A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP⁺ Reductase Fusion System for Light-Driven NADPH...

Area of Science:

• Biochemistry
• Photosynthesis research
• Synthetic biology

Background:

• Photosynthetic organisms utilize light to regenerate dihydronicotinamide dinucleotide phosphate (NADPH) via a complex electron transfer chain.
• Key components include photosystem II (PSII), cytochrome b6f complex, photosystem I (PSI), ferredoxin (Fd), and ferredoxin NADP+ reductase (FNR).
• Efficient in vitro NADPH regeneration is crucial for various biotechnological applications.

Purpose of the Study:

• To establish a novel clickable fusion system for in vitro NADPH regeneration.
• To demonstrate the modular assembly and functionality of engineered protein complexes.
• To compare the efficiency of the fused system with free protein systems for NADPH regeneration.

Main Methods:

• Fusion of immunity protein 7 (Im7) to the PSI-PsaE subunit in Synechocystis sp. PCC 6803.
• Expression of colicin DNase E7 (E7) fusion chimeras of Fd and FNR in Escherichia coli.
• Assembly of Im7-PSI with E7-Fd or E7-Fd-FNR via high-affinity E7/Im7 binding.
• Testing the NADPH regeneration capacity of the assembled complexes.

Main Results:

• Successfully established a clickable fusion system for in vitro NADPH regeneration.
• Demonstrated high-affinity binding between Im7-PSI and E7-Fd/E7-Fd-FNR fusion proteins.
• The assembled complexes showed comparable NADPH regeneration capacity to free protein systems.
• Validated the general applicability of the strategy for creating functional protein complexes.

Conclusions:

• The developed clickable fusion system provides a versatile platform for in vitro NADPH regeneration.
• This approach allows for modular assembly of photosynthetic protein complexes.
• The strategy holds promise for optimizing NADPH production in biotechnological systems.