A Cell-Free Platform to Rapidly Optimize Synthetic Enzymes for Cellular Design

The Science

Researchers will develop an in vitro approach to substantially accelerate the design and optimization of biosynthetic pathways to introduce into engineered cells. The iPROBE cell-free framework was developed in which cell lysates are enriched with selected enzymes by overexpression or in vitro translation. Lysates are mixed to assemble multiple enzymatic pathway combinations. Productivity is then quantified and ranked to select the best pathways to implement in vivo.


  • Using iPROBE, 36 pathway combinations were tested for high production of 3-hydroxybutyrate in Clostridium, reducing from months to weeks the time needed to develop engineered strains.
  • Integrated with machine learning, iPROBE could rapidly test a large number of enzyme combinations to optimize a six-step n-butanol pathway.
  • Demonstrated strong correlation between in vivo and cell-free pathway performance.
  • With iPROBE, synthetic pathways can be designed and tested at high throughput, enabling fast engineering of new industrial organisms.


Karim, A.S. et al, “In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.” Nature Chemical Biology 16, 912–919 (2020). DOI:10.1038/s41589-020-0559-0