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A Step-by-Step Protocol for COMPASS, a Synthetic Biology Tool for Combinatorial Gene Assembly

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DNA Cloning and Assembly

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2205))

Abstract

For industry-scale production of high-value chemicals in microbial cell factories, the elimination of metabolic flux imbalances is a critical aspect. However, a priori knowledge about the genetic design of optimal production pathways is typically not available. COMPASS, COMbinatorial Pathway ASSembly, is a rapid cloning method for the balanced expression of multiple genes in biochemical pathways. The method generates thousands of individual DNA constructs in modular, parallel, and high-throughput manner. COMPASS employs inducible artificial transcription factors derived from plant (Arabidopsis thaliana) regulators to control the expression of pathway genes in yeast (Saccharomyces cerevisiae). It utilizes homologous recombination for parts assembly and employs a positive selection scheme to identify correctly assembled pathway variants after both in vivo and in vitro recombination. Finally, COMPASS is equipped with a CRISPR/Cas9 genome modification system allowing for the one-step multilocus integration of genes. Although COMPASS was initially developed for pathway engineering, it can equally be employed for balancing gene expression in other synthetic biology projects.

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References

  1. Nielsen J, Fussenegger M, Keasling J, Lee SY, Liao JC, Prather K, Palsson B (2014) Engineering synergy in biotechnology. Nat Chem Biol 10(5):319–322. https://doi.org/10.1038/nchembio.1519

    Article  CAS  PubMed  Google Scholar 

  2. Nielsen J (2013) Production of biopharmaceutical proteins by yeast: advances through metabolic engineering. Bioengineered 4(4):207–211. https://doi.org/10.4161/bioe.22856

    Article  PubMed  Google Scholar 

  3. Pinto JP, Pereira R, Cardoso J, Rocha I, Rocha M (2013) TNA4OptFlux – a software tool for the analysis of strain optimization strategies. BMC Res Notes 6:175–188

    Article  Google Scholar 

  4. Du J, Yuan Y, Si T, Lian J, Zhao H (2012) Customized optimization of metabolic pathways by combinatorial transcriptional engineering. Nucleic Acids Res 40(18):e142. https://doi.org/10.1093/nar/gks549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rodrigues R (2015) Improving microbial chemical production strains through transcriptional regulatory network rewiring. PhD Dissertaion, Imperial College London, Department of Life Sciences

    Google Scholar 

  6. Subtil T, Boles E (2012) Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 5:14. https://doi.org/10.1186/1754-6834-5-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Li YJ, Wang MM, Chen YW, Wang M, Fan LH, Tan TW (2017) Engineered yeast with a CO2-fixation pathway to improve the bio-ethanol production from xylose-mixed sugars. Sci Rep 7:43875. https://doi.org/10.1038/srep43875

    Article  PubMed  PubMed Central  Google Scholar 

  8. Thodey K, Galanie S, Smolke CD (2014) A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat Chem Biol 10(10):837–844. https://doi.org/10.1038/nchembio.1613

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pfleger BF, Pitera DJ, Smolke CD, Keasling JD (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol 24(8):1027–1032. https://doi.org/10.1038/nbt1226

    Article  CAS  PubMed  Google Scholar 

  10. Bond-Watts BB, Bellerose RJ, Chang MC (2011) Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat Chem Biol 7(4):222–227. https://doi.org/10.1038/nchembio.537

    Article  CAS  PubMed  Google Scholar 

  11. Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC (2011) Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol 77(9):2905–2915. https://doi.org/10.1128/AEM.03034-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bujara M, Panke S (2010) Engineering in complex systems. Curr Opin Biotechnol 21:586–591

    Article  CAS  Google Scholar 

  13. Holtz WJ, Keasling JD (2010) Engineering static and dynamic control of synthetic pathways. Cell 140(1):19–23. https://doi.org/10.1016/j.cell.2009.12.029

    Article  CAS  PubMed  Google Scholar 

  14. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343. https://doi.org/10.1093/nar/gkt135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mitchell LA, Chuang J, Agmon N, Khunsriraksakul C, Phillips NA, Cai Y, Truong DM, Veerakumar A, Wang Y, Mayorga M, Blomquist P, Sadda P, Trueheart J, Boeke JD (2015) Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic Acids Res 43(13):6620–6630. https://doi.org/10.1093/nar/gkv466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Jones JA, Vernacchio VR, Lachance DM, Lebovich M, Fu L, Shirke AN, Schultz VL, Cress B, Linhardt RJ, Koffas MA (2015) ePathOptimize: a combinatorial approach for transcriptional balancing of metabolic pathways. Sci Rep 5:11301. https://doi.org/10.1038/srep11301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460(7257):894–898. https://doi.org/10.1038/nature08187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lian J, HamediRad M, Hu S, Zhao H (2017) Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nat Commun 8(1):1688. https://doi.org/10.1038/s41467-017-01695-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhou YJ, Gao W, Rong Q, Jin G, Chu H, Liu W, Yang W, Zhu Z, Li G, Zhu G, Huang L, Zhao ZK (2012) Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 134(6):3234–3241. https://doi.org/10.1021/ja2114486

    Article  CAS  PubMed  Google Scholar 

  20. Noskov VN, Chuang R-Y, Gibson DG, Leem S-H, Larionov V, Kouprina N (2010) Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies. Nat Protoc 6(1):89–96

    Article  Google Scholar 

  21. de Boer CG, Hughes TR (2012) YeTFaSCo: a database of evaluated yeast transcription factor sequence specificities. Nucleic Acids Res 40(Database issue):D169–D179. https://doi.org/10.1093/nar/gkr993

    Article  CAS  PubMed  Google Scholar 

  22. Karim AS, Curran KA, Alper HS (2013) Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res 13(1):107–116. https://doi.org/10.1111/1567-1364.12016

    Article  CAS  PubMed  Google Scholar 

  23. Naseri G, Balazadeh S, Machens F, Kamranfar I, Messerschmidt K, Mueller-Roeber B (2017) Plant-derived transcription factors for orthologous regulation of gene expression in the yeast Saccharomyces cerevisiae. ACS Synth Biol 6(9):1742–1756. https://doi.org/10.1021/acssynbio.7b00094

    Article  CAS  PubMed  Google Scholar 

  24. Naseri G, Behrend J, Rieper L, Mueller-Roeber B (2019) COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors. Nat Commun 10(1):2615. https://doi.org/10.1038/s41467-019-10224-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40(8):e55. https://doi.org/10.1093/nar/gkr1288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:349–361. https://doi.org/10.1016/B978-0-12-385120-8.00015-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kouprina N, Larionov V (2016) Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology. Chromosoma 125(4):621–632. https://doi.org/10.1007/s00412-016-0588-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hochrein L, Machens F, Gremmels J, Schulz K, Messerschmidt K, Mueller-Roeber B (2017) AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies. Nucleic Acids Res 45(10):e80. https://doi.org/10.1093/nar/gkx034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jang CW, Magnuson T (2013) A novel selection marker for efficient DNA cloning and recombineering in E. coli. PLoS One 8(2):e57075. https://doi.org/10.1371/10.1371/journal.pone.0057075.t001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bao Z, Xiao H, Liang J, Zhang L, Xiong X, Sun N, Si T, Zhao H (2015) Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol 4(5):585–594. https://doi.org/10.1021/sb500255k

    Article  CAS  PubMed  Google Scholar 

  31. Zwolshen JH, Bhattacharjee JK (1981) Genetic and biochemical properties of thialysine-resistant mutants of Saccharomyces cerevisiae. J Gen Microbiol 122:281–287

    CAS  PubMed  Google Scholar 

  32. Dorfman BZ (1969) The isolation of adenylosuccinate synthetase mutants in yeast by selection for constitutive behavior in pigmented strains. Genetics 61:377–389

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

COMPASS was previously developed through funding by the Federal Ministry of Education and Research of Germany (BMBF; grant numbers FKZ 031A172 and FKZ 031B0223). G.N. received a fellowship from the Potsdam Graduate School, University of Potsdam.

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Correspondence to Bernd Mueller-Roeber .

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Naseri, G., Mueller-Roeber, B. (2020). A Step-by-Step Protocol for COMPASS, a Synthetic Biology Tool for Combinatorial Gene Assembly. In: Chandran, S., George, K. (eds) DNA Cloning and Assembly. Methods in Molecular Biology, vol 2205. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0908-8_16

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  • DOI: https://doi.org/10.1007/978-1-0716-0908-8_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0907-1

  • Online ISBN: 978-1-0716-0908-8

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