In vitro biotransformation is an innovative biomanufacturing platform that involves the design of efficient enzymatic pathways utilizing natural or artificial enzymes, coenzymes, and their analogues. This approach holds significant potential across diverse fields such as pharmaceuticals, agriculture, food production, energy, and materials (Zhang et al., 2023). Despite significant advancements in in vitro biotransformation over recent decades, the energy supply remains a critical challenge. The supplementation of stoichiometric amounts of ATP is necessary unless an ATP regeneration module is used. Taking starch synthesis from CO2 as an example (Cai et al., 2021), ATP supplementation modules, such as polyphosphate kinase (PPK, EC 2.7.4.1), which catalyzes the conversion of inorganic polyphosphate (poly P) and ADP to ATP, are essential for two steps: dihydroxyacetone (DHA) phosphorylation by dihydroxyacetone kinase and glucose-6-phosphate (G-1-P) activation by glucose pyrophosphorylase. However, this supplementation leads to an accumulation of free phosphate ions in the solution. Moreover, excessive ATP may inhibit the ability of fructose-bisphosphatase to convert d-fructose-1,6-bisphosphate (F-1,6-BP) to d-fructose-6-phosphate (F-6-P). In such cases, it is preferable to adopt ATP-free or ATP-balancing strategies involving the selection of ATP-independent enzymes or the introduction of ATP regeneration modules during pathway design.

Living cells naturally synthesize ATP through three mechanisms: substrate-level phosphorylation (SLP), oxidative phosphorylation, and photophosphorylation. In SLP, ATP or GTP is synthesized from ADP or GDP and a phosphoryl group, which is typically donated by other phosphorylated compounds such as 1,3-bisphosphoglycerate, phosphoenolpyruvate, acetyl phosphate, and phosphocreatine (Chen and Zhang, 2021). However, their high cost renders them economically impractical for ATP-involved in vitro biotransformation. In contrast, inorganic poly P, generated by the dehydration of orthophosphate at elevated temperatures (Brown and Kornberg, 2004), has emerged as a highly attractive phosphate source for ATP regeneration due to its low cost and stability compared to other phosphate-containing chemicals. Nevertheless, dealing with excess phosphate ions after the reaction remains a challenge. Photophosphorylation and oxidative phosphorylation are mechanisms adopted by organisms for continuous ATP regeneration during photosynthesis and cell respiration. In these processes, ATPases play a central role in ATP synthesis, driven by a proton electrochemical gradient established via in vivo redox reactions and electron transfer along the electron transport chain (ETC) (Junge and Nelson, 2015; Nesci et al., 2021). Unlike the universal application of SLP in in vitro biotransformation (Chen and Zhang, 2021), significant barriers exist for the wide application of ATPase-based ATP regeneration. For instance, isolating massive ATPase molecules from natural membranes is challenging (Varco-Merth et al., 2008), as is precisely manipulating the orientation of ATPase when assembling it with artificial membranes (Jia and Li, 2019).

In vitro ATPase-based ATP regeneration involves the assembly of ATPases and artificial membranes into ATPase-loaded liposomes. To generate the proton electrochemical gradient, bioproton pumps (membrane proteins (MPs) in the electron transfer chain of photosynthesis and respiration) must be co-assembled into the ATPase-liposomes to achieve self-driven ATP synthesis (Choi and Montemagno, 2005; Feng et al., 2016; Otrin et al., 2017; Richard et al., 1995). To date, in vitro ATPase-based ATP regeneration modules have been widely integrated into artificial or natural cells to mimic the energy supply, cell metabolism, cell communication, growth/division, replication, protein expression and so on (Biner et al., 2020; Jiang et al., 2022). The effectiveness of the in vitro ATPase-based ATP regeneration modules strongly depends on both the characteristics (activity, distribution density, etc.) of ATPases and the robustness/durability of the constructed configuration.

This review provides an illustration of ATPases, including their structure, working principle, driving forces, and distribution in nature. The primary factors influencing the effectiveness and efficiency of in vitro ATPase-based ATP regeneration were subsequently identified and analyzed. Various strategies concerning each involved component or factor are summarized and discussed to assemble in vitro ATPase-based ATP regeneration modules and facilitate their broader application in biotransformation. Finally, the challenges inherent in this technology and possible solutions are proposed. Addressing these issues may lead to breakthroughs in in vitro synthetic biology and pave the way for wider applications of ATPase-based ATP regeneration in in vitro biotransformation processes.