optimizing metabolic pathways,and establishing an effective genetic operating system in R.toruloide.In the DNA repair pathway of R.toruloides,the non-homologous end joining(NHEJ)pathway is dominant,and homologous recombination(HR)pathway is extremely inefficient,making it difficult to knockout the target gene by HR.In view of the above problems,we attempted to weaken the NHEJ mechanism and strengthen the HR mechanism in the R.toruloides by knocking down or knocking out the non-homologous terminal ligation-related KU70 gene and over-expressing the key protein Rad52 involved in the HR mechanism,to achieve the regulation of the DNA fracture repair mechanism.The main research contents and results are as follows:In this paper,RNA interference(RNAi)method was used to knock down the NHEJ related gene KU70 of R.toruloides haploid strain NP11,and ku70-RNAi strains were obtained.The RT-PCR results showed that the transcription level of KU70 gene was significantly reduced.Using the ku70-RNAi as starting strains,the strains with CRT knockout was obtained by introducing the knockout cassettes via electroporation transformation.Furthermore,the color phenotype can be complemented by introducing the CRT gene into the CRT gene knockout strain,indicating that the HR efficiency of ku70-RNAi strain was improved.Besides,KU70 knockout vectors with different target sites were constructed,and the CRISPR-cas9 system was used to knock out the KU70 gene of R.toruloides NP11.An engineered strain with the deletion of 35 bp bases in KU70 was obtained(?KU70(1005 bp–1039 bp),ku70~-).Using the ku70~-strain as the starting strain,the URA5 and MET15 knockout cassettes were introduced by electroporation transformation respectively.More phenotypic correct engineered strains were obtained.The combination of the above work and the CRISPR-Cas9 editing technology established by the research group,it would be of great significance for the regulation of metabolic pathways and engineered strains of R.toruloides.