One-Step Preparation of Cell-Free ATP Regeneration Module Based on Non-Oxidative Glycolysis Using Thermophilic Enzymes

Adenosine triphosphate (ATP) is an essential cofactor for energy-dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non-oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597-5601). However, the 12 enzymes required for this system hampered its practical usability and further testing potential. Here, we addressed this issue by constructing co-expression vectors for the simultaneous gene expression of the 12 enzymes in a single expression strain. All enzymes were sourced from (hyper)thermophiles, which enabled a one-step purification via a heat-treatment process. We showed that the combination of the two enabled the ATP regeneration system to function in a single recombinant Escherichia coli strain. Additionally, this work provides a strategy to rationally design and control proteins expression levels in the co-expression vectors.