1. Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides.
- Author
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Sun W, Yang X, Wang X, Jiao X, Zhang S, Luan Y, and Zhao ZK
- Subjects
- Basidiomycota metabolism, Biotechnology, Fungal Proteins genetics, Promoter Regions, Genetic genetics, Transformation, Genetic genetics, Basidiomycota genetics, DNA Nucleotidyltransferases genetics, Genetic Engineering methods, Homologous Recombination genetics
- Abstract
Objectives: To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides., Results: Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium., Conclusions: We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.
- Published
- 2018
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