Synthetic biochemistry molecular purge valve module that maintain co-factor balance

Abstract

The disclosure provides a metabolic pathway for producing a metabolite, the metabolic pathway having a co-factor purge valve system for recycling a cofactor used in the metabolic pathway.

What is claimed is: 
     
         1 . A recombinant, artificial or engineered metabolic pathway comprising a plurality of enzymatic steps that converts a substrate to a product, wherein the pathway produces an unbalanced production of a cofactorcofactors, said pathway comprising:
 a first cofactor-dependent enzyme that is capable of converting a first substrate to a second substrate, said first cofactor-dependent enzyme producing the unbalanced production of a first cofactor; and   a second cofactor-dependent enzyme comprising that uses a second cofactor different from the first cofactor-dependent enzyme and that converts the first substrate to the second substrate and wherein said second cofactor-dependent enzyme produces an unbalanced production of the second cofactor, and wherein the pathway comprises an NADH or an NADPH oxidase that recycles the an NADH or NADPH cofactor.   
     
     
         2 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the first and second co-factor is anco-factors are oxidizing/reducing co-factorco-factors. 
     
     
         3 . The recombinant, artificial or engineered pathway of  claim 2 , wherein the oxidizing/reducing co-factor isco-factors are selected from the group consisting of NAD + /NADH, NADP + /NADPH and FAD + /FADH. 
     
     
         4 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the cofactorfirst and second cofactors comprises NAD + /NADH orand NADP + /NADPH. 
     
     
         5 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the first cofactor-dependent enzyme comprises an NADH or an NADPH dehydrogenase. 
     
     
         6 . The recombinant, artificial or engineered pathway of  claim 5 , wherein the NADH dehydrogenase is a NADH pyruvate dehydrogenase complex. 
     
     
         7 . The recombinant, artificial or engineered pathway of  claim 6 , wherein the NADH pyruvate dehydrogenase complex comprises a pyruvate dehydrogenase subunit a, a pyruvate dehydrogenase subunit b, a dihydrolipoamide acetyltransferase, and a dihydrolipoamide dehydrogenase. 
     
     
         8 . The recombinant, artificial or engineered pathway of  claim 7 , wherein the pyruvate dehydrogenase subunit a comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1, wherein the pyruvate dehydrogenase subunit b comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 2, wherein the dihydrolipoamide acetyltransferase has at least 90% sequence identity to SEQ ID NO: 3, and wherein the dihydrolipoamide dehydrogenase has at least 90% sequence identity to SEQ ID NO: 5, wherein the complex converts pyruvate to acetyl-CoA. 
     
     
         9 . The recombinant, artificial or engineered pathway of  claim 7 , wherein the pyruvate dehydrogenase subunit a comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1, wherein the pyruvate dehydrogenase subunit b comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 2, wherein the dihydrolipoamide acetyltransferase has at least 90% sequence identity to SEQ ID NO: 3, and wherein the dihydrolipoamide dehydrogenase has at least 90% sequence identity to SEQ ID NO: 7 and preferentially uses NADP + , wherein the complex converts pyruvate to acetyl-CoA. 
     
     
         10 . The recombinant, artificial or engineered pathway of  claim 5 , wherein the NADPH dehydrogenase is a member of a NADPH pyruvate dehydrogenase complex. 
     
     
         11 . The recombinant, artificial or engineered pathway of  claim 10 , wherein the NADPH pyruvate dehydrogenase complex comprises a pyruvate dehydrogenase subunit a, a pyruvate dehydrogenase subunit b, a dihydrolipoamide acetyltransferase, and a mutant dihydrolipoamide dehydrogenase thethat preferentially uses NADP + . 
     
     
         12 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the NADH or the NADPH oxidase is a NoxE or homolog thereof. 
     
     
         13 . The recombinant, artificial or engineered pathway of  claim 12 , wherein the NADH or the NADPH oxidase comprises a sequence that has at least 50% sequence identity to SEQ ID NO: 10. 
     
     
         14 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the pathway is in a cell-free system. 
     
     
         15 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the pathway is in a living cell. 
     
     
         16 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the recombinant, artificial or engineered pathway produces PHB. 
     
     
         17 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the recombinant, artificial or engineered pathway produces ethanol. 
     
     
         18 . The recombinant, artificial or engineered pathway of  claim 1 , wherein the recombinant, artificial or engineered pathway produces lactate. 
     
     
         19 . An enzymatic system comprising a metabolic pathway including a plurality of enzymes for converting a substrate to a product, the metabolic pathway having an unbalanced utilization of reducing/oxidizing cofactors, wherein the enzymatic system comprises
 a first cofactor-dependent enzyme that is capable of converting the substrate to the product, said first cofactor-dependent enzyme producing the unbalanced production of a first cofactor; and    a second cofactor-dependent enzyme that uses a second cofactor than the first cofactor-dependent enzyme and that converts the substrate to the product and wherein said second cofactor-dependent enzyme produces an unbalanced production of the second cofactor, and a metabolic purge valve comprising an NADH pyruvate dehydrogenase and a NADH/NADPHan NADH or an NADPH oxidase.   
     
     
         20 . A recombinant polypeptide comprising a sequence that has at least 99% sequence identity to SEQ ID NO: 5 and comprising the mutations E206V, G207R, A208K, and S213R, wherein the polypeptide has dihydrolipoamide dehydrogenase activity.