WO2010085712A2 - Methods for producing dodecanedioic acid and derivatives thereof - Google Patents

Abstract

Methods for producing biosourced dodecanedioic acid and compositions comprising biosourced dodecanedioic acid are provided. In some embodiments, the method comprises first forming muconic acid biologically from a renewable carbon source, reducing the muconic acid to hexenedioic acid, and then reacting the hexenedioic acid with an unsaturated fatty acid, typically a Δ⁹ unsaturated fatty acid, in a metathesis reaction to produce dodecenedioic acid. Dodecenedioic acid is then reduced to dodecanedioic acid. Dodecanedioic acid is can be used to form polymers, such as polyamides. Examples of polyamides include nylon, such as nylon 6,12. Nylon 6,12 can be formed by reacting dodecanedioic acid with 1,6-hexamethylene diamine.

Description

METHODS FOR PRODUCING DODECANEDIOIC ACID AND DERIVATIVES

THEREOF

RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 61/146,545, filed January 22, 2009, which application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[002] The invention relates generally to the production of dodecanedioic acid from renewable feedstock and subsequent uses thereof, such as for forming polyamides.

BACKGROUND OF THE INVENTION

[003] Nylon is a generic designation for a family of synthetic thermoplastic polyamides that are used to make fabrics, musical strings, rope, screws and gears, to name just a few examples. Nylon is available with fillers too, such as glass- and molybdenum sulfϊde-fϊlled variants. [004] Nylon 6 is the most common commercial grade of molded nylon. The numerical suffix specifies the numbers of carbon atoms donated by the monomers; the diamine first and the diacid second. For nylon 6,6, the diamine typically is hexamethylenediamine and the diacid is adipic acid. Each of these monomers donates 6 carbons to the polymer chain. [005] Another example of a useful nylon is nylon 6,12, which is a copolymer of a 6-carbon diamine and a 12-carbon dicarboxylic acid. One method for making nylon 6,12 comprises forming a polycondensation product of 1,6-hexamethylene diamine and dodecanedioic acid. For commercial production of such polymeric materials, the starting materials are virtually solely obtained from hydrocarbon sources.

[006] Dodecanedioic acid is thus a very important chemical. It is used in a variety of industrial applications, such as plasticizers for polymers, epoxy curing agents, adhesive and powder coatings, engineering plastics, perfumery and pharmaceutical products, etc. Annually, 15,000,000,000 pounds of dodecanedioic acid are synthesized from petrochemical feedstock. Such petrochemical feedstocks are a predominantly depleting natural resource, and the use of such feedstocks has been linked to detrimental changes to the environment on a global scale. [007] Such feedstock materials useful for the production of nylon have therefore limited availability and are subject to substantial price fluctuations. As a result, there has been a growing interest and need for alternative methods to produce dodecanedioic acid as well as polyamides that are renewable, sustainable and less harmful for the environment.

SUMMARY OF THE INVENTION

[008] Aspects of the invention relate to the production of useful commercial product, such as polyamides, starting with materials produced by a biological process from renewable feedstock, as opposed to using starting materials derived from non-renewable feedstock such as petroleum or other fossil carbon resources. Aspects of the invention relates more particularly to the production of dicarboxylic acid, as well as derivatives thereof, from renewable biomass-derived carbon source. More particularly, some aspects of the invention relate to the production of dodecanedioic acid, as well as precursors and derivatives thereof, from renewable biomass-derived carbon source. More specifically, the methods of the invention make use of a metathesis step with olefinic compounds in order to produce biosourced dicarboxylic acid such as dodecanedioic acid from renewable biosourced feedstock. The resulting renewable dodecanedioic acid can be separated from other products of the metathesis reaction and from any remaining starting materials. Dodecanedioic acid and derivatives thereof have utility in the production of polyamides and other polymers. [009] In some aspects, the invention provides a method of producing first muconic acid biologically from renewable feedstock. In preferred embodiments, the muconic acid is reduced to an isomer or isomers of hexenedioic acid. Reduction of the muconic acid can be performed using methods known in the art such as a zinc halide reagent, electrochemical reduction, or selective hydrogenation. The hexenedioic acid may be present as a derivative such as an ester, amide, or salt. [0010] In some embodiments, the hexenedioic acid is reacted with an unsaturated fatty acid in a metathesis reaction to produce dodecenedioic acid, which is then reduced to dodecanedioic acid. The reaction typically involves using a metathesis catalyst, such as a Grubbs catalyst, including benzylidene-bis(tricyclohexylphosphine)dichlororuthenium or benzylidene[1,3- bis(2,4,6- trimethylphenyl)-2- imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium. [0011] Forming muconic acid biologically may comprise forming the muconic acid bacterially using prokaryotes belonging to the genera Escherichia, Klebsiella, Corynebacterium, Brevibacterium, Arthrobacter, Bacillus, Pseudomonas, Streptomyces, Staphylococcus, or Serratia, or by using yeasts of the genus Saccharomyces or Schizosaccharomyces. [0012] Muconic acid is reduced to an isomer of hexenedioic acid, such as 3-hexenedioic acid, using any suitable reagent. One suitable reagent is a zinc halide reagent, such as zinc chloride in pyridine.

[0013] The hexenedioic acid or derivative thereof is reacted with an unsaturated fatty acid to form an unsaturated dicarboxylic acid or derivative thereof. In preferred embodiments, the unsaturated fatty acid is first reacted in a self metathesis reaction to produce Δ⁹ octadecenedioic acid. Δ⁹ octadecenedioic acid then reacts with the hexenedioic acid to produce dodecenedioic acid. Preferably, the unsaturated fatty acid is a Δ⁹ unsaturated fatty acid. Examples, without limitation, of the Δ⁹ unsaturated fatty acid include myristoleic acid, palmitoleic acid, elaidic acid, and oleic acid. The unsaturated dicarboxylic acid or derivative thereof which is produced by the metathesis reaction can then be reduced to a saturated dicarboxylic acid. In preferred embodiments, the unsaturated dicarboxylic acid which is formed is dodecenedioic acid is then reduced to its saturated analog dodecanedioic acid. This can be accomplished by, for example, hydrogenating the dodecenedioic acid using a precious metal hydrogenation catalyst.

[0014] In another embodiment, the Δ⁹ unsaturated fatty acid is first transformed to the symmetric Δ⁹ unsaturated dicarboxylic acid octadecenedioic acid via a self-metathesis reaction. The symmetric Δ⁹ octadecenedioic acid can then be used in a cross-metathesis reaction with the symmetric 3-hexenedioic acid to give the desired dodecenedioic acid as a single product of the metathesis reaction.

[0015] In some embodiments, the dodecanedioic acid is used for forming polymers, such as polyamides. Examples of polyamides include a nylon, such as nylon 6,12. Nylon 6,12 can be formed by reacting 1,6-hexamethylene diamine with dodecanedioic acid.

[0016] A particular embodiment of the disclosed invention comprises first forming muconic acid biologically using prokaryotes belonging to the genera Escherichia, Klebsiella, Corynebacterium, Brevibacterium, Arthrobacter, Bacillus, Pseudomonas, Streptomyces, Staphylococcus, ox Serratia or yeasts of the genus Saccharomyces or Schizosaccharomyces. The muconic acid is reduced to 3-hexenedioic acid using a zinc halide reagent. The 3-hexenedioic acid is reacted with a Δ⁹ unsaturated fatty acid, such as myristoleic acid, palmitoleic acid, elaidic acid, oleic acid, or combinations thereof, in a metathesis reaction using a metathesis catalyst, to produce dodecenedioic acid. The dodecenedioic acid is reduced by hydrogenation to form dodecanedioic acid. The dodecanedioic acid is then used to form a poly amide, such a nylon 6,12, by reaction with a suitable diamine, such as 1,6-hexamethylene diamine.

[0017] Aspects of the invention relate to compositions comprising biosourced unsaturated dicarboxylic acid or derivative thereof and their saturated analogs which have been produced from renewable feedstock derived from biomass. In some embodiments, the composition comprises a biosourced dodecenedioic acid or dodecenedioic acid derivatives thereof. In preferred embodiments, disclosed are biosourced products with a carbon isotope distribution or a ¹⁴C/¹²C ratio characteristic of product synthesized from renewable carbon sources. In some embodiments, the renewable isolated dodecenedioic acid or dodecenedioic acid derivatives thereof are characterized by a ¹⁴C/¹²C ratio greater than 0, greater than 0.9 x 10^'12, or of about 1.2 x 10^'12. In some embodiments, the dodecenedioic acid derivative is a dimethyl dodecenedioic acid. In other embodiments, compositions comprising biosourced dodecanedioic acid or derivatives thereof are disclosed. Other aspects of the invention relates to compositions comprising a renewable isolated 3- hexenedioic and 3-hexenedioic derivatives thereof. In some embodiments, the biosourced 3- hexenedioic and 3-hexenedioic derivatives thereof is characterized by a ¹⁴C/¹²C isotopic ratio of about 1.2 x 10^-12. Further aspects of the invention relate to compositions comprising biosourced polyamide. In some embodiments, the polyamide is a nylon 6,12 polymer and at least 12 carbon atoms per monomer units derived from renewable carbon sources. In some embodiments, the nylon 6,12 comprises detectable traces of carbon 14. In some embodiments, the renewable compounds disclosed herein contains up to about 1 part per trillion of carbon 14.

[0018] Aspects of the invention relates to compositions comprising a dodecenedioic acid or dodecenedioic acid derivative and at least one unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproduct derived from the dodecenedioic acid or dodecenedioic acid derivative . In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 byproducts derived from the dodecenedioic acid or dodecenedioic acid derivative. In some embodiments, the at least one unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproduct comprises an alkene chain having from about 7 to about 16 carbon atoms. In some embodiments, the compositions comprises at least 9 byproducts derived from the dodecenedioic acid or dodecenedioic acid derivative, the byproducts comprising an alkene chain having from about 7 to about 16 carbon atoms. In some embodiments, the dodecenedioic acid derivative is a dodecenedioic acid diester. In preferred embodiments, the dodecenedioic acid or dodecenedioic acid derivative contains up to about 1 part per trillion of Carbon 14. Preferably, the alkene chain comprises a carbon double bond in a position of C3-C4. [0019] Other aspects of the invention relate to compositions comprising 9-octadecenedioic acid or 9-octadecenedioic acid derivative and at least one octadecenedioic acid or octadecenedioic acid derivative byproduct derived from 9-octadecenedioic acid or 9-octadecenedioic acid derivative. In some embodiments, the composition comprises at least 1, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 16 byproducts derived from 9- octadecenedioic acid or 9-octadecenedioic acid derivative. In some embodiments, the octadecenedioic acid or octadecenedioic acid derivative byproducts comprise a carbon double bond in Cl-C2, C2-C3, C3-C4, C4-C5, C5-C6,, C6-C7, C7-C8, C8-C9, ClO-CI l, C11-C12, C12-C13, C 13-Cl 4, C 14- Cl 5, C 15-Cl 6, C 16-Cl 7, or C 17-Cl 8 position. In some embodiments, the 9- octadecenedioic acid or 9-octadecenedioic acid derivative contains up to about 1 part per trillion of Carbon 14.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The advantages of the invention described above, together with further advantages, can be better understood by referring to the following description taken in conjunction with the accompanying drawings. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention.

[0021] FIG. 1 shows the common pathway of aromatic amino acid biosynthesis and the divergent pathway synthesizing cis,cis-muconic acid from 3-dehydroshikimate.

[0022] FIG. 2 shows a process flow diagram of an embodiment of the dodecanedioic acid synthesis. Fig. 2 shows the self-metathesis reaction forming the symmetric Δ⁹ octadecenedioic acid and the subsequent cross metathesis reaction with 3-hexenedioic acid.

[0023]

[0024] FIG. 3 shows the effect of double bond migration on self metathesis reaction.

[0025] FIG. 4 shows the effect of double bond migration on cross metathesis reaction. DETAILED DESCRIPTION OF THE INVENTION

[0026] Aspects of the invention relates to methods and compositions to produce dodecenedioic acid derivatives thereof and/or dodecanedioic acid and derivatives thereof. In some aspects, the invention relates to the methods and compositions for the production of dodecenedioic acid from 3-hexenedioic acid and octadecenedioate. In preferred embodiment, dodecenedioic acid is produced from dimethyl hexenedioates and dimethyl octadecenedioate. In preferred embodiments, reduction of dimethyl dodecenedioic acid produces dimethyl dodecanedioic acid. As used herein the terms hexenedioic acid and hexanedioate are used interchangeably and refer to a molecule comprising six carbon atoms, eight hydrogen atoms and four oxygen atoms having the formula

AS used herein the terms dodecenedioic acid and dodecenedioate are used interchangeably and refer to a molecule comprising twelve carbon atoms, twenty hydrogen atoms and four oxygen atoms having the formula

COOH . As used herein the terms dodecanedioic acid and dodecanedioic acid are used interchangeably and refer to a molecule comprising twelve carbon atoms, twenty two hydrogen atoms and four oxygen atoms having the formula

[0027] Several multistep chemical process have been employed for the preparation of dodecanedioic acid, usually from cyclohexanone, cyclododecadiene or cyclododecatriene. Dodecanedioic acid can be produced from epoxidation of the 1,5,9 cyclodecatriene using hydrogen peroxide and acetic acid to form the corresponding epoxy compound which is subsequently hydrogenated to form the alcohol and oxidized to form the desired product. In another chemical process, the dodecanedioic acid is prepared by the epoxidation of 1,5,9 cyclododecatriene with an organic hydroperoxide to form 1,2-epoxy-5,9-cyclododecadiene followed by conversion of this compound to a cycloderivative oxidizable to dodecanedioic acid. In some aspects, the invention uses oils or fats and muconic acid as an alternative starting material for the production of dicarboxylic acids, oxo chemicals such as oxo aldehydes and oxo esters.

[0028] As used herein, the words "preferred" and "preferably" refer to embodiments of the invention that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention. [0029] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0030] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included.

[0031] The term "including" is used to mean "including but not limited to". "Including" and

"including but not limited to" are used interchangeably.

[0032] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

[0033] Any numerical values recited herein include all values from the lower value to the upper value. All possible combinations of numerical values between the lowest value and the highest value enumerated herein are expressly included in this application.

[0034] As used herein, the term "aldehyde" refers to a carbonyl-bearing functional group having a formula

where R is virtually any group including, by way of example and without limitation, aliphatic, substituted aliphatic, aryl, arylalkyl, heteroaryl, etc.

[0035] As used herein the term "aliphatic" refers to a substantially hydrocarbon-based compound, or a radical thereof

, for a hexane radical), including alkanes, alkenes and alkynes, and further including straight- and branched-chain arrangements, and as well as all stereo and position isomers.

[0036] As used herein the term "alkyl" refers to a hydrocarbon arranged in a chain in a homologous series having the general formula

Alkyl substituents include methyl,

and so on. The structure of an alkyl group is like that of its alkane counterpart, but with one less hydrogen atom.

[0037] As used herein the term "aryl" refers to a substantially hydrocarbon-based aromatic compound, or a radical thereof as a substituent bonded to another group, particularly

other organic groups, having a ring structure as exemplified by benzene, naphthalene, phenanthrene, anthracene, and the like. [0038] As used herein the term "arylalkyl" refers to a compound, or a radical thereof (e.g.

C₇H₇ for toluene) as a substituent bonded to another group, particularly other organic groups, containing both aliphatic and aromatic structures.

[0039] As used herein the term "carboxylic acid" refers to a compound having a formula R-

COOH, where R can be virtually any group including, by way of example and without limitation, aliphatic, substituted aliphatic, aryl, arylalkyl, heteroaryl, and the like.

[0040] As used herein the term "cyclic" refers to a substantially hydrocarbon, closed-ring compound, or a radical thereof. Cyclic compounds or substituents also can include one or more sites of unsaturation. Exemplary cyclic compounds include compounds typically having 3 or more, more typically 4 or more, and even more typically 5 or more carbon atoms in the ring including, without limitation, cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, and conjugated derivatives thereof, such as compounds having olefins conjugated with carbonyl functionalities, such as carboxylic acids, amides and esters.

[0041] As used herein the term "derivative" refers to a molecule that differs in chemical structure from a parent compound. Examples of derivatives include, without limitation: homologs, which differ incrementally from the chemical structure of the parent, such as a difference in the length of an aliphatic chain; molecular fragments; structures that differ by one or more functional groups from the parent compound, such as can be made by transforming one or more functional groups of a parent, such as by changing an acid functional group of a parent molecule into an acid halide, amide, or ester; a change in ionization state of a parent, such as ionizing an acid to its conjugate base; isomers, including positional, geometric and stereoisomers; and combinations thereof.

[0042] As used herein the term "ester" refers to a compound having a formula

where R and R¹ are independently selected from virtually any group, including aliphatic, substituted aliphatic, aryl, arylalkyl, heteroaryl, etc.

[0043] As used herein the term "heteroaryl" refers to an aromatic, closed-ring compound, or radical thereof as a substituent bonded to another group, particularly other organic groups, where at least one atom in the aromatic ring is other than carbon, such as oxygen, sulfur and/or nitrogen. [0044] As used herein the term "heterocyclic" refers to a cyclic, i.e. closed-ring, aliphatic compound, or radical thereof as a substituent bonded to another group, particularly other organic groups, where at least one atom in the ring structure is other than carbon, such as oxygen, sulfur and/or nitrogen.

[0045] As used herein the term "ketone" refers to a compound having a formula

where R and R' are independently selected from virtually any group including, without limitation, aliphatic, substituted aliphatic, aryl, arylalkyl, heteroaryl, etc.

[0046] As used herein the term "lower" organic compounds refers to organic compounds or radicals thereof having 10 or fewer carbon atoms in a chain, including all branched and stereochemical variations thereof, particularly including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

[0047] As used herein the term "substituted" refers to a fundamental compound, such as an aliphatic, aryl, arylalϊphatic, heterocyclic, heteroaryl, or heteroarylaliphatic compound, or a radical thereof, having coupled thereto, typically in place of a hydrogen atom, a second atom, substituent, functional group, etc. For example, substituted aryl compounds or substituents may have an aliphatic group coupled to the closed ring of the aryl base, such as with toluene, which has a methyl group substituted for a hydrogen atom of benzene. Again solely by way of example and without limitation, a long-chain hydrocarbon may have, without limitation, an atom or substituent bonded thereto, such as a halide, a heteroatom, a functional group, an aryl group, a cyclic group, a heteroaryl group or a heterocyclic group.

[0048] As used herein, the term "unsaturated fatty acid" refers to compounds that have an alkene chain with a terminal carboxylic acid group.

[0049] As used herein, the term "unsaturated dicarboxylic acid" refers to a compound with a carboxylic acid at each end of an unbranched carbon chain, which also includes at least one double bond in the carbon chain.

Muconic acid [0050] The description of the invention uses the terms "muconate" and "muconic acid." The term "muconic acid" refers to chemical species in which both carboxylic acid functions are protonated, and the molecule is formally a neutral species. The term "muconate" refers to the chemical species in which one or both of the carboxylic acid functions is deprotonated to give the anionic or doubly-anionic form of muconic acid which would be the predominate chemical species at physiological pH values. However, as the terms "muconic acid" and "muconate" refer to the protonated or deprotonated forms of the same molecule, the terms are used synonymously where the difference between protonated and deprotonated (e.g., non-ionized and ionized) forms of the molecule is not usefully distinguished.

[0051] There are three isomers of hexa-2,4-dienedioic acids, commonly referred to as muconic acids: these are the trans,trans (2E,4E), cis,trans (2Z,4E) and cis.cis (2Z,4Z) isomers:

[0052] Cis cis-muconic acid and trans, trans-muconic acid are available commercially in small quantities (e.g., from Sigma- Aldrich), but are quite expensive. However, cis.cis-muconic acid also is produced by some bacteria through the enzymatic degradation of aromatic compounds. In some embodiments, cis.cis-muconic acid is synthesized biologically in certain bacteria as disclosed in U.S. Patent Nos. 5,487,987 and 5,616,496, which are incorporated herein by reference. Industrial-scale quantities of Cis cis-muconic acid can be produced by such biosynthesis. Bacteria that are capable of the biosynthesis of Cis cis-muconic acid are members of genera having an endogenous common pathway of aromatic amino acid biosynthesis. Suitable bacteria include prokaryotes belonging to the genera Escherichia, Klebsiella, Corynebacterium, Brevibacterium, Arthrobacter, Bacillus, Pseudomonas, Streptomyces, Staphylococcus, or Serratia. Eukaryotic host cells can also be utilized, particularly yeasts of the genus Saccharomyces or Schizosaccharomyces. [0053] Suitable prokaryotic species include Escherichia coli, Klebsiella pneumonia,

Corynebacterium glutamicum, Corynebacterium herculis, Brevibacterium divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus lichenformis, Bacillus megaterium, Bacillus mesentericus, Bacillus pumilis, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas angulata, Pseudomonas fluorescens, Pseudomonas tabaci, Streptomyces aureofaciens, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces kasugensis, Streptomyces lavendulae, Streptomyces lipmanii, Streptomyces Iividans, Staphylococcus epidermis, Staphylococcus saprophyticus, and Serratia marcescens. Suitable eukaryotic species include Saccharomyces cerevisiae and Saccharomyces carlsbergensis.

[0054] In some embodiments, the methods include microbial biosynthesis of biosourced muconic acid from readily available, renewable carbon sources (see, for example U.S. Patent No. 5,616,496 which is incorporated herein by reference). As used herein, the term "biosourced" refers to a material derived using a biological process as opposed to a non-biological process such as a synthetic, chemical process. For example, "biosourced" muconic acid is derived from a fermentation process utilizing a fermentable carbon source. A "biosourced" compound or product refers to a product comprised in whole or in part from biosourced material. In some embodiments, preferred host cells for use in this invention are able to convert carbon sources into D-erythrose-4- phosphate (E4P) and phosphoenolpyruvate (PEP). In some embodiments, E4P and PEP are subsequently converted to amino acids via a metabolic pathway ultimately producing aromatic amino acids. Fermentable carbon sources can include essentially any carbon source capable of being biocatalytically converted into D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP), two precursor compounds to the common pathway of aromatic amino acid biosynthesis. Suitable carbon sources include, but are not limited to, biomass-derived, renewable sources such as starches, cellulose, polyols such as glycerol, pentose sugars such as arabinose and xylose, hexose sugars such as glucose, and fructose, disaccharides such as sucrose and lactose, as well as other carbon sources capable of supporting microbial metabolism, for example, carbon monoxide. Exemplary carbon sources include glucose, glycerol, sucrose, xylose and arabinose. In one embodiment, D-glucose is the biomass-derived carbon source.

[0055] Host cells suitable for use in the present invention include members of genera that can be utilized for biological production of desired intermediates in the biosynthesis of aromatic compounds such as amino acids. In some embodiments, such host cells are suitable for industrial- scale biosynthetic or biological production of industrially useful aromatic compounds, and intermediates leading to such useful compounds. One intermediate in the pathway of aromatic amino acid biosynthesis is 3-dehydroshikimate (DHS). In particular, suitable host cells can have an endogenous common pathway of aromatic amino acid biosynthesis that is functional at least to the production of DHS. Common pathways for the biosynthesis of aromatic amino acids are endogenous in a wide variety of microorganisms, and can be used for the production of various aromatic compounds. In certain embodiments, auxotrophic mutant cell lines having a mutation blocking the conversion of 3-dehydroshikimate (DHS) to chorismate are used. Such mutants have a mutation in one or more of the genes encoding shikimate dehydrogenase, shikimate kinase, EPSP synthase or chorismate synthase. These mutants accumulate elevated intracellular levels of DHS. Suitable mutant cell lines include Escherichia coli strains AB2834, AB2829 and AB2849. [0056] For example, E. coli AB2834 has a mutation in the aroE locus which encodes shikimate dehydrogenase, preventing the cells from converting DHS into shikimic acid. As a result, the carbon flow directed into aromatic amino acid biosynthesis is not processed beyond DHS. Similarly E. coli AB2829 accumulates DHS because it cannot convert shikimate 3-phosphate (S3P) into 5-enoipyruvylshikimate-3-phosphate (EPSP) due to a mutation in the aroA locus which encodes EPSP synthase. E. coli AB2849 is unable to catalyze the conversion of EPSP into chorismic acid due to a mutation in the aroC locus which encodes chorismate synthase also result in increased intracellular levels of DHS.

[0057] Host cells can be transformed so that the intracellular DHS can be used as a substrate for biocatalytic conversion to catechol, which can thereafter be converted to muconic acid. For example, host cells can be transformed with recombinant DNA to force carbon flow away from the common pathway of aromatic amino acid biosynthesis after DHS is produced and into a divergent pathway to produce muconic acid.

[0058] A mechanism for transforming the host cell to direct carbon flow into the divergent pathway can involve the insertion of genetic elements including expressible sequences coding for 3- dehydroshikimate dehydratase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase. Regardless of the exact mechanism utilized, it is contemplated that the expression of these enzymatic activities will be effected or mediated by the transfer of recombinant genetic elements into the host cell. Genetic elements as herein defined include nucleic acids (generally DNA or RNA) having expressible coding sequences for products such as proteins, apoproteins, or antisense RNA, which can perform or control pathway enzymatic functions. The expressed proteins can function as enzymes, repress or derepress enzyme activity, or control expression of enzymes. The nucleic acids coding these expressible sequences can be either chromosomal (e.g., integrated into a host cell chromosome) or extrachromosomal (e.g., carried by plasmids, cosmids, and the like). [0059] The genetic elements of the present invention can be introduced into a host cell by plasmids, cosmids, phages, yeast artificial chromosomes or other vectors that mediate transfer of the genetic elements into a host cell. These vectors can include an origin of replication along with cis- acting control elements that control replication of the vector and the genetic elements carried by the vector. Selectable markers can be present on the vector to aid in the identification of host cells into which the genetic elements have been introduced. For example, selectable markers can be genes that confer resistance to particular antibiotics such as tetracycline, ampicillin, chloramphenicol, kanamycin, or neomycin.

[0060] Introducing genetic elements into a host cell can utilize an extrachromosomal multicopy plasmid vector into which genetic elements are inserted. Plasmid borne introduction of the genetic element into host cells involves an initial cleaving of a plasmid with a restriction enzyme, followed by ligation of the plasmid and genetic elements in accordance with the invention. Upon recircularization of the ligated recombinant plasmid, transduction or other mechanism (e.g., electroporation, microinjection, and the like) for plasmid transfer is utilized to transfer the plasmid into the host cell. Plasmids suitable for insertion of genetic elements into the host cell include, but are not limited to, pBR322, and its derivatives such as pAT153, pXf3, pBR325, pBr327, pUC vectors, pACYC and its derivatives, pSClOl and its derivatives, and CoIEl. In addition, cosmid vectors such as pLAFR3 are also suitable for the insertion of genetic elements into host cells. Examples of plasmid constructs include, but are not limited to, p2-47, pKDS.243A, pKD8.243B, and pSUaroZYl 57-27, which carry the aroZ and aroY loci isolated from Klebsiella pneumoniae, which respectively encode 3-dehydroshikimate dehydratase and protocatechuate decarboxylase. Additional examples of plasmid constructs include pKDS.292, which carries genetic fragments endogenous to Acinetobacter calcoaceticus catA, encoding catechol 1 ,2-dioxygenase. [0061] Methods for transforming a host cell can also include insertion of genes encoding for enzymes, which increase commitment of carbon into the common pathway of aromatic amino acid biosynthesis. The expression of a gene is primarily directed by its own promoter, although other genetic elements including optional expression control sequences such as repressors, and enhancers can be included to control expression or derepression of coding sequences for proteins, apoproteins, or antisense RNA. In addition, recombinant DNA constructs can be generated whereby the gene's natural promoter is replaced with an alternative promoter to increase expression of the gene product. Promoters can be either constitutive or inducible. A constitutive promoter controls transcription of a gene at a constant rate during the life of a cell, whereas an inducible promoter's activity fluctuates as determined by the presence (or absence) of a specific inducer. For example, control sequences can be inserted into wild type host cells to promote overexpression of selected enzymes already encoded in the host cell genome, or alternatively can be used to control synthesis of extrachromosomally encoded enzymes.

[0062] Control sequences to promote overproduction of DHS can be used. As previously noted, DHS is synthesized in the common pathway by the sequential catalytic activities of the tyrosine-sensitive isozyme of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthase (encoded by aroF) and 3-dehydroquinate (DHQ) synthase (encoded by aroB) along with the pentose phosphate pathway enzyme transketolase (encoded by tkt). The expression of these biosynthetic enzymes can be amplified to increase the conversion of D-glucose into DHS. Increasing the in vivo catalytic activity of DAHP synthase, the first enzyme of the common pathway, increases the flow of D-glucose equivalents directed into aromatic biosynthesis. However, levels of DAHP synthase catalytic activity are reached beyond which no further improvements are achieved in the percentage of D-glucose that is committed to aromatic biosynthesis. At this limiting level of aromatic amino acid biosynthesis, amplification of the catalytic levels of the pentose phosphate pathway enzyme transketolase achieves sizable increases in the percentage of D-glucose siphoned into the pathway. [0063] Amplified transketolase activity can increase D-erythrose 4-phosphate concentrations. As one of the two substrates for DAHP synthase, limited D-erythrose 4-phosphate availability can limit DAHP synthase catalytic activity. Therefore, one method for amplifying the catalytic activities of DAHP synthase, DHQ synthase and DHQ dehydratase is to overexpress the enzyme species by transforming the microbial catalyst with a recombinant DNA sequence encoding these enzymes.

[0064] Amplified expression of DAHP synthase and transketolase can create a surge of carbon flow directed into the common pathway of aromatic amino acid biosynthesis, which is in excess of the normal carbon flow directed into this pathway. If the individual rates of conversion of substrate into product catalyzed by individual enzymes in the common aromatic amino acid pathway are less than the rate of DAHP synthesis, the substrates of these rate-limiting enzymes can accumulate intracellularly.

[0065] Microbial organisms such as E. coli frequently cope with accumulated substrates by exporting such substrates into the external environment, such as the bulk fermentation medium. This results in a loss of carbon flow from the common pathway since exported substrates are typically lost to the microbe's metabolism. DHQ synthase is an example of a rate-limiting common pathway enzyme. Amplified expression of DHQ synthase removes the rate-limiting character of this enzyme, and prevents the accumulation of DAHP and its nonphosphorylated analog, DAH. DHQ dehydratase is not rate-limiting. Therefore, amplified expression of aroF-encoded DAHP synthase, tkt-encoded transketolase and aroB-encoded DHQ synthase increases production of DHS, which in the presence of DHS dehydratase and protocatechuate decarboxylase is converted to catechol, which is subsequently biocatalytically converted to cis,cis-muconic acid.

[0066] Thus, as a preferred embodiment of the present invention, a heterologous strain of

Escherichia coli expressing genes encoding DHS dehydratase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase was constructed enabling the biocatalytic conversion of D-glucose to cis.cis-muconic acid. Efficient conversion of D-glucose to DHS was accomplished upon transformation of the host cell with pKD136. The strain E. coli AB2834/pKD136 was then transformed with plasmids pKD8.243A and pKDS.292. The result was E. coli AB2834/pKD136/pKDS.243A/pKDS.292 that expresses the enzymes 3-dehydroshikimate dehydratase (aroZ), protocatechuate decarboxylase (aroY) and catechol 1,2-dioxygenase (catA). This bacterial cell line was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville MD 20852, on Aug. 1, 1995 and assigned accession number 69875. [0067] In another embodiment, E. coli AB2834/pKD 136 is transformed with plasmids p2-47 and pKD8.292 to generate E. coli AB2834/pKD136/p2-47/pKDS.292. In another embodiment, E. coli AB2834/pKD136 is transformed with plasmids pKD8.243B and pKDS.292 to generate E. coli AB2834/pKD136/p2-47/pKDS.292. Each of these heterologous host cell lines catalyzes the conversion of D-glucose into cis,cis-muconic acid. Synthesized cis,cis-muconic acid accumulates extracellularly and can be separated from the cells. Subsequently, the cis.cis-muconic acid can be isomerized into cisjrans-muconic acid and further to trans,trans-muconic acid as desired. [0068] Some aspects to the invention thus relates to a transformant of a host cell having an endogenous common pathway of aromatic amino acid biosynthesis. The transformant is characterized by the constitutive expression of heterologous genes encoding 3-dehydroshikimate dehydratase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase. In one embodiment, the cell transformant is further transformed with expressible recombinant DNA sequences encoding the enzymes transketolase, DAHP synthase, and DHQ synthase. In another embodiment, the host cell is selected from the group of mutant cell lines including mutations having a mutation in the common pathway of amino acid biosynthesis that blocks the conversion of 3-dehydroshikimate to chorismate. In yet another embodiment, the genes encoding 3-dehydroshikimate dehydratase and protocatechuate decarboxylase are endogenous to Klebsiella pneumoniae. In a further embodiment, the heterologous genes encoding catechol 1,2-dioxygenase are endogenous to Acinetobacter calcoaceticus.

[0069] As shown in FIG. 1, the intermediates in the divergent pathway are protocatechuate, catechol, and Cis cis-muconic acid. The enzyme responsible for the biocatalytic conversion of DHS to protocatechuate is the enzyme 3-dehydroshikimate dehydratase, labeled "aroZ" in FIG. 1. The enzyme responsible for the decarboxylation of protocatechuate to form catechol is protocatechuate decarboxylase, labeled "aroY" in FIG. 1. Lastly, the enzyme catalyzing the oxidation of catechol to produce cis.cis-muconic acid is catechol 1,2-dioxygenase, labeled "catA" in FIG. 1. In accordance with standard notation, the genes for the expression of these enzymes are denoted using italics and are thus aroZ, aroY, and cat A respectively. The cis,cis-muconic acid can subsequently be isomerized (not shown). In one embodiment of the invention, host cells may exhibit constitutive expression of the genes aroZ, aroY, and catA. In another embodiment, host cells may exhibit constitutive expression of any one or more of the genes aroZ, aroY and catA; or any combination of two thereof In yet another embodiment, host cells may exhibit constitutive expression of none of aroZ, aro Y and catA.

[0070] The enzymes 3-dehydroshikimate dehydratase and protocatechuate decarboxylase are recruited from the ortho cleavage pathways which enable microbes such as Neurospora, Aspergillus, Adnetobacter, Klebsiella, and Pseudomonas to use aromatics (benzoate and p-hydroxybenzoate) as well as hydroaromatics (shikimate and quinate) as sole sources of carbon for growth. DHS dehydratase plays a critical role in microbial catabolism of quinic and shikimic acid. Protocatechuate decarboxylase was formulated by Patel to catalyze the conversion of protocatechuate into catechol during catabolism of p-hydroxybenzoate by Klebsiella aerogenes. Reexamination of Patel's strain (now referred to as Enterobacter aerogenes) [(a) Grant, D. J. W.; Patel, J. C. Antonie van Leewenhoek 1969, 35, 325. (b) Grant, D. J. W. Antonie van Leewenhoek 1970, 36, 161] recently led Ornstonto conclude that protocatechuate decarboxylase was not metabolically significant in catabolism of p-hydroxybenzoate [Doten, R. C; Ornston, N. J. Bacteriol. 1987, 169, 5827].

Renewable compounds

[0071] Compounds of interest comprising at least one carbon atom, such as muconic acid, dodecanedioic acid, dodecenedioic acid, 3-hexenedioic acid and derivatives thereof produced from renewable, biologically derived carbon sources will be composed of carbon from atmospheric carbon dioxide which has been incorporated by plants (e.g., from a carbon source such as glucose, sucrose, glycerin, or plant oils). Therefore, such compounds include renewable carbon rather than fossil fuel-based or petroleum-based carbon in their molecular structure. Accordingly, the biosourced dodecanedioic acid or products synthesized from dodecanedioic acid, and associated derivative products, will have a smaller carbon footprint than dodecanedioic acid and associated products produced by conventional methods because they do not deplete fossil fuel or petroleum reserves and because they do not increase the amount of carbon in the carbon cycle (e.g., life cycle analysis shows no net carbon increase to the global carbon balance).

[0072] The biosourced dodecanedioic acid and associated products as well as the starting compounds (such as muconic acid) or intermediate products (such as 3-hexenedioic acid) can be distinguished from products produced from a fossil fuel or petrochemical carbon source by methods known in the art, such as dual carbon-isotopic finger printing. This method can distinguish otherwise chemically-identical materials, and distinguishes carbon atoms in the material by source, that is biological versus non-biological, using the ¹⁴C and ¹³C isotope ratios. The carbon isotope ¹⁴C is unstable, and has a half life of 5730 years. Measuring the relative abundance of the unstable ¹⁴C isotope relative to the stable ¹³C isotope allows one to distinguish specimen carbon between fossil (long dead) and biospheric (alive and thus renewable) feedstock (See Currie, L. A. "Source Apportionment of Atmospheric Particles," Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3-74). The basic assumption in radiocarbon dating is that the constancy of ¹⁴C concentration in the atmosphere leads to the constancy of ¹⁴C in living organisms. [0073] When dealing with an isolated sample, the age of a sample can be deduced approximately by the relationship t = (-5730/0.693)ln(A/Ao) where t = age, 5730 years is the half- life of the unstable ¹⁴C isotope, and A and A₀ are the specific ¹⁴C activity of the sample and of the modern standard, respectively (Hsieh, Y., Soil ScL Soc. Am J., 56, 460, (1992)). However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, ¹⁴C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO₂, and hence in the living biosphere, approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (¹⁴C/¹²C) of ca. 1.2 x 10^-12, with an approximate relaxation half-life of 7-10 years. (This latter half- life must be distinguished from the isotopic half-life, that is, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric ¹⁴C since the onset of the nuclear age.) It is this latter biospheric ¹⁴C time characteristic that holds out the promise of annual dating of recent biospheric carbon. ¹⁴C can be measured by accelerator mass spectrometry (AMS), with results given in units of fraction of modern carbon (f_M). f_M is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). For the current living biosphere (plant material), fM ≡ 1.1.

[0074] The ratio of the stable carbon isotopes ¹³C and ¹²C provides a complementary route to source discrimination and apportionment. The ¹³C/¹²C ratio in a given biosourced material is a consequence of the ¹³C/¹²C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed and also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C₃ plants (the broadleaf), C₄ plants (the grasses), and marine carbonates all show significant differences in ¹³C/¹²C and the corresponding differences in the ¹³C values, that is the δ¹³C values. The ¹³C measurement scale was originally defined by a zero set by pee dee belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The

values are in parts per thousand (per mil), abbreviated %₀ , and are calculated as follows:

Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is δ¹³C. Measurements are made on CO₂ by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45 and 46. Furthermore, lipid matter of C₃ and C₄ plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway. Within the precision of measurement, ¹³C shows large variations due to isotopic fractionation effects, the most significant of which for the instant invention is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (e.g., the initial fixation of atmospheric CO₂). Two large classes of vegetation are those that incorporate the C₃ (or Calvin-Benson) photosynthetic cycle and those that incorporate the C₄ (or Hatch-Slack) photosynthetic cycle. C₃ plants, such as hardwoods and conifers, are dominant in the temperate climate zones. In C₃ plants, the primary CO₂ fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase and the first stable product is a 3-carbon compound. C₄ plants, on the other hand, include such plants as tropical grasses, corn and sugar cane. In C₄ plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid, which is subsequently decarboxylated. The CO₂ thus released is refixed by the C₃ cycle. Both C₄ and C₃ plants exhibit a range of ¹³C¹²C isotopic ratios, but typical values are ca. -10 to -14 per mil (C₄) and -21 to -26 per mil (C₃) (Weber et al, J. Agric. Food Chem., 45, 2942 (1997)). Coal and petroleum fall generally in this latter range. [0075] Therefore, the biosourced muconic acid, the dodecanedioic acid and compositions including dodecanedioic acid of the invention can be distinguished from their ancient fossil-fuel and petrochemical derived counterparts on the basis of ¹⁴C (fM) and dual carbon-isotopic fingerprinting, indicating new compositions of matter (e.g., U.S. Patent Nos. 7,169,588, 7,531,593, and 6,428,767). The ability to distinguish these products is beneficial in tracking these materials in commerce. For example, products comprising both new and old carbon isotope profiles can be distinguished from products made only of ancient materials. Hence, the biosourced dodecanedioic acid and derivative materials can be followed in commerce on the basis of their unique profile.

[0076] One would therefore appreciate that a molecule or compound containing at least one carbon atom, such as muconic acid or derivatives thereof, fatty acid or derivative thereof, 3- hexenedioic acid or derivatives thereof, dodecenedioic acid or derivatives thereof, dodecanedioic acid or derivatives thereof, dicarboxylic acids or derivatives thereof, polyamides or derivatives thereof, nylon or derivatives thereof can be described by their carbon isotope distribution (for example, ¹⁴C, ¹³C, or ¹²C) or by their ¹⁴C/¹²C ratio. As each single carbon atom within compound comes from a naturally occurring carbon isotope, the source of carbon atoms will affect the carbon isotope distribution of the compound or the ¹⁴C/¹²C ratio. More specifically, the carbon isotope distribution or ¹⁴C/¹²C ratio of a compound synthesized from petrochemical feedstock is distinguishable form the carbon isotope distribution or ¹⁴C/¹²C ratio of a compound produced from renewable carbon sources. The basic assumption is that the constancy of ¹⁴C concentration in the atmosphere leads to the constancy of ¹⁴C in living organisms whereas in inorganic carbon sources (such as petrochemical feedstock) all the ¹⁴C has decayed. Thus, a compound produced from renewable carbon source can be distinguished from a product produced from inorganic carbon source. In some embodiments, the ¹⁴C/¹²C ratio is measured using a ASTM test method D 6866-05 Determining the Biobased Content of Natural Range Materials Using Radiocarbon and Isotope Ratio Mass Spectrometry Analysis, incorporated by reference. Assessment of the renewably based carbon in a compound can be performed through standard test methods. Using radiocarbon and isotope ratio mass spectrometry analysis, the biobased content of materials can be determined. ASTM International, formally known as the American Society for Testing and Materials, has established a standard method for assessing the biobased content of materials. The ASTM method is designated ASTM-D6866. This test method measures the ¹⁴C/¹²C isotope ratio in a sample and compares it to the ¹⁴C/¹²C isotope ratio in a standard 100% biosourced material to give percent biosourced content of the sample.

[0077] In some embodiments, the compositions including biosourced dodecanedioic acid, dodecenedioic acid, 3-hexenedioic acid, polyamide can be distinguished from non-renewable corresponding compounds using a ¹⁴C distribution or a ¹⁴C/¹²C ratio. In some embodiments, the ¹⁴C/¹²C ratio is indicative of the fraction of carbon atoms coming from renewable carbon source. In an exemplary embodiment, the starting materials for the production of dodecanedioic acid are muconic acid and Δ⁹ unsaturated fatty acid such as oleic acid. In a preferred embodiment, muconic acid is produced from biologically derived carbon sources and the Δ⁹ unsaturated fatty acid is derived from a vegetable or animal source. The resulting dodecanedioic acid product from cross metathesis reaction will therefore has a ¹⁴C/¹²C ratio indicative that all carbon atoms are coming from renewable carbon sources and no carbon atoms are coming from ancient carbon (e.g. carbon from coal, oil, or natural gas). Synthesis of compounds from starting materials coming from a renewable carbon source containing ¹⁴C and from an ancient carbon source containing no radiocarbon, will result in a decrease of the ¹⁴C/¹²C ratio when compared to a ¹⁴C/¹²C ratio of a compound comprised in whole from biosourced material. By presuming that 1.2 x 10^-12 value represents the ¹⁴C/¹²C ratio of a compound comprised in whole from biosourced material and the 0 value corresponds to the ¹⁴C/¹²C ratio of compound derived from ancient carbon, a compound formed in part from biosourced material will have a radiocarbon signature or fingerprint lower than 1.2 x 10^-12. In an exemplary embodiment, nylon 6, 12 results in the condensation of 1 ,6- hexamethylene diamine and dodecanedioic acid. If 1,6-hexamethylene diamine is produced from petrochemical feedstocks (e.g. from coal, oil, and natural gas) and doceanedioic acid is formed in whole from biosourced materials, two third of the carbon atoms content will come from renewable carbon source. In some embodiment, the desired product's ¹⁴C/¹²C ratio is greater than 0, greater than 0.9x 10^-12 In some embodiments, the biosourced carbon content of a compound can be analyzed and a ratio of the amount of ¹⁴C can be reported to that of a biosourced reference standard (percent of modern carbon). In some embodiments, the carbon content of a biosourced product is 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%.

[0078] Aspects of the invention relate to the products prepared by the processes described herein. In some embodiments wherein the starting muconic acid is prepared from biomass, the resulting products of the process contain a significant percentage of carbon derived from renewable resources. Such products are unique because the products contain a detectable trace or amount of carbon 14, and preferably up to about 1 part per trillion, as determined according to ASTM D6866- 08. The resulting products preferably contain 3 or greater carbons, more preferably 9 or greater carbons, more preferably 12 carbons or greater carbons derived from renewable resources, such as biomass, preferably by microbial synthesis. The resulting products are prepared from renewable resources prepared by microbial synthesis under fermentor-controlled conditions. In embodiments wherein the products are utilized to prepare polymers, the monomer units preferably contain 12 or greater carbons, derived from renewable resources, such as biomass.

Muconic Acid Derivatives

[0079] In some aspects of the invention, muconic acids are reduced to 3-hexenedioic acid.

In some embodiments, Cis cis-muconic acid and derivatives thereof produced biologically by a recombinant host cell culture are first reduced to 3-hexenedioic acid. In some embodiments, 3- hexenedioic acid is used in a metathesis reaction to form desired compounds. General Formula 1 exemplifying muconic acids and muconic acid derivatives within the scope of the present invention is provided below.

[0080] With reference to Formula 1, Ri-R₆ typically are independently selected from aliphatic, substituted aliphatic, alkoxy, amino, amines, substituted amines, protected amines, aryl, substituted aryl, arylalkyl, substituted arylalkyl, carbonyl-containing moieties (such as aldehydes, amides, carboxylic acids, esters, ketones and thioesters), cyclic, substituted cyclic, ethers, substituted ethers, halide, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, hydrogen, hydroxyl, hydroxylamine, and nitrogen-containing moieties, such as nitrile (RCN), nitro (NO₂) and nitroso (RNO). Preferably, Ri-R₆ are independently selected from aliphatic, typically lower aliphatic, and even more preferably lower alkyl, and hydrogen. Ri and R₆ are most typically independently hydrogen or lower alkyl. R₂-R₅ are most typically hydrogen. Formula 1, and certain other structural formulas provided herein, includes bonds indicated by a wavy, as opposed to a straight line, to indicate that all possible stereoisomers are included in that particular general formula. [0081] In some embodiments, the primary derivatives made from muconic acids are (1) conjugate bases, (2) various stereoisomers produced by an isomerization reaction of a parent compound, and/or (3) compounds formed by transforming one or more carboxylic acid functional groups to another functional group. In some embodiments, cis,cis-muconic acid can be isomerized to trans,trans-muconic acid by dissolving Cis cis-muconic acid in methanol along with traces of iodine and exposing the reaction mixture to light. The methane solubilities of cis,cis-mxxcom^'c acid and trans, trans-mucomc acid differ substantially. As a result, trans, trans-mucomc acid precipitates from solution as it forms.

[0082] In some embodiments, the Cis cis-muconic acid can be isomerized to cisjrans- muconic acid using methods known in the art. One should appreciate that because the cis, trans- mucomc acid is more soluble in both organic solvent and aqueous media than either of the cis, cis- or trans, trans- isomers, cisjrans-muconic acid is an advantageous starting compound allowing easy processing and recovery.

[0083] In various embodiments, the method includes culturing recombinant cells that express 3-dehydroshikimate dehydratase, protocatechuate decarboxylase and catechol 1,2- dioxygenase in a medium comprising a renewable carbon source and under conditions in which such renewable carbon source is converted to DHS by enzymes found in the common pathway of aromatic amino acid biosynthesis of the cell, and the resulting DHS is biocatalytically converted to cis.cis-mucorύc acid. In certain embodiments, the fermentation broth is provided in a vessel such as a fermenter vessel and the isomerization reaction may be carried out in the vessel. [0084] The production of Cis cis-muconic acid by the fermentation of the renewable carbon source can produce a broth comprising the recombinant cells and extracellular cis,c is-muconic acid. The production can also include the step of separating the recombinant cells, cell debris, insoluble proteins and other undesired solids from the broth to give a clarified fermentation broth containing substantially all, or most of, the cis,cis-mucoxύc acid formed by the fermentation. After muconic acid has been produced it may accumulate in the extracellular medium (e.g. fermentation broth) and can be separated from the cells by centrifugation, filtration, or other methods known in the art. In some embodiments, cic, cis-muconic acid is first isomerized to cis,trans-muconic acid and which is then separated from the fermentation broth or cell free fermentation broth by precipitation, extraction, filtration, or other methods known in the art

[0085] In some aspects of the invention, the carboxylic acid functional group of the muconic acid is converted to various different functional groups that facilitate the cycloaddition reaction. This conversion promotes desired physical properties of compounds made by the cycloaddition reaction, or derivatives or polymers made therefrom using the compounds produced by cycloaddition as monomers, or both. Exemplary carboxylic acid functional group transformations include forming an acid halide, such as an acid chloride, using suitable reagents known to a person of ordinary skill in the art, such as thionyl chloride, phosphorous pentachloride or pentbromide. The carboxylic acid functional group of the muconic acid also can be converted to an ester, such a methyl or ethyl ester. Examples of methods for forming esters, particularly lower alkyl esters, include: the alcohol/H* protocol, such as using methanol or ethanol and catalytic sulfuric acid to form the corresponding methyl and ethyl esters, which is convenient to execute, but may be accompanied by double bond isomerization; methylation with diazomethane to form the methyl ester; or treating the acid with an alkyl iodide, such as MeI/ Et₄NOH, in a dark protocol. In preferred embodiments, a dimethyl ester derivative is obtained starting from cis,cis-rmiconic acid and using dimethyl sulfate and potassium carbonate.

[0086] Table 1 below provides a partial list of exemplary muconic acids and lower alkyl muconic acid esters, particularly methyl and ethyl muconic acid esters, as well as their melting points and solvents useful for purification by recrystallization.

Reduction of Muconic Acid

[0087] Scheme 1 below illustrates one embodiment of a method for reducing muconic acid, or a muconic acid derivative, to 3-hexenedioic acid, or a derivative thereof, such as a mono- or diester. As shown in Scheme 1, the muconic acid diene can be reduced to 3-hexenedioic acid using a zinc halide reagent in a suitable solvent, such as pyridine. See, Kotora et al., Chem. Lett., p. 236- 237 (2000).

Olefin Metathesis

[0088] As used herein the term "olefin" refers to is an unsaturated chemical compound containing at least one carbon-to-carbon double bond. In an exemplary embodiment, the simplest olefin with only one double bond and no other functional groups, form a homologous series of hydrocarbons with the general formula C_nH_2n. The term "lower olefin" refers to an organic compound having less than about 10 carbon atoms and containing at least one carbon-carbon double bond. Lower olefin may have one, two or more unsaturated bonds. Preferably, the lower olefin has a single unsaturated bond. Lower olefins may be substituted at any position along the carbon chain with one or more substituents, provided that the one or more substituents are substantially inert with respect to the metathesis reaction. Suitable substituents include, but are not limited to alkyl, preferably methyl, as well as hydroxy, ether, keto, and aldehyde.

[0089] Olefin metathesis is an important reaction used in organic synthesis. Olefin metathesis is also known as transalkylidenation organic reaction and entails cleavage of alkene double bond followed by redistribution of alkylene fragments. The reaction was developed by Yves Chauvin, Richard R. Schrock and Robert H. Grubbs, who shared a Nobel Prize in Chemistry in 2005. Olefin metathesis reactions proceed in the presence of a catalytically effective amount of metathesis catalyst. Exemplary metathesis catalysts include metal carbene catalysts based upon catalytic transition metals, such as ruthenium, nickel, tungsten, osmium, chromium, rhenium, and molybdenum. Metathesis catalysts, which can be employed in the process according to the invention, are all metathesis catalysts which are known to the person skilled in the art and which are suitable for metathesis reactions, or a mixture of at least two catalysts. In some embodiments, the olefin metathesis reaction uses first generation Grubbs catalysts, variant or derivative of the first generation Grubbs-type catalyst. In other embodiments, the olefin metathesis reaction uses second generation Grubbs catalysts, variant or derivative of the Grubbs-type catalyst. Examples of Grubbs catalysts include but are not limited to, benzylidene-bis(tricyclohexylphosphine)dichlororuthenium and benzylidene[1,3- bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium. In other embodiments, Schrock catalysts or a variant or derivative of the Schrock catalysts are used as catalysts for the metathesis reactions. Yet in other embodiments, the catalyst is a Hoveyda-Grubbs catalyst or variant of the Hoveyda-Grubbs catalyst. In some embodiments, at least one metathesis catalyst selected from carbene or carbyne complexes or a mixture of these complexes, is employed. The term "complex" as used herein refers to a metal atom with at least one ligand or complexing agent bound thereto. Metathesis catalysts are used using techniques known to those skilled in the art.

[0090] As illustrated in Scheme 2, 3-hexenedioic acids, or derivatives thereof, such as their lower alkyl esters, may be reacted with an unsaturated dicarboxylic acid, particularly a Δ⁹ dicarboxylic fatty acid, in the presence of a metathesis catalyst to produce dodecenedioic acid. However, any suitably unsaturated fatty acid can be suitably employed in the process of this invention.

[0091] The alkene chain of the unsaturated fatty acid may be a linear or branched and may optionally include one or more functional groups in addition to the carboxylic acid group. For example, some carboxylic acids include one or more hydroxy 1 groups. The alkene chain typically contains about 4 to about 30 carbon atoms, more typically about 4 to about 22 carbon atoms. In many embodiments, the alkene chain contains 18 carbon atoms (i.e., a Cl 8 fatty acid). The unsaturated fatty acids have at least one carbon-carbon double bond in the alkene chain (i.e., a monounsaturated fatty acid), and may have more than one double bond (i.e., a polyunsaturated fatty acid) in the alkene chain. In exemplary embodiments, the unsaturated fatty acid is a Δ⁹ unsaturated fatty acid. Δ⁹ unsaturated fatty acids have a carbon-carbon double bond located between the C9 and ClO in the alkene chain of the unsaturated fatty acid. In determining this position, the alkene chain is numbered beginning with the carbon atom in the carbonyl group of the unsaturated fatty acid. In preferred embodiments, the Δ⁹ unsaturated starting materials have a straight alkene chain. Examples, without limitation of suitable Δ⁹ unsaturated fatty acids, include myristoleic acid,

palmitoleic acid,

, elaidic acid,

and oleic acid,

each of which has a C9-C10 unsaturated bon (Δ⁹ olefin). In many embodiments, useful Δ⁹ unsaturated fatty acids are derived from natural oils such as plant-based oil or animal fats. Representative examples of renewable plant based oils include olive oil, peanut oil, grape seed oil, sea buckthorn oil, and sesame oil, poppyseed oil, nutmeg butter, palm oil, coconut oil, Macadamia oil, Sea Buckthorn oil. Representative examples of animal fats include lard and tallow fats. Unsaturated fatty acids can be obtained commercially or synthesized by saponification of fatty acid esters using methods known to those skilled in the art.

[0092] In a preferred embodiment, dodecenedioic acid or derivative thereof (e.g. (Z)- dimethyldodecenedioic acid) is synthesized by a cross-metathesis reaction using a metathesis catalyst as shown in Fig. 2.

[0093] In an exemplary embodiment, oleic acid is converted to dimethyl octadecenedioate in a two-step synthesis process as described inNgo and Foglia (JAOCS, 1985, 84:777-784). For example, oleic acid is placed in presence of a metathesis catalyst to form dimethyl 9- octadecenedioate. Octadecenedioic acid can then be purified using techniques known in the art. [0094] In some embodiments, the starting materials include dimethyl-hexenedioate and dimethyl-octadecenedioate. After the cross metathesis reaction of dimethyl-hexenedioate in presence of dimethyl-octadecenedioate, dimethyl-dodecenedioate is formed. The dodecenedioic acid can be converted into dodecanedioic acid by hydrogenation to saturate the olefin. In preferred embodiments, dimethyl dodecenedioate is reduced to form dimethyl dodecanedioate. In preferred embodiments, biosourced dimethyl-hexenedioate is produced by reduction of muconic acid derivatives. The resulting product from the metathesis reaction is a composition comprising a biosourced dimethyl dodecanedioate compound.

[0095] Metathesis reactions are conducted under appropriate conditions to produce the desired metathesis product(s). In some embodiments, metathesis reactions may be performed under an inert atmosphere. Preferably, the inert atmosphere is an inert gas that does not interfere with the metathesis catalyst. Examples of inert gases include, but are not limited to, nitrogen, argon, neon, helium and combination thereof. In some embodiments, the metathesis reactions are processed under any desired pressure. In some embodiments, the metathesis reactions are conducted in an inert solvent that does not impede catalysis. For example, inert solvent include, but are not limited to, aromatic hydrocarbon such as benzene or toluene, halogenated aromatic hydrocarbons, aliphatic hydrocarbons such as methanol. A solvent may be desirable where the reactants are not entirely miscible and both can be solubilized in a suitable solvent. Preferably, the solvent is thermally stable, and does not decompose at the process temperature. Yet, in some other embodiments, metathesis reactions proceed in a solvent-free reaction. The metathesis reactions are performed at a temperature selected to form the desired product(s) and lower the formation of undesired products. Typically, the temperature is above O°C, above 2O°C, above 4O°C, above 5O°C. In an exemplary embodiment, the metathesis temperature reaction is from about 2O°C to about 100°C. For example, the process temperature may be about 45°C or about 50°C. In some embodiments, the amount of catalyst used in the metathesis reaction is selected to form the desired product and lower the formation of undesired products. For example, the molar ratio of the dioic acid to the catalyst may rage from 5:l to 20:1 or to 100:1 or to 1,000:1, or to 100,000: 1. In some embodiments, the reaction time is about 1 hour, about 2 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours or longer. [0096] After cross metathesis, the products are separated from starting material and from the catalyst. Useful techniques to separate and purify the desired product(s) from starting material or other undesired product(s) include, but are not limited to, distillation, chromatography, fractional crystallization, liquid/liquid extraction, or any combination thereof. Preferably, the desired products are purified to a high degree, for example to 90% or greater. In some embodiments, the conversion can be checked by gas chromatography-mass spectrometry. For example, filtrate of the reaction mixture (cross metathesis of dimethyl hexenedioate and dimethyl octadecenedioates and subsequent hydrogenation) can be checked by gas chromatography-mass spectrometry and compared with standard dimethyl dodecanedioates calibration curve.

[0097] One should appreciate that oleic acid, octadoc-9-enedioic acid and the 3-hexenedioic acid are symmetric molecules with respect to the carboxylic or ester terminal groups and therefore self metathesis reactions of the oleic acid or cross metathesis of the octadoc-9-enedioic acid and the 3-hexenedioic acid will result, theoretically, in the formation of fewer products than if an asymmetric molecule was used. For example, cross metathesis reaction of 3-hexenedioic acid with dimethyl octadoc-9-enedioic acid, will form theoretically only dimethyl dodecenedioic acid. Whether a cis isomer or trans isomer is formed in this type of reaction is determined by the orientation the molecules assume when they coordinate to the catalyst, as well as the sterics of the substituents on the carbon-carbon double bond of the newly forming molecule. [0098] If the metathesis catalyst used for metathesis processes promotes double bond migration, that is, if the metathesis catalyst causes the double bond to move from its original position in the unsaturated fatty acid to a position either closer to, or further from, the carboxylic acid function, the metathesis reaction will produce a mixture of dicarboxylic acid products. For example, if the metathesis catalyst promotes the central double bond migration of the octadec-9- enedioate to form octadec-8-enedioate, octadec-7-enedioate and octadec-6-enedioate etc. (as shown in Fig. 3), these octadecenedioates may undergo cross-metathesis with dimethyl hexenedioate to form dimethyl unsaturated fatty acid esters byproducts with different backbone length from 7 carbons to 16 carbons (as shown in Fig. 4). In some embodiments, reaction conditions are modified to prevent double bond migration. In some embodiments, the ratio of lower olefin to unsaturated fatty acid or derivative thereof is selected to prevent double-bond migration. In one embodiment, the dimethyl hexenedioic acid is added in excess of the dimethyl octadecendioic acid. For example, the molar ratio of reactants dimethyl hexenedioate/ dimethyl octadecenedioate can be 2:1, 3:1, 4:1 or greater. In other embodiments, an acidic additive is added to inhibit double bond migration and undesired product formation. Examples of acidic additive include, but are not limited to, benzoic acid and salts, phosphoric acid and salts.

[0099] Aspects of the invention relates to compositions comprising a dodecenedioic acid or dodecenedioic acid derivative and at least one unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproduct derived from the dodecenedioic acid or dodecenedioic acid derivative . In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 byproducts derived from the dodecenedioic acid or dodecenedioic acid derivative. In some embodiments, the at least one unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproduct comprises an alkene chain having from about 7 to about 16 carbon atoms. In some embodiments, the compositions comprises at least 9 byproducts derived from the dodecenedioic acid or dodecenedioic acid derivative, the byproducts comprising an alkene chain having from about 7 to about 16 carbon atoms. In some embodiments, the dodecenedioic acid derivative is a dodecenedioic acid diester. Preferably, the alkene chain comprises a carbon double bond in a position of C3-C4.

[00100] Other aspects of the invention relate to compositions comprising 9-octadecenedioic acid or 9-octadecenedioic acid derivative and at least one octadecenedioic acid or octadecenedioic acid derivative byproduct derived from 9-octadecenedioic acid or 9-octadecenedioic acid derivative. In some embodiments, the composition comprises at least 1, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 16 byproducts derived from 9- octadecenedioic acid or 9-octadecenedioic acid derivative. In some embodiments, the octadecenedioic acid or octadecenedioic acid derivative byproducts comprise a carbon double bond in

position.

Polyamide Production

[00101] As used herein, a "polyamide" is a polymer containing amide monomers joined by amide bonds that are produced by reacting an amine and a carboxylic acid or derivative thereof. As with the polyesters, industrial polyamides have many uses, including aramids, nylons, and polyaspartates. Aramid (pictured below) is an aromatic polyamide that is made by polymerizing terephthalic acid or a derivative thereof, such as terephthaloyl chloride, and a diamine, such as 1 ,4- phenyldiamine (para-phenylenediamine). [00102] Nylon is a generic designation for a family of synthetic thermoplastic polyamides that are used to make fabrics, musical strings, rope, screws and gears, to name just a few examples.

Nylon is available with fillers too, such as glass- and molybdenum sulfide-filled variants. Nylon 6 is the most common commercial grade of molded nylon. The numerical suffix specifies the numbers of carbon atoms donated by the monomers; the diamine first and the diacid second. For nylon 6,6, the diamine typically is hexamethylenediamine and the diacid is adipic acid. Each of these monomers donates 6 carbons to the polymer chain. [00103] Another example of a useful nylon is nylon 6,12, which has a glass transition temperature of 46 °C, and a molecular weight of repeat units of 310.48 g/mol. The structural formula of nylon 6,12 is as shown below.

[00104] One method for making nylon 6, 12 comprises forming a polycondensation product of

1,6-hexamethylene diamine H₂N-(CH₂)₆-NH₂ and dodecanedioic acid HOOC-(CH₂)i₀-COOH (See for example U.S. Patent No. 3,903,152, which is incorporated herein by reference). US Patent No. 3,903,152 provides a method for the production of nylon 6,12 from dodecanedioic acid in which 20 g of dodecanedioic acid was heated to 70 °C together with 20 grams of water and neutralized with a 50 wt. % hexamethylenediamine aqueous solution. The pH was then adjusted to 8 to prepare a 50% nylon salt aqueous solution. Then, the nylon salt aqueous solution was heated from room temperature to 250 °C in a salt bath under nitrogen atmosphere followed by polymerization at 250 °C for 5 hours under ordinary pressure. WO/2000/009586 provides another method for making nylon 6,12. According to this document, nylon 6,12 can be prepared by combining decanedioic and an aqueous 1,6-hexane diamine solution in water with stirring in an autoclave for 30 minutes at 90 °C such that a 55 % by weight salt solution is obtained. Water is removed by distillation by first raising the temperature in 10 minutes to 180 °C, removing half of the amount of water through distillation and then raising the temperature to 200 °C and removing an amount of water through distillation such as to obtain a 90 % by weight aqueous salt solution. The reactor is completely closed, the distillation is stopped and the temperature is raised to 227 °C and prepolymerization begins. The water present and the high temperature cause the pressure to rise slowly . The pressure at the end of the prepolymerization is about 12x105 Pa. The prepolymerization is performed during 1/2 hour at a constant temperature, after which the content of the autoclave is flashed in a nitrogen atmosphere. The prepolymer is cooled in a nitrogen atmosphere. The prepolymer granules obtained are sieved so that the fraction having a diameter of between 1 and 2 mm is obtained. This fraction is introduced into either a static bed (capacity approximately 50 g of solid substance) or a tumble dryer (capacity approximately 10 liters) and postcondensed at an elevated temperature (about 25 °C below the polymer's melting point) in a nitrogen/water vapor (75/25 % by volume) atmosphere for 24 hours. Then the polymer granules were cooled to room temperature. From the polymer thus prepared a number of rods and plates were injection-molded.

[00105] While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

CLAIMSWe claim:

1. A method for producing dodecanedioic acid, the method comprising: reducing muconic acid to hexenedioic acid; reacting the hexenedioic acid with an unsaturated fatty acid in a metathesis reaction to produce dodecenedioic acid; and reducing the dodecenedioic acid to dodecanedioic acid.

2. The method of claim 1 further comprising reacting the unsaturated fatty acid in a self metathesis reaction to produce Δ⁹ octadecenedioic acid and reacting the hexenedioic acid with the Δ⁹ octadecenedioic acid to produce dodecenedioic acid.

3. The method of claim 1 comprising providing muconic acid wherein muconic acid is cis,trans-muconic acid.

4. The method of claim 1 wherein muconic acid is produced from a renewable carbon source through biocatalytic conversion.

5. The method of claim 4, further comprising culturing recombinant cells that express 3-dehydroshikimate dehydratase, protocatechuate decarboxylase and catechol 1,2-dioxygenase in a medium comprising the renewable carbon source and under conditions in which the renewable carbon source is converted to 3-dehydroshikimate by enzymes in the common pathway of aromatic amino acid biosynthesis of the cell, and the 3- dehydroshikimate is biocatalytically converted to cis,cis-muconic acid.

6. The method of claim 4 further isomerizing cis,cis-muconic acid to cis,trans-mucomc acid under reaction conditions in which substantially all of the cis,cis-muconic acid is isomerized to cis,trans-muconic acid.

7. The method of claim 5 wherein the recombinant cell is a prokaryotic cell or a yeast cell.

8. The method of claim 7 wherein the prokaryotic cell belongs to the genera Escherichia, Klebsiella, Corynebacterium, Brevibacterium, Arthrobacter, Bacillus, Pseudomonas, Streptomyces, Staphylococcus, or Serratia.

9. The method of claim 7 wherein the yeast cell belongs to the genus Saccharomyces or Schizosaccharomyces.

10. The method of claim 5, wherein the culturing step produces a broth comprising the recombinant cells and extracellular muconic acid, and further comprising the step of removing the recombinant cells from the broth.

11. The method of claim 1 wherein reducing the muconic acid to hexenedioic acid comprises reacting the muconic acid with a zinc halide.

12. The method of claim 1 wherein the unsaturated fatty acid is a Δ⁹ unsaturated fatty acid.

13. The method of claim 12 where the Δ⁹ unsaturated fatty acid is myristoleic acid, palmitoleic acid, elaidic acid, oleic acid, or combinations thereof.

14. The method of claim 1 comprising using a metathesis catalyst.

15. The method of claim 14 where the catalyst is a Grubbs catalyst.

16. The method of claim 15 where the catalyst is benzylidene- bis(tricyclohexylphosphine)dichlororuthenium or benzylidene[1,3- bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium.

17. The method of claim 1 comprising hydrogenating the dodecenedioic acid to form dodecanedioic acid.

18. The method of claim 1 further comprising using the dodecanedioic acid to form a poly amide.

19. The method of claim 18 where the polyamide is a nylon 6,12.

20. The method of claim 18 comprising reacting 1,6-hexamethylene diamine with dodecanedioic acid.

21. The method of claim 1 wherein the hexenedioic acid is an isomer of 3-hexenedioic acid.

22. The method of claim 1 wherein the renewable carbon source is D-glucose.

23. A method for producing dodecanedioic acid, the method comprising: reacting a hexenedioic acid with an unsaturated fatty acid in a metathesis reaction thereby producing dodecenedioic acid; and reducing the dodecenedioic acid to dodecanedioic acid.

24. The method of claim 23 comprising reacting the unsaturated fatty acid in a self metathesis reaction to produce Δ⁹ octadecenedioic acid before reacting the hexenedioic acid with the Δ⁹ octadecenedioic acid in a metathesis reaction.

25. The method of claim 23wherein the hexenedioic acid is an isomer of 3-hexenedioic acid and the unsaturated fatty acid is a Δ⁹ unsaturated fatty acid.

26. The method for producing dodecanedioic acid, the method comprising: providing muconic acid produced from a renewable carbon source through biocatalytic conversion; reducing the muconic acid to hexenedioic acid using a zinc halide reagent; reacting the hexenedioic acid with a Δ⁹ unsaturated fatty acid in a metathesis reaction to produce dodecenedioic acid; reducing the dodecenedioic acid to dodecanedioic acid; and forming a polyamide using the dodecanedioic acid.

27. The method of claim 26 further comprising reacting the Δ⁹ unsaturated fatty acid in a self metathesis reaction to produce Δ⁹ octadecenedioic acid and reacting the hexenedioic acid with the Δ⁹ octadecenedioic acid to produce dodecenedioic acid.

28. The method of claim 26 where the Δ⁹ unsaturated fatty acid is myristoleic acid, palmitoleic acid, elaidic acid, oleic acid, or combinations thereof.

29. The method of claim 26 comprising using a metathesis catalyst.

30. The method of claim 29 where the catalyst is benzylidene- bis(tricyclohexylphosphine)dichlororuthenium or benzylidene[1,3- bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium.

31. The method of claim 26 where the polyamide is nylon 6,12.

32. A composition comprising a biosourced dicarboxylic acid represented by the following formula HOOC-(CH₂)_n-COOH, wherein n is an integer of 4 to 22.

33. The composition of claim 32 wherein the dicarboxylic acid is a dodecanedioic acid or derivative thereof.

34. A composition of claim 32 wherein the biosourced dicarboxylic acid comprises a 1⁴C/^I2C ratio greater than 0.

35. A composition of claim 32 wherein the biosourced dicarboxylic acid comprises a 1⁴C/¹²C ratio of about 1.2 x 10^-12.

36. The composition of claim 33 wherein the dodecanedioic acid derivative is a dimethyl dodecanedioic acid.

37. A composition comprising a biosourced dicarboxylic acid diester of formula Ri -

wherein Rl and R2 are each, independently, hydrogen or an aliphatic group, wherein n is an integer of 4 to 22.

38. The composition of claim 37 wherein the dicarboxylic acid diester comprises a 1⁴C/¹²C ratio greater than 0.

39. The composition of claim 37 wherein the dicarboxylic acid diester comprises a 1⁴C/¹²C ratio of about 1.2 x 10^-12.

40. A composition comprising a biosourced dodecenedioic acid represented by the following formula

41. The composition of claim 40 wherein the dodecenedioic acid comprises a ¹⁴C/¹²C ratio greater than 0.

42. The composition of claim 40 wherein the dodecenedioic acid comprises a ¹⁴C/¹²C ratio of about 1.2 x 10^-12.

43. A composition comprising a biosourced dodecenedioic acid diester represented by the following formula

wherein Rl and R2 are each, independently, hydrogen or an aliphatic group.

44. The composition of claim 43 wherein the dodecenedioic acid diester comprises a 1⁴C/¹²C ratio greater than 0.

45. The composition of claim 43 wherein the dodecenedioic acid diester comprises a ¹⁴C¹²C ratio of about 1.2 x 10^-12.

46. A composition comprising a biosourced 3-hexenedioic or 3-hexenedioic derivative thereof, represented by the following formula R₁-OOC-CH₂-CH=CH-CH₂-COO-R₂, wherein Ri and R₂ are each, independently, hydrogen or an aliphatic group.

47. The composition of claim 46 wherein the 3-hexenedioic or 3-hexenedioic derivative thereof comprises a ¹⁴C/¹²C ratio of about 1.2 x 10^-12.

48. A composition comprising a biosourced nylon 6,12 wherein the nylon 6,12 comprises a ¹⁴C/¹²C ratio greater than O.

49. The composition of claim 48 wherein the ¹⁴C/¹²C ratio is greater than 0.9 x 10^-12.

50. A composition comprising a biosourced dicarboxylic acid represented by the following formula HOOC-(CH₂)_n-COOH, wherein n is an integer of 4 to 22 and wherein the biosourced dicarboxylic acid contains up to about 1 part per trillion of Carbon 14.

51. A composition comprising a biosourced dicarboxylic acid diester of formula Ri- OOC-(CH₃)_n-COOR₂ wherein Rl and R2 are each, independently, hydrogen or an aliphatic group, wherein n is an integer of 4 to 22 and wherein the biosourced dicarboxylic acid contains up to about 1 part per trillion of Carbon 14.

52. A composition comprising a biosourced dodecanedioic acid diester of formula Ri- OOC-(CH₂)_n-COO-R₂ wherein Rl and R2 are each, independently, hydrogen or an aliphatic group, wherein the biosourced dodecanedioic acid diester contains up to about 1 part per trillion of Carbon 14.

53. A composition comprising a biosourced 3-hexenedioic acid or 3-hexenedioic derivative thereof, represented by the following formula

wherein Ri and R₂ are each, independently, hydrogen or an aliphatic group and wherein the biosourced 3-hexenedioic acid contains up to about 1 part per trillion of Carbon 14.

54. A composition comprising a biosourced nylon 6,12 wherein the nylon 6,12 contains detectable traces of Carbon 14.

55. A dicarboxylic acid produced by a process comprising the steps of: reducing muconic acid to hexenedioic acid; reacting the hexenedioic acid with an unsaturated fatty acid in a metathesis reaction to produce an unsaturated dicarboxylic acid; and reducing the unsaturated dicarboxylic acid to form a dicarboxylic acid wherein the dicarboxylic acid is represented by the following formula )_n-

and wherein n is an integer of 4 to 22.

56. The dicarboxylic acid of claim 55 wherein the muconic acid is produced from a renewable carbon source through biocatalytic conversion.

57. The dicarboxylic acid of claim 55 wherein the unsaturated fatty acid is a Δ⁹ unsaturated C18 fatty acid and wherein the dicarboxylic acid is a dodecanedioic acid.

58. The dodecanedioic acid of claim 57 wherein the dodecanedioic acid contains up to about 1 part per trillion of Carbon 14.

59. A method for producing a dicarboxylic acid, the method comprising: reducing muconic acid to hexenedioic acid; reacting the hexenedioic acid with an unsaturated fatty acid in a metathesis reaction to produce dodecenedioic acid; and reducing the unsaturated dicarboxylic acid to form the dicarboxylic acid.

60. The method of claim 59 further comprising reacting the unsaturated fatty acid in a self metathesis reaction to produce Δ⁹ octadecenedioic acid and reacting the the hexenedioic acid with the Δ⁹ octadecenedioic acid to produce dodecenedioic acid.

61. A composition comprising a dodecenedioic acid or dodecenedioic acid derivative and at least one unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproduct derived from the dodecenedioic acid or dodecenedioic acid derivative .

62. The composition of claim 61 comprising at least two unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproducts.

63. The composition of claim 61 comprising at least nine unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproducts.

64. The composition of claim 61 wherein the at least one unsaturated dicarboxylic acid or unsaturated dicarboxylic acid derivative byproduct comprises an alkene chain having from about 7 to about 16 carbon atoms.

65. The composition of claim 61 wherein the dodecenedioic acid derivative is a dodecenedioic acid diester.

66. The composition of claim 61 wherein the dodecenedioic acid or dodecenedioic acid derivative contains up to about 1 part per trillion of Carbon 14.

67. The composition of claim 61 wherein the alkene chain comprises a carbon double bond in a position of C3-C4.

68. A composition comprising 9-octadecenedioic acid or 9-octadecenedioic acid derivative and at least one octadecenedioic acid or octadecenedioic acid derivative byproduct comprising a carbon double bond in C1-C2, C2-C3, C3-C4, C4-C5, C5-C6,, C6-C7, C7-C8, C8-C9, ClO-Cl 1, Cl 1-C12, C12-C13, C13-C14, C14- C15, C15-C16, C16-C17, or C17-C18 position and wherein the at least one byproduct derived from 9-octadecenedioic acid or 9-octadecenedioic acid derivative.

69. The composition of claim 68 comprising at least two byproducts derived from 9- octadecenedioic acid or 9-octadecenedioic acid derivative.

70. The composition of claim 68 wherein the 9-octadecenedioic acid or 9- octadecenedioic acid derivative contains up to about 1 part per trillion of Carbon 14.