Development of an enzymatic cascade for semi de novo ATP production using thermophilic enzymes

Industrial production of ATP has mostly relied on extraction from living cells. Although microbial and enzymatic ATP production have also been developed, the former suffers from complexity in product separation, while the latter requires expensive substrates, making their practical use difficult. To tackle these problems, we newly developed an enzymatic cascade for ATP production, which does not use expensive substrates, by assembling 16 thermophilic enzymes prepared through a heat-purification from the crude extract of recombinant Escherichia coli. This cascade consists of two modules: an ATP regeneration module based on a non-oxidative glycolysis and an ADP supply module. The ATP regeneration module can provide the energy required for phosphorylation of AMP and ADP to ATP while simultaneously supplying ribose-5-phosphate, a building block of adenosine phosphates, from inexpensive starch and inorganic phosphate. Ribose-5-phosphate is then adenylated with exogenously supplied adenine in the ADP supply module and further phosphorylated to ATP. This ATP production cascade is not accompanied by CO₂ emission and is expected to be a novel ATP manufacturing platform with less environmental impact. In the present study, ATP production with 100 % molar conversion yield was achieved from 1 mM adenine. However, increasing the initial adenine concentration resulted in lower yields. Enzyme characterization and docking simulations revealed that this decline was due to non-competitive inhibition of certain enzymes by ATP, which could potentially be mitigated through protein engineering.