Engineering Escherichia coli Pyruvate Metabolism to Generate Noncanonical Reducing Power.

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Biocatalysis

Background:

• Biomanufacturing relies on rewiring biological systems for sustainable chemical production.
• Current biomanufacturing is limited by reliance on native redox cofactors (NAD(P)+), causing resource competition.
• Noncanonical redox cofactors, like nicotinamide mononucleotide (NMN+), offer a solution for dedicated electron transfer.

Purpose of the Study:

• To engineer the Escherichia coli pyruvate dehydrogenase complex (PDHc) to utilize NMN+ as a redox cofactor.
• To overcome limitations of native cofactor competition in biomanufacturing processes.
• To expand the toolkit for biomanufacturing with an insulated NMN+-dependent electron source.

Main Methods:

• Rational and computational enzyme design of the Lpd E3 subunit of PDHc.
• Directed evolution and protein engineering to enhance NMN+ affinity and specificity.
• Molecular simulations to track cofactor binding evolution.
• Functional characterization of engineered PDHc in E. coli.

Main Results:

• Discovered a cofactor promiscuous variant (Lpd Penta) with a 2500-fold improved NMN+ turnover.
• Engineered an NMN+-specific variant (Lpd Ortho) with a 2.4 × 10^5-fold increased specificity for NMN+.
• Demonstrated functional NMN+-dependent PDHc activity in E. coli, sustaining essential pyruvate metabolism.

Conclusions:

• Successfully engineered a PDHc that exclusively uses NMN+, creating an insulated electron pathway.
• This work expands the NMN+ cofactor toolkit for biomanufacturing applications.
• The engineered NMN+-specific PDHc provides a high-flux, irreversible electron source for industrial biotechnology.