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



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


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.


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.


Agrobacterium-mediated transformation Carotenoids Homologous gene targeting Rhodosporidium toruloides 



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)


  1. Braunwald T, Schwemmlein L, Graeff-Honninger S, French WT, Hernandez R, Holmes WE, Claupein W (2013) Effect of different C/N ratios on carotenoid and lipid production by Rhodotorula glutinis. Appl Microbiol Biotechnol 97:6581–6588CrossRefPubMedGoogle Scholar
  2. Buzzini P, Innocenti M, Turchetti B, Libkind D, van Broock M, Mulinacci N (2007) Carotenoid profiles of yeasts belonging to the genera Rhodotorula, Rhodosporidium, Sporobolomyces, and Sporidiobolus. Can J Microbiol 53:1024–1031CrossRefPubMedGoogle Scholar
  3. Evans CT, Ratledge C (1984) Effect of nitrogen-source on lipid-accumulation in oleaginous yeasts. J Gen Microbiol 130:1693–1704Google Scholar
  4. Hu C, Zhao X, Zhao J, Wu S, Zhao ZK (2009) Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides. Bioresour Technol 100:4843–4847CrossRefPubMedGoogle Scholar
  5. Koh CMJ, Liu Y, Moehninsi, Du M, Ji L (2014) Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides. BMC Microbiol 14:50CrossRefPubMedCentralPubMedGoogle Scholar
  6. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol 9:963–967CrossRefGoogle Scholar
  7. Lin X, Wang Y, Zhang S, Zhu Z, Zhou YJ, Yang F, Sun W, Wang X, Zhao ZK (2014) Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides. FEMS Yeast Res 14:547–555CrossRefPubMedGoogle Scholar
  8. 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
  9. Liu Y, Koh CMJ, Ngoh ST, Ji L (2015) Engineering an efficient and tight d-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species. Microb Cell Fact 14:170CrossRefPubMedCentralPubMedGoogle Scholar
  10. Ma S, Wang Y, Jiao X, Zhang S, Zhao ZK (2015) Phosphate starvation derepressed expression vector for engineering oleaginous yeast Rhodosporidium toruloides. Acta Microbiol Sin 55:1505–1511Google Scholar
  11. Park SY, Choi SR, Lim SH, Yeo Y, Kweon SJ, Bae YS, Kim KW, Im KH, Ahn SK, Ha SH, Park SU, Kim JK (2014) Identification and quantification of carotenoids in paprika fruits and cabbage, kale, and lettuce leaves. J Korean Soc Appl Biol Chem 57:355–358CrossRefGoogle Scholar
  12. Sabri S, Steen JA, Bongers M, Nielsen LK, Vickers CE (2013) Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci. Microb Cell Fact 12:60CrossRefPubMedCentralPubMedGoogle Scholar
  13. Takahashi S, Okada H, Abe K, Kera Y (2014) Genetic transformation of the yeast Rhodotorula gracilis ACTT 26217 by electroporation. Appl Biochem Microbiol 50:624–628CrossRefGoogle Scholar
  14. Tully M, Gilbert HJ (1985) Transformation of Rhodosporidium toruloides. Gene 36:235–240CrossRefPubMedGoogle Scholar
  15. Van den Ent F, Lowe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74CrossRefPubMedGoogle Scholar
  16. Verdoes JC, Sandmann G, Visser H, Diaz M, van Mossel M, van Ooyen AJJ (2003) Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Appl Environ Microbiol 69:3728–3738CrossRefPubMedCentralPubMedGoogle Scholar
  17. Wang Y, Lin X, Zhang S, Sun W, Ma S, Zhao ZK (2016a) Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides. Yeast 33:99–106CrossRefPubMedGoogle Scholar
  18. Wang Y, Zhang S, Pötter M, Sun W, Li L, Yang X, Jiao X, Zhao ZK (2016b) Overexpression of Δ12-fatty acid desaturase in the oleaginous yeast Rhodosporidium toruloides for production of linoleic acid-rich lipids. Appl Biochem Biotechnol 180:1497–1507CrossRefPubMedGoogle Scholar
  19. Zhang C, Shen H, Zhang X, Yu X, Wang H, Xiao S, Wang J, Zhao ZK (2016a) Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids. Biotechnol Lett 38:1733–1738CrossRefPubMedGoogle Scholar
  20. Zhang S, Ito M, Skerker JM, Arkin AP, Rao CV (2016b) Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation. Appl Microbiol Biotechnol 100:9393–9405CrossRefPubMedGoogle Scholar
  21. Zhu Z, Zhang S, Liu H, Shen H, Lin X, Yang F, Zhou YJ, Jin G, Ye M, Zou H, Zhao ZK (2012) A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nat Commun 3:1112CrossRefPubMedCentralPubMedGoogle Scholar

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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