It is pertinent now more than ever that we find sustainable alternatives to produce chemicals. For decades, scientists and engineers have turned to biological systems to help meet societal needs in energy, medicine, materials, and more—especially when chemical synthesis is untenable. Often, biologically-produced small molecules are insufficient for production at the optimal titer, rate, or yield because natural sources are difficult to optimize and are simply not scalable (e.g., plants grow slowly). Thus, engineers seek to design enzymatic reaction schemes in microorganisms to meet manufacturing criteria. Unfortunately, this is difficult because design-build-test (DBT) cycles—iterations of re-engineering organisms to test new sets of enzymes—are detrimentally slow due to the constraints of cell growth. As a result, a typical project today might only explore dozens of variants of an enzymatic reaction pathway. This is often insufficient to identify a commercially relevant solution, consequently requiring more DBT iterations. In this work, we establish an in vitro prototyping and rapid optimization of biosynthetic enzymes approach (termed iPROBE) to inform cellular metabolic engineering. In our approach, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates selectively enriched with pathway enzymes. The rapid ability to build pathways in vitro using iPROBE allows us to generate large amounts of data describing pathway operation under several operating conditions. We reduce that data into a quantitative metric that combines product titer, production rate, and enzyme expression (TREE score). By simplifying the complexity of available cell-free data to one value we can now quickly screen and rank pathways in the cell-free environment and provide useful information for cellular metabolic engineering. We demonstrate iPROBE for the production of 3-hydroxybutyrate and n-butanol in Clostridium, an industrially relevant non-model organism. We anticipate that integrating cell-free systems into industrial metabolic engineering DBT cycles will facilitate and accelerate efforts to define, manipulate, and understand metabolic pathways before engineering organisms for industrial operations.

Integrating cell-free systems into industrial metabolic engineering design-build-test cycles

256th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.