Cell-free production of geranyl pyrophosphate from glycerol in a cell-free manufacturing system

A method of converting glycerol to geranyl pyrophosphate (GPP) and additional secondary metabolites in a cell-free medium, the method comprising:

• a) adding glycerol to a reaction mixture;
• b) adding a plurality of enzymes to the reaction mixture from (a);
• wherein the plurality of enzymes comprises an enzyme that comprises SEQ ID NO:
• 1 (maltose binding protein alditol oxidase (MPD-ALDO) or an optimized form thereof, and one or more enzymes selected from the group consisting of:
• an enzyme that comprises SEQ ID NO:
• 2 (dihydroxy-acid dehydratase (DHAD)) or an optimized form thereof, an enzyme that comprises SEQ ID NO:
• 3 (acetyl-phosphate transferase (PTA)) or an optimized form thereof, an enzyme that comprises SEQ ID NO:
• 4 (acetyl-CoA acetyltransferase (PhaA)) or an optimized form thereof, an enzyme that comprises SEQ ID NO:
• 5 (HMG-CoA Synthase A110G (HMGS)) or an optimized form thereof, an enzyme that comprises SEQ ID NO:
• 6 (HMG-CoA Reductase (HMGR)) or an optimized form thereof, an enzyme that comprises SEQ ID NO:
• 7 (mevalonate kinase (MVK)) or an optimized form thereof, an enzyme that comprises SEQ ID NO:
• 8 (phosphomevalonate kinase (PMVK)) or an optimized form thereof, an enzyme that comprises SEQ ID NO:
• 9 (diphosphomevalonate kinase (MDC)) or an optimized form thereof, and an enzyme that comprises SEQ ID NO:
• 10 (isopentyl-PP Isomerase (IDI)) or an optimized form thereof;
• c) removing a supernatant from the reaction mixture from (b);
• and
• d) isolating GPP.