Transhydrogenase Promotes the Robustness and Evolvability of Metabolic Networks

Homeostasis of redox cofactors NAD(P)H is crucial for metabolism but inevitably disturbed when microbes switch growth substrates, evolve biochemical pathways, or are engineered to produce valuable chemicals. To illuminate how metabolic systems endure NADPH perturbations brought instantly by pathway modification and restore homeostasis in the long run, we studied laboratory evolution of two pathway-engineered bacteria, Escherichia coli and Methylobacterium extorquens, which underproduced NADPH during growth on glucose and on methanol, respectively. Literature suggests multiple metabolic strategies to restore NADPH homeostasis. Surprisingly, genetic dissection of isolates from twelve populations of E. coli evolved on glucose and eight populations of M. extorquens evolved on methanol revealed merely two solutions: (1) enhancing the NADH/NADPH exchange reaction by boosting the expression of membrane-bound transhydrogenase (mTH) in every population of E. coli and M. extorquens; (2) simultaneously consuming glucose with acetate, an disfavored byproduct normally excreted during glucose catabolism, in two E. coli sub-populations. Notably, mTH is spontaneously upregulated upon pathway modification in both bacteria and distributes broadly among prokaryotes and eukaryotes. Convergent evolution of two phylogenetically and metabolically distinct bacterial species suggests mTH as a conserved buffering mechanism that promotes the robustness and evolvability of metabolism. Moreover, adaptive diversification of E. coli via evolving dual substrate consumption highlights the physiological flexibility to exploit ecological opportunities and points to a bioengineering alternative to fuel the redox currency production.