Microorganisms and methods for the biosynthesis of (2-hydroxy-3methyl-4-oxobutoxy) phosphonate

Abstract

The invention provides non-naturally occurring microbial organisms having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, p-toluate pathway, and/or terephthalate pathway. The invention additionally provides methods of using such organisms to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, p-toluate pathway or terephthalate pathway.

What is claimed is: 
     
         1 . A non-naturally occurring microbial organism having carbon monoxide dehydrogenase or hydrogenase activity and a reductive tricarboxylic acid (rTCA) pathway, wherein the microbial organism comprises (i) at least one exogenous nucleic acid encoding a rTCA pathway enzyme expressed in a sufficient amount to convert CO, CO 2  or H 2  to acetyl-CoA, wherein the rTCA enzyme is selected from the group consisting of an ATP citrate-lyase, a citrate lyase, an aconitase, an isocitrate dehydrogenase, an alpha-ketoglutarate:ferredoxin oxidoreductase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarate reductase, a fumarase, and a malate dehydrogenase; and (ii) at least one exogenous nucleic acid encoding a biosynthetic product pathway enzyme expressed in a sufficient amount to produce the biosynthetic product; and wherein the microbial organism requires a carbon source as an energy source. 
     
     
         2 . The non-naturally occurring microbial organism of  claim 1 , wherein the biosynthetic product is p-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. 
     
     
         3 . The non-naturally occurring microbial organism of  claim 1 , wherein the microbial organism does not require light for growth. 
     
     
         4 . The non-naturally occurring microbial organism of  claim 1  further comprises a ferredoxin. 
     
     
         5 . The non-naturally occurring microbial organism of  claim 1  further comprises a reducing equivalent producing pathway comprising at least one exogenous nucleic acid encoding a reducing equivalent producing enzyme selected from the group consisting of a carbon monoxide dehydrogenase, a hydrogenase and a NAD(P)H:ferredoxin oxidoreductase. 
     
     
         6 . The non-naturally occurring microbial organism of  claim 1 , wherein the rTCA pathway enzyme is selected from the group consisting of an ATP citrate-lyase, a citrate lyase, an alpha-ketoglutarate:ferredoxin oxidoreductase, and a fumarate reductase. 
     
     
         7 . The non-naturally occurring microbial organism of  claim 1 , wherein the rTCA pathway enzyme is an aconitase or an isocitrate dehydrogenase. 
     
     
         8 . The non-naturally occurring microbial organism of  claim 1 , wherein the microbial organism is a species of bacteria, yeast or fungus. 
     
     
         9 . A non-naturally occurring microbial organism comprising:
 a rTCA pathway comprising at least one exogenous nucleic acid encoding a rTCA pathway enzyme expressed in a sufficient amount to convert (i) CO, (ii) CO 2  and H 2 , (iii) CO and CO 2 , (iv) synthesis gas comprising CO and H 2 , or (v) synthesis gas comprising CO, CO 2 , and H 2 ; to acetyl-CoA; wherein the rTCA pathway enzyme is selected from the group consisting of an ATP citrate-lyase, a citrate lyase, an aconitase, an isocitrate dehydrogenase, an alpha-ketoglutarate:ferredoxin oxidoreductase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarate reductase, a fumarase and a malate dehydrogenase;   a reducing equivalent producing pathway comprising at least one exogenous nucleic acid encoding a reducing equivalent producing enzyme selected from the group consisting of a carbon monoxide dehydrogenase, a hydrogenase and a NAD(P)H:ferredoxin oxidoreductase; and   at least one exogenous nucleic acid encoding a biosynthetic product pathway enzyme expressed in a sufficient amount to produce a biosynthetic product; and wherein the microbial organism does not require acetyl-CoA synthase.   
     
     
         10 . The non-naturally occurring microbial organism of  claim 9 , wherein the biosynthetic product is p-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. 
     
     
         11 . The non-naturally occurring microbial organism of  claim 9  further comprises a ferredoxin. 
     
     
         12 . The non-naturally occurring microbial organism of  claim 9 , wherein the rTCA pathway enzyme is selected from the group consisting of an ATP citrate-lyase, a citrate lyase, an alpha-ketoglutarate:ferredoxin oxidoreductase, and a fumarate reductase. 
     
     
         13 . The non-naturally occurring microbial organism of  claim 9 , wherein the rTCA pathway enzyme is an aconitase or an isocitrate dehydrogenase. 
     
     
         14 . The non-naturally occurring microbial organism of  claim 9 , wherein the microbial organism is a species of bacteria, yeast or fungus. 
     
     
         15 . A non-naturally occurring microbial organism comprising:
 a reducing equivalent producing pathway comprising at least one exogenous nucleic acid encoding a reducing equivalent producing enzyme selected from the group consisting of a carbon monoxide dehydrogenase, a hydrogenase and a NAD(P)H:ferredoxin oxidoreductase; and   at least one exogenous nucleic acid encoding a biosynthetic product pathway enzyme expressed in a sufficient amount to produce the biosynthetic product, wherein the microbial organism does not require acetyl-CoA synthase.   
     
     
         16 . The non-naturally occurring microbial organism of  claim 15 , wherein the biosynthetic product is p-toluate, terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. 
     
     
         17 . The non-naturally occurring microbial organism of  claim 15  comprising at least one exogenous nucleic acid encoding a reducing equivalent producing enzyme carbon monoxide dehydrogenase. 
     
     
         18 . The non-naturally occurring microbial organism of  claim 15  comprising at least one exogenous nucleic acid encoding a reducing equivalent producing enzyme carbon monoxide dehydrogenase and a ferredoxin. 
     
     
         19 . A method of producing biosynthetic products comprising:
 culturing the non-naturally occurring microbial organism of  claim 1  under conditions and for a sufficient period of time to produce the biosynthetic products, wherein the non-naturally occurring organism is cultured on a carbon source as an energy source; and   separating the biosynthetic products produced by the non-naturally occurring microbial organism.   
     
     
         20 . A method of  claim 19 , wherein the culturing is under anaerobic or substantially anaerobic conditions.