Cell-free metabolic pathway for glucose metabolism with a molecular purge valve

A cell-free in vitro metabolic pathway comprising a plurality of enzymatic steps that converts a substrate to acetyl-phosphate, pyruvate, glyceraldehyde-3-phosphate, or acetyl-CoA, wherein the pathway includes an unbalanced production and utilization of a co-factor, the pathway comprising a non-naturally occurring purge valve pathway that recycles the co-factor, wherein the non-naturally occurring purge valve pathway comprises (i) a wild-type enzyme that utilizes NADP+, (ii) a mutant of the wild-type enzyme that utilizes NAD+ and (iii) an NADH oxidase, wherein the wild-type enzyme of (i) is a glucose-6-phosphate dehydrogenase and/or a 6-phosphogluconate dehydrogenase and wherein the pathway comprises pentose phosphate pathway enzymes including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase,

• wherein (1) the wild-type enzyme of (i) has a sequence that is 95% identical to SEQ ID NO:23 and has glucose-6-phosphate dehydrogenase activity and uses NADP+ as a cofactor and the mutant enzyme of (ii) has a sequence that is 95% identical to SEQ ID NO:24 and has glucose-6-phosphate dehydrogenase activity and uses NAD+ as a cofactor;
• and/or (2) wherein the wild-type enzyme of (i) has a sequence that is 95% identical to SEQ ID N0:27 and has 6-phosphogluconate dehydrogenase activity and uses NADP+ as a cofactor and the mutant enzyme of (ii) has a sequence that is 95% identical to SEQ ID NO:28 and has 6-phosphogluconate dehydrogenase activity and uses NAD+ as a cofactor;
• and
• wherein the NADH oxidase of (iii) has a sequence that is at least 80% identical to SEQ ID NO:18 and has NADH oxidase activity.