Systematic evaluation of the DNA repair machinery facilitated the development of efficient genome editing tools for the red yeast Rhodotorula toruloides.

The red yeast Rhodotorula toruloides is a robust lipid producer, and its biotechnology applications have been expanded through genetic engineering based on the non-homologous end joining (NHEJ) pathway. As the NHEJ pathway leads to random integration, it remains laborious in phenotypic assessment and challenging for rational strain design. To develop more efficient genetic tools for R. toruloides based on the homologous recombination (HR) pathway, we conducted a systematic evaluation of its DNA repair machinery. Overexpression of HR-related proteins, such as ScRad52, RtRad54 and ScSae2, increased the HR efficiency from less than 0.1% to 3.6%. In addition, knockout of RtKU70 and RtKU80 elevated the HR efficiency to 4.2% and 5.3%, respectively, but reduced resistance to mutagenic stress. Accordingly, we combined the two aforementioned strategies, which yielded an HR efficiency of 5.9%. Although this combined strategy failed to further elevate HR efficiency as expected, it partially alleviated the hypersensitivity to mutagenic stress caused by Ku protein inactivation. Remarkably, when the CRISPR-Cas9 system was used to introduce double-strand breaks (DSBs), HR efficiency was dramatically increased to 16%, and further overexpression of ScRAD52 led to higher HR efficiency of 75-95% across multiple genomic loci. These results will facilitate advanced genetic engineering of R. toruloides for producing value-added compounds and provide a valuable reference for the genetic manipulation of other oleaginous basidiomycetous yeasts.