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

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 (Eric); Quattlebaum, Amy; Jensen, Rasmus O.; Stuart, Jeffrey A.; Simpson, Sean D.; Köpke, Michael; Jewett, Michael C.

doi:10.1038/s41589-020-0559-0

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

Journal Article · Mon Jun 15 00:00:00 EDT 2020 · Nature Chemical Biology

DOI:https://doi.org/10.1038/s41589-020-0559-0· OSTI ID:1659129

^[1]; Dudley, Quentin M. ^[1]; Juminaga, Alex ^[2]; Yuan, Yongbo ^[2];

^[1]; Heggestad, Jacob T. ^[1]; Garg, Shivani ^[2]; Abdalla, Tanus ^[2]; Grubbe, William S. ^[1];

^[1]; Coar, David N. ^[3]; Torculas, Maria ^[3]; Krein, Michael ^[3]; Liew, FungMin (Eric) ^[2]; Quattlebaum, Amy ^[2]; Jensen, Rasmus O. ^[2]; Stuart, Jeffrey A. ^[3]; Simpson, Sean D. ^[2];

^[2];

^[4]

Northwestern Univ., Evanston, IL (United States)
LanzaTech Inc., Skokie, IL (United States)
Lockheed Martin Advanced Technology Lab., Cherry Hill, NJ (United States)
Northwestern Univ., Evanston, IL (United States); Northwestern Univ., Chicago, IL (United States)

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.

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Research Organization:: Northwestern Univ., Evanston, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0018249

OSTI ID:: 1659129

Alternate ID(s):: OSTI ID: 2298994

Journal Information:: Nature Chemical Biology, Vol. 16, Issue 8; ISSN 1552-4450

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 102 works

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