In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design

Area of Science:

• Synthetic biology
• Metabolic engineering
• Biotechnology

Background:

• Designing biosynthetic pathways in non-model organisms is difficult due to limited genetic tools and high-throughput methods.
• Challenges include transformation variability and a scarcity of validated genetic parts.

Purpose of the Study:

• To develop a platform for rapid in vitro prototyping and optimization of biosynthetic enzymes (iPROBE).
• To accelerate the design-build-test cycle for industrial biotechnology applications.

Main Methods:

• iPROBE utilizes cell-free protein synthesis to enrich cell lysates with enzymes.
• Metabolic pathways are assembled using a mix-and-match approach to evaluate performance.
• Data-driven design was employed to optimize a six-step butanol pathway through 205 permutations.

Main Results:

• Screened 54 cell-free pathways for 3-hydroxybutyrate production.
• Demonstrated a strong correlation (r=0.79) between in vitro and in vivo pathway performance.
• Achieved a 20-fold improvement in in vivo 3-hydroxybutyrate production in Clostridium, reaching 14.63 g/L.

Conclusions:

• The iPROBE platform effectively accelerates the optimization of biosynthetic pathways.
• The correlation between cell-free and cellular performance validates iPROBE's predictive power.
• This approach significantly enhances the production of target molecules in industrial microorganisms.