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.
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Acknowledgement
This work was supported by National Natural Science Foundation of China (51761145014, 21602218).
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Sun, W., Yang, X., Wang, X. et al. Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides. Biotechnol Lett 40, 933–940 (2018). https://doi.org/10.1007/s10529-018-2542-3
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DOI: https://doi.org/10.1007/s10529-018-2542-3