TWI461537B - Preparation of 6-aminocaproic acid from α-ketopimelic acid - Google Patents

Description

Technique for preparing 6-amine hexanoic acid from α-keto pimelic acid

The present invention relates to a process for preparing 6-aminocaproic acid (hereinafter also referred to as "6-ACA"). The present invention further relates to a process for producing ε-caprolactam (hereinafter referred to as "caprolactam") from 6-ACA. The invention further relates to host cells useful for the preparation of 6-ACA or caprolactam.

Caprolactam is an internal guanamine which can be used in the manufacture of polyamines such as nylon-6 or nylon-6,12 (copolymer of caprolactam and laurylamine). A variety of ways are known in the art for the preparation of caprolactone from bulk chemicals, including the preparation of caprolactam from cyclohexanone, toluene, phenol, cyclohexanol, benzene or cyclohexane. These intermediate compounds are usually derived from mineral oil. In view of the growing demand for materials prepared using greener resiliency techniques, it is desirable to provide a process wherein caprolactone is prepared from an intermediate compound derived from a biorenewable source, or caprolactone is derived at least from It can be converted to an intermediate compound of caprolactam using biochemical methods. It is further desirable to provide a method of comparing the conventional chemical methods utilizing bulk chemicals derived from petrochemical sources that require less energy.

It is known to prepare caprolactam from 6-ACA, for example as described in US-A 6,194,572. Biochemically prepared 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, for example, from lysine by biochemical means or by purification synthesis. Although it is feasible to prepare 6-ACA by reduction of 6-AHEA by the method disclosed in WO 2005/068643, the inventors have found that under the reducing reaction conditions, 6-AHEA may spontaneously and substantially irreversibly cyclize to form an undesired A by-product, notable for beta-protonic acid. Such cyclization can be a bottleneck in the manufacture of 6-ACA and can result in significant loss in yield.

The object of the present invention is to provide a novel process for the preparation of 6-ACA or caprolactam, which can be used for the preparation of intermediates in the preparation of polyamido or 6-ACA or caprolactam, and can be used as An alternative to knowing the method.

Yet another object is to provide a novel method that overcomes one or more of the above disadvantages.

One or more other objects that can be solved in accordance with the present invention are obtained by the following description.

It is known today to prepare 6-ACA from a specific starting material, in other words, it has been found that 6-aminocaproic acid (6-ACA) can be prepared, wherein 6-aminocaproic acid is also known as 2-ketopimelic acid as α-keto Diacid (AKP) preparation. In particular, the process can be carried out in two or more reaction steps. For example, a method is provided in which AKP is first converted to 5-methylvalerate (5-mevalonate, 5-FVA), which is converted to 6-ACA. Further provided is a method wherein AKP is first converted to alpha-amine pimelic acid (AAP). The AAP is then converted to 6-ACA.

The inventors understand that in principle, 6-ACA can be prepared from AKP by means of a complete chemical (in other words a biocatalyst). Examples of suitable chemical means for carrying out individual reaction steps are listed below. However, the inventors also understand that 6-ACA can be prepared biochemically by AKP.

Thus, the invention relates in particular to a process for the preparation of 6-ACA wherein the 6-ACA is prepared from AKP using at least one biocatalyst.

The invention further relates to a process wherein 6-ACA is prepared from 5-methylvalerate (5-mevalonate, 5-FVA) using a biocatalyst. As indicated above, 5-FVA is available from AKP.

In one embodiment, the 6-ACA prepared in the process of the invention can be used to prepare caprolactam. Such a process comprises cyclizing 6-amine hexanoic acid in the presence of a biocatalyst, as desired.

The carboxylic acid or carboxylic acid ester such as 6-ACA, 2-amine pimelic acid (α-amine pimelic acid, hereinafter also abbreviated as "AAP"), other amino acids, 5-FVA or AKP are mentioned herein. These terms are meant to include protonated carboxylic acid groups (i.e., neutral groups), their corresponding carboxylates (its conjugate bases), and salts thereof. When reference is made herein to amino acids such as 6-ACA, the term is meant to include amino acids in their zwitterionic form (wherein the amine group is in protonated form and the carboxylic acid group is in deprotonated form), wherein the amine The amino acid which is protonated and the carboxylic acid is in its neutral form, and the amino acid in which the intermediate group is in its neutral form and the carboxylic acid group is in a deprotonated form, and salts thereof.

According to the present invention, no undesired cyclization of the intermediate product which causes loss of yield was observed when 6-ACA was formed and if decylamine was formed as needed.

The process of the invention is expected to allow comparison of the process described in WO 2005/68643 to obtain comparable or even better yields. It is expected that the method of the present invention is particularly advantageous when utilizing organisms, particularly in the context of growth and maintenance methods of organisms.

It is further contemplated that in the examples of the present invention, the productivity of 6-ACA in the process of the present invention (g/liter ‧ hours formed) can be improved.

The word "or" as used herein is defined as "and/or" unless otherwise specified.

The term "一" as used herein is defined as "at least one" unless otherwise specified.

When a noun (for example, a compound, an additive, etc.) is used in the singular, it is meant to include a plural.

When referring to a compound in which a stereoisomer is present, the compound can be any one of such stereoisomers or a combination thereof. Thus, when referring to, for example, the presence of an amino acid of the enantiomer, the amino acid can be the L-enantiomer, the D-enantiomer or a combination thereof. Where a natural stereoisomer is present, the compound is preferably a natural stereoisomer.

When an enzyme is mentioned and the enzyme class (EC) is included, the enzyme class is based on the enzyme system or may be based on the "enzyme nomenclature" provided by the International Society for Biochemistry and Molecular Biology Names Committee (NC-IUBMB). For the classified category, refer to http://www.chem.qmul.ac.uk/iubmb/enzyme/. Intentions include other suitable enzymes that have not (not yet) been classified into a particular class but can be classified in this manner.

The term "homolog" as used herein particularly means having 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%, and even more preferably. At least 80%, particularly preferably at least 85%, more preferably 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% sequence homologous polynucleotide or polypeptide. The term "homolog" also refers to a nucleotide sequence (polynucleotide sequence) that differs from another nucleotide sequence but encodes the same polynucleotide sequence due to the simplicity 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 nucleotide sequences via a comparison of two sequences. Usually the sequence identity or similarity compares the full length of the sequence, but it is also possible to compare only the portions of the sequence aligned to each other. In the industry, "identity" or "similarity" also indicates the degree of sequence correlation between a multi-peptide sequence or a nucleotide sequence (as the case may be) determined by the matching between such sequences. Preferably, the assay of identity or similarity is designed to achieve the largest match between the sequences tested. In the context of the present invention, preferred computer program methods for the degree of similarity and similarity between specific sequences include BLASTP and BLASTN (Altschul, SF et al, J. Mol. Biol. 1990, 215, 403-410, publicly available from NCBI). And other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH, Beira, Maryland, 20894)). Preferably, the parameters for multi-peptide sequence comparison 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 gap opening 10.0, gap extension 0.5, DNA full matrix (DNA identity matrix).

According to the invention, a biocatalyst is used, in other words a reaction step of the method 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, it is isolated from the natural environment (isolated from the organism in which the enzyme has been produced) using, for example, a solution, an emulsion, a dispersion, a lyophilized cell (a suspension), a lysate, or a brake on a support. One or more enzymes. In one embodiment, the one or more enzymes form part of a living organism (eg, 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 spores) or cells in a stationary phase. It is also possible to use an enzyme-forming portion of a permeabilized cell (in other words, a matrix precursor of a permeabilizable enzyme or a matrix precursor of an enzyme).

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

In one embodiment, the biocatalyst is derived from an animal, particularly from a portion of an animal such as the liver, pancreas, brain, kidney, heart or other organ. The animal may in particular be selected from the group consisting of mammals, more particularly from the group consisting of Leporidae, Muridae, Suidae and Bovidae. .

Suitable plants include, inter alia, from the genus Asplenium; Cucurbitaceae, especially Cucurbita such as Cucurbita moschata (squash), or Cucumis; Mercurialis such as perennial Mercurianis perennis; Hydnocarpus; and a group of plants of the genus Ceratonia.

Suitable bacteria are particularly selected from the group consisting of Vibrio, Pseudomonas, Bacillus, Corynebacterium, Brevibacterium, Enterococcus. ), Streptococcus, Klebsiella, Lactococcus, Lactobacillus, Clostridium, Escherichia, Habitat Thermus, Mycobacterium, Zymomonas, Proteus, Agrobacterium, Geobacillus, Acinetobacter ), Ralstonia, Rhodobacter, Paracoccus, Novosphingobium, Nitrosomonas, Legionella , a group consisting of Neisseria, Rhodopseudomonas, Staphylococcus, Deinococcus, and Salmonella.

Suitable archaea may be particularly selected from the group consisting of Archaeoglobus, Aeropyrum, Halobacterium, Methanosarcina, Methanococcus, and heat. Thermoplasma, Pyrobaculum, Methanocaldococcus, Methanobacterium, Methanosphaera, Methanopyrus, and Methanobrevibacter ) the group consisting of.

Suitable fungi are particularly selected from the group consisting of Rhizopus, Neurospora, Penicillium and Aspergillus.

Suitable yeasts are particularly selected from the group consisting of Candida, Hansenula, Kluyveromyces, and Saccharomyces.

It will be apparent to those skilled in the art that a mutant having a suitably active natural biocatalyst (wild type) or a natural biocatalyst can be utilized in the method according to the present invention. The nature of natural biocatalysts can be improved by biotechnology known to those skilled in the art, such as molecular evolution or theoretical design. The preparation method of the mutant strain of the wild type biocatalyst can be modified, 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 as a biocatalyst or a biocatalytic moiety. Prepared by encoding the DNA of an organism such as an enzyme. In particular, DNA can be modified to allow the DNA to encode an enzyme that differs from the wild-type enzyme by at least one amino acid, thus encoding a wild-type enzyme that contains one or more amino acid substitutions, deletions, and/or insertions, or mutations. The strain combines two or more parental enzyme sequences, or by affecting the performance of such modified DNA in appropriate (host) cells. The latter can be achieved by methods known to those skilled in the art, such as codon optimization methods or codon pair optimization methods, for example based on the method described in WO 2008/000632.

The mutant biocatalyst may have improved properties in terms of one or more of the following phases: selectivity to the enzyme substrate, activity, stability, solvent tolerance, pH profile data, temperature profile data, enzyme matrix profile data, inhibition Susceptibility, cofactor utilization and enzyme matrix affinity. Mutants with improved properties can be identified by applying, for example, a suitable high throughput screening or selection method based on methods known to those skilled in the art.

When referring to a biocatalyst derived from a particular source, in particular an enzyme, it is meant in particular that the recombinant biocatalyst, which is derived from the first organism but is actually produced by the genetically modified second organism, is specifically enzymatically included. The biocatalyst from the first organism is in particular an enzyme.

In a preferred process of the invention, the preparation is carried out in the presence of a biocatalyst which catalyzes the decarboxylation of an alpha-keto acid or an amino acid (i.e., a compound comprising at least one carboxylic acid group and at least one amine group) Biocatalytic (usually enzyme catalyzed) reactions. The enzymes having such catalytic activity are thus referred to as α-keto acid decarboxylase or amino acid decarboxylase, respectively.

The acid is preferably a dibasic acid wherein the biocatalyst is selective for the acid group adjacent to the keto group or the amine group.

In general, a suitable decarboxylase has alpha-ketopimelate decarboxylase activity, which catalyzes the conversion of AKP to 5-FVA; or has alpha-amine pimelic acid decarboxylase activity, which catalyzes the conversion of AAP to 6-ACA.

The enzyme which decarboxylates the α-keto acid or the amino acid is selected in particular from the decarboxylase group (EC 4.1.1), preferably from the grasshopper acetic acid decarboxylase (EC 4.1.1.3). ), diamine pimelic acid decarboxylase (EC 4.1.1.20), branched chain α-keto acid decarboxylase (EC 4.1.1.72), α-ketoisovalerate decarboxylase, α-ketoglutaric acid A group consisting of a carboxylase (EC 4.1.1.71) and a pyruvate decarboxylase (EC 4.1.1.1).

One or more other suitable decarboxylases may be selected from the group consisting of oxalate decarboxylase (EC 4.1.1.2), acetamidine decarboxylase (EC 4.1.1.4), proline decarboxylase/leucine decarboxylation Enzyme (EC 4.1.1.14), glutamic acid decarboxylase (EC 4.1.1.15), aspartate 1-decarboxylase (EC 4.1.1.11), 3-hydroxyglutamate decarboxylase (EC 4.1.1.16) ), alginate decarboxylase (EC 4.1.1.17), lysine decarboxylase (EC 4.1.1.18), arginine decarboxylase (EC 4.1.1.19), 2-ketoglutarate decarboxylase (EC 4.1.1.71), and a group consisting of diamine butyrate decarboxylase (EC 4.1.1.86).

The decarboxylase may especially be an organism decarboxylase selected from the group consisting of pumpkin; courgette; yeast; fungi such as Saccharomyces cerevisiae, Candida flareri, Hansenula species ( Hansenula sp.), Kluyveromyces marxianus, Rhizopus javanicus, and Neurospora crassa; mammals are particularly derived from mammalian brains and bacteria such as Escherichia coli. , a group consisting of Lactococcus lactis, Mycobacterium tuberculosis, Pseudomonas sp., and Zymomonas mobilis.

The pyruvate decarboxylase may be derived from Saccharomyces cerevisiae or Z. mobilis. In particular, pyruvate decarboxylase mutant I472A from Z. mobilis can be used.

A glutamate decarboxylase, a diamine pimelic acid decarboxylase or an aspartate decarboxylase derived from E. coli can be used.

A glutamate decarboxylase derived from Rhizopus oryzae, Mycobacterium leprae, Clostridium perfringens, Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus may be used. Examples of the genus Lactococcus species which can obtain glutamate decarboxylase include, inter alia, Lactococcus lactis such as Lactococcus lactis B1157, Lactococcus lactis IFPL730, and more particularly Lactococcus lactis variant maltigenes (formerly known as lactic acid) Streptococcus lactis var. maltigenes).

In particular, a grasshopper acetic acid decarboxylase derived from Pseudomonas can be used.

A branched chain α-keto acid decarboxylase derived from Lactococcus lactis can be used. More specifically, an α-ketoisovalerate decarboxylase derived from Lactococcus lactis can be used.

In particular, alpha-ketoglutarate decarboxylase from Mycobacterium tuberculosis can be used.

In a preferred method of the invention, the preparation of 6-ACA comprises catalyzing a transamination reaction in the presence of an amine-based donor and enzymatically catalyzing the reaction in the presence of an enzyme selected from the group of transaminase (EC 2.6.1) .

In general, the appropriate transaminase has 6-aminohexanoic acid 6-transaminase activity, which can catalyze the conversion of 5-FVA to 6-ACA; or has α-amine pimelic acid 2-transaminase activity, which can catalyze AKP. Convert to AAP.

The transaminase is particularly selected from the group consisting of β-amine isobutyric acid: α-ketoglutarate transaminase, β-alanine transaminase, aspartate transaminase, 4-amine butyrate transaminase ( EC 2.6.1.19), L-lysine 6-transaminase (EC 2.6.1.36), 2-amine adipate transaminase (EC 2.6.1.39), 5-amine valerate transaminase (EC 2.6) .1.48), a group consisting of 2-aminohexanoate transaminase (EC 2.6.1.67) and lysine: pyruvate 6-transaminase (EC 2.6.1.71).

In one embodiment, the transaminase may be selected from the group consisting of alanine transaminase (EC 2.6.1.2), leucine transaminase (EC 2.6.1.6), alanine-ketoacid transaminase (EC 2.6) .1.12), β-alanine-pyruvate transaminase (EC 2.6.1.18), (S)-3-amine-2-methylpropionic acid transaminase (EC 2.6.1.22), L, L-II A group consisting of amine pimelic acid transaminase (EC 2.6.1.83).

The transaminase is particularly selected from the group consisting of mammals; the genus Hawthorn is especially a perennial hawthorn, more particularly the perennial hawthorn; the genus Pteridium is more particularly the unilateral hornbeam fern (Asplenium unilaterale) or the north Aspergillus fern (Asplenium septentrionale); Jiale tree is more particularly Ceratonia siliqua; Rhodobacter genus is especially Rhodobacter sphaeroides; Staphylococcus is Staphylococcus aureus Vibrio genus is Vibrio fluvialis; Pseudomonas aeruginosa; Pseudomonas aeruginosa; Bacillus weihenstephanensis And Bacillus subtilis; Legionella; nitrous genus; Neisseria; or yeast, especially the transaminase of Saccharomyces cerevisiae.

Where the enzyme is a mammalian enzyme, it may be derived in particular 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 β-amine isobutyric acid derived from a mammalian kidney: α-ketoglutarate transaminase, especially β-amine isobutyric acid derived from pig kidney: α-ketoglutaric acid Aminease; beta-alanine transaminase from mammalian liver, especially beta-alanine transaminase from rabbit liver; aspartate transaminase from mammalian heart, especially from pigs Heart aspartate transaminase; 4-amine butyrate transaminase from mammalian liver, especially 4-amine butyrate transaminase from pig liver; 4-amine butyl from mammalian brain Acid transaminase, especially 4-amine butyrate transaminase obtained from human, porcine or rabbit brain; α-ketoadipate-glutamic acid transaminase obtained from the genus Rhodochrous, especially from rough red Mildew alpha-ketoadipate-glutamic acid transaminase; 4-amine butyrate transaminase from Escherichia coli, or alpha-amine adipate transaminase from the genus Thermus, especially The α-amine adipic acid transaminase obtained from Thermus thermophilus, and the 5-aminovalerate transaminase derived from Clostridium, especially from Clostridium aminovalericum A group consisting of 5-amine valerate transaminase. A suitable 2-amine adipate transaminase may, for example, also be provided by Pyrobaculum islandicum.

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

In a more preferred embodiment, the method for preparing 6-ACA comprises an enzyme capable of catalytically reductive amination in the presence of an ammonia source selected from the group consisting of an oxidoreductase (EC 1.4) acting on the CH-NH ₂ group of the donor. The group is particularly subjected to a biocatalytic reaction in the presence of an enzyme selected from the group consisting of amino acid dehydrogenases (EC 1.4.1). In general, an appropriate amino acid dehydrogenase has 6-aminohexanoic acid 6-dehydrogenase activity, which catalyzes the conversion of 5-FVA to 6-ACA; or has α-amine pimelic acid 2-dehydrogenase activity. It can catalyze the conversion of AKP to AAP. In particular, the appropriate amino acid dehydrogenase may be selected from the group consisting of diamine pimelate dehydrogenase (EC 1.4.1.16), lysine 6-dehydrogenase (EC 1.4.1.18), glutamic acid. A group consisting of hydrogenase (EC 1.4.1.3; EC 1.4.1.4) and leucine dehydrogenase (EC 1.4.1.9).

In one embodiment, the amino acid dehydrogenase may be selected from the group consisting of glutamate dehydrogenase (EC 1.4.1.3) classified as a receptor with NAD or NADP, and acting as a receptor with NADP. Gluten glutamate dehydrogenase (EC 1.4.1.4), leucine dehydrogenase (EC 1.4.1.9), diamine pimelic acid dehydrogenase (EC 1.4.1.16), and 6-dehydrogenation of lysine Amino acid dehydrogenase of the enzyme (EC 1.4.1.18).

The amino acid dehydrogenase is particularly derived from an organism selected from the group consisting of: Corynebacterium glutamicum, particularly of the genus Corynebacterium; (Proteus vulgaris); Agrobacterium is particularly Agrobacterium tumefaciens; Geobacterium genus is Geobacillus stearothermophilus; Acinetobacter is a genus ADP1, of the genus Acinetobacter; In particular, it is Ralstonia solanacearum; Salmonella is Salmonella typhimurium; Saccharomyces is especially Saccharomyces cerevisiae; Brevibacterium is Brevibacterium flavum; and Bacillus is special. It is Bacillus sphaericus, Bacillus cereus, and Bacillus subtilis. For example, a suitable amino acid dehydrogenase may be selected from the group consisting of diamine pimelic acid dehydrogenase derived from Bacillus, particularly Bacillus licheniformis; diamine pimelic acid dehydrogenase obtained from Brevibacterium; a diamine pimelic acid dehydrogenase from Corynebacterium, in particular a diamine pimelic acid dehydrogenase derived from Corynebacterium glutamicum; a diamine pimelic acid dehydrogenase derived from Proteus, in particular a diamine pimelic acid dehydrogenase derived from Proteus vulgaris; an lysine 6-dehydrogenase derived from Agrobacterium, particularly Agrobacterium tumefaciens; obtained from the genus Geobacillus, particularly from Bacillus licheniformis Leucine 6-dehydrogenase; glutamate dehydrogenase (EC 1.4.1.3) derived from Acinetobacter with NADH or NADPH as a cofactor, especially glutamine derived from Acinetobacter sp. ADP1 Acid dehydrogenase; glutamate dehydrogenase (EC 1.4.1.3) from the genus Rasta, in particular glutamate dehydrogenase from R. solani; from Salmonella A glutamate dehydrogenase (EC 1.4.1.4) that acts as a cofactor with NADPH, in particular a glutamate dehydrogenase from Salmonella typhi; a glutamine from the genus Saccharomyces Acid dehydrogenase (EC 1.4.1.4), in particular glutamate dehydrogenase from Saccharomyces cerevisiae; glutamate dehydrogenase from the genus Brevibacterium (EC 1.4.1.4), especially from yellow Bacillus glutamate dehydrogenase; and leucine dehydrogenase derived from Bacillus, particularly leucine dehydrogenase derived from Bacillus cereus or Bacillus subtilis.

In a particular embodiment, AKP is biocatalyzed to 5-methylvaleric acid (5-FVA) in the presence of a decarboxylase or other biocatalyst that catalyzes such conversion. The decarboxylase used according to the invention is in particular selected from the group consisting of Lactobacillus lactis, Lactobacillus lactis variant malt genes or Lactobacillus lactis subsp. cremoris. Α-keto acid decarboxylase; branched-chain α-keto acid decarboxylase obtained from Lactobacillus lactis B1157 or Lactobacillus lactis IFPL730; obtained from Saccharomyces cerevisiae, Candida faecalis, Actinobacillus mobilis, Han Pyruvate decarboxylase from the genus Zygophyllum, Rhizopus oryzae, Rhizoctonia solani, or Kluyveromyces cerevisiae; α-ketoglutarate decarboxylase from Mycobacterium tuberculosis; obtained from Escherichia coli, short lactic acid A glutamate decarboxylase of Bacillus, Leprosy, Rhizopus or Pseudomonas aeruginosa; and aspartate decarboxylase from Escherichia coli.

It has been found that decarboxylase derived from Escherichia coli, Z. mobilis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Pseudomonas species or Lactobacillus lactis is suitable for catalyzing the conversion of AKP to 5-FVA. More specifically, a biocatalyst having a decarboxylase having an amino acid sequence recognized by sequence ID 31, sequence ID 34, sequence ID 37, sequence ID 40, sequence ID 43, sequence ID 46 or a homolog thereof can be used. It is also contemplated that such decarboxylase can be used to prepare 6-ACA from AAP.

The 5-FVA is then converted to 6-ACA. It can be carried out in a chemical manner: by using a hydrogenation catalyst such as nickel in a SiO ₂ /Al ₂ O ₃ support, by a reductive amination reaction of 5-FVA with ammonia, 6-ACA can be produced in high yield, such as EP-A 628 535 or DE. 4 322 065 is described for 9-aminononanoic acid (9-amine geranyl acid) and 12-aminedodecanoic acid (12-amine lauric acid). In addition, 6-ACA can be prepared by using PtO ₂ hydrogenated 6-mercapto acid by using 6-mercapto acid prepared by reacting 5-FVA with hydroxylamine (for example, refer to FO Ayorinde, EY Nana, PD Nicely, AS Woods, EO). Price, CP Nwaonicha J. Am. Oil Chem. Soc. 1997, 74, 531-538 for the synthesis of the homologous 12-amine dodecanoic acid.

In one embodiment, the conversion of 5-FVA to 6-ACA is carried out in (i) an amine donor and (ii) a transaminase, an amino acid dehydrogenase or other biocatalyst capable of catalyzing such conversion. The next step is carried out in a biocatalytic manner. In particular, in such embodiments, the transaminase may be selected from the group consisting of: transaminase from Vibrio fluvialis, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus veii, or Escherichia coli ; β-amine isobutyric acid derived from pig kidney: α-ketoglutarate transaminase; β-alanine transaminase obtained from rabbit liver; transaminase obtained from perennial hawthorn shoots; Porcine liver or 4-amine butyrate transaminase obtained from human, rabbit or pig brain; β-alanine transaminase obtained from rabbit liver; and L-lysine: α-ketoglutarate-ε- Transaminase. In the case of the use of an amino acid dehydrogenase, such an amino acid dehydrogenase is particularly selected from the group consisting of lysine 6-dehydrogenase derived from Agrobacterium tumefaciens or B. thermophilus. group. Other suitable amino acid dehydrogenases may be selected from the group consisting of diamine pimelate dehydrogenases derived from Bacillus sphaericus, Brevibacterium species, Corynebacterium glutamicum, or Proteus vulgaris; a group consisting of glutamate dehydrogenase (EC 1.4.1.3) derived from Acinetobacter sp. ADP1 or R. solani with NADH or NADPH as a cofactor; a group consisting of glutamate dehydrogenase (EC 1.4.1.4) with NADPH as a cofactor; consisting of glutamate dehydrogenase (EC 1.4.1.4) obtained from Saccharomyces cerevisiae or Brevibacterium flavum a group; or a group consisting of leucine dehydrogenase derived from Bacillus cereus or Bacillus subtilis.

In a particular embodiment, the transaminase comprising an amino acid sequence identified by sequence ID 2, sequence ID 5, sequence ID 8, sequence ID 65, sequence ID 67, sequence ID 69, or any homolog of such sequences The biocatalyst catalyzes the conversion of 5-FVA to 6-ACA.

In a particular embodiment, the AKP is chemically converted to 5-FVA. By removing the azeotropic water and simultaneously losing carbon dioxide, the second amine is, for example The phenyl group is subjected to the formation of an intermediate enamine, and the 2-ketocarboxylic acid can be effectively chemically decarboxylated to a corresponding aldehyde. The method is, for example, based on the method described in the tetrahedron 1982, 23(4), 459-462. The intermediate end group eneamine is then hydrolyzed to the corresponding aldehyde. 5-FVA is subsequently converted to 6-biocatalytic by transamination in the presence of a transaminase or by enzymatic reductive amination via an amino acid dehydrogenase or other biocatalyst that catalyzes such conversion. ACA. Such a transaminase or amino acid dehydrogenase may be particularly selected from the above-described biocatalysts described when 5-FVA is converted to 6-ACA.

Alternatively, the conversion of 5-FVA to 6-ACA can be carried out by chemical methods such as those described above.

In a particular embodiment, AKP is biocatalyzed to AAP in the presence of (i) a transaminase, an amino acid dehydrogenase, or other biocatalyst that catalyzes such conversion and (ii) an amine-based donor. . Such a transaminase for converting AKP to AAP according to the present invention is particularly selected from the group consisting of the transaminase described above, and more particularly selected from the group consisting of: aspartic acid from pig heart Aminease; alpha-ketoadipate from glutamic acid or yeast: glutamate transaminase; transaminase from perennial hawthorn shoots; 4-amine butyrate transaminase from Escherichia coli ; a-arene adipate transaminase obtained from the thermophilic thermophilus; a transaminase obtained from the northern fern or unilateral iron fern; and a transaminase derived from the long pod.

In a preferred embodiment, the transaminase for converting AKP to AAP is selected from the group consisting of Vibrio, Pseudomonas, Bacillus, Legionella, nitrite, A group consisting of a transaminase of Neisseria, Rhodobacter, Escherichia, and Rhodopseudomonas.

It is particularly found that a transaminase derived from an organism selected from the group consisting of the following is suitable for catalyzing the conversion of AKP to AAP: Bacillus subtilis, Rhodobacter sphaeroides, Legionella vulgaris, European nitrous acid bacteria, Neisseria gonorrhoeae, Pseudomonas syringae, Rhodopseudomonas palustris, Vibrio fluvialis, Escherichia coli and Pseudomonas aeruginosa.

In a particular embodiment, in order to convert AKP to AAP, use includes according to sequence ID 2, sequence ID 8, sequence ID 12, sequence ID 15, sequence ID 17, sequence ID 19, sequence ID 21, sequence ID 23, sequence ID 25. A transaminase of the amino acid sequence of sequence ID 27, sequence ID 29 or a homologue of any of these sequences.

In still another embodiment, the method for preparing AAP comprises the enzyme capable of catalytically reducing the amination reaction in the presence of an ammonia source selected from the group consisting of an oxidoreductase (EC 1.4) acting on the CH-NH ₂ group of the donor. The group, in particular, is subjected to a biocatalytic reaction in the presence of an enzyme selected from the group consisting of amino acid dehydrogenases (EC 1.4.1). In general, an appropriate amino acid dehydrogenase has alpha-amine pimelic acid 2-dehydrogenase activity which catalyzes the conversion of AKP to AAP.

Particularly suitable amino acid dehydrogenases may be selected from the group consisting of diamine pimelate dehydrogenase (EC 1.4.1.16), glutamate dehydrogenase (EC 1.4.1.3; EC 1.4.1.4) and leucine A group consisting of hydrogenase (EC 1.4.1.9).

In one embodiment, the amino acid dehydrogenase is selected from the group consisting of glutamate dehydrogenase (EC 1.4.1.3), which functions as a receptor with NAD and NADP, and acts as a receptor with NADP. Amino acid dehydrogenase of glutamate dehydrogenase (EC 1.4.1.4), leucine dehydrogenase (EC 1.4.1.9), and diamine pimelate dehydrogenase (EC 1.4.1.16).

The amino acid dehydrogenase is particularly derived from an organism selected from the group consisting of: Corynebacterium, in particular Corynebacterium glutamicum; Proteus, in particular Proteus vulgaris; Agrobacterium, especially nodule Agrobacterium; the genus of the genus A. faecalis; in particular, the Acinetobacter spp. ADP1; the genus Astragalus, particularly the genus R. solani; the genus Salmonella, especially the genus Salmonella; In particular, it is Saccharomyces cerevisiae; Brevibacterium genus is particularly Brevibacterium flavum; and Bacillus is in particular Bacillus cerevisiae, Bacillus cereus and Bacillus subtilis.

For example, a suitable amino acid dehydrogenase may be selected from the group consisting of diamine pimelic acid dehydrogenase derived from Bacillus, particularly Bacillus licheniformis; diamine pimelic acid dehydrogenase obtained from Brevibacterium; a diamine pimelic acid dehydrogenase from Corynebacterium, in particular a diamine pimelic acid dehydrogenase derived from Corynebacterium glutamicum; a diamine pimelic acid dehydrogenase derived from Proteus, in particular Diamine pimelic acid dehydrogenase derived from Proteus vulgaris; glutamate dehydrogenase (EC 1.4.1.3) derived from Acinetobacter with NADH or NADPH as a cofactor, especially from Acinetobacter a glutamic acid dehydrogenase of the genus ADP1; a glutamate dehydrogenase (EC 1.4.1.3) derived from the genus Russini, in particular dehydrogenating glutamic acid from the genus An enzyme; a glutamate dehydrogenase (EC 1.4.1.4) derived from Salmonella with NADPH as a cofactor, particularly a glutamate dehydrogenase from Salmonella typhi; a glutamic acid obtained from the genus Saccharomyces Hydrogenase (EC 1.4.1.4), especially glutamate dehydrogenase from Saccharomyces cerevisiae; glutamate dehydrogenase (EC 1.4.1.4) from Brevibacterium, especially from yellow Bacteria of glutamate dehydrogenase; and available from the genus Bacillus leucine dehydrogenase, especially for the leucine dehydrogenase from Bacillus subtilis or Bacillus subtilis of Opuntia.

Another suitable amino acid dehydrogenase may be selected from the group consisting of lysine 6-dehydrogenase from Agrobacterium tumefaciens or B. thermophilus; or from Bacillus cereus or Bacillus subtilis a group consisting of leucine dehydrogenase.

The AAP prepared by the method of the present invention is further useful for the preparation of 6-ACA. The inventors have realized that AAP made from AKP can be converted to 6-ACA by a carboxylation reaction. This reaction can be carried out chemically, for example by heating in a high boiling solvent in the presence of a ketone or aldehyde catalyst. For example, the amino acid is decarboxylated with cyclohexanol at 150-160 ° C using 1-2 v/v% cyclohexenone in good yields, such as M. Hashimoto, Y. Eda, Y. Osanai, T. Iwai And S. Aoki et al., Chem. Lett. 1986, 893-896. A similar method is described in Eiso. Pat. Appl. 1586553 by Daiso, and S. D. Brandt, D. Mansell, S. Freeman, I. A. Fleet, J. F. Alder J. Pharm. Biomed. Anal. 2006, 41, 872-882.

Alternatively, decarboxylation of AAP to 6-ACA can be carried out in a biocatalytical manner in the presence of a decarboxylase or other biocatalyst that catalyzes such decarboxylation.

The decarboxylase may be selected from decarboxylase which catalyzes the decarboxylation of an a-amino acid. The enzyme which catalyzes the α-amino acid is particularly selected from the group consisting of decarboxylase (EC 4.1.1), preferably selected from pyruvate decarboxylase (EC 4.1.1.1), diamine gly. Diacid decarboxylase (EC 4.1.1.20), diamine pimelic acid decarboxylase (EC 4.1.1.20), branched chain α-keto acid decarboxylase (EC 4.1.1.72) which includes α-ketoisovalerate A group consisting of decarboxylase and alpha-ketoglutarate decarboxylase (EC 4.1.1.71).

One or more other suitable decarboxylases may be selected in particular from the group consisting of oxalic acid decarboxylase (EC 4.1.1.2), oxalic acid decarboxylase (EC 4.1.1.3), deacetoacetic acid decarboxylation Enzyme (EC 4.1.1.4), aspartic acid 1-decarboxylase (EC 4.1.1.11), proline decarboxylase/leucine decarboxylase (EC 4.1.1.14), glutamate decarboxylase ( EC 4.1.1.15), 3-hydroxyglutamic acid decarboxylase (EC 4.1.1.16), alanine decarboxylase (EC 4.1.1.17), lysine decarboxylase (EC 4.1.1.18), spermine Acid decarboxylase (EC 4.1.1.19), 2-ketoglutarate decarboxylase (EC 4.1.1.71), and diamine butyrate decarboxylase (EC 4.1.1.86).

The decarboxylase is in particular a decarboxylase selected from the group consisting of: pumpkin, for example pumpkin; courgette; yeast; fungi such as Saccharomyces cerevisiae, Candida faecalis, Hansenula species, Markscroft Saccharomyces cerevisiae, Rhizopus oryzae, and Rhizopus oryzae; mammals are specially composed of mammalian brain and bacteria such as Escherichia coli, Lactococcus lactis, Mycobacterium tuberculosis, Pseudomonas species and Active Zymomonas Group of groups.

The pyruvate decarboxylase may be derived from Saccharomyces cerevisiae or Z. mobilis. In particular, pyruvate decarboxylase mutant I472A from Z. mobilis can be used. In particular, a grasshopper acetic acid decarboxylase derived from Pseudomonas can be used. A glutamate decarboxylase, diamine pimelic acid decarboxylase or aspartate decarboxylase derived from E. coli, or from Rhizopus oryzae, M. leprae, Clostridium perfringens, A glutamate decarboxylase of Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus. Examples of the genus Lactococcus species which can obtain glutamate decarboxylase include, inter alia, Lactococcus lactis, such as Lactococcus lactis B1157, Lactococcus lactis IFPL730, and more particularly Lactococcus lactis variant malt gene (formerly known as Streptococcus mutans) Malt gene). The diamine pimelic acid decarboxylase can be obtained, for example, from an organism in which an amine acid can be synthesized from diamine pimelic acid. Such organisms are particularly derived from bacteria, archaea and plants. A particular diamine pimelic acid decarboxylase can be obtained from a Gram-negative bacterium such as E. coli. A branched chain α-keto acid decarboxylase derived from Lactobacillus lactis can be used. More specifically, a branched chain α-keto acid decarboxylase derived from Lactobacillus lactis and an α-ketoisovalerate decarboxylase can be used.

In particular, alpha-ketoglutarate decarboxylase from Mycobacterium tuberculosis can be used. The inventors have found that alpha-ketoglutarate decarboxylase (Kgd) from Mycobacterium tuberculosis can be used to convert AAP to 6-ACA. In particular, the inventors have found that such a decarboxylase comprising a sequence as shown in SEQ ID NO: 46 or a functional analog thereof catalyzes the formation of 6-ACA from AAP.

The glutamine decarboxylase is particularly selected from the group consisting of pumpkin, courgette, yeast, or calf brain; and diamine pimelic acid decarboxylase (EC 4.1.1.20).

The diamine pimelic acid decarboxylase can be obtained, for example, from an organism in which an amine acid can be synthesized from diamine pimelic acid. Such organisms are particularly derived from bacteria, archaea and plants.

A particular diamine pimelic acid decarboxylase can be obtained from a Gram-negative bacterium such as E. coli.

In a particular embodiment, the AKP is chemically converted to AAP. AAP can be prepared from 2-keto pimelic acid by catalyzing the Leukart-Wallach reaction as described for similar compounds. This reaction was carried out using ammonium formate in methanol and [RhCp*Cl ₂ ] ₂ as a homogeneous catalyst (M. Kitamura, D. Lee, S. Hayashi, S. Tanaka, M. Yoshimura J. Org. Chem. 2002, 67, 8685-8687). In addition, the Rukat-Wara reaction can be used as aqueous catalyst for aqueous ammonium formate using [Ir ^III Cp*(bpy)H ₂ O]SO ₄ as a catalyst, such as S. Ogo, K. Uehara and S. Fukuzumi at J. Am. Chem. Soc . 2004, 126, 3020-3021. Conversion of an α-keto acid to an (enantiomerically enriched) amino acid by reaction with a (preferable) benzylamine followed by hydrogenation of the intermediate imine with Pd/C or Pd(OH) ₂ /C May be reached. See, for example, RG Hiskey, RC Northrop J. Am. Chem. Soc. 1961, 83, 4798.

AAP is then biocatalytically converted to 6-ACA in the presence of decarboxylase or other biocatalyst that can undergo such decarboxylation. Such a decarboxylase may be particularly selected from the foregoing biocatalysts described when describing a biocatalyst for the conversion of AAP to 6-ACA.

Alternatively, the conversion of AAP to 6-ACA can be carried out by chemical methods such as the methods previously described.

In a particular embodiment, AKP is converted to 5-FVA in a biocatalytical manner in the presence of a decarboxylase or other biocatalyst that catalyzes such conversion; and subsequently in a transaminase, amino acid dehydrogenase, or other In the presence of a biocatalyst that catalyzes such conversion, 5-FVA is converted to 6-ACA. Decarboxylases suitable for use in such reactions are particularly selected from the group consisting of the decarboxylase described above for the catalysis of AKP to 5-FVA. Suitable transaminase or amino acid dehydrogenases for the conversion of 5-FVA are particularly selected from those previously described for the biocatalytic conversion of 5-FVA biocatalytic conversion to 6-ACA.

In a particular embodiment, AKP is biocatalyzed to AAP in the presence of a transaminase, an amino acid dehydrogenase, or other biocatalyst that catalyzes such conversion; and subsequently catalyzed by decarboxylase or other In the presence of a transformed biocatalyst, AAP is converted to 6-ACA.

Enzymes suitable for use in such reactions are selected, inter alia, from transaminase, amino acid dehydrogenase and decarboxylation described above when describing biotransformation of AKP to AAP and biotransformation of AAP to 6-ACA. a group consisting of enzymes.

The AKP used to prepare 6-ACA can in principle be obtained in any manner. For example, AKP can be based on H. Et al., obtained by the method described in Chem. Ber. 1959, 92, 2492-2499. AKP can be prepared by alkylating cyclopentanone with diethyl oxalate as a base, refluxing the resulting product in a strong acid (2M hydrochloric acid), and recovering the product, for example, by crystallization of toluene, to prepare AKP.

It can also be derived from natural sources such as methane-producing archaea, from the northern hornbeam fern, or from the AKP of Hydnocarpus anthelminthica. The AKP can, for example, be extracted from such organisms or parts thereof such as deworming zephyr seeds. Suitable extraction methods are, for example, based on the method described by A. I. Virtanen and A. M. Berg in Acta Chemica Scandinavica 1954, 6, 1085-1086, which describes the extraction of amino acids and AKP from the hornwood fern using 70% ethanol.

In a particular embodiment, the AKP is prepared by a process comprising converting alpha-ketoglutaric acid (AKG) to alpha-ketoadipate (AKA) and converting alpha-ketoadipate to alpha-keto-glycol acid. This reaction can be catalyzed by a biocatalyst. AKG can be prepared, for example, in a biocatalytic manner from a carbon source such as a carbohydrate, in a manner known in the art.

Suitable catalyst for the preparation of AKP from AKG in particular optionally catalyzed C-ketoglutarate ₁ of alpha] - Extension ketoadipate be alpha] and / or ketone adipic acid alpha] C _1-- be extended alpha] Biocatalyst for ketopimelic acid.

In a particular embodiment, the preparation of AKP is catalyzed by a biocatalyst comprising

a. AksA enzyme or a homolog thereof;

b. at least one enzyme selected from the group consisting of AksD enzyme, AksE enzyme, AksD enzyme homologue, and AksE enzyme homolog; and

c. AksF enzyme or a homolog thereof.

One or more of AksA, AksD, AksE, AksF enzymes or homologues thereof may be obtained from an organism selected from the group consisting of methane-producing archaea, preferably selected from the group consisting of Methanococcus, M. thermophilus , a group consisting of M. oxysporum, Methanothermobacter, Methanococcus, Methane archaea and Brevibacterium methane.

In a particular embodiment, the biocatalyst that catalyzes the preparation of AKP from alpha-ketoglutarate (AKG) comprises an enzyme system that catalyzes the conversion of alpha-ketoglutarate to alpha-ketoadipate, wherein the enzyme is formed. Part of the alpha-amine adipic acid pathway in the biosynthesis of amino acids. The term "enzyme" is used herein specifically to mean a single enzyme or group of enzymes that catalyze a particular transformation.

Preparation of AKP from AKG involves the conversion of one or more biocatalytic reactions, such as AKG to AKA or AKA, to AKP using known or unknown intermediates. These systems may be present inside the cell or may be isolated by the cell. The enzyme system is particularly selected from the group consisting of yeast, fungi, archaea and bacteria, in particular from the group consisting of Penicillium, Cephalosporium, and Bacillus Genus (Paelicomyces), Trichophytum, Trichophyton, Phanerochaete, Emericella, Ustilago, Schizosaccharomyces, Yeast Genus, Candida, Yarrowia, Pichia, Kluyveromyces, Thermus, Dinoococcus, Pyrococcus, Thiobacillus (Sulfolobus), Thermococcus, Methanococcus, M. thermophilus, Methanococcus, Methane archaea, Brevibacterium methane, M. ocella, and M. thermophilus.

In a particular embodiment, the biocatalyst for catalyzing the production of AKP from alpha-ketoglutaric acid comprises an enzyme system that catalyzes the conversion of alpha-ketoglutarate to alpha-ketoadipate, wherein at least one of the enzymes in the enzyme system One is derived from a nitrogen-fixing bacteria selected from the group consisting of cyanobacteria, Rhizobium, γ-proteobacteria and Actinobacillus, and is particularly selected from the group consisting of Anabaena and Microcystis. ), Synechocystis, Rhizobium, Bradyrhizobium, Pseudomonas, Azotobacter, Klebsiella, and Frankishia A group of Frankia).

Examples of homologues of such Aks enzymes and genes encoding such enzymes are listed in Tables 1A and 1B on the following two pages.

If desired, the 6-ACA obtained in accordance with the present invention can be cyclized to form caprolactam as described in US-A 6,194,572.

The reaction conditions for any of the biocatalytic steps in the context of the present invention can be selected based on the biocatalyst, particularly the known conditions of the enzyme, the information disclosed herein, and optionally 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 pH conditions. Basic conditions, neutral conditions, and acidic conditions can be used depending on the biocatalyst and other factors. Where the method comprises a microorganism, for example for expressing an enzyme that catalyzes the method of the invention, the pH system is selected to allow the microorganism to perform its desired function. In the case of a predominantly aqueous system at 25 ° C, the pH may in particular be selected from the range of four pH units below neutral pH to two pH units above neutral pH, ie selected from pH 3 to pH 9. If water is the sole solvent or primary solvent (>50 wt.%, especially >90 wt.%, based on total liquid), the system is considered aqueous, with a small amount of alcohol or other solvent (<50 wt.%, especially <10 wt) .%, based on the total liquid, may be dissolved (e.g., as a carbon source) at a concentration that maintains the presence of microbial activity. Particularly in the case of using yeast and/or fungi, it is generally an aqueous system based on 25 ° C, preferably acidic, particularly pH ranging from pH 3 to pH 8. If desired, the pH can be adjusted using an acid and/or base or buffered with an appropriate combination of acid and base.

In principle, the culture conditions can be selected in a wide range as long as the biocatalyst exhibits sufficient activity and/or growth. This includes aerobic, microaerobic, oxygen-limited, and anaerobic conditions.

Anaerobic conditions are defined herein as being free of any oxygen or substantially free of any oxygen being consumed by the biocatalyst, particularly microorganisms, typically equivalent to less than 5 millimoles per liter of oxygen consumed, especially The oxygen consumption is less than 2.5 millimoles per liter ‧ hours or less than 1 millimol / liter ‧ hours

Aerobic conditions are those in which the high oxygen concentration for unrestricted growth is dissolved in the medium to support at least 10 millimoles per liter ‧ hours, more preferably greater than 20 millimoles per liter ‧ hours, and even more preferably greater than 50 millimoles / liter ‧ hours, and optimal conditions for oxygen consumption rates greater than 100 millimoles per liter ‧ hours

Oxygen limiting conditions are defined as conditions under which oxygen consumption is limited by the conversion of oxygen from gas to liquid. The lower limit of the oxygen limiting condition is determined by the upper limit of the anaerobic conditions, in other words, usually at least 1 millimol/liter ‧ hours, and particularly at least 2.5 millimoles per liter ‧ hours or at least 5 millimoles per liter ‧ hours The upper limit of the oxygen limiting 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 Less than 10 millimoles / liter ‧ hours

The conditions are aerobic, anaerobic or oxygen-free, depending on the conditions under which the process is carried out, in particular the quantity and composition of the influent gas stream, the actual mixing/mass transfer properties of the equipment used, and the type of microorganisms and microbial density used. Decide.

In principle, the temperature to be used is not particularly limited as long as the biocatalyst exhibits substantial activity especially for the enzyme. The temperature is substantially at least 0 ° C, especially at least 15 ° C, more particularly at least 20 ° C. The maximum temperature desired is determined based on the biocatalyst. Typically such maximum temperatures are known to the art, for example, in the case of commercially available biocatalysts, as indicated in the product data sheet, or may be routinely determined 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.

Particularly if the biocatalytic reaction is carried out on the outside of the host organism, a high concentration (for example, more than 50 wt.% or more than 90 wt.%) of the reaction medium containing the organic solvent can be used when the enzyme having sufficient activity in the medium is used. .

In a preferred method, 6-ACA is prepared using whole cell biotransformation of a 6-ACA enzyme substrate or an intermediate for the formation of 6-ACA (AKP, AAP or 5-FVA), including a method in which a catalytic bioconversion is produced. Or a microorganism of a plurality of biocatalysts (usually one or more enzymes), such as one or more biocatalysts selected from the group consisting of: a biocatalyst that catalyzes the conversion of AKP to AAP, which catalyzes the conversion of AAP to 6-ACA The biocatalyst can catalyze the conversion of AKP into a 5-FVA biocatalyst and a biocatalyst which can catalyze the conversion of 5-FVA to 6-ACA. In a preferred embodiment, the microorganism can produce a decarboxylase and/or at least one enzyme selected from the group consisting of amino acid dehydrogenase and transaminase; catalyzing the aforementioned reaction steps, 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, carboxylic acids, carbon dioxide, fatty acids, glycerides, including mixtures comprising any of these compounds. Suitable monohydric alcohols include methanol and ethanol. Suitable polyols include glycerin and carbohydrates. Suitable fatty acids or glycerides are especially provided in the form of edible oils, preferably in plant form.

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

Cells comprising one or more biocatalysts (usually one or more enzymes) for use in the catalytic reaction step of the method of the invention, particularly recombinant cells, can be constructed using molecular biotechnology known in the art. For example, if one or more biocatalysts are produced in a recombinant cell (which may be a non-homogeneous system), such techniques can be used to provide a vector (such as a recombinant vector) comprising one or more genes encoding one or more biocatalysts. One or more vectors each comprising one or more genes can be used. Such vectors comprise one or more regulatory elements, such as one or more promoter genes, linked in a working manner to a gene encoding a biocatalyst.

As used herein, the term "working link" refers to a functional relationship of a polynucleotide element (or coding sequence or nucleic acid sequence). A nucleic acid sequence is a "workable link" when placed in a functional relationship with another nucleic acid sequence. For example, if the promoter gene or the booster gene affects the transcription of the coding sequence, it is functionally linked to the coding sequence.

As used herein, the term "initiating gene" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, upstream of the direction of transcription relative to the start of transcription of the gene, and structurally dependent on DNA. The location of the RNA polymerase, the location of the transcription initiation, and the presence of any other DNA sequence, including but not limited to transcription factor binding sites, repressor genes, and activation gene protein binding sites, and those skilled in the art are known to Directly or indirectly acting on any other nucleotide sequence that modulates the amount of transcription from the promoter gene. A "constitutive" promoter gene is a promoter that is active under most environmental conditions and developmental conditions. An "inducible" promoter is a promoter that is active under environmental regulation or developmental regulation. The term "homologous", when used to indicate the relationship of a given (recombinant) or multi-peptide molecule to a given host organism or host cell, is understood to be expressed in nature as the nucleic acid or multi-peptide peptide. Made from host cells or host organisms of the same species and preferably of the same variety or of the same germline.

A promoter gene useful for achieving the expression of a nucleic acid sequence encoding an enzyme for use in the methods of the invention, particularly a transaminase, amino acid dehydrogenase or decarboxylase, such as described above, which may be a nucleic acid encoding the enzyme to be expressed A non-homologous promoter gene that is either a sequence or a nucleic acid sequence (coding sequence) that is a working link. Preferably, the promoter gene is the homologity of the host cell, that is, the host cell endogenous.

If a non-homologous promoter gene is used (non-homologous to the nucleic acid sequence encoding the enzyme of interest), the non-homologous promoter gene preferably compares the original promoter gene of the coding sequence to produce a higher concentration The transcript of the coding sequence (or more transcript molecules per unit time, ie, mRNA molecules). Such promoter genes in this context include constitutive native promoter genes 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 mRNA to start at a higher frequency than the native 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 the inducible promoter gene in Gram-positive microorganisms include the IPTG-inducible Pspac promoter gene, the xylose-inducible Pxy1A promoter gene.

Examples of constitutive promoter genes and inducible promoter genes in Gram-negative microorganisms include, but are not limited to, 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 .

The promoter gene of (filamentous) fungal cells is known in the art and may be, for example, a glucose-6-phosphate dehydrogenase gpdA promoter gene, a protease promoter gene such as pepA, pepB, pepC, a glucoamylase glaA promoter gene, amylase. amyA, amyB promoter gene, catalytic enzyme catR or catA promoter gene, glucose oxidase goxC promoter gene, β-galactosidase lacA promoter gene, α-glucosidase aglA promoter gene, translation elongation factor tefA promoter gene, xylan Enzyme-initiating genes such as xlnA, xlnB, xlnC, xlnD, cellulase promoter genes such as eglA, eglB, cbhA, transcriptional regulatory gene promoter genes such as areA, creA, xlnR, pacC, prtT, or other promoter genes can be found on the NCBI website. ( http://www.ncbi.nlm.nih.gov/entrez/ ).

When used in reference to a nucleic acid (DNA or RNA) or protein, the term "homologous" refers to a nucleic acid or protein that does not naturally occur as part of an organism, cell, genome, or a DNA or RNA sequence thereof; or A cell or protein that appears in a different cellular or genetic location or DNA or RNA sequence than occurs in nature. A non-homologous nucleic acid or protein is not endogenous to the cell into which the nucleic acid or protein is introduced, but is derived from other cells or produced synthetically or recombinantly. Roughly, but not necessarily, such nucleic acids encode proteins that are not naturally produced by cells transcribed or expressed by the DNA. Similarly, exogenous RNA encodes a protein in which the exogenous RNA is not naturally expressed by the cell. Non-homologous nucleic acids and proteins are also referred to as foreign nucleic acids or proteins. Those skilled in the art will recognize that any nucleic acid or protein that is recognized as non-homologous or foreign to a performance cell is encompassed herein by a term that is not a homologous nucleic acid or protein.

The method according to the invention can be carried out in a host organism which can be novel.

Thus, the present invention is also directed to a host cell comprising one or more biocatalysts which catalyze at least one of the reaction steps of the method of the invention, particularly for the conversion of AKP, AAP or 5-FVA to 6-ACA. At least one reaction step. The invention also relates to novel vectors comprising one or more genes encoding one or more enzymes which catalyze at least one of the reaction steps of the method of the invention, in particular to catalyze at least one of conversion of AKP to 6-ACA Reaction step; and the present invention relates to a novel host cell comprising one or more genes encoding one or more enzymes which catalyze at least one of the reaction steps of the method of the invention, particularly for the conversion of AKP to 6-ACA At least one reaction step (the one or more genes may form part of one or more vectors).

In a specific embodiment, the host cell according to the present invention is a recombinant cell comprising a nucleic acid sequence encoding a biocatalyst which catalyzes a transamination reaction or a reductive amination reaction to form α-by-ketopimelate. Amine pimelic acid. Such a sequence may be part of a vector or may be inserted into chromosomal DNA.

In particular, the host cell or vector according to the invention comprises at least one nucleic acid sequence selected from the group consisting of, in particular at least two nucleic acid sequences encoding an enzyme having alpha-ketopimelate decarboxylase activity. a nucleic acid sequence, a nucleic acid sequence encoding an enzyme having 5-methylvalerate transaminase activity, a nucleic acid sequence encoding an enzyme having alpha-ketopimelate transaminase activity, encoding an alpha-ketopimelate dehydrogenase A nucleic acid sequence of an active enzyme, and a nucleic acid sequence encoding an enzyme having alpha-amine pimelic acid decarboxylase activity. Of such sequences, typically one or more, in particular, two or more belong to a recombination sequence.

In a preferred embodiment, the host cell according to the invention is typically a recombinant host cell or vector comprising a nucleic acid sequence encoding at least one biocatalyst having alpha-ketopimelate decarboxylase activity, and/or selected from the At least one nucleic acid sequence of a biocatalyst sequence of 5-methylvalerate transaminase activity.

In such an embodiment, the nucleic acid sequence encoding an enzyme having alpha-ketopimelate decarboxylase activity comprises, inter alia, sequence ID 31, sequence ID 34, sequence ID 37, sequence ID 40, sequence ID 43 or sequence ID 46. Or an amino acid sequence of a homologue of any such sequence; and/or a nucleic acid sequence encoding an enzyme having 5-methylvalerate transaminase activity, particularly comprising according to sequence ID 2, sequence ID 5, sequence ID 8, sequence ID 65, Sequence ID 67, Sequence ID 69 or the amino acid sequence of its homologue. One or more of the nucleic acid sequences may form part of one or more recombinant vectors.

In still further embodiments, the vector or host cell comprises a nucleic acid sequence encoding alpha-ketopimelate transaminase activity and/or a nucleic acid sequence encoding alpha-amine pimelic acid decarboxylase activity. The nucleic acid sequence encoding alpha-ketopimelate transaminase activity specifically comprises according to sequence ID 2, sequence ID 8, sequence ID 12, sequence ID 15, sequence ID 17, sequence ID 19, sequence ID 21, sequence ID 23, sequence ID 25, sequence ID 27, sequence ID 29 or the amino acid sequence of its homologue. One or more of the nucleic acid sequences can form part of one or more recombinant vectors.

In a particularly preferred embodiment, the host cell according to the invention comprises an amino acid sequence encoding alpha-amine pimelic acid 2-dehydrogenase activity and an amine group encoding alpha-amine pimelic acid decarboxylase activity. Acid sequence.

In a particularly preferred embodiment, the host cell according to the present invention comprises a nucleic acid sequence encoding an enzyme having 6-amine adipic acid 6-dehydrogenase activity and a nucleic acid encoding an enzyme having alpha-ketopimelate decarboxylase activity. sequence.

One or more suitable genes of the host cell or vector according to the invention are in particular selected from the genes encoding the enzymes described above.

In a particular embodiment, the host cell is a recombinant cell comprising at least one nucleic acid sequence selected from the group consisting of: sequence ID 1, sequence ID 3, sequence ID 4, sequence ID 6, sequence ID 7 , sequence ID 11, sequence ID 13, sequence ID 14, sequence ID 16, sequence ID 18, sequence ID 20, sequence ID 22, sequence ID 24, sequence ID 26, sequence ID 28, sequence ID 30, sequence ID 32, sequence ID 33, sequence ID 35, sequence ID 36, sequence ID 38, sequence ID 39, sequence ID 41, sequence ID 42, sequence ID 44, sequence ID 45, sequence ID 47, sequence ID 64, sequence ID 66, sequence ID 68 A sequence recognized by any of its functional analogs.

A nucleic acid sequence encoding an enzyme having 5-FVA transaminase activity is particularly selected from any one of the functional analogs of sequence ID 1, 3, 4, 6, 7, 64, 66, 68 and any such sequences. A sequence of one of the groups consisting of the sequences represented by the person.

As used herein, the term "functional analog" includes at least the other sequences encoding the enzymes having the same amino acid sequence and other sequences encoding the homologs of such enzymes.

The nucleic acid sequence encoding an enzyme having AKP decarboxylase activity is particularly selected from the group consisting of sequence IDs 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47 and any such sequences. A sequence of one of the groups consisting of the sequences represented by any of the similarities.

In a preferred embodiment, the host cell comprises a nucleic acid sequence encoding an enzyme that catalyzes the conversion of AAP to AKP, based on sequence IDs 1, 3, 7, 11, 13, 14, 16, 18, 20 , 22, 24, 26, 28, or a functional analog thereof and may be a wild-type sequence or a non-wild-type sequence.

In a particular embodiment, the host cell comprises at least one nucleic acid sequence encoding a biocatalyst having alpha-amine pimelic acid decarboxylase activity, which sequence may be homologous or non-homologous to the host cell. In particular such biocatalysts may be selected from the group consisting of decarboxylase (EC 4.1.1), more particularly from the group consisting of glutamate decarboxylase (EC 4.1.1.15). , diamine pimelic acid decarboxylase (EC 4.1.1.20), aspartic acid 1-decarboxylase (EC 4.1.1.11), branched chain α-keto acid decarboxylase, α-ketoisovalerate decarboxylation Enzyme, α-ketoglutarate decarboxylase, pyruvate decarboxylase (EC 4.1.1.1) and oxaloacetate decarboxylase (EC 4.1.1.3).

In a particular embodiment, the host cell comprises one or more enzymes that catalyze the formation of AKP by AKG (also referred to above). An enzyme system that forms part of the alpha-amine adipic acid pathway for the biosynthesis of the amino acid can be used. The term "enzymatic system" as used herein, in particular, refers to a single enzyme or a group of enzymes that catalyze a particular transformation reaction. Such transformation involves one or more chemical reactions with known intermediates or unknown intermediates, such as conversion of AKG to AKA or AKA to AKP. Such a system can exist within or be separated from cells. Transaminase is known to have a broad range of enzyme matrices. If it is desired to reduce the activity of one or more of these enzymes in the host cell in the presence of an enzyme matrix, the conversion activity of AKA to alpha-amine adipic acid (AAA) is reduced while maintaining other amino acids or cellular components. Catalytic functions related to biosynthesis. Furthermore, host cells which do not contain any other enzymatic activity will result in the conversion of AKA to an undesirable by-product.

Preferred host cells suitable for use in the preparation of AAP by whole cell biotransformation encode one or more biocatalysts which catalyze at least one of the alpha-ketopimelic acid preparation steps from alpha-ketoglutarate. Suitable biocatalysts are for example those discussed in the preparation of AKP.

The host cell can be selected from bacteria, yeast or fungi. In particular, the host cell may be selected from the group consisting of a genus of the genus Trichophyton, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus, and rods. A group consisting of Bacillus, Pseudomonas, Gluconobacter, Methanococcus, Mycobacterium, M. thermophilus, and the genus Escherichia and Escherichia. Here, it has generally been selected and expressed one or more of the encoding nucleic acid sequences as previously described.

In particular, host cells and host cells so suitable for biosynthesis of 6-ACA can be selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, bran Corynebacterium glutamicum, Aspergillus niger, Penicillium chrysogenum, Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, lactic acid Kluyveromyces lactis, Pichia stipitis, Pichia pastoris, Methanobacterium thermoautothrophicum ΔH, Methanococcus maripaludis, Volt Host cells such as Methanococcus voltae, Methanosarcina acetivorans, Methanosarcina barker, and Methanosarcina mazei. In a preferred embodiment, the host cell can produce an isoleic acid (as a precursor).

The host cell is in principle a naturally occurring organism or may be a genetically engineered organism. Such organisms can be genetically engineered using mutation screening strategies or metabolic genetic modification strategies known in the art. In a particular embodiment, the host cell naturally comprises (or can be made) one or more enzymes suitable for catalyzing the reaction step in the method of the invention, such as a decarboxylase selected from the group of reactions which catalyze the process of the invention One or more activities in a group consisting of a transaminase and an amino acid dehydrogenase. For example, E. coli naturally produces an enzyme that catalyzes the transamination reaction in the methods of the invention. It is also possible to provide a recombinant host cell carrying two recombinant genes, a recombinant gene encoding a transaminase or an amino acid dehydrogenase which catalyzes a reaction step of the method of the invention, and a recombinant gene encoding which catalyzes the method of the invention The decarboxylase gene of the reaction step.

For example, the host cell may be selected from the group consisting of Corynebacterium, particularly Corynebacterium glutamicum, enterobacteria, in particular Escherichia coli, Bacillus, especially Bacillus subtilis and B. methanolicus, and Saccharomyces cerevisiae, especially Saccharomyces cerevisiae . Particularly suitable are corynebacterium glutamicum or Bacillus licheniformis which have been developed for the industrial manufacture of lysine.

The present invention further relates to a microorganism which may be a wild type microorganism or a recombinant microorganism isolated from the natural environment, comprising, for example, selected from Sequence ID No. 3, Sequence ID No. 6, Sequence ID No. 13, Sequence. The DNA of the nucleic acid sequence recognized by any one of the sequence IDs of ID No. 32, Sequence ID No. 35, Sequence ID No. 41, Sequence ID No. 44, Sequence ID No. 47, and a functional analog thereof.

A functional analog of a nucleotide sequence as described herein is particularly such a nucleotide sequence that encodes the same amino acid sequence as the nucleotide sequence or a homologue encoding the nucleotide sequence. In particular, a preferred functional analog is a nucleotide sequence having a similar, or better, degree of expression than a nucleotide sequence referred to as a functional analog thereof in a host cell of interest.

The present invention further relates to a polynucleotide or vector comprising, selected from the group consisting of Sequence ID No. 3, Sequence ID No. 6, Sequence ID No. 13, Sequence ID No. 32, Sequence ID No. 35, Sequence ID No. 41. A nucleic acid sequence recognized by any one of sequence IDs of sequence ID No. 44, sequence ID No. 47, and a non-wild type functional analog thereof. Such a polynucleotide or vector provides a host cell, more particularly an E. coli host cell, or other at least one transformation step that can catalyze the conversion of AKP to 6-ACA in high yield, in comparison to the corresponding wild-type gene. Other host cells.

Optionally, the polynucleotide or vector comprises one or more nucleic acid sequences encoding one or more other biocatalysts suitable for catalyzing the reaction step in the process according to the invention, in particular one of the catalysts described above Or more.

The invention further relates to a process for the preparation of alpha-amine pimelic acid (AAP) comprising converting AKP to AAP which is catalyzed by a biocatalyst.

For use in such methods, in particular, a biocatalyst having transaminase activity or reductive amination activity as described above can be used.

As previously indicated, AAP can then be used for the preparation of 6-ACA. In addition, AAP can be used as such, for example as a chemical for biochemical research or as a pH buffering compound, for example for preparative separation techniques or analytical separation techniques such as liquid chromatography or capillary electrophoresis.

Further, the AAP prepared by the method of the present invention can be further used for the preparation of other compounds, for example, AAP can be converted to caprolactam. As explained above, based on the exemplification in the following examples, AAP can be converted to caprolactam by chemical means such as exposure to elevated temperatures. Without wishing to be bound by theory, it is expected that in this reaction, 6-ACA may form a short-lived intermediate.

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

Instance Rough method Molecular and genetic techniques

Standard genetic and molecular biology techniques are generally known to the art world and are described above (Maniatis et al., 1982, "Molecular Selection: Laboratory Handbook", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Miller, 1972" Molecular Genetics Experiment", Cold Spring Harbor Laboratory, Cold Spring Harbor; Sambrook and Russell 2001 "Molecular Selection: Laboratory Manual" (3rd Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; F. Ausubel et al. , "Popular Programs in Molecular Biology", Green Press and Willy Technology, New York, 1987).

Plastid and germline

The pBAD/Myc-His C line was obtained from Invitrogen (Casper, CA). The plastid pBAD/Myc-His-DEST consisting of the composition described in WO2005/068643 is used for protein expression. E. coli TOP10 (Invitrogen, Inc., Casper, CA) was used for all selection procedures and for the performance of target genes.

Medium

LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) was used for the growth of E. coli. Antibiotics (50 μg/ml carbenicillin) were added to maintain the plastids. To induce gene expression in the plastids derived from pBAD/Myc-His-DEST under the control of the P _BAD promoter, L-arabinose was added to a final concentration of 0.2% (w/v).

Plasm recognition

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

HPLC-MS analytical method for the determination of 5-FVA

5-FVA was detected via selective reaction monitoring (SRM)-MS, measuring transitions 129→83. The 5-FVA concentration was calculated by measuring the peak area of the 5-FVA peak eluted by about 6 minutes. Calibrate using an external standard program. All LC-MS experiments were performed on an Agilent 1200 LC system containing a fourth stage pump, autosampler and column oven coupling Aegean 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 (Alltech).

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, the flow splits 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

HPLC-MS analysis for the determination of AAP

AAP was detected by measuring the protonated molecule of AAP with m/z 176 by selected ion monitoring (SIM)-MS. The AAP concentration was calculated by measuring the peak area of the AAP peak eluted in the sample at a residence time of 2.7 minutes. Calibrate via an external standard procedure. All LC-MS experiments are performed on the Aijilan 1100 LC system coupled with a fourth stage pump, deaerator, autosampler and column oven. API 2000 Triple Quaternary MS (Applied Biosystems) Biosystems)).

The LC conditions are as follows:

Column: 50*4 Newquay Sil C18, 5 micron (McKelly-Nagy) + 250x4.6 Pver C18, 5 micron (Aer Technology), all at room temperature (RT).

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

B = 0.1% (v / v) formic acid in acetonitrile (pa, Merck)

Flow rate: 1.2 ml/min, the flow splits 1:3 before entering MS.

Gradient: The gradient was started at 90% (v/v) A at t = 0 minutes and changed to 50% (v/v) A within 6 minutes. The gradient was changed to the original condition at 6.1 minutes.

Injection volume: 2 microliters

MS conditions: positive ion electrospray for ionization.

Detection: SIM mode is at m/z 176 and residence time is 100 milliseconds.

HPLC-MS analysis for the determination of 6-ACA

calibration:

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 the Aijilan 1100, which was equipped with a fourth stage pump, deaerator, autosampler, column oven, and single quaternary MS (Aijilan, Germany) Bonn). The LC-MS conditions are:

Column: 50*4 Newquay (McKentley-Nage) +250 x 4.6 Pavel C18 (Aer Technology), 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, the flow was split 1:3 before entering MS.

Gradient: The gradient started 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 to 31 minutes.

Injection volume: 5 microliters

MS detection: ESI(+)-MS

Electrospray ionization (ESI) was performed in a positive scan mode under the following conditions: m/z 50-500, 50V segmenter, 0.1 m/z step size, 350 °C dry gas temperature, 10 liters nitrogen/min dry gas, 50 psig nebulizer pressure and 2.5 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 the Gateway technology (Invitrogen, Inc., Casper, CA).

Gene synthesis and composition of plastids

Synthetic genes are derived from DNA2.0 and codons that are optimized for expression in E. coli according to standard procedures for DNA 2.0. Vibrio fluvialis JS17 The amino acid sequence of ω-transaminase [SEQ ID No. 2] and the amino acid sequence of B. faecalis KBAB4 transaminase (ZP_01186960) [SEQ ID No. 5] was obtained from Vibrio fluvialis JS17 [SEQ. ID No.1] and Weiss bud The transaminase gene of Bacillus sp. KBAB4 [SEQ ID No. 4] was codon-optimized, and the resulting sequences [SEQ ID No. 3] and [SEQ ID No. 6] were obtained by DNA synthesis.

E. coli diamine pimelate decarboxylase LysA [SEQ ID No. 31], Saccharomyces cerevisiae pyruvate decarboxylase Pdc [SEQ ID No. 34], Z. mobilis Pyruvate decarboxylase Pdc1472A [SEQ ID No. 37], Lactobacillus lactis branched-chain alpha-keto acid decarboxylase KdcA [SEQ ID No. 40] and α-ketoisovalerate decarboxylase KivD [SEQ ID No.] 43], and Mycobacterium tuberculosis α - Amino acid sequence of ketoglutarate decarboxylase Kgd [SEQ ID No. 46] from Escherichia coli [SEQ ID No. 30], Saccharomyces cerevisiae [SEQ ID No. 33], Zymomonas activeis [ SEQ ID No. 36], Lactobacillus lactis The decarboxylase genes of [SEQ ID No. 39], [SEQ ID No. 42], and Mycobacterium tuberculosis [SEQ ID No. 45] were also codon-optimized, and the resulting sequence [SEQ ID No. 32] , [SEQ ID No. 35], [SEQ ID No. 38], [SEQ ID No. 41], [SEQ ID No. 44], and [SEQ ID No. 47] were obtained by DNA synthesis, respectively.

The gene composition was introduced into pBAD/ using the introduced attB position and pDONR201 (Invitrogen) as an entry vector, using Jiawei technology ( Invitrogen ) as described in the manufacturer's protocol ( www.invitrogen.com ). Myc-His-DEST expression vector. Thereby, the expression vectors pBAD-Vfl_AT and pBAD-Bwe_AT are obtained respectively. Corresponding performance lines were obtained by using individual pBAD expression vector transformation chemistry to competent E. coli TOP10 (Invitrogen).

Colonization by PCR

Multiple genes encoding biocatalysts were amplified by genomic DNA by PCR using primers listed in the table below using PCR Supermix High Fidelity (Invitrogen) according to the manufacturer's specifications. .

The PCR reaction was analyzed by agarose gel electrophoresis, and the 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- using the introduced attB position and pDONR-zeo as the entry vector using Jiawei Technology (Invitrogen). His-DEST expression vector. The gene sequence cloned by PCR was confirmed by DNA sequencing. In this way, the performance vectors pBAD-Pae_gi9946143_AT, pBAD-Bsu_gi16078032_AT, pBAD-Bsu_gi16080075_AT, pBAD-Bsu_gi16077991_AT, pBAD-Rsp_AT, pBAD-Lpn_AT, pBAD-Neu_AT, pBAD-Ngo_AT, pBAD-Pae_gi9951299_AT, pBAD-Pae_gi9951072_AT, pBAD-Pae_gi9951630_AT and pBAD-Rpa_AT. Corresponding performance lines were obtained by using the pBAD constitutively transformed chemically competent E. coli TOP10 (Invitrogen).

Growth of E. coli for protein expression

Small scale growth was performed on 96 deep well plates using 940 microliters of medium containing 0.02% (w/v) L-arabinose. Use a 96-well puncher from the cells that are frozen from the spare culture (Kuna ( ), Bofeden, Switzerland) Transfer cells for inoculation. The plates were incubated at 25 ° C for 48 hours on an orbital shaker (300 rpm, 5 cm amplitude). Typical OD _620nm is 2-4.

Preparation of cell lysate Preparation of dissolution buffer

The lysis buffer contains the following ingredients:

The solution was prepared just prior to use.

Preparation of cell-free extract by dissolving

The cells obtained from small-scale growth (refer to the previous paragraph) were obtained by centrifugation, 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. Five microliters of freshly prepared lysis buffer was added to each well and the cells were resuspended by vigorous vortexing for 2-5 minutes. To achieve cell lysis, 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.

Preparation of cell-free extracts by sonication

The cells obtained from the growth of the medium (refer to the preceding paragraph) were harvested by centrifugation and discarded. 1 ml of potassium phosphate buffer pH 7 was added to 0.5 g of wet cell pellets, and the cells were again suspended by vigorous vortexing. To achieve cell lysis, the cells were sonicated for 20 minutes. To remove cell debris, the lysate was centrifuged at 4 ° C and 6000 g for 20 minutes. The supernatant was transferred to a new tube and chilled at -20 °C until further use.

Preparation of 5-methylvaleric acid by chemical hydrolysis of methyl 5-methylvalerate

The enzyme substrate for the transaminase reaction, ie 5-methylvaleric acid, is prepared by chemical hydrolysis of methyl 5-methylvalerate. The following is 10% (w/v) methyl 5-methylvalerate used in aqueous solution. Sodium oxide was fixed at pH 14.1. After incubation at 20 ° C for 24 hours, the pH was fixed to 7.1 using hydrochloric acid.

Enzymatic reaction of 5-methylvaleric acid to 6-ACA

Unless otherwise specified, the preparation reaction mixture contained 10 mM 5-mevalonate, 20 mM racemic α-methylbenzylamine, and 200 μM pyridoxal 5'-phosphate in 50 mM potassium phosphate buffer, pH 7.0. 100 μl The reaction mixture was dispensed to each well of the well plate. To initiate the reaction, 20 microliters of cell free extract was added to each well. The reaction mixture was incubated 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.

6-ACA was formed from 5-FVA in the presence of a transaminase.

Enzymatic reaction of AKP to 5-methylvaleric acid

The reaction mixture was prepared to contain 50 mM AKP, 5 mM magnesium chloride, 100 μM pyridoxal 5'-phosphate (for LysA) and 1 mM thiamine diphosphate (for all other enzymes) in 100 mM potassium phosphate buffer, pH 6.5. 4 ml reaction mixture Distribute into the reaction vessel. To initiate the reaction, 1 ml of the cell-free extract obtained by the sonication treatment was added to each well. 50 units were used in the case of commercial grass 醯 acetic acid decarboxylase (Sigma-Aldrich product number 04878). The reaction mixture was incubated at 37 ° C for 48 hours using a magnetic stirrer. 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. Samples from different time points during the reaction were analyzed by HPLC-MS. The results are summarized in the table below.

5-FVA is formed from AKP in the presence of decarboxylase.

Enzymatic reaction of converting AKP into 6-ACA in the presence of recombinant decarboxylase

The preparation reaction mixture contained 50 mM AKP, 5 mM magnesium chloride, 100 μM pyridoxal 5'-phosphate (for LysA) and 1 mM thiamine diphosphate (biocatalyst for all other tests) in 100 mM potassium phosphate buffer, pH 6.5. The milliliter of the reaction mixture is dispensed into the reaction vessel. To initiate the reaction, 1 ml of the cell-free extract obtained by the sonication treatment was added to each well. The reaction mixture was incubated at 37 ° C for 48 hours using a magnetic stirrer. 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. Samples from different time points during the reaction were analyzed by HPLC-MS. The results are summarized in the table below.

It is shown that 6-ACA is formed from AKP in the presence of decarboxylase. E. coli is expected to contain native 5-FVA transaminase activity.

Enzymatic reaction of AKP to 6-ACA in the presence of recombinant decarboxylase and recombinant transaminase

The reaction mixture was prepared to contain 50 mM AKP, 5 mM magnesium chloride, 100 μM pyridoxal 5'-phosphate, 1 mM thiamine diphosphate, and 50 mM racemic α-methylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml reaction mixture Distribute into the reaction vessel. To initiate the reaction, 0.2 ml of decarboxylase containing cell-free extract and 0.2 ml of transaminase containing cell-free extract were added to each reaction vessel. The reaction mixture was incubated at 37 ° C for 48 hours with a magnetic stirrer. 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. Samples from different time points during the reaction were analyzed by HPLC-MS. The results are summarized in the table below.

No 6-ACA was detected in the chemical blank group and the biological blank group. Furthermore, the results show that the conversion of 6-ACA is improved by comparing the example in which the host cell contains only the recombinant decarboxylase (but not the recombinant transaminase).

a plastid for the expression of transaminase and decarboxylase in Saccharomyces cerevisiae

Phusion DNA polymerase (Finnzymes) was used according to manufacturer's specifications and using specific primers [SEQ ID No. 76 and 77], amplified by pBAD-Vfl_AT [SEQ ID No. 3] by PCR. The transaminase gene encoding the amino acid sequence [SEQ ID No. 2] of Vibrio fluvialis JS17 ω-transaminase from Vibrio fluvialis JS17.

Pseudomonas aeruginosa transaminase derived from Pseudomonas aeruginosa amplified from pBAD-Pae_AT by PCR using Füssen DNA polymerase (Finzan) according to manufacturer's specifications and using specific primers [SEQ ID No. 78 and 79] The transaminase gene of [SEQ ID No. 8] [SEQ ID No. 7].

The resulting PCR product was transfected into the vector pAKP-41 using SpeI and BamHI restriction enzymes to obtain pAKP-79 and pAKP-80, respectively, and today contained the transaminase gene under the Saccharomyces cerevisiae gal10 promoter gene and the Saccharomyces cerevisiae adh2 terminator gene.

Saccharomyces cerevisiae pyruvate decarboxylase from Saccharomyces cerevisiae was amplified by PCR from pBAD-Pdc using Fussen DNA polymerase (Finzan) according to the manufacturer's specifications and using specific primers [SEQ ID No. 80 and 81] Pdc [SEQ ID No. 34] decarboxylase gene [SEQ ID No. 33].

The Lactobacillus lactis branched-chain α derived from Lactobacillus lactis was amplified by PCR from FBAD-KdcA according to the manufacturer's specifications and using a specific primer [SEQ ID No. 82 and 83] using Fussen DNA polymerase (Finzan). a decarboxylase gene of keto acid decarboxylase KdcA [SEQ ID No. 40] [SEQ ID No. 39].

The resulting PCR product was transfected into vector pAKP-44 using AscI and BamHI restriction enzymes to obtain pAKP-81 and pAKP-82, respectively, and today contained the transaminase gene under the Saccharomyces cerevisiae gal2 promoter and the S. cerevisiae pma1 terminator gene.

The plastids pAKP-79 and pAKP-80 were digested with SacI and XbaI by restriction enzymes; the plastids pAKP-81 and pAKP-82 were digested with SalI and XbaI by restriction enzymes. The SacI/XbaI transaminase fragment and the fragment were combined with the SalI/XbaI decarboxylase fragment to form the S. cerevisiae low-replication free gene vector pRS414, which was digested with SalI and SacI.

The resulting plastid is:

pAKP-85: Pgal40-Pae_AT-Tadh2 Pgal2-Pdc_DC-Tpma1

pAKP-86: Pgal10-Pag_AT-Tadh2 Pgal2-KdcA_DG-Tpma1

pAKP-87: Pgal10-Vfl_AT-Tadh2 Pgal2-Pdc_DC-Tpma1

pAKP-88: Pgal10-Vfl_AT-Tadh2 Pgal2-KdcA_DC-Tpma1

Transformation and growth of Saccharomyces cerevisiae

The Saccharomyces cerevisiae line CEN.PK113-3C is according to the method described by Gietz and Woods (Gietz, R.D. and Woods, R.A. (2002)). Yeast transformation is performed by Liac/SS vector DNA/PEG method. Enzymology Method 350: 87-96), transformed with 1 microgram of plastid DNA. The cells were seeded on agar plates containing 1 x yeast nitrogen bases without amino acids and 2% glucose.

The resulting strain was aerobically grown for 48 hours at 30 ° C in Verduyn minimal medium containing 0.05% glucose and 4% galactose.

Preparation of cell-free extract

1 ml of potassium phosphate buffer (pH 7) was added to 0.5 g of cell pellets. This mixture was added to a 2 ml eppendorf test tube having a diameter of 0.4-0.5 mm containing 0.5 g of glass beads. The sample was shaken vigorously for 20 seconds with the Ibn VIBRAX-VXR. The resulting cell-free extract was centrifuged at 14,000 rpm and 4 ° C for 5 minutes. The supernatant was used for enzyme activity assay analysis.

Enzymatic reaction of converting AKP into 6-ACA in the presence of decarboxylase and transaminase in Saccharomyces cerevisiae

The reaction mixture was prepared and contained 50 mM AKP, 5 mM magnesium chloride, 100 μM pyridoxal 5'-phosphate, 1 mM thiamine diphosphate and 50 mM racemic α-methylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml reaction The mixture is dispensed into the reaction vessel. To initiate the reaction, 0.4 ml of cell-free extract from Saccharomyces cerevisiae containing decarboxylase and transaminase was added to each reaction vessel. The reaction mixture was incubated at 37 ° C with a magnetic stirrer. In addition, the chemical blank group mixture (without cell-free extract) and the biological blank group (Saccharomyces cerevisiae) were cultured under the same conditions. The resulting sample after 19 hours of incubation was analyzed by HPLC-MS. The results are summarized in the table below.

Enzymatic reaction of α-keto pimelic acid to α-amine pimelic acid

The reaction mixture was prepared to contain 10 mM α-ketopimelic acid, 20 mM L-alanine, and 50 μM pyridoxal 5'-phosphoric acid in 50 mM potassium phosphate buffer, pH 7.0. 800 μl of the reaction mixture was dispensed into the wells of the well plate. . To initiate the reaction, 200 microliters of cell lysate was added to each well. The reaction mixture was incubated at 37 ° C for 24 hours on a shaker. 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 AAP is formed by AKP and catalyzed by a biocatalyst.

Chemical conversion of AAP to caprolactam

To 1.5 ml of a suspension of D,L-2-amine pimelic acid in 21 ml of cyclohexanone was added 0.5 ml of cyclohexenone. The mixture was heated for about 20 hours (about 160 ° C) from about reflux. After cooling to room temperature, the reaction mixture was decanted and evaporated under reduced pressure to remove a clear solution. The remaining 2 g of brown oil was analyzed by ¹ H-NMR and HPLC to contain a cyclic oligomer of 0.8 wt% of caprolactam and 6 wt% of caprolactam.

Claims (19)

A method for preparing 6-amine hexanoic acid, wherein the 6-amine hexanoic acid is used by at least one biocatalyst and α-keto Diacid preparation, wherein the biocatalyst comprises a transaminase comprising according to sequence ID 2, sequence ID 5, sequence ID 8, sequence ID 12, sequence ID 15, sequence ID 17, sequence ID 19, sequence ID twenty one , Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ID 29, Sequence ID 65, Sequence ID 67, Sequence ID 69 or the amino acid sequence of a homologue of any such sequence. A process for the preparation of 6-amine hexanoic acid, wherein the 6-amine hexanoic acid is prepared from α-keto pimelic acid, and wherein the α-ketopimelic acid First converted to 5-methylvalerate, and the 6-aminocaproic acid is then prepared from 5-methylvalerate using at least one biocatalyst comprising a transaminase, the transaminase Contains according to sequence ID 2 , sequence ID 5, sequence ID 8, sequence ID 12, sequence ID 15, sequence ID 17, sequence ID 19, sequence ID 21, sequence ID 23, sequence ID 25, sequence ID 27, sequence ID 29, sequence ID 65, sequence ID 67. Sequence ID 69 or an amino acid sequence of a homologue of any such sequence. The method of claim 1 or 2, wherein the biocatalyst comprises an enzyme that catalyzes the decarboxylation of an alpha-keto acid or an amino acid. The method of claim 3, wherein the enzyme that catalyzes decarboxylation is a decarboxylase (E.C. 4.1.1). The method of claim 4, wherein the decarboxylase is selected from the group consisting of glutamic acid decarboxylase (EC 4.1.1.15) and diamine pimelic acid. Carboxase (EC 4.1.1.20), aspartic acid 1-decarboxylase (EC 4.1.1.11), branched chain α-keto acid Decarboxylase, α-ketoisovalerate decarboxylase, α-ketoglutarate decarboxylase, pyruvate decarboxylase (EC 4.1.1.1) and grasshopper acetic acid decarboxylase (EC 4.1.1.3) group. The method of claim 3, wherein the catalyzable decarboxylation enzyme is obtained from a family selected from the group consisting of Cucurbitaceae and leaven. Saccharomyces, Candida, Hansenula, Kluyveromyces, Rhizopus, Neurospora, Fermentation Zymomonas, Escherichia, Mycobacterium, Clostridium, Lactobacillus, Streptococcus An enzyme of an organism of the group of (Streptococcus), Pseudomonas, and Lactococcus or a part thereof. The method of claim 4, wherein the catalyzable decarboxylation enzyme is obtained from a family selected from the group consisting of Cucurbitaceae and leaven. Saccharomyces, Candida, Hansenula, Kluyveromyces, Rhizopus, Neurospora, Fermentation Zymomonas, Escherichia, Mycobacterium, Clostridium, Lactobacillus, Streptococcus An enzyme of an organism of the group of (Streptococcus), Pseudomonas, and Lactococcus or a part thereof. The method of claim 5, wherein the catalyzed decarboxylation The enzyme is obtained from Cucurbitaceae, Saccharomyces, Candida, Hansenula, Kluyveromyces, Rhizopus. Rhizopus, Neurospora, Zymomonas, Escherichia, Mycobacterium, Clostridium, Lapara An enzyme of an organism or a part thereof of a group of Lactobacillus, Streptococcus, Pseudomonas, and Lactococcus. The method of claim 3, wherein the catalyzable decarboxylation enzyme comprises according to sequence ID 31, sequence ID 34, sequence ID 37. Amino acid sequence of sequence ID 40, sequence ID 43 or sequence ID 46 or a homolog of any such sequence. The method of claim 1, wherein the α-ketopimelic acid is in the presence of a biocatalyst capable of catalyzing the decarboxylation of the α-keto acid Biocatalytic conversion to 5-methylvalerate, and 5-methylvalerate is converted to 6-amine hexanoic acid by biocatalysis in the presence of at least one amine donor and at least one transaminase. The method of claim 1, wherein the α-ketopimelic acid is in the presence of at least one amine donor and at least one transaminase Biocatalytic conversion to alpha-amine pimelic acid, and alpha-amine pimelic acid is converted to 6-amine caproic acid in a biocatalytical manner in the presence of a biocatalyst that catalyzes the decarboxylation of the amino acid. The method of claim 1, wherein the α-keto pimelic acid has From natural sources. A method for preparing caprolactam comprising cyclizing 6-amine hexanoic acid prepared by the method of claim 1 to form a Endoamine. A recombinant host cell comprising a nucleic acid sequence encoding an alpha-ketopimelate decarboxylase activity and/or encoding a 5-methylidene A nucleic acid sequence of one of the enzymes of acid transaminase activity, wherein the enzyme having 5-methylvalerate transaminase activity comprises according to sequence ID 2, sequence ID 5, sequence ID 8, sequence ID 65, sequence ID 67, sequence ID 69 or its etc. An amino acid sequence of one of the homologs; and the enzyme having alpha-ketopimelate decarboxylase activity comprises according to sequence ID 31, sequence ID 34, sequence ID 37, sequence ID 40, sequence ID 43 or sequence ID 46 or Any such order One of the homologues of the amino acid sequence. A recombinant host cell comprising a nucleic acid sequence encoding an enzyme having alpha-ketopimelate transaminase activity or alpha-ketopimerate dehydrogenase activity And a nucleic acid sequence encoding one of the enzymes having alpha-amine pimelic acid decarboxylase activity, wherein the transaminase comprises according to sequence ID 2, sequence ID 8, sequence ID 12, sequence ID 15, sequence ID 17, Sequence ID 19, sequence An amino acid sequence of one of the homologs of ID 21, sequence ID 23, sequence ID 25, sequence ID 27, sequence ID 29 or the like. The recombinant host cell of any one of claims 14 and 15 comprising one or more of one or more biocatalysts A plurality of nucleic acid sequences, and the biocatalysts catalyze at least one of the reaction steps of preparing alpha-ketopimelic acid from alpha-ketoglutaric acid. Recombination host fine as claimed in any of claims 14 and 15 a cell, wherein the host cell line is selected from the group consisting of Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Pichia, a group of Candida, Hansenula, Bacillus, Corynebacterium, and Escherichia. The recombinant host cell according to any one of claims 14 and 15, which comprises a substance selected from the group consisting of selected from the group consisting of DNA of one of the sequence groups represented by any sequence: sequence ID 1, sequence ID 3, sequence ID 4, sequence ID 6, sequence ID 7, sequence ID 11, sequence ID 13, sequence ID 14, sequence ID 16 ,sequence Column ID 18, sequence ID 20, sequence ID 22, sequence ID 24, sequence ID 26, sequence ID 28, sequence ID 30, sequence ID 32, sequence ID 33, sequence ID 35, sequence ID 36, sequence ID 38, sequence ID 39 , a sequence ID 41, a sequence ID 42, a sequence ID 44, a sequence ID 45, a sequence ID 47, a sequence ID 64, a sequence ID 66, a sequence ID 68, and the like. a polynucleotide comprising: selected from the group consisting of sequence ID 3, sequence ID 6, sequence ID 13, sequence ID 32, sequence ID 35, sequence A nucleic acid sequence in a sequence group of column ID 38, sequence ID 41, sequence ID 44, sequence ID 47, and functional analogs thereof.