TWI628282B - Adipate (ester or thioester) synthesis - Google Patents

Abstract

This invention relates to a process for the preparation of adipates or thioesters. The invention further relates to a process for the preparation of adipic acid from the ester or thioester. Further the invention provides a variety of methods for preparing intermediates of such esters or thioesters. Still another invention relates to a method for preparing 6-aminocaproic acid (6-ACA), a method for preparing 5-methylvaleric acid (5-FVA), and a method for preparing caprolactam method. Furthermore, the invention relates to a host cell for use in the method according to the invention.

Description

Synthesis technology of adipic acid (ester or thioester) Field of invention

This invention relates to a process for the preparation of adipates or thioesters. The invention further relates to a process for the preparation of adipic acid from the ester or thioester. Further the invention provides a variety of methods for preparing intermediates of such esters or thioesters. Still another invention relates to a method for preparing 6-aminocaproic acid (6-ACA), a method for preparing 5-methylvaleric acid (5-FVA), and a method for preparing caprolactam method. Furthermore, the invention relates to a host cell for use in the method according to the invention.

Background of the invention

Adipic acid (hexane diacid) can be used for the preparation of polyamines and the like. In addition, esters of adipic acid can be used in plasticizers, lubricants, solvents, and various polyurethane resins. Other uses of adipic acid are food acidulants, adhesives, insecticides, suede and dyeing applications. Known preparation methods include oxidation of cyclohexanol or cyclohexanone with nitric acid or a mixture thereof (KA oil).

Caprolactam is an internal guanamine which can also be used in the manufacture of polyamines such as nylon-6 or caprolactam-lauric acid amide copolymer (nylon-6, 12). A variety of ways to prepare caprolactone from bulk chemicals are known in the art, including the preparation of caprolactam from cyclohexanone, toluene, phenol, cyclohexanol, benzene or cyclohexane.

Intermediate compounds for the preparation of adipic acid or caprolactam such as cyclohexanol, cyclohexanone or phenol are typically derived from mineral oil, in view of the increased demand for materials prepared using better green resiliency techniques, A method is provided wherein adipic acid or caprolactam is prepared from an intermediate compound obtainable from a biorenewable source or at least from an intermediate compound which can be converted to adipic acid or caprolactam using biochemical methods .

US 5,487,987 describes a process for the preparation of adipic acid in which bacterial cells are used, wherein the carbon source is converted to 3-dehydrohumulic acid by enzymatic biosynthesis of bacterial cells or aromatic amino acid biosynthesis, The cis, cis-hexadiene diacid is produced by biocatalytic conversion of 3-dehydrohumulic acid. The cis, cis-hexadiene diacid is then chemically reduced (using a rhodium catalyst) to produce adipic acid. As such, the last step requires chemical catalysis. The inventors further expect that the aromatic intermediate formed by the bacterial cells is toxic to the cells and may require a low concentration in vivo and in cell culture.

It is known to prepare caprolactam from 6-aminocaproic acid (6-ACA), as described, for example, in US-A 6,194,572. Biochemical synthesis of 6-ACA via conversion of 6-amine hex-2-enoic acid (6-AHEA) in the presence of an enzyme having alpha, beta-enoate reductase activity as disclosed in WO 2005/068643 . 6-AHEA can be prepared from an amide acid, for example, biochemically or by purification. Although the method 6-ACA disclosed in WO 2005/068643 can be prepared by reduction of 6-AHEA, the inventors have found that under reducing reaction conditions, 6-AHEA may spontaneously and irreversibly cyclize to form an undesired By-products, notable, are beta-homoamine. Cyclization becomes a bottleneck in the manufacture of 6-ACA, resulting in considerable loss of yield.

Summary of invention

It is an object of the present invention to provide a novel process for the preparation of intermediate compounds which can be used in the preparation of polydecylamine and adipic acid or caprolactone or for adipic acid or caprolactam, which can be used as An alternative to knowing the method.

A further object is to provide a novel method that overcomes one or more of the aforementioned disadvantages.

One or more objects that can be solved in accordance with the present invention will be described hereinafter.

The inventors have made it possible to prepare adipic acid (or its ester or thioester) from succinic acid (or its ester or thioester). In particular, the inventors have concluded that adipic acid can be prepared from succinic acid (thio) esters and acetic acid (thio) esters through a series of specific reactions such as reverse β-oxidation in living cells and biosynthesis of fatty acids. Sulfur) ester, as shown in Figure 2. Here, each R represents a reactive group (assisting reaction), for example, as will be described later. Each X represents S or O, respectively. ED/EDH ₂ represents an oxidized/reduced electron donor such as NAD/NADH, NADP/NADPH, FAD/FADH ₂ , or oxidized ferredoxin/reduced ferredoxin. The actual transfer of electrons can occur directly or via an intermediate electron carrier such as a coenzyme or electron transport flavin (ETF) medium. Y-NH ₂ represents an amine-based donor, for example, as described later.

Thus, the inventors have concluded that adipic acid (or its ester or ester) can be prepared biocatalytically through a reaction cascade starting from succinic acid (or its ester or thioester) and acetic acid (or its ester or thioester) Thioester). Further, adipic acid can be converted into 5-methylvaleric acid ("5-FVA", 5-carbenic acid) by biocatalysis, which is an intermediate in the preparation of 6-ACA, and is used for the present transformation. A specific biocatalyst can be used.

Thus, the present invention relates to a process for preparing an adipate or a thioester from a succinate or a thioester by a plurality of reactions, wherein at least one of the reactions is catalyzed by a biocatalyst.

In particular, the present invention relates to a process for the preparation of adipate or thiodicarboxylate comprising 2,3-dehydroabietic acid (IUPAC name: 5-carboxy-2-pentanoic acid in the presence of a biocatalyst) The ester or 2,3-dehydroadipate thioester is converted to an adipate or thioester.

Detailed description of the preferred embodiment

When a carboxylic acid or a carboxylic acid salt such as 6-ACA, other amino acids, 5-FVA, adipic acid/adipate, succinic acid/succinate, acetic acid/acetate is mentioned herein, unless Unless otherwise specified, these terms are meant to include protonated carboxylic acids (free state acids), corresponding carboxylates (its conjugate bases), and salts thereof. When an amino acid such as 6-ACA is recited herein, the term is meant to include amino acids in their zwitterionic form (wherein the amine group is in a protonated form and the carboxylic acid group is in a deprotonated form), The amino group which is protonated and the carboxyl group is in a neutral form, and the amino acid in which the intermediate group is in a neutral form and the carboxylic acid group is in a deprotonated form, and a salt thereof.

When referring to esters or thioesters of carboxylic acids such as adipates or thioesters, acetates or thioesters, succinates or thioesters, these terms are meant to include any reactive group, particularly any biological activity. The group includes Coenzyme A (also known as CoA), Phosphopantothiol which binds to a thiol or peptide carrier protein (ACP or PCP, respectively), N-Ethyl-cysteine, Methyl- Thioglycolate, methyl phthalate, ethyl-deuteropropionate, methyl-deuterated butyrate, methyl-deuterated butyrate, deuterated propionate and may provide the same Or other esters or thioesters of similar function. In the case of using living cells as biocatalysts, the ester or thioester, in particular CoA, can be produced using a biocatalyst or derived from an organism which can also produce the appropriate enzyme for catalyzing the reaction. CoA-binding enzymes and CoA-transferases have been identified in a variety of organisms and can provide the desired activated ester or thioester.

The preparation of adipates or thioesters from succinates or thioesters specifically comprises the following reaction steps (the number between brackets also corresponds to the number in Figure 1):

(1) providing a succinate or a thioester and reacting the ester or thioester with an acetate or a thioester, thereby forming a 3-keto adipate or a thioester;

(2) hydrogenating the 3-keto adipate or the 3-keto group of the thioester thereby forming a 3-hydroxyadipate or a thioester;

(3) dehydrogenating the 3-hydroxyadipate or thioester to form 2,3-dehydroadipate or thioester;

(4) Hydrogenating the C-C double bond of the 2,3-dehydroadipate or thioester, thereby forming an adipate or a thioester.

The invention also relates to a process suitable for the preparation of intermediate compounds suitable for use in the manufacture of adipic acid, comprising one or more of the reaction steps 1-4 in the presence of a biocatalyst which catalyzes such a reaction step.

In one embodiment, the adipate or thioester is converted to 5-FVA (5).

The adipate or thioester can be converted to adipic acid if desired. (7) can be achieved by hydrolyzing an ester bond or a thioester bond, thereby forming adipic acid; or by a transfer reaction, wherein an "alcohol" moiety or a "thiol" moiety (such as CoA) is derived from the adipate. Or the thioester is transferred to an acid different from adipic acid, thereby forming adipic acid and an acid (thio) ester (7) different from adipic acid. If succinic acid or acetic acid is used as the different acid, the advantage of this reaction is that the alcohol moiety or the thiol moiety such as CoA can be recovered, for example, hexamethylene-CoA+ succinate or acetate can be converted (usually to CoA). In the presence of a transferase), butadiene-CoA or acetamidine-CoA + adipic acid is formed. Butadiene-CoA or acetamidine-CoA can then be used as the starting compound for the process of the invention.

Adipic acid (or an ester or thioester thereof) can be converted, for example, to 5-FVA (8).

In one embodiment, the 5-FVA obtained by the process of the invention is converted to 6-ACA (6). The 6-ACA can then be converted to caprolactam, for example, by methods known in the art.

In yet another embodiment, the adipic acid or caprolactam obtained in accordance with the process of the present invention is used in the preparation of polyamide.

Words such as "a" as used herein are defined as "at least one" unless otherwise specified.

When a noun (for example, a compound, an additive, etc.) is recited in the singular, the plural also includes the plural.

When referring to compounds in which several isomers are present (e.g., cis and trans isomers, R and S enantiomers), in principle, such compounds include all pairs of such compounds which are particularly useful in the methods of the invention. Opposites, diastereomers and cis/trans isomers.

When the enzyme is described in reference to the enzyme class (EC) in parentheses, the enzyme class is classified or categorizable based on the enzyme nomenclature provided by the International Federation of Biochemistry and Molecular Biology Committee (NC-IUBMB). The name can be found at http://www.chem.qmul.ac.uk/iubmb/enzyme / . Other suitable enzymes that have not been classified as a particular class but can be classified as such are also included herein.

When a protein is referred to herein as an accession number, the number is specifically used to refer to a protein having a sequence appearing in Uniprot on March 11, 2008, unless otherwise specified.

The term "homolog" is used herein particularly to have at least 30%, preferably at least 40%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more Preferably at least 80%, especially at least 85%, more particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 % of the sequence of the same degree of polynucleotide or multi-peptide. The term homologue also refers to a nucleic acid sequence (polynucleotide sequence) encoding the same multi-peptide sequence, which differs from other nucleic acid sequences due to degeneracy of the genetic code.

Sequence identity or similarity is defined herein as determining the relationship between two or more multi-peptide sequences or two or more nucleic acid sequences by comparing the two sequences. Generally, sequence identity or similarity compares the full length of the sequence, but it is also possible to compare only the portions of the sequence that are calibrated to each other. In the art world, "identity" or "similarity" also refers to the degree of matching between sequences, the sequence correlation between multiple peptide sequences or nucleic acid sequences, as the case may be. Preferably, the method of determining the degree of identity or similarity is designed to achieve the maximum degree of matching between the sequences tested. In the context of the present invention, preferred computer programs for determining the degree of identity and similarity between two sequences include BLASTP and BLASTN (Altschul, SF et al, J. Mol. Biol. 1990, 215, 403-410, by NCBI and other sources). Available from work (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Basta, Maryland 20894)). Preferred parameters for the comparison of multi-peptide sequences using BLASTP are gap opening 10.0, gap extension 0.5, and Blosum 62 matrix. Preferred parameters for nucleic acid sequence comparison using BLASTN are sequence opening 10.0, sequence extension 0.5, DNA full matrix (DNA identity matrix).

In the process of the invention, a biocatalyst is used, in other words, at least one of the reaction steps is catalyzed by a biological material or biological moiety derived from a biological source such as an organism, or a biomolecule derived from an organism. Biocatalysts in particular comprise one or more enzymes. The biocatalyst can be used in any form. In one embodiment, one or more enzymes isolated from the natural environment (from the organism from which the enzyme is produced) are used, such as in solution, emulsion, dispersion, lyophilized cells (suspension), lysate, or brake The form of the body is used. The use of an enzyme isolated from the organism from which it is derived is particularly useful because the flexibility of the adjustment of the reaction conditions is increased and the equilibrium of the reaction moves towards the desired end.

In one embodiment, the one or more enzymes form part of a living organism, such as a living whole cell. The enzyme can perform catalytic functions inside the cell. It is also possible that the enzyme can be secreted into the medium in which the cells are present.

The living cells can be growing cells, resting resting cells or dormant cells (such as spores) or cells in stationary phase. Enzymes that form part of a permeabilizable cell (i.e., a precursor that becomes a permeabilizable enzyme substrate or an enzyme substrate) can also be used.

The biocatalyst used in the process of the invention is in principle any organism or may be derived or derived from any organism. The organism can be a eukaryote, a prokaryote or an archaea. The particular organism may be selected from the group consisting of animals (including humans), plants, bacteria, archaea, yeast, and fungi.

Suitable bacteria are particularly selected from the group consisting of Absidia, Achromobacter, Acinetobacter, Agrobacterium, Aeromonas. Aeromonas, Alcaligenes, Arthrobacter, Arzoarcus, Azomonas, Azospirillum, Azotobacter ), Bacillus, Beijerinckia, Bradyrhizobium, Burkholderia, Byssochlamys, Citrobacter, Clostridium (Clostridium), Comamonas, Corynebacterium, Deinococcus, Escherichia, Enterobacter, Flavobacterium , Fusobacterium, Gossypium, Klebsiella, Lactobacillus, Listeria, Megasphaera, Micro Micrococcus, branch rod Genus (Mycobacterium), Nocardia (Norcadia), Porphyromonas (Porphyromonas), Propionibacterium, Pseudomonas, Rastaea ( Ralstonia), Rhizobium, Rhodopseudomonas, Rhodospirillum, Rodococcus, Rosbergia, Shewanella, chain Streptomycetes, Xanthomonas, Xylella, Yersinia, Treponema, Vibrio, Streptococcus ), Lactococcus, Zymomonas, Staphylococcus, Salmonella, Brucella, Microscilla.

Suitable eukaryotes may in particular be selected from the group consisting of fungi; multicellular animals; Viridiplantae (especially Arabidopsis and Chlamydomonadales; Diplomonads) Special Giardiinae; Entamoebidae (especially Entaboeba); Euglenozoa (especially Euglena); Pelopinantida (especially roots) Mastigamoeba; and Alveolata (especially Cryptosporidium).

Suitable fungi include, in particular, fungi and yeasts selected from the group consisting of Rhizopus, Neurospora, Penicillium, Aspergillus, Piurobacter Genus (Piromyces), Trichosporon, Candida, Hansenula, Kluyveromyces, Saccharomyces, Rhodotorula , Schizosaccharomyces, Yarrowia (such as Yarrowia lypolytica).

Suitable multicellular animals include, in particular, multicellular animals selected from the group consisting of mammals, including humans, more particularly selected from the group consisting of Leporidae, Muridae, and Suidae. ), Bovidae, hominidae. The biocatalyst can be derived from any part of a multicellular organism such as the liver, pancreas, brain, kidney, heart or other organs. Suitable multicellular animals also include, in particular, Caenorhabditis and Drosophila.

An organism that specifically provides a suitable biocatalyst for a particular reaction step when describing a particular reaction step of the process of the invention is illustrated below.

It will be apparent to those skilled in the art that a mutant of a natural biocatalyst (wild type) or natural biocatalyst having suitable activity for the method according to the present invention can be used. The nature of the natural biocatalyst can be improved by biotechnological improvements known to those skilled in the art, such as molecular evolution or theoretical design. The mutant strain of the wild-type biocatalyst can be used as a biocatalyst or a biocatalytic moiety via modification, for example, by using a mutation-generating technique (random mutation occurrence, position-directed mutation occurrence, directed evolution, genetic recombination, etc.) known to those skilled in the art. Manufactured from the DNA encoding the organism (such as an enzyme). In particular, the DNA is modified such that the DNA encodes an enzyme that differs from the wild-type enzyme by at least one amino acid, thus encoding a wild-type enzyme comprising one or more amino acid substitutions, deletions and/or insertions, or a mutation The strain combines two or more parental enzyme sequences, or by performing such modified DNA on appropriate (host) cells. The latter can be achieved by methods known to those skilled in the art, such as codon optimization or codon pair optimization methods, for example based on the method described in WO 2008/000632.

Mutant biocatalysts have improved properties, for example, improved properties for one or more of the following phases: selectivity to enzyme matrix, activity, stability, solvent tolerance, pH profile data, temperature profile data, enzyme matrix profile Data, susceptibility to inhibition, cofactor utilization, and enzyme matrix affinity. Mutants with improved properties can be identified based on methods known to those skilled in the art, such as appropriate high throughput screening or selection methods.

The enzyme substrate specificity of the enzyme acting on the alkyl or alkyl ester or thioester can be modified. Molecular evolution can be utilized to form diversity, followed by screening of desired mutants and/or rationally engineered enzyme matrix binding pockets. Enzyme matrix specific modification techniques for the enzymes used in the methods of the invention can be based on methods known to those skilled in the art. For example, the rational design of structural and sequence information to design a specific mutant strain has been used to modify the enzyme matrix specificity of the thiol transferase site 4 derived from erythromycin polyketide synthase. Other sputum bases. It has been shown to modify the suggested enzyme matrix binding sites, resulting in modified binding pockets that can be combined with other enzyme matrices to achieve different product ratios (Reeves, CD; Murli, S.; Ashley, GW; Piagentini, M.; Hutchinson , CR; McDaniel, R. Biochemistry 2001, 40 (51), 15464-15470). Both the theoretical design method and the molecular evolution method have been used to change the enzyme matrix specificity of the biocatalyst BM3, resulting in the replacement of the natural enzyme matrix of the medium chain fatty acid (such as myristic acid) or the medium chain fatty acid natural enzyme matrix, and a large amount of oxidizable. Mutant strains of various different alkenes, cycloolefins, arylenes and heteroarylenes (Peters, MW; Meinhold, P.; Glieder, A.; Arnold, FH American Chemical Society Journal 2003, 125(44), 13424 -13450; Appel, D.; Lutz-Wahl, S.; Fischer, P.; Schwaneberg, U.; Schmid, RD Biotechnology Journal 2001, 88(2), 167-171 and references therein).

When referring to a biocatalyst derived from a particular source, in particular an enzyme, it is specifically meant to include a recombinant biocatalyst, in particular an enzyme, derived from the first organism but actually in a (genetically modified) second organism. The biocatalyst from the first organism is in particular an enzyme.

Preparation of 3-ketoadipate (ester or thioester) ("Reaction 1")

In the examples of the present invention, 3-ketoadipate (ester or thioester) is prepared from succinate and acetate, and the succinate and acetate actually have an activating group, and in particular, an ester or Thioester to aid in the reaction.

3-ketoadipate (ester or thioester) can be achieved in a biocatalytical or chemical manner, in particular by a Claisen condensation reaction in which an acetate or thioester is coupled with a succinate or thioester. .

In a preferred process of the invention, the biocatalytic reaction is carried out in the presence of a biocatalyst capable of catalyzing sulfhydryl transfer. An enzyme having such catalytic activity can thus be referred to as a transferase.

In a preferred method, the thiol transfer is carried out between two thiol esters or oxime esters. Preferably, the thiol esters are acetamidine-CoA and butyl ruthenium-CoA. Preferably, the catalyst is selective for the guanidine thioester.

The biocatalyst comprises in particular an enzyme which is capable of undergoing thiol transfer, the enzyme being selected from the group consisting of a transductase (EC 2.3.1), preferably selected from the group consisting of acetamidine-CoA: acetamidine-CoAC -Acetyltransferase (EC 2.3.1.9), 醯-CoA: acetamidine-CoAC-transferase (EC 2.3.1.16) and succinyl-CoA: acetamidine-CoAC-butane transferase (EC 2.3) .1.174, also known as the group consisting of β-ketohexanide-CoA thiolase. The chymase activity is, for example, described in β-oxidation, fatty acid biosynthesis, biosynthesis of polyketide, or butyric acid metabolism in the KEGG (Kyoto Encyclopedia of Genes and Genomes) gene pool.

The transferase is particularly a transferase selected from the group consisting of an organism consisting of archaea, bacteria and eukaryotes.

In particular, the enzyme may be derived from a microorganism which decomposes an organic compound comprising an aromatic ring structure or a cycloaliphatic ring structure, particularly a 5, 6, or 7 membered ring structure. The organic compound may optionally contain one or more heteroatoms or as part of a substituent or a substituent in the ring. For example, the organic moiety can be an aromatic compound, particularly an aromatic compound comprising a 6-membered ring. In particular, the aromatic compound may be selected from the group consisting of phenyl acetate, benzoate, catechol, cucurbitate and gentisate. The organic compound may be a cycloaliphatic compound, especially a cyclic alcohol such as cyclohexanol, a cyclic ketone such as cyclohexanone, or a cycloalkane such as cyclohexane. The organic compound can be an internal guanamine such as caprolactam. In one embodiment, the unsaturated dicarboxylic acid such as adipic organism decomposable enzyme derived from dicarboxylic acids (typically C ₆ -C _10), particularly a linear embodiment.

In yet another embodiment, the enzyme is derived from an enzyme that can synthesize 3-keto-adipate, for example, as part of a secondary metabolite (eg, malonomycin), for example, derived from a chain. A genus of genus (particularly Streptomyces rimosus, from Actinomyces, from other Actinobacteria or other known secondary metabolite manufacturers).

Preferred microorganisms for providing a biocatalyst which catalyzes the preparation of 3-ketoadipate (ester or thioester) further include Acinetobacter (particularly Acinetobacter sp. ADP1 and A. calcoaceticus) , Agrobacterium (especially A. tumefaciens), Alcaligenes (especially for Alcaligenes strain D2 and A. eutrophus), Arthrobacter spp. Azulosis (specially A. evansii), Azotobacter, Azotobacter, Bacillus (especially B. halodurans), Bayein Genus, genus Rhizobium, Burkholder, Clostridium (especially C. kluyveri, acetobutylicum, Clostridium palustris (C. Beijerinckii)), C. genus, Corynebacterium (especially C. glutamicum and C. aurantiacum), E. coli, Enterobacter, Flavobacterium, Mycobacterium (particularly M. elsdenii), Nocardia, Pseudomonas (especially P. pulcherii) P. putida, P. aeruginosa and Pseudomonas strain B13), Ralstonia (especially R. eutropha), Rhizobium, Rhodopseudomonas (especially R. palustris), Rhodococcus (especially Rh. erythropolis, R. opacus) And Rhodococcus sp. RHA1), Fusarium (especially A. niger), Euglena (especially Euglena gracilis), Rhodobacter genus (especially Rough red mold (N . crassa)), Penicillium (particularly P. chrysogenum), Rhodotorula, Saccharomyces, Trichosporon (especially T. cutaneum).

In a particular embodiment, the biocatalyst comprises an enzyme comprising an amino acid sequence identified by any of Sequence IDs 1-13 or any of its homologs.

Preparation of 3-hydroxyadipate (ester or thioester) ("Reaction 2")

In one embodiment, 3-hydroxyadipate (ester or thioester) is prepared from 3-ketoadipate (ester or thioester). Typically 3-ketoadipate is provided as a reactive group as previously described.

In principle 3-hydroxyadipate (ester or thioester) can be prepared chemically, for example via selective hydrogenation of a 3-keto group in 3-ketoadipate (ester or thioester).

This reaction is carried out in particular in the presence of a biocatalyst which catalyzes the reaction step, in particular a biocatalyst which catalyzes the reduction of the ketone group, more particularly the reduction of the carbonyl group to a hydroxyl group.

In a specific embodiment, the 3-ketoadipate is present in the form of a thioester having a coenzyme A (hereinafter, the thioester of 3-ketoadipate and coenzyme A will be referred to as 3-ketohexanide-CoA ).

In a preferred method of the invention, the preparation comprises a biocatalytic reaction in the presence of a biocatalyst which catalyzes the reduction of 3-ketooxime (ester or thioester) to 3-oxindole (ester or thioester).

An enzyme having such catalytic activity is thus referred to as a 3-hydroxyindole (ester or thioester) dehydrogenase. Therefore, an enzyme having such catalytic activity for 3-hydroxyindole CoA-thioester is referred to as 3-hydroxyindole CoA-dehydrogenase. Preferably, the 3-hydroxyindole CoA-dehydrogenase is selective for the enzyme substrate 3-ketohexanide-CoA.

An enzyme which catalyzes the reduction of 3-ketooxime (ester or thioester) to 3-oxindole (ester or thioester) is selected in particular from the group consisting of dehydrogenase (EC 1.1.1), preferably Selected from 3-hydroxyindole-CoA dehydrogenase (EC 1.1.1.35 and EC 1.1.1.36), 3-hydroxybutyrene-CoA dehydrogenase (EC 1.1.1.157), 3-hydroxyheptafluorene-CoA A group consisting of dehydrogenase (EC 1.1.1.259) and long chain 3-hydroxyindole-CoA dehydrogenase (EC 1.1.1.211). These enzymes may use NADH or NADPH as a cofactor. According to the KEGG (Kyoto Gene and Genome Encyclopedia) database, 3-hydroxyindole-CoA dehydrogenase activity has been described, for example, in fatty acid metabolism, polyketide biosynthesis, polyhydroxyalkanoate metabolism, butyric acid metabolism, and aromatic compounds. break down.

Specifically, the microorganism can decompose the organic compound (referred to as "Reaction 1") as previously identified, and is particularly an aromatic compound, a cycloaliphatic compound or a dicarboxylic acid.

Other preferred organisms for providing biocatalysts which catalyze the preparation of 3-hydroxyadipate (ester or thioester) include: Acinetobacter (particularly Acinetobacter sp. ADP1 and A. calcoaceticus) ), Alcaligenes (especially for Alcaligenes genus D2 and A. eutrophus), denitrifying bacteria (specially denitrifying bacteria), Bacillus (particularly B. halodurans, Bordetella (especially B. pertussis, Burkholderia (especially B. pseudomallei and B. xenovorans), Corynebacterium ( Especially for Corynebacterium glutamicum, Corynebacterium sphaeroides and C. efficiens, Dinocoides (especially D. radiodurans), Escherichia coli, Flavobacterium, gram Klebsiella (particularly K. pneumonia), Pseudomonas (especially Pseudomonas platensis and P. fluorescens), red fake Cyanobacteria (especially R. palustris), Rhodococcus (particularly Rhodococcus erythropolis, milk) Rhodococcus (R. opacus and Rhodococcus sp. RHA1), Fusarium (especially A. niger), Rhizopus (especially N. crassa), Penicillium ( In particular, it is P. chrysogenum, and the genus Saccharomyces (especially S. cerevisiae).

Suitable organisms for providing an EC 1.1.1.35 enzyme acting on 3-ketohexano-CoA may be obtained from any organism including mammals, particularly selected from the group consisting of European cattle (Bos taurus), brown rats (Rattus norvegicus), A mammal in a group consisting of European pigs (Sus scrofa) and humans (Homo sapiens).

Suitable biocatalysts involving (anaerobic) fatty acid synthesis, polyketide biosynthesis or polyhydroxyalkanoic acid metabolism can be obtained from any organism, in particular microorganisms including: Clostridium (especially Clostridium butyricum and Kluwer) Clostridium), Euglena (especially for the genus Euglena), Megacute (especially for the genus Erythromycin), genus Rasta (especially for the genus Rasmusia), and fungal micelles (Zoogloea) ) (especially for Zoogloea ramigera).

In a particular embodiment, the biocatalyst (catalyzed "Reaction 2") comprises an enzyme comprising an amino acid sequence as recognized by any of Sequence IDs 15-26, 29 or a homolog thereof. It is specifically contemplated that an enzyme comprising an amino acid sequence according to sequence ID 26 catalyzes "Reaction 2" and "Reaction 3".

Preparation of 2,3-dehydroabietic acid (ester or thioester) ("Reaction 3")

In one embodiment, 2,3-dehydroadipate (5-carboxy-2-pentanoic acid) (ester or thioester) is prepared from 3-hydroxyadipate (ester or thioester). Optionally, 2,3-dehydroadipate and 3-hydroxyadipate are coupled to a coenzyme, ACP or other reactive group as previously described.

In one embodiment of the invention, 2,3-dehydrohexanedicarboxylic acid can be prepared by chemically converting 3-hydroxyadipate (ester or thioester), for example, in the presence of water in an anhydrous environment, for example, in the presence of sulfuric acid. Ester or thioester).

2,3-Dehydroabietate is prepared in particular from 3-hydroxyadipate using at least one biocatalyst.

Preferred biocatalysts are biocatalysts which catalyze the dehydration of 3-hydroxydecyl ester or thioester to its 2-enyl ester or its 2-enyl thioester.

In a particular embodiment, the 2,3-dehydroadipate is present as a thioester of Coenzyme A (hereinafter, 2, 3-thiodeadipate and Coenzyme A thioester will be referred to as 5-carboxy- 2-pentene fluorene-CoA).

In a particular embodiment, the biocatalyst catalyzes the dehydration of 3-hydroxyhexamidine-CoA to 5-carboxy-2-pentenyl-CoA.

In particular, the biocatalytic reaction can be carried out in the presence of a biocatalyst which catalyzes the dehydration of 3-oxoquinone (thio) ester to 2,3-dehydroabietyl ester.

Therefore, an enzyme having such activity is referred to as a 3-hydroxyindole (ester or thioester) dehydratase. An enzyme having such catalytic activity for 3-hydroxyindole CoA-thioester is thus referred to as a 3-hydroxyindole-CoA dehydrating enzyme. Preferably, the 3-hydroxyindole-CoA dehydrating enzyme system is selective for the enzyme substrate 3-hydroxyhexamidine-CoA.

An enzyme which catalyzes the dehydration of 3-hydroxyindole (ester or thioester) to 2,3-dehydroindole (ester or thioester) is especially selected from the group consisting of dehydratase (EC 4.2.1). The best is selected from the group consisting of olefin-CoA hydratase (EC 4.2.1.17), 3-hydroxybutyrene-CoA dehydratase (EC 4.2.1.55) and long-chain olefin-CoA hydratase (EC 4.2.1.74). ) the group consisting of. According to the KEGG database, 3-hydroxyindole-CoA dehydroenzyme activity has been described, for example, in fatty acid metabolism, synthesis of polyketides, or metabolism of butyric acid, and decomposition of aromatic compounds.

The 3-hydroxyindole (ester or thioester) dehydrating enzyme may be one selected from the group consisting of archaea, bacteria, and eukaryotes, for example, selected from the group consisting of yeast, fungi, and mammals. 3-Hydroxyindole (ester or thioester) dehydratase of the organism.

In particular, the decomposable organic compound (referred to as "Reaction 1") identified above is particularly a catalyst of an aromatic compound, a cycloaliphatic compound or a dicarboxylic acid to catalyze 2,3-dehydroadipate (ester or thioester) A preferred source of biocatalyst for the preparation.

The microorganism capable of decomposing an aromatic compound, a cycloaliphatic compound, or a dicarboxylic acid includes Acinetobacter (particularly Acinetobacter sp. ADP1 and Acinetobacter calcoacetic), Alcaligenes (particularly Alcaligenes sp. D2), and alfalfa. Genus (in particular, black bacillus), denitrifying genus (especially for the denitrifying bacteria), azotobacter, Azotobacter, Bacillus (particularly Bacillus halophilus), Corynebacterium (especially for C. glutamicum and C. annua), Escherichia coli, Flavobacterium, Rhizopus (especially Rough red mold), Penicillium (especially P. chrysogenum), Pseudomonas (special It is Pseudomonas platensis and Pseudomonas fluorescens, Rhodopseudomonas (especially Rhodopseudomonas palustris), Rhodococcus (particularly Rhodococcus germ line RHA1).

Preferred organisms which provide an EC4.2.1.17 enzyme for 3-hydroxyhexanthene-CoA include organisms selected from the group consisting of mammals and microorganisms. The EC4.2.1.17 enzyme suitably derived from mammals is obtained in particular from a group consisting of European cattle, humans, brown rats and European pigs. Suitable enzymes derived from microorganisms of EC 4.2.1.17 are in particular selected from the group consisting of Aeromonas (Specific A. caviae), Clostridium (Special Acetyls) Clostridium butyricum), Gossypium (G. hirsutum), Rhodospirillum (R. rubrumi) and Rastaella (special A group consisting of Rasporium ssp.

It is also preferably a microorganism capable of performing (anaerobic) fatty acid biosynthesis. Such microorganisms include Clostridium (especially Clostridium butyricum and Clostridium clostridium), Euglena (specially thin eyeworm), Macrococcal (especially Escherichia coli), and Saccharomyces (especially Saccharomyces cerevisiae).

Suitable enzymes specifically comprise an amino acid sequence according to any one of the sequence IDs 14, 27, 28, 30-41, 92 or a homolog thereof.

Preparation of adipic acid (ester or thioester) from 2,3-dehydroadipate (ester or thioester) ("Reaction 4")

In one embodiment, adipic acid (ester or thioester) is prepared from 2,3-dehydroadipate (ester or thioester). Adipic acid (ester or thioester) can be prepared chemically from 2,3-dehydroadipate (ester or thioester), for example by selective hydrogenation of C ₂ -C ₃ double bonds or by biocatalysis .

Typically 2,3-dehydrohexanedioic acid has an active group as indicated above.

Adipic acid (ester or thioester) is preferably a 2,3-dehydrogenated catalyst using at least one biocatalyst that catalyzes the hydrogenation of a carbon-carbon double bond of 5-carboxy-2-pentanoic acid (ester or thioester) Preparation of adipic acid (ester or thioester).

In a preferred method of the invention, the preparation comprises a biocatalytic reaction in the presence of a biocatalyst which catalyzes the reduction of a cis or trans-2-ene oxime (ester or thioester) to a oxime (ester or thioester). The biocatalyst can use a range of electron donors, such as an electron donor selected from the group consisting of NADH, NADPH, FADH _2, and reduced ferredoxin. The electrons can be transferred directly from the electron donor to the biocatalyst, or otherwise transferred from the electron donor to the biocatalyst, particularly by means of the so-called electron transfer flavin (ETF) medium. Therefore, an enzyme having such catalytic activity is referred to as a 2-olefin (ester or thioester) reductase (ER). Therefore, an enzyme having such catalytic activity for 2-enoxan-CoA thioester is referred to as 2-ene fluorene-CoA reductase. Preferably, the 2-enyl-CoA reductase is selective for the enzyme substrate 2,3-dehydrohexamethylene-CoA.

The enzyme which catalyzes the reduction of 2-ene oxime (ester or thioester) is especially selected from the group consisting of oxidoreductases (EC 1.3.1 and EC 1.3.99), preferably selected from the group consisting of a group consisting of 醯-CoA reductase (EC 1.3.1.8, EC 1.3.1.38, and EC 1.3.1.44); selected from the group consisting of olefin-[醯-carrier-protein] reductase EC 1.3.1.9, EC a group consisting of 1.3.1.10 and EC 1.3.1.39; and selected from the group consisting of Dingshao-CoA dehydrogenase (EC 1.3.99.2), 醯-CoA dehydrogenase (1.3.99.3) and long chain 醯-CoA A group consisting of dehydrogenase (EC 1.3.99.13). Trans-2-enyl (ester or thioester) reductase activity has been described, for example, in fatty acid metabolism, polyketide synthesis, butyrate metabolism, and mitochondrial fatty acid biosynthesis according to the KEGG database.

The 2-olefin (ester or thioester) reductase can in principle be obtained or derived from any organism. The particular organism may be selected from bacteria, archaea or eukaryotes, such as a group selected from the group consisting of yeast, fungi, protists, plants and animals, including humans.

In one embodiment, the organism may be selected from the group consisting of Escherichia coli, Vibrio, Bacillus (particularly Bacillus subtilis), Clostridium (particularly Kluyveromyces cerevisiae, Clostridium butyric acid) , C. perfringens and C. perfringens, Streptomyces (especially S. coelicolor and S. avermitilis) ), Pseudomonas (special Pseudomonas platensis and Pseudomonas aeruginosa), Schwannella, Xanthomonas, xylobacter, Yarrowia, Treponema (especially for tooth T. denticola), Aeromonas (especially Aeromonas hydrophila), Trachomatis (especially Microscilla marina), Megacoccal ( Especially for the genus Erythromycin, Dinobacter (especially for the radiation of Dinoococcus), Yarrowia (especially for the decomposition of A. faecalis) and Eubacterium (especially for the genus Pyruvate (E. pyruvativorans)).

In one embodiment, the 2-enyl (ester or thioester) reductase is derived from an organism selected from the group consisting of the eye worm (particularly Euglena).

In one embodiment, the 2-enyl (ester or thioester) reductase is selected from the group consisting of Saccharomyces (Special Saccharomyces cerevisiae), Kluyveromyces (Special K. lactis, An organism in a group consisting of Schizosaccharomyces (particularly S. pombe) and Candida (C. tropicalis).

In one embodiment, the 2-enyl (ester or thioester) reductase is selected from the group consisting of the genus Trichophyton (specially, the genus A. nidulans) and the genus Penicillium (particularly P. chrysogenum) ) the organisms in the group formed.

In one embodiment, the 2-enyl (ester or thioester) reductase is derived from an organism selected from the group consisting of Arabidopsis, particularly A. thaliana.

In one embodiment, the 2-enyl (ester or thioester) reductase is selected from the group consisting of human, brown rat, European cattle, Cavia sp., Caenorhabditis elegans, and black belly. An organism in a group of Drosophila melanogaster.

Suitable enzymes in particular comprise an amino acid sequence according to any of the sequence IDs 42-67, 94, 96, 98, 100, 105, 107, 109, 111, 113 or a homolog thereof, more particularly according to the sequence ID 60, The amino acid sequence of any of 63, 96, 100 or a homolog thereof. Examples of nucleotide sequences encoding appropriate enzymes for catalyzing "Reaction 4" are represented by 2-enyl (ester or thioester) reductases 93, 95, 97, 99, 104, 106, 108, 110 and 112.

In a preferred embodiment, the use of ETF facilitates the activity of the reductase in addition to the 2-ene oxime (ester or thioester) reductase. Such ETFs may be derived or derived from organisms obtained or derived from reductase as previously identified. It may in particular be derived or derived from an organism of the same species as the reductase used, more particularly an organism of the same species. A particular ETF comprises the amino acid sequence represented by sequence IDs 102, 103, 115, 116. Sequence IDs 101 and 114 represent nucleotide sequences encoding a particular ETF. Typically such ETFs comprise two subunits (etfA and etfB) encoded by two different genes. Typically used in combination to make active ETF proteins. For example, the following composition can be used: sequence ID 102 and sequence ID 103 or a combination of sequence ID 116 and sequence ID 115. Skilled people can choose other suitable ETF compositions known to the art world.

In embodiments of the invention, a biocatalyst having a desired activity or enzyme matrix specificity can be modified by methods known in the art, such as by theoretical design or molecular evolution modification to form a catalyst that can be rationally or reasonably selective 2 3-Dehydroabietate (ester or thioester) is converted to a mutant of adipic acid (ester or thioester). It is preferred to use a biocatalyst having activity with a 2-ether oxime-CoA derivative having a chain length of 6, which is obtained from Clostridium clostridium, European cattle, Euglena, guinea pig species, Saccharomyces cerevisiae, Candida tropicalis Biocatalysts for humans and P. pyruvate are particularly good.

Preparation of adipic acid ("Reaction 7")

According to the invention, the adipate or thioester can be used to prepare adipic acid by hydrolysis of an ester bond or a thioester bond. It can be achieved chemically, for example in the presence of an acid or a base or by chemical catalysis.

In a preferred method of the invention, the biocatalytic reaction is carried out in the presence of a biocatalyst which catalyzes the hydrolysis of hydrazine (thio) ester.

Therefore, the catalytically active enzyme can be referred to as a thiol ester hydrolase. An enzyme having such catalytic activity for a ruthenium-CoA thioester is thus referred to as a ruthenium-CoA hydrolase. Preferably, the 醯-CoA hydrolase is selective for the enzyme substrate hexamethylene-CoA.

The enzyme which catalyzes the hydrolysis of hydrazine (thio) ester is selected in particular from the group consisting of hydrolase (EC 3.1.2), preferably selected from the group consisting of hydrazine-CoA hydrolase (EC 3.1.2.20), Acetyl-CoA hydrolase (EC 3.1.2.1), long-chain fat-醯-CoA hydrolase (EC 3.1.2.2), butadiene-CoA hydrolase (EC 3.1.2.3) and 醯-[醯-carrier a group consisting of -protein]-hydrolase (EC 3.1.2.14).

Biocatalysts comprise enzymes derived from any organism, including archaea, bacteria or eukaryotes.

The special biocatalyst comprises an enzyme selected from the group consisting of: Escherichia coli, Brucella (Special Brucella melitensis), Agrobacterium (special nodules) Agrobacterium), Xanthomonas, Sinorhizobium (Specially Sinorhizobium meliloti), Mesorhizobium (Special Mesorhizobium loti) , Vibrio, Streptomyces (especially Streptomyces aureus and A. streptococci), Rhodopseudomonas (specially Rhodopseudomonas pallidum), xylobacter, Yersinia, Pseudomonas (special Pseudomonas and Pseudomonas aeruginosa), Schwannella, Shigella, Salmonella, Corynebacterium, Mycobacterium, Tuenomonas (Hyphomonas) (specially Hyphomonas neptunium) and oleic acid genus.

Suitable biocatalysts are particularly found in yeasts selected from the group consisting of Saccharomyces (Special Saccharomyces cerevisiae) and Kluyveromyces (Specific Kluyveromyces lactis).

Suitable biocatalysts are particularly found in fungi selected from the group consisting of the genus Fusarium (specially, the genus A. fumigatus and the genus) and the genus Penicillium (particularly P. chrysogenum). .

In yet another embodiment, the organic system is selected from the group consisting of Arabidopsis (particularly Arabidopsis), Muridae (particularly brown rat, mouse (Mus musculus), bovidae (special European) A group of cattle, sheep (Ovis aries), humans, and Caenorhabditis (Caenorhabditis elegans).

In one embodiment of the invention, a biocatalyst that does not itself have the desired activity or enzyme matrix specificity can be formed by techniques known in the art, such as by theoretical design or molecular evolution modification, to form an adipate or thioester. A mutant that is efficiently converted to adipic acid. A biocatalyst having an initial activity with a ruthenium-CoA derivative of a C4-C8 acid, particularly a dicarboxylic acid, is preferred. For example, a mutant strain can be formed based on a sputum-CoA-thioesterase obtained from a mouse (for example, listed in Seq ID 73).

In a particular embodiment of the invention, the preparation procedure comprises a biocatalytic reaction in the presence of a biocatalyst which catalyzes the transfer of the active agent, particularly an ester or thioester, most particularly CoA.

Therefore, an enzyme having such catalytic activity can be referred to as a CoA transferase. Preferably, the CoA transferase is selective for dicarboxylic acid-CoA which is an enzyme substrate for CoA administration. More preferably, the dicarboxylic acid-CoA is hexamethylene-CoA. Preferably, the CoA system is selective for acetic acid as a CoA-receptor substrate.

The enzyme which catalyzes the transfer of the CoA group is particularly selected from the group consisting of CoA transferase (EC 2.8.3), preferably selected from the dicarboxylic acid-CoA: dicarboxylic acid CoA transferase, Diacid: butyl dioxime-CoA CoA transferase, 3-keto acid CoA-transferase (EC 2.8.3.5), 3-ketoadipate CoA-transferase (EC 2.8.3.6) and acetic acid CoA-transferase ( Group of EC 2.8.3.8).

CoA transferases can in principle be derived or derived from any organism. The organism can be a bacterium, an archaea or a eukaryote. In particular, it is preferred that the organism which decomposes the diacid to specifically decompose adipic acid is preferred.

The organism is particularly a bacterium selected from the group consisting of Acinetobacter (Special Acinetobacter sp. ADP1, Acinetobacter calcoaceticus), Clostridium (Special Kluyveromyces cerevisiae, acetylbutyrate) Phytophthora or Clostridium variabilis), Pseudomonas (especially Pseudomonas prgenskii and Pseudomonas fluorescens), Agrobacterium, Alcaligenes, Arthritis, Azotobacter Genus, Azospirillum, Azotobacter, Bacillus, P. sclerophylla, Rhizobium, Burkholder, Coronaria, Corynebacterium, Nocardia, Rhizobium, Rhodotorula, Rhodococcus, Trichosporon, Roseburia sp.

The organism is particularly a yeast or fungus selected from the group consisting of the genus Fusarium (particularly, the genus Phytophthora), the genus Penicillium (particularly P. chrysogenum), and the genus Rhodochrid.

Particularly suitable CoA transferases may be obtained or derived from one of the human family (Hominidea), more particularly from humans.

An enzyme suitable for use in Reaction 7 specifically comprises an amino acid sequence according to any one of Sequence IDs 68-73, 85, 116, 117, 119-124 or a homolog thereof.

The preparation of adipic acid from a thioester is particularly catalyzed by a biocatalyst comprising a ruthenium-CoA hydrolase comprising an amino acid sequence according to one of the sequence IDs 68-73, 117, 119 or a homolog thereof.

The preparation of adipic acid from a thioester may in particular be carried out by a biocatalyst comprising a CoA transferase comprising an amino acid sequence according to one of the sequence IDs 85, 121, 122, 123, 124, 125, 126 or a homologue thereof. catalytic.

A CoA transferase can be encoded by a single gene or by more than one gene. For example, several CoA transferases comprise two subunits encoded by two different genes. These subunits are commonly used together to make the CoA transferase protein active. For example, the following composition may be used: a combination of sequence ID 121 and sequence ID 122 or a combination of sequence ID 125 and sequence ID 126.

Preparation of 5-FVA from adipate or thiodicarboxylate ("Reaction 5")

In one embodiment, 5-methylvaleric acid (5-FVA) is prepared from adipate or thiodicarboxylate. The preparation can be carried out chemically or biocatalytically. The adipate is specifically coupled to CoA or coupled to other reactive groups as indicated above.

In particular, the present invention also provides a method for preparing 5-FVA from adipate or thiodicarbonate in the presence of a biocatalyst capable of catalyzing the reduction of oxime ester or guanidine thioester to aldehyde, particularly for use in hexamethylene -CoA thioester method for preparing 5-FVA.

Therefore, an enzyme having such catalytic activity can be referred to as an aldehyde dehydrogenase. Thus, an enzyme having such catalytic activity for an oxime ester or a sulfonyl ester such as a ruthenium-CoA thioester can be referred to as an aldehyde dehydrogenase (acetamidine). Preferably, the biocatalyst comprising aldehyde dehydrogenase (acetamidine) is selective for the enzyme matrix adipate or thiodicarboxylate.

The enzyme which catalyzes the reduction of thiol (thio) ester is selected in particular from the group consisting of oxidoreductases (EC 1.2.1), preferably selected from the group consisting of aldehyde dehydrogenase (acetylation) (EC) 1.2.1.10), fat 醯-CoA reductase (EC 1.2.1.42), long-chain fat 醯-CoA reductase (EC 1.2.1.50), butyraldehyde dehydrogenase (EC 1.2.1.57) and succinic semialdehyde A group consisting of dehydrogenase (e.g., see Sohling et al., 1996, J Bacteriol. 178: 871-880).

Aldehyde dehydrogenases can in principle be derived or derived from any organism. It is understood that the enzyme is obtained from a multi-genome source by direct isolation of the encoding nucleic acid and subsequent determination of activity in a non-homologous host, or by sequence homology found in multi-source genomic DNA. The organism can be a bacterium, an archaea or a eukaryote. The particular organism may be selected from bacteria, more particularly selected from the group consisting of Escherichia coli, Clostridium (Special Kluyveromyces, Clostridium cloacae, Clostridium butyricum, C. botylicum, C. tetani, Clostridium perfringens and C. novyi, Porphyromonas gingivalis, Lis Listeria, oleic acid genus (especially Phytophthora fuliginea), Enterococcus, Clostridium, Lactobacillus (especially Lactobacillus lactis), Bacillus (especially for B. thuringiensis), Burkholderia (especially B. thailandensis and B. mallei), Pseudomonas (especially P. sinensis) Monocytogenes), Rhodococcus (particularly Rhodococcus species RHA1) and Salmonella (especially Salmonella typhi). The organism may also be selected from eukaryotes, more particularly from Giardia (especially G. lamblia), Entamoeba. (especially for E. Histolytica), Mastigamoeba balamuthi, Chlamydomonas reinhardtii, Polytomella, Piromyces, Cryptosporidium Genus (Cryptosporidium), and cristospores (Spironucleus barkhanus).

Suitable dehydrogenases, in particular, comprising an amino acid sequence according to any of Sequence IDs 74-81, 139-148, or a homolog thereof, in the examples of the present invention, do not themselves have the desired activity or enzyme matrix specificity The biocatalyst can be modified by theoretical methods or molecular evolution to form a mutant capable of converting an adipate or a thioester into a 5-FVA. It is preferred to have a biocatalyst which can degrade aldehyde dehydrogenase activity using a ruthenium-CoA derivative having a chain length of 4-8, including but not limited to, from Kluyveromyces cerevisiae (sequence ID 74) or porphyrin Succinic acid (sequence ID 75) succinic acid semialdehyde dehydrogenase (acetylated) and obtained from acetamidine butyral acetal (sequence ID 80, 81) or P. stipitis (sequence ID 79) A biocatalyst of butyraldehyde dehydrogenase (acetamidine) is preferred.

Preparation of 5-FVA from adipic acid ("Reaction 8")

According to the present invention, adipic acid can be used to prepare 5-FVA by reducing one of the carboxylic acid groups. This item can be achieved chemically, for example by selective chemical reduction, which may be achieved by a protonation reaction of a carboxylic acid group or by biocatalysis. In a preferred method of the invention, the preparation comprises a biocatalytic reaction in the presence of a biocatalyst which catalyzes the reduction of the carboxylic acid. The biocatalyst can use NADH or NADPH as an electron donor.

Therefore, an enzyme having such catalytic activity is referred to as an aldehyde dehydrogenase. Preferably, the aldehyde dehydrogenase is selective for the enzyme substrate adipic acid.

The enzyme which catalyzes the reduction of the carboxylic acid is selected in particular from the group consisting of oxidoreductases (EC 1.2.1), preferably selected from the group consisting of aldehyde dehydrogenase (EC 1.2.1.3, EC 1.2.1.4). And EC 1.2.1.5), malonate semialdehyde dehydrogenase (EC 1.2.1.15), succinic acid semialdehyde dehydrogenase (EC 1.2.1.16 and EC 1.2.1.24); glutamic acid semialdehyde dehydrogenase (EC 1.2.1.20), amine adipic acid semialdehyde dehydrogenase (EC 1.2.1.31), adipic acid semialdehyde dehydrogenase (EC 1.2.1.63), also known as 6-ketohexanoate dehydrogenase group. The adipic acid semialdehyde dehydrogenase activity has been described in the KEGG database for the decomposition of caprolactam. In particular, 6-ketohexanoate dehydrogenase can be used. An example of a 6-ketohexanoate dehydrogenase is an enzyme comprising a sequence represented by the sequence ID 127, 128 or a homolog thereof.

The aldehyde dehydrogenase can in principle be derived or derived from any organism. The organism can be prokaryotic or eukaryotic. Particular organisms may be selected from the group consisting of bacteria, archaea, yeast, fungi, protists, plants and animals (including humans).

In one embodiment, the bacterium is selected from the group consisting of: Acinetobacter (particularly Acinetobacter species NCIMB9871), Rasta genus, Bordetella, Burkholderia, Mycobacterium, Flavobacterium, Rhizobium, Rhizobium, Nitrobacter, Brucella (especially Brucella melanogaster), Pseudomonas, Agrobacterium (especially nodules) Agrobacterium), Bacillus, Listeria, Alcaligenes, Corynebacterium, and Flavobacterium.

In one embodiment, the organic system is selected from the group consisting of yeast and fungi, and is particularly selected from the group consisting of the genus Fusarium (particularly, the sputum and the genus) and the genus Penicillium (especially the genus Phaeocystis) The group consisting of.

In one embodiment, the organism is a plant, particularly Arabidopsis, more particularly Arabidopsis.

Preparation of 6-ACA ("Reaction 6")

In one embodiment of the invention, 5-FVA is used to prepare 6-ACA.

In one embodiment, 6-ACA is obtained by hydrogenating P-O ₂ with 6-mercaptohexanoic acid obtained by reacting 5-FVA with hydroxylamine (FO Ayorinde, EY Nana, PD Nicely, AS Woods, EO Price, CP Nwaonicha) J. Am. Oil Chem. Soc. 1997, 74, 531-538 also refers to the synthesis of the homolog 12-amine dodecanoic acid).

6-ACA can be prepared in high yield via a reductive amination reaction of 5-FVA with a hydrogenation catalyst such as Ni in a SiO ₂ /Al ₂ O ₃ support, as in EP-A 628 535 or DE 4 322 065 9-amine decanoic acid (9-amine geranyl acid) and 12-amine dodecanoic acid (12-amine lauric acid).

In yet another embodiment, 6-ACA is prepared in a biocatalytic manner. In a preferred method, the preparation of 6-ACA from 5-FVA comprises enzymatic catalyzed reaction in the presence of an enzyme selected from the group consisting of a transaminase (E.C. 2.6.1) which catalyzes a transamination reaction in the presence of an amine donor.

In general, an appropriate transaminase has 6-ACA 6-transaminase activity which catalyzes the conversion of 5-FVA to 6-ACA.

The transaminase is particularly selected from the group consisting of the following transaminase: mammals; the genus Hawthorn is especially perennial hawthorn, more particularly the perennial hawthorn; the genus Tetradon, more particularly the unilateral fern Or Northern fern; Ceratonia, more particularly Ceratonia siliqua; Rhodobacter sphaeroides; Rhizobacter sphaeroides; Staphylococcus aureus; Vibrio genus is particularly Vibrio fluvialis; Pseudomonas is especially Pseudomonas aeruginosa; Rhodopseudomonas; Bacillus, especially Bacillus vemurai and Bacillus subtilis; Pneumocystis; nitrous acid; Neisseria; or yeast, especially Saccharomyces cerevisiae.

Where the enzyme is a mammalian enzyme, the enzyme is particularly derived from a mammalian kidney, a mammalian liver, a mammalian heart or a mammalian brain. For example, a suitable enzyme may be selected from the group consisting of beta-amine isobutyric acid derived from mammalian kidneys: alpha-ketoglutarate transaminase, especially beta-amine isobutyric acid derived from porcine kidney : α-ketoglutarate transaminase; β-alanine transaminase obtained from mammalian liver, especially β-alanine transaminase obtained from rabbit liver; derived from mammalian heart aspartate The transaminase is especially aspartic acid transaminase obtained from pig heart; the 4-amine-butyric acid transaminase obtained from mammalian liver is especially 4-amine-butyric acid transaminase obtained from pig liver; 4-amine-butyric acid transaminase from mammalian brain, especially 4-amine-butyrate transaminase obtained from human, pig or rat brain; α-ketoadipate obtained from Rhodotorium- A glutamic acid transaminase, particularly derived from the alpha-ketoadipate-glutamic acid transaminase of Rhizopus oryzae; 4-amine-butyrate transaminase from Escherichia coli, or from the genus The α-amine adipic acid transaminase is especially an α-amine adipic acid transaminase obtained from Thermomyces faecalis, and 5-amine pentyl derived from Clostridium genus Clostridium faecalis. Acid transaminase. For example, a suitable 2-amine adipic acid transaminase can be provided by Pyrobaculum islandicum.

In a particular embodiment, the transaminase used comprises one of the homologues according to sequence ID 82, sequence ID 83, sequence ID 84, sequence ID 134, sequence ID 136, sequence 138, or any of these sequences. Amino acid sequence. Sequence ID 86 (wild type) and 88 (codon optimization) represent the coding sequence of the enzyme represented by sequence ID 82 (=87). Sequence ID 89 (wild type) and 91 (codon optimization) represent the coding sequence of the enzyme represented by sequence ID 82 (=87). Sequence ID 133, sequence ID 135, and sequence ID 137 represent the sequence of sequence ID 134, sequence ID 136, and sequence 138, respectively.

The particular amine-based donor may be selected from the group consisting of ammonia, ammonium ions, amines, and amino acids. Suitable amines are the first amine and the second amine. Amino acids have a D-configuration or an L-configuration. Examples of amine donors are alanine, glutamic acid, isopropylamine, 2-amine butane, 2-amine heptane, benzylamine, 1-phenyl-1-amine ethane, glutamine, tyrosine, amphetamine Acid, aspartic acid, β-amine isobutyric acid, β-malonic acid, 4-amine butyric acid, and α-amine adipic acid.

In still another more preferred embodiment, the method for preparing 6-ACA comprises performing a biocatalytic reaction in the presence of an enzyme capable of catalytically reductive amination in the presence of an ammonia source, the enzyme being selected from the group consisting of CH applied to the donor. A group consisting of -NH ₂ -based oxidoreductase (EC 1.4), in particular selected from the group consisting of amino acid dehydrogenases (EC 1.4.1). In general, a suitable amino acid dehydrogenase has 6-aminocaproic acid 6-dehydrogenase activity to catalyze the conversion of 5-FVA to 6-ACA; or has alpha-amine pimelic acid 2-dehydrogenase activity to catalyze AKP Convert to AAP. Particularly suitable amino acid dehydrogenases may be selected from the group consisting of diamine pimelate dehydrogenase (EC 1.4.1.16), lysine 6-dehydrogenase (EC 1.4.1.18), glutamate dehydrogenase ( EC 1.4.1.3; EC 1.4.1.4) and a group consisting of leucine dehydrogenase (EC 1.4.1.9).

In one embodiment, the amino acid dehydrogenase may be selected from the group consisting of amino acid dehydrogenases classified as follows: glutamate dehydrogenase (EC 1.4.1.3) that acts as a receptor with NAD or NADP, A glutamate dehydrogenase (EC 1.4.1.4), leucine dehydrogenase (EC 1.4.1.9), diamine pimelic acid dehydrogenase (EC 1.4.1.16), which functions as a receptor with NADP, and Amino acid 6-dehydrogenase (EC 1.4.1.18).

The amino acid dehydrogenase may in particular be derived from an organism selected from the group consisting of: Corynebacterium, in particular Corynebacterium glutamicum; Proteus, in particular Proteus vulgaris; Agrobacterium, in particular Agrobacterium tumefaciens; the genus of the genus Bacillus is particularly a fixed thermophilic bacterium; the genus Acinetobacter is particularly ADP1 of the Acinetobacter species; the genus Astragalus is particularly a Ralstonia solanacearum; the genus Salmonella is particularly a typhoid bacillus; Genus, especially Saccharomyces cerevisiae; Brevibacterium genus, especially Brevibacterium flavum; and Bacillus, especially Bacillus sphaericus, Bacillus cereus or Bacillus subtilis. For example, a suitable amino acid dehydrogenase may be selected from the group consisting of diamine pimelic acid dehydrogenase from Bacillus sphaeroides; diamine pimelic acid dehydrogenase from Brevibacterium; from Corynebacterium a diamine pimelic acid dehydrogenase of the species, in particular a diamine pimelic acid dehydrogenase derived from Corynebacterium glutamicum; a diamine pimelic acid dehydrogenase obtained from the Proteus species, in particular Diamine pimelate dehydrogenase from common Proteus; lysine 6-dehydrogenase from Agrobacterium tumefaciens; Amino acid 6-derived from thermophilic Bacillus Dehydrogenase; glutamate dehydrogenase (EC 1.4.1.3) derived from Acinetobacter with NADH or NADPH as a cofactor, especially from the glutamate dehydrogenase of the Acinetobacter sp. ADP1; The glutamate dehydrogenase of the genus Russula (EC 1.4.1.3), especially from the glutamate dehydrogenase of the bacterial wilt; the glutamic acid derived from the genus Salmonella with NADPH as a cofactor Dehydrogenase (EC 1.4.1.4), especially from glutamic acid dehydrogenase of Salmonella typhi; glutamate dehydrogenase from yeast (EC 1.4.1.4), Not derived from glutamic acid dehydrogenase from brewer's yeast; glutamate dehydrogenase from Brevibacterium (EC 1.4.1.4), especially from glutamate dehydrogenase from Brevibacterium flavum; and from spores The leucine dehydrogenase of Bacillus is particularly derived from the leucine dehydrogenase of Bacillus cereus or Bacillus subtilis.

In one embodiment, the 6-ACA prepared in the process of the invention is used to prepare caprolactam. This method involves recycling the 6-amine hexanoic acid in the presence of a biocatalyst as needed.

The reaction conditions for any of the biocatalytic steps of the present invention may be selected based on the known conditions of the biocatalyst, particularly the conditions for the enzyme, the information disclosed herein, and optionally via several routine experiments.

In principle, the pH of the reaction medium used can be selected from a wide range as long as the biocatalyst is active under such pH conditions. Alkaline, neutral or acidic conditions can be used depending on the biocatalyst and other factors. Where the method comprises the use of a microorganism, for example, for expressing an enzyme that catalyzes the method of the invention, the pH is selected such that the microorganism can perform its intended function. The pH may in particular be selected from 4 pH units below neutral pH and 2 pH units above neutral pH, i.e. at 25 ° C in the range of pH 3 to pH 9 in the case of a substantially aqueous system. If the water is the sole solvent or the main solvent (greater than 50 wt.%, especially greater than 90 wt.% based on the total liquid) then the system is considered to be aqueous, wherein for example a small amount can be dissolved (less than 50 wt.% based on the total liquid) The concentration of less than 10 wt.%) alcohol or other solvent (e.g., as a carbon source) maintains the activity of the microorganisms present. Particularly in the case of using yeast and/or fungi, acidic conditions are preferred, particularly based on a substantially aqueous system at 25 ° C and a pH in the range of pH 3 to pH 8. If desired, the pH can be adjusted using an acid and/or base or the pH can be buffered with a suitable combination of acid and base.

In principle, the culture conditions can be selected from a wide range as long as the biocatalyst exhibits sufficient activity and/or growth. Such conditions include aerobic, micro-oxygen, oxygen-limited and anaerobic conditions.

An anaerobic condition is defined herein as a condition free of any oxygen, or wherein substantially no oxygen is consumed by the biocatalyst, particularly by microorganisms, typically equivalent to less than 5 millimoles per liter. The oxygen consumption of the hour is particularly equivalent to less than 2.5 millimoles per liter. hours or less than 1 millimol per liter.

Aerobic conditions are those in which sufficient oxygen concentration for unrestricted growth is dissolved in the medium and thus supports at least 10 millimoles per liter. hour, more preferably greater than 20 millimoles per liter. hour, and even more preferably greater than 50. The conditions of millimolar/liter. hour and optimum oxygen consumption rate greater than 100 millimoles per liter.

Oxygen finite conditions are defined as conditions in which the consumption of oxygen is limited by the oxygen transferred from the gas to the liquid. The lower limit of the oxygen-limited condition is determined by the upper limit of the anaerobic condition, that is, usually at least 1 millimole/liter hour and particularly at least 2.5 millimoles per liter hour or at least 5 millimoles per liter. . The upper limit of the oxygen limited condition is determined by the lower limit of the aerobic condition, that is, less than 100 millimoles per liter. hour, less than 50 millimoles per liter. hour, less than 20 millimoles per liter. hour or low. At 10 millimoles per liter. hour.

The conditions are aerobic conditions, anaerobic conditions or limited oxygen conditions depending on the conditions under which the process is carried out, in particular the amount and composition of the feed gas stream, the actual mixing/mass transfer properties of the equipment used, and the microorganisms used. The category and microbial density are determined.

In principle, the temperature to be used is not particularly limited as long as the biocatalyst exhibits substantial activity, particularly for the enzyme. The temperature is substantially at least 0 ° C, especially at least 15 ° C, more particularly at least 20 ° C. The highest temperature desired is determined by the biocatalyst. Typically such elevated temperatures are known to the art, for example, as indicated by the product information sheet in the case of commercially available biocatalysts, or may be determined routinely based on common knowledge and information disclosed herein. The temperature is usually 90 ° C or less, preferably 70 ° C or less, particularly 50 ° C or less, more specifically 40 ° C or less.

In addition, solvents, additional reagents, and other auxiliaries such as cofactors (eg, FAD/FADH and/or NAD/NADH cofactors) can be selected to achieve or accelerate specific reactions based on known reaction principles, and/or steps can be taken to The balance moves to the desired side. Particularly if the biocatalytic reaction is carried out outside the host organism, a reaction medium containing a high concentration (for example, more than 50 wt.% or more than 90 wt.%) of an organic solvent can be used in the case of using an enzyme which can maintain sufficient activity in such a medium. .

The succinic acid (ester or thioester) and acetic acid (ester or thioester) used in the process of the invention can in principle be obtained in any manner.

Succinic acid, for example, is naturally formed as an intermediate of the citric acid cycle (Krebs cycle) or as an end product of cellular metabolism. This can be obtained from a renewable carbon source using a suitable biocatalyst. Biocatalysts, particularly microorganisms, can be used to produce succinic acid from a suitable carbon source. The microorganism can be a prokaryote or a eukaryote. The microorganism can be recombinant or wild type.

In recombinant microorganisms, metabolism can be altered to increase succinic acid production and productivity above a suitable carbon source. Methods for increasing succinic acid production have been described for prokaryotes in Song and Lee, Enzymes and Microbial Technology, 2006, 39: 352-361. Succinic acid can also be produced by eukaryotes. Additionally and additionally, an adaptation adaptation evolution may be applied, such as illustrated in U.S. Patent No. 2007/111294.

The succinate or thioester can be obtained from succinic acid in either manner. A particular succinate or thioester can be obtained from succinic acid using a biocatalyst. Particularly via the use of an enzyme comprising a group selected from the group consisting of acid thiol ligases (EC 6.2.1), preferably selected from the group consisting of diacetyl-CoA synthetase (EC 6.2.1.4 and EC 6.2.1.5) A catalyst for the enzyme consisting of the group, which can obtain butadiene-CoA from succinic acid. Additionally or alternatively, butadiene-CoA can be obtained from succinic acid via the use of a biocatalyst comprising an enzyme selected from the group consisting of CoA transferase (EC 2.8.3), as specified for Reaction 7.

The succinate or thioester can also be obtained from molecules other than succinic acid in either manner. Specifically, butadiene-CoA can be obtained from 2-ketoglutaric acid using a biocatalyst comprising a 2-ketoglutarate dehydrogenase complex. It is known to those skilled in the art that the ketoglutarate dehydrogenase complex is a multi-enzyme complex that participates in the TCA cycle. Additionally or alternatively, butadiene-CoA can be obtained from 2-ketoglutaric acid using a biocatalyst comprising 2-ketoglutaric acid: ferredoxin oxidoreductase (EC 1.2.7.3).

Acetic acid is the natural intermediate or end product of cellular metabolism. Thus acetic acid can be obtained from a renewable carbon source via the use of a suitable biocatalyst. Biocatalysts, particularly microorganisms, can be used to produce succinic acid from a suitable carbon source. The microorganism can be a prokaryote or a eukaryote. The microorganism can be recombinant or wild type.

Acetate or thioacetate can be obtained from acetic acid in either manner. In particular, by using an enzyme comprising an enzyme selected from the group consisting of acid thiol ligase (EC 6.2.1), preferably acetyl-CoA synthetase (EC 6.2.1.1 and EC 6.2.1.13) Catalyst, acetic acid-CoA can be obtained from acetic acid. Additionally or alternatively, acetamidine-CoA can be obtained from acetic acid using a biocatalyst comprising an enzyme selected from the group consisting of CoA transferase (EC 2.8.3) as defined for Reaction 7.

The acetate or thioacetate may also be obtained from molecules other than acetic acid in any manner. In particular, the use comprises or consists of a pyruvate dehydrogenase complex, pyruvate dehydrogenase (NADP+) (EC 1.2.1.51), pyruvate formate lysing enzyme (EC 2.3.1.54) or pyruvate A biocatalyst for converting into a group of enzymes consisting of a biocatalyst or an enzyme of acetamidine-CoA, which can be obtained from pyruvic acid, as known to those skilled in the art, pyruvate dehydrogenase complex is used to convert pyruvate A multi-enzyme complex of Chengyi-CoA.

Using a biocatalyst comprising an enzyme selected from the group consisting of oxidoreductases (EC 1.2.1), acetamidine-CoA can also be obtained from acetaldehyde, preferably the enzyme is selected from the group consisting of aldehyde dehydrogenase ( Acetylation (EC 1.2.1.10), fatty acid acetamidine-CoA reductase (EC 1.2.1.42), butyraldehyde dehydrogenase (EC 1.2.1.57) and succinic acid semialdehyde dehydrogenase (acetylation) A group consisting of (described in Sohling et al. 1996. J Bacteriol. 178: 871-880).

When the biocatalyst is a eukaryote, the supply of Ethyl-CoA can be increased by over-characterizing a homologous gene and/or a non-homologous gene encoding an enzyme that converts a catalytic precursor molecule into an Ethyl-CoA molecule, preferably increased in Supply of acetaminophen-CoA in the cytosolic compartment of the host cell. The precursor molecule is, for example, acetic acid as described by Shiba et al., Metabolic Engineering, 2007, 9: 160-8.

In the excellent process of the present invention, particularly for the preparation of 6-ACA adipic acid or an intermediate compound for 6-ACA or adipic acid, for use in 6-ACA, adipic acid or an intermediate thereof Whole cell biotransformation of an enzyme substrate comprising the use of a microorganism in which one or more enzymes that catalyze any of the foregoing reactions are produced and a carbon source for the microorganism.

The carbon source particularly contains at least one compound selected from the group consisting of monohydric alcohols, polyhydric alcohols, carbonic acid carboxylic acids, fatty acids, glycerides, and mixtures comprising any of the compounds. Suitable monohydric alcohols include methanol and ethanol. Suitable polyols include glycerin and carbohydrates. Suitable fatty acids or glycerides are in particular provided in the form of edible oils, preferably vegetable-derived edible oils.

Carbohydrates are particularly used because in general carbohydrates can be obtained in large quantities from biorenewable sources, such as agricultural products such as agricultural waste. Preferably, the carbohydrate used is selected from the group consisting of glucose, fructose, sucrose, lactose, sucrose, starch, cellulose, and hemicellulose. Particularly preferred are glucose, oligosaccharides containing glucose, and polysaccharides containing glucose.

In a particular method of the invention, the method is a fermentation process. Such methods, in particular, comprise a cell comprising a biocatalyst, optionally contacting a host cell as described herein with a fermentable carbon source, wherein the carbon source comprises any compound that will be converted to a pre-prepared compound, or wherein the cells are prepared The compound to be converted into a compound to be prepared from the carbon source.

Cells comprising one or more enzymes for catalyzing the reaction steps in the methods of the invention can be constructed using molecular biotechnology known in the art. For example, if one or more biocatalysts are to be produced in a non-homologous system, such techniques can be used to provide a vector comprising one or more genes encoding one or more of the catalysts. A vector comprising one or more of the genes can comprise one or more regulatory elements, such as one or more promoter genes, operably linked to a gene encoding one of the biocatalysts.

As used herein, the term "working link" means that a polynucleotide element (or coding sequence or nucleic acid sequence) is functionally linked. When a nucleic acid sequence is functionally related to other nucleic acid sequences, the nucleic acid sequence is a "workable link." For example, if the promoter gene or the booster gene affects the transcription of the coding sequence, the promoter gene or the booster gene line is functionally linked to the coding sequence.

As used herein, the term "initiating gene" refers to a nucleic acid fragment that is used to control the transcription of one or more genes upstream of the transcriptional orientation relative to the transcriptional origin of the gene, structurally DNA-dependent. Identification of the binding site of the RNA polymerase, the location of transcription initiation, and the presence of any other DNA, including but not limited to transcription factor binding sites, repressor proteins and activated protein binding sites, and those skilled in the art knowing directly or Indirectly acting to mediate any other nucleotide sequence that is transcribed by the promoter gene. A "constitutive" promoter gene is a promoter that is active under most environmental and developmental conditions. An "inducible promoter gene" is a promoter that is active under environmental or developmental regulation. The term "homologous", when used to indicate the relationship between a given (recombinant) nucleic acid or a multi-peptide molecule and a given host organism or host cell, is understood to be indicative of the nucleic acid or the multi-peptide in nature. Molecules are made from host cells or organisms of the same species, preferably of the same variety or of the same germline.

The promoter gene which can be used to express an encoding nucleic acid sequence for an enzyme of the method of the invention (such as those described above) may be the nucleic acid sequence encoding the enzyme to be expressed; or may be the nucleic acid sequence (coding sequence) Homology, through a working link. Preferably, the promoter gene is homologous, that is, the promoter gene is endogenous to the host cell.

If a non-homologous promoter gene is used (relative to the nucleic acid sequence encoding the enzyme of interest), the non-homologous promoter gene preferably produces a promoter gene that is comparable to the coding sequence, with a higher steady state concentration including the coding sequence. The transcript of the sequence (or a larger number of transcript molecules, i.e., mRNA molecules) can be produced per unit time. Suitable promoter genes in this context include constitutive and inducible natural promoter genes as well as genetically engineered promoter genes, which are well known to those skilled in the art.

A "potent constitutive promoter gene" is a promoter gene that causes the mRNA to start at a higher frequency than the natural host cell. Examples of such potent constitutive promoter genes in Gram-positive microorganisms include SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter gene) and amyE.

Examples of inducible promoter genes in Gram-positive microorganisms include the IPTG-inducible Pspac promoter gene, the xylose-inducible PxylA promoter gene.

Examples of constitutive promoter genes and inducible promoter genes for Gram-negative microorganisms include tac, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara (P) _BAD ), SP6, λ-P _R , and λ-P _L .

When used in a nucleic acid (DNA or RNA) or protein, the term "non-homologous" refers to a nucleic acid or protein that does not naturally occur as part of the organism, cell, genome or DNA sequence or RNA sequence in which it is present. Or the nucleic acid or protein is present at a different location or different DNA or RNA sequence from a different cell or genome than the naturally occurring one. A non-homologous nucleic acid or a non-homologous protein is not the endogenous of the cell into which it is introduced, but instead is obtained from other cells or produced synthetically or recombinantly. Roughly, but not necessarily, such nucleic acids encode proteins in which the cells in which the DNA is transcribed or expressed are not normally produced. Similarly, exogenous RNA encodes a protein that is not normally expressed by cells in which the exogenous RNA is present. Non-homologous nucleic acids and proteins can also be referred to as foreign nucleic acids or proteins. A non-homologous nucleic acid or foreign nucleic acid or protein that is known to those skilled in the art to be recognized as a cell of the nucleic acid is encompassed herein by the term non-homologous nucleic acid or protein.

The process according to the invention can be carried out using an organism which can be a host organism, in particular a host microorganism or a wild-type microorganism. Thus, the invention also relates to a novel (host) cell which can be a microorganism comprising a biocatalyst which catalyzes at least one reaction step of the method of the invention, preferably the cell can produce an enzyme or a plurality of enzymes, thereby catalyzing Two or more reaction steps in the process of the invention. The invention also relates to a novel vector comprising one or more genes encoding one or more enzymes that catalyze at least one of the reaction steps of the methods of the invention.

In one embodiment, a cell or a vector comprising a nucleic acid sequence encoding a recombinant nucleic acid sequence encoding 5-carboxy-2-pentenyl ester or thioester hydrogenase activity, particularly 5- An enzyme of carboxy-2-pentene hydrazine hydrogenase activity.

Preferably, the cell further comprises a nucleic acid sequence encoding at least one enzyme (the recombinant vector comprises one) selected from the group consisting of catalyzable conversion of hexamethylene diester or thioester, especially hexamethylene hydride-CoA, to 5-FVA. The enzyme and the group of enzymes which can catalyze the conversion of hexamethylene diester or thioester, especially hexamethylene di-CoA to adipic acid.

In particular, in an embodiment, wherein the cell comprises an enzyme that catalyzes the conversion of hexamethylene diester or thioester to 5-FVA, the cell preferably comprises (a recombinant vector comprising) encoding to convert 5-FVA to 6- A nucleic acid sequence of one of the enzymes of ACA. Such an enzyme may in particular be an enzyme having 5-FVA transaminase activity.

Additionally or alternatively, the (host) cell or vector comprises at least one of the following nucleic acid sequences:

Encoding a nucleic acid sequence which catalyzes the formation of 3-ketohexyl adipate or thioester by reaction of butyric acid ester or thioester with an acetate or thioester;

Encoding a nucleic acid sequence which catalyzes the formation of one of 3-hydroxyhexanediester or thioester from 3-ketohexyl adipate or thioester;

- The code can catalyze the formation of 5-carboxyl from 3-hydroxyhexanedicarboxylate or thioester

a nucleic acid sequence of one of the enzymes of 2-pentene oxime ester or thioester;

- A nucleic acid sequence encoding one of the enzymes which catalyze the formation of hexamethylene or thioester from 5-carboxy-2-pentene oxime ester or thioester.

One or more suitable genes are selected in particular from a gene encoding an enzyme as described above, more particularly from the coding according to sequence ID 1-67, 94, 96, 98, 100, 102, 103, 105, 107, 109. The gene of the enzyme of any of 111, 113, 115, 116 or a homolog thereof.

The host cell can be a prokaryotic cell or a eukaryotic cell. The particular host cell can be selected from the group consisting of bacteria, archaea, yeast, fungi, protists, plants and animals including humans.

In particular, the host cell according to the present invention may be selected from the group consisting of genus Fusarium, Bacillus, Corynebacterium, Escherichia, Saccharomyces, Pseudomonas, Gluconobacter, Penicillium, Bi A group consisting of the genus Saccharomyces. Particularly for the host germline, the host cell may be selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Corynebacterium glutamicum, Melastia, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae group.

Host cells which can produce short chain fatty acids such as succinic acid and/or acetic acid and/or esters thereof or thioesters thereof are preferred. Organisms that achieve this function are usually present in the tumor position of the anti-bred animals. In particular, it is preferred to use an organism which can co-produce succinic acid and acetic acid or an ester thereof or a thioester thereof.

The microorganism can be a recombinant or wild type microorganism. In particular, microorganisms capable of producing succinic acid include Escherichia coli, Acinetobacter (especially A. succinogenes), Mannheimia (especially for succinic acid Mannheim) M. succiniciproducens), Saccharomyces cerevisiae, genus Fusarium (especially black sputum), Penicillium (especially P. simplicissimum) and Kaemwich Jantama, MJ Haupt, Spyros A. Svoronos , Xueli Zhang, JC Moore, KT Shanmugam LO Ingram. Other organisms described in Biotechnology and Bioengineering (2007) 99, 5: 1140-1153.

Particularly suitable microorganisms for the production of acetic acid include Enterobacteriaceae (especially Escherichia coli, Salmonella and Shigella), Acetic acid bacteria (including Acetobcter (especially Acetobacter aceti), and Lactobacillus (especially for Gluconobacter oxidans), Acidomonas, Gluconacetobacter, Asaia, Kozakia, and Shaming Swaminathania, Saccharibacter, Neoasaia, Granulibacter, Clostridium (especially C. aceticum, Clostridium thermoacetum (C) Thermoaceticum), C. thermoautotrophicum, C. formicoaceticum, Kluyveromyces clostridium, C. propionicum, and the genus Coccidia (especially Ai Aspergillus, Acetobacterium (especially A. woodii and A. wieringae), Lactobacillus (especially L. plantarum, L. brevum, Bifidobacterium (Bif) Idobacterium) (especially B. bifidum) and Leuconostoc.

The invention further relates to novel multi-peptides and to nucleotide sequences encoding such multi-peptides. In particular, the invention further relates to a multi-peptide comprising an amino acid sequence according to any of Sequence ID 57, 68-72, 79, 85 and homologs thereof. In particular, the invention further relates to a polynucleotide encoding a polypeptide comprising an amino acid sequence according to any one of Sequence ID 57, 68-72, 79, 85 and its homologs.

Next, the present invention will be exemplified by the following examples.

Instance Example 1: Summary method Molecular and genetic techniques

Standard genetic and molecular biology techniques are generally known to the art world and have been described previously (Maniatis et al. 1982 "Molecular Transduction: A Laboratory Manual". Cold Harbor Laboratory, Cold Spring Harbor, New York; Miller 1972, "Molecular Genetics Experiment" , Cold Spring Harbor Laboratory, Cold Spring Harbor; Sambrook and Russell 2001 "Molecular Transduction: Laboratory Manual" (Third Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; F. Ausubel et al. The popular program, Green Publishing and Willy Technology, New York, 1987).

Plastid and germline

pBAD/Mc-His C and pET21d were obtained from Invitrogen (Casper Bed, California, USA) and EMD Biosciences (Danstedt, Germany). pF113 (a derivative of pJF119EH ( , JP, W. Pansegrau, R. Frank, H. Blocker, P. Scholz, M. Bagdasarian and E. Lanka. 1986 Molecular selection of the RP4 primer region of the colon in the multi-host tacP expression vector, gene 48: 131) It contains two notI positions at positions 515 and 5176, starting with the tac promoter gene as a starting point), pACYC-tac ( , M. (2000). Untersuchungen zum Einfluss Bereitstellung von Erythrose-4-Phosphat und Phosphosenolpyurvat auf den Kohlenstofffluss in den Aromatenbiosyntheseweg von Escherichia coli. Berichte des Forschungszentrums , 3824. ISSN 0944-2952 (Ph.D. thesis, Russdorf University) and pMS470 (Balzer, D.; Ziegelin, G.; Pansegrau, W.; Kruft, V.; Lanka, E. Nucleic Acids Research 1992, 20 (8) ), 1851-1858) has been explained as before. E. coli TOP10 (British Microgenes, Inc., Kasperd, CA) was used for all selection procedures. E. coli germ line Top 10 (British Microgenes, Inc., Kasperd, CA), Rv308 (ATCC 31608), Rv308 ΔaraB, and BL21 A1 (British Microgenes, Inc., Kasperd, CA) were used for protein expression.

All carriers are allowed to have the same selection strategy by inserting a common link adjustment. The adjustment plan and the general selection plan are shown in Figure 2.

Medium

2xTY medium (16 g/L tryptic, 10 g/L yeast extract, 5 g/L NaCl) was used for the growth of E. coli. Antibiotics (100 μg/ml ampicillin) were added to maintain the plastids. For the induction of gene expression, using arabinose (for pBAD derivatives), IPTG (for pMS470 and pF113 derivatives), and a combination of arabinose and IPTG (for pET21d derivatives in E. coli BL21-A1) The final concentrations were between 0.005-0.2% (arabinose) and 0.1-0.5 mM (IPTG), respectively.

Plasm recognition

The system of genes carrying different genes is identified by genetic, biochemical and/or phenotypic means generally known in the art, such as the resistance of the transgenic strain to antibiotics, PCR diagnostic analysis of transformed plants or purification of plastid DNA, Restriction analysis or DNA sequence analysis of purified plastid DNA.

HPLC-MS analytical method for the determination of CoA derivatives

The concentrations of hexamethylene-CoA and 6-carboxy-2,3-enhexanyl-CoA were determined by LC-MS. Separation was performed using an Agilent SB-C18 2.1*50 mm column using 750 mg/L octyl ammonium acetate buffered acetonitrile/water (pH = 7.5) as the mobile phase. The flow rate was 300 μl/min, with a gradient (start: 70% water, reduced to 58% after 3 minutes, changed to 45% in stages, further reduced to 20% after 1.5 minutes, then re-equilibrated the column) In the mode, the operation time is 7 minutes) and elution is performed. The LTQ orbitap was scanned by m/z 765-900 using electrospray free mode. Hexane-CoA and 6-carboxy-2,3-enhexanyl-CoA were eluted at 2.25 minutes and 2.5 minutes, respectively. By observing the exact protonated molecule of the desired compound (hexamethylene-CoA; 894, 15123-894, 16017, 6-carboxy-2,3-enhexanyl-CoA: 892. 13437-892. 14329), the method The selectivity is enhanced. To determine the concentration, a standard curve of the synthetically prepared compound is calculated to calculate the response factor of the individual ions. Used to calculate the concentration in an unknown sample.

Detection and quantification of adipic acid is described in Kippenberge, M.; Winterhalter, R.; M oortgat, G. K. Anal. Bioanal. Chem. 2008, 392(7-8), 1459-1470.

Example 2: Determination of 6-carboxy-2,3-enhexanyl-CoA reductase activity Performance component

The putative 6-carboxy-2,3-enhexanyl-CoA reductase was selected from the database (Table 1).

The target gene encoding the selected protein is optimized by codon pairing (using the method described in WO08000632) and synthesized synthetically (Geneart, Reynoldsberg, Germany). Target genes (eg, secretion signals or peroxisome/mitochondrial target sequences) are removed from the amino acid sequence prior to optimization. This target sequence can be identified by bioinformatics tools known to the artisan, such as described in Emanuelsson et al. Nature 2: 953-971. In the optimization procedure, avoiding the internal limited shear position, the common cut-off position is introduced at the start and end points to allow for colonization according to the strategy shown in Figure 2. Such modifications may result in small changes in individual protein sequences and are not expected to alter the properties of individual proteins in any way. There is an isotopic ribosome binding position and leader sequence in front of each ORF to drive translation of pF113, pMS470 and pET21d. In pBAD, the translation initiation signal is provided by the vector. The target genes 'Adi4', 'Adi5', 'Adi8' and 'Adi9' were cloned with and without all four plastids read to the C-terminal His-tag provided by the linker sequence.

Protein expression in E. coli

The initial culture was grown overnight in 96-well plates at 200 microliters of medium per well. Transfer 40-160 microliters to a 24-deep well plate containing 4 ml of medium. For the pBAD composition of E. coli TOP10 or E. coli Rv308 ΔaraB, this medium directly contains 0.005% arabinose for induction. The plates were incubated at 25 ° C on an orbital shaker (Infors, 550 rpm). After 4-6 hours, an inducing group (0.5 mM IPTG for pF113 and pMS470 in E. coli Rv308, E. coli BL21 or E. coli TOP10; 0.5 mM IPTG and 0.2% arabinose for pET21d in E. coli BL21A1 was added. ), the well plates were cultured for an additional 4-48 hours to harvest the cells by centrifugation.

Preparation of cell-free extract and purification of His-tag

The cells obtained by small-scale growth (refer to the previous paragraph) obtained by centrifugation were discarded and the supernatant was discarded. The pellets formed during centrifugation were frozen at -20 ° C for at least 16 hours and then thawed on ice. 2 ml of freshly prepared lysis buffer (50 mM potassium phosphate pH 7.5, 0.1 mg/ml DNAse I (Roche, Almere, NL), 2 mg/ml lysozyme, 0.5 mM MgSO ₄ , 1 mM dithiosul Sugar alcohol and protease inhibitors (completely mini-free EDTA ingots, Roche, Almere, NL, according to the manufacturer's specifications) were added to each well and the cells were resuspended by vigorous vortex plate for 2-5 minutes. To achieve dissolution, the plates were incubated for 30 minutes at room temperature. To remove cell debris, the plates were centrifuged at 4 ° C and 6000 g for 20 minutes. The supernatant was transferred to a new well plate and maintained on ice until further use. For the purification of His-tagged proteins, His Multitrap HP filter plates (GE Helthcare bioscience AB, Uppsala, Sweden) were used according to the manufacturer's instructions.

Synthesis of enzyme matrix

Enzyme matrix (JR Stern, A. del Campillo, J. Biol. Chem., 1956, 985. AK Das, MD Uhler, AK Hajra, J. Biol. Chem., 2000, 24333. H. Oku, N. Futamori, K. Masuda, Y. Shimabukuro, T. Omine, H. Iwasaki, Biosc. Biotech. Biochem., 2003, 2107. Elvidge et al., J. Chem. Soc., 1953, 1793. F. Liu, HY. Zha, ZJ. Yao, J. Org. Chem., 2003, 6679-6684) and the desired biochemical reaction product (WO 2004/106347) were synthesized by Synom (Groningen, NL) according to the published procedure.

Enzyme catalyzed olefin-CoA assay

The reaction mixture was prepared to contain 50 mM potassium buffer (pH 7.5), 0.7 mM NADH and NADPH, and about 20 μM enzyme substrate 6-carboxy 2,3-enhexanyl-CoA. 190 microliters of the reaction mixture was dispensed into each well of a 96-well plate. The same procedure was used for the control reaction by preparing 10 microliters of cell free extract from individual lines carrying a blank vector. To initiate the reaction, 10 microliters of cell free extract or purified protein was added to each well. The reaction mixture was incubated at room temperature (20-25 ° C) for 15 minutes to 24 hours, and the line was monitored for UV absorption at 340 nm. At the end, the reaction was stopped and the sample was centrifuged by adding an equal amount of methanol. The supernatant was transferred to a new well plate and stored at -80 °C until further analysis by HPLC-MS. The diterpene-CoA was found as shown in Table 1.

Example 3: Production of adipic acid by non-homologous microorganisms Composition of adipic acid biosynthesis pathway

The design is synthesized by one of the enzyme catalytic activities shown in Figure 3. Enzymes encoding these activities are identified in the database. The target genes encoding these enzymes are optimized by codon pair and synthesized synthetically (Centre, Inc., Reynoldsberg, Germany). In the optimization procedure, the internal clipping position, undesired target sequences (such as secretion signals or peroxygen target sequences) are removed, and the clipping position is introduced at the start and end points to allow performance according to Figure 4. The carrier is assembled in the path. Such modifications can result in minor changes in the protein sequence, but it is expected that the properties of individual proteins will never be altered. Each ORF is preceded by an omnidirectional ribosome binding position and a leader sequence to drive translation.

For reactions 1, 2 and 3, use a combination of adi21+22+23 or adi26+27+28. For reaction 7, use a combination of adi29, adi30 or adi24+25. For reaction 4, adi4, adi5, adi9 can be used with adi8.

Composition of adipic acid-producing Escherichia coli

To form an adipic acid-producing E. coli germ line, the plastid encoded by the complete adipic acid pathway is transformed into a suitable host species for expression of the cloned gene. The pMS470 composition containing the complete pathway was transformed into E. coli BL21, TOP10 and Rv308. pBAD/Myc-His C was transformed into E. coli TOP10 and Rv308 △araB. The composition in pF113 or pACYC-tac is co-transformed with a compatible pF113, pACYC-tac or pMS470 composition to allow the final germline to contain the complete adipic acid pathway. These plastids were co-transformed into E. coli TOP10, BL21 and Rv308.

Manufacture of adipic acid

To produce adipic acid, the initial culture was grown overnight at 30 °C in 96-well plates containing 200 microliters of medium. Fifty microliters were transferred to a new 24-well plate containing 4 ml of medium and grown at 25 °C for 4-6 hours, followed by the addition of an inducer that induces adipic acid pathway performance. Incubate for another 12 hours to 72 hours at 25 °C. The plates were centrifuged and the samples were prepared for LC-MS analysis. The supernatant was mixed with methanol in 1:1 to precipitate the protein and then directly analyzed. The metabolites obtained from the cells were extracted by resuspending the pellets in 1 ml of ethanol. The cell suspension was transferred to a test tube with a screw cap and heated in a boiling water bath for 3 minutes. After centrifugation, the supernatant was transferred to a new tube and evaporated in a speed-vac. The dry sample was resuspended in 100 microliters of mobile phase followed by analysis.

Example 4: Preparation of 5-FVA from hexamethylene-CoA HPLC-MS analytical method for the determination of 5-FVA

5-FVA was detected by selective reaction monitoring (SRM)-MS, measuring transition m/z 129→83. The concentration of 5-FVA was calculated by measuring the peak area of the 5-FVA peak eluted in about 6 minutes. Calibrate using an external standard program. All LC-MS experiments were performed on the Ajicool 1200 LC system, which consisted of a fourth-stage pump, an autosampler, and a column oven coupled to the Yajikong 6410 QQQ triple quaternary MS.

LC conditions:

Column: 50 x 4.6 mm (Nucleosil C18, 5 micron (Machery & Nagel) pre-column coupled to 250 x 4.6 mm inner diameter Prevail C18 , 5 micron (Altech)

Column temperature: room temperature

Eluent: A: water containing 0.1% formic acid

B: Acetonitrile containing 0.1% formic acid

Flow rate: 1.2 ml/min split into 1:3 before entering MS

Injection volume: 2 microliters

MS conditions:

Ionization: negative ion electric spraying

Source condition: ion spray voltage: 5kV

Temperature: 350 ° C

Segmenter voltage and collision energy optimization

Scan mode: select the reaction mode: transition m / z 129 → 83

Performance component

Putative hexamethylene-CoA reductase (SeqID 74, 75, 77, 79, 80, 139-148) was selected. The expression constructs were designed and prepared in the same manner as previously described in Example 2 using pBAD/Myc-His C and pET21d.

Protein expression, extraction and purification

All steps are performed as described in Example 2.

Enzyme-catalyzed assay for the determination of hexamidine-CoA reductase

The reaction mixture was prepared to contain 50 mM potassium buffer (pH 7.5), 0.7 mM NADH and NADPH, and 10 μm to 10 mM enzyme substrate hexanedi-CoA. 190 microliters of the reaction mixture was dispensed into each well of a 96-well plate. The hexamethylene-CoA system was prepared as described in Example 2. To initiate the reaction, 10 microliters of cell free extract or purified protein was added to each well. Ten microliters of cell-free extract prepared in the same manner from the germline carrying the blank vector was used for the control reaction. The reaction mixture was incubated at room temperature (20-25 ° C) for 15 minutes to 24 hours with on-line monitoring of 340 nm ultraviolet light absorption. At the end, the reaction was stopped by adding an equal amount of methanol, and the sample was centrifuged. The supernatant was transferred to a fresh well plate and stored at -80 ° C until 5-FVA was detected by HPLC-MS. The measurement of 5-FVA verified that the selected enzyme had hexamethylene-CoA reductase activity.

Example 5 Preparation of 6-ACA from 5-FVA HPLC-MS analysis for the determination of 6-ACA calibration:

The calibration was performed by an external calibration line of 6-ACA (m/z 132→m/z 114, room temperature 7.5 minutes). All LC-MS experiments were performed on Aji Cool 1100, which was equipped with a fourth-stage pump, deaerator, autosampler, column oven and a single quaternary MS (Yage Cold, Germany ). The LC-MS conditions are:

Column: 50*4 New Zealand (Maggie and Naja) + 250×4.6 Puwell C18 (Asian), all at room temperature (RT)

Eluent: A=0.1(v/v) formic acid in ultrapure water

B = acetonitrile (pa, Merck)

Flow rate: 1.0 ml / min, before the MS enters the stream to split 1:3

Gradient: starting at 100% (v/v) A at t = 0 minutes, maintained for 15 minutes, and changed to 80% (v/v) B (t = 30 minutes) in 15 minutes. The gradient was maintained constant at 80% (v/v) B from 30 minutes to 31 minutes.

Injection volume: 5 microliters

MS detection: ESI (+)-MS electrospray ionization (ESI) was performed in positive scan mode using the following conditions: m/z 50-500, 50 V segmenter, 0.1 m/z step size 350 ° C dry gas temperature, 10 liters of nitrogen per minute of dry gas, 50 psig atomizer pressure and 2.5 kV capillary voltage.

Colonization of target genes Performance component design

The attB position was added to the ribosome binding site and all genes downstream of the initiation codon and downstream of the initiation codon to assist in the selection of Gatway technology (British Microgenes, Inc., Kasbet, CA).

Gene synthesis and composition of plastids

The synthetic gene line was obtained from DNA 2.0 and the codon was optimized for E. coli expression according to the standard procedure of DNA 2.0. Vibrio fluvialis JS17[SEQ ID No. 86] was encoded by Vibrio fluvialis JS17 ω-transaminase [SEQ ID No. 82] and B. sphaericus KBAB4 transaminase (ZP_01186960) [SEQ ID No. 83], respectively. The transaminase gene of B. sphaericus KBAB4 [SEQ ID No. 89] was codon-optimized, respectively, and the obtained sequences [SEQ ID No. 88] and [SEQ ID No. 91] were obtained by DNA synthesis.

The cells to which these genes are supplied are hereinafter referred to as Escherichia coli TOP10/pBAD-Vfl_AT and Escherichia coli TOP10/pBAD_Bwe_AT, respectively.

Colonization by PCR

According to the manufacturer's specifications, a plurality of genes encoding biocatalysts were amplified from the genomic DNA by PCR using PCR Supermix High Fidelity (British Microgene).

The PCR reaction was analyzed by agarose gel electrophoresis, and a PCR product of the correct size was eluted from the gel using a QIAquick PCR purification kit set (Qiagen, Hilden, Germany). As described in the manufacturer's protocol, the purified PCR product was cloned into pBAD/Myc-Hs using the introduced attB position and pDONR-zeo as an entry vector using the Gateway technology (British Microgene). -DEST performance vector. The sequence of the gene cloned by PCR was confirmed by DNA sequencing. In this way, the expression vector, pBAD-Bsu-gi16077991_AT (containing the gene represented by sequence ID 133, the peptide represented by coding sequence ID 134), pBAD-Pae_gi9946143_AT (using primers identified as sequence IDs 130 and 131), pBAD-Pae_gi9951072_AT was obtained. (Including the gene represented by SEQ ID NO: 135, the peptide represented by SEQ ID NO: 136), pBAD-Pae_gi9951630_AT (containing the gene represented by SEQ ID NO: 137, and the peptide represented by SEQ ID NO: 138). E. coli TOP10 (British Microgene), which is a chemically competent pBAD constitutive body, was obtained to obtain a corresponding germline.

Enzymatic reaction for converting 5-methylvaleric acid to 6-ACA

Unless otherwise specified, the preparation reaction mixture contained 10 mM 5-methylvaleric acid, 20 mM racemic alpha methylmercaptoamine and 200 μM pyridoxal 5'-phosphoric acid in 50 mM potassium phosphate buffer, pH 7.0. 100 μl reaction The mixture is dispensed into the wells of the well plate. To initiate the reaction, add 20 microliters of cell free extract to each well. The reaction mixture was shaken on a shaker at 37 ° C for 24 hours. In addition, a chemical blank group mixture (without cell-free extract) and a biological blank group (E. coli TOP10 containing pBAD/Myc-His C) were cultured under the same conditions. The samples were analyzed by HPLC-MS. The results are summarized in the table below.

It is shown that 5-ACA is formed from 5-FVA in the presence of a transaminase, and E. coli can catalyze this formation reaction.

<210> 102

<211> 334

<212> PRT

<213> Clostridium kluyveri(strain ATCC 8527/DSM 555/NCIMB 10680)

<400> 102

<210> 103

<211> 259

<212> PRT

<213> Clostridium kluyveri(strain ATCC 8527/DSM 555/NCIMB 10680)

<400> 103

Figure 1 shows the preparation of adipate or thioester from succinate or thioester;

Figure 2 shows a summary of the transfer strategy, showing only the relevant features and the limited shear position;

Figure 3 shows the biosynthetic pathway of adipic acid;

Figure 4 shows the transfer strategy for assembling the adipic acid pathway.

Claims (23)

A method for preparing an adipate or a thioester of adipate, comprising converting 2,3-dehydroadipate or 2,3-dehydroadipate thioester into a diamide in the presence of a biocatalyst An acid ester or a thioester of adipate, wherein the biocatalyst comprises an enzyme comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by any of the following: sequence ID 42-67, 94, 96, 98, 100, 102, 103, 105, 107, 109, 111, 113, 115, 116 and homologs thereof, the homologues having at least 90% sequence with any of these sequences The same degree. The method of claim 1, wherein the amino acid sequence is selected from the group consisting of amino acid sequences represented by any one of the following: sequence IDs 60, 63, 96, 100 and A homologue that has at least 90% sequence identity to any of these sequences. A method for preparing an adipate or a thioester of adipate, comprising converting 2,3-dehydroadipate or 2,3-dehydroadipate thioester into a diamide in the presence of a biocatalyst An acid ester or a thioester of adipate wherein the 3-hydroxyl group is biocatalyzed in the presence of a biocatalyst which catalyzes the dehydration of 3-hydroxydecyl ester or 3-hydroxyindole thioester to a 2-enyl oxime ester or a thioester. Conversion of adipate or thioester to 2,3-dehydroadipate or 2,3-dehydroadipate thioester, wherein the 3-hydroxyadipate or 3-hydroxyadipate thioester Prepared by converting 3-ketoadipate or 3-ketoadipate thioester to 3-hydroxyadipate or 3-hydroxyadipate thioester, wherein the 3-keto adipate or 3 - keto adipate thioester is prepared by reacting succinate or succinate with acetate or thioester in the presence of a biocatalyst as described below, the biocatalyst comprising a An enzyme of the amino acid sequence identified by any of Sequence IDs 1-13 and any of its homologues having at least 90% sequence identity to any of these sequences. A process for the preparation of adipic acid, which comprises preparing adipic acid ester or adipic acid thioester by a method according to any one of claims 1-3, and hydrolyzing as in the scope of claims 1-3 The adipate or hexane dicarboxylate obtained in the process of any of the above. The method of claim 4, wherein the hydrolysis is catalyzed by a biocatalyst. The method of claim 5, wherein the biocatalyst is an enzyme selected from the group of hydrolase (EC 3.1.2). A method for producing adipic acid, which comprises preparing an adipate or a thiodicarboxylate as described in any one of claims 1-3, and transferring as described in claim 1-3 The active group of the adipate or thiodicarboxylate obtained by the method of any one of the methods, wherein the transfer of the reactive group is catalyzed by a biocatalyst. The method of claim 7, wherein the biocatalyst comprises an enzyme that catalyzes the transfer of a sulfur-containing group (EC 2.8). The method of claim 8, wherein the enzyme is selected from the group consisting of CoA transferase (EC 2.8.3). A process for the preparation of 5-methylvaleric acid, which comprises preparing adipic acid ester or adipic acid thioester by the method of any one of claims 1-3, or as in the scope of the patent application A method according to any one of the preceding claims for the preparation of adipic acid and conversion of the adipate, thiodicarboxylate or adipic acid to 5-methylhydrazone Valeric acid. The method of claim 10, wherein the conversion to 5-methylvaleric acid is catalyzed by a biocatalyst. The method of claim 11, wherein the biocatalyst comprises an enzyme selected from the group consisting of oxidoreductases (EC 1.2.1). The method of claim 12, wherein the enzyme is selected from the group consisting of aldehyde dehydrogenase (EC 1.2.1.3, EC 1.2.1.4 and EC 1.2.1.5), malonate semialdehyde dehydrogenase (EC 1.2) .1.15), succinic acid semialdehyde dehydrogenase (EC 1.2.1.16 and EC 1.2.1.24); glutamic acid semialdehyde dehydrogenase (EC 1.2.1.20), amine adipic acid semialdehyde dehydrogenase (EC) 1.2.1.31), a group consisting of adipic acid semialdehyde dehydrogenase (EC 1.2.1.63); or selected from aldehyde dehydrogenase (acetylation) (EC 1.2.1.10), fat 醯- CoA reductase (EC 1.2.1.42), long-chain fat 醯-CoA reductase (EC 1.2.1.50), butyraldehyde dehydrogenase (EC 1.2.1.57) and succinic semialdehyde dehydrogenase (acetylation) The group formed. A process for the preparation of 6-aminocaproic acid, which comprises the process of preparing 5-methylvaleric acid and converting the 5-methylvaleric acid to a 6-amine by the method of any one of claims 10-13 Hexanoic acid. The method of claim 14, wherein the conversion reaction is catalyzed by a biocatalyst. The method of claim 15, wherein the biocatalyst is capable of catalyzing transamination and/or reductive amination, comprising one selected from the group consisting of a transaminase (EC 2.6.1) and an amino acid dehydrogenase ( The enzyme in the group of EC 1.4.1). The method of claim 16, wherein the enzyme is selected from the group consisting of β-amine isobutyric acid: α-ketoglutarate transaminase, β-alanine transaminase, and day Aspartate transaminase, 4-amine-butyrate transaminase (EC 2.6.19), L-lysine 6-transaminase (EC 2.6.1.36), 2-amine adipate transaminase (EC) 2.6.1.39), 5-aminopentanoate transaminase (EC 2.6.1.48), 2-aminohexanoate transaminase (EC 2.6.1.67), lysine: pyruvate 6-transaminase (EC 2.6. 1.71), and a group consisting of lysine-6-dehydrogenase (EC 1.4.1.18). The method of claim 17, wherein the transaminase used comprises any one of sequence ID 82, sequence ID 83, sequence ID 84, sequence ID 134, sequence ID 136, sequence ID 138, or a homolog thereof. An amino acid sequence having at least 90% sequence identity to any of these sequences. The method of claim 15, wherein the biocatalyst comprises an enzyme derived from an organism selected from the group consisting of Vibrio; Pseudomonas ( Pseudomonas); Bacillus; Mercurialis; Asplenium; Ceratonia; Mammals; Neurospora; Escherichia ); Thermus; Saccharomyces; Brevibacterium; Corynebacterium; Proteus; Agrobacterium; Geobacillus Acinetobacter; Ralstonia and Salmonella. A method for preparing caprolactam, comprising preparing a 6-amine hexanoic acid by the method of any one of claims 14 to 19, and cyclizing the 6-amine hexanoic acid, thereby forming a hexene Guanamine. A host cell for use in the preparation of an adipate or adipic acid thioester and/or a conversion of an adipate or adipic acid thioester to 5-carulic acid, comprising one or more nucleic acid sequences encoding one An amino acid sequence represented by sequence IDs 42-59, 61, 62, 64-67, sequence ID 74-81, or a homolog thereof, having at least 90% sequence identity to any of these sequences. . A host cell for preparing an adipate or adipic acid thioester and/or converting an adipate or adipic acid thioester to 5-caruvinoic acid, comprising at least two nucleic acid sequences each encoding one Different amino acid sequences as represented by sequence IDs 42-67, sequence IDs 74-81, 94, 96, 98, 100, 102, 103, 105, 107, 109, 111, 113, 115, 116 and their homologues The homologue has at least 90% sequence identity to any of these sequences. A fermentation process for preparing an adipate or a thioester of adipate, comprising the method of any one of claims 1 to 20, wherein a cell comprising a biocatalyst is contacted with a fermentable carbon source The biocatalyst may comprise a host cell according to claim 21 or 22, wherein the carbon source comprises any one of the compounds to be converted into a compound to be prepared, or wherein the cell preparation is to be A compound that is converted to a compound to be prepared from the carbon source.