Semi-synthetic terephthalic acid via microorganisms that produce muconic acid

Abstract

The invention provides a non-naturally occurring microbial organism having a muconate pathway having at least one exogenous nucleic acid encoding a muconate pathway enzyme expressed in a sufficient amount to produce muconate. The muconate pathway including an enzyme selected from the group consisting of a beta-ketothiolase, a beta-ketoadipyl-CoA hydrolase, a beta-ketoadipyl-CoA transferase, a beta-ketoadipyl-CoA ligase, a 2-fumarylacetate reductase, a 2-fumarylacetate dehydrogenase, a trans-3-hydroxy-4-hexendioate dehydratase, a 2-fumarylacetate aminotransferase, a 2-fumarylacetate aminating oxidoreductase, a trans-3-amino-4-hexenoate deaminase, a beta-ketoadipate enol-lactone hydrolase, a muconolactone isomerase, a muconate cycloisomerase, a beta-ketoadipyl-CoA dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA transferase, a 2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA ligase, a muconate reductase, a 2-maleylacetate reductase, a 2-maleylacetate dehydrogenase, a cis-3-hydroxy-4-hexendioate dehydratase, a 2-maleylacetate aminoatransferase, a 2-maleylacetate aminating oxidoreductase, a cis-3-amino-4-hexendioate deaminase, and a muconate cis/trans isomerase. Other muconate pathway enzymes also are provided. Additionally provided are methods of producing muconate.

1 . A non-naturally occurring microbial organism, comprising a microbial organism having a muconate pathway comprising at least one exogenous nucleic acid encoding a muconate pathway enzyme expressed in a sufficient amount to produce muconate, said muconate pathway comprising an enzyme selected from the group consisting of a beta-ketothiolase, a beta-ketoadipyl-CoA hydrolase, a beta-ketoadipyl-CoA transferase, a beta-ketoadipyl-CoA ligase, a 2-fumarylacetate reductase, a 2-fumarylacetate dehydrogenase, a trans-3-hydroxy-4-hexendioate dehydratase, a 2-fumarylacetate aminotransferase, a 2-fumarylacetate aminating oxidoreductase, a trans-3-amino-4-hexenoate deaminase, a beta-ketoadipate enol-lactone hydrolase, a muconolactone isomerase, a muconate cycloisomerase, a beta-ketoadipyl-CoA dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA transferase, a 2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA ligase, a muconate reductase, a 2-maleylacetate reductase, a 2-maleylacetate dehydrogenase, a cis-3-hydroxy-4-hexendioate dehydratase, a 2-maleylacetate aminoatransferase, a 2-maleylacetate aminating oxidoreductase, a cis-3-amino-4-hexendioate deaminase, and a muconate cis/trans isomerase. 
     
     
         2 . The non-naturally occurring microbial organism of  claim 1 , wherein said muconate pathway comprises a set of muconate pathway enzymes, said set of muconate pathway enzymes selected from the group consisting of:
 A) (1) beta-ketothiolase, (2) an enzyme selected from beta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase, and beta-ketoadipyl-CoA ligase, (3) beta-ketoadipate enol-lactone hydrolase, (4) muconolactone isomerase, (5) muconate cycloisomerase, and (6) a muconate cis/trans isomerase;   B) (1) beta-ketothiolase, (2) an enzyme selected from beta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase and beta-ketoadipyl-CoA ligase, (3) 2-maleylacetate reductase, (4) 2-maleylacetate dehydrogenase, (5) cis-3-hydroxy-4-hexendioate dehydratase, and (6) muconate cis/trans isomerase;   C) (1) beta-ketothiolase, (2) an enzyme selected from beta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase and beta-ketoadipyl-CoA ligase, (3) 2-maleylacetate reductase, (4) an enzyme selected from 2-maleylacetate aminotransferase and 2-maleylacetate aminating oxidoreductase, (5) cis-3-amino-4-hexenoate deaminase, and (6) muconate cis/trans isomerase;   D) (1) beta-ketothiolase, (2) beta-ketoadipyl-CoA dehydrogenase, (3) 3-hydroxyadipyl-CoA dehydratase, (4) an enzyme selected from 2,3-dehydroadipyl-CoA transferase, 2,3-dehydroadipyl-CoA hydrolase and 2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase;   E) (1) beta-ketothiolase, (2) an enzyme selected from beta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase and beta-ketoadipyl-CoA ligase, (3) 2-fumarylacetate reductase, (4) 2-fumarylacetate dehydrogenase, and (5) trans-3-hydroxy-4-hexendioate dehydratase;   F) (1) beta-ketothiolase, (2) an enzyme selected from beta-ketoadipyl-CoA hydrolase, beta-ketoadipyl-CoA transferase and beta-ketoadipyl-CoA ligase, (3) 2-fumarylacetate reductase, (4) an enzyme selected from 2-fumarylacetate aminotransferase and 2-fumarylacetate aminating oxidoreductase, and (5) trans-3-amino-4-hexenoate deaminase.   
     
     
         3 . The non-naturally occurring microbial organism of  claim 2 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         4 . The non-naturally occurring microbial organism of  claim 2 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         5 . The non-naturally occurring microbial organism of  claim 2 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         6 . The non-naturally occurring microbial organism of  claim 2 , wherein said microbial organism comprises five exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         7 . The non-naturally occurring microbial organism of  claim 2 , wherein said microbial organism comprises six exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         8 . The non-naturally occurring microbial organism of  claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         9 . The non-naturally occurring microbial organism of  claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         10 . A method for producing muconate, comprising culturing the non-naturally occurring microbial organism according to  claim 1 , under conditions and for a sufficient period of time to produce muconate according to  claim 1 . 
     
     
         11 .- 18 . (canceled) 
     
     
         19 . A non-naturally occurring microbial organism, comprising a microbial organism having a muconate pathway comprising at least one exogenous nucleic acid encoding a muconate pathway enzyme expressed in a sufficient amount to produce muconate, said muconate pathway comprising an enzyme selected from the group consisting of a 4-hydroxy-2-ketovalerate aldolase, a 2-oxopentenoate hydratase, a 4-oxalocrotonate dehydrogenase, a 2-hydroxy-4-hexenedioate dehydratase, a 4-hydroxy-2-oxohexanedioate oxidoreductase, a 2,4-dihydroxyadipate dehydratase (acting on 2-hydroxy), a 2,4-dihydroxyadipate dehydratase (acting on 4-hydroxyl group) and a 3-hydroxy-4-hexenedioate dehydratase. 
     
     
         20 . The non-naturally occurring microbial organism of  claim 19 , wherein said muconate pathway comprises a set of muconate pathway enzymes, said set of muconate pathway enzymes selected from the group consisting of:
 A) (1) 4-hydroxy-2-ketovalerate aldolase, (2) 2-oxopentenoate hydratase, (3) 4-oxalocrotonate dehydrogenase, (4) 2-hydroxy-4-hexenedioate dehydratase;   B) (1) 4-hydroxy-2-ketovalerate aldolase, (2) 4-hydroxy-2-oxohexanedioate oxidoreductase, (3) 2,4-dihydroxyadipate dehydratase (acting on 2-hydroxy), (4) 3-hydroxy-4-hexenedioate dehydratase; and   C) (1) 4-hydroxy-2-ketovalerate aldolase, (2) 4-hydroxy-2-oxohexanedioate oxidoreductase, (3) 2,4-dihydroxyadipate dehydratase (acting on 4-hydroxyl group), (4) 2-hydroxy-4-hexenedioate dehydratase.   
     
     
         21 . The non-naturally occurring microbial organism of  claim 20 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         22 . The non-naturally occurring microbial organism of  claim 20 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         23 . The non-naturally occurring microbial organism of  claim 20 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         24 . The non-naturally occurring microbial organism of  claim 19 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         25 . The non-naturally occurring microbial organism of  claim 19 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         26 . A method for producing muconate, comprising culturing the non-naturally occurring microbial organism according to  claim 19 , under conditions and for a sufficient period of time to produce muconate. 
     
     
         27 .- 32 . (canceled) 
     
     
         33 . A non-naturally occurring microbial organism, comprising a microbial organism having a muconate pathway comprising at least one exogenous nucleic acid encoding a muconate pathway enzyme expressed in a sufficient amount to produce muconate, said muconate pathway comprising an enzyme selected from the group consisting of an HODH aldolase, an OHED hydratase, an OHED decarboxylase, an HODH formate-lyase, an HODH dehydrogenase, an OHED formate-lyase, an OHED dehydrogenase, a 6-OHE dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase, a 2,3-dehydroadipyl-CoA hydrolase, a 2,3-dehydroadipyl-CoA transferase, a 2,3-dehydroadipyl-CoA ligase, and a muconate reductase. 
     
     
         34 . The non-naturally occurring microbial organism of  claim 33 , wherein said muconate pathway comprises a set of muconate pathway enzymes, set of muconate pathway enzymes selected from the group consisting of:
 A) (1) HODH aldolase, (2) OHED hydratase, (3) OHED decarboxylase, (4) 6-OHE dehydrogenase, and (5) muconate reductase;   B) (1) HODH aldolase, (2) OHED hydratase, (3) an enzyme selected from OHED formate-lyase and OHED dehydrogenase, (4) an enzyme selected from 2,3-dehydroadipyl-CoA hydrolase, 2,3-dehydroadipyl-CoA transferase and 2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase; and   C) (1) HODH aldolase, (2) an enzyme selected from HODH formate-lyase and HODH dehydrogenase, (3) 3-hydroxyadipyl-CoA dehydratase, (4) an enzyme selected from 2,3-dehydroadipyl-CoA hydrolase, 2,3-dehydroadipyl-CoA transferase and 2,3-dehydroadipyl-CoA ligase, and (5) muconate reductase   
     
     
         35 . The non-naturally occurring microbial organism of  claim 34 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         36 . The non-naturally occurring microbial organism of  claim 34 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         37 . The non-naturally occurring microbial organism of  claim 34 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         38 . The non-naturally occurring microbial organism of  claim 34 , wherein said microbial organism comprises five exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         39 . The non-naturally occurring microbial organism of  claim 33 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         40 . The non-naturally occurring microbial organism of  claim 33 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         41 . A method for producing muconate, comprising culturing the non-naturally occurring microbial organism according to  claim 33 , under conditions and for a sufficient period of time to produce muconate. 
     
     
         42 .- 47 . (canceled) 
     
     
         48 . A non-naturally occurring microbial organism, comprising a microbial organism having a muconate pathway comprising at least one exogenous nucleic acid encoding a muconate pathway enzyme expressed in a sufficient amount to produce muconate, said muconate pathway comprising an enzyme selected from the group consisting of a lysine aminotransferase, a lysine aminating oxidoreductase, a 2-aminoadipate semialdehyde dehydrogenase, a 2-aminoadipate deaminase, a muconate reductase, a lysine-2,3-aminomutase, a 3,6-diaminohexanoate aminotransferase, a 3,6-diaminohexanoate aminating oxidoreductase, a 3-aminoadipate semialdehyde dehydrogenase, and a 3-aminoadipate deaminase. 
     
     
         49 . The non-naturally occurring microbial organism of  claim 48 , wherein said muconate pathway comprises a set of muconate pathway enzymes, set of muconate pathway enzymes selected from the group consisting of:
 A) (1) lysine aminotransferase, (2) lysine aminating oxidoreductase, (3) 2-aminoadipate semialdehyde dehydrogenase, (4) 2-aminoadipate deaminase, and (5) muconate reductase   B) (1) lysine-2,3-aminomutase, (2) 3,6-diaminohexanoate aminotransferase, (3) 3,6-diaminohexanoate aminating oxidoreductase, (4) 3-aminoadipate semialdehyde dehydrogenase, (5) 3-aminoadipate deaminase, and (6) muconate reductase.   
     
     
         50 . The non-naturally occurring microbial organism of  claim 49 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         51 . The non-naturally occurring microbial organism of  claim 49 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         52 . The non-naturally occurring microbial organism of  claim 49 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         53 . The non-naturally occurring microbial organism of  claim 49 , wherein said microbial organism comprises five exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         54 . The non-naturally occurring microbial organism of  claim 49 , wherein said microbial organism comprises six exogenous nucleic acids each encoding a muconate pathway enzyme. 
     
     
         55 . The non-naturally occurring microbial organism of  claim 48 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         56 . The non-naturally occurring microbial organism of  claim 48 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         57 . A method for producing muconate, comprising culturing the non-naturally occurring microbial organism according to  claim 48 , under conditions and for a sufficient period of time to produce muconate. 
     
     
         58 .- 65 . (canceled) 
     
     
         66 . A semi-synthetic method for synthesizing terephthalate (PTA) comprising preparing muconic acid by culturing an organism of any one of claims,  2 ,  20 ,  34 , or  49 , reacting said muconic acid with acetylene to form a cyclohexadiene adduct, and oxidizing said cyclohexadiene adduct to form PTA. 
     
     
         67 . The method of  claim 66 , further comprising isolating muconic acid prior to reacting with acetylene. 
     
     
         68 . The method of  claim 66 , wherein the step of reacting said muconic acid with acetylene comprises adding acetylene to the culture broth. 
     
     
         69 . The method of  claim 68 , wherein the culture broth is filtered prior to adding acetylene. 
     
     
         70 . The method of  claim 68 , wherein said step of oxidizing said cyclohexadiene adduct comprises adding air or oxygen to the culture broth.