Skip to main content
SpringerLink
Log in

Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping

  • Protocol
  • First Online:
Cell-Free Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2433))

Abstract

Biological systems provide a sustainable and complimentary approach to synthesizing useful chemical products. Metabolic engineers seeking to establish economically viable biosynthesis platforms strive to increase product titers, rates, and yields. Despite continued advances in genetic tools and metabolic engineering techniques, cellular workflows remain limited in throughput. It may take months to test dozens of unique pathway designs even in a robust model organism, such as Escherichia coli. In contrast, cell-free protein synthesis enables the rapid generation of enzyme libraries that can be combined to reconstitute metabolic pathways in vitro for biochemical synthesis in days rather than weeks. Cell-free reactions thereby enable comparison of hundreds to thousands of unique combinations of enzyme homologs and concentrations, which can quickly identify the most productive pathway variants to test in vivo or further characterize in vitro. This cell-free pathway prototyping strategy provides a complementary approach to accelerate cellular metabolic engineering efforts toward highly productive strains for metabolite production.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nielsen J, Keasling JD (2016) Engineering cellular metabolism. Cell 164(6):1185–1197

    Article  CAS  PubMed  Google Scholar 

  2. Wu G et al (2016) Metabolic burden: cornerstones in synthetic biology and metabolic engineering applications. Trends Biotechnol 34(8):652–664

    Article  CAS  PubMed  Google Scholar 

  3. Bowie JU et al (2020) Synthetic biochemistry: the bio-inspired cell-free approach to commodity chemical production. Trends Biotechnol

    Google Scholar 

  4. Wilding KM et al (2018) The emerging impact of cell-free chemical biosynthesis. Curr Opin Biotechnol 53:115–121

    Article  CAS  PubMed  Google Scholar 

  5. Swartz JR (2018) Expanding biological applications using cell-free metabolic engineering: an overview. Metab Eng 50:156–172

    Article  CAS  PubMed  Google Scholar 

  6. Lim HJ, Kim DM (2019) Cell-free metabolic engineering: recent developments and future prospects. Methods Protoc 2(2)

    Google Scholar 

  7. Laohakunakorn N et al (2020) Bottom-up construction of complex biomolecular systems with cell-free synthetic biology. Front Bioeng Biotechnol 8:213

    Article  PubMed  PubMed Central  Google Scholar 

  8. Borkowski O et al (2020) Large scale active-learning-guided exploration for in vitro protein production optimization. Nat Commun 11(1):1872

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Petroll K et al (2020) A novel framework for the cell-free enzymatic production of glucaric acid. Metab Eng 57:162–173

    Article  CAS  PubMed  Google Scholar 

  10. Kopp D, Willows RD, Sunna A (2019) Cell-free enzymatic conversion of spent coffee grounds into the platform chemical lactic acid. Front Bioeng Biotechnol 7:389

    Article  PubMed  PubMed Central  Google Scholar 

  11. Guterl JK et al (2012) Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. ChemSusChem 5(11):2165–2172

    Article  CAS  PubMed  Google Scholar 

  12. Sherkhanov S et al (2020) Isobutanol production freed from biological limits using synthetic biochemistry. Nat Commun 11(1):4292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Korman TP, Opgenorth PH, Bowie JU (2017) A synthetic biochemistry platform for cell free production of monoterpenes from glucose. Nat Commun 8:15526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Opgenorth PH, Korman TP, Bowie JU (2016) A synthetic biochemistry module for production of bio-based chemicals from glucose. Nat Chem Biol 12(6):393–395

    Article  CAS  PubMed  Google Scholar 

  15. Valliere MA et al (2019) A cell-free platform for the prenylation of natural products and application to cannabinoid production. Nat Commun 10(1):565

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dudley QM, Karim AS, Jewett MC (2015) Cell-free metabolic engineering: biomanufacturing beyond the cell. Biotechnol J 10(1):69–82

    Article  CAS  PubMed  Google Scholar 

  17. Gregorio NE, Levine MZ, Oza JP (2019) A user’s guide to cell-free protein synthesis. Methods Protoc 2(1)

    Google Scholar 

  18. Silverman AD, Karim AS, Jewett MC (2020) Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet 21(3):151–170

    Article  CAS  PubMed  Google Scholar 

  19. Kay JE, Jewett MC (2020) A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds. Metab Eng Commun 10

    Google Scholar 

  20. Kay JE, Jewett MC (2015) Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol. Metab Eng 32:133–142

    Article  CAS  PubMed  Google Scholar 

  21. O’Kane PT et al (2019) High-throughput mapping of CoA metabolites by SAMDI-MS to optimize the cell-free biosynthesis of HMG-CoA. Sci Adv 5(6)

    Google Scholar 

  22. Dudley QM, Anderson KC, Jewett MC (2016) Cell-free mixing of Escherichia coli crude extracts to prototype and rationally engineer high-titer mevalonate synthesis. ACS Synth Biol 5(12):1578–1588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dudley QM, Nash CJ, Jewett MC (2019) Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates. Synth Biol (Oxf) 4(1):ysz003

    Article  CAS  Google Scholar 

  24. Niu FX et al (2020) Enhanced production of pinene by using a cell-free system with modular cocatalysis. J Agric Food Chem 68(7):2139–2145

    Article  CAS  PubMed  Google Scholar 

  25. Karim AS, Rasor BJ, Jewett MC (2019) Enhancing control of cell-free metabolism through pH modulation. Synth Biol 5(1)

    Google Scholar 

  26. Karim AS et al (2018) Controlling cell-free metabolism through physiochemical perturbations. Metab Eng 45:86–94

    Article  CAS  PubMed  Google Scholar 

  27. Karim AS, Jewett MC (2016) A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metab Eng 36:116–126

    Article  CAS  PubMed  Google Scholar 

  28. Karim AS, Jewett MC (2018) Cell-free synthetic biology for pathway prototyping. Methods Enzymol 608:31–57

    Article  CAS  PubMed  Google Scholar 

  29. Kwon YC, Jewett MC (2015) High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci Rep 5:8663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sun ZZ et al (2014) Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. ACS Synth Biol 3(6):387–397

    Article  CAS  PubMed  Google Scholar 

  31. Kightlinger W et al (2018) Design of glycosylation sites by rapid synthesis and analysis of glycosyltransferases. Nat Chem Biol 14(6):627–635

    Article  CAS  PubMed  Google Scholar 

  32. Kelwick R et al (2018) Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics. Synth Biol 3(1)

    Google Scholar 

  33. Karim AS et al (2020) In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design. Nat Chem Biol 16:912–919

    Article  CAS  PubMed  Google Scholar 

  34. Grubbe WS et al (2020) Cell-free biosynthesis of styrene at high titers. Metab Eng 61:89–95

    Article  CAS  PubMed  Google Scholar 

  35. Zhuang L et al (2020) Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin. Metab Eng 60:37–44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Khatri Y et al (2020) Multicomponent microscale biosynthesis of unnatural cyanobacterial indole alkaloids. ACS Synth Biol 9(6):1349–1360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jeschek M, Gerngross D, Panke S (2017) Combinatorial pathway optimization for streamlined metabolic engineering. Curr Opin Biotechnol 47:142–151

    Article  CAS  PubMed  Google Scholar 

  38. Ojima-Kato T, Nagai S, Nakano H (2017) Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation. Sci Rep 7(1):13979

    Article  PubMed  PubMed Central  Google Scholar 

  39. Dopp JL et al (2019) Rapid prototyping of proteins: mail order gene fragments to assayable proteins within 24 hours. Biotechnol Bioeng 116(3):667–676

    Article  CAS  PubMed  Google Scholar 

  40. Rasor BJ et al (2021) Toward sustainable, cell-free biomanufacturing. Curr Opin Biotechnol 69:136–144

    Article  CAS  PubMed  Google Scholar 

  41. Kelwick RJR, Webb AJ, Freemont PS (2020) Biological materials: the next frontier for cell-free synthetic biology. Front Bioeng Biotechnol 8:399

    Article  PubMed  PubMed Central  Google Scholar 

  42. Bogart JW et al (2020) Cell-free exploration of the natural product chemical space. Chembiochem

    Google Scholar 

  43. Yin G et al (2017) RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates. Sci Rep 7(1):3026

    Article  PubMed  PubMed Central  Google Scholar 

  44. Garcia DC et al (2021) A lysate proteome engineering strategy for enhancing cell-free metabolite production. Metab Eng Commun 12:e00162

    Article  PubMed  PubMed Central  Google Scholar 

  45. Silverman AD et al (2019) Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry. ACS Synth Biol 8(2):403–414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cole SD et al (2020) Methodologies for preparation of prokaryotic extracts for cell-free expression systems. Synth Syst Biotechnol 5(4):252–267

    Article  PubMed  PubMed Central  Google Scholar 

  47. Jewett MC et al (2008) An integrated cell-free metabolic platform for protein production and synthetic biology. Mol Syst Biol 4:220

    Article  PubMed  PubMed Central  Google Scholar 

  48. Hershewe JM et al (2021) Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles. Nat Commun 12(1):2363

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Cabantous S, Waldo GS (2006) In vivo and in vitro protein solubility assays using split GFP. Nat Methods 3(10):845–854

    Article  CAS  PubMed  Google Scholar 

  50. Liu H, Naismith JH (2009) A simple and efficient expression and purification system using two newly constructed vectors. Protein Expr Purif 63(2):102–111

    Article  CAS  PubMed  Google Scholar 

  51. Boute N et al (2016) NanoLuc luciferase—a multifunctional tool for high throughput antibody screening. Front Pharmacol 7:27

    Article  PubMed  PubMed Central  Google Scholar 

  52. Hook B, Schagat T (2011) Non-radioactive detection of proteins expressed in cell-free expression systems. PRO. https://www.promega.com/resources/pubhub/tpub_049-nonradioactive-detection-of-proteins-expressed-in-cell-free-expression-systems

Download references

Acknowledgments

This work was supported by the U.S. Department of Energy (DOE) Biological and Environmental Research Division (BER), Genomic Science Program (GSP) for funding of this project under Contract No. DE-SC0018249 and the Office of Energy Efficiency and Renewable Energy Grant DE-EE0008343. B.J.R. is supported by an NDSEG Fellowship (Award ND-CEN-017-095). B.V. was supported by a SNSF Early Postdoc Mobility fellowship (P2SKP3_184036).

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Center for Synthetic Biology, Robert H. Lurie Comprehensive Cancer Center, and Simpson Querrey Institute, Northwestern University, Evanston, IL, USA

    Michael C. Jewett

  2. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, and Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Ashty S. Karim

  3. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    Blake J. Rasor & Bastian Vögeli

  4. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Blake J. Rasor & Bastian Vögeli

  5. Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Blake J. Rasor & Bastian Vögeli

Authors
  1. Blake J. Rasor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bastian Vögeli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael C. Jewett
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ashty S. Karim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Michael C. Jewett or Ashty S. Karim .

Editor information

Editors and Affiliations

  1. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, and Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Ashty S. Karim

  2. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Center for Synthetic Biology, Robert H. Lurie Comprehensive Cancer Center, and Simpson Querrey Institute, Northwestern University, Evanston, IL, USA

    Michael C. Jewett

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Rasor, B.J., Vögeli, B., Jewett, M.C., Karim, A.S. (2022). Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping. In: Karim, A.S., Jewett, M.C. (eds) Cell-Free Gene Expression. Methods in Molecular Biology, vol 2433. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1998-8_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1998-8_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1997-1

  • Online ISBN: 978-1-0716-1998-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation