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Title: In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design

Abstract

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l -1. Finally, we expect iPROBE to accelerate design–build–test cycles for industrial biotechnology.

Authors:
ORCiD logo [1];  [1];  [2];  [2]; ORCiD logo [1];  [1];  [2];  [2];  [1]; ORCiD logo [1];  [3];  [3];  [3];  [2];  [2];  [2];  [3];  [2]; ORCiD logo [2]; ORCiD logo [4]
  1. Northwestern Univ., Evanston, IL (United States)
  2. LanzaTech Inc., Skokie, IL (United States)
  3. Lockheed Martin Advanced Technology Lab., Cherry Hill, NJ (United States)
  4. Northwestern Univ., Evanston, IL (United States); Northwestern Univ., Chicago, IL (United States)
Publication Date:
Research Org.:
Northwestern Univ., Evanston, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1659129
Grant/Contract Number:  
SC0018249
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Chemical Biology
Additional Journal Information:
Journal Volume: 16; Journal Issue: 8; Journal ID: ISSN 1552-4450
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
metabolic engineering; synthetic biology

Citation Formats

Karim, Ashty S., Dudley, Quentin M., Juminaga, Alex, Yuan, Yongbo, Crowe, Samantha A., Heggestad, Jacob T., Garg, Shivani, Abdalla, Tanus, Grubbe, William S., Rasor, Blake J., Coar, David N., Torculas, Maria, Krein, Michael, Liew, FungMin, Quattlebaum, Amy, Jensen, Rasmus O., Stuart, Jeffrey A., Simpson, Sean D., Köpke, Michael, and Jewett, Michael C. In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design. United States: N. p., 2020. Web. doi:10.1038/s41589-020-0559-0.
Karim, Ashty S., Dudley, Quentin M., Juminaga, Alex, Yuan, Yongbo, Crowe, Samantha A., Heggestad, Jacob T., Garg, Shivani, Abdalla, Tanus, Grubbe, William S., Rasor, Blake J., Coar, David N., Torculas, Maria, Krein, Michael, Liew, FungMin, Quattlebaum, Amy, Jensen, Rasmus O., Stuart, Jeffrey A., Simpson, Sean D., Köpke, Michael, & Jewett, Michael C. In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design. United States. doi:10.1038/s41589-020-0559-0.
Karim, Ashty S., Dudley, Quentin M., Juminaga, Alex, Yuan, Yongbo, Crowe, Samantha A., Heggestad, Jacob T., Garg, Shivani, Abdalla, Tanus, Grubbe, William S., Rasor, Blake J., Coar, David N., Torculas, Maria, Krein, Michael, Liew, FungMin, Quattlebaum, Amy, Jensen, Rasmus O., Stuart, Jeffrey A., Simpson, Sean D., Köpke, Michael, and Jewett, Michael C. Mon . "In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design". United States. doi:10.1038/s41589-020-0559-0.
@article{osti_1659129,
title = {In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design},
author = {Karim, Ashty S. and Dudley, Quentin M. and Juminaga, Alex and Yuan, Yongbo and Crowe, Samantha A. and Heggestad, Jacob T. and Garg, Shivani and Abdalla, Tanus and Grubbe, William S. and Rasor, Blake J. and Coar, David N. and Torculas, Maria and Krein, Michael and Liew, FungMin and Quattlebaum, Amy and Jensen, Rasmus O. and Stuart, Jeffrey A. and Simpson, Sean D. and Köpke, Michael and Jewett, Michael C.},
abstractNote = {The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. Finally, we expect iPROBE to accelerate design–build–test cycles for industrial biotechnology.},
doi = {10.1038/s41589-020-0559-0},
journal = {Nature Chemical Biology},
issn = {1552-4450},
number = 8,
volume = 16,
place = {United States},
year = {2020},
month = {6}
}

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