Emergence of NADP⁺-reducing enzymes inEscherichia colicentral metabolism via adaptive evolution

Abstract

The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation towards the emergence of novel cofactor regeneration routes was not previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotroph strain. We continuously cultivated this strain under selective conditions. After 500-1100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wildtype variant with NAD+. In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP+ thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve towards reducing NADP+, which we attribute to several biochemical constraints such as unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context towards changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries.

Protein cost minimization promotes the emergence of coenzyme redundancy

“…More generally, specificity for NAD(H) or NADP(H) for some enzymes can strongly influence intracellular reaction flux distributions (10), serving as the basis for metabolic engineering strategies to dramatically improve yields of valuable metabolic byproducts (10, 16–18). Importantly, recent work has demonstrated that specificity for NAD(P)H can emerge in laboratory evolution studies, suggesting that coenzyme preference might be a highly adaptable feature of metabolic networks (19). However, it is unclear if most enzymes are typically constrained to use only NAD(H) or NADP(H), and whether a single coenzyme could support the functioning of metabolism.…”