DNA Double-Strand Break-Induced Gene Amplification in Yeast

  • Tomas Strucko
  • Michael Lisby
  • Uffe Hasbro MortensenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2153)


Precise control of the gene copy number in the model yeast Saccharomyces cerevisiae may facilitate elucidation of enzyme functions or, in cell factory design, can be used to optimize production of proteins and metabolites. Currently, available methods can provide high gene-expression levels but fail to achieve accurate gene dosage. Moreover, strains generated using these methods often suffer from genetic instability resulting in loss of gene copies during prolonged cultivation. Here we present a method, CASCADE, which enables construction of strains with defined gene copy number. With our present system, gene(s) of interest can be amplified up to nine copies, but the upper copy limit of the system can be expanded. Importantly, the resulting strains can be stably propagated in selection-free media.

Key words

CEN.PK DNA double-strand break Gene amplification Gene targeting Homology-directed recombination I-SceI nuclease Metabolic engineering Saccharomyces cerevisiae 



This work was supported by the Villum Foundation to ML, grant DNRF99 from the Danish National Research Foundation, and by grant 0603-00323B from the Danish Council for Strategic Research to UHM.


  1. 1.
    Nandy SK, Srivastava RK (2018) A review on sustainable yeast biotechnological processes and applications. Microbiol Res 207:83–90CrossRefGoogle Scholar
  2. 2.
    Nielsen J (2019) Yeast systems biology: model organism and cell factory. Biotechnol J 14(9):e1800421CrossRefGoogle Scholar
  3. 3.
    Borodina I, Nielsen J (2014) Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals. Biotechnol J 9:609–620CrossRefGoogle Scholar
  4. 4.
    Hong K-K, Nielsen J (2012) Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell Mol Life Sci 69:2671–2690CrossRefGoogle Scholar
  5. 5.
    Kim I-K, Roldão A, Siewers V, Nielsen J (2012) A systems-level approach for metabolic engineering of yeast cell factories. FEMS Yeast Res 12:228–248CrossRefGoogle Scholar
  6. 6.
    Nielsen J, Keasling JD (2016) Engineering cellular metabolism. Cell 164:1185–1197CrossRefGoogle Scholar
  7. 7.
    Borodina I, Zhao ZK (2017) Editorial: yeast cell factories for production of fuels and chemicals. FEMS Yeast Res 17(8):fox082Google Scholar
  8. 8.
    Siewers, V.; Mortensen, U.F.; Nielsen, J. Genetic engineering tools for Saccharomyces cerevisiae. Manual of industrial microbiology and biotechnology. 3rd edn. Baltz RH, Demain AL, Davies JE; 2010; Washington, DC: ASM PressGoogle Scholar
  9. 9.
    Kilonzo PM, Margaritis A, Bergougnou MA (2009) Plasmid stability and kinetics of continuous production of glucoamylase by recombinant Saccharomyces cerevisiae in an airlift bioreactor. J Ind Microbiol Biotechnol 36:1157–1169CrossRefGoogle Scholar
  10. 10.
    Zhang Z, Moo-Young M, Chisti Y (1996) Plasmid stability in recombinant Saccharomyces cerevisiae. Biotechnol Adv 14:401–435CrossRefGoogle Scholar
  11. 11.
    Caunt P, Impoolsup A, Greenfield PF (1988) Stability of recombinant plasmids in yeast. J Biotechnol 8:173–192CrossRefGoogle Scholar
  12. 12.
    Mikkelsen MD, Buron LD, Salomonsen B, Olsen CE, Hansen BG, Mortensen UH, Halkier BA (2012) Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab Eng 14:104–111CrossRefGoogle Scholar
  13. 13.
    Jensen N, Strucko T et al (2014) EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res 14(2):238–248CrossRefGoogle Scholar
  14. 14.
    Stovicek V, Borja GM, Forster J, Borodina I (2015) EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains. J Ind Microbiol Biotechnol 42:1519–1531CrossRefGoogle Scholar
  15. 15.
    Lopes TS, de Wijs IJ, Steenhauer SI, Verbakel J, Planta RJ (1996) Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae. Yeast 12:467–477CrossRefGoogle Scholar
  16. 16.
    Lopes TS, Klootwijk J, Veenstra AE, Van Der Aarb PC, Van H, Rauc HA, Planta RJ (1989) High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector. Gene 79:199–206CrossRefGoogle Scholar
  17. 17.
    Lopes TS, Hakkaart GAJ, Koerts BL, Rauc HA, Planta RJ (1991) Mechanism of high-copy-number Saccharomyces cerevisiae. Gene 105:83–90CrossRefGoogle Scholar
  18. 18.
    Parekh R, Shaw M, Wittrup K (1996) An integrating vector for tunable, high copy, stable integration into the dispersed Ty δ sites of Saccharomyces cerevisiae. Biotechnol Prog 12(1):16–21CrossRefGoogle Scholar
  19. 19.
    Semkiv MV, Dmytruk KV, Sibirny AA (2016) Development of a system for multicopy gene integration in Saccharomyces cerevisiae. J Microbiol Methods 120:44–49CrossRefGoogle Scholar
  20. 20.
    Sakai A, Shimizu Y, Hishinuma F (1990) Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty. Appl Microbiol Biotechnol 33:302–306CrossRefGoogle Scholar
  21. 21.
    Wang X, Wang Z, Da Silva NA (1996) G418 selection and stability of cloned genes integrated at chromosomal delta sequences of Saccharomyces cerevisiae. Biotechnol Bioeng 49:45–51CrossRefGoogle Scholar
  22. 22.
    Lee FW, Da Silva NA (1996) Ty1-mediated integration of expression cassettes: host strain effects, stability, and product synthesis. Biotechnol Prog 12:548–554CrossRefGoogle Scholar
  23. 23.
    Maury J, Germann SM, Baallal Jacobsen SA, Jensen NB, Kildegaard KR, Herrgård MJ, Schneider K, Koza A, Forster J, Nielsen J et al (2016) EasyCloneMulti: a set of vectors for simultaneous and multiple genomic integrations in Saccharomyces cerevisiae. PLoS One 11:e0150394CrossRefGoogle Scholar
  24. 24.
    Jessop-Fabre MM, Jakočiūnas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I (2016) EasyClone-MarkerFree: a vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnol J 11(8):1110–1117CrossRefGoogle Scholar
  25. 25.
    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 33:19–27CrossRefGoogle Scholar
  26. 26.
    Jakočiūnas T, Jensen M, Keasling J (2016) CRISPR/Cas9 advances engineering of microbial cell factories. Metab Eng 34:44–59CrossRefGoogle Scholar
  27. 27.
    Strucko T, Buron LD, Jarczynska ZD, Nødvig CS, Mølgaard L, Halkier BA, Mortensen UH (2017) CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast. Sci Rep 7:41431CrossRefGoogle Scholar
  28. 28.
    Haber J (2000) Partners and pathways: repairing a double-strand break. Trends Genet 16:259–264CrossRefGoogle Scholar
  29. 29.
    Sherman F (2002) Getting started with yeast. Methods Enzymol 350:3–41CrossRefGoogle Scholar
  30. 30.
    Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34CrossRefGoogle Scholar
  31. 31.
    Gibson DDG, Young L, Chuang RR-Y, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Tomas Strucko
    • 1
  • Michael Lisby
    • 2
  • Uffe Hasbro Mortensen
    • 1
    Email author
  1. 1.Department of Biotechnology and BiomedicineTechnical University of DenmarkKongens LyngbyDenmark
  2. 2.Department of BiologyUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations