Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides
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.
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.
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.
KeywordsFLP/FRT system Homologous recombination Inducible expression Nuclear localization signal sequence Rhodosporidium toruloides
This work was supported by National Natural Science Foundation of China (51761145014, 21602218).
Compliance with ethical standards
Conflict of interest
The authors declare no financial or commercial conflict of interest.
- Liu Y, Koh CMJ, Sun L, Hlaing M, Du M, Peng N, Ji L (2013) Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides. Appl Microbiol Biotechnol 97:719–729CrossRefPubMedGoogle Scholar
- Liu H, Jiao X, Wang Y, Yang X, Sun W, Wang J, Zhang S, Zhao ZK (2017) Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation. FEMS Yeast Res 17:fox017Google Scholar
- Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN Jr, McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) ‘GM-gene-deletor’: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–274CrossRefPubMedGoogle Scholar
- Yaegashi J, Kirby J, Ito M, Sun J, Dutta T, Mirsiaghi M, Sundstrom ER, Rodriguez A, Baidoo E, Tanjore D, Pray T, Sale K, Singh S, Keasling JD, Simmons BA, Singer SW, Magnuson JK, Arkin AP, Skerker JM, Gladden JM (2017) Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts. Biotechnol Biofuels 10:241CrossRefPubMedPubMedCentralGoogle Scholar