MICROORGANISMS AND PROCESSES FOR PRODUCING TEREPHTHALIC ACID AND ITS SALTS
The invention provides non-naturally occurring microbial organisms having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) pathway, p-toluate pathway, and/or terephthalate pathway. The invention additionally provides methods of using such organisms to produce 2H3M4OP, p-toluate or terephthalate. Also provided herein are processes for isolating bio-based aromatic carboxylic acid, in particular, p-toluic acid or terephthalic acid, from a culture medium, wherein the processes involve contacting the culture medium with sufficient carbon dioxide (CO 2 ) to lower the pH of the culture medium to produce a precipitate comprised of the aromatic carboxylic acid.
1 . A non-naturally occurring microbial organism, said microbial organism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M4OP) pathway a p-toluate pathway and a terephthalate pathway and comprising at least one exogenous nucleic acid encoding a 2H3M4OP pathway enzyme expressed in a sufficient amount to produce 2H3M4OP, wherein said 2H3M4OP pathway comprises a pathway selected from:
(1) 1A, 1B, 1C, 1D, 1E and 1F;
(2) 2A, 2B and 2C; and
(3) 2D, 2E and 2C,
wherein 1A is an erythrose-4-phosphate dehydrogenase, wherein 1B is a 4-phosphoerythronate dehydrogenase, wherein 1C is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate synthase, wherein 1D is a 2-acetyl-2,3-dihydroxy-4-phosphobutanoate reductoisomerase, wherein 1E is a 2,3,4-trihydroxy-3-methyl-5-phosphopentanoate dehydratase, wherein 1F is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2A is a 4,5-dihydroxy-2-oxopentanoate methyltransferase, wherein 2B is a 4,5-dihydroxy-3-methyl-2-oxopentanoate kinase, wherein 2C is a 4-hydroxy-3-methyl-2-oxo-5-phosphopentanoate decarboxylase, wherein 2D is a 4,5-dihydroxy-2-oxopentanoate kinase, wherein 2E is a 4-hydroxy-2-oxo-5-phosphopentanoate methyltransferase,
wherein said p-toluate pathway comprises at least one exogenous nucleic acid encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p-toluate, wherein said p-toluate pathway comprises 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H, wherein 4A is a 2-dehydro-3-deoxyphosphoheptonate synthase; wherein 4B is a 3-dehydroquinate synthase; wherein 4C is a 3-dehydroquinate dehydratase; wherein 4D is a shikimate dehydrogenase; wherein 4E is a shikimate kinase; wherein 4F is a 3-phosphoshikimate-2-carboxyvinyltransferase; wherein 4G is a chorismate synthase and wherein 4H is a chorismate lyase; and
wherein said microbial organism has a terephthalate pathway and comprises at least one exogenous nucleic acid encoding a terephthalate pathway enzyme expressed in a sufficient amount to produce terephthalate, wherein said terephthalate pathway comprises 5A, 5B and 5C, wherein 5A is a p-toluate methyl-monooxygenase reductase, wherein 5B is a 4-carboxybenzyl alcohol dehydrogenase and wherein 5C is a 4-carboxybenzyl aldehyde dehydrogenase.
2 . The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding at least one enzyme from a 2H3M4OP, a p-toluate or a terephthalate pathway enzyme.
3 . The non-naturally occurring microbial organism of claim 2 , wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from the 2H3M4OP (1)-(3).
4 . The non-naturally occurring microbial organism of claim 1 , wherein said exogenous nucleic acid is a heterologous nucleic acid.
5 . The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
6 .- 15 . (canceled)
16 . The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is a species of bacteria, yeast or fungus.
17 .- 21 . (canceled)
22 . Culture medium comprising bio-based terephthalate, wherein said culture medium is separated from said microbial organism of claim 1 .
23 .- 24 . (canceled)
25 . A composition comprising bio-based terephthalate and a compound other than said bio-based terephthalate, wherein said compound is a trace amount of a cellular portion of said microbial organism of claim 1 .
26 . A process for isolating a bio-based aromatic carboxylic acid from a culture medium comprising:
a) culturing a non-naturally occurring microbial organism in a culture medium to produce an aromatic carboxylate anion at a pH sufficient to maintain the aromatic carboxylate anion in soluble form;
b) separating the culture medium from non-soluble materials;
c) contacting the culture medium with sufficient carbon dioxide (CO 2 ) to lower the pH of the culture medium to produce an aromatic carboxylic acid precipitate, wherein the culture medium is substantially depleted of the aromatic carboxylate anion; and
d) separating the aromatic carboxylic acid from the culture medium;
wherein the aromatic carboxylic acid is p-toluic acid or terephthalic acid.
27 .- 52 . (canceled)
53 . A process for obtaining a bio-derived polymer using said bio-based terephthalate produced by the non-naturally occurring microbial organism of claim 1 .
54 . The bio-derived polymer of claim 53 , where said polymer is polyethylene terephthalate (PET), polybutyl terephthalate (PBT), or polytrimethylene terephthalate (PTT).
55 .- 88 . (canceled)