Malic enzyme-based system for transhydrogenation between nicotinamide cofactors.

Area of Science:

• Biochemistry
• Metabolic Engineering
• Synthetic Biology

Background:

• Nicotinamide cofactors like NAD and NADP are crucial for cellular redox reactions.
• A non-natural cofactor, nicotinamide cytosine dinucleotide (NCD), expands the cofactor repertoire.
• Efficient transfer of reducing equivalents between cofactors remains a metabolic engineering challenge.

Purpose of the Study:

• To develop a novel strategy for enabling reducing equivalent exchange among different cofactors.
• To utilize malic enzyme (ME)-mediated transhydrogenation for cofactor interconversion.
• To demonstrate the feasibility of this approach both in vitro and in vivo.

Main Methods:

• Employed wild-type and engineered malic enzymes (ME and ME*) favoring different cofactors (NAD, NADP, NCD).
• Established an in vitro system with NADH, NCD, ME, ME*, and pyruvate to assess cofactor conversion.
• Implemented the system in NCD-self-sufficient Escherichia coli cells to observe reducing equivalent flux.

Main Results:

• In vitro experiments showed up to 65% NADH consumption and 57% NCDH generation within 2 hours.
• In Escherichia coli, the system successfully directed reducing equivalents towards NCDH-linked lactate formation.
• Demonstrated cofactor interconversion using ME-mediated transhydrogenation.

Conclusions:

• Developed an effective strategy for regulating intracellular reducing equivalents via cofactor transhydrogenation.
• The ME-mediated approach provides a novel tool for metabolic engineering and synthetic biology applications.
• Facilitates the rational transfer of reducing equivalents, overcoming a long-standing challenge.