Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides

  • Wenyi Sun
  • Xiaobing Yang
  • Xueying Wang
  • Xiang Jiao
  • Sufang Zhang
  • Yushi Luan
  • Zongbao K. Zhao
Original Research Paper

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.

Keywords

FLP/FRT system Homologous recombination Inducible expression Nuclear localization signal sequence Rhodosporidium toruloides 

Notes

Acknowledgement

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.

Supplementary material

10529_2018_2542_MOESM1_ESM.doc (1.1 mb)
Supplementary material 1 (DOC 1099 kb)

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Biotechnology, Dalian Institute of Chemical PhysicsCASDalianChina
  2. 2.School of Life Science and BiotechnologyDalian University of TechnologyDalianChina

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