Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation

Metabolic engineering of Rhodosporidium toruloides, a robust lipid and caroteinoid producer, is of great importance for oleochemicals and carotenoids production. However, the Agrobacterium-mediated gene transformation is tedious and time consuming. Here, we described a fast and efficient genetic transformation of R. toruloides using electroporation with linear DNA fragments, and the process was optimized. The results showed that 2 x 10(3) transformants can be obtained at 0.7 kV/mu g linear DNA by using hygromycin and bleomycin as selection markers after the competent cells pretreated with 25 mM DTT and 100 mM LiAc. Our results would facilitate mutant library construction and metabolic engineering of R. toruloides for production of oleochemicals and carotenoids. We further demonstrated that all transformants arose due to illegitimate integration of transforming DNA fragments by colony PCR.