A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery

Area of Science:

• Biochemical Engineering
• Synthetic Biology
• Metabolic Engineering

Background:

• Accelerating design-build-test (DBT) cycles is crucial in biochemical engineering.
• Current methods for metabolic engineering often require extensive organism re-engineering, slowing down research and development.

Purpose of the Study:

• To introduce a novel cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework.
• To enable rapid prototyping of biosynthetic pathways in a cell-free environment.

Main Methods:

• Assembled cell-free cocktails using a mix-and-match approach from crude cell lysates.
• Utilized lysates with either enriched heterologous enzymes or enzymes produced via cell-free protein synthesis (CFPS).
• Modeled the framework using the 17-step CoA-dependent n-butanol biosynthesis pathway in *Escherichia coli* lysates.

Main Results:

• Demonstrated a highly active in vitro n-butanol pathway using *Escherichia coli* lysates.
• Showcased the flexibility of the cell-free environment for manipulating conditions and accessing enzymatic nodes.
• Successfully prototyped enzyme sets using linear DNA templates to assess pathway performance.

Conclusions:

• The CFPS-ME framework significantly accelerates the prototyping of metabolic pathways.
• This approach bypasses the need for organism re-engineering, streamlining DBT cycles.
• CFPS-ME facilitates the definition, manipulation, and understanding of metabolic pathways for faster innovation.