Method of producing and processing diamines

Abstract

To provide a method of producing, isolating and purifying diamine.SOLUTION: There is provided a method of producing and isolating diamine produced by microbial fermentation that minimizes undesirable salt formation to provide a lower cost process.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Claim of Priority) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 211,315, filed Aug. 28, 2015; U.S. Provisional Patent Application No. 62 / 193,693, filed Jul. 17, 2015; and U.S. Provisional Patent Application No. 62 / 096,309, filed Dec. 23, 2014, the disclosures of each of which are incorporated herein by reference.

[0002] The present invention generally provides methods for producing, isolating, and purifying diamines (including hexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine, and heptamethylenediamine). More specifically, the present invention relates to methods for culturing microorganisms that produce diamines (such as HMD), and methods for isolating diamines from cultures or culture media containing diamines. Such diamine products are used to make diamine-containing polymers (including polyamides).

Background Art

[0003] Hexamethylenediamine, also known as 1,6-diaminohexane or 1,6-hexanediamine (abbreviated as HMD or HMDA), has the chemical formula H2N(CH2)6NH2. HMD is important as a raw material in the chemical industry. HMD is used, for example, in the preparation of polyamides, polyureas or polyurethanes, and copolymers of these materials. Cadaverine (also called 1,5-diaminopentane) is used as a monomer for polyamine production. Putrescine (also called 1,4-diaminobutane) is used as a monomer for polyamine production. Heptamethylenediamine (also called 1,7-diaminoheptane) is used as a monomer for polyamine production. Ethylenediamine is used as a monomer for polyamine production and as a precursor for other chemical products. Microorganisms engineered for the fermentative production of these compounds and other diamines or their direct precursors have been reported. Typically, the processes for their fermentation and isolation require acids and bases that produce salts as by-products. Summary of the Invention

[0004] Embodiments of the present invention utilize carbon dioxide (added externally or generated by metabolism) during a culture or fermentation process for producing at least one or more of diamine chemical species (diamine carbonates, diamine bicarbonates, and / or diamine bis-bicarbonates (collectively referred to herein as "carbonates"), and optionally diamine carbamates or diamine bis-carbamates (collectively referred to herein as "carbamates")). When carbonates and / or carbamates are formed, the diamine chemical species are neutralized and the pH of the fermentation is controlled. A carbon source for the growth of the microorganism and its diamine production is provided as follows. Optionally, carbon dioxide (or carbonate, bicarbonate) is a carbon source for the microorganism (through CO2 fixation) and also a compound for neutralizing the diamine. The diamine can be, for example, hexamethylenediamine (HMD), dimethylenediamine, trimethylenediamine, cadaverine, putrescine, or heptamethylenediamine (a diamine having 2 to 7 carbon atoms (C2 to C7), C3 to C7, preferably C4 to C7 or C4 to C12 or C2 to C12). Thus, the diamine chemical species are, for example, in the case of HMD, HMD carbonate, HMD bicarbonate, HMD bis-bicarbonate. The carbamate and bis-carbamate are, for example, in the case of HMD, HMD carbamate and HMD bis-carbamate. Chemical formulas for HMD chemical species are shown below.

[0005]

Table 1

[0006] In one embodiment, the present invention provides an improved isolation of carbonates and / or carbamates from a culture or fermentation medium, solution or broth, which comprises carbonates and carbamates of HMD, cadaverine, putrescine and heptamethylenediamine. In another embodiment, the carbonates and carbamates are treated such that carbon dioxide and diamine free bases (e.g., HMD free base, cadaverine free base, putrescine free base, heptamethylenediamine free base) are released, and then the diamine free bases can be extracted with a suitable organic solvent. It has been found that HMD carbonates and HMD carbamates (such as HMD carbonate, bicarbonate, bis-bicarbonate, as well as carbamate and bis-carbamate) generated during pseudo-fermentation conditions release CO2 or other fragments and generate HMD free base (which is then solvent extracted). If necessary, the diamine free base-rich fraction is subjected to further purification processes.

[0007] In another embodiment, the present invention a) culturing a genetically modified microorganism in a medium under suitable conditions for a sufficient period of time to form in the medium one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate (carbonates) and / or DA carbamate or DA bis-carbamate (carbamates), wherein carbon dioxide, carbonate, bicarbonate or carbonic acid mainly controls the pH of the medium as the cultured medium; b) converting the DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate to HMD free base and carbon dioxide; and c) isolating the DA free base: to provide a method for producing a diamine (DA).

[0008] In another embodiment, the present invention a) Culturing a genetically modified microorganism in a medium under appropriate conditions for a sufficient period of time to form one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate, wherein the dissolved inorganic carbon percentage (DIC) is determined by the formula: DIC / TDCA×100 (TDCA is the total dissolved counter anion, which is the sum of DIC and other anions), and the DIC% is 40% or more; b) Converting the above DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate to DA free base and carbon dioxide; and c) Isolating the above DA free base: A method for producing diamine (DA) is provided, which includes the above steps.

[0009] In another embodiment, the present invention provides a) Culturing a genetically modified microorganism in a medium under appropriate conditions for a sufficient period of time to produce diamine and form one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate (DA carbonates), and / or DA carbamate or DA bis-carbamate (DA carbamates) in the medium, wherein at least 40% of the carbonates or carbamates in the medium contains one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate; b) Converting the above carbonates or carbamates to DA free base and carbon dioxide; and c) Isolating the above DA free base: A method for producing diamine (DA) is provided, which includes the above steps.

[0010] In some embodiments, an enzyme carbonic anhydrase (CA) is added to the fermentation broth to increase the amount or proportion of gaseous CO2 converted to soluble ions (thereby providing a greater amount or availability of soluble ions available to the diamine or HMD), thereby catalyzing or increasing the formation of diamine carbonates and / or diamine carbamates (e.g., HMDA carbonates). CA can also be used to increase the formation of diamine carbonates (including carbonates, bicarbonates or bis-bicarbonates) and diamine carbamates (including carbamates or bis-carbamates) or any mixture thereof when the diamine contains a C2-C7 methylene segment, a C2-C12 methylene segment, or a C4-C7 methylene segment (which can be, for example, hexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine or heptamethylenediamine). In some embodiments, carbonic anhydrase is used to form one or more of HMD carbonate, HMD bicarbonate, HMD bis-bicarbonate, HMD carbamate or HMD bis-carbamate. Carbonic anhydrase is a reversible enzyme and thus, in other embodiments, is used to catalyze the conversion of DA carbonates or DA carbamates to the DA free base and carbon dioxide.

[0011] In some embodiments, carbonic anhydrase is present in an amount sufficient to (a) promote the formation of DA carbonates or DA carbamates by converting carbon dioxide to bicarbonate and / or carbonate ions, (b) promote the release of carbon dioxide from a solution of DA carbonates or DA carbamates by converting bicarbonate and / or carbonate ions to carbon dioxide, or (c) do both (a) and (b).

[0012] CA may be added exogenously or may be produced by a genetically modified microorganism. In some embodiments, CA is part of a microorganism that expresses a DA synthesis pathway, such as the HMD synthesis pathway. In other embodiments, CA is introduced as a recombinant microorganism having the ability to produce CA.

[0013] CA or a variant is expressed in an amount sufficient to facilitate either the desired conversion of CO2 to ions, or ions to CO2, or both (which can be compared to the conversion(s) in the absence of CA or the variant). The amount of the CA or variant protein can depend on its carbonic anhydrase activity and the desired facilitation. Typical amounts can range from at least 0.001 g / L to at least 5 g / L, and can be from at least 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L or 1 g / L to at least 5 g / L, and can be, for example, 0.05 to 0.2 g / L.

[0014] Alternative embodiments are methods in which at least 50% of the carbonates and / or carbamates in the medium comprise one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate (e.g., HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDA carbamate or HMDA bis-carbamate), or at least 60% of the carbonates and / or carbamates in the medium comprise one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate (e.g., HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDA carbamate or HMDA bis-carbamate), or at least 70% of the carbonates and / or carbamates in the medium comprise one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate (e.g., HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDA carbamate or HMDA bis-carbamate), or at least 80% of the carbonates and / or carbamates in the medium comprise one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate (e.g., HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDA carbamate or HMDA bis-carbamate), or at least 90% of the carbonates and / or carbamates in the medium comprise one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate (e.g., HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDA carbamate or HMDA bis-carbamate), or at least 99.9% of the carbonates and / or carbamates in the medium comprise one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate (e.g., HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDA carbamate or HMDA bis-carbamate).In some embodiments, the carbonates are the dominant diamine species and can contain at least 50% to at least 90% of the DA species.

[0015] In some embodiments, the genetically modified microorganism further forms one or more of carbon dioxide, carbonate, bicarbonate, or carbonic acid. The carbon dioxide, carbonate, bicarbonate, or carbonic acid formed by the genetically modified microorganism can include stoichiometric carbon dioxide from the formation of carbonates and / or carbamates, or the carbon dioxide, carbonate, bicarbonate, or carbonic acid formed by the genetically modified microorganism can include respiratory carbon dioxide or byproduct carbon dioxide. In certain embodiments, the respiratory carbon dioxide is formed from at least one pathway selected from, for example, via completion of the TCA cycle, via the glyoxylate pathway, via the pentose phosphate pathway (e.g., gnd (6-phosphogluconate dehydrogenase that converts 6-phosphogluconate to ribulose-5-phosphate and CO2)), or via the Entner-Doudoroff pathway. In other embodiments, the byproduct carbon dioxide is associated with the formation of byproducts including acetone, ethanol, succinate, 3-oxoadipate, and 3-hydroxyadipate.

[0016] In some embodiments, a genetically engineered microorganism that comprises a diamine synthesis pathway and optionally produces CO2 may further comprise a CA enzyme (in particular, when the microorganism has a nucleic acid sequence capable of expressing CA), as described herein. Thus, a recombinant microorganism comprising a synthetic pathway for producing a diamine comprising a C2-C7 methylene segment, a C2-C12 methylene segment, or a C4-C7 methylene segment (e.g., the diamine is hexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine or heptamethylenediamine) may further comprise a CA enzyme (in particular, when the microorganism has been engineered to have a nucleic acid sequence capable of expressing CA). In some embodiments, the genetically engineered microorganism comprises a hexamethylenediamine synthesis pathway and a sequence capable of expressing CA. The CA can be natural or genetically engineered (such as increased activity or stability (including thermal stability and alkaline pH stability)). Preferably, the alkaline pH is about pH 8-13, pH 8.5-13, pH 9-13, pH 10-13, pH 8-12, pH 8.5-12, pH 9-12, pH 8-11, pH 8.5-11, pH 9-11, pH 10-11 and pH 10-12.

[0017] In some embodiments, the genetically engineered microorganism forms carbon dioxide and hexamethylenediamine in a ratio of about 0.05:1 to about 7:1. In other embodiments, the genetically engineered microorganism forms carbon dioxide and hexamethylenediamine in a ratio of about 0.05:1 to about 5:1, in a ratio of about 0.05:1 to about 3.5:1, in a ratio of about 0.05:1 to about 3:1, in a ratio of about 0.05:1 to about 2:1, in a ratio of about 0.05:1 to about 1.5:1, in a ratio of about 0.05:1 to about 1:1, or in a ratio of about 0.2:1 to about 3:1.

[0018] In some embodiments, the genetically engineered microorganism comprises an HMD synthesis pathway together with at least one exogenous nucleic acid encoding at least one enzyme of the HMD synthesis pathway, which is expressed in an amount sufficient to produce at least one HMD carbonate and / or carbamate compound. In yet other embodiments, the genetically engineered microorganism comprises an HMD synthesis pathway together with at least two, three, four, five, six, seven, eight, nine, ten or eleven exogenous nucleic acids encoding at least two, three, four, five, six, seven, eight, nine, ten or eleven enzymes of the HMD synthesis pathway, which are expressed in an amount sufficient to produce at least one HMD carbonate and / or carbamate compound.

[0019] In some embodiments, the HMD synthesis pathway comprises an intermediate compound selected from the group consisting of 3-oxoadipyl-CoA, adipic acid semialdehyde, 6-aminocaproate (6-ACA), 6-ACA semialdehyde, 2-aminopimelate, 3,6-dihydroxyhexanoyl-CoA, and homolyseine.

[0020] In some embodiments, the HMD synthesis pathway comprises an enzyme selected from the group consisting of 3-oxoadipyl-CoA thiolase, 6-ACA transaminase or dehydrogenase, 6-aminocaproy-CoA reductase, 6-ACA reductase, adipyl-CoA reductase, adipic acid reductase, 6-hydroxy-3-oxohexanoyl-CoA dehydrogenase, 2-aminopimelate decarboxylase, and homolyseine decarboxylase.

[0021] In some embodiments, the HMD synthesis pathway comprises an enzyme and a substrate-product pair selected from the group consisting of 3-oxoadipyl-CoA thiolase, which acts on succinyl-CoA and acetyl-CoA to form 3-oxoadipyl-CoA; 6-ACA transaminase, which acts on adipyl-CoA to form 6-ACA; 6-aminocaproyl-CoA reductase, which acts on 6-aminocaproyl-CoA to form 6-ACA semialdehyde; 6-ACA reductase, which acts on 6-ACA to directly convert it to 6-ACA semialdehyde; adipyl-CoA reductase, which acts on adipyl-CoA to form adipic semialdehyde; adipate reductase, which acts on adipate to directly convert it to adipic semialdehyde; 6-hydroxy-3-oxohexanoyl-CoA dehydrogenase, which reduces 6-hydroxy-3-oxohexanoyl-CoA to form 3,6-dihydroxyhexanoyl-CoA; 2-aminopimelate decarboxylase, which decarboxylates 2-aminopimelate to form 6-ACA; and homoly sine decarboxylase, which decarboxylates homoly sine to form HMD.

[0022] In some embodiments, the HMD synthesis pathway is selected from the group consisting of pathways (a) through (m): (a) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, and 6-ACA-CoA reductase, or 6-ACA reductase, HMDA transaminase or dehydrogenase; (b) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (c) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA transferase, hydrolase or transferase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (d) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA transferase, hydrolase or transferase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (e) 3-oxoadipyl-CoA thiolase, 3-oxoadipic acid dehydrogenase, 3-hydroxyadipic acid dehydratase, 5-carboxy-2-pentenoic acid reductase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (f) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (g) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (h) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (i) 4-hydroxy-2-oxoheptane-1,7-dioate (HODH aldolase); 2-oxohepta-4-ene-1,7-dioate (OHED) hydratase; OHED formate lyase and pyruvate formate-lyase activating enzyme or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; or adipic semialdehyde aminotransferase or adipic semialdehyde oxidoreductase (amination); (j) β-ketothiolase or acetyl-CoA carboxylase and acetoacetyl-CoA synthetase, 3-hydroxyacyl CoA dehydrogenase or 3-oxoacyl-CoA reductase, enoyl-CoA hydratase, and trans-2-enoyl-CoA reductase for generating hexanoyl-CoA, one or more thioesterases, aldehyde dehydrogenase, or butanal dehydrogenase, hexanal or hexanoates produced by the host; one or more monooxygenases, alcohol dehydrogenase, aldehyde dehydrogenase, 6-hydroxyhexanoate dehydrogenase, 5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase, 6-oxohexanoate dehydrogenase, or 7-oxoheptanoate dehydrogenase, adipic acid or adipic acid semialdehyde produced by the host; one or more monooxygenases, transaminase, 6-hydroxyhexanoate dehydrogenase, 5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase, and alcohol dehydrogenase, 6-aminohexanoate produced by the host; one or more carboxylate reductase, ω-transaminase, deacetylase, N-acetyltransferase, alcohol dehydrogenase, hexamethylenediamine produced by the host; (k) acetyltransferase or thiolase for forming 6-hydroxy-3-oxo-hexanoyl-CoA, 6-hydroxy-3-oxo-hexanoyl-CoA dehydrogenase, 3,4-dihydroxyhexanoyl-CoA dehydrogenase, 6-hydroxy-2-hexanoyl-CoA reductase, 6-hydroxyhexanoyl-CoA hydrolase for forming 6-ACA, 6-hydroxycaproic acid dehydrogenase and transaminase for forming HMDA; (l) Homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase for forming 2-ketopimelate, 2-ketodecarboxylase that catalyzes the conversion of α-ketopimelate to adipic semialdehyde, 2-aminopimelate transferase that catalyzes the conversion of α-ketopimelate to 2-aminopimelate, 2-aminopimelate decarboxylase for decarboxylating 2-aminopimelate to form 6-ACA, aldehyde dehydrogenase that catalyzes the conversion of 6-ACA to 6-aminohexanal and aminotransferase that catalyzes the conversion of 6-aminohexanal to 6-hexamethylenediamine; and (m) Glutamyl-CoA transferase and / or ligase, β-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA reductase, 6-amino-7-carboxyhepta-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde forming), 2-amino-7-oxoheptanoate aminotransferase and / or aminating oxidoreductase, homolysine decarboxylase, 6-aminopimeloyl-CoA hydrolase, transferase and / or ligase, 2-aminopimelate decarboxylase.

[0023] In any embodiment of the above alternative pathways, suitable enzymes may be selected from the group consisting of 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 3-oxoadipyl-CoA: acyl-CoA transferase, 3-oxoadipic acid dehydrogenase, 3-hydroxyadipic acid dehydratase, 5-carboxy-2-pentanoate reductase, adipyl-CoA transferase, ligase or hydrolase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, or 6-ACA reductase.

[0024] In some embodiments, the genetically modified microorganisms include, for the genus Escherichia, Klebsiella; for the family Succinivibrionaceae of the order Aeromonadales including the genus Anaerobiospirillum; for the family Pasteurellaceae of the order Pasteurellales including the genera Actinobacillus and Mannheimia; for the family Bradyrhizobiaceae of the order Rhizobiales including the genus Rhizobium; for the family Bacillaceae of the order Bacillales including the genus Bacillus; for the family Corynebacteriaceae and Streptomycetaceae of the order Actinomycetales each including the genus Corynebacterium and Streptomyces, respectively; for the family Acetobacteraceae of the order Rhodospirillales including the genus Gluconobacter; for the family Sphingomonadaceae of the order Sphingomonadales including the genus Zymomonas; for the family Lactobacillaceae and Streptococcaceae of the order Lactobacillales each including the genus Lactobacillus and Lactococcus, respectively; for the family Clostridiaceae of the order Clostridiales including the genus Clostridium; for the family Pseudomonadaceae of the order Pseudomonadales including the genera Pseudomonas, Alkaliphilus, Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis and Hyphomicrobium; for the family Saccaromycetaceae of the order Saccharomycetales including the genera Saccharomyces, Kluyveromyces and Pichia; for the family Dipodascaceae of the order Saccharomycetales including the genus Yarrowia; for the family Schizosaccaromycetaceae of the order Schizosaccharomycetales including the genus Schizosaccharomyces; for the family Trichocomaceae of the order Eurotiales including the genus Aspergillus;and the Mucoraceae family of the order Mucorales including the genus Rhizopus are mentioned;

[0025] In some embodiments, the genetically modified microorganisms include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida, Bacillis pseudofirmus, Bacillus halodurans, Bacillus alcalophilus, Clostridium paradoxum, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and Issatchenkia orientalis.

[0026] Some embodiments of the alkaliphilic bacteria are Bacillis pseudofirmus, Bacillus halodurans, Bacillus alcalophilus, Clostridium paradoxum, Arthrospira platensis, Bacillus clausii, Oceanobacillus iheyensis, Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Bacillus selentireducens, Desulfovibrio alkaliphiles, Dethiobacter alkaliphiles, Thioalkalivibrio sp., Natranaerobius thermophilus, Alkalilimnicola ehrlichii, and Desulfonatronospira thiodismutans.

[0027] In some embodiments, the culture medium fermentation may be substantially free of buffer, may be substantially free of inorganic or organic acids, may be substantially free of externally added inorganic or organic acids, or may be substantially free of DIC.

[0028] In some embodiments, the pH of the medium and / or the cultured medium is controlled by the amount of carbon dioxide added to the cultured medium, or the pH of the cultured medium is controlled by the amount of carbon dioxide formed by the genetically modified microorganism. In one embodiment, the medium has a pH of less than 11, less than 10, less than 9 or less than 8, and / or the cultured medium is controlled to a pH of less than 11, less than 10, less than 9 or less than 8. In other embodiments, the medium has a pH of at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7, and / or the cultured medium is controlled to a pH of at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7. In yet other embodiments, the medium has a pH of about 6 to 9.5, about 6 to 9, about 6 to 8, about 7 to 9 or about 8 to 9, and / or the cultured medium is controlled to a pH of about 6 to 9.5, about 6 to 9, about 6 to 8, about 7 to 9 or about 8 to 9.

[0029] In yet other embodiments, the medium comprises a sugar carbon source for the genetically modified microorganism selected from the group consisting of sucrose, glucose, galactose, fructose, starch, mannose, isomaltose, xylose, panose, maltose, arabinose, cellobiose, and their 3-, 4- or 5-oligomers, or the medium comprises an alcohol carbon source for the genetically modified microorganism selected from the group consisting of methanol, ethanol, glycerol, formate, and fatty acids, or the medium comprises a carbon source obtained from a gas for the genetically modified microorganism selected from the group consisting of synthesis gas, waste gas, methane, CO, CO2, and any mixture of CO or CO2 and H2.

[0030] In some embodiments, the carbonates and / or carbamates are converted to a free base (e.g., hexamethylenediamine free base) by producing carbon dioxide. In certain embodiments, the carbonates and / or carbamates are converted to the free base by heat, or the carbonates and / or carbamates are converted to the free base by reduced pressure, or the carbonates and / or carbamates are converted to the free base by pressure, or the carbonates and / or carbamates are converted to the free base by ion exchange, or the carbonates and / or carbamates are converted to the free base by steam stripping, or the carbonates and / or carbamates are converted to the free base by electrodialysis using a bipolar membrane. In yet other embodiments, the conversion to the free base is accelerated or facilitated by the addition of a carbonic anhydrase enzyme. Carbonic anhydrase can also be used to accelerate or facilitate the release of the free base when heat or other processes are used to convert DA carbonates and / or DA carbamates to free DA and carbon dioxide.

[0031] In some embodiments, the diamine free base (e.g., HMD) is isolated from the culture medium using an extraction solvent, and the extracted diamine is separated from the extraction solvent by distillation. In certain embodiments, the extraction solvent is selected from the group consisting of alcohols, amines, ethers, and ketones. Suitable extraction solvents include C4 - C8 monohydric alcohols (such as butanol, hexanal, 1 - hexanol, isopentanol, or cyclohexanol), or toluene or ethyl ether or mixtures thereof. Alkanes are suitable solvents as demonstrated in the examples, especially for the HMD free base. Alkanes (especially hexane) were screened and subsequently tested due to their very low water solubility. Hexane hardly extracts even in the presence of water and gave an acceptable recovery rate of the available free base. Thus, alkanes are suitable solvents for use in the recovery of diamine free bases. Suitable alkanes include C5 - C12 straight - chain or branched alkanes. In one embodiment, both the extracted diamine and the alkane selected as the solvent can have the same number of carbon atoms. Heptane is another suitable alkane particularly suitable for HMD and is further supported by the in silico modeling studies described below. Isomers of hexane and heptane are suitable. Isomers of hexane are 2 - methylpentane, 3 - methylpentane, 2,2 - dimethylbutane, and 2,3 - dimethylbutane. Isomers of heptane are 2 - methylhexane, 3 - methylhexane, 2,2 - dimethylpentane, 2,3 - dimethylpentane, 2,4 - dimethylpentane, 3,3 - dimethylpentane, 3 - ethylpentane, and 2,2,3 - trimethylbutane.

[0032] In some embodiments, the genetically engineered microorganism is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Bacillis pseudofirmus, Bacillus halodurans, Bacillus alcalophilus, Clostridium paradoxum, Saccharomyces cerevisiae. In other embodiments, the genetically engineered microorganism is modified for improved alkali resistance.

[0033] In some embodiments, the DA (e.g., HMD) produced by the present invention contains impurities of one or more DA carbonates, DA bicarbonates, DA bis-bicarbonates or DA carbamates.

[0034] In some embodiments, a polymer (e.g., a polyamide for PA66) containing a diamine (e.g., HMD) produced by the present invention contains one or more of DA carbonate, DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA bis-carbamate (e.g., HMD carbonate, HMD bicarbonate, HMD bis-bicarbonate, HMD carbamate or HMD bis-carbamate) as impurities.

[0035] Another embodiment of the present invention can be a genetically modified microorganism comprising at least one exogenous nucleic acid encoding at least one enzyme of a diamine synthesis pathway (e.g., an HMD synthesis pathway), and at least one genetic modification that promotes or increases CO2 utilization ability, wherein the genetic modification increases the production of diamine carbonates and / or carbamates (e.g., HMD carbonates and / or carbamates) as compared to a genetically modified microorganism without the genetic modification, together with a diamine synthesis pathway (e.g., a hexamethylenediamine synthesis pathway). In one embodiment, a genetically modified microorganism comprising a diamine (e.g., HMD) synthesis pathway together with at least one exogenous nucleic acid encoding at least one enzyme of the diamine (e.g., HMD) synthesis pathway and at least one carbonic anhydrase enzyme is used to increase the production of diamine (e.g., HMD) carbonate and / or carbamate as compared to a genetically modified microorganism without a CA enzyme. A microorganism expressing CA may further comprise at least one genetic modification that increases CO2 utilization ability.

[0036] In any embodiment of the present invention, the released carbon dioxide, extraction solvent and / or water can be recycled. In other embodiments, CA can be recycled.

[0037] In addition to the culturing, conversion, and isolation process steps described in the above embodiments, the present invention also includes alternative and optional process steps. In some embodiments, the cultured medium or solution can be treated to remove solids and water either before isolating the DA free base and / or before converting carbonates and / or carbamates to the DA free base during the method. In other embodiments, the cultured medium can be treated to remove water, preferably before isolating the DA free base. In yet other embodiments, the DA free base can be distilled directly from the cultured medium or solution. In still other embodiments, after the extraction solvent is removed by distillation, the DA free base is further treated and / or purified. In still other embodiments, the removal or reduction of water and the conversion of carbonates and / or carbamates to the DA free base (e.g., the HMD free base) occur simultaneously and / or sequentially during the same unit operation. For example, in the presence of a stripper unit (e.g., an inert gas or steam), it can be used to remove or reduce both water and CO2 to produce the free base. In a further example, in the presence of a water evaporation unit, it can be used to remove or reduce both water and CO2 to produce the free base. CA may be present during the fermentation for the formation of diamine carbonates and / or carbamates, during the process for the release of CO2, or in either or both of these processes. In some embodiments, CA can be recycled. These alternative and / or optional process steps are described in detail below.

Brief Description of the Drawings

[0038]

Figure 1

Figure 2

Figure 3

Figure 4

[0039] 3: Removal of solids 4: Conversion of carbonate / carbamate to carbon dioxide and free bases. Carbon dioxide is optionally recycled to the fermenter.

[0040] 5: Removal of water. Water is optionally recycled to the production fermentation step.

[0041] 6: Solvent extraction. The aqueous raffinate is optionally recycled to the carbonate / carbamate conversion step.

[0042] 7: Purification: including distillation to return the organic solvent to box 6 for recycling; may include more distillation columns for purifying HMD and may also include other steps for removing compounds that form color, etc.

[0043] 8: Purified HMD 9: Any sterilization that does not release carbon dioxide.

[0044] 10: Optional removal of water. Water is optionally recycled to the production fermentation step. If the water removal step includes a state where carbon dioxide and free bases can be released, carbon dioxide can also be recycled.

[0045] 11: Any direct purification from the aqueous phase, with or without the release of carbon dioxide. This may include distillation, ion exchange, electrodialysis, etc. If water and carbon dioxide are produced in these steps, they can be recycled.

[0046] 12: Alkalization (NaOH or CaOH) or other processes (ion exchange, electrodialysis, etc.) to remove carbonate from HMD. If CaOH is used, precipitation occurs.

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[0047] Disclosed is a method for the production and isolation of diamines. In this context, "diamine" shall include those containing C2-C7 methylene segments such as hexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine, and heptamethylenediamine. The number of carbon atoms that a diamine can have is C2-C7, C3-C7, preferably C4-C7, more preferably C4-C12 or C2-C12. For ease of reading, HMD may be described in detail, but it should be understood that the disclosed method is applicable to any of the diamines such as cadaverine, putrescine, ethylenediamine, and heptamethylenediamine.

[0048] Hexamethylenediamine (abbreviated as HMD), also known as 1,6-diaminohexane or 1,6-hexanediamine, can be represented by the chemical formula H2N(CH2)6NH2. HMD is an important raw material in the chemical industry. For example, HMD is used as a raw material for polyamides, polyureas or polyurethanes, as well as copolymers based on these.

[0049] Cadabaline, also known as 1,5-diaminopentane, is used as a monomer for polyamine synthesis. Engineered microorganisms suitable for cadabaline fermentation production have been reported. For example, methods for producing and recovering biologically derived amines (such as cadabaline) have been reported as follows: U.S. Patent No. 8906653; Kind et al., “From zero to hero - Production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum,” (Metabolic Engineering, 25 (2014) pp. 113-123); Kind et al., “Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane,” (Metabolic Engineering 12 (2010) 341-351). These reported methods involve the active neutralization of the fermentation broth or the cultured medium with inorganic salts (such as sulfuric acid). After fermentation, the cultured medium or broth is alkalized using a strong base for deprotonation of the amine. Then, the amine is extracted with an organic solvent and subsequently obtained by distillation. In these methods, a large amount of unnecessary salt by-products are generated along with the amine.

[0050] In another method, as reported in International Publication No. WO2006 / 123778, lysine carbonate prepared in vitro is enzymatically decarboxylated with the addition of a dicarboxylate to maintain a pH suitable for the decarboxylation reaction to produce cadaverine carbonate, and cadaverine and cadaverine-dicarboxylate are produced by concentration. In yet another method, as reported in International Publication No. WO2010 / 002000, lysine carbonate prepared in vitro is enzymatically decarboxylated for the purpose of producing cadaverine carbonate, and cadaverine is obtained from the prepared cadaverine carbonate by heat treatment and subsequent distillation.

[0051] Putrescine, also known as 1,4-diaminobutane, is used as a monomer for polyamine synthesis. Engineered microorganisms suitable for putrescine fermentation production have been reported. For example, Schneider et al. “Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addition system,” (Appl Microbiol Biotechnol. 2012; 95(1):169-78); and U.S. Patent A1-20140004577 “Microorganisms for producing putrescine and method for producing putrescine using same.”

[0052] Heptamethylenediamine, also known as 1,7-diaminopentane, is used as a monomer for polyamine synthesis. Engineered microorganisms suitable for putrescine fermentative production have been reported. For example, International Publication No. WO2014 / 105790A2 “Methods of producing 7-carbon chemicals via c1 carbon chain elongation associated with coenzyme b synthesis.”

[0053] Ethylenediamine is used as a monomer for polyamine synthesis, similar to being a precursor for other chemicals. Engineered microorganisms suitable for ethylenediamine fermentative production have been reported. For example, International Publication No. WO2014 / 049382A2 “Ethylenediamine fermentative production by a recombinant microorganism.”

[0054] Hexamethylenediamine (abbreviated as HMD), also known as 1,6-diaminohexane or 1,6-hexanediamine, can be represented by the chemical formula H2N(CH2)6NH2. HMD is an important raw material in the chemical industry. For example, HMD is used as polyamide, polyurethane or copolymers based on these. Cadaverine, also known as 1,5-diaminopentane, is used as a monomer for polyamine synthesis. Putrescine, also known as 1,4-diaminobutane, is used as a monomer for polyamine synthesis. Heptamethylenediamine, also known as 1,7-diaminopentane, is used as a monomer for polyamine synthesis. Ethylenediamine is used as a monomer for polyamine synthesis, similar to being a precursor for other chemicals. Engineered microorganisms that fermentatively produce these compounds and other diamines or their immediate precursors have been reported. Generally, in these fermentation and separation processes, acids and bases are required, and the acids and bases produce salts of by-products.

[0055] During the fermentation process, this utilizes carbon dioxide, but ultimately produces one or more diamine carbonates, diamine bicarbonates, and / or diamine bis-bicarbonates (collectively referred to herein as "diamine carbonates"), and optionally diamine carbamates or diamine bis-carbamates (collectively referred to herein as "carbamates"). The disclosed method further provides an increased yield of diamine and an improvement in the purification of the desired diamine using organic solvent-based extraction.

[0056] Figure 1 is a schematic diagram of an embodiment according to the present invention, including the steps of culturing microorganisms; producing one or more HMD carbonates and / or HMD carbamates using the cultured microorganisms; producing a neutral charged or free base of HMD having the chemical formula H2N-(CH2)6-NH2 and having higher solubility in a hydrophobic organic solvent compared to salts of HMD or HMD carbonates and / or HMD carbamates; and isolating the neutral charged or free base of HMD.

[0057] Figure 2 is a schematic diagram of another embodiment according to the present invention, including the steps of preparing a source of HMD biosynthesis mixed with impurities; producing one or more HMD carbonates and / or carbamates, which are charged compounds, in the presence of CO2; isolating or separating HMD carbonates and / or carbamates from unwanted by-products or substances; producing a compound of a neutral charged or free base of HMD having the chemical formula H2N(CH2)6NH2 and having higher solubility in a hydrophobic organic solvent compared to the charged salts of HMD or HMD carbonates and / or HMD carbamates; and isolating the neutral charged or free base of HMD.

[0058] Figure 3 is a schematic diagram of one embodiment of a fermentation system that can be used in the production of HMD. The genetically modified microorganism is cultured or grown in a suitable culture or fermentation medium containing a nitrogen source and a carbohydrate source in reaction vessel 10. In one embodiment according to the present invention, typical culture or fermentation media and growth conditions are shown below in Example 3. During the fermentation of the genetically modified microorganism to produce the desired diamine (e.g., HMD), carbon dioxide is used to control the pH of the cultured medium. The carbon dioxide may be metabolically produced by the microorganism, or may be artificially produced, or added from an external source. In one embodiment, the growth state of the microorganism and the concentration of CO2 in the culture or fermentation medium are controlled such that the pH is maintained at a predetermined level for a selected period during the fermentation cycle. Throughout the fermentation process, the pH will rise from near neutral to a stable pH of 8.5, which is due, for example, to the buffer created by HMD and carbon dioxide. When fermentation is complete, or when HMD-carbonates and / or carbamates are produced, the cells can be removed (e.g., by membrane filtration 20), and the unpurified or impurity-laden aqueous solution containing the raw material of charged HMD-carbonates and / or carbamates, e.g., the salt of HMD, is separated from the unwanted by-products that would be contained in the concentrated water. At least after cell removal, the aqueous fermentation solution containing at least the raw material of charged HMD-carbonates and / or carbamates can be decarbonated by water vapor via inert gas or steam stripping. The water vapor can be added from an external source, or can be produced in situ by the apparatus 30 by boiling the broth under high temperature and high pressure. Removal of CO2 will produce HMD that is neutrally charged or in free base form in the aqueous solution. By removal of carbon dioxide, the pH of the solution rises and solvent extraction can be used to remove HMD in free base form. Neutrally charged or free base HMD can be extracted from the aqueous solution by an extractor 40 that separates the organic components from the water-soluble raffinate.The organic components and HMD containing at least a solvent can be separated by a distillation apparatus, and the distilled HMD is further purified by being distilled by the distillation apparatus 60. The CO2 returned from the apparatus 30 and the solvent returned from the distillation apparatus can be recycled in the above-mentioned process, improving the process and economic characteristics of the illustrated fermentation process.

[0059] Culturing the microorganism under suitable conditions and for a sufficient period of time results in the formation of one or more diamine carbonates and / or carbamates. The produced compounds in the cultured medium contain at least 40% carbonates and / or carbamates in the cultured medium. In other embodiments, the carbonates and / or carbamates can be included in the cultured medium at least 50%, 60%, 70%, 80%, 90% or 99.9%. According to the above definitions, this does not mean that the carbonates or carbamates of the desired diamine (e.g., HMD) contain at least 40% or more of all the carbonates and / or carbamates in the cultured medium.

[0060] [Culture medium] An appropriate culture medium will be used depending on the desired microorganism or strain employed. For example, various culture media can be found in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). In this book, when referring to the growth source, "medium" refers to the starting medium in solid or liquid form. On the other hand, in this book, "cultured medium" refers to a medium (e.g., liquid medium) containing fermentable growing microorganisms and possibly other cellular biomass. The medium generally contains one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and / or trace elements.

[0061] Specific examples of carbon sources include sugars such as sucrose, glucose, galactose, fructose, mannose, isomaltose, xylose, pannose, maltose, arabinose, cellobiose, and their 3-, 4-, or 5-oligomers. Other carbon sources include alcohol carbon sources such as methanol, ethanol, glycerol, formate, and fatty acids. Still other carbon sources include gas-derived carbon sources such as synthesis gas, exhaust gas, methane, CO, CO2, and mixtures of CO or CO2 and H2. Other carbon sources may include recycled feedstocks and biomass. Specific examples of recycled feedstocks include cellulose-derived biomass, hemicellulose-derived biomass, and lignin feedstocks.

[0062] In some embodiments, the state of the medium includes anaerobic or substantially anaerobic growth, or a maintenance state. The anaerobic state is specifically as described above, which is well known to those skilled in the art. The anaerobic state of a specific fermentation process is disclosed in US Patent Application Publication No. 2009 / 0047719 (filed on August 10, 2007). Any of these states is used in association with microorganisms in the same manner as other anaerobic states well known to those skilled in the art.

[0063] The state of the medium includes, for example, the procedure of liquid culture, and the procedure is similar to fermentation and other large-scale culture procedures. As described herein, particularly beneficial production yields are obtained in anaerobic or substantially anaerobic culture states.

[0064] Specific growth conditions for achieving the objective include hexamethylenediamine in an anaerobic culture or fermentation state. In one embodiment, the microorganism is nourished, cultured, or fermented in an anaerobic or substantially anaerobic state. In short, the anaerobic state refers to an environment without any oxygen. A substantially anaerobic state includes, for example, culturing, fed-batch fermentation, or continuous fermentation where the dissolved oxygen concentration in the medium is between 0 and 10% of the saturation state. A substantially anaerobic state also includes growing or stationary cells in a liquid medium or on solid agar in a sealed chamber maintained by an atmosphere with less than 1% oxygen. The oxygen ratio is maintained, for example, by sparging the medium with a mixture of N2 and CO2 or other suitable oxygen-free gas.

[0065] The state of the medium can be scaled up and continuously grown for the production of hexamethylenediamine. Specific growth procedures include, for example, fed-batch culture and batch separation, fed-batch culture and continuous separation, or continuous culture and continuous separation. All of these processes are well known to those skilled in the art. The fermentation process is particularly beneficial for the production of large quantities of hexamethylenediamine. Generally, and as a non-continuous culture procedure, the production of hexamethylenediamine in a continuous and / or near-continuous manner involves culturing hexamethylenediamine such that, during the exponential growth phase, sufficient nutrients for the organism and medium are produced to support growth by nutrient replenishment and / or near it. Continuous culture in such a state can include, for example, 1 day, 2, 3, 4, 5, 6, or 7 days or more. Additionally, continuous culture can include up to 1 week, 2, 3, 4, or 5 or more weeks and months. Also, when suitable for a particular application, the desired microorganism is cultured for several hours. It should be understood that continuous and / or near-continuous culture states can also include all of these exemplified periods. It is further understood that the time for culturing the microorganism is sufficient to produce a sufficient amount of product for the desired purpose.

[0066] The cultivation procedures are well-known in this field. Briefly, the procedures for the biosynthesis of hexamethylenediamine can be utilized, for example, in fed-batch culture and batch separation; fed-batch culture and continuous separation, or continuous culture and continuous immediate separation. The procedures for fed-batch culture and continuous culture are well-known in this field.

[0067] The culture medium at the start of cultivation has a pH of about 5 to 7. The pH can be 11 or less, 10 or less, 8 or less, 8 or less. In other embodiments, the pH can be at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7. In still other embodiments, the pH of the medium can be about 6 to 9.5, about 6 to 9, about 6 to 8, or about 8 to 9.

[0068] [CO2 Source and Seed] As described above, CO2 is added to produce the desired diamine (e.g., HMD) and to adjust the pH of the culture medium. The source of CO2 can be, for example, CO2, carbonate, bicarbonate, or carbonic acid. In one embodiment, CO2 can be added externally to the cultured medium. In other embodiments, CO2 can be produced by respiration or by microorganisms as a byproduct. For example, respiratory CO2 can be generated by the conversion of the tricarboxylic acid (TCA) cycle via the glyoxylate pathway, the pentose phosphate pathway (e.g., gnd (phosphogluconate dehydrogenase that converts phosphogluconic acid to ribulose-5-phosphate and CO2)), or the Entner-Doudoroff pathway. In still other embodiments, byproduct CO2 can be generated from acetone, ethanol, succinate, 3-oxoadipate, and 3-hydroxyadipate.

[0069] Culturing the microorganism in a suitable condition for a sufficient time also results in the microorganism producing one or more CO2 sources containing CO2, carbonate, bicarbonate, or carbonic acid. In one embodiment, the microorganism produces CO2 stoichiometrically with the diamine.

[0070] The stoichiometric CO2 mentioned here is the amount of CO2 related to the formation of products from a given substrate based on stoichiometry. An example of the stoichiometry for HMD production from glucose is as follows.

[0071] 1.47 C6H12O6+0.321 O2+ 2NH3 → C6H16N2+2.821 CO2+3.821 H2O The stoichiometric amount of CO2 produced per mole of HMD is 2.821 moles. Any CO2 produced in excess of this amount is produced either by respiration or by by - product formation.

[0072] Some respiratory CO2 sources are the TCA cycle, the glyoxylate pathway, the pentose phosphate pathway (e.g., zwf), and the Entner - Doudoroff pathway, etc. These pathways produce NAD(P)H from ATP which is then used via the electron transport chain. The by - product CO2 is defined as the CO2 produced as a by - product. For example, 2 moles of acetate are produced from each 1 mole of glucose, which is related to the release of 2 moles of CO2. Thus, the by - product CO2 related to the formation of each 1 mole of acetate is 1 mole.

[0073] In another example, the production of HMD from methanol can have multiple stoichiometries depending on whether only the oxidative branches of the TCA that have oxidative power are used to produce HMD or both the oxidative and reductive branches of the TCA are used to produce HMD.

[0074] 6.31 CH3OH+2 NH3+0.97 O2+C6H 16 N2 +7.62 H2O+0.31 CO2 7CH3OH+2 NH3+2 O2+C6H 16 N2+9 H2O+CO2 When CO2 is used because the pH drops (e.g., to 7), the solubility of CO2 in the medium can be increased by increasing the back pressure at the top of the fermenter to at least 2 bar (but 10 bar or less). In other embodiments, the solubility of CO2 in the medium can be increased, so the temperature can be lowered. In still other embodiments, the temperature is 37 °C or lower to increase the solubility of CO2.

[0075] In one embodiment, the microorganism that produces diamine is cultured in the liquid medium of the fermenter, but an inlet gas containing carbon dioxide is provided into the fermenter, and the back pressure at the top of the fermenter is increased to at least 2 bar (but 10 bar or less), whereby the solubility of carbon dioxide increases.

[0076] In other embodiments, in the process of fermentative production of diamine, the microorganism that produces diamine is cultured in the liquid medium of the fermenter, but an inlet gas containing carbon dioxide is provided into the fermenter, and its temperature is 37 °C or lower to increase the solubility of CO2.

[0077] In some embodiments, an enzyme, carbonic anhydrase (CA), can be added to the broth or medium to catalyze or enhance the formation of diamine carbonates and / or diamine carbamates (e.g., HMDA carbonates) by increasing the amount or rate of conversion of gaseous CO2 to soluble ions. In this way, a greater amount or higher availability of soluble ions can be made available for diamine or HMD.

[0078] Carbonic anhydrase catalyzes a reversible reaction. In the forward reaction, CA binds to carbon dioxide with water:

[0079]

Chemical formula

[0080] Then, H2CO3 releases bicarbonate (HCO3-) and a proton. In the reverse reaction, CA combines with bicarbonate and a proton, and the proton provides carbon dioxide and water. Thus, in the reverse reaction, certain reverse reactions can be used as reactions to take or release CO2 after DA carbonate is formed. In some embodiments, CA can be used as hydrate CO2 in the form of bicarbonate and a proton, which can be subsequently converted to DA. Depending on the direction of the reaction, certain suitable conditions can be selected that favor the absorption of carbon dioxide into the solution (e.g., via the hydration of carbon dioxide to bicarbonate) and / or the desorption of carbon dioxide from the solution (e.g., via the dehydration of bicarbonate to carbon dioxide and water).

[0081] Carbonic anhydrase can be provided exogenously by providing the CA directly to the culture solution or introduced via a microorganism capable of producing carbonic anhydrase. Carbonic anhydrase can also be provided as a recombinant or engineered CA. The recombinant CA is part of a microorganism that contains a diamine pathway (e.g., the HMD synthesis pathway) or has been introduced into a microorganism that can express other CAs. A native CA of the microorganism can be used, e.g., it is overexpressed or engineered for excretion into the broth or solution or for secretion into the periplasm of the microorganism. The CA can be of the EC4.2.1.1. enzyme family. The CA can be of Escherichia, e.g., the Can gene or b1026 (KEGG nomenclature) or other host strains, including those described herein. In some embodiments, the CA is obtained from the following genera: Methanobacterium, Desulfovirbio, Methanosarcina, Thiomicrospira, Acetobacterium, Clostridium, Methylobacterium, Rhizobium, Rhodobacter, Rhodospirillum, Staphylococcus, Methanococcus, Methanosaeta, Methanospirillum, and Sulfolobus. 1999 PNAS 96(26):15184 - 15189)。

[0082] Examples of microorganisms from which CA can be obtained include Neisseria gonorrhoeae (Jo et al., 2013 Appl. Environ. Microbiol. 79(21):6697 - 6705), Methanosarcina, Thiomicrospira, Acetobacterium woodii, Clostridium thermoaceticum, Methylobacterium extorquens, Rhizobium meliloti, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Staphylococcus aureus, Methanococcus jannaschii, Methanosaeta concilii, Methanosarcina barkeri, Methanosarcina thermophila, Methanospirillum hungateii, Sulfolobus solfataricus. (Smith et al., 1999 PNAS 96(26):15184 - 15189). CA is obtained from Neisseria gonorrhoeae. (Jo et al., 2013 Appl. Environ. Microbiol. 79(21):6697 - 6705).

[0083] In some embodiments, the gene encoding the β-type CA is obtained from the genus Desulfovirbio (such as Desulfovirbio fructosivorans, Desulfovirbio Tom C, Desulfovirbio magneticus, Desulfovirbio alcholivorans, etc.). In still other embodiments, the CA is from the genus Desulfomonile such as Desulfomonile tiedjei, the genus Methanobacterum such as Methanobacterium thermoautotrophicum, the genus Metanoacina such as Metanoacina thermophilia, Thiomicrospira such as Thiomicrospira crunogena.

[0084] In other embodiments, the CA shown in Table A below can be used.

[0085] [Table 2-1]

[0086] [Table 2-2]

[0087] [Table 2-3]

[0088] [Table 2-4]

[0089] [Table 2-5]

[0090] CA can be encoded by Desulfovibrio vulgaris. Desulfovibrio vulgaris has unique properties and has high activity in 4.2 M N-methyldiethanolamine (MDEA) at high temperatures and pH > 10. The CA of D. vulgaris has evolved to have activity at 100 °C for a long period (8 weeks) in high concentrations of MDEA for use in carbon capture technology (Alvizo, et. al. 2014 PNAS 111(46): 16436-16441). In some embodiments, the engineered CA is from Desulfovibrio vulgaris (GenBank accession ACL09337.1 GI:218758438). In some embodiments, Desulfo The carbonic anhydrase of Vibrio vulgaris str “Miyazaki F” has amino acid substitutions that stabilize the activity of carbonic anhydrase at high temperatures and alkaline pH, which are due to one or more of the following identified substitutions: A56S, T30R, A40L, A84Q, G120R, T139M, K37R, E68AQ, A95V, Q119M, N145WFC, N213E, A219T, R31P, Q43M, V70I, H124T, H148T, V157A, M170F, H44L, M129F, S144R, Y49F, S126N, D196S, P136R, P174E, D195A, G89A, D96E, V100T, A121Q, A181K, M207A, S216D.

[0091] In some embodiments, the CA is encoded by E. coli (EG10176 (EcoCyc), or EG12319 (EcoCyc), Can gene, b1026 (KEGG designation)).

[0092] The disclosed enzyme may also be in the form of a fusion protein, which is a recombinant or engineered CA fused to an antibody label (e.g., myc epitope), a purification sequence (e.g., His tag for binding to metals and cell localization signals such as secretion or export signals). Secretion signals such as Sec or Tat tags can be used to help express the desired protein in the periplasmic space. In a preferred embodiment, the CA is exported into the culture medium rather than secreted into the periplasmic space. In some embodiments, the secretion or export signal is a secretion label genetically encoded and fused to the N-terminus of the protein that is fused to the carbonic anhydrase DNA sequence to help expression into the periplasmic space or export extracellularly from a microorganism (e.g., E. coli). In another embodiment, the CA can be fused to the E. coli protein OmpF, which is transported (exported) into the culture medium (Nagahari et al., 1985 The EMBO J. 4(13A):3589-3592; Jeong and Lee, 2002 Appl. Environ. Microbiol. 68:4979-4985). In yet another embodiment, the CA can be fused to the E. coli protein YebF, which has been seen to support the export of the protein into the culture medium, which is an extracellular protein with an unknown function (Zhang et al., 2006 Nat. Biotech. 24:100-10). The N-terminal sorting signal peptide label is determined using the SignalP 4.1 Server (http: / / www.cbs.dtu.dk / services / SignalP / ).

[0093] Carbonic anhydrase can be provided by genetically engineered microorganisms in the fermentation broth. In other embodiments, carbonic anhydrase is provided by genetically engineered microorganisms, but the microorganisms produce DA, optionally excrete it into the broth, and optionally are present in the periplasm of the microorganisms. In other embodiments, carbonic anhydrase is a native gene or enzyme, optionally engineered to be secreted into the broth, and optionally engineered to be secreted into the periplasmic space of the microorganism.

[0094] In some embodiments, CA can be excreted into the fermentation broth, and in other embodiments, CA can be present in the periplasm of the microorganism.

[0095] Depending on the direction or rate of the reaction, in some embodiments, the native CA gene sequence (e.g., the CA encoded by E. coli) can be overexpressed, for example, by altering its promoter and / or by creating it through secretion or peptide excretion or fusion.

[0096] In some embodiments, variants of CA that can perform the forward and / or reverse reactions are contemplated. Variants can include homologs, paralogs, or genetically engineered ones, for example, those with increased stability to alkaline pH and heat.

[0097] CAs that can support the forward and / or reverse reactions and have improved properties (e.g., thermal stability, solubility stability, and / or base stability) can be selected and used. In some embodiments, variants of CA that are active and / or stable against high concentrations of DA (e.g., HMD) can be selected and used. In other embodiments, CAs and their variants that are active and / or stable against high concentrations of methylene segments from C2 to C7, such as hexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine, and heptamethylenediamine, can be selected and used.

[0098] Depending on the selected conditions, CA can be heat-resistant CA, or alkaline pH-resistant CA, or both. Preferably, the alkaline pH is from pH 8 to 13. In some embodiments, the pH can be in the range of pH 8 to 13, pH 8.5 to 13, pH 9 to 13, pH 10 to 13, pH 8 to 12, pH 8.5 to 12, pH 9 to 12, pH 8 to 11, pH 8.5 to 11, pH 9 to 11, pH 10 to 11, and pH 10 to 12. Temperature and basic pH resistance can be beneficial when the CA releases CO2 from the DA carbonate or carbamate. This process results in an increase in pH as CO2 is released and free base is formed. Additionally, in some embodiments, temperatures higher than room temperature and typical fermentation temperatures can be used to promote the release of CO2.

[0099] Depending on whether the forward or reverse reaction is the preferred condition, CA enzymes with different activities are provided to the culture solution. In some embodiments, CA has optimal activity for the forward reaction, and in other embodiments, the CA enzyme has optimal activity for the reverse reaction. In some embodiments, mixtures of different enzymes with varying optimal activities and / or the improved properties disclosed herein can be used.

[0100] In some embodiments, the carbonic anhydrase described above can perform the method when, under suitable conditions, it has improved properties such that the activity of hydrated carbon dioxide or dehydrated bicarbonate is at least 1.2 times, at least 1.3 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 25 times higher. To that end, in some embodiments, the concentration of the carbonic anhydrase polypeptide in the suitable conditions used in the method is from about 0.1 g / L to about 10 g / L, from about 0.25 g / L to about 7.5 g / L, from about 0.5 g / L to about 5 g / L, less than about 10 g / L, less than about 5 g / L, or less than about 2.5 g / L.

[0101] In some embodiments, CA can be provided exogenously. In other embodiments, the CA enzyme can be provided directly to the broth or fermentation solution. In other embodiments, CA is provided by a genetically engineered microorganism in the fermentation broth or solution, and CA can be excreted into the broth or present in the periplasm of the microorganism. In other embodiments, the enzyme is provided immobilized on particles. The recombinant carbonic anhydrase polypeptide can be immobilized on the surface, for example, the enzyme described above is bound to the surface of the solid phase portion in solution. Methods for binding the enzyme to a solid phase portion (e.g., porous or non-porous beads, or solid support), either covalently or non-covalently, and retaining activity for use in a bioreactor are well known in the art. Methods for handling gas streams using immobilized enzymes are disclosed, for example, in U.S. Patent No. 6,143,556, U.S. Patent Publication 2007 / 0004023 A1, and International Publication Numbers WO98 / 55210 A1, WO2004 / 056455 A1, and WO2004 / 028667 A1, which are incorporated herein by reference.

[0102] Thus, in some embodiments, a method for enhancing the release of CO2 from a solution of DA carbonates or DA carbamates can be carried out by immobilizing the engineered carbonic anhydrase polypeptide on a surface, specifically, by binding the enzyme to the surface of a solid phase portion (e.g., beads). In some embodiments, the method using the immobilized polypeptide can further be carried out including the step of isolating or separating the immobilized carbonic anhydrase from the broth or fermentation solution. After separating the immobilized carbonic anhydrase from the broth or fermentation solution, the broth or fermentation solution can be handled in such a way that, for example, the enzyme can be inactivated by desorbing CO2 at a high temperature. Further, the separately retained and immobilized enzyme can be added to another solution and reused. [CO2:DA ratio] In the disclosed process, the microorganism (e.g., a genetically modified microorganism) forms CO2 and DA (e.g., HMD) in a ratio of from about 0.05:1 to about 5:1 to about 7:1. Other suitable ratios include from 0.2:1 to about 3:1. In other embodiments, the ratio of CO2 to DA (e.g., HMD) is from about 0.05:1 to about 3:1, from about 0.05:1 to about 2.5:1, from about 0.05:1 to about 2:1, from about 0.05:1 to about 1.5:1, from about 0.05:1 to about 1:1.

[0103] The disclosed ratios can be determined by measuring the CO2 in the form of all dissolved inorganic carbon (DIC) in the cultured medium. DIC includes carbonates and / or carbamates and can be measured, for example, by the "Handbook of Methods for the Analysis of the Various Parameters of the Carbon Dioxide System in Sea Water" Prepared for the U. S. Department of Energy, Special Research Grant Program 89-7A: Global survey of carbon dioxide in the oceans. Version 2 - September 1994 Edited by Andrew G. Dickson & Catherine Goyet (referred to as the Handbook). For example, DIC can be measured by "SOP 2: Determination of total dissolved inorganic carbon in sea water, p. 1-18" on pages 38-55 of the Handbook.

[0104] In other embodiments, the fraction of DIC (which is DIC relative to total dissolved counter anions (TDCA)) can be measured. TDCA is the sum of DIC and other anions. Other anions other than DIC (e.g., Cl - , SO -2, PO4 -3 , NO3 - , NO2 - ) can also be determined by using any suitable method such as ion exchange chromatography. For example, ion exchange chromatography with a commercially available conductivity detector (and ion suppressor) from DIONEX can be used.

[0105] The produced compound contained in the cultured medium contains DIC / TDCA in an amount of at least 40%. In other embodiments, the proportion of DIC in the cultured medium can be at least 50%, 60%, 70%, 80, 90% or 99.9%. In some embodiments, DIC can be at least 40%, 50%, 60%, 70%, 80, 90% or 99.9% of TDCA in the cultured medium at pH 9.

[0106] [Fermentation pH] As described above, the starting culture medium can have a pH of about 5 to about 7. When microorganisms grow on the culture medium and produce the desired diamine (e.g., HMD carbonates and / or HMD carbamates), and before the diamine carbonates and / or carbamates are converted to the diamine free base, the pH of the cultured medium is less than 11, less than 10, less than 10, less than 9, or less than 8. In other embodiments, the pH can be at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7. In other embodiments, the pH of the medium is from about 6 to about 9.5, from about 6 to about 9, from about 6 to about 8, from about 6.5 to about 7.5, from about 7.5 to about 9.5, or from about 8 to about 9.

[0107] The medium (e.g., the starting medium) can be adjusted by an inorganic acid, a base or a buffer to adjust the pH, while the culture medium contains substantially no buffer, substantially no inorganic or organic acid, or no inorganic or organic acid is added from the outside. As used herein, "substantially free" refers to the culture medium. In other words, salts, buffers, acids (excluding CO2) or bases can be used to adjust the pH of the starting medium. When these salts, buffers, acids (excluding CO2) or bases are used to adjust the pH variation of the medium due to fermentation and microbial growth, a minimal amount is used. However, salts, buffers, acids (excluding CO2) or bases are not used to neutralize diamine carbonates and / or carbamates. It should be understood that microorganisms produce by-products such as acetates, succinates, other salts and / or organic acids. The pH adjustment during fermentation is by using carbon dioxide that can be added from the outside or produced by microbial growth.

[0108] [Release of CO2 and free base of diamine] Once the diamine carbonates and / or carbamates are formed, the diamine is obtained by converting the free base of the diamine (e.g., the free base of HMD). In some embodiments, the DA-carbonates and / or carbamates are separated from the microorganisms in the initially cultured medium before being converted to the free base of the DA. Examples for converting DA carbonates and / or carbamates to the free base of the DA include heat, reduced pressure, ion exchange, or electrodialysis. In one embodiment, the diamine is the free base of HMD that can be converted by the released carbon dioxide. In some embodiments, to enhance the release of carbon dioxide from the DA carbonates or DA carbamates by converting bicarbonate and / or carbonate ions to carbon dioxide, an enzyme of carbonic anhydrase (regarding the forward and reverse reactions described in more detail in the above text) can be provided. In such embodiments, the enzyme can be provided exogenously and can be, for example, part of a microorganism or an engineered enzyme that can be added exogenously. In some embodiments, the engineered CA can be engineered to be excreted into the fermentation broth or fermentation solution. In other embodiments, the engineered CA can be engineered to be present in the periplasm of the microorganism. In some embodiments, the DA carbonates and / or carbamates are first separated from the microorganisms in the cultured medium before being converted to the free base of the DA. In such embodiments, the CA can be provided by adding CA exogenously. In other embodiments, the CA can be immobilized. In other embodiments, the CA is part of an engineered microorganism having the activity of carbonic anhydrase. In other embodiments, the activity of carbonic anhydrase is provided by an engineered microorganism including the DA synthesis pathway such as the HMD synthesis pathway and the activity of carbonic anhydrase.

[0109] If heat is used to convert diamine carbonates and / or carbamates (e.g., HMD) to the free base of the diamine, the temperature includes a temperature greater than 70°C, greater than 80°C, or greater than 105°C. In some embodiments, the temperature can be greater than 200°C. In still other embodiments, the temperature can be about 315°C. In some embodiments, the temperature is lower than 315°C, lower than 250°C, or lower than 215°C. In still other embodiments, the temperature is greater than 20°C, greater than 30°C, greater than 40°C, and reduced pressure is used. In some embodiments, the diamine carbonates and / or carbamates converted to the free base at the temperatures disclosed heretofore can be HMD. CA is also added for the heating step to enhance the release of carbon dioxide from the solution of DA carbonates or DA carbamates by converting bicarbonate and / or carbonate ions to carbon dioxide and the free base. Thus, in some embodiments, CA having enhanced properties (e.g., thermal stability, dissolution stability, enhanced stability or activity in high concentrations of DA, and / or base stability) that support the reverse reaction is provided. Therefore, in some embodiments, the method of catalyzing the reverse reaction by carbonic anhydrase is carried out at a temperature greater than 70°C, greater than 80°C, or greater than 105°C. In some embodiments, the temperature can be greater than 200°C. In still other embodiments, the temperature can be about 315°C. In some embodiments, the temperature is lower than 315°C, lower than 250°C, or lower than 215°C. In still other embodiments, the temperature is greater than 20°C, greater than 30°C, greater than 40°C, and reduced pressure is used. Greater than 70°C, greater than 80°C, or greater than 105°C. In some embodiments, the temperature can be greater than 200°C. In still other embodiments, the temperature can be about 315°C. In some embodiments, the temperature is lower than 315°C, lower than 250°C, or lower than 215°C. In still other embodiments, the temperature is greater than 20°C, greater than 30°C, greater than 40°C, and reduced pressure is used.

[0110] The conversion can also be carried out under a reduced pressure lower than atmospheric pressure (e.g., from 0.01 to 1 atm). In other embodiments, the atmospheric pressure can include from about 1 to about 10 bar (inside the container, not the inlet air pressure) or from about 1 to about 3 bar. When temperature and atmospheric pressure are used in combination, the atmospheric pressure can be from about 1 to about 3 bar. In some embodiments, the temperature is lower than 315, lower than 250, or lower than 215. The CO2 released can be recycled into the system (e.g., into the cultured medium).

[0111] As other examples of converting diamine carbonates and / or carbamates (e.g., HMD) to diamine free bases (e.g., the free base of HMD), sparging with a gas (e.g., air, or an inert gas such as nitrogen or helium) or steam distillation is included. Steam from an external source or generated by boiling the broth in situ can be added. In some embodiments, the diamine (e.g., HMD) carbonates and / or carbamates converted to free bases also include those by heat and gas sparging. In one embodiment, stripping can be carried out at an atmospheric pressure of about 1 to about 10 bar. In some embodiments, the conversion step can result in at least about 20% to about 99% diamine free base. In other embodiments, the conversion step can result in at least about 20 - 30%, 30 - 40%, or 40 - 50%.

[0112] When sufficient heat is applied to generate steam from the medium in situ in the stripping column, both water and carbon dioxide can be removed in an amount sufficient to obtain a solution in which the diamine (e.g., HMD) free base is condensed, and subsequent recovery of the diamine becomes effective. In some embodiments, CA is also added to enhance the release of carbon dioxide from the solution of DA carbonates or DA carbamates by conversion of bicarbonate and / or carbonate ions to carbon dioxide. Enhancing the effectiveness of the CO2 removed from the DA carbonate solution allows for a low purification cost by reducing the size of the stripping column.

[0113] In some embodiments, the diamine recovered from the diamine free base can be greater than 40%, or can be 50% greater. In still other embodiments, the recovered diamine can be greater than 50% of the free base formed from the stripping process at atmospheric pressure, air sparging, and elevated temperature (e.g., less than 315 °C, less than 215 °C, or around 115 °C).

[0114] After the formation of the diamine (e.g., HMD) free base and the removal of CO2, a strong base (e.g., sodium or calcium hydroxide) can be added to raise the pH, thereby improving extraction. When calcium hydroxide is utilized, a carbonate precipitate is formed and can be separated from the liquid phase.

[0115] [Solid removal before conversion] Before the conversion of diamine (e.g., HMD) carbonates and / or carbamates to the diamine free base, solids can be separated from the cultured medium. Such solids can include cells and other organisms and impurities of the cultured medium by the product. The resulting liquid fraction is rich in diamine (e.g., HMD) carbonates and / or carbamates.

[0116] Separation can be achieved by centrifugation, filtration, rotary drum, or combinations thereof. Examples of centrifugation include disk stack centrifugation, or decanter centrifugation or solid bowl type centrifugation. It should be understood that any combination of the type of centrifugation or centrifugation and the number thereof can be used to achieve the separation of the desired solids from the culture medium. If the solids are not separated by centrifugation or further separation required, separation by filtration can be used. Filtration can be achieved by ultrafiltration.

[0117] [Reduction or removal of water] In some embodiments, water can be removed or reduced after solid removal and before conversion. Any known suitable method for water removal or reduction (e.g., evaporation, reverse osmosis, electrodialysis, etc.) can be used.

[0118] In other embodiments, water can be removed before the step of isolating the diamine free base. One advantage of removing water before the isolation step is an increase in pH. Methods for reducing or removing water by reducing or removing water after solid removal and before conversion, as previously disclosed, can also be used. The amount of water to be removed (e.g., the upper limit of water to be removed) is determined by the solubility limit of the components of the medium or by-products or salts or carbamates of the diamine. In one embodiment, the water to be removed depends on whether it hinders the insolubility of the components of the medium or by-products containing salts or carbamates of the diamine.

[0119] Evaporation can be carried out by multi-effect evaporation, thermal vapor recompression, or mechanical vapor recompression. The evaporator is a heat exchanger in which the liquid is boiled to obtain steam, which is also a low-pressure steam generator. This steam can be used to further heat other evaporators called other "effects". Thus, for example, when two evaporators are connected, the steam line from one is connected to the steam box of the other, providing a two- or multi-effect evaporator. This arrangement can be increased by adding a third evaporator, for example, to make a triple-effect evaporator.

[0120] In one embodiment, the amount of water to be removed is 10% by mass. The removed water is further recovered and recycled to step a) of the culture process or as shown in Figure 4.

[0121] [Simultaneous Removal of Water and Carbon Dioxide] Removal or reduction of water enables a smaller solvent extraction column and lower dissolution power, and removal of carbon dioxide enables alkalization (e.g., filtration osmosis) of the diamine compound to increase solvent extraction efficiency. In one embodiment described above, the evaporator is used to remove any remaining carbon dioxide (and also any water that can be removed) following a stripping column that removes sufficient water (and also removable CO2). Simultaneous removal of water and carbon dioxide in one step to a sufficient degree for the next extraction has the advantage of reducing costs and downtime associated with multiple steps. Thus, in other embodiments described above, one step or unit operation is used for the purpose of removing sufficient water and carbon dioxide (e.g., DIC) to enhance recovery of the diamine by solvent extraction in downstream processes. Thus, the cost of one step or unit operation (e.g., a water evaporator or stripping column) and the associated preparation, maintenance, use and downtime risks can be eliminated or reduced. In one embodiment, simultaneous removal of water and carbon dioxide enhances recovery of HMD in downstream processes.

[0122] Removal of water also enables stripping of carbon dioxide. Conditions for removal of water enable sufficient removal of carbon dioxide without the need for a CO2 removal step. Thus, in one embodiment, simultaneous removal of water and CO2 is effectively achieved by both a stripping unit or an evaporator unit. As an example, an evaporator, e.g., a multiple effect evaporation method, a self vapor mechanical compressor, can be used to remove sufficient water and carbon dioxide to obtain a solution condensed in the diamine free base, thereby enabling effective next diamine recovery. For example, in Figure 4, Step 4 or 5 can be eliminated when sufficient removal of both water and carbon dioxide is achieved by the retained steps. As demonstrated in the examples, the step of using an evaporator can be advantageous compared to a water vapor stripping step.

[0123] It should be understood that carbonic anhydrase may be present in the carbon dioxide release and free DA base formation steps to enhance the release of carbon dioxide from solutions of DA carbonates or DA carbamates. In some embodiments, CA may be present in the water removal or evaporation step, in other embodiments, CA may be present in the CO2 stripping step, and in still other embodiments, CA may be present in both the water removal or evaporation step and the CO2 stripping step.

[0124] [Isolation of diamine free base] Once converted, the DA (e.g., HMD) free base can be isolated from the cultured medium together with an organic solvent. The isolated DA is separated from the organic solvent by a process such as distillation. Examples of extraction solvents include alcohols, amines, ethers, alkanes, and ketones. Examples of alcohols include monohydric alcohols from C4 to C8. In some embodiments, examples of extraction alcohols include hexanol, particularly 1 - hexanol, isopentanol, or cyclohexanol, toluene, ethyl ether, or mixtures thereof. As shown in the examples, alkanes are suitable solvents, particularly when HMD is a diamine. Alkanes, particularly hexane, can be used because of its extremely low water solubility. If there is even a little water, very little hexane is extracted, providing a reasonable recovery of the available free base. Alkanes are therefore suitable solvents for use in the recovery of diamine free bases (e.g., HMD). Suitable alkanes include straight - chain or branched ones from C5 to C12. In one embodiment, both the diamine being extracted and the alkane selected as the solvent can have the same number of carbon atoms. Heptane is another alkane particularly suitable for HMD, which is further supported by the in silico modeling studies below. Isomers of hexane and heptane are suitable. Isomers of hexane are 2 - methylpentane, 3 - methylpentane, 2,2 - dimethylbutane, and 2,3 - dimethylbutane. Isomers of heptane are 2 - methylhexane, 3 - methylhexane, 2,2 - dimethylpentane, 2,3 - dimethylpentane, 2,4 - dimethylpentane, 3,3 - dimethylpentane, 3 - ethylpentane, and 2,2,3 - trimethylbutane. In some embodiments, the DA (e.g., HMD) free base can be directly distilled from the cultured medium.

[0125] Any suitable solvent can be used. In some embodiments, the solvent can have a boiling point higher than the HMD free base or the desired diamine free base and lower than water, or any boiling point can be between the HMD free base (or the desired diamine free base) and water (intermediate boiling point).

[0126] The DA (e.g., HMD) free base can be isolated from the medium or the DA-rich fraction using an extraction solvent to provide an aqueous phase and an organic phase containing the DA free base (e.g., when solids and / or water are removed before isolation).

[0127] The organic phase (extract) can contain at least 10%, at least 20%, at least 30%, at least 40%, or less than 50% by mass of the DA (e.g., HMD) carbonate and / or carbamine form in the free base, with the majority in the form of the free base. Depending on the number of extractions, in some embodiments, the DA (e.g., HMD) in the extract can be greater than 90% by weight.

[0128] The amount of the extracted DA free base is greater than about 90% by weight. In some embodiments, the DA free base is the HMD free base, which is greater than 90% by weight.

[0129] The efficiency of solvent extraction of the diamine, e.g., the free base of HMD, increases with a decrease in the carbon dioxide concentration such as DIC as shown in the examples. The decrease in carbon dioxide results in a higher pH and a higher concentration of the recoverable free base form. In some embodiments, the aqueous diamine solution before solvent extraction contains undetectable carbon dioxide, carbon dioxide lower than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 1%, or lower than 5%. In some embodiments, the aqueous diamine solution before solvent extraction contains undetectable DIC, DIC lower than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 1%, or lower than 5%. In other embodiments, the aqueous DA solution before solvent extraction contains undetectable DIC, DIC lower than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 1%, or lower than 5%.

[0130] In one embodiment, CO2, which is stoichiometrically produced by an enzymatic pathway, along with DA, e.g., HMD, is used to neutralize DA, e.g., HMD, and to maintain a pH suitable for fermentation, generally a pH of about 9 or less. Additional pH adjustment can be achieved by supplementing with CO2 produced as a byproduct by microorganisms (e.g., converting pyruvate from formate to CO2), or externally derived CO2, if desired or necessary. The external CO2 can be purchased or can be CO2 recycled from the fermentation / isolation process. The presence of CO2 during fermentation forms HMD carbonate, HMD bicarbonate, HMD bisbicarbonate and small amounts of HMD carbamate and HMD biscarbamate. At the end of fermentation, the cells are optionally removed and the culture medium is treated under reduced pressure to reduce HMD carbonate or carbamate compounds (or DA carbonate or carbamate compounds) that release gaseous CO2 and the free base HMD (neutralized by 2NH-(CH2)6-NH2) (or DA free base) that raises the pH of the cultured medium. The free base or neutral HMD (or DA) can be isolated via solvent extraction. During this process, the released CO2 can be recycled. In one embodiment, the disclosed method does not require the use of an acid for pH adjustment and the subsequent addition of a base to neutralize the acid to produce a solvent extractable free base HMD (or DA).

[0131] In other embodiments, the culture or the cultured medium is heat treated with heat at a reduced temperature, e.g., both batch or continuous, e.g., 90 - 100 °C. At atmospheric pressure or at an overpressure at even higher temperature.

[0132] In the process of other embodiments, DA (e.g., HMD) is extracted with an organic solvent that has a solubility gap with water and is stable at alkaline pH, particularly polar, more specifically bipolar protic organic solvents, etc. Suitable solvents are as indicated above.

[0133] In one embodiment, the extraction and / or subsequent phase separation of DA (e.g., HMD) is performed batchwise at elevated temperatures.

[0134] Before or after removal of the microorganism, the cultured medium is concentrated or concentrated by known methods such as, for example, reverse osmosis or nanofiltration with the aid of a rotary evaporator, thin film evaporator, falling film evaporator. If necessary, salts that can be precipitated for the concentration procedure can be removed by filtration or centrifugation. This concentrated cultured medium is then purified to obtain DA (e.g., HMD) by the methods disclosed herein. Such concentration procedures are suitable but not necessarily required for production according to the disclosed methods.

[0135] According to one embodiment, DA (e.g., HMD) is extracted from the cultured medium with the aid of an organic solvent. The organic solvent can be, for example, one that has a solubility gap with water and is stable at alkaline pH, especially a polar, bipolar protic organic solvent. Suitable solvents, especially cyclic or acyclic, optionally branched alkanols having 3 to 8 carbon atoms, especially n- and iso-propanol, n-, sec- and iso-butanol, or cyclohexanol, and n-pentanol, n-hexanol -n-heptanol, n-octanol, 2-octanol, and mono- or their multi-branched isomers.

[0136] In one embodiment, and / or subsequent phase separation is performed batchwise at elevated temperatures limited to the boiling point of water and the extraction solvent or an azeotrope formed thereby. By way of example, the extraction solvent n-butanol extraction and phase separation can be performed, for example, at about 25-90 °C, preferably 40-70 °C. For extraction, the two phases are stirred for, for example, 10 seconds to 2 hours, or 5 to 15 minutes, until a partition equilibrium is established. The phases are then left until they are completely separated so as to be stable, which takes, for example, 10 seconds to 5 hours, for example, especially at a temperature in the range of about 25-90 °C, 15 to 120 or 30 to 90 minutes. Or 40-70 °C. In the case of n-butanol.

[0137] In a further embodiment, the DA (e.g., HMD) is continuously extracted from the cultured medium, either continuously in a multi-step process (e.g., a combination of mixing - stirring) or continuously in an extraction column.

[0138] One skilled in the art can establish the form of the extraction column that is used according to the methods disclosed as part of the optimization procedure for the phases to be separated in each case. Suitable extraction columns can in principle be either without or with an electrical input, for example, a pulsed column or a rotating - internal column. Also, one skilled in the art can choose, for routine work parts, the type of method suitable for optimizing phase separation and the internal materials such as sieve trays and column trays. The basic theory of liquid - liquid extraction of small molecules is well known (see, for example, H.-J. Rehm and G. Reed, Eds., (1993), Biotechology, Volume 3 Bioprocessing, Chapter 21, VCH, Weinheim). An industrially practical arrangement of extraction columns is desirable, for example, as in Lo et al., Eds., (1983) Handbook of Solvent Extraction, John Wiley & Sons, New York. The later reference is provided for the disclosure of the above - mentioned text.

[0139] After phase separation, the DA (e.g., HMD) is isolated and purified from the separated phase containing the DA in essentially known methods. Possible procedures for recovering the DA (e.g., HMD) include, but are not limited to, distillation, precipitation of salts with suitable organic or inorganic acids, or a combination of these suitable procedures.

[0140] [Distillation] Distillation can be carried out in a continuous or batch mode. A single distillation column or a plurality of distillation columns combined with each other can be used. Establishing the arrangement and operating parameters of a set of distillation column apparatuses is the responsibility of those skilled in the art. The distillation columns used in each case are designed in an essentially known manner (see, for example, Sattler, Thermische Trennverfahren [Thermal separation methods], 2nd Edition 1995, Weinheim, p. 135ff; Perry's Chemical Engineers Handbook, 7th Edition 1997, New York, Section 13). Thereby, the distillation columns used have internals effective for separation, such as separation trays, for example, perforated plates, bubble cap trays or valve trays, arranged packings, for example, sheet metal or fabric packings, or random beds of packings. The number of plates required in the columns used and the reflux ratio are essentially determined by the purity requirements and the relevant boiling positions for the separation of the liquid, and those skilled in the art can determine the specific design and operating data by known methods.

[0141] In some embodiments, the distillation step substantially removes water and solvent. The temperature of the distillation can be less than 170 °C, less than 160 °C, less than 150 °C, or less than 140 °C.

[0142] Precipitation of salts can be achieved by suitable organic or inorganic acids (such as sulfuric acid, hydrochloric acid, phosphoric acid, formic acid, carbonic acid, oxalic acid, etc.). In other embodiments, the organic dicarboxylic acid is used in the polycondensation to obtain the resulting polyamide, in the form of a salt (for example, by recrystallization) used either directly or after purification. More specifically, such dicarboxylic acids are C4 - C12 dicarboxylic acids.

[0143] The organic DA (e.g., HMD) phase produced by the extraction procedure is also chromatographically concentrated. For chromatography, the DA phase is applied to a suitable resin. Examples of resins are strong or weak acidic ion exchangers (Lewatit 1468 S, Dowex Marathon C, Amberlyst 119 Wet or others) on which the desired product or contaminants are partially or completely retained on the chromatography resin. These chromatography steps can be repeated if necessary using the same or other chromatography resins. A person skilled in the art is accustomed to choosing suitable chromatography resins and their most effective uses. The purified product is concentrated by filtration or ultrafiltration and stored at an appropriate temperature.

[0144] The identity and purity of the isolated compound are determined by known techniques. These include high performance liquid chromatography (HPLC), gas chromatography (GC), methods using spectrometers, staining methods, thin layer chromatography, NIRS, enzyme quantification, or microorganism quantification. These analytical methods are summarized below: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17。

[0145] The disclosed method may include various combinations of steps or processes, as shown in Figure 4. Referring to Figure 4, the system and process include the following.

[0146] 1. A fermenter or any container in which the microorganism can be cultured or grown in a suitable medium under suitable conditions and for a sufficient time in a culture medium in the presence of carbon dioxide, carbonate, bicarbonate, or carbonic acid to form one and more diamine carbonates and / or carbamates. 2. Microbial heat sterilization / conversion: Many processes require the cultivation of microorganisms after fermentation. Once the diamine carbonates and / or carbamates are formed, they can be converted to free bases when sterilized / heat sterilized at high temperatures, where process CO2 is released.

[0147] 3: Solid removal: Solids from the cultured medium are optionally removed before the diamine carbonates and / or carbamates are converted to release CO2.

[0148] 4: Conversion (all possible ways to release CO2). The released CO2 can be recycled to the fermenter.

[0149] 5: Water removal: Remove water from the DA free base mixture and optionally recycle water and / or carbon dioxide to the fermenter.

[0150] 6: Solvent extraction, extract the DA mixture with an organic solvent in an extractor to obtain an organic phase DA solution and an aqueous raffinate, and the aqueous raffinate is optionally recycled to the conversion step; 7: Purification: Include distillation to optionally recycle the organic solvent back to the solvent extraction step. Purification includes producing diamine and other steps to remove color-forming compounds and the like in additional distillation columns.

[0151] 8: Purified DA: The purified DA free base obtained from the above steps 9: Heat sterilization of any microorganisms that do not release CO2 10: Any water removal, water recycling and, if released, possible CO2. Remove water from the mixture of carbonates and / or carbamates, and water and / or carbon dioxide are optionally recycled to the fermenter.

[0152] 11:Direct purification from any aqueous phase, with or without the evolution of CO2, involves distillation, ion exchange, electrodialysis and other suitable processes or steps. Water and CO2 (if generated) can be recycled in these steps: any direct conversion of carbonates and / or carbamates for the formation of the DA free base mixture from the cultured medium, and any evolution of water and / or carbon dioxide recycled to the fermenter; 12:Alkalinization (NaOH and CaOH) or other steps to remove carbonates from HMD (ion exchange, electrodialysis, etc.): An aqueous base is added to remove carbonates and / or carbamates from the DA free base mixture.

[0153] Referring to Figure 4, there are several different combinations of steps as follows: 1, 2, 3, 4, 5, 6, 7, 8 1, 3, 4, 5, 6, 7, 8 1, 9, 3, 4, 5, 6, 7, 8 1, 2, 3, 10, 4, 5, 6, 7, 8 1, 3, 10, 4, 5, 6, 7, 8 1, 9, 3, 10, 4, 5, 6, 7, 8 1, 2, 3, 10, 11, 8 1, 3, 10, 11, 8 1, 9, 3, 10, 11, 8 1, 2, 3, 11, 8 1, 3, 11, 8 1, 9, 3, 11, 8 1, 2, 3, 4, 11, 8 1, 2, 3, 10, 4, 11, 8 1, 3, 10, 4, 11, 8 1, 3, 4, 11, 8 1, 9, 3, 4, 11, 8 1, 9, 3, 10, 4, 11, 8 1, 2, 3, 4, 12, 6, 7, 8 1, 2, 3, 10, 4, 12, 6, 7, 8 1, 3, 4, 12, 6, 7, 8 1, 3, 10, 4, 12, 6, 7, 8 1, 9, 3, 4, 12, 6, 7, 8 1, 9, 3, 10, 4, 12, 6, 7, 8 1, 2, 3, 4, 5, 12, 6, 7, 8 1, 2, 3, 10, 4, 5, 12, 6, 7, 8 1, 3, 4, 5, 12, 6, 7, 8 1, 3, 10, 4, 5, 12, 6, 7, 8 1, 9, 3, 4, 5, 12, 6, 7, 8 1, 9, 3, 10, 4, 5, 12, 6, 7, 8.

[0154] In principle, the disclosed method can be applied using any cultured medium containing diamine (e.g., a cultured medium containing HMD). There are also few restrictions in principle regarding the microorganisms used for culture or fermentation. The microorganisms can be naturally occurring microorganisms; microorganisms improved by mutation and selection, and microorganisms created or genetically engineered by recombination, such as bacteria and fungi. These microorganisms can produce any of DA or DA derivatives, HMD and / or HMD derivatives such as HMD carbonate or HMD bicarbonate. Furthermore, the recombinant microorganisms used are disclosed in the following and in U.S. Patent No. 8,377,680 or other references shown in this document (these disclosures are incorporated herein by reference in their entirety), and for example, DA biosynthesis (e.g., HMD biosynthesis) is possible via the HMD pathway (the "HMD pathway").

[0155] In some embodiments, a genetically engineered microorganism having a DA pathway comprising at least one exogenous nucleic acid encoding at least one enzyme of the DA pathway may also contain an exogenous nucleic acid encoding an enzyme of carbonic anhydrase. In other embodiments, the genetically engineered microorganism comprises a DA synthesis pathway (preferably an HMD synthesis pathway), at least one enzyme of the DA synthesis pathway (preferably an HMD synthesis pathway), and at least two exogenous nucleic acids encoding a carbonic anhydrase enzyme or variant that is expressed in an amount sufficient to produce at least one DA carbonate and / or DA carbamate compound (preferably an HMD carbonate and / or carbamate compound). In other embodiments, the genetically engineered microorganism comprises a DA synthesis pathway (preferably an HMD synthesis pathway), at least one enzyme of the DA synthesis pathway (preferably an HMD synthesis pathway), and at least two exogenous nucleic acids encoding a carbonic anhydrase enzyme or variant that is expressed in an amount sufficient to produce at least one or more DA free bases (preferably an HMD free base) and carbon dioxide. It should be understood that a process for diamine production may include: a genetically engineered microorganism as described above, and a DA pathway (preferably an HMD synthesis pathway) and a carbonic anhydrase enzyme or variant that is expressed in an amount sufficient to produce at least one DA carbonate and / or DA carbamate (preferably an HMD carbonate and / or carbamate) compound, or a genetically engineered microorganism capable of producing at least one or more DA free bases (preferably an HMD free base) and carbon dioxide or both.

[0156] Examples of the HMD synthesis pathway include the pathways shown in FIGS. 10, 11, 13, 20, 21, 22, 24, 25, and 26. Disclosed are various pathways for producing HMD. For example, the HMD pathway includes the following: а) The steps shown as A - N in FIG. 13 b) Steps A / L / N / C in FIG. 13 c) Steps M / N / C in FIG. 13 d) Steps D / E / F / G / H in Figure 13 e) Steps D / I / J / G / H in Figure 13 f) Steps D / E / K / J / G in Figure 13 g) Steps A - H in Figure 15 h) Steps A / B / C / D / E / R / S in Figure 16 i) Steps A / B / F / G / D / E / R / S in Figure 16 j) Steps A / B / H / I / D / E / R / S in Figure 16 k) Steps A / B / C / AB / Z / R / S in Figure 16 l) Steps A / B / H / I / AB / Z / R / S in Figure 16 m) Steps A / B / F / G / AB / Z / R / S in Figure 16 n) Steps A / B / / J / O / P / Q / S in Figure 16 o) Steps A / B / J / M / N / P / Q / S in Figure 16 p) Steps A / B / J / K / L / P / Q / S in Figure 16 q) Steps A / B / J / O / Z / R / S in Figure 16 r) Steps A / B / J / K / L / Z / R / S in Figure 16 s) Steps A / B / J / M / N / Z / R / S in Figure 16 t) Steps A / B / J / T / W / Q / S in Figure 16 u) Steps A / B / J / T / U / X / Q / S in Figure 16 v) Steps A / B / J / T / V / Y / Q / S in Figure 16 w) Steps A - G in Figure 17 x) Step O / C in Figure 19 or Steps D / P / G / H y) Steps A / B / C / G / H / I / J / K / L / M in Figure 21 z) Steps K / L / H in Figure 21 аа) Steps I / J / H in Figure 21 bb); Step I / G / C in Figure 21 cc) Steps A / B / C in Figure 21 dd) Steps A / M / H in Figure 21 ee) Steps A / B / C in Figure 22 ff) Steps A / E / F / G / AA in Figure 20

[0157] Any of the disclosed HMD synthesis pathways can be used to generate a genetically modified microorganism that produces that pathway, an intermediate of the pathway, or a product as disclosed. For example, the genetically modified microorganism can have an HMD pathway that contains at least one exogenous nucleic acid encoding at least one enzyme of the HMD synthesis pathway that is expressed in an amount sufficient to produce at least one HMD carbonate and / or carbamate compound. The genetically modified microorganism can have an HMD pathway that contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven exogenous nucleic acids encoding enzymes of the HMD synthesis pathway. The exogenous nucleic acid can encode a polypeptide, where the polypeptide is an enzyme or a protein, and the protein can convert a desired substrate, intermediate, and produce a product of the desired HMD synthesis pathway.

[0158] In some embodiments, the genetically modified microorganism having an HMD synthesis pathway has an HMD pathway that contains at least one exogenous nucleic acid of an HMD enzyme and can also contain an exogenous nucleic acid encoding an enzyme of carbonic anhydrase.

[0159] For example, the HMD synthesis pathway can include 3-oxoadipyl-CoA, adipic acid semialdehyde, 6-aminocaproic acid (6-ACA), 6-ACA semialdehyde, 2-aminopimelate, 3,6-dihydroxyhexanoyl-CoA, and homolyseine.

[0160] In some embodiments, the HMD synthesis pathway can include the following enzymes: 3-oxoadipyl-CoA thiolase, 6-ACA transaminase or dehydrogenase, 6-aminocaproyl-CoA reductase, 6-ACA reductase, adipyl-CoA reductase, adipic acid reductase, 6-hydroxy-3-oxohexanoyl-CoA dehydrogenase, 2-aminopimelic acid decarboxylase, and homolyseine decarboxylase.

[0161] In other embodiments, the HMD synthetic pathway may include the following enzymes and substrate-product pairs: 3-oxoadipyl-CoA thiolase, which acts on succinyl-CoA and acetyl-CoA to form 3-oxoadipyl-CoA; 6-ACA transaminase, which acts on adipyl-CoA to form 6-ACA; 6-aminocaproyl-CoA reductase, which acts on 6-aminocaproyl-CoA to form 6-ACA semialdehyde; 6-ACA reductase, which acts on 6-ACA to directly convert it to 6-ACA semialdehyde; adipyl-CoA reductase, which acts on adipyl-CoA to form adipic semialdehyde; adipate reductase, which acts on adipate to directly convert it to adipic semialdehyde; 6-hydroxy-3-oxohexanoyl-CoA dehydrogenase, which reduces 6-hydroxy-3-oxohexanoyl-CoA to form 3,6-dihydroxyhexanoyl-CoA; 2-aminopimelate decarboxylase, which decarboxylates 2-aminopimelate to form 6-ACA; and homolysine decarboxylase, which decarboxylates homolysine to form HMDA.

[0162] In some embodiments, the microorganism can produce the desired diamine (e.g., HMD) via a desired synthetic pathway that may include: (a) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase and 6-ACA-CoA reductase, or 6-ACA reductase, HMDA transaminase or dehydrogenase; (b) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (c) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA transferase, hydrolase or transferase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (d) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA transferase, hydrolase or transferase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (e) 3-oxoadipyl-CoA thiolase, 3-oxoadipic acid dehydrogenase, 3-hydroxyadipic acid dehydratase, 5-carboxy-2-pentenoic acid reductase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (f) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (g) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (h) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (i) 4-hydroxy-2-oxoheptane-1,7-dioate (HODH aldolase); 2-oxohepta-4-ene-1,7-dioate (OHED) hydratase; OHED formate lyase and pyruvate formate lyase activating enzyme or OHED dehydrogenase; 2,3-dehydroadipyl-CoA reductase; adipyl-CoA dehydrogenase; or adipic acid semialdehyde aminotransferase or adipic acid semialdehyde oxidoreductase (amination); (j) β-ketothiolase or acetyl-CoA carboxylase and acetoacetyl-CoA synthetase, 3-hydroxyacyl CoA dehydrogenase or 3-oxoacyl-CoA reductase, enoyl-CoA hydratase, and trans-2-enoyl-CoA reductase for generating hexanoyl-CoA, one or more thioesterases, aldehyde dehydrogenase, or butanal dehydrogenase, hexanal or hexanoates produced by the host; one or more monooxygenases, alcohol dehydrogenase, aldehyde dehydrogenase, 6-hydroxyhexanoate dehydrogenase, 5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase, 6-oxohexanoate dehydrogenase, or 7-oxoheptanoate dehydrogenase, adipic acid or adipic acid semialdehyde produced by the host; one or more monooxygenases, transaminase, 6-hydroxyhexanoate dehydrogenase, 5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase, and alcohol dehydrogenase, 6-aminohexanoate produced by the host; one or more carboxylate reductase, ω-transaminase, deacetylase, N-acetyltransferase, alcohol dehydrogenase. Such pathways are disclosed in UK Patent No. 20140186302; (k) acetyltransferase or thiolase for forming 6-hydroxy-3-oxo-hexanoyl-CoA, 6-hydroxy-3-oxo-hexanoyl-CoA dehydrogenase, 3,4-dihydroxyhexanoyl-CoA dehydrogenase, 6-hydroxy-2-hexanoyl-CoA reductase, 6-hydroxyhexanoyl-CoA hydrolase for forming 6-ACA, 6-hydroxycaproic acid dehydrogenase and transaminase for forming HMDA. Such pathways are disclosed in International Publication No. WO2014 / 047407A1; (l) Homocitrate synthase, homaconitase and homoisocitrate dehydrogenase for forming 2-ketopimelate, 2-ketodecarboxylase that catalyzes the conversion of α-ketopimelate to adipic semialdehyde, 2-aminopimelate transferase that catalyzes the conversion of α-ketopimelate to 2-aminopimelate, 2-aminopimelate decarboxylase for decarboxylating 2-aminopimelate to form 6-ACA, aldehyde dehydrogenase that catalyzes the conversion of 6-ACA to 6-aminohexanal and aminotransferase that catalyzes the conversion of 6-aminohexanal to 6-hexamethylenediamine. Such a pathway is disclosed in International Publication No. WO / 2010 / 068944; and (m) Glutamyl-CoA transferase and / or ligase, β-ketothiolase, 3-oxo-6-aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA reductase, 6-amino-7-carboxyhepta-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde-forming), 2-amino-7-oxoheptanoate aminotransferase and / or aminating oxidoreductase, homolysine decarboxylase, 6-aminopimeloyl-CoA hydrolase, transferase and / or ligase, 2-aminopimelate decarboxylase. Such a pathway is disclosed in International Publication No. WO2010 / 129936.

[0163] In some embodiments, the HMD synthesis pathway comprises at least one enzyme such as 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentanoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 3-oxoadipyl-CoA:acyl CoA transferase, 3-oxoadipic acid dehydrogenase, 3-hydroxyadipic acid dehydratase, 5-carboxy-2-pentanoate reductase, adipyl-CoA transferase, ligase, hydrolase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase, adipic acid reductase, 6-ACA transaminase or dehydrogenase, or 6-ACA reductase, and a nucleic acid encoding one or more of the enzymes.

[0164] Suitable microorganisms that can be used as hosts for containing one or more exogenous nucleic acids of the HMD synthesis pathway include, for example, prokaryotes such as bacteria, and eukaryotes such as fungi (e.g., yeast), or any other microorganisms suitable for fermentation processes or capable of withstanding pH conditions of pH 4 or less.

[0165] In some embodiments, the genetically modified microorganism is from the order Aeromonadales, family Succinivibrionaceae, including the genus Escherichia, Klebsiella, Anaerobiospirillum; the order Pasteurellales, family Pasteurellaceae, including the genus Actinobacillus and Mannheimia; the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, family Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and Streptomyces, respectively; the order Rhodospirillales, family Acetobacteraceae, including the genus Gluconobacter; the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, family Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and Lactococcus, respectively; the order Clostridiales, family Clostridiaceae, including the genus Clostridium; the order Pseudomonadales, family Pseudomonadaceae, including the genus Pseudomonas, Alkaliphilus, Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis, and Hyphomicrobium; the order Saccharomycetales, family Saccaromycetaceae, including the genus Saccharomyces, Kluyveromyces, and Pichia; the order Saccharomycetales, family Dipodascaceae, including the genus Yarrowia; the order Schizosaccharomycetales, family Schizosaccaromycetaceae, including the genus Schizosaccharomyces; the order Eurotiales, family Trichocomaceae, including the genus Aspergillus;and is Mucoraceae family of the order Mucorales including the genus Rhizopus;

[0166] In other embodiments, the genetically engineered microorganisms include, without limitation, species of host bacteria including Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida, Bacillis pseudofirmus, Bacillus halodurans, Bacillus alcalophilus, Clostridium paradoxum, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and Issatchenkia orientalis.Some preferred alkaliphilic bacteria are: Bacillis pseudofirmus, Bacillus halodurans, Bacillus alcalophilus, Clostridium paradoxum, Arthrospira platensis, Bacillus clausii, Oceanobacillus iheyensis, Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Bacillus selentireducens, Desulfovibrio alkaliphiles, Dethiobacter alkaliphiles, Thioalkalivibrio sp., Natranaerobius thermophilus, Alkalilimnicola ehrlichii, and Desulfonatronospira thiodismutans.

[0167] In some embodiments, the fungal or yeast species can be selected from, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and Issatchenkia orientalis.

[0168] In some embodiments, the genetically engineered microorganism is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Bacillis pseudofirmus, Bacillus halodurans, Bacillus alcalophilus, Clostridium paradoxum, Saccharomyces cerevisiae.

[0169] For example, E. coli is a particularly useful host organism because it is a well-characterized microorganism suitable for genetic engineering. Other particularly useful host organisms include yeasts such as Saccharomyces cerevisiae. In some embodiments, the genetically engineered microorganism is modified to have improved alkali tolerance.

[0170] Using adaptive evolution, alkali tolerance can be introduced into organisms that normally do not exhibit alkali tolerance. Cells are grown in a gradually increasing pH state until the cellular mechanism is adapted to optimally grow at the desired high pH (alkaline pH). Typically, a small amount of cells that are still in the exponential growth phase are transferred to fresh medium at a given pH until they reach a given biomass concentration. These cells are then diluted in fresh medium while maintaining a gradually increasing pH. This process selects for cells that are more suitable for growth at higher pH. The process of transferring a small amount of cells in the exponential growth phase to fresh medium at a higher pH is continued until the cells evolve to grow at the highest pH level.

[0171] Adaptive evolution has been used for the evolution of strains that grow on non-native substrates (Lee and Palsson, Appl Environ Microbiol. 2010 Jul;76(13):4158-68, Adaptive evolution of Escherichia coli K-12 MG1655 during growth on a Nonnative carbon source, L-1,2-propanediol), for improvement of salt tolerance (Ketola and Hiltunen, Ecol Evol. 2014 Oct;4:3901-8, Rapid evolutionary adaptation to elevated salt concentrations in pathogenic freshwater bacteria Serratia marcescens), for improvement of product tolerance (Kildegaard KR et al., Metab Eng. 2014 Sep 28;26C:57-66, Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance), for growth at high temperatures (Sandeberg et al., Mol Biol Evol. 2014 Oct;31(10):2647-62, Evolution of Escherichia coli to 42 °C and subsequent genetic engineering reveals adaptive mechanisms and novel mutations), for evolution of aerobic fermentation (Portnoy et al., Appl Environ Microbiol. 2008 Dec;74(24), Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain), or for several other purposes.

[0172] For example, one such route involves the activation of 6-aminocaproate to 6-aminocaproyl-CoA by a transferase or synthetase enzyme (Figure 10, step Q or R), followed by the formation of caprolactam by the spontaneous cyclization of 6-aminocaproyl-CoA (Figure 10, step T). Another route described involves the formation of HMD by the activation of 6-aminocaproate to 6-aminocaproyl-CoA (Figure 10, step Q or R), followed by reduction (Figure 10, step U) and amination (Figure 10, step V or W). 6-Aminocaproic acid can alternatively be activated to 6-aminocaproyl-phosphate rather than 6-aminocaproyl-CoA. 6-Aminocaproyl-phosphate cyclizes spontaneously to form caprolactam. Alternatively, 6-aminocaproyl-phosphate can be reduced to 6-aminocaproic acid semialdehyde. This can then be converted to HMD as depicted in Figures 10 and 11. In either case, the amination reaction must occur relatively rapidly to minimize the spontaneous formation of the cyclic imine of 6-aminocaproic acid semialdehyde. The binding or scaffold of the enzymes involved represents a potentially powerful option to ensure that the 6-aminocaproic acid semialdehyde intermediate is effectively channeled from the reductase enzyme to the amination enzyme.

[0173] Another option to minimize and even eliminate the formation of cyclic imines or caprolactam during the conversion of 6-aminocaproic acid to HMD involves adding a functional group (e.g., acetyl, succinyl) that protects against the cyclization of 6-aminocaproic acid to the amino group of 6-aminocaproic acid. This is similar to ornithine formation from L-glutamate in Escherichia coli. Specifically, glutamate is first converted to N-acetyl-L-glutamate by N-acetylglutamate synthase. N-acetyl-L-glutamate is then activated to N-acetylglutamyl-phosphate, which forms N-acetyl-L-ornithine by reduction and transamination. Subsequently, the acetyl group is removed from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylase to form L-ornithine. Such a route is essential because the formation of glutamate-5-phosphate from glutamate and the subsequent reduction to glutamate-5-semialdehyde leads to (S)-1-pyrroline-5-carboxylate. (S)-1-pyrroline-5-carboxylate is a cyclic imine that forms spontaneously from glutamate-5-semialdehyde. When forming HMD from 6-aminocaproic acid, it can involve the steps of forming acetyl-6-aminocaproic acid by acetylation of 6-aminocaproic acid, activation of the carboxylic acid group by CoA or a phosphate group, reduction, amination, and deacetylation.

[0174] Note that 6-aminocaproate can be formed from various starting molecules. For example, the carbon backbone of 6-aminocaproate can be derived from succinyl-CoA and acetyl-CoA, as depicted in FIG. 10 and also as depicted in FIGS. 2, 3, and 8. Alternatively, 6-aminocaproate can be derived from alpha-ketoadipate, which is converted to adipyl-CoA (see FIG. 9), and as shown in FIG. 10, adipyl-CoA is converted to 6-aminocaproate.

[0175] Figure 11 provides two additional metabolic pathways starting from 4-aminobutyryl-CoA and acetyl-CoA and leading to 6-aminocaproate or 6-aminocaproyl-CoA. The first route involves the formation of 3-oxo-6-aminohexanoyl-CoA by condensation of 4-aminobutyryl-CoA and acetyl-CoA (step A), followed by reduction (step B), dehydration (step C), and reduction (step D) to form 6-aminocaproyl-CoA. 6-Aminocaproyl-CoA can be converted to 6-aminocaproate by a transferase enzyme (step K), a synthetase enzyme (step L), or a hydrolase enzyme (step M). Alternatively, 6-aminocaproyl-CoA can be converted to caprolactam by spontaneous cyclization (step Q), or can be converted to HMD followed by reduction (step N) and amination (steps O or P). The second route depicted in Figure 11 involves the formation of 3-oxo-6-aminohexanoyl-CoA by condensation of 4-aminobutyryl-CoA and acetyl-CoA (step A), followed by conversion to 3-oxo-6-aminohexanoate by a transferase (step E), a synthetase (step F), or a hydrolase (step G). 3-Oxo-6-aminohexanoate is then reduced (step H), dehydrated (step I), reduced (step J), and 6-aminocaproate is formed.

[0176] The starting molecule, 4-aminobutyryl-CoA, can be formed from a variety of common major metabolites. For example, glutamate can be decarboxylated to 4-aminobutyrate, which can then be activated by CoA-transferase or synthetase to 4-aminobutyryl-CoA. Alternatively, succinic semialdehyde is formed either by reduction of succinyl-CoA or decarboxylation of alpha-ketoglutarate, and prior to being activated by CoA-transferase or synthetase to form 4-aminobutyryl-CoA, aminotransfer to 4-aminobutyrate can occur. Note that 4-aminobutyryl-CoA, and some intermediates in the pathway from 4-aminobutyryl-CoA to 6-aminocaproyl-CoA, can cyclize spontaneously to the corresponding lactam. Therefore, the attachment of a protecting functional group to the terminal amino group of 4-aminobutyryl-CoA and / or some amino-CoA intermediates can be used to minimize the formation of unwanted cyclic by-products. In this case, although two additional steps (e.g., acetylase and deacetylase) can be added to the pathway, the same general set of conversions depicted in Figure 11 will apply.

[0177] All of the conversions depicted in Figures 10 - 11 are classified into 12 general categories of conversions shown in Table 8. Below, an explanation of some candidate genes that have been biochemically characterized in each category is provided. The genes specifically listed are those that, when cloned and expressed, can be applied to catalyze the appropriate conversions in Figures 10 - 11.

[0178] [Table 3]

[0179] 1.1.1.a Oxidoreductase. The four conversions depicted in FIGS. 10 and 11 require an oxidoreductase that converts a ketone functionality to a hydroxyl group. Step B in both FIGS. 10 and 11 involves the conversion of 3-oxoacyl-CoA to 3-hydroxyacyl-CoA. Step H in both FIGS. 1 and 2 involves the conversion of 3-oxoacid to 3-hydroxyacid.

[0180] Typical examples of enzymes that can convert 3-oxoacyl-CoA molecules, such as 3-oxoadipyl-CoA and 3-oxo-6-aminohexanoyl-CoA, to 3-hydroxyacyl-CoA molecules, such as 3-hydroxyadipyl-CoA and 3-hydroxy-6-aminohexanoyl-CoA, respectively, include enzymes whose native physiological role is fatty acid beta oxidation or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes of E. coli encoded by fadB and fadJ function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71:403-411 (1981)). Furthermore, phaC of Pseudomonas putida U (Olivera et al., Proc. The gene products encoded in Natl. Acad. Sci. USA 95:6419-6424 (1998) and the gene product encoded in paaC of Pseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007)) catalyze the reverse reaction of step B in Figure 10, namely, the formation of 3-oxoadipyl-CoA by the oxidation of 3-hydroxyadipyl-CoA, in the catabolism of phenylacetate or styrene. Note that the reactions catalyzed by these enzymes are reversible. Furthermore, in E. coli, considering that paaH is close to the phenylacetate degradation operon (Nogales et al., Microbiology 153:357-365 (2007)) and that the paaH mutant cannot grow on phenylacetate (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003)), the paaH gene of E. coli is predicted to encode 3-hydroxyacyl-CoA dehydrogenase.

[0181]

Table 4

[0182] Further typical examples of oxo-reductases that can convert 3-oxoacyl-CoA molecules to the corresponding 3-hydroxyacyl-CoA molecules include 3-hydroxybutyryl-CoA dehydrogenase. The enzyme derived from Clostridium acetobutylicum encoded by hbd has been cloned and functionally expressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807 (1989)). Further gene candidates include Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) of Clostridium kluyveri (Hillmer et include those in al., FEBS Lett. 21:351-354 (1972), and HSD17B10 in Bos taurus (Wakil et al., J. Biol. Chem. 207:631-638 (1954)). Another gene candidate for reducing acetoacetyl-CoA to 3-hydroxybutyryl-CoA is phbB from Zoogloea ramigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol 61:297-309 (2006)). The former gene candidate is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al., Mol. Microbiol. 3:349-357 (1989)), and the gene has been expressed in E. coli. Studies on the substrate specificity of this gene have led to the conclusion that in addition to acetoacetyl-CoA, 3-oxopropionyl-CoA can also be accepted as a substrate (Ploux et al., supra).

[0183]

Table 5

[0184] Several similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)).

[0185]

Table 6

[0186] Various alcohol dehydrogenases are promising candidates for converting 3-oxoadipate to 3-hydroxyadipate (Figure 10, step H) or 3-oxo-6-aminohexanoate to 3-hydroxy-6-aminohexanoate (Figure 11, step H). Two such enzymes capable of converting oxo acids to hydroxy acids are encoded by the malate dehydrogenase (mdh) gene and the lactate dehydrogenase (ldhA) gene of E. coli. Furthermore, lactate dehydrogenase from Ralstonia eutropha has been shown to exhibit high activity against substrates of various chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoate, and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)). The conversion of alpha-ketoadipate to alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase. The enzyme has been reported to be found in rat and human placentas (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). Further candidates for these steps are mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from human heart, which has been cloned and characterized (Marks et al., J. Biol. Chem. 267:15459-15463 (1992)). This enzyme is a dehydrogenase that acts on 3-hydroxy acids. Another typical example of alcohol dehydrogenase is the conversion of acetone to isopropanol as shown in C. beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993) and T. brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al., Biochemistry 28:6549-6555 (1989)).

[0187]

Table 7

[0188] 1.2.1.b Oxidoreductase (from acyl-CoA to aldehyde). The conversion of adipyl-CoA to adipic semialdehyde (step N in Figure 10) and the conversion of 6-aminocaproyl-CoA to 6-aminocaproic semialdehyde (step U in Figure 10; step N in Figure 11) require acyl-CoA dehydrogenase, which can reduce acyl-CoA to the corresponding aldehyde. Typical examples of genes encoding such enzymes include acrl of Acinetobacter calcoaceticus encoding fatty acid acyl-CoA reductase (Reiser et al., J. Bacteriology 179:2969-2975 (1997)), the fatty acid acyl-CoA reductase of Acinetobacter sp. M-1 (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and the CoA-dependent and NADP-dependent succinic semialdehyde dehydrogenase encoded by the sucD gene of Clostridium kluyveri (Sohling et al., J. Bacteriol. 178:871-880 (1996)) is included. SucD of P. gingivalis is another succinic semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). Since it has been demonstrated that the enzyme encoded by bphG acylates acetaldehyde dehydrogenase in Pseudomonas sp., which oxidizes and acylates acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, and formaldehyde, it is another candidate (Powlowski et al., J Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize isobutyraldehyde, a branched-chain compound, to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett. 27:505-510 (2005)).

[0189]

Table 8

[0190] A further type of enzyme that converts acyl-CoA to the corresponding aldehyde is malonyl-CoA reductase, which converts malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaea (Berg et al., supra; Thauer R. K., Science 318:1732-1733 (2007)). This enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol. 184: 2404-2410 (2002)). This enzyme is encoded by Msed-0709 in Metallosphaera sedula (Alber et al., supra; Berg et al., supra). The gene encoding malonyl-CoA reductase from Sulfolobus tokodaii has been cloned and heterologously expressed in E. coli (Alber et al., supra). This enzyme has been shown to catalyze the conversion of methylmalonyl-CoA to the corresponding aldehyde (WO / 2007 / 141208). The aldehyde dehydrogenase functionality of these enzymes is similar to that of the bifunctional dehydrogenase from Chloroflexus aurantiacus, but the sequence similarity is low. Both candidates for the malonyl-CoA reductase enzyme have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme that catalyzes the reduction of aspartyl-4-phosphate to aspartic semialdehyde and the concomitant dephosphorylation. Further gene candidates can be found in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius, as well as other organisms listed below, by sequence homology to the protein.Another candidate for Co-A acylated aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE, which encodes acetaldehyde dehydrogenase in Salmonella typhimurium and E. coli (Toth et al., supra).

[0191]

Table 9

[0192] 1.3.1.a Oxidoreductases acting on CH-CH donors. Referring to Figure 10, step D refers to the conversion of 5-carboxy-2-pentanoyl-CoA to adipyl-CoA by 5-carboxy-2-pentenoyl-CoA reductase. Referring to Figure 11, step D refers to the conversion of 6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA. Enoyl-CoA reductase enzymes are suitable for any of these conversions. One typical example of enoyl-CoA reductase is the gene product of bcd from C. acetobutyli (Boynton et al., J Bacteriol. 178:3015-3024 (1996); Atsumi et al., Metab. Eng. 2008 10(6):305-311 (2008)(Epub Sep. 14, 2007), which catalyzes the reduction of crotonyl-CoA to butyryl-CoA in vivo. The activity of this enzyme can be enhanced by expressing bcd along with the expression of the etfAB gene of C. acetobutylicum, which encodes an electron transfer flavoprotein. A further candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence with its mitochondrial targeting leader sequence removed has been cloned into E. coli and is an active enzyme (Hoffmeister et al., supra). This approach is well known to those skilled in the art in the field of expressing eukaryotic genes, especially eukaryotic genes having leader sequences that can target gene products to specific intracellular compartments, in prokaryotes. TDE0597 from the prokaryote Treponema denticola, a homolog closely related to this gene, is a third enoyl-CoA reductase that has been cloned and expressed in E. coli (Tucci et al., FEBS Letters 581:1561-1566 (2007)).

[0193]

Table 10

[0194] In both steps J of FIGS. 10 and 11, a 2-enoyl-CoA reductase enzyme is required. 2-Enoyl-CoA reductase (EC 1.3.1.31) is known to catalyze the NAD(P)H-dependent reduction of a wide range of α,β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). 2-Enoyl-CoA reductase is encoded by enr in several species of Clostridia, including C. tyrobutyricum and C. thermoaceticum (now called Moorella thermoaceticum) (Rohdich et al., supra) (Giesel et al., Arch Microbiol 135:51-57 (1983)). In the published genome sequence of C. kluyveri, nine coding sequences for enoyl-CoA reductase have been reported, and one of these has been characterized (Seedorf et al., Proc. Natl. Acad. Sci. USA, 105:2128-2133 (2008)). Both the enr gene from C. tyrobutyricum and the enr gene from C. thermoaceticum have been cloned, sequenced, and shown to be 59% identical to each other. The former gene has also been found to have approximately 75% similarity to the gene characterized in C. kluyveri (Giesel et al., supra). Based on the results of these sequences, enr has been reported to be very similar to dienoyl-CoA reductase (fadH) in E. coli (Rohdich et al., supra). The enr gene of C. thermoaceticum has also been expressed in an enzymatically active form in E. coli (Rohdich et al., supra).

[0195] [Table 11]

[0196] 1.4.1.a Oxidoreductases acting on amino acids. Figure 10 depicts two reductive aminations. Specifically, step P in Figure 10 involves the conversion of adipic acid semialdehyde to 6-aminocaproate, and step W in Figure 10 causes the conversion of 6-aminocaproic acid semialdehyde to hexamethylenediamine. The latter conversion is also required in step P of Figure 11.

[0197] Most oxidoreductases acting on amino acids use NAD+ or NADP+ as acceptors and catalyze the oxidative deamination of alpha-amino acids. However, this reaction is typically reversible. Typical examples of oxidoreductases acting on amino acids include glutamate dehydrogenase (which deaminates), encoded by gdhA, leucine dehydrogenase (which deaminates), encoded by ldh, and aspartate dehydrogenase (which deaminates), encoded by nadX. The gdhA gene product from Escherichia coli (McPherson et al., Nucleic. Acids Res. 11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273 (1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles 1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998); Lebbink et al., J. Mol. Biol. 289:357-369 (1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2-oxoglutarate and ammonia, where NADP(H), NAD(H), or both are preferred respectively. The ldh gene of Bacillus cereus encodes the LeuDH protein, It has a broad substrate range including leucine, isoleucine, valine and 2-aminobutyrate (Stoyan et al., J. Biotechnol 54:77-80 (1997); Ansorge et al., Biotechnol Bioeng. 68:557-562 (2000)). The nadX gene from Thermotoga maritime encoding aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al., J. Biol. Chem. 278:8804-8808 (2003)).

[0198] [Table 12]

[0199] The lysine 6-dehydrogenase (deaminating) encoded by lysDH catalyzes the oxidative deamination of the ε-amino group of L-lysine to form 2-aminoadipic acid-6-semialdehyde, which then cyclizes non-enzymatically to form Δ1-piperidine-6-carboxylate (Misono et al., J. Bacteriol. 150:398-401 (1982)). Typical enzymes can be found in Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem 106:76-80 (1989); Misono et al., supra), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)). Considering the structural similarity between adipic acid semialdehyde and 2-aminoadipic acid-6-semialdehyde, these enzymes are particularly good candidates for the conversion of adipic acid semialdehyde to 6-aminocaproate.

[0200]

Table 13

[0201] 2.3.1.b Acyltransferase. Referring to FIG. 10, step A involves 3-oxoadipyl-CoA thiolase, or equivalently, succinyl CoA:acetyl CoA acyltransferase (β-ketothiolase). The gene products encoded by pcaF in Pseudomonas B13 strain (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., supra), paaE in Pseudomonas fluorescens ST (Di Gennaro et al., supra), and paaJ from E. coli (Nogales et al., supra) catalyze the conversion of 3-oxoadipyl-CoA to succinyl-CoA and acetyl CoA during the degradation of aromatic compounds such as phenylacetate or styrene. Since β-ketothiolase enzymes catalyze reversible conversions, these enzymes can be used for the synthesis of 3-oxoadipyl-CoA. For example, the ketothiolase phaA from R. eutropha binds two molecules of acetyl-CoA to produce acetoacetyl-CoA (Sato et al., J Biosci Bioeng 103:38-44 (2007)). Similarly, in R. eutropha, it has been reported that β-ketothiolase (bktB) catalyzes the condensation of acetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA (Slater et al., J. Bacteriol. 180:1979-1987 (1998)). In addition to the possibility of retaining 3-oxoadipyl-CoA thiolase activity, all such enzymes, in their native form or once appropriately engineered, have been shown to be excellent candidates for condensing 4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (step A in FIG. 11).

[0202]

Table 14

[0203] 2-Amino-4-oxopentanoic acid (AKP) thiolase enzyme or AKP thiolase (AKPT) enzyme is a further candidate for performing step A in FIGS. 10 and 11. AKPT is a pyridoxal phosphate-dependent enzyme involved in ornithine degradation in Clostridium sticklandii (Jeng et al., Biochemistry 13:2898-2903 (1974); Kenklies et al., Microbiology 145:819-826 (1999)). A gene cluster encoding the alpha and beta subunits of AKPT (i.e., -2 (ortA) and -3 (ortB)) has been identified in recent years, and the biochemical properties of this enzyme have been characterized (Fonknechten et al., J. Bacteriol. In Press (2009)). This enzyme is operable in both directions and inherently reacts with the D-isomer of alanine. AKPT from Clostridium sticklandii has been characterized, but the protein sequence has not yet been published. Enzymes with high sequence homology have been found in Clostridium difficile, Alkaliphilus metalliredigenes QYF, Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis MB4 (Fonknechten et al., supra).

[0204]

Table 15

[0205] 2.6.1.a Aminotransferase. In steps O of FIGS. 10 and 11 and step V of FIG. 10, the transfer of an amino group from 6-aldehyde to an amine is required. These aminotransfers can be catalyzed by gamma-aminobutyric acid aminotransferase (GABA aminotransferase). One E. coli GABA aminotransferase is encoded by gabT and transfers an amino group from glutamate to the terminal aldehyde of succinyl semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042 (1990)). The gene product of puuE catalyzes another 4-aminobutyric acid aminotransferase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)). GABA aminotransferases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); Scott et al., J. Biol. Chem. 234:932-936 (1959)).

[0206]

Table 16

[0207] Additional enzyme candidates include putrescine aminotransferase or other diamine aminotransferases. Such enzymes are particularly well-suited for performing the conversion from 6-aminocaproic acid semialdehyde to hexamethylenediamine. E. The putrescine aminotransferase of E. coli is encoded by the ygjG gene, and the resulting enzyme was also able to transfer the amino groups of cadverine and spermidine (Samsonova et al., BMC Microbiol 3:2 (2003)). In addition, the activity of this enzyme towards 1,7-diaminoheptane and the activity of this enzyme with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutyrate) have been reported (Samsonova et al., supra; Kim, K. H., J Biol Chem 239:783-786 (1964)). The putrescine aminotransferase with pyruvate as the amino acceptor, which has higher activity than alpha-ketoglutarate, is the spuC gene of Pseudomonas aeruginosa (Lu et al., J Bacteriol 184:3765-3773 (2002)).

[0208]

Table 17

[0209] 1. Yet another candidate enzyme includes beta-alanine / alpha-ketoglutarate aminotransferase, which produces malonate semialdehyde from beta-alanine (WO08027742). The gene product of SkPYD4 in Saccharomyces kluyveri selectively uses beta-alanine as an amino group donor (Andersen et al., FEBS. J. 274:1804-1817 (2007)). SkUGA1 encodes a homolog of the GABA aminotransferase UGA1 in Saccharomyces cerevisiae (Ramos et al., Eur. J. Biochem., 149:401-404 (1985)), while SkPYD4 encodes an enzyme involved in both beta-alanine amino transfer and GABA amino transfer (Andersen et al., supra). 3-Amino-2-methylpropionate aminotransferase catalyzes the transfer of 3-amino-2-methylpropionate from methylmalonate semialdehyde. This enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat (Tamaki et al, Methods Enzymol, 324:376-389 (2000)).

[0210]

Table 18

[0211] 2.8.3.a Coenzyme-A transferase. CoA transferase catalyzes reversible reactions involving the transfer of the CoA moiety from one molecule to another. For example, step E in Figure 10 is catalyzed by 3-oxoadipyl-CoA transferase. In this step, 3-oxoadipate is formed by the transfer of the CoA group from 3-oxoadipyl-CoA to succinate, acetate, or another CoA acceptor. In step E of Figure 11, it causes the transfer of the CoA moiety from 3-oxo-6-aminohexanoyl-CoA, another 3-oxoacyl-CoA. One candidate enzyme for these steps is the 2-unit enzyme encoded by pcaI and pcaJ of Pseudomonas, which has been shown to have 3-oxoadipyl-CoA / succinate transferase activity (Kaschabek et al., supra). Similar enzymes based on homology are present in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces coelicolor. Additional typical succinyl-CoA:3-oxoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein. Expr. Purif. 53:396-403 (2007)).

[0212]

Table 19

[0213] 3-oxoacyl-CoA transferase, which utilizes acetate as a CoA acceptor, is acetylacetyl-CoA transferase and is encoded by the atoA (alpha subunit) and atoD (beta subunit) genes of E. coli (Vanderwinkel et al., Biochem. Biophys. Res Commun. 33:902-908 (1968); Korolev et al., Acta Crystallogr. D Biol Crystallogr. 58:2116-2121 (2002)). This enzyme has also been shown to transfer the CoA moiety from various branched-chain and straight-chain acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra), and butyrate (Vanderwinkel et al., supra), to acetate. Similar enzymes are present in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl Environ Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583 (1990)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

[0214]

Table 20

[0215] The above-mentioned enzymes may also exhibit the desired activity towards adipyl-CoA and adipate (Figure 10, step K), or towards 6-aminocaproate and 6-aminocaproyl-CoA (Figure 10, step Q; Figure 2, step K). Nevertheless, additional typical transferase candidates have been shown to be catalyzed by the gene products of cat1, cat2, and cat3 of Clostridium kluyveri, which exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activities, respectively (Seedorf et al., supra; Sohling et al., Eur. J. Biochem. 212:121-127 (1993); Sohling et al., J. Bacteriol. 178:871-880 (1996)).

[0216]

Table 21

[0217] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from the anaerobic bacterium Acidaminococcus fermentans reacts with the dicarboxylic acid glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has a reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA, and acrylyl-CoA (Buckel et al., Eur. J. Biochem. 118:315-321 (1981)). This enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).

[0218]

Table 22

[0219] 3.1.2.a Thiolester hydrolase (CoA-specific). Some eukaryotic acetyl-CoA hydrolases have broad substrate specificities and are therefore suitable candidates for enzymes to hydrolyze 3-oxoadipyl-CoA, adipyl-CoA, 3-oxo-6-aminohexanoyl-CoA, or 6-aminocaproyl-CoA (Steps G and M in FIGS. 10 and 11). For example, an enzyme derived from the brain of Rattus norvegicus (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA, and malonyl-CoA.

[0220] [Table 23]

[0221] Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase, which has been reported to effectively catalyze the conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during the degradation of valine (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al., supra; Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and hibch of Homo sapiens (Shimomura et al., supra). Candidate genes based on sequence homology include hibch of Saccharomyces cerevisiae and BC 2292 of Bacillus cereus.

[0222] [Table 24]

[0223] Another candidate hydrolase shows activity against glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA and dodecanedioyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132 (2005)), is acot8, a human dicarboxylic acid thioesterase, and tesB, the most closely related E. coli homolog, which can also hydrolyze a broad range of CoA thioesters (Naggert et al., J Biol Chem 266:11044-11050 (1991)). Similar enzymes have been identified in rat liver (Deana R., Biochem Int 26:767-773 (1992)).

[0224]

Table 25

[0225] Other potential E. coli thiolester hydrolases include tesA (Bonner et al., J Biol Chem 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); Zhuang et al., FEBS Lett 516:161-163 (2002)), paaI (Song et al., J Biol Chem 281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol 189:7112-7126 (2007)).

[0226]

Table 26

[0227] 6.3.1.a / 6.3.2.a Amid synthase / peptide synthase. The direct conversion of 6-aminocaproic acid to caprolactam (step S in Figure 10; step R in Figure 11) requires the formation of an intramolecular peptide bond. Ribosomes that assemble amino acids into proteins during translation are the most naturally abundant peptide bond-forming catalysts. Nonribosomal peptide synthetases are peptide bond-forming catalysts that do not contain messenger mRNA (Schwarzer et al., Nat. Prod. Rep. 20:275-287 (2003)). Additional enzymes capable of forming peptide bonds include acyl-CoA synthetase from Pseudomonas chlororaphis (Abe et al., J Biol Chem 283:11312-11321 (2008)), gamma-glutamylputrescine synthase from E. coli (Kurihara et al., J Biol Chem 283:19981-19990 (2008)), and beta-lactam synthase from Streptomyces clavuligerus (Bachmann et al., Proc Natl Acad Sci USA 95:9082-9086 (1998); Bachmann et al., Biochemistry 39:11187-11193 (2000); Miller et al., Nat. Struct. Biol 8:684-689 (2001); Miller et al., Proc Natl Acad Sci USA 99:14752-14757 (2002); Tahlan et al., Antimicrob. Agents. Chemother. 48:930-939 (2004)) are included.

[0228]

Table 27

[0229] 4.2.1.a Hydrolase. Most dehydratases catalyze the α,β-elimination of water. This involves the activation of the α-hydrogen by an electron-withdrawing carbonyl, carboxylate or CoA-thiol ester group, and the removal of the hydroxyl group from the β-position. Enzymes showing activity towards substrates with an electron-withdrawing carboxylate functional group are excellent candidates for dehydrating 3-hydroxyadipate (step I in Figure 10) or 3-hydroxy-6-aminohexanoate (step I in Figure 11).

[0230] For example, the fumarase enzyme naturally catalyzes the reversible dehydration of malate to fumarate. E. coli has three fumarases: FumA, FumB, and FumC, which are regulated by growth conditions. FumB is oxygen-sensitive and active only under anaerobic conditions. FumA is active under microaerobic conditions, and FumC is the only enzyme active in aerobic growth (Tseng et al., J Bacteriol 183:461-467 (2001); Woods et al., Biochim Biophys Act a 954:14-26 (1988); Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Further enzyme candidates are found in Campylobacter jejuni (Smith et al., Int. J Biochem. Cell Biol 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequence homology include fuml from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).

[0231]

Table 28

[0232] Two additional dehydratase candidates are 2-(hydroxymethyl)glutamate dehydratase and dimethylmaleate dehydratase, enzymes that have been studied for their role in nicotinate catabolism in Eubacterium barkeri (Alhapel et al., Proc Natl Acad Sci USA 103:12341-6 (2006)). 2-(Hydroxymethyl)glutamate dehydratase is a [4Fe-4S]-containing enzyme that dehydrates 2-(hydroxymethyl)glutamate to 2-methylene-glutamate. This enzyme is encoded by hmd in Eubacterium barkeri (Alhapel et al., supra). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius. These enzymes are homologs of the alpha and beta subunits of [4Fe-45]-containing bacterial serine dehydratases (e.g., the E. coli enzymes encoded by tdcG, sdhB, and sdaA).

[0233]

Table 29

[0234] Dimethylmaleate hydratase (EC 4.2.1.85) is an Fe 2+ -dependent and oxygen-sensitive reversible enzyme belonging to the aconitase family that hydrates dimethylmaeate to (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857 (1984)).

[0235]

Table 30

[0236] A further enzyme candidate is 2-methylmalate dehydratase, also known as citramalate hydratase, a reversible hydrolase that catalyzes the alpha, beta-elimination of water from citramalate to form mesaconate. This enzyme has been isolated and identified in Clostridium tetanomorphum (Wang et al., J. Biol. Chem. 244:2516-2526 (1969)). The activity of this enzyme has been detected in several bacteria of the genera Citrobacter and Morganella in the context of the glutamate degradation VI pathway (Kato et al., Arch. Microbiol 168: 457-463 (1997)). The gene encoding this enzyme has not, as yet, been identified in any organism. at all.

[0237] Enzymes showing activity towards substrates with an electron-withdrawing CoA-thioester group adjacent to the alpha-hydrogen are excellent candidates for performing the dehydration of 3-hydroxyadipyl-CoA (Figure 10, step C) or 3-hydroxy-6-aminohexanoyl-CoA (Figure 11, step C). The enoyl-CoA hydratases phaA and phaB of P. putida are thought to perform the hydroxylation of the double bond during phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). paaA and paaB from P. fluorescens catalyze similar conversions (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). Finally, maoC (Park et al., J. Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., supra; Park et al., Appl. Biochem. Biotechnol 113-116: In several Escherichia coli genes, including paaA (Ismail et al., supra; Park et al., Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., supra; Park et al., Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681-686 (2004)), demonstration of enoyl-CoA hydratase functionality has been shown. The crotonase enzyme is a further candidate for dehydrating the essential 3-hydroxyacyl-CoA molecule represented in FIGS. 10 and 11. These enzymes are required for n-butanol formation in several organisms, particularly Clostridial species, and also include one step of the 3-hydroxypropionate / 4-hydroxybutyrate cycle in the thermoacidophilic archaea of the genera Sulfolobus, Acidianus, and Metallosphaera. Typical genes encoding the crotonase enzyme are found in C. acetobutylicum (Boynton et al., supra), C. kluyveri (Hillmer et al., FEBS Lett. 21:351-354 (1972)), and Metallosphaera sedula (Berg et al., supra), but the sequence of the latter gene is not known. Enoyl-CoA hydratase, which is involved in fatty acid beta-oxidation and / or various amino acid metabolisms, can also catalyze the hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA (Roberts et al., Arch. Microbiol 117:99-108 (1978); Agnihotri et al., Bioorg. Med. Chem. 11:9-20 (2003); Conrad et al., J Bacteriol. 118:103-111 (1974)).

[0238]

Table 31

[0239] 6.2.1.a Acid-thiol ligase. Steps F, L, and R in FIG. 10, and steps F and L in FIG. 11, functionally require an acid-thiol ligase or synthetase (the terms ligase, synthetase, and synthase are used interchangeably herein and refer to the same enzyme classification). Exemplary genes encoding the enzyme appear to perform these conversions and include the sucCD gene of E. coli that naturally forms the succinyl-CoA synthetase complex. This enzyme complex naturally catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP. This reaction is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Considering the structural similarities between succinate and adipate, namely that both are linear dicarboxylic acids, it is reasonable to expect that the sucCD enzyme has some activity towards adipyl-CoA.

[0240]

Table 32

[0241] Additional exemplary CoA-ligases include the rat dicarboxylate-CoA ligase whose sequence has not yet been identified (Vamecq et al., Biochemical Journal 230:683-693 (1985)), both of two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), phenylacetate-CoA ligase derived from Pseudomonas putida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and 6-carboxyhexanoate-CoA ligase derived from Bacilis subtilis (Bower et al., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidate enzymes include those from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., is acetoacetyl-CoA synthetase from Biochem Pharmacol 65:989-994 (2003), which naturally catalyzes the ATP-dependent conversion of acetoacetate to acetoacetyl CoA.

[0242]

Table 33

[0243] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of the corresponding acid of acyl-CoA esters to the concomitant ATP synthesis. A variety of enzymes with broad substrate characteristics have been described in the literature. ACD I from Archaeoglobus fulgidus encoded by AF1211 has been shown to act on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyrate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as succinyl-CoA synthetase) has been shown to accept propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates and act in both the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum shows the broadest substrate range among all characterized ACDs and reacts with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra).

[0244]

Table 34

[0245] Still other options are to use a set of enzymes having net ligase or synthetase activity. For example, phosphotransadipylase and adipate kinase enzymes are catalyzed by the gene products of buk1, buk2, and ptb from C. acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol. Microbiol. Biotechnol. 2:33-38 (2000)). The ptb gene encodes an enzyme that can convert butyryl-CoA to butyryl-phosphate, which is then converted to butyrate via both buk gene products with concomitant ATP production.

[0246]

Table 35

[0247] Enzyme-free - spontaneous cyclization. 6-Aminocaproyl-CoA cyclizes spontaneously to caprolactam, thus obviating the need for a dedicated enzyme for this step. A similar spontaneous cyclization has been observed in 4-aminobutyryl-CoA, which forms pyrrolidone (Ohsugi et al., J Biol Chem 256:7642-7651 (1981)).

[0248] Microorganisms capable of producing hexamethylenediamine from acetyl-CoA and succinyl-CoA and starting from acetyl-CoA and succinyl-CoA, as shown in FIG. 10, can also be produced. E. coli is a good host for producing non-naturally occurring microorganisms that can produce hexamethylenediamine. Although E. coli is used to illustrate this pathway, it should be understood that any microorganism can be adapted to produce such a pathway. To produce an E. coli strain designed to produce hexamethylenediamine, the nucleic acids encoding the essential enzymes are expressed in E. coli using well-known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ (NP-415915.1), paaH (NP-415913.1), and maoC (NP-415905.1) genes encoding 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydratase activities are each cloned under the PA1 / lacO promoter of the pZE13 vector (Expressys, Ruelzheim, Germany). In addition, the bcd (NP-349317.1) and eftAB (NP-349315.1 and NP-349316.1) genes encoding 5-carboxy-2-pentanoyl-CoA reductase activity are cloned under the PA1 / lacO promoter of the pZA33 vector (Expressys, Ruelzheim, Germany).Finally, clone the acrl (YP-047869.1), gabT (NP-417148.1), bioW (NP-390902.2), and ygjG (NP-417544) genes, which encode adipyl-CoA reductase (aldehyde-forming), 6-aminocaproyl-CoA reductase (aldehyde-forming), 6-aminocaproate transaminase, 6-aminocaproyl-CoA synthetase, and hexamethylenediamine transaminase activities, under the PA1 / lacO promoter of the third compatible plasmid, pZS23. pZS23 can be obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. Transform three sets of plasmids into E. coli strain MG1655 to express the proteins and enzymes required for hexamethylenediamine synthesis.

[0249] In other examples, hexamethylenediamine can be produced by a pathway for converting acetyl-CoA and 4-aminobutyryl-CoA to 6-aminocaproyl-CoA.

[0250] Other pathways for producing hexamethylenediamine are from acetyl-CoA and 4-aminobutyryl-CoA.

[0251] The paaJ (NP-415915.1), paaH (NP-415913.1), and maoC (NP-415905.1) genes encoding 3-oxo-6-aminohexanoyl-CoA thiolase, 3-oxo-6-aminohexanoyl-CoA reductase, and 3-hydroxy-6-aminohexanoyl-CoA dehydratase activities can be cloned, respectively, under the PA1 / lacO promoter of the pZE13 vector (Expressys, Ruelzheim, Germany). In addition, the bcd (NP-349317.1), etfAB (NP-349315.1 and NP-349316.1), acrl(YP-047869.1), and ygjG (NP-417544) genes encoding 6-aminohex-2-enoyl-CoA reductase, 6-aminocaproyl-CoA reductase (aldehyde-forming), and hexamethylenediamine transaminase activities are cloned under the PA1 / lacO promoter of the pZA33 vector (Expressys, Ruelzheim, Germany). Finally, to enhance the accessibility of 4-aminobutyryl-CoA, the sucD (NP-904963.1), gabT(NP-417148.1), and cat2(P38942.2) genes encoding succinyl-CoA reductase (aldehyde-forming), GABA transaminase, and 4-aminobutyryl-CoA / acyl-CoA transferase activities are cloned under the PA1 / lacO promoter of a third compatible plasmid, pZS23. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module using well-known molecular biology techniques. To express the proteins and enzymes required for hexamethylenediamine synthesis, three sets of plasmids are transformed into the E. coli strain MG1655.

[0252] Hexamethylenediamine may also be produced from 6-aminocaproate (6-ACA). This route involves activation of the acid group by phosphorylation and / or acrylation. Acetylation of the terminal amino group can prevent spontaneous cyclization of the intermediates in the route.

[0253] Several routes for producing HMD from 6-aminocaproate are shown in detail in Figure 13. In all routes, activation of the carboxylic acid group is required, followed by reduction and aminotransfer. In three routes, 6-aminocaproate is directly activated, while in other routes, the terminal amine group is protected from spontaneous cyclization by N-acetylation.

[0254] In one route, 6-aminocaproate is phosphorylated to 6-AHOP by 6-aminocaproate kinase (Figure 13, step A). 6-AHOP is then reduced to 6-aminocaproate semialdehyde (Figure 13, step B) and subsequently undergoes aminotransfer by aminotransferase or aminooxidoreductase (Figure 13, step C).

[0255] Alternatively, 6-AHOP is converted to 6-aminocaproyl-CoA by acyltransferase (Figure 13, step L). 6-aminocaproyl-CoA is then reduced to 6-aminocaproate semialdehyde by CoA-dependent aldehyde dehydrogenase (Figure 13, step N). HMD is then formed by aminotransfer of 6-aminocaproate semialdehyde by aminotransferase or aminooxidoreductase (Figure 13, step C).

[0256] In other routes, 6-aminocaproate is first activated to a CoA derivative by CoA transferase or CoA ligase (Figure 13, step M). The product, 6-aminocaproy-CoA, either cyclizes spontaneously or is converted to 6-aminocaproic semialdehyde by aldehyde-forming CoA-dependent aldehyde dehydrogenase (Figure 13, step N). 6-aminocaproic semialdehyde is converted to HMD by aminotransferase or aminating oxidoreductase (Figure 13, step C).

[0257] An additional route proceeds from 6-acetamidohexanoate, the acetylated product of 6-aminocaproate N-acetyltransferase. 6-acetamidohexanoate is converted to 6-acetamidohexanal by different routes (described below). In the last two steps of these routes, 6-acetamidohexanal is first converted to 6-acetamidohexanamine by aminotransferase or aminating oxidoreductase (Figure 13, step G). 6-acetamidohexanamine is then converted to HMD by amidohydrolase or N-acetyltransferase (Figure 13, step H).

[0258] In one route, 6-acetamidohexanoate is phosphorylated by 6-acetamidohexanoate kinase (Figure 13, step E). The product, 6-AAHOP, is reduced to form 6-acetamidohexanal (Figure 13, step F), which is then converted to HMD as described above.

[0259] In another route, 6 - acetamidohexanoate is activated to 6 - acetamidohexanoyl - CoA by CoA - transferase or CoA - ligase (Figure 13, step I). The CoA derivative is then reduced to 6 - acetamidohexanal by aldehyde - forming CoA - dependent oxidoreductase (Figure 13, step J). 6 - acetamidohexanal is then converted to HMD as described above.

[0260] Alternatively, 6 - acetamidohexanoate is phosphorylated to 6 - AAHOP (Figure 13, step E) and then converted to 6 - acetamidohexanoyl - CoA by acyl - transferase (Figure 13, step K). 6 - acetamidohexanoyl - CoA is then reduced to HMD as described above.

[0261] The conversions represented in Figures 12 and 13 are classified into the general categories shown in Table 9. Below, several genes biochemically characterized in each category will be described. The specifically listed genes are those that can be applied to catalyze the appropriate conversions in Figures 12 - 13 when properly cloned and expressed.

[0262] Table 9 shows the types of enzymes useful for converting common central metabolic intermediates to 6 - aminocaproate and hexamethylenediamine. The first three digits of each label correspond to the first three digits of the enzyme number indicating the general type of conversion independent of substrate specificity.

[0263]

Table 36

[0264] 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). The conversion of 6-aminohexanal to 6-aminohexanoyl-CoA (Figure 13, step J), and the conversion of 6-aminocaproyl-CoA to 6-aminocaproic acid semialdehyde (Figure 13, step N) are catalyzed by CoA-dependent oxidoreductase enzymes of EC classification 1.2.1. Adipyl-CoA is converted to adipic acid semialdehyde by adipyl-CoA oxidoreductase, an enzyme with similar functionality (Figure 12, step O). Succinate semialdehyde dehydrogenase, the enzyme that forms succinate semialdehyde, the precursor in Figure 12, from succinyl-CoA, is also a CoA-dependent oxidoreductase. Oxidoreductases of EC classification 1.2.1 - can reduce acyl-CoA to its corresponding aldehyde. Exemplary genes encoding such enzymes include Acinetobacter calcoaceticus acrl, which encodes an aliphatic acyl-CoA reductase (Reiser and Somerville, Journal of Bacteriology 179:2969-2975 (1997)), the aliphatic acyl-CoA reductase of Acinetobacter sp. M-1 (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)), and the CoA- and NADP-dependent succinate semialdehyde dehydrogenase encoded by the sucD gene of Clostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710(2000)).The acylated acetaldehyde dehydrogenase encoded by bphG of Pseudomonas sp has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, and formaldehyde, and other candidates (Powlowski et al., J. Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE of Leuconostoc mesenteroides has been shown to oxidize the branched-chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol... 18:43-55 (1972); and Koo et al., Biotechnol Lett. 27:505-510 (2005)).

[0265]

Table 37

[0266] ​An additional enzyme that converts acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase, which converts malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation by the 3-hydroxypropionate cycle in thermoacidophilic archaea (Berg et al., Science 318:1782-1786 (2007); and Thauer, R. K., Science. 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus sp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is Msed of Metallosphaera sedula _Encoded at 0709 (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al., Science. 318:1782-1786 (2007)). The gene encoding malonyl-CoA reductase from Sulfolobus tokodaii has been cloned and heterologously expressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (WIPO Patent Application WO / 2007 / 141208 Kind Code: A2). The functionality of these enzymes as aldehyde dehydrogenases is similar to that of the bifunctional dehydrogenase from Chloroflexus aurantiacus, but the sequence similarity is low. Both candidates for the malonyl-CoA reductase enzyme have high sequence similarity to aspartate semialdehyde dehydrogenase, an enzyme that catalyzes the reduction of aspartyl-4-phosphate to aspartate semialdehyde and the accompanying dephosphorylation. Additional gene candidates can be found by sequence homology to proteins from other organisms, including Sulfolobus solfataricus and Sulfolobus acidocaldarius, as well as the organisms listed below. Still other candidates for CoA-acylating aldehyde dehydrogenase are the ald gene from Clostridium beijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE, which encodes acetaldehyde dehydrogenase in Salmonella typhimurium and E. coli (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999))。

[0267]

Table 38

[0268] 1.2.1.c Oxidoreductase (from 2-keto acid to acyl-CoA). Some of the conversions in FIG. 12 require the conversion of 2-keto acid to acyl-CoA (steps L, P, and Q) by enzymes of EC classification 1.2.1. Such reactions are catalyzed by multi-enzyme complexes that catalyze a series of partial reactions, resulting in the acylating oxidative decarboxylation of 2-keto-acid. Exemplary enzymes include: 1) branched-chain 2-keto-acid dehydrogenase, 2) alpha-ketoglutarate dehydrogenase, and 3) pyruvate dehydrogenase multi-enzyme complex (PDHC). Each of the 2-keto-acid dehydrogenase complexes occupies a key position in intermediate metabolism, and enzyme activity is typically tightly regulated (Fries et al., Biochemistry 42:6996-7002 (2003)). The enzymes share a complex but common structure composed of multiple copies of three catalytic components. The three catalytic components are as follows: alpha-keto acid decarboxylase (E1), dihydrolipoamide acyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The E3 component is shared among all 2-keto-acid dehydrogenase complexes within a single organism, while the E1 and E2 components are encoded by different genes. The components of the enzyme are present in multiple copies in the complex and utilize a number of cofactors to catalyze the directed arrangement of the reaction via substrate channelling. The overall size of these dehydrogenase complexes is very large, with a molecular weight between 4 and 10 million Da (i.e., larger than ribosomes).

[0269] Under anaerobic conditions in E. coli, the activity of enzymes in the 2-keto-acid dehydrogenase family is usually low or limited. An increase in the production of NADH (or NADPH) can lead to redox imbalance, and NADH itself functions as an inhibitor to enzyme function. Through engineering efforts, the anaerobic activity of the pyruvate dehydrogenase complex in E. coli has been increased (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008); and Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effect of NADH can be overcome by engineering the H322Y mutation in the E3 component (Kim et al., J. Bacteriol. 190:3851-3858 (2008)). Structural studies of the individual components and how they function together as a complex provide insights into the catalytic mechanism and architecture of the enzymes in this family (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); and Zhou et al., Proc. Natl. Acad. Sci. U.S. A 98:14802-14807 (2001)). Dehydrogenase The substrate specificity of the dehydrogenase complex varies among different organisms, but generally, branched-chain keto-acid dehydrogenase has the broadest substrate range.

[0270] Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate to succinyl-CoA and is a major site for controlling the metabolic flux through the TCA cycle (Hansford, Curr. Top. Bioenerg. 10:217-278 (1980)). The expression of the AKGD genes encoded by the genes sucA, sucB, and lpd in E. coli is down-regulated under anaerobic conditions and during growth on glucose (Park et al., Mol. Microbiol. 15:473-482 (1995)). Although the substrate range of AKGD is narrow, specific residues responsible for substrate specificity have been identified by structural studies of the catalytic center of the E2 component (Knapp et al., J. Mol. Biol. 280:655-668 (1998)). The AKGD (E1 and E2) of Bacillus subtilis encoded by odhAB, and pdhD (E3, shared domain) are controlled at the transcriptional level and are dependent on the carbon source and the growth rate of the organism (Resnekov et al., Mol. Gen. Genet. 234:285-296 (1992)). In yeast, the LPD1 gene encoding the E3 component is controlled by glucose at the transcriptional level (Roy and Dawes, J. Gen. Microbiol. 133:925-933 (1987)). The E1 component encoded by KGD1 is also controlled by glucose and is activated by the products of HAP2 and HAP3 (Repetto and Tzagoloff, Mol. Cell. Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, which is inhibited by the products NADH and succinyl-CoA, has been well studied in mammalian systems as a functional impairment associated with several neurological diseases (Tretter and dam-Vizi, Philos. Trans. R. Soc. Lond B Biol. Sci. 360:2335-2345 (2 005)).

[0271]

Table 39

[0272] The branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as 2-oxoisovalerate dehydrogenase, is involved in the branched-chain amino acid catabolic pathway that converts the 2-keto acid derivatives valine, leucine, and isoleucine to their acyl-CoA derivatives and CO2. The complex has been studied in a variety of organisms including Bacillus subtilis (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)), Rattus norvegicus (Namba et al., J. Biol. Chem. 244:4437-4447 (1969)) and Pseudomonas putida (Sokatch et al., J. Bacteriol. 148:647-652 (1981)). In Bacillus subtilis, the enzyme is encoded by the genes pdhD (E3 component), bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)). In mammals, the complex is regulated by phosphorylation by specific phosphatases and protein kinases. The complex has been studied in rat hepatocytes (Chicco et al., J. Biol. Chem. 269:19427-19434 (1994)), genes Bckdha (E1 alpha), Bckdhb (E1 beta), Dbt (E2), and are encoded by Dld (E3). The E1 and E3 components of the BCKAD complex of Pseudomonas putida have been crystallized (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); and Mattevi.et al., Science. 255:1544-1550 (1992)), and the enzyme complex has been studied (Sokatch et al., J. Bacteriol. 148:647-652 (1981)). Transcription of the P. putida BCKAD gene is activated by the gene product of bkdR (Hesslinger et al., Mol. Microbiol. 27:477-492 (1998)). In several organisms including Rattus norvegicus (Paxton et al., Biochem. J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al., Biochem. Mol. In several organisms including Rattus norvegicus (Paxton et al., Biochem. J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al., Biochem. Mol. Biol. Int. 31: 911-922 (1993)), this complex has been shown to have a broad substrate width, including straight-chain oxo-acids such as 2-oxobutanoate and alpha-ketoglutarate in addition to branched-chain amino acid precursors. The active site of bovine BCKAD has been engineered to prefer the alternative substrate acetyl-CoA (Meng and Chuang, Biochemistry. 33:12879-12885 (1994)).

[0273]

Table 40

[0274] The pyruvate dehydrogenase complex that catalyzes the conversion of pyruvate to acetyl-CoA has also been widely studied. In the E. coli enzyme, specific residues of the E1 component are responsible for substrate specificity (Bisswanger, J Biol Chem. 256:815-822 (1981); Bremer, Eur. J Biochem. 8:535-540 (1969); and Gong et al., J Biol Chem. 275:13645-13653 (2000)). As described above, efforts to engineer the enzyme resulted in improved enzymatic activity of E. coli PDH under anaerobic conditions (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008)); and Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The PDH of Klebsiella pneumoniae, which is specific to the period of growth on glycerol, is also active under anaerobic conditions (Menzel et al., J. Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme complex from bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U.S.A 98:14802-14807 (2001)) and the E2 catalytic domain from Azotobacter vinelandii are available (Mattevi.et al., Science. 255:1544-1550 (1992)). Comparative kinetics of Rattus norvegicus PDH and BCKAD show that BCKAD has higher activity towards 2-oxobutanoate as a substrate, although some mammalian PDH enzyme complexes can react with alternative substrates such as 2-oxobutanoate (Paxton et al., Biochem. J. 234:295-303 (1986)).

[0275]

Table 41

[0276] As an alternative to the above-described multienzyme 2-keto-acid dehydrogenase complex, some anaerobic organisms utilize enzymes in the 2-keto-acid oxidoreductase family (OFOR) to catalyze the acylating oxidative decarboxylation reaction of 2-keto-acids. Unlike dehydrogenase complexes, these enzymes contain iron-sulfur clusters, utilize different cofactors, and use ferredoxin or flavodoxin as the electron acceptor instead of NAD(P)H. Most enzymes in this family are specific for pyruvate as the 2-keto-acid substrate (POR), but ferredoxin oxidoreductase has been shown to accept a wide range of 2-keto-acids, including alpha-ketoglutaric acid and 2-oxobutyrate, as substrates (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al., J. Biochem. 120:587-599 (1996)). One such enzyme is OFOR from the hyperthermophilic and acidophilic archaeon Sulfolobus tokodaii 7, which contains alpha and beta subunits encoded by the gene T2300 (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al., J. Biochem. 120:587-599 (1996)). In E. coli, a plasmid-based expression system for efficient expression of this protein has been developed (Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)), and residues related to substrate specificity have been determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str. K1 have also been cloned, characterized, and found to react with a wide range of 2-oxoacids in E. coli in recent years (Nishizawa et al., FEBS Lett. 579:2319-2322 (2005)).The gene sequences of these OFOR candidates are available, but they have not been assigned GenBank identifiers to date. There is bioinformatics evidence that similar enzymes are present in all archaea, some anaerobic bacteria, and amitochondrial eukaryotes (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). Enzymes of this classification are also interesting from an energy perspective because reduced ferredoxin can also be used for the production of NADH by ferredoxin-NAD reductase (Petitdemange et al., Biochim. Biophys. Acta 421:334-337 (1976)). Also, since most enzymes are designed to function under anaerobic conditions, manipulation of the enzymes for activity in anaerobic environments is less necessary compared to enzymes of the 2-keto-acid dehydrogenase complex family.

[0277]

Table 42

[0278] 1.2.1.d Oxidoreductase (phosphonic acid to aldehyde). The reduction of phosphonic acid to its corresponding aldehyde is catalyzed by an oxidoreductase of EC class 1.2.1. Steps B and F of Figure 13 require such an enzyme for the reduction of 6-AHOP and 6-AAHOP to their corresponding aldehydes. These reactions are not catalyzed by known enzymes, although a similar reaction is catalyzed by aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11): the NADPH-dependent reduction of 4-aspartyl-phosphate to aspartate-4-semialdehyde. ASD is involved in amino acid biosynthesis and has recently been studied as an antibacterial target (Hadfield et al., Biochemistry 40:14475-14483 (2001)). The structure of E. coli ASD has been elucidated (Hadfield et al., J. Mol. Biol. 289:991-1002 (1999)), and the enzyme has been shown to accept the alternative substrate beta-3-methylaspartyl-phosphate (Shames et al., J. Biol. Chem. 259:15331-15339 (1984)). Altering the substrate binding affinity at the active site of the Haemophilus influenzae enzyme is a topic in enzyme engineering studies (Blanco et al., Acta Crystallogr. D. Biol. Crystallogr. 60:1388-1395 (2004); and Blanco et al., Acta Crystallogr. D. Biol. Crystallogr. 60:1808-1815 (2004)). Other ASD candidates are Mycobacterium tuberculosis (Shafiani.e.t al., J Appl Microbiol 98:832-838 (2005)), Methanococcus jannaschii (Faehnle et al., J Mol. Biol. 353:1055-1068 (2005)), and in the infectious microorganisms Vibrio cholera and Heliobacter pylori (Moore et al., Protein Expr. Purif. 25:189-194 (2002)). Candidate related enzymes are acetylglutamyl phosphate reductase (EC 1.2.1.38), an enzyme that naturally reduces acetylglutamyl phosphate to acylglutamate-5-semialdehyde, which has been found in S. cerevisiae (Pauwels et al., Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly and Devine, Microbiology 140 (Pt 5):1023-1025 (1994)) and other organisms.

[0279]

Table 43

[0280] 1.3.1.a Oxidoreductase (alkene to alkane). Various conversions are classified into the category of oxidoreductases (EC 1.3.1.-) that reduce alkenes to alkanes. For example, the steps catalyzed by OHED reductase, 6-OHE reductase, 2-AHE reductase and 2,3-dihydroadipyl-CoA reductase in steps C, G, K and N of Figure 12 are classified into this category. Enone reductase, alkenal reductase, and enoate reductase enzymes are candidate enzymes suitable for catalyzing the conversions in steps C, G and K. The enoyl-CoA reductase enzyme catalyzes the conversion from 2,3-dihydroadipyl-CoA to adipyl-CoA (step N).

[0281] Enzymes with enone reductase activity have been identified in prokaryotes, eukaryotes, and plants (Shimoda et al., Bulletin of the chemical society of Japan 77:2269-2 (2004); and Wanner and Tressl, Eur. J Biochem. 255:271-278 (1998)). Two enone reductases were purified and characterized from the cytoplasmic fraction of Saccharomyces cerevisiae and were found to accept various alkenals (similar to 6-OHE) and enoyl ketones (similar to OHED) as substrates (Wanner and Tressl, Eur. J Biochem. 255:271-278 (1998)). The genes encoding these enzymes have not been identified to date. Cell extracts of the cyanobacterium Synechococcus sp. PCC7942 reduced numerous enone substrates to their corresponding alkyl ketones (Shimoda et al., Bulletin of the chemical society of Japan 77:2269-2 (2004)). No genes have been associated with this activity in this organism. Enone reductases from other organisms can also catalyze this conversion.

[0282] The recombinant NADPH-dependent enone reductase derived from Nicotiana tabacum, encoded by NtRed1, has been functionally expressed and characterized in E. coli (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). This reductase is functional against exocyclic enoyl ketone pregone (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). The enzyme candidate at the YML131W locus in S. cerevisiae has 30% sequence similarity with NtRed1 (e-value = 1e-26). The amino acid sequence of NtRed1 has significant homology with 2-alkenal reductase from Arabidopsis thaliana, zeta-crystallin homolog from A. thaliana, pregone reductase from Menthe piperita, and phenylpropenal alkene reductase from Pinus taeda. These enzymes are known to catalyze the reduction of alkenes of α,β-unsaturated ketones and aldehydes.

[0283]

Table 44

[0284] 2-Alkenal reductase catalyzes the reduction of the α,β-unsaturated double bond of aldehydes and ketones. The alkenal hydrogenase ALH1 of barley was identified to have various activities against α,β-unsaturated ketones and aldehydes including trans-2-nonenal, 2-hexenal, traumatine, and 1-octen-3-one (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)). The ALH1 cDNA of Hordeum vulgare has been cloned and expressed in E. coli (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)).

[0285]

Table 45

[0286] 2-Enoyl-CoA reductase enzymes are known to catalyze the NAD(P)H-dependent reduction of a wide variety of α,β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). In the genome sequence of C. kluyveri published in recent years, nine sequences encoding enoyl-CoA reductase have been reported, and one of them has been characterized (Seedorf et al., Proc. Natl. Acad. Sci U.S. A 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and M. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene has also been found to have approximately 75% similarity to the gene characterized in C. kluyveri (Giesel and Simon, Arch. Microbiol 135:51-57 (1983)). Based on these sequences, it has been reported that enr is very similar to the dienoyl-CoA reductase (fadH) of E. coli (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). The enr gene of C. thermoaceticum has also been expressed in E. coli in a catalytically active form (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)).

[0287] [Table 46]

[0288] Another candidate, enoate reductase, is 3-oxoadipate oxidoreductase (maleylacetate reductase), an enzyme that catalyzes the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. Enzyme activity has been identified and characterized in Pseudomonas sp. strain B13 (Kaschabek and Reineke, J. Bacteriol. 177:320-325 (1995); and Kaschabek. and Reineke, J. Bacteriol. 175:6075-6081 (1993)), and the coding gene has been cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)). Candidate genes for 3-oxoadipate oxidoreductase include the clcE gene from Pseudomonas sp. strain B13 (Kasberg et al., J. Bacteriol. 179:3801-3803 (1997)), the macA gene from Rhodococcus opacus (Seibert et al., J. Bacteriol. 180:3503-3508 (1998)), and the macA gene from Ralstonia eutropha (also known as Cupriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)).

[0289]

Table 47

[0290] Enoyl-CoA reductase enzymes are suitable for catalyzing the reduction of 2,3-dehydroadipyl-CoA to adipyl-CoA (Figure 12, step N). One exemplary enoyl-CoA reductase is the gene product of bcd from C. acetobutylicum, which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA (Atsumi et al., Metab Eng 10:305-311 (2008); and Boynton et al., J. Bacteriol. 178:3015-3024 (1996)). The activity of this enzyme can be enhanced by expressing bcd in conjunction with the expression of the etfAB gene of C. acetobutylicum, which encodes the electron carrier flavoprotein. An additional candidate for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence was cloned in E. coli following removal of its mitochondrial targeting leader sequence, resulting in an active enzyme (Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). This approach is well known to those skilled in the art for expressing eukaryotic genes, particularly those having leader sequences that target the gene product to specific intracellular compartments, in prokaryotes. TDE0597, a close homolog of this gene from the prokaryote Treponema denticola, represents a third enoyl-CoA reductase and has been cloned and expressed in E. coli (Tucci and Martin, Febs Letters 581:1561-1566 (2007)).

[0291]

Table 48

[0292] Additional candidates for enoyl-CoA reductase enzymes have been found in organisms that degrade aromatic compounds. Rhodopseudomonas palustris, a model organism for benzoate degradation, has the enzymatic ability to degrade pimelate via beta-oxidation of pimeloyl-CoA. The adjacent genes pimC and pimD within the pim operon have sequence homology to C. acetobutylicum bcd and are predicted to encode flavin-containing pimeloyl-CoA dehydrogenase (Harrison and Harwood, Microbiology 151:727-736 (2005)). The genome of Bradyrhizobium japonicum, a symbiont of legumes that fixes nitrogen, also contains a pim operon composed of genes with high sequence similarity to pimC and pimD of R. palustris including the pim operon composed of genes with high sequence similarity to pimC and pimD of R. palustris (Harrison and Harwood, Microbiology 151:727-736 (2005)).

[0293] [Table 49]

[0294] Additional candidates are 2-methyl-branched-chain enoyl-CoA reductase (EC 1.3.1.52), which catalyzes the reduction of a sterically encumbered trans-enoyl-CoA substrate. This enzyme is involved in branched-chain fatty acid synthesis in the nematode Ascarius suum and can reduce a variety of linear and branched-chain substrates, including 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA, and pentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993)). Two isoforms of this enzyme, encoded by the genes acad1 and acad, have been characterized.

[0295] [Table 50]

[0296] 1.4.1.a Oxidoreductase (ketone or aldehyde to amine). Oxidoreductases of EC class 1.4.1 that convert an aldehyde or a ketone to its corresponding amine group catalyze various biosynthetic steps of the disclosed pathways. In Figure 12, the conversions from OHED to 2-AHE (step J), from 2-OHD to 2-AHD (step H), and from adipic acid semialdehyde to 6-aminocaproate (step E) are catalyzed by OHED aminating oxidoreductase, 2-OHD aminating oxidoreductase, and adipic acid semialdehyde aminating oxidoreductase. In Figure 13, the conversions from 6-aminocaproic acid semialdehyde to HMD (step H), and from 6-acetamidohexanal to 6-acetamidohexanamine (step G) are also catalyzed by aminating oxidoreductases.

[0297] Most aminoxidoreductases catalyze the reversible oxidative deamination of alpha - amino acids with NAD+ or NADP+ as acceptor, and the reaction is generally reversible. Exemplary enzymes include glutamate dehydrogenase (deamination) encoded by gdhA, leucine dehydrogenase (deamination) encoded by ldh, and aspartate dehydrogenase (deamination) encoded by nadX. The gdhA gene product from Escherichia coli (Korber et al., J. Mol. Biol. 234:1270 - 1273 (1993); and McPherson et al., Nucleic Acids Res. 11:5257 - 5266 (1983)), gdh from Thermotoga maritime (Kort et al., Extremophiles. 1:52 - 60 (1997); Lebbink et al., J Mol. Biol. 280:287 - 296 (1998); and Lebbink et al., J Mol. Biol. 289:357 - 369 (1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al., Gene 349:237 - 244 (2005)) catalyze the reversible interconversion of glutamate to 2 - oxoglutarate and ammonia, while NADP(H), NAD(H), or both are each conveniently available. The ldh gene of Bacillus cereus encodes a LeuDH protein with a broad substrate range including leucine, isoleucine, valine, and 2 - aminobutyrate (Ansorge and Kula, Biotechnol Bioeng 68:557 - 562 (2000); and Stoyan et al., J Biotechnol. 54:77 - 80 (1997)). The nadX gene from Thermotoga maritime encoding aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al., J Biol. Chem. 278:8804 - 8808 (2003)).

[0298]

Table 51

[0299] The lysine 6-dehydrogenase (deaminating) encoded by lysDH catalyzes the oxidative deamination of the 6-amino group of L-lysine to form 2-aminoadipic acid-6-semialdehyde. 2-Aminoadipic acid-6-semialdehyde then cyclizes non-enzymatically to form Δ 1 -piperidine-6-carboxylate (Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found in Geobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem. 106:76-80 (1989); and Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)). Such enzymes are particularly good candidates for the conversion of adipic acid semialdehyde to 6-aminocaproate, given the structural similarity of adipic acid semialdehyde and 2-aminoadipic acid-6-semialdehyde.

[0300]

Table 52

[0301] 2.3.1.a Acyltransferases (transferring CoA to phosphate). Acyltransferases that exchange a portion of CoA for phosphate are found in the EC classification 2.3.1. Conversions in this category include the conversion from 6-AAHOP to 6-acetamidohexanoyl-CoA (Figure 13, step K), and from 6-AHOP to 6-aminocaproyl-CoA (Figure 13, step L). Exemplary phosphate-transfer acyltransferases include phosphotransacetylase (EC 2.3.1.8) encoded by pta, and phosphotransbutyrylase (EC 2.3.1.19) encoded by ptb. The pta gene from E. coli encodes an enzyme that reversibly converts acetyl-CoA to acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme also utilizes propionyl-CoA as a substrate and forms propionate in the process (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Similarly, the ptb gene from C. acetobutylicum encodes phosphotransbutyrylase, an enzyme that reversibly converts butyryl-CoA to butyryl-phosphate (Walter et al., Gene 134:107-111 (1993); and Wiesenborn et al., Appl Environ. Microbiol 55:317-322 (1989)). Additional ptb genes have been found in the butyrate-producing bacterium L2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez et al., Curr. Microbiol 42:345-349 (2001)).

[0302]

Table 53

[0303] 2.3.1.c Acyltransferase (N-acetyltransferase). N-acetyltransferase transfers an N-acetyl group to an amine to form an N-acetyl group. N-acetylation is involved in diverse functions in biological systems, including transcriptional control, nuclear translocation, chromosomal assembly, and nucleosome remodeling (Kouzarides, EMBO J 19:1176-1179 (2000)). N-acetylation of metabolic intermediates in the arginine biosynthetic pathway serves both to protect reactive intermediates from spontaneous cyclization and to sequester pathway intermediates from competing pathways (Caldovic and Tuchman, Biochem. J 372:279-290 (2003)). The acetylation of 6-ACA (Figure 13, step D) serves a role similar to that of the proposed HMD biosynthetic route in Figure 13, protecting reactive intermediates from spontaneous cyclization. The acetylation of 6-ACA serves a role similar to that of the proposed HMD biosynthetic route in Figure 13, protecting reactive intermediates from spontaneous cyclization.

[0304] One candidate enzyme for acetylation of 6-ACA is lysine N-acetyltransferase (EC 2.3.1.32), an enzyme that selectively transfers part of the acetyl group from acetyl-phosphate to the terminal amino group of L-lysine, beta-L-lysine, or L-ornithine. Although it is not known that this enzyme acetylates 6-ACA, this substrate is structurally similar to natural substrates. Lysine N-acetyltransferase has been characterized in Bos taurus (Paik. and Kim, Arch. Biochem. Biophys. 108:221-229, 1964) and Methanosarcina mazei (Pfluger et al., Appl Environ. Microbiol 69:6047-6055 (2003)). Methanogenic archaea, M. maripaludis, M. acetivorans, M. barkeri, and M. jannaschii are also predicted to encode an enzyme with this functionality (Pfluger et al., Appl Environ. Microbiol 69:6047-6055 (2003)).

[0305]

Table 54

[0306] Alternatively, 6-ACA acetylation can be catalyzed by enzymes of the GNAT family of N-acetyltransferases. Such enzymes transfer an N-acetyl group from acetyl-CoA to a primary amine. The enzyme spermidine N-acetyltransferase (SSAT), also known as diamine N-acetyltransferase (EC 2.3.1.57), can acetylate a variety of small molecule substrates. Enzymes purified from Ascaris suum and Onchocerca volvulus show a broad substrate width including HMD (Davids et al., Mol. Biochem. Parasitol. 64:341-344 (1994); and Wittich and Walter, Mol. Biochem. Parasitol. 38:13-17 (1990)), but the related genes have not been identified to date. Other enzymes with this functionality have been found in Bacillus subtilis (Forouhar et al., J. Biol. Chem. 280:40328-40336 (2005)) and Homo sapiens (Casero and Pegg, FASEB J 7: 653-661 (1993)). A closely related enzyme is the thialidine N-acetyltransferase of C. elegans, an enzyme that accepts a variety of substrates including lysine, ornithine, thialidine and other substrates (bo-Dalo et al., Biochem. J 384:129-137 (2004)). Amino acid residues involved in substrate binding have been identified in the thialidine N-acetyltransferase from Leishmania major (Luersen, K., FEBS Lett. 579:5347-5352 (2005)).Additional candidates are diaminobutyrate acetyltransferase (EC 2.3.1.178), an enzyme involved in ectoine biosynthesis in Methylomicrobium alcaliphilum (Reshetnikov et al., Arch. Microbiol 184:286-297 (2006)) and C. salexigens (formerly Halomonas elongata) (Canovas et al., Syst. Appl Microbiol 21:487-497 (1998)).

[0307]

Table 55

[0308] Additional enzyme candidates for acetylating 6-ACA (Figure 13, step D) and deacetylating 6-acetamidohexanoic amine (Figure 13, step H) are bifunctional enzymes that catalyze two steps of arginine biosynthesis (Figure 14A), ornithine acetyltransferase (OAT, They are EC 2.3.1.35 and EC 2.3.1.1). The first step in arginine biosynthesis (Figure 14A, step 1) is the N-acetylation of N-acetylglutamate catalyzed by OAT, with acetyl-CoA as the acetyl group donor (O'Reilly and Devine, Microbiology 140 (Pt 5):1023-1025 (1994)). OAT also catalyzes the fifth step in arginine biosynthesis (Figure 14A, step 2), in which the N-acetyl group is transferred from N-acetyl-L-ornithine to L-glutamate, the first metabolite in the arginine biosynthetic pathway. This conversion serves to recycle the acetyl group and regenerate N-acetylglutamate, conserving energy and thereby turning a linear pathway into a cyclic route. A similar strategy is used in HMD biosynthesis from 6-aminocaproate, where a single enzyme acetylates 6-aminocaproate and de-acetylates 6-acetamidohexanamine to form HMD (Figure 14B). Exemplary OAT enzymes are encoded by argJ of Bacillus subtilis (O'Reilly and Devine, Microbiology 140 (Pt 5):1023-1025 (1994); and Sakanyan et al., Journal of General Microbiology 138:125-130 (1992)) and ECM40 of S. cerevisiae (Abadjieva et al., J Biol. Chem. 275:11361-11367 (2000); and Liu et al., Eur. J Biochem. 228:291-296 (1995)).Crystal structures of enzymes from yeast (Maes et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 62:1294-1297 (2006)) and Mycobacterium tuberculosis (Sankaranarayanan et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 65:173-176 (2009)) are available. Although encoded by a single open reading frame, the OAT enzyme has separate alpha and beta subunit peptides (Liu et al., Eur. J Biochem. 228:291-296 (1995)).

[0309]

Table 56

[0310] 2.3.1.d Acyltransferase (formate C-acyltransferase). The acylation of ketoacids HODH, OHED and 2-OHD to their corresponding CoA derivatives (Figure 12, steps L, P and Q), and the accompanying release of formate are catalyzed by the formate C-acyltransferase enzyme of EC classification 2.3.1. Enzymes in this classification include pyruvate formate-lyase and ketoacid formate-lyase. Pyruvate formate-lyase (PFL, EC 2.3.1.54) encoded by pflB of E. coli converts pyruvate to acetyl-CoA and formate. The active site of PFL is , after translation, it contains a glycyl radical essential for catalysis that is activated by the PFL-activating enzyme (PFL-AE, EC 1.97.1.4) encoded by pflA under anaerobic conditions (Knappe et al., Proc. Natl. Acad. Sci U.S. A 81:1332-1335 (1984); and Wong et al., Biochemistry 32:14102-14110 (1993)). The pyruvate formate-lyase from Archaeglubus fulgidus encoded by pflD has been cloned, expressed in E. coli, and characterized (Lehtio, L. and A. Goldman, Protein Eng Des Sel 17:545-552 (2004)). The crystal structures of the enzymes from A. fulgidus and E. coli have been elucidated (Lehtio et al., J Mol. Biol. 357:221-235 (2006)). Additional PFL and PFL-AE candidates have been found in Clostridium pasteurianum (Weidner and Sawers, J. Bacteriol. 178:2440-2444 (1996)) and the eukaryotic alga Chlamydomonas reinhardtii (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)). Keto-acid formate-lyase (EC 2.3.1.-), also known as 2-ketobutyrate formate-lyase (KFL) and pyruvate formate-lyase 4, is the gene product of tdcE in E. coli. This enzyme catalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formate during anaerobic threonine degradation and serves as an alternative to pyruvate formate-lyase in anaerobic catabolism (Simanshu et al., J Biosci. 32:1195-1206 (2007)).The enzyme is oxygen-sensitive and, like PflB, requires post-translational modification by PFL-AE to activate the glycyl radical at the active site (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)).

[0311]

Table 57

[0312] 2.6.1.a Aminotransferase. Steps E, H, and J in Figure 12, and steps C and G in Figure 13 require the conversion of an aldehyde or ketone to an amino group. This conversion can be achieved by an aminotransferase (EC 2.6.1.-). The conversion of an aldehyde to a terminal amine (Figure 12, step E; Figure 13, steps C and G) can be catalyzed by gamma-aminobutyrate transaminase (GABA transaminase). One E. coli GABA transaminase is encoded by gabT and transfers an amino group from glutamate to the terminal aldehyde of succinic semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042 (1990)). This enzyme exhibits a broad substrate range (Liu et al., Biochemistry 43:10896-10905 (2004)). The gene product of puuE encodes another 4-aminobutyrate transaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)). The GABA transaminases of Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); and Scott and Jakoby, J Biol. Chem. 234:932-936 (1959)).

[0313]

Table 58

[0314] Additional enzyme candidates include putrescine aminotransferase or other diamine aminotransferases. Such enzymes are particularly well-suited to perform the conversion from 6-aminocaproic acid semialdehyde to HMD. E. coli putrescine aminotransferase is encoded by the ygjG gene, and the purified enzyme was also able to transfer the amino groups of cadaverine and spermidine (Samsonova et al., BMC. Microbiol 3:2 (2003)). In addition, the activity of this enzyme towards 1,7-diaminoheptane and with amino acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutyrate) has been reported (Kim, J Biol. Chem. 239:783-786 (1964); and Samsonova et al., BMC. Microbiol 3:2 (2003)). A putrescine aminotransferase that has higher activity when using pyruvate as the amino acceptor than alpha-ketoglutaric acid is the spuC gene of Pseudomonas aeruginosa (Lu et al., J. Bacteriol. 184:3765-3773 (2002)).

[0315]

Table 59

[0316] Additional candidate enzymes include beta-alanine / alpha-ketoglutarate aminotransferase, which produces malonic semialdehyde from beta-alanine (WO08027742). The gene product of SkPYD4 of Saccharomyces kluyveri has been shown to preferentially use beta-alanine as the amino group donor (Andersen and Hansen, Gene 124:105-109 (1993)). SkUGA1 encodes a homolog of the GABA aminotransferase UGA1 of Saccharomyces cerevisiae (Ramos et al., Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both beta-alanine and GABA amino group transfer (Andersen and Hansen, Gene 124:105-109 (1993)). 3-Amino-2-methylpropionate transaminase catalyzes the conversion of methylmalonate semialdehyde to 3-amino-2-methylpropionate. The enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat 1968 (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); and Tamaki et al., Methods Enzymol. 324:376-389 (2000)).

[0317]

Table 60

[0318] Steps J and H of Figure 12 are catalyzed by aminotransferases that convert an amino acid to an oxo-acid. In step J, OHED is transaminated by OHED aminotransferase to form 2-AHE. The transamination of 2-OHD to 2-AHD by 2-OHD aminotransferase (step H) is a similar reaction. An exemplary enzyme candidate that catalyzes these reactions is aspartate aminotransferase, an enzyme that naturally transfers an oxo group from oxaloacetate to glutamate to form alpha-ketoglutarate and aspartate. Aspartate is structurally similar to OHED and 2-AHD. Aspartate aminotransferase activity is catalyzed, for example, by the gene products of aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84, (1979); and Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi.e.t al., J. Biochem. 92:35-43 (1982)), and ASP5 from Arabidopsis thaliana (de la Torre et al., Plant J 46:414-425 (2006); Kwok and Hanson, J Exp. Bot. 55:595-604 (2004); and Wilkie and Warren, Protein Expr. Purif. 12:381-389 (1998)). Enzymes from Rattus norvegicus have been shown to transfer the amino group of alternative substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferases that act on other amino-acid substrates can catalyze this conversion. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine.The avtA gene, which is an E. coli gene, encodes one such enzyme (Whalen and Berg, C. J. Bacteriol. 150:739-746 (1982)). This gene product also catalyzes the aminotransfer of α-ketobutyrate to produce α-aminobutyrate, but the amine donor in this reaction has not been identified (Whalen and Berg, J. Bacteriol. 158:571-574 (1984)). E. coli. The serC gene product catalyzes two reactions, phosphoserine aminotransferase and phosphohydroxythreonine aminotransferase (Lam and Winkler, J. Bacteriol. 172:6518-6528 (1990)), and no reaction with a non-phosphorylated substrate has been detected (Drewke et al., FEBS. Lett. 390:179-182 (1996)).

[0319]

Table 61

[0320] 2.7.2.a Phosphotransferase (carboxy acceptor). Phosphotransferase enzymes of EC class 2.7.2 convert carboxylic acids to phosphonic acids with the hydrolysis of an accompanying molecule of ATP. Steps A and E in Figure 13 require phosphotransferase to activate the carboxyl groups of 6-ACA (step A) and 6-acetamidohexanoate (step E) to their corresponding phosphonic acids. Butyrate kinase performs the reversible conversion from butyryl-phosphate to butyrate during acidogenesis in C. acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990)). This enzyme is encoded by either of two buk gene products (Huang et al., J. Mol. Microbiol Biotechnol 2:33-38 (2000)). The related enzyme isobutyrate kinase from Thermotoga maritima has also been expressed and crystallized in E. coli (Diao et al., Acta Crystallogr. D. Biol. Crystallogr. 59:1100-1102 (2003); and Diao and Hasson, J. Bacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzes the ATP-dependent phosphorylation of aspartate and is involved in several amino acid syntheses. The Escherichia coli aspartokinase III enzyme encoded by lysC has a broad substrate width and catalytic residues related to substrate specificity have been elucidated (Keng and Viola, Arch. Biochem. Biophys. 335:73-81 (1996)). Two additional kinases in E. coli are also good candidates: acetate kinase and gamma-glutamyl kinase.The acetate kinase of E. coli encoded by ackA (Skarstedt and Silverstein, J. Biol. Chem. 251:6775-6783 (1976)) phosphorylates propionate in addition to acetate (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). The gamma-glutamyl kinase of E. coli encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)) phosphorylates the gamma-carbonyl group to glutamate.

[0321]

Table 62

[0322] Acylglutamate kinase phosphorylates acetylated glutamate during arginine biosynthesis and is a good candidate for the phosphorylation of 6-acetamidohexanoate (Figure 13, Engineering E). This enzyme is not known to accept alternative substrates; however, some residues of the E. coli enzyme involved in substrate binding and phosphorylation have been elucidated by site-directed mutagenesis (Marco-Martin et al., J Mol. Biol. 334:459-476 (2003); and Ramon-Maiques et al., Structure. 10:329-342 (2002)). The enzyme is encoded by argB in Bacillus subtilis and E. coli (Parsot et al., Gene 68:275-283 (1988)), and by ARG5,6 in S. cerevisiae (Pauwels et al., Eur. J Biochem. 270:1014-1024 (2003)). The ARG5,6 gene of S. cerevisiae encodes a polyprotein precursor that is matured in the mitochondrial matrix to be a candidate enzyme for the reduction of 6-AAHOP (Figure 13, step F), an acylglutamate kinase and an acetylglutamyl phosphate reductase.

[0323]

Table 63

[0324] 2.8.3.a Coenzyme-A transferase. Coenzyme-A (CoA) transferase catalyzes the reversible transfer of one molecule of a part of CoA from one to another. In step M of Figure 13, 3-aminocaproyl-CoA is formed by the transfer of a CoA group from acetyl-CoA, succinyl-CoA, or another CoA donor. A similar conversion is catalyzed by 6-acetamidohexanoate CoA-transferase as shown in step I of Figure 13. Candidate exemplary CoA transferases are catalyzed by the gene products of cat1, cat2, and cat3 of Clostridium kluyveri, which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activities, respectively (Seedorf et al., Proc. Natl. Acad. Sci U.S. A 105:2128-2133 (2008); and Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)).

[0325]

Table 64

[0326] The CoA transferase that can utilize acetyl-CoA as a CoA donor is the acetoacetyl-CoA transferase encoded by the atoA (alpha subunit) and atoD (beta subunit) genes of E. coli (Korolev et al., Acta Crystallogr. D. Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek and Frerman, Arch. Biochem. Biophys. 171:14-26 (1975)) and has been shown to transfer a portion of CoA from various branched-chain and straight-chain acyl-CoA substrates, including isobutyrate (Matthies and Schink, Appl Environ. Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)) and butyrate (Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968)), to acetate. Since this enzyme is induced by acetoacetate at the transcriptional level, alterations in regulatory control would be required to engineer the enzyme into a pathway (Pauli and Overath, Eur. J Biochem. 29:553-562 (1972)).Similar enzymes are present in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ. Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583 (1990); and Wiesenborn et al., Appl. Environ. Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

[0327]

Table 65

[0328] The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from the anaerobic bacterium Acidanimococcus fermentans reacts with glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51(1994)). The genes encoding this enzyme are gctA and gctB. This enzyme shows decreased but detectable activity towards other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).

[0329]

Table 66

[0330] Still other CoA transferases are 2 units of succinyl-CoA: the 3-oxoacid-CoA transferase encoded by pcaI and pcaJ of Pseudomonas putida (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)). Similar enzymes based on homology are present in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)). Additional exemplary succinyl-CoA:3-oxoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403 (2007)).

[0331]

Table 67

[0332] 3.5.1.a Hydrolase (acting on linear amides). The deacetylation of linear acetamide is catalyzed by amide hydrolases of the 3.5.1 family of enzymes. Such enzymes are required for the deacetylation of 6-acetamidohexaneamine to HMD (Figure 13, step H). An enzyme that catalyzes a similar conversion is 4-acetamidobutyrate deacetylase (EC 3.5.1.63), which naturally deacetylates 4-acetamidobutyrate. The enzyme is Candida In B. boidinii, its role in putrescine degradation has been studied (Gillyon et al., Journal of General Microbiology 133:2477-2485 (1987)), and it has been shown to deacetylate various substrates including 6-acetamidohexanoate (Haywood and Large, Journal of General Microbiology 132:7-14 (1986)). 6-Acetamidohexanoate is structurally similar to the preferred substrate, but deacetylation of this compound (Figure 13, step D, reverse reaction) hinders efficient production of HMD. Protein engineering or directed evolution may be required to improve specificity for 6-acetamidohexanamine. The gene involved in this activity has not been identified to date.

[0333] 2. Acetylpolyamine amidohydrolase (EC 3.5.1.62) is another candidate enzyme for forming diamines putrescine and cadaverine from their acetylated precursors. Acetylpolyamine deacetylase (AphA) from Mycoplana ramosa has been cloned and characterized in E. coli (Sakurada et al., J. Bacteriol. 178:5781-5786 (1996)), and its crystal structure is available (Fujishiro et al., Biochem. Biophys. Res. Commun. 157:1169-1174 (1988)). This enzyme has also been studied in Micrococcus luteus, but the related gene has not been elucidated to date (Suzuki et al., Biochim. Biophys. Acta 882:140-142 (1986)). A protein of the histone deacetylase superfamily with high sequence similarity to AphA was identified in the M. luteus genome (e-value = 1e-18, 37% identity). N-Acetyl-L-ornithine deacetylase from E. coli is another candidate for amidohydrolase (EC 3.5.1.16). The E. coli enzyme encoded by the argE gene (McGregor et al., J Am. Chem. Soc. 127:14100-14107 (2005); and Meinnel et al., J. Bacteriol. 174:2323-2331 (1992)) removes the N-acetyl group from various substrates including ornithine, lysine, glutamine, and other amino acids (Javid-Majd and Blanchard, Biochemistry 39:1285-1293 (2000)).

[0334]

Table 68

[0335] 4.1.1.a Carboxy-lyase. Steps D and F in Figure 12 are catalyzed by a 2-keto acid decarboxylase enzyme that produces 6-OHE and adipic semialdehyde from OHED (step F) and 2-OHD (step D). In addition, alpha-ketoglutaric acid is decarboxylated by alpha-ketoglutaric acid decarboxylase, which is a keto-acid decarboxylase, to form succinic semialdehyde, a pathway precursor. The decarboxylation reaction of keto-acids is catalyzed by various enzymes with different substrate specificities, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutaric acid decarboxylase, and branched-chain alpha-keto acid decarboxylase. Pyruvate decarboxylase (PDC), also known as keto-acid decarboxylase, is a key enzyme in alcohol fermentation and catalyzes the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate width for aliphatic 2-keto acids, including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate, and 2-phenylpyruvate (22). This enzyme has been extensively studied, engineered for modified activity, and functionally expressed in E. coli (Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001); Li, H. and F. Jordan, Biochemistry. 38:10004-10012 (1999); and ter Schure et al., Appl. Environ. Microbiol. 64:1303-1307 (1998)). The PDC from Zymomonas mobilus encoded by pdc also has a broad substrate range and has been the subject of directed engineering studies to modify its affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al., Eur. J. Biochem. 268: 1698-1704 (2001)). Other well-characterized PDC candidates include enzymes from Acetobacter pasteurians (Cha ndra et al., Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263 (2002)).

[0336]

Table 69

[0337] Similar to PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and is a target for enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and its crystal structure is available (Hasson et al., Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry 42:1820-1830 (2003)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme has been used to modify the affinity (Km) for natural and non-natural substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726 (2003)). The enzyme from Pseudomonas aeruginosa encoded by mdlC has also been experimentally characterized (Barrowman et al., FEMS Microbiology Letters 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using the growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol. 72:7510-7517 (2006)).

[0338]

Table 70

[0339] A third enzyme capable of decarboxylating 2-oxo acids is alpha-ketoglutarate decarboxylase (KGD). The substrate breadth of enzymes in this classification has not been studied to date. KDC from Mycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci US.A 102:10670-10675 (2005)) has been cloned and functionally expressed in other Genomatica in-house projects. However, it is large (~130 kD) and GC-rich, so it is not an ideal candidate for strain engineering. KDC enzyme activity has been detected in several species of rhizobia, including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844 (2000)). The (multiple) genes encoding KDC have not been isolated in these organisms, but the genomic sequences are available and several genes in each genome are annotated as putative KDCs. The KDC from Euglena gracilis has also been characterized, but the genes involved in this activity have not been identified to date (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The first 20 amino acids starting from the N-terminus were sequenced as MTYKAPVKDVKFLLDKVFKV (SEQ ID NO:1) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene can be identified by verifying the KDC activity of candidate genes containing this N-terminal sequence.

[0340]

Table 71

[0341] A fourth candidate enzyme that catalyzes this step is branched-chain alpha-keto acid decarboxylase (BCKA). Enzymes of this class have been shown to act on a variety of compounds that differ in chain length between 3 and 6 carbons (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988); and Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). Lactococcus lactis enzymes have been characterized with respect to a variety of branched and straight-chain substrates, including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate, and isocaproate (Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318:1782-1786 (2007)). Sequence alignment between the Lactococcus lactis enzyme and pyruvate decarboxylase from Zymomonas mobilus has shown that the active and substrate recognition residues are almost identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme is a promising candidate for the directed manipulation of the decarboxylation reaction of alpha-ketoglutaric acid by BCKA detected in Bacillus subtilis; however, compared to its activity towards other branched-chain substrates, this activity is low (5%) (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988)), and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the protein sequence of Lactococcus lactis. Many of those that hit with a high score in BLASTp for this enzyme are annotated as indolepyruvate decarboxylase (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

[0342]

Table 72

[0343] Branched-chain ketoacid dehydrogenase of mitochondria from Homo sapiens and Bos taurus A recombinant branched-chain alpha-keto acid decarboxylase enzyme derived from the E1 subunit of the complex has been cloned and functionally expressed in E. coli (Davi.e. et al., J. Biol. Chem. 267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887 (1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). In these studies, the authors found that co-expression of the chaperones GroEL and GroES increased the activity of the specific decarboxylase by 500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). These enzymes are composed of two alpha subunits and two beta subunits.

[0344]

Table 73

[0345] The decarboxylation of 2-AHD to 6-aminocaproate (Figure 12, step I) is catalyzed by an amino acid decarboxylase such as aspartate decarboxylase. Aspartate decarboxylase is involved in pantothenate biosynthesis and is encoded by the Escherichia coli gene panD (Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999); Merke and Nichols, FEMS Microbiol Lett. 143:247-252 (1996); Ramjee et al., Biochem. J 323 (Pt 3):661-669 (1997); and Schmitzberger et al., EMBO J 22:6193-6204 (2003)). Mycobacterium tuberculosis (Chopra et al., Protein Expr. Purif. Similar enzymes from Pseudomonas aeruginosa (Nakazawa et al., J. Bacteriol. 184:533-540 (2002)) and Corynebacterium glutamicum (Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999)) have been expressed and characterized in E. coli.

[0346]

Table 74

[0347] 4.1.2.a Aldehyde-lyase. Also known as HHED aldolase, HOHD aldolase catalyzes the conversion of 4-hydroxy-2-oxopentane-1,7-dioate (HOHD) to pyruvate and succinic semialdehyde (Figure 12, step A). The enzyme is a divalent metal ion-dependent class II aldolase and catalyzes the last step of 4-hydroxyphenylacetic acid degradation in E. coli C, E. coli W, and other organisms. In the native context, the enzyme functions in the degradative direction. The reverse (condensation) reaction is thermodynamically unfavorable; however, the equilibrium can be shifted by linking the HOHD aldolase with a downstream pathway enzyme that acts efficiently on the reaction products. Such a strategy has been effective in shifting the equilibrium of other aldolases in the direction of condensation (Nagata et al., Appl Microbiol Biotechnol 44:432-438 (1995); and Pollard et al., Appl Environ. Microbiol 64:4093-4094 (1998)). The E. coli C enzyme encoded by hpcH has been extensively studied and recently crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007); and Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme is encoded by hpaI (Prieto et al., J. Bacteriol. 178:111-120 (1996)).

[0348]

Table 75

[0349] 4.2.1.a Hydro-lyase. The enzyme OHED hydratase is involved in the degradation of 4-hydroxyphenylacetic acid and converts 2-oxohepta-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate (HODH) using magnesium as a cofactor (Burks et al., J. Am. Chem. Soc. 120 (1998)) (Figure 12, step B). OHED hydratase Enzyme candidates have been identified and characterized in E. coli C (Izumi et al., J Mol. Biol. 370:899-911 (2007); and Roper et al., Gene 156:47-51 (1995)) and E. coli W (Prieto et al., J. Bacteriol. 178:111-120 (1996)). Sequence comparison reveals homologs in various bacteria, plants, and animals. Enzymes with highly similar sequences included, among others, Klebsiella pneumonia (91% identity, e-value = 2e-138) and Salmonella enterica (91% identity, e-value = 4e-138).

[0350]

Table 76

[0351] The dehydration of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (Figure 12, step M) is catalyzed by an enzyme with enoyl-CoA hydratase activity. 3-Hydroxybutyryl-CoA dehydrogenase (EC 4.2.1.55), also known as crotonase, dehydrates 3-hydroxyisobutyryl-CoA to form crotonoyl-CoA (Figure 14, step 2). Crotonase enzymes are required for n-butanol formation in some organisms, particularly Clostridium species, and also constitute one step of the 3-hydroxypropionate / 4-hydroxybutyrate cycle in the thermoacidophilic archaea of the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genes encoding crotonase enzymes have been found in C. acetobutylicum (Atsumi.et al., Metab Eng 10:305-311 (2008); and Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), C. kluyveri (Hillmer and Gottschalk, FEBS Lett. 21:351-354 (1972)), and Metallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007)), but the sequence of the latter gene is not known.

[0352]

Table 77

[0353] Enoyl-CoA hydratase (EC 4.2.1.17) also catalyzes the dehydration of 3-hydroxyacyl-CoA substrates (Agnihotri and Liu., J. Bacteriol. 188:8551-8559 (2003); Conrad et al., J. Bacteriol. 118:103-111 (1974); and Roberts et al., Arch. Microbiol 117:99-108 (1978)). The enoyl-CoA hydratase of Pseudomonas putida encoded by ech catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonoyl-CoA (Roberts et al., Arch. Microbiol 117:99-108 (1978)). Additional enoyl-CoA hydratase candidates are phaA and phaB from P. putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc. Natl. Acad. Sci U.S. A 95:6419-6424 (1998)). Rhodopseudomonas palustris' gene product of pimF is predicted to encode an enoyl-CoA hydratase involved in pimeloyl-CoA degradation (Harrison and Harwood, Microbiology 151:727-736 (2005)). Finally, maoC (Park and Lee, J. Bacteriol. 185:5391-5397 (2003)), paaF(Ismail et al., J. Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., J. Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686 (2004)) have shown that a number of Escherichia coli genes, including

[0354]

Table 78

[0355] Alternatively, the E. coli gene products of fadA and fadB exhibit enoyl-CoA hydratase activity and encode a multienzyme complex involved in fatty acid oxidation (Nakahigashi and Inokuchi, Nucleic acid Res. 18:4937 (1990); Yang, J. Bacteriol. 173:7405-7406 (1991); and Yang et al., Biochemistry 30:6788-6795 (1991)). The knockout of the negative regulator encoded by fadR can be utilized to activate the fadB gene product (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805 (2003)).

[0356]

Table 79

[0357] 6.2.1.a Acid - thiol ligase (also referred to as CoA synthetase). Steps I and M in Figure 13 functionally require acid - thiol ligase or CoA synthetase to convert 6 - ACA and 6 - acetamidohexanoate to their corresponding CoA derivatives (the terms ligase, synthetase, and synthase are used interchangeably here and refer to the same enzyme classification). Enzymes that catalyze these exact conversions have not been characterized to date; however, several enzymes with broad substrate specificities have been described in the literature. ADP - forming acetyl - CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of an acyl - CoA ester to its corresponding acid with the synthesis of the accompanying ATP. ACD I from Archaeoglobus fulgidus encoded by AF1211 has been shown to act on various linear and branched - chain substrates, including isobutyrate, isopentanoate, and fumarate (Musfeldt and Schonheit, J. Bacteriol. 184:636 - 644 (2002)). A second reversible ACD from Archaeoglobus fulgidus encoded by AF1983 has also been shown to have a broad substrate range and has been shown to have high activity towards the cyclic compounds phenylacetate and indoleacetate (Musfeldt and Schonheit, J. Bacteriol. 184:636 - 644 (2002)). The enzyme from Haloarcula marismortui (annotated as succinyl - CoA synthetase) has been shown to accept as substrates propionate, butyrate, and branched - chain acids (isovalerate and isobutyrate) and to act in both the forward and reverse directions (Brasen and Schonheit, Arch. Microbiol 182:277 - 287 (2004)).The ACD from Pyrobaculum aerophilum, a hyperthermophilic clavaminate synthase encoded by PAE3250, showed the broadest substrate range among all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate), and phenylacetyl-CoA (Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004)). This enzyme can be modified using directed evolution or engineering to act at the physiological temperature of the host organism. Enzymes from A. fulgidus, H. marismortui, and P. aerophilum have all been cloned, functionally expressed in E. coli, and characterized (Brasen. and Schonheit, Arch. Microbiol 182:277-287 (2004); and Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). An additional candidate is the enzyme encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the consumption of one molecule of ATP in an associated reaction, and the reaction is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)).

[0358]

Table 80

[0359] Another candidate enzyme for this step is 6-carboxyhexanoate-CoA ligase, also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activates pimelate to pimeloyl-CoA during biotin biosynthesis in Gram-positive bacteria. The enzyme from Pseudomonas mendocina cloned into E. coli was shown to accept the alternative substrates hexanedioate and nonanedioate (Binieda et al., Biochem. J 340 (Pt 3):793-801 (1999)). Other candidates have been found in Bacillus subtilis (Bower et al., J. Bacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem. J 287 (Pt 3):685-690 (1992)).

[0360]

Table 81

[0361] Additional CoA-ligases include a rat dicarboxylating-CoA ligase whose sequence has not yet been characterized (Vamecq et al., Biochem. J 230:683-693 (1985)), either of two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J 395:147-155 (2006); and Wang et al., Biochem. Biophys. Res. Commun. 360:453-458 (2007)), and a phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturally catalyze the ATP-dependent conversion of acetoacetate to acetoacetyl-CoA.

[0362] [Table 82]

[0363] The present invention will be described in more detail hereinafter, based on the following non-limiting examples, with reference to the accompanying drawings. [Examples]

[0364] [Example 1] This experiment explains the HMD / CO2pH equilibrium under changing conditions. In this example, a 10% w / w aqueous solution of HMD was prepared (70% HMD purchased from Sigma Aldrich). The HMD solution was heated to 30 °C and the initial pH was recorded. Then, while monitoring the pH, CO2 (approx. 98% purity) was bubbled into the solution for 60 minutes. The sparging of CO2 was stopped. While monitoring the pH, air was sparged into the solution for 30 minutes. While maintaining the sparging of air, the solution was heated to 80 °C for 40 minutes. The pH was measured by sampling 3 mL into a Falcon tube and cooling to 30 °C. The solution was heated to 88 °C for 20 minutes. The solution was cooled to 30 °C and the final pH was recorded. The measured pH values are plotted in the following graphs 1-1, 1-2, and 1-3. Note that the temperature probe in the pH meter showed 44.4 °C at 300 minutes and cooled back to 30 °C by the end of 60 minutes. This was most likely caused by an exothermic acid / base reaction. This temperature increase could also be the cause of the non-linearity in graph 1-3. Because the pH decreases with increasing temperature.

[0365]

Table 83

[0366]

Table 84

[0367]

Table 85

[0368]

Table 86

[0369] Table 1-2 shows the pseudo molar ratio of CO2:HMD based on pH. It is observed that 1.8 equivalents of CO2 go into the solution very rapidly. The absorption rate starts to become significantly slower when it reaches 2 equivalents.

[0370]

Table 87

[0371] The results explain that air and heat can fully return the pH to 11.02. Extraction should be possible at this pH. Aeration during fermentation may be able to remove some of the dissolved CO2.

[0372] 〔Example 2〕 This example reports the results of a pseudo fermentation process. Briefly, CO2 (about 98% purity) and HMD (70% HMD purchased from Sigma Aldrich) were fed into an MM9 solution with the following composition: · 0.68% (6.8 g / L) disodium hydrogen phosphate Na2HPO4 · 0.3% (3 g / L) potassium dihydrogen phosphate KH2PO4 · 0.15% (1.5 g / L) ammonium chloride NH4Cl · 0.1% (1 g / L) ammonium sulfate (NH4)2SO4 · 0.05% (0.5 g / L) sodium chloride NaCl The experimental conditions for this example are listed in Table 2-1.

[0373]

Table 88

[0374] The measured pH of the solution and the concentration of HMD added over time are described in Graph 2-1.

[0375]

Table 89

[0376] The feed rate used in this example corresponds to an average rate of 3.31 g / L / hr (a reasonable production rate in an industrial fermentation process). The final titer and the feed concentration of HMD were confirmed by LCMS. HMD and CO2 reach dynamic equilibrium near pH 8.5. When more HMD is added, more CO2 is absorbed. The pseudo HMD:CO2 ratio is 1.95 at pH = 8.53. This ratio indicates that the major chemical species in the solution is HMD 2+ (HCO3 - )2 salt (hexamethylenediamine bis bicarbonate). Figures 2-2 and 2-3 illustrate the relative concentrations of chemical species as a function of pH for HMD and H2CO3, respectively.

[0377]

Table 90

[0378]

Table 91

[0379] At pH 8.5 (black dashed line), the major chemical species in the solution are deprotonated HMD and bicarbonate. A minor presence of HMD carbamate may also be present.

[0380] 〔Example 3〕 This example demonstrates the formation of HMD as a free base from protonated and / or carbonate / carbamate compounds formed by the contact of an aqueous solution of HMD (which was MM9 medium) with CO2 gas.

[0381] A condenser, a temperature probe, and an air sparging needle were attached to a 250 mL four-necked flask. The system was opened to air through the top of the condenser. The feed was an aqueous solution of HMD in MM9, and the pH was adjusted to 8.68 using CO2. The HMD concentration of this feed solution was analyzed by LCMS, and the pH was measured. This solution was sparged with air and refluxed at 85 °C for 3 hours. The pH of the resulting solution was obtained, and the concentration of HMD was measured by LCMS.

[0382]

Table 92

[0383] The positive pH charges of 8.68 - 11.26 shown in Table 3-1 indicate that CO2 has been removed from the solution. This concentration increased slightly as some water escaped through the condenser and the solution became concentrated. The fraction of HMD in the free base form at pH 11.26 is 57%.

[0384] 〔Example 4〕 This example describes the solvent extraction of HMD from the CO2-removed solution prepared in Example 3 above, and the extraction of HMD from an aqueous solution without pH adjustment as a control for the performance of the solvent. In this example, the following protocol was used: The pH of the aqueous feed was measured. In a 50 mL Falcon tube, 20 g of the solvent and 20 g of the feed were mixed. This mixture was mixed more strongly for 5 minutes in the Falcon tube and vortexed for 1 minute. The tube containing the mixed solvent and feed was allowed to stand until phase separation was complete. The volume of the lower (aqueous layer) was recorded. The upper layer sample was carefully pipetted out of the upper layer. A sample of the lower layer was also taken and the pH of the lower layer was measured. The recovery rate, distribution coefficient, and selectivity were calculated based on the mass balance.

[0385] The extraction data for three solvents for extracting HMD from water are described in Table 4-1.

[0386]

Table 93

[0387] Based on the screening reported in Table 4-1, in order to extract the product of the HMD regeneration solution prepared in Example 3 above, 1-hexanol was first used. This is because 1-hexanol has lower water solubility than isopentanol and cyclohexanol. Alkanes (especially hexane) were screened and subsequently tested due to their very low water solubility. Hexane extracted little even in the presence of water and gave a satisfactory recovery of available free base.

[0388] Table 4-2 shows that at a solvent-to-feed ratio of 1:1, HMD can be extracted at an overall recovery of 25.8% or 4% in the case of hexane. Based on the pH before extraction, only 60% of the free base HMD is available. Therefore, approximately 43% of the available free base HMD is extracted by the 1-hexanol solvent and approximately 7% of the available free base HMD is extracted by hexane. Hexane extracted little even in the presence of water and gave a satisfactory recovery of available free base.

[0389]

Table 94

[0390] 〔Example 5〕 Comparative Example When the modeled HMD fermentation was controlled at pH 7 with H2SO4, assuming that 88% of the glucose was converted to HMD on a mass basis and the final HMD titer was 116 g / L, 0.843 g of H2SO4 per gram of HMD was required to maintain the pH at 7. Due to the amount of sulfuric acid used, carbon dioxide is not absorbed immediately. This results in a final DIC / TDCA value of less than 0.5%. The fermentation model takes into account, among other things, cell growth and respiration, by-product formation, and the required medium composition.

[0391] Example 5A When modeling HMD fermentation at pH 8.5, assuming that 88% of glucose is converted to HMD on a mass basis and the final HMD titer is 116 g / L, sulfuric acid is not required to maintain a pH of 8.5 (based on experimental results). Carbon dioxide is immediately absorbed by HMD. During the seed fermentation, DIC / TDCA increased from <1% to approximately 54%. During the production fermentation, the value increased from approximately 54% to approximately 96%. The fermentation model takes into account, among other things, cell growth and respiration, by-product formation, and the required medium composition.

[0392] Example 5B When controlling the modeled HMD fermentation to pH 7 with CO2 only, assuming that 88% of glucose is converted to HMD on a mass basis and the final HMD titer is 116 g / L, it is shown that 2.4 moles of CO2 per mole of HMD need to be absorbed. It has been experimentally shown in other examples that pH 7 can be achieved using CO2 as the only acid for pH control. During the seed fermentation model, DIC / TDCA increased from <1% to approximately 82%. During the production fermentation model, the value increased from approximately 82% to approximately 97%. The fermentation model takes into account, among other things, cell growth and respiration, by-product formation, and the required medium composition.

[0393] Example 6 As a solvent suitable for recovering HMD free base from an aqueous solution, solvents such as alkanes were evaluated. ASPEN (Aspen Plus ver 8.6; Aspen Technology, Inc., USA) software was used to model the percentage of HMD recovered from a 50% HMD aqueous solution by solvent extraction using hexane or heptane. The components included in the Aspen model were water, HMD, DIC (dissolved inorganic carbon), and the solvent (hexane or heptane). The electrolyte NRTL model (ENRTL-RK) in ASPEN was used. The 50% HMD solution was modeled because it is a concentration that can be achieved (using the methods described in this document, especially for water and CO2 removal) and is also thought to be a concentration that avoids or reduces precipitation of HMD. The modeled CO2 content in the 50% HMD solution was 0.3%. However, since the efficiency of solvent extraction increases as the HMD concentration increases, the limit of the HMD concentration for solvent extraction can be even higher.

[0394] In this example, the extraction column had 10 theoretical stages. In this model, since HMD contained approximately 96.6% free base (solvent-extractable form), the plotted values slightly underestimated the percentage recovery of HMD in the recoverable form. These in silico modeling results demonstrate that alkanes can be effective solvents for HMD recovery from aqueous solutions in the range of the ratio of solvent to HMD solution.

[0395]

Table 95

[0396] As shown below, as the DIC concentration decreases, the efficiency of solvent extraction increases, supporting the importance of CO2 removal. An increase in DIC results in an increase in pH and an increase in the concentration of the recoverable free base form.

[0397]

Table 96

[0398] Example 7 ASPEN was used to model the sole use of either an evaporator (multiple effect evaporator) or a steam stripping column to achieve water and CO2 removal prior to HMD recovery. The conditions were as described above, and the components included in the ASPEN model were water, HMD, DIC species, and hexane, and the electrolyte NRTL model was used. While any process can be removed, the use of an evaporator results in a reduction in utility costs because it is more efficient in water removal than a stripping column, even though the use of electricity and steam in the evaporator increases when more water is evaporated. The figure below shows the use of steam and electricity in the absence of a stripping column as a function of the total water removed. The plotted utility costs relate to the evaporator process. The maximum steam and electricity use relates to the point at which a 50 wt% HMD aqueous solution is achieved. As described above, this HMD concentration was selected for the model because it was thought to be suitable for extraction, avoiding further water and CO2 removal that could cause precipitation formation as the solution is concentrated. The efficiency of solvent extraction increases with increasing HMD concentration, and further concentration may be beneficial.

[0399]

Table 97

[0400] The figure below demonstrates the use of steam in a stripping column in the absence of an evaporator. The concentration limit is a 50 wt% HMD solution. As more water evaporates from the feed, the duty and steam use of the reboiler increase significantly. However, this is partially offset by the lower capital cost of the stripping column compared to the evaporation unit. The "maximum" point on the graph represents the point at which a 50% HMD solution is obtained.

[0401]

Table 98

[0402] Example 8 Preparation of HMDA-producing microorganisms having carbonic anhydrase Escherichia coli is used as a target organism for engineering to produce HMDA, which has nucleic acids encoding enzymes utilized in the HMDA pathway and carbonic anhydrase.

[0403] The gene encoding Desulfovibrio vulgais (GenBank accession ACL09337.1 GI:218758438, SEQ ID) is codon-optimized for expression in E. coli and cloned into an expression vector under the control of a constitutive promoter. This vector also contains an origin of replication and an antibiotic resistance gene. Also cloned into the expression vector or integrated into the host (E. coli in this example) is a gene encoding an enzyme for the production of diamine (e.g., HMD).

[0404] The resulting plasmid is introduced into E. coli (e.g., MG1655 or ATCC 8739) by chemical transformation or electroporation. For chemical transformation, the cells are grown to mid-logarithmic growth phase, determined by the optical density at 600 nm (0.5 - 0.8). The cells are harvested, washed, and finally treated with CaCl2. To chemically transform these E. coli cells, the purified plasmid DNA is mixed with the cell suspension in a microcentrifuge tube on ice. The mixture is heat shocked and then recovery incubated in rich culture medium for 30 - 60 minutes. For electroporation, E. coli grown to mid-logarithmic growth phase is washed several times with water and finally resuspended in a 10% glycerol solution. To electroporate DNA into these cells, the mixture of cells and DNA is pipetted into a disposable plastic cuvette equipped with electrodes. A short electrical pulse is then applied to the cells to form small pores through which the DNA can enter the membrane. The cell suspension is then incubated in rich liquid medium and subsequently plated onto solid agar plates. Detailed protocols are described in Molecular Cloning: A Laboratory Manual Third Edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press, 3rd Edition.

[0405] The resulting genetically engineered E. coli is cultured in a glucose-containing medium according to procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of the carbonic anhydrase and HMDA genes is confirmed using methods well known in the art for determining polypeptide expression or enzyme activity (examples include Northern blotting, PCR amplification of mRNA, immunoblotting, etc.). The enzyme activity of the expressed enzyme is confirmed using assays specific for individual activities. The viability of the E. coli strain engineered to produce carbonic anhydrase can be confirmed using a phenolphthalein pH indicator to monitor the pH change. This is because carbonic anhydrase converts carbonate to CO2, increasing the pH of the resulting solution containing DA. The color change of phenolphthalein can be monitored by absorption at 550 nm. The assay is performed by adding 300 mM - 1000 mM DA to cell lysates or extracellular media containing 300 mM - 400 mM KHCO3 and 400 μM - 1100 μM phenolphthalein (Alvizo, et. al. 2014 PNAS 111(46): 16436 - 16441). Another assay for carbonic anhydrase activity is a colorimetric assay using 4-nitrophenyl acetate (3 mM 4-nitrophenyl acetate) as a substrate (Verpoorte et. al. 1967 J. Biol. Chem. 242: 4221 - 4229). Carbonic anhydrase can also be monitored by the production of CO2. HMD can be confirmed by HPLC.

Claims

【Claim 1】 a) culturing a genetically modified microorganism in a medium in a fermenter for a sufficient period of time under appropriate conditions to form a cultured medium containing one or more of DA carbonate, DA bicarbonate or DA bisbicarbonate, wherein the DA in the DA carbonate, the DA bicarbonate or the DA bisbicarbonate is selected from the group consisting of hexamethylenediamine, cadaverine, putrescine, ethylenediamine, and heptamethylenediamine, carbon dioxide, carbonate, bicarbonate, or carbonic acid mainly controls the pH of the medium which is the cultured medium, the microorganism contains at least one exogenous nucleic acid encoding at least one enzyme of a DA synthesis pathway selected from the group consisting of a hexamethylenediamine synthesis pathway, a cadaverine synthesis pathway, a putrescine synthesis pathway, an ethylenediamine synthesis pathway, and a heptamethylenediamine synthesis pathway; b) removing the cells from the medium to form an unpurified aqueous solution containing DA carbonate, DA bicarbonate or DA bisbicarbonate; c) converting the DA carbonate, DA bicarbonate or DA bisbicarbonate to DA free base and carbon dioxide; and d) isolating the DA free base: A method for producing diamine (DA) comprising the above steps.

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