A Hitchhiker's Guide to Supplying Enzymatic Reducing Power into Synthetic Cells

Area of Science:

• Synthetic biology
• Biochemistry
• Cellular engineering

Background:

• Synthetic cells aim to replicate life's functions using molecular building blocks.
• Maintaining an out-of-equilibrium state requires continuous energy supply, primarily through electron carriers like nicotinamide cofactors (NAD(H) and NADP(H)).
• These cofactors are essential for metabolic redox reactions in living systems.

Purpose of the Study:

• To propose guidelines for establishing a minimal enzymatic module that continuously regenerates reduced nicotinamide cofactors within synthetic cells.
• To identify suitable external electron donors and corresponding enzymes for cofactor reduction.
• To address challenges like cofactor specificity and integration with other cellular modules.

Main Methods:

• Literature review of potential electron donors and enzymes.
• Analysis of biochemical and structural features of candidate proteins.
• Evaluation of reaction feasibility and transport into vesicle-based compartments.
• Discussion of strategies to overcome cofactor specificity limitations.

Main Results:

• A shortlist of potentially suitable electron donors and enzymes was identified based on key requirements.
• Biochemical and structural characteristics of promising proteins were reviewed.
• The limitation of enzyme specificity towards either NAD(H) or NADP(H) was highlighted.
• The necessity of transhydrogenases for efficient cofactor regeneration was discussed.

Conclusions:

• Guidelines are proposed for selecting electron donors and enzymes to regenerate nicotinamide cofactors in synthetic cells.
• The integration of these modules requires careful consideration of reaction kinetics and transport.
• Transhydrogenases are crucial for overcoming cofactor specificity bottlenecks in synthetic metabolism.