Details

Autor(en) / Beteiligte

• 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

Titel

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

Ist Teil von

• Nature chemical biology, 2020-08, Vol.16 (8), p.912-919

Ort / Verlag

United States

Erscheinungsjahr

2020

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 . We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.