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

The Science

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.

The foundational principle is that researchers can construct discrete enzymatic pathways through modular assembly of cell lysates, containing pathway enzymes produced by cell-free protein synthesis, making the design-build-test unit cellular lysates rather than genetic constructs or a re-engineered organism. This reduces the overall time to build pathways from weeks (or months) to a few days, providing an increased capability to test numerous pathways with large numbers of enzyme combinations. Researchers demonstrated iPROBE by optimizing biosynthetic pathways for the production of 3-hydroxybutyrate (3-HB) and n-butanol in Clostridium autoethanogenum, revealing a strong correlation (r = 0.79) between in-cell and cell-free pathway performance.

The Impact

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. An in vitro approach could substantially accelerate the design and optimization of biosynthetic pathways to introduce into engineered cells. iPROBE can be used to engineer and improve small-molecule biosynthesis in non-model organisms that can be arduous to manipulate. In one example, iPROBE enabled the construction of a strain of C. autoethanogenum that produces high titers and yields of 3-HB in continuous fermentations.

Researchers expect iPROBE will facilitate DBT cycles to decrease the number of strains that need to be engineered in vivo and the time required to achieve desired process objectives. This will increase the flexibility of biological processes to adapt to new markets, expand the range of fossil-derived products that can be displaced with bioderived alternatives and enhance the economic benefits for co-produced fuels.

Summary

• 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.

Principal Investigator

Michael C. Jewett
Northwestern University

References

Karim, A.S., Q. M. Dudley, A. Juminaga, Y. Yuan, S. A. Crowe, J. T. Heggestad, S. Garg, T. Abdalla, W. S. Grubbe, B. J. Rasor, D. N. Coar, M. Torculas, M. Krein, F. Liew, A. Quattlebaum, R. O. Jensen, J. A. Stuart, S. D. Simpson, M. Köpke, and M. C. Jewett. 2020. “In vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes for Cell Design,” Nature Chemical Biology 16, 912–9. DOI:10.1038/s41589-020-0559-0.