Exploiting cell-free systems: Implementation and debugging of a system of biotransformations

Area of Science:

• Biochemistry
• Synthetic Biology
• Metabolic Engineering

Background:

• Cells utilize complex enzyme networks for challenging chemical reactions.
• In vitro biotransformations aim to replicate cellular catalytic efficiency.
• Dihydroxyacetone phosphate (DHAP) is a versatile metabolic building block.

Purpose of the Study:

• To construct a multi-step catalytic system for synthesizing unnatural monosaccharides.
• To insulate a biotransformation pathway from central carbon metabolism in E. coli.
• To demonstrate the feasibility of assembling in vitro enzymatic systems from metabolic pathways.

Main Methods:

• Assembly of a 10-step system of biotransformations (SBT) using DHAP.
• Insulation of the production pathway by modifying glycolysis (omitting triose-phosphate isomerase) in E. coli.
• Inactivation of the AMP nucleosidase (amn gene) to prevent glucose-independent DHAP production.
• Addition of lactate dehydrogenase to regenerate NAD+.

Main Results:

• Successful synthesis of unnatural monosaccharides, including 5,6,7-trideoxy-D-threo-heptulose-1-phosphate from DHAP and butanal.
• Demonstrated accumulation of DHAP by insulating the pathway.
• Established a functional multi-step enzymatic system outside of native metabolic regulation.

Conclusions:

• A 10-step system of biotransformations (SBT) can be effectively assembled for unnatural monosaccharide synthesis.
• Pathway insulation is crucial for efficient DHAP accumulation and controlled biotransformations.
• This approach facilitates the construction of complex in vitro enzymatic systems from central carbon metabolism.