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Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR

  • Tadas Jakočiūnas
  • Emil D. Jensen
  • Michael K. JensenEmail author
  • Jay D. Keasling
Part of the Methods in Molecular Biology book series (MIMB, volume 1671)

Abstract

Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building.

Key words

Genome engineering CRISPR/Cas9 Metabolic engineering In vivo assembly DNA assembly CasEMBLR Homologous recombination 

References

  1. 1.
    Li M, Borodina I (2015) Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae. FEMS Yeast Res. doi: 10.1111/1567-1364.12213
  2. 2.
    Giaever G, Chu AM, Ni L et al (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391. doi: 10.1038/nature00935 CrossRefPubMedGoogle Scholar
  3. 3.
    Lam FH, Ghaderi A, Fink GR, Stephanopoulos G (2014) Biofuels. Engineering alcohol tolerance in yeast. Science 346:71–75. doi: 10.1126/science.1257859 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zhang Z, Moo-Young M, Chisti Y (1996) Plasmid stability in recombinant Saccharomyces cerevisiae. Biotechnol Adv 14:401–435. doi: 10.1016/S0734-9750(96)00033-X CrossRefPubMedGoogle Scholar
  5. 5.
    Özaydin B, Burd H, Lee TS, Keasling JD (2013) Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production. Metab Eng 15:174–183. doi: 10.1016/j.ymben.2012.07.010 CrossRefPubMedGoogle Scholar
  6. 6.
    Shao Z, Zhao H, Zhao H (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37:e16. doi: 10.1093/nar/gkn991 CrossRefPubMedGoogle Scholar
  7. 7.
    Storici F, Lewis LK, Resnick MA (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol 19:773–776. doi: 10.1038/90837 CrossRefPubMedGoogle Scholar
  8. 8.
    Storici F, Resnick MA (2003) Delitto perfetto targeted mutagenesis in yeast with oligonucleotides. Genet Eng 25:189–207Google Scholar
  9. 9.
    Kuijpers NGA, Chroumpi S, Vos T et al (2013) One-step assembly and targeted integration of multigene constructs assisted by the I-SceI meganuclease in Saccharomyces cerevisiae. FEMS Yeast Res 13:769–781. doi: 10.1111/1567-1364.12087 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Siddiqui MS, Choksi A, Smolke CD (2014) A system for multilocus chromosomal integration and transformation-free selection marker rescue. FEMS Yeast Res 14:1171–1185. doi: 10.1111/1567-1364.12210 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wach A, Brachat A, Pöhlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808. doi: 10.1002/yea.320101310 CrossRefPubMedGoogle Scholar
  12. 12.
    Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A 78:6354–6358. doi: 10.1073/pnas.78.10.6354 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW (1983) The double-strand-break repair model for recombination. Cell 33:25–35. doi: 10.1016/0092-8674(83)90331-8 CrossRefPubMedGoogle Scholar
  14. 14.
    DiCarlo JE, Norville JE, Mali P et al (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. doi: 10.1093/nar/gkt135
  15. 15.
    Jakočiūnas T, Bonde I, Herrgård M et al (2015) Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae. Metab Eng 28:213–222. doi: 10.1016/j.ymben.2015.01.008 CrossRefPubMedGoogle Scholar
  16. 16.
    Bao Z, Xiao H, Liang J et al (2014) A homology integrated CRISPR-Cas (HI-CRISPR) system for one-step multi-gene disruptions in Saccharomyces cerevisiae. ACS Synth Biol. doi: 10.1021/sb500255k
  17. 17.
    Ryan OW, Skerker JM, Maurer MJ et al (2014) Selection of chromosomal DNA libraries using a multiplex CRISPR system. Elife:e03703. doi: 10.7554/eLife.03703
  18. 18.
    Verwaal R, Wang J, Meijnen JP et al (2007) High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol 73:4342–4350. doi: 10.1128/AEM.02759-06 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Rodriguez A, Kildegaard KR, Li M et al (2015) Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis. Metab Eng 31:181–188. doi: 10.1016/j.ymben.2015.08.003 CrossRefPubMedGoogle Scholar
  20. 20.
    Jakoči\( \overline{\mathrm{u}} \)nas T, Rajkumar AS, Zhang J et al (2015) CasEMBLR: Cas9-facilitated multiloci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae. ACS Synth Biol. doi: 10.1021/acssynbio.5b00007

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Tadas Jakočiūnas
    • 1
  • Emil D. Jensen
    • 1
  • Michael K. Jensen
    • 1
    Email author
  • Jay D. Keasling
    • 1
    • 2
    • 3
    • 4
    • 5
  1. 1.The Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkKgs. LyngbyDenmark
  2. 2.Joint BioEnergy InstituteEmeryvilleUSA
  3. 3.Physical Biosciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  4. 4.Department of Chemical and Biomolecular EngineeringUniversity of California, BerkeleyBerkeleyUSA
  5. 5.Department of BioengineeringUniversity of California, BerkeleyBerkeleyUSA

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