Synthetic NAD(P)(H) Cycle for ATP Regeneration

Abstract

ATP is the energy currency of the cell and new methods for ATP regeneration will benefit a range of emerging biotechnology applications including synthetic cells. We designed and assembled a membraneless ATP-regenerating enzymatic cascade by exploiting the substrate specificities of selected NAD­(P)­(H)-dependent oxidoreductases combined with substrate-specific kinases. The enzymes in the NAD­(P)­(H) cycle were selected to avoid cross-reactions, and the cascade was driven by irreversible fuel oxidation. As a proof-of-concept, formate oxidation was chosen as the fueling reaction. ATP regeneration was accomplished via the phosphorylation of NADH to NADPH and the subsequent transfer of the phosphate to ADP by a reversible NAD+ kinase. The cascade was able to regenerate ATP at a high rate (up to 0.74 mmol/L/h) for hours, and >90% conversion of ADP to ATP using monophosphate was also demonstrated. The cascade was used to regenerate ATP for use in cell free protein synthesis reactions, and the ATP production rate was further enhanced when powered by the multistep oxidation of methanol. The NAD­(P)­(H) cycle provides a simple cascade for the in vitro regeneration of ATP without the need for a pH-gradient or costly phosphate donors.

Revisiting Solar Energy Flow in Nanomaterial-Microorganism Hybrid Systems

“…334−336 ATP production is achieved through oxidative phosphorylation 285,328,330,331,337,338 and substrate-level phosphorylation. 335,339,340 Upon light activation, semiconductor nanomaterials have been observed to modulate the levels of reducing equivalents and/or ATP. This modulation may, in turn, influence the metabolic pathways that require these energy forms.…”

“…Such reactions can be further tailored to integrate with metabolic pathways for ATP regeneration, ensuring a balanced intracellular energy profile. 335,339,340 In NMHSs that bypass the intracellular energy pool using QDs and artificial energy carriers such as MV and NR, those with redox potentials and E CB that are thermodynamically capable of activating the target enzymes could be selected. Furthermore, a systematic approach involving high-throughput in vitro and in vivo screening of various combinations of carriers/QDs and target enzymes is proposed to identify those exhibiting optimal catalytic activities for efficient energy conversion.…”

“…As previously mentioned, living cells intricately regulate the levels of reducing equivalents and ATP through various metabolic pathways. Intracellular NADH is primarily generated from glucose metabolism, encompassing processes such as the TCA cycle and glycolysis. ,, NADPH production, on the other hand, involves photosynthetic light reactions in phototrophs, ,, pentose phosphate pathway in nonphototrophs, , and direct conversion of NADH to NADPH catalyzed by specific NADH kinases, etc . − ATP production is achieved through oxidative phosphorylation ,,,,, and substrate-level phosphorylation. ,, Upon light activation, semiconductor nanomaterials have been observed to modulate the levels of reducing equivalents and/or ATP. This modulation may, in turn, influence the metabolic pathways that require these energy forms.…”

“…Numerous nanomaterials have been shown to enhance intracellular reducing equivalents (NAD(P)H). Such reactions can be further tailored to integrate with metabolic pathways for ATP regeneration, ensuring a balanced intracellular energy profile. ,, …”