Systems and methods for atp regeneration using a synthetic enzyme cascade and nadh oxidation

A method of generating products via a synthetic enzymatic cascade, comprising:

• preparing a reaction medium including a concentration of a fuel source, a first concentration of NAD+, and one or more NADH-dependent dehydrogenases;
• converting at least a portion of the NAD+ to a concentration of NADH;
• contacting one or more polyphosphate NAD+ kinases (PPNK) with the concentration of NADH;
• converting at least a portion of the NADH to a concentration of NADPH;
• contacting one or more NADPH oxidases with the concentration of NADPH;
• converting at least a portion of the NADPH to a concentration of NADP+;
• contacting one or more ATP-NAD+ kinases (NADK) with the concentration of NADP+ and a concentration of adenosine diphosphate (ADP);
• and
• converting at least a portion of the ADP to adenosine triphosphate (ATP).

A method for continuously producing adenosine triphosphate (ATP), comprising:

• expressing in a reaction medium a plurality of proteins including:
• a formate dehydrogenase, a formaldehyde dehydrogenase, an alcohol dehydrogenase, pigeon ATP-NAD+ kinase (NADK), a polyphosphate NAD+ kinase (PPNK) from B. subtilis, and triphosphopyridine nucleotide oxidase (TPNOX);
• administering one or more fuel sources, monohydrogen phosphate, and adenosine diphosphate (ADP) to the reaction medium;
• converting the ADP to a concentration of ATP via the NADK;
• and
• providing at least a portion of the concentration of ATP to an in vitro process,
• wherein the one or more fuel sources includes methanol.