In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.
- Karim AS
Nature chemical biology[Nat Chem Biol] 2020 Aug; Vol. 16 (8), pp. 912-919. Date of Electronic Publication: 2020 Jun 15.
- Publication Date:
- 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 . We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.
- Academic Journal
- Nature chemical biology
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