Microorganisms and methods for the biosynthesis of p-toluate and terephthalate

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.

1 . A non-naturally occurring microbial organism, comprising a microbial organism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising at least one exogenous nucleic acid encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme expressed in a sufficient amount to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, said (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising 2-C-methyl-D-erythritol-4-phosphate dehydratase. 
     
     
         2 . The non-naturally occurring microbial organism of  claim 1 , wherein said (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway further comprises 1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         3 . The non-naturally occurring microbial organism of  claim 1 , wherein said (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway further comprises 1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         4 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme. 
     
     
         5 . The non-naturally occurring microbial organism of  claim 4 , wherein said three exogenous nucleic acids encode 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         6 . The non-naturally occurring microbial organism of  claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         7 . The non-naturally occurring microbial organism of  claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         8 . A method for producing (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, comprising culturing the non-naturally occurring microbial organism of  claim 1  under conditions and for a sufficient period of time to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate. 
     
     
         9 . The method of  claim 8 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         10 . The method of  claim 8 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme. 
     
     
         11 . The method of  claim 10 , wherein said three exogenous nucleic acids encode 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         12 . The method of  claim 8 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         13 . A non-naturally occurring microbial organism, comprising a microbial organism having a p-toluate pathway comprising at least one exogenous nucleic acid encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p-toluate, said p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; or chorismate lyase. 
     
     
         14 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         15 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         16 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         17 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism comprises five exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         18 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism comprises six exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         19 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism comprises seven exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         20 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism comprises seven exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         21 . The non-naturally occurring microbial organism of  claim 19 , wherein said eight exogenous nucleic acids encode 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 
     
     
         22 . The non-naturally occurring microbial organism of  claim 13 , wherein said microbial organism further comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. 
     
     
         23 . The non-naturally occurring microbial organism of  claim 22 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         24 . The non-naturally occurring microbial organism of  claim 23 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         25 . The non-naturally occurring microbial organism of  claim 13 , wherein at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         26 . The non-naturally occurring microbial organism of  claim 13 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         27 . A method for producing p-toluate, comprising culturing the non-naturally occurring microbial organism of  claim 13  under conditions and for a sufficient period of time to produce p-toluate. 
     
     
         28 . The method of  claim 27 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         29 . The method of  claim 27 , wherein said microbial organism comprises seven exogenous nucleic acids each encoding a p-toluate pathway enzyme. 
     
     
         30 . The method of  claim 29 , wherein said seven exogenous nucleic acids encode 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 
     
     
         31 . The method of  claim 27 , wherein said microbial organism further comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. 
     
     
         32 . The method of  claim 31 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         33 . The method of  claim 32 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         34 . The method of  claim 27 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         35 . A non-naturally occurring microbial organism, comprising a microbial organism having a terephthalate pathway comprising at least one exogenous nucleic acid encoding a terephthalate pathway enzyme expressed in a sufficient amount to produce terephthalate, said terephthalate pathway comprising p-toluate methyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl aldehyde dehydrogenase; and wherein said microbial organism further comprises a p-toluate pathway, wherein said p-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; or chorismate lyase. 
     
     
         36 . The non-naturally occurring microbial organism of  claim 35 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a terephthalate pathway enzyme. 
     
     
         37 . The non-naturally occurring microbial organism of  claim 35 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a terephthalate pathway enzyme. 
     
     
         38 . The non-naturally occurring microbial organism of  claim 37 , wherein said three exogenous nucleic acids encode p-toluate methyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; and 4-carboxybenzyl aldehyde dehydrogenase. 
     
     
         39 . The non-naturally occurring microbial organism of  claim 35 , wherein said p-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 
     
     
         40 . The non-naturally occurring microbial organism of  claim 35 , wherein said microbial organism further comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. 
     
     
         41 . The non-naturally occurring microbial organism of  claim 40 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         42 . The non-naturally occurring microbial organism of  claim 41 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         43 . The non-naturally occurring microbial organism of  claim 35 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         44 . The non-naturally occurring microbial organism of  claim 35 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         45 . A method for producing terephthalate, comprising culturing the non-naturally occurring microbial organism of  claim 35  under conditions and for a sufficient period of time to produce terephthalate. 
     
     
         46 . The method of  claim 45 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         47 . The method of  claim 45 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a terephthalate pathway enzyme. 
     
     
         48 . The method of  claim 47 , wherein said three exogenous nucleic acids encode p-toluate methyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl aldehyde dehydrogenase. 
     
     
         49 . The method of  claim 45 , wherein said p-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 
     
     
         50 . The method of  claim 45 , wherein said microbial organism further comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway. 
     
     
         51 . The method of  claim 50 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         52 . The method of  claim 51 , wherein the (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprises 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. 
     
     
         53 . The method of  claim 45 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.