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Selectable marker recycling in the nonconventional yeast Xanthophyllomyces dendrorhous by transient expression of Cre on a genetically unstable vector

  • Ning Zhang
  • Jiaxin Li
  • Fuli Li
  • Shi’an Wang
Methods and protocols
  • 69 Downloads

Abstract

Selectable marker recycling is a basic technique in bioengineering. However, this technique is usually unavailable in non-model microorganisms. In this study, we proposed a simple and efficient method for selectable marker recycling in the astaxanthin-synthesizing yeast Xanthophyllomyces dendrorhous. This method was based on a Cre-loxP system, in which the transient expression of the Cre recombinase was controlled by a genetically unstable vector independent of episomal plasmids and inducible promoters. The selectable markers in single-gene locus and multigene loci were removed along with the loss of the Cre vector with a ratio of 100% and 29%, respectively. The significance of the method was highlighted by the finding that stable autotrophic mutants were not readily obtained in X. dendrorhous. Comparative studies in X. dendrorhous and the non-homologous end joining dominant yeast Yarrowia lipolytica suggested that the method could be universally used in homologous recombination dominant yeasts.

Keywords

Cell factory Yeast Cre-loxP system Marker recycling Homologous recombination 

Notes

Acknowledgments

We thank the Natural Science Foundation of China and Shandong Provincial Natural Science Foundation for financial support.

Funding

This study was supported by the Natural Science Foundation of China (No. 31670054 and No. 21676159) and Shandong Provincial Natural Science Foundation (ZR2017ZB0209).

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9496_MOESM1_ESM.pdf (762 kb)
ESM 1 (PDF 761 kb)

References

  1. Adrio JL, Veiga M, Casqueiro J, Lopez M, Fernandez C (1993) Isolation of Phaffia rhodozyma auxotrophic mutants by enrichment methods. J Gen Appl Microbiol 39:303–312CrossRefGoogle Scholar
  2. Alcalde E, Fraser PD (2018) Extending our tools and resources in the non-conventional industrial yeast Xanthophyllomyces dendrorhous through the application of metabolite profiling methodologies. Metabolomics 14:30CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ambati RR, Phang SM, Ravi S, Aswathanarayana RG (2014) Astaxanthin: sources, extraction, stability, biological activities and its commercial applications − a review. Mar Drugs 12:128–152CrossRefPubMedPubMedCentralGoogle Scholar
  4. An GH, Schuman DB, Johnson EA (1989) Isolation of Phaffia rhodozyma mutants with increased astaxanthin content. Appl Environ Microbiol 55:116–124PubMedPubMedCentralGoogle Scholar
  5. Bassalo MC, Liu RM, Gill RT (2016) Directed evolution and synthetic biology applications to microbial systems. Curr Opin Biotechnol 39:126–133CrossRefPubMedGoogle Scholar
  6. Borgel D, van den Berg M, Huller T, Andrea H, Liebisch G, Boles E, Schorsch C, van der Pol R, Arink A, Boogers I, van der Hoeven R, Korevaar K, Farwick M, Köhler T, Schaffer S (2012) Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases. Metab Eng 14:412–426CrossRefPubMedGoogle Scholar
  7. Cao MF, Gao MR, Lopez-Garcia CL, Wu YT, Seetharam AS, Severin AJ, Shao Z (2017) Centromeric DNA facilitates nonconventional yeast genetic engineering. ACS Synth Biol 6:1545–1553CrossRefPubMedGoogle Scholar
  8. Chen DC, Beckerich JM, Gaillardin C (1997) One-step transformation of the dimorphic yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 48:232–235CrossRefPubMedGoogle Scholar
  9. Cordova P, Alcaino J, Bravo N, Barahona S, Sepulveda D, Fernandez-Lobato M, Baeza M, Cifuentes V (2016) Regulation of carotenogenesis in the red yeast Xanthophyllomyces dendrorhous: the role of the transcriptional co-repressor complex Cyc8-Tup1 involved in catabolic repression. Microb Cell Factories 15:193CrossRefGoogle Scholar
  10. Darvishi F, Ariana M, Marella ER, Borodina I (2018) Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals. Appl Microbiol Biotechnol 102:5925–5938CrossRefPubMedGoogle Scholar
  11. Dennison PMJ, Ramsdale M, Manson CL, Brown AJP (2005) Gene disruption in Candida albicans using a synthetic, codon-optimized Cre-loxP system. Fungal Genet Biol 42:737–748CrossRefPubMedGoogle Scholar
  12. Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud JM (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55:727–737CrossRefPubMedGoogle Scholar
  13. Florea S, Andreeva K, Machado C, Mirabito PM, Schardl CL (2009) Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid. Fungal Genet Biol 46:721–730CrossRefPubMedGoogle Scholar
  14. Galanie S, Thodey K, Trenchard IJ, Filsinger Interrante M, Smolke CD (2015) Complete biosynthesis of opioids in yeast. Science 349:1095–1100CrossRefPubMedPubMedCentralGoogle Scholar
  15. Gassel S, Breitenbach J, Sandmann G (2014) Genetic engineering of the complete carotenoid pathway towards enhanced astaxanthin formation in Xanthophyllomyces dendrorhous starting from a high-yield mutant. Appl Microbiol Biotechnol 98:345–350CrossRefPubMedGoogle Scholar
  16. Güldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524CrossRefPubMedPubMedCentralGoogle Scholar
  17. Guo XG, Chavez A, Tung A, Chan Y, Kaas C, Yin Y, Cecchi R, Garnier SL, Kelsic ED, Schubert M, DiCarlo JE, Collins JJ, Church GM (2018) High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast. Nat Biotechnol 36:540–546CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hara KY, Morita T, Mochizuki M, Yamamoto K, Ogino C, Araki M, Kondo A (2014) Development of a multi-gene expression system in Xanthophyllomyces dendrorhous. Microb Cell Factories 13:175CrossRefGoogle Scholar
  19. Iwaki T, Takegawa K (2004) A set of loxP marker cassettes for cre-mediated multiple gene disruption in Schizosaccharomyces pombe. Biosci Biotechnol Biochem 68:545–550CrossRefPubMedPubMedCentralGoogle Scholar
  20. Jullesson D, David F, Pfleger B, Nielsen J (2015) Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals. Biotechnol Adv 33:1395–1402CrossRefPubMedGoogle Scholar
  21. Kawano F, Okazaki R, Yazawa M, Sato M (2016) A photoactivatable Cre-loxP recombination system for optogenetic genome engineering. Nat Chem Biol 12:1059–1064CrossRefPubMedGoogle Scholar
  22. Lee C, Kim J, Shin SG, Hwang S (2006) Absolute and relative qPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123:273–280CrossRefPubMedGoogle Scholar
  23. Li Y, Li S, Thodey K, Trenchard I, Cravens A, Smolke CD (2018) Complete biosynthesis of noscapine and halogenated alkaloids in yeast. Proc Natl Acad Sci 115:E3922–E3931CrossRefPubMedGoogle Scholar
  24. Liu L, Redden H, Alper HS (2013) Frontiers of yeast metabolic engineering: diversifying beyond ethanol and Saccharomyces. Curr Opin Biotechnol 24:1023–1030CrossRefPubMedGoogle Scholar
  25. Liu W, Luo ZQ, Wang Y, Pham NT, Tuck L, Perez-Pi I, Liu LY, Shen Y, French C, Auer M, Marles-Wright J, Dai JB, Cai YZ (2018) Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods. Nat Commun 9:1936CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lutz KA, Svab Z, Maliga P (2006) Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system. Nat Protoc 1:900–910CrossRefPubMedGoogle Scholar
  27. Martinez-Moya P, Niehaus K, Alcaino J, Baeza M, Cifuentes V (2015) Proteomic and metabolomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous using different carbon sources. BMC Genomics 16:289CrossRefPubMedPubMedCentralGoogle Scholar
  28. Pan XS, Wang BB, Gerken H, Lu YH, Ling XP (2017) Proteomic analysis of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to low carbon levels. Bioprocess Biosyst Eng 40:1091–1100CrossRefPubMedGoogle Scholar
  29. Patel RD, Lodge JK, Baker LG (2010) Going green in Cryptococcus neoformans: the recycling of a selectable drug marker. Fungal Genet Biol 47:191–198CrossRefPubMedGoogle Scholar
  30. Pollmann H, Breitenbach J, Sandmann G (2017) Engineering of the carotenoid pathway in Xanthophyllomyces dendrorhous leading to the synthesis of zeaxanthin. Appl Microbiol Biotechnol 101:103–111CrossRefPubMedGoogle Scholar
  31. Qian W, Song H, Liu Y, Zhang C, Niu Z, Wang H, Qiu B (2009) Improved gene disruption method and Cre-loxP mutant system for multiple gene disruptions in Hansenula polymorpha. J Microbiol Methods 79:253–259CrossRefPubMedGoogle Scholar
  32. Ribeiro O, Gombert AK, Teixeira JA, Domingues L (2007) Application of the Cre-loxP system for multiple gene disruption in the yeast Kluyveromyces marxianus. J Biotechnol 131:20–26CrossRefPubMedGoogle Scholar
  33. Sharma R, Gassel S, Steiger S, Xia X, Bauer R, Sandmann G, Thines M (2015) The genome of the basal agaricomycete Xanthophyllomyces dendrorhous provides insights into the organization of its acetyl-CoA derived pathways and the evolution of Agaricomycotina. BMC Genomics 16:233CrossRefPubMedPubMedCentralGoogle Scholar
  34. Shen Y, Wang Y, Chen T, Gao F, Gong J, Abramczyk D, Walker R, Zhao H, Chen S, Liu W, Luo Y, Muller CA, Paul-Dubois-Taine A, Alver B, Stracquadanio G, Mitchell LA, Luo Z, Fan Y, Zhou B, Wen B, Tan F, Wang Y, Zi J, Xie Z, Li B, Yang K, Richardson SM, Jiang H, French CE, Nieduszynski CA, Koszul R, Marston AL, Yuan Y, Wang J, Bader JS, Dai J, Boeke JD, Xu X, Cai Y, Yang H (2017) Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science 355:eaaf4791CrossRefPubMedPubMedCentralGoogle Scholar
  35. Shi S, Liang Y, Zhang MM, Ang EL, Zhao H (2016) A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae. Metab Eng 33:19–27CrossRefPubMedGoogle Scholar
  36. Steensma HY, Linde JJMT (2001) Plasmids with the Cre-recombinase and the dominant nat marker, suitable for use in prototrophic strains of Saccharomyces cerevisiae and Kluyveromyces lactis. Yeast 18:469–472CrossRefPubMedGoogle Scholar
  37. Tuteja N, Verma S, Sahoo RK, Raveendar S, Reddy IN (2012) Recent advances in development of marker-free transgenic plants: regulation and biosafety concern. J Biosci 37:167–197CrossRefPubMedGoogle Scholar
  38. Wagner JM, Alper HS (2016) Synthetic biology and molecular genetics in non-conventional yeasts: current tools and future advances. Fungal Genet Biol 89:126–136CrossRefPubMedGoogle Scholar
  39. Wery J, Verdoes JC, van Ooyen AJJ (1998) Efficient transformation of the astaxanthin-producing yeast Phaffia rhodozyma. Biotechnol Tech 12:399–405CrossRefGoogle Scholar
  40. Xu P, Qiao KJ, Ahn WS, Stephanopoulos G (2016) Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals. Proc Natl Acad Sci U S A 113:10848–10853CrossRefPubMedPubMedCentralGoogle Scholar
  41. Xu P, Qiao KJ, Stephanopoulos G (2017) Engineering oxidative stress defense pathways to build a robust lipid production platform in Yarrowia lipolytica. Biotechnol Bioeng 114:1521–1530CrossRefPubMedGoogle Scholar
  42. Yamamoto K, Hara KY, Morita T, Nishimura A, Sasaki D, Ishii J, Ogino C, Kizaki N, Kondo A (2016) Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes. Microb Cell Factories 15:155CrossRefGoogle Scholar
  43. Yu BJ, Sung BH, Koob MD, Lee CH, Lee JH, Lee WS, Kim MS, Kim SC (2002) Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nat Biotechnol 20:1018–1023CrossRefPubMedGoogle Scholar
  44. Zhang N, Fan Y, Li C, Wang Q, Leksawasdi N, Li F, Wang SA (2018) Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts. Appl Microbiol Biotechnol 102:4183–4191CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess TechnologyChinese Academy of SciencesQingdaoChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

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