In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.
Journal
Nature chemical biology
ISSN: 1552-4469
Titre abrégé: Nat Chem Biol
Pays: United States
ID NLM: 101231976
Informations de publication
Date de publication:
08 2020
08 2020
Historique:
received:
25
10
2019
accepted:
06
05
2020
revised:
10
04
2020
pubmed:
17
6
2020
medline:
24
11
2020
entrez:
17
6
2020
Statut:
ppublish
Résumé
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
Identifiants
pubmed: 32541965
doi: 10.1038/s41589-020-0559-0
pii: 10.1038/s41589-020-0559-0
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
912-919Références
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