Biotechnology Letters

, Volume 39, Issue 7, pp 1001–1007 | Cite as

Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation

  • Wenyi Sun
  • Xiaobing Yang
  • Xueying Wang
  • Xinping Lin
  • Yanan Wang
  • Sufang Zhang
  • Yushi Luan
  • Zongbao K. Zhao
Original Research Paper

Abstract

Objectives

To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.

Results

The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.

Conclusions

Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

Keywords

Agrobacterium-mediated transformation Carotenoids Homologous gene targeting Rhodosporidium toruloides 

Notes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21325627, 31370128).

Supporting information

Supplementary Table 1—Strains and plasmids used.

Supplementary Table 2—Primers used.

Sequence data—(a) The sequences of ADH2 (RHTO_03062) promoter region.

(b) The sequences of GPD (RHTO_3746) promoter region.

(c) The sequences of the RHTO_04602 locus.

(d) Homologous arm amplification sequences:CRTI-3225-R and CRTI-5695-R.

Supplementary material

10529_2017_2324_MOESM1_ESM.doc (84 kb)
Supplementary material 1 (DOC 84 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Wenyi Sun
    • 1
    • 2
    • 3
  • Xiaobing Yang
    • 2
  • Xueying Wang
    • 2
  • Xinping Lin
    • 4
  • Yanan Wang
    • 2
  • Sufang Zhang
    • 2
  • Yushi Luan
    • 1
  • Zongbao K. Zhao
    • 2
  1. 1.School of Life Science and BiotechnologyDalian University of TechnologyDalianChina
  2. 2.Division of BiotechnologyDalian Institute of Chemical Physics, CASDalianChina
  3. 3.School of Life SciencesJilin Normal UniversitySipingChina
  4. 4.School of Food Science and TechnologyDalian Polytechnic UniversityDalianChina

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