457539 A Cell-Free Framework for Rapid Biosynthetic Pathway Prototyping and Enzyme Discovery

Tuesday, November 15, 2016: 9:06 AM
Continental 6 (Hilton San Francisco Union Square)
Ashty S. Karim and Michael C. Jewett, Chemical and Biological Engineering, Northwestern University, Evanston, IL

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. Further, we leverage robotic liquid-handling systems to access combinatorial design space exceeding typical pipelines pursued in cells. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.

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See more of this Session: Advances in Metabolic Engineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division