Controlling cell-free metabolism through physiochemical perturbations.

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Biotechnology

Background:

• Cellular metabolism complexities hinder efficient biosynthetic pathway design and optimization.
• Traditional methods for pathway construction and testing are time-consuming.
• A novel cell-free framework offers a simplified approach to pathway engineering.

Purpose of the Study:

• To demonstrate probing biosynthetic pathway performance within a cell-free framework by perturbing physiochemical conditions.
• To optimize n-butanol synthesis as a model system.
• To compare different pathway construction methods and identify factors affecting metabolic efficiency.

Main Methods:

• Utilized a cell-free system with enriched crude-cell extracts for rapid pathway construction.
• Employed a robotic liquid-handler to map physiochemical landscapes and identify key factors (NAD, CoA) for n-butanol metabolism.
• Compared metabolic profiles from heterologous expression versus cell-free protein synthesis for pathway construction.

Main Results:

• Identified NAD and CoA as critical factors for maximizing n-butanol biosynthesis yields in the cell-free system.
• Discovered that phosphate from PEP utilization inhibits optimal cell-free n-butanol metabolism when coupled with cell-free protein synthesis.
• Demonstrated the use of non-phosphorylated secondary energy substrates to fuel cell-free protein synthesis and n-butanol biosynthesis.

Conclusions:

• The cell-free framework facilitates rapid exploration of physiochemical perturbations for pathway optimization.
• Findings highlight the impact of specific reagents (e.g., phosphate) and energy substrates on cell-free metabolic performance.
• Suggests the development of more controllable, multi-step, separated cell-free systems for future pathway prototyping and enzyme discovery.