Patent Grant
10087472
A computer readable text file, entitled “SequenceListing.txt,” created on or about Mar. 11, 2016 with a file size of about 40 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
The present invention relates to a recombinant microorganism for the biological synthesis of 6-aminocaproic acid in the microorganism so as to synthesize caprolactam.
Caprolactam, which is an organic compound, is a lactam of 6-aminohexanoic acid (ε-aminohexanoic acid, 6-aminocaproic acid). Unlike other compounds, caprolactam can be considered as a cyclic amide of caproic acid. One use of caprolactam is as a monomer in the production of nylon 6. Base materials that are most widely used in the production of caprolactam are aromatic compounds such as benzene, phenol, toluene, etc. Caprolactam is ultimately synthesized via the Beckmann rearrangement using sulfur catalysts by preparing oxime compounds via oximation, which drives a reaction of cyclohexanone with hydroxylamine obtained from the base materials of the aromatic compounds. When caprolactam is synthesized via such process, it is difficult to avoid formation of ammonium sulfate as a byproduct. In the production of caprolactam, the yield of caprolactam decreases as more ammonium sulfate is produced, and therefore, caprolactam can be obtained in high yield only if the formation of ammonium sulfate is suppressed.
Recent development trends for production techniques of caprolactam are divided into developing processes for reducing or eliminating the formation of ammonium sulfate or developing alternative base materials therefor. One example of the former development includes a caprolactam production facility recently developed by Sumitomo Chemical, Japan. This involves the Beckmann rearrangement in a gas phase using fluid bed gas-phase zeolite-catalysts and an ammoximation reaction using hydrogen peroxide catalysts (Enichem). Also, the base materials developed as alternatives for caprolactam include hexamethylene diamine (HMDA) and tetramethylene diamine (TMDA). HMDA can be produced from adiponitrile, propylene, and acrylonitrile. However, the process for HMDA production using adiponitrile can only be used by BASF, Solutia, Butachimie, and DuPont. Adiponitrile is produced by reacting butadiene with hydrogen cyanide. Butadiene may be used as a base material for adipic acid, which is a base material for nylon 4 and nylon 6. Most of the intermediates used in the production of nylon have their origins in butadiene, and such tendency is increasingly spreading.
As such, much more attention has been drawn to the production of these chemicals and materials from renewable non-food biomass via biorefinery as concerns about environmental problems and the availability of fossil resources increase. With the development of the biorefinery processes, microorganisms have been used as core biocatalysts capable of successfully producing chemicals, plastics, and fuels from renewable resources. However, non-manipulated natural microorganisms are not suitable for efficiently producing target products at the industrial level due to their reduced metabolism. Therefore, techniques that improve the metabolism of microorganisms have been actively studied to efficiently produce target products. Many studies are being conducted to solve the optimization of these microorganisms via systems metabolic engineering at the system level.
Accordingly, the present inventors have constructed a transformed microorganism capable of biosynthesizing 6-aminocaproic acid in the microorganism by expressing genes of enzymes used in the biosynthetic pathway of 6-aminocaproic acid, which is a precursor of caprolactam, to produce caprolactam.
An object of the present invention is to provide a method for producing 6-aminocaproic acid.
Another object of the present invention is to provide an expression vector for biosynthesis of 6-aminocaproic acid comprising HpaI (4-hydroxy-2-oxoheptane-1,7-dioate aldolase)-HpaH (2-oxohept-3-ene-1,7-dioate dehydratase) gene, nemA (N-ethylmaleimide reductase) gene, KIVD (alpha-ketoisovalerate decarboxylase) gene; and at least one of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase).
A further object of the present invention is to provide a transformant transformed with the expression vector above.
Yet another object of the present invention is to provide a method for producing caprolactam further comprising converting 6-aminocaproic acid produced by the method for producing 6-aminocaproic acid to caprolactam.
In order to accomplish the above objects, the present invention provides a method for producing 6-aminocaproic acid.
Also, the present invention provides an expression vector for the biosynthesis of 6-aminocaproic acid comprising HpaI (4-hydroxy-2-oxoheptane-1,7-dioate aldolase)-HpaH (2-oxohept-3-ene-1,7-dioate dehydratase) gene, nemA (N-ethylmaleimide reductase) gene, KIVD (alpha-ketoisovalerate decarboxylase) gene; and at least one of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase).
Further, the present invention provides a transformant transformed with the expression vector above.
Furthermore, the present invention provides a method for producing caprolactam further comprising converting 6-aminocaproic acid produced by the method for producing 6-aminocaproic acid to caprolactam.
A microorganism transformed into an expression vector comprising HpaI (4-hydroxy-2-oxoheptane-1,7-dioate aldolase)-HpaH (2-oxohept-3-ene-1,7-dioate dehydratase) gene, nemA (N-ethylmaleimide reductase) gene, and KIVD (alpha-ketoisovalerate decarboxylase) gene; and at least one of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) genes encoding enzymes used in the biosynthetic pathway of 6-aminocaproic acid can efficiently biosynthesize 6-aminocaproic acid, and thus, it can be used in the synthesis of caprolactam.
first chromatogram: total ion current plot (TIC);
second chromatogram: selected ion monitoring (SIM); and
third chromatogram: selected reaction monitoring (SRM).
L1: 1 M 6-aminocaproic acid;
L2: negative control group (chemical blank);
L3: negative control group (biological blank with KIVD-His);
L4: KIVD-PdAT; and
L5: KIVD-PdAT-BcAT.
first chromatogram: total ion current plot (TIC);
second chromatogram: selected ion monitoring (SIM); and
third chromatogram: selected reaction monitoring (SRM).
SM: 6-aminocaproic acid;
L1: negative control group (pACYC184); and
L2: response group pACYCWG.
first chromatogram: total ion current plot (TIC);
second chromatogram: selected ion monitoring (SIM); and
third chromatogram: selected reaction monitoring (SRM).
L1: pACYCWG total proteins;
L2: pACYCWG water soluble proteins;
L3: pACYCWG purified proteins, diluted by 1/5;
L4: pACYCWG purified proteins;
(1): aldolase-dehydratase (HpaI-HpaH)-58KD;
(2): decarboxylase (KIVD)-55KD;
(3): aminotransferase 1 (PdAT)-46KD;
(4): reductase (nemA)-40KD; and
(5): aminotransferase 1 (BcAT)-38KD.
(from top) first arrow: HpaI-H;
second arrow: Kivd;
third arrow: PdAT; and
fourth arrow: nemA.
Hereinbelow, the present invention is described in detail.
As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” have identical meaning, and represent nucleotide polymers having any length. Such terms also include “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” may include nucleotide derivatives or may represent oligonucleotides or polynucleotides having a general system or a different system between nucleotides, and they are used interchangeably.
As used herein, the term “polynucleotide” is used interchangeably with nucleic acid, oligonucleotide, and polynucleotide, and may include cDNA, mRNA, genome DNA, etc. The polynucleotide used herein is included by the term “genes”. A polynucleotide encoding gene sequences may include “splicing variants”. Similarly, specific proteins encoded by nucleic acid may include proteins encoded by splicing variants encoded thereby. As the name indicates, the term “splicing variants” represents a product of alternative splicing variants. The first nucleic acid transcriptome after transcription is spliced by encoding other polypeptides which are identical to a product of distinct nucleic acid variants. They may include exon-selective splicing even if a mechanism of splicing variant formation is different. Other polypeptides derived from identical nucleic acids by incorrect transcription may include such definitions. Products (including splicing products in the form of a recombinant) of a splicing reaction may be included in the definitions.
As used herein, the term “expression” of a gene product, such as a polynucleotide and a polypeptide, may represent transformation of a gene into another form under the influence of predetermined action in vivo. Preferably, the term “expression” represents transcription of genes, polynucleotides, etc. and translation into polypeptides. In an embodiment of the present invention, genes are transcribed into mRNA. More preferably, the polypeptides undergo post-translation modifications. Accordingly, a “reduction” in “expression” of genes, polynucleotides, and polypeptides used herein represents a significant reduction when an agent of the present invention is applied, compared to when no agent is applied. Preferably, the reduction in expression may include a reduction in expression level of polypeptides. More specifically, the reduction in expression level represents a reduction in expression by at least 10%, preferably by at least 20%, more preferably by at least 30%, still more preferably by at least 40%, still more preferably by at least 50%, still more preferably by at least 75%, still more preferably by at least 90%, and most preferably by at least 100%, when compared before and after applying the agent. An “increase” in “expression” of genes, polynucleotides, and polypeptides used herein represents a significant increase when an agent of the present invention is applied, compared to when no agent is applied. Preferably, the increase in expression may include an increase in an expression level of polypeptides. More specifically, the increase in the expression level represents an increase by at least 10%, preferably by at least 20%, more preferably by at least 30%, still more preferably by at least 40%, still more preferably by at least 50%, still more preferably by at least 75%, still more preferably by at least 90%, still more preferably by at least 100%, and most preferably by at least 200%, when compared before and after applying the agent, or an occurrence of expression before the agent is applied in which no expression occurs.
The present invention provides a method for preparing 6-aminocaproic acid comprising: preparing an expression vector comprising HpaI (4-hydroxy-2-oxoheptane-1,7-dioate aldolase)-HpaH (2-oxohept-3-ene-1,7-dioate dehydratase) gene, nemA (N-ethylmaleimide reductase) gene, and KIVD (alpha-ketoisovalerate decarboxylase) gene; and at least one of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) (step 1); and transforming the expression vector of step 1 into a microorganism (step 2).
The HpaI-HpaH gene preferably includes a polynucleotide represented by SEQ ID NO: 3 encoding aldolase-dehydratase, which converts pyruvate and/or succinic semialdehyde (SSA) into 2-oxohept-3-enedioic acid, but is not limited thereto.
The nemA gene preferably includes a polynucleotide represented by SEQ ID NO: 4 encoding reductase, which converts 2-oxohept-3-enedioic acid into 2-ketopimelic acid, but is not limited thereto.
The KIVD (alpha-ketoisovalerate decarboxylase) gene preferably includes a polynucleotide represented by SEQ ID NO: 5 encoding decarboxylase, which converts 2-ketopimelic acid into adipate semialdehyde, but is not limited thereto.
The PdAT (beta-alanine-pyruvate transaminase) gene preferably includes a polynucleotide represented by SEQ ID NO: 6 encoding transaminase, which converts adipate semialdehyde into 6-aminocaproic acid, but is not limited thereto.
The BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) gene preferably includes a polynucleotide represented by SEQ ID NO: 7 encoding transaminase, which converts adipate semialdehyde into 6-aminocaproic acid, but is not limited thereto.
The expression vector of step 1 may further include nucleic acid sequences encoding GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, 6HIS, STREP, CBP, CBD, or S-tag affinity tag, but is not limited thereto.
The expression vector of step 1 may further include nucleic acid sequences encoding kex2p in yeasts, purine in mammals, Factor Xa, enterokinase, subtilisin, tobacco etch virus protease, thrombin, or ubiquitin hydrolase, but is not limited thereto.
The microorganisms of step 2 may preferably be a bacterium, yeast, or fungus.
The method may further include producing and secreting 6-aminocaproic acid by culturing the transformed microorganism of step 2 in a fed-batch fermentation, but is not limited thereto.
The method may further include purifying the protein secreted above, but is not limited thereto.
The polynucleotides of the present invention may include amino acid sequences having a homology to each of the above-listed amino acid sequences of more than 70%, more preferably more than 80%, even more preferably more than 90%, and most preferably more than 95%. The “sequence homology percentage” may be confirmed through comparison between two sequences having an optimal arrangement and a region to be compared, and some parts of polynucleotide sequence in the region to be compared may include addition or deletion (i.e., gaps), compared to reference sequences (where addition or deletion is not included) for the optimal arrangement of the two sequences.
Also, the present invention provides an expression vector for the biosynthesis of 6-aminocaproic acid comprising HpaI-HpaH gene, nemA gene, KIVD (alpha-ketoisovalerate decarboxylase) gene, and at least one of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase).
The expression vector may preferably be pACYCWG illustrated in
The recombinant vector of the present invention may be obtained by inserting the genes or fragments thereof into the expression vector via a general cloning method (Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Specifically, an appropriate adaptor may be connected to a gene construct in order to facilitate the cloning of the gene construct.
The terms “vector”, “expression vector”, or “recombinant vector” are used to refer to DNA fragment(s) and nucleic acid molecules that transfer into cells. Vectors can duplicate DNA and can be reproduced independently in host cells. The vectors may refer to recombinant DNA molecules including proper nucleic acid sequences essential for expressing coding sequences operably linked to target coding sequences in specific host organisms. Promoters, enhancers, termination signals, and polyadenylation signals which can be used in cells of microorganisms are known in the art.
The vector of the present invention may typically be constructed as a vector for cloning or expression. Also, the vector of the present invention may be constructed with prokaryotic cells or eukaryotic cells serving as host cells. For example, in a case in which the recombinant vector of the present invention is an expression vector with prokaryotic cells serving as a host cell, it is common to include strong promoters (e.g. pLλ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.), ribosome binding sites, and transcription/translation termination sequences for initiation of translation.
Meanwhile, the vector that can be used in the present invention may be constructed by manipulating plasmids (e.g. pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series, pACYC184, pUC19, etc.), phages (e.g. λgt4˜λB, λ-Charon, λΔz1, M13, etc.), or viruses (e.g. SV40, etc.) often used in the art.
The expression vector may preferably include at least one selective marker. The marker, which is generally a nucleic acid sequence having properties that can be selected by a chemical method, include every gene that can distinguish a transformed cell from a non-transformed cell. The examples thereof include herbicide-resistant genes such as glyphosate, glufosinate ammonium, or phosphinothricin, and antibiotic-resistant genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, but are not limited thereto.
In the vector of the present invention, a promoter may be promoters of CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone, but is not limited thereto. The term “promoter” may refer to an upstream region of DNA in a gene structure and DNA molecules where RNA polymerase binds to initiate transcription. A “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states, or during cell differentiation. As a transformant may be selected by various tissues in various steps, the constitutive promoter may be preferred in the present invention. Therefore, the constitutive promoter may be selected without limitation.
In the vector of the present invention, a general terminator may be used, and the examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, terminator of octopine gene of Agrobacterium tumefaciens, etc., but are not limited thereto.
Also, the present invention provides a transformant as the recombinant vector described above.
The transformant may preferably be selected from the group consisting of bacteria, yeasts, and fungi, more preferably, it may be bacteria, and most preferably it may be E. coli, but is not limited thereto.
The transformant may convert pyruvate and/or succinic semialdehyde (SSA) to 6-aminocaproic acid, but is not limited thereto.
In order to carry the vector of the invention into a host cell, the vector may be injected into a host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, Agrobacterium-mediated transfection, DEAE-dextran treatment, gene bombardment, etc.
Further, the present invention provides a method for producing caprolactam further including transforming 6-aminocaproic acid produced by the method for producing 6-aminocaproic acid to caprolactam.
In a specific embodiment of the present invention, the present inventors isolated HpaI gene encoding aldolase, HpaH gene encoding dehydratase, nemA gene encoding reductase, KIVD gene encoding decarboxylase, and BcAT and PdAT genes encoding transaminase, which are enzymes related to the biosynthetic pathway of 6-aminocaproic acid (
Therefore, as the biosynthesis of 6-aminocaproic acid, which is a precursor of caprolactam, is possible in the transformant which is transformed into the vector in which the genes of the present invention are introduced, the transformant can be used in the biosynthesis of 6-aminocaproic acid.
Hereinafter, the present invention will be described in more detail with reference to the following examples to have a better understanding of the present invention. However, the following examples are provided for illustrative purposes only, and the scope of the present invention should not be limited thereto in any manner. The examples of the present invention are provided in order to fully describe the invention to those skilled in the art.
<1-1> Cloning of Each Enzyme
The genes encoding enzymes in the pathway biosynthesizing 6-aminocaproic acid from pyruvate were cloned from E. coli.
Specifically, HpaI gene (SEQ ID NO: 1) encoding aldolase, which converts pyruvate and/or succinic semialdehyde to 4-hydroxy-2-oxoheptanedioic acid; HpaH gene (SEQ ID NO: 2) encoding dehydratase, which converts 4-hydroxy-2-oxoheptanedioic acid to 2-oxohept-3-enedioic acid; nemA gene (SEQ ID NO: 4) encoding reductase, which converts 2-oxohept-3-enedioic acid to 2-ketopimelic acid; KIVD (alpha-ketoisovalerate decarboxylase) gene (SEQ ID NO: 5) encoding decarboxylase, which converts 2-ketopimelic acid to adipate semialdehyde; and PdAT (beta-alanine-pyruvate transaminase) gene (SEQ ID NO: 6) and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) gene (SEQ ID NO: 7) encoding transaminase, which convert adipate semialdehyde to 6-aminocaproic acid from E. coli described in Table 1 below, were amplified using primers (SEQ ID NOs: 15 to 26) described in Table 2, respectively, using PCR (95° C., 30 seconds, [95° C., 30 seconds, TM value for each primer: 30 seconds and 72° C., 60 seconds, total 30 cycles], 72° C., 5 minutes). Among the primers in Table 2, the parts written in italic refer to restriction enzyme sites, and the primers that were introduced into an In-Fusion™ Advantage PCR cloning kit (Clontech, USA) are underlined. After the amplification, HpaI and HpaH were ligated to be co-expressed using a ligase, and were assigned as HpaI-HpaH (SEQ ID NO: 3). The PCR products which were amplified above, having a ligase, were introduced into PET28(b+) vector decomposed by restriction enzymes (NdeI and BamHI), and the expression vectors introduced were assigned as pETHpaI, pETHpaH, pETnemA, pETKIVD, pETBcAT, and pETPdAT (Table 4).
E. coli strain used for standard cloning procedures
E. coli strain used for heterologous gene expression
E. coli strain used for heterologous gene expression
CGCGCGGCAGCCATATGATGGAAAACAGTTT
Escherichia
coli
GGTGGTGGTGCTCGAGATACACGCCGGGCTT
CGCGCGGCAGCCATATGATGTTCGACAAACA
Escherichia
coli
GGTGGTGGTGCTCGAGAACAAAGCGGCAGCT
CGCGCGGCAGCCATATGATGTCATCTGAAAA
Escherichia
coli
GGTGGTGGTGCTCGAGCAACGTCGGGTAATC
Escherichia
coli
CGCGCGGCAGCCATATG
ATGTATACAGTAGG
Lactococcus
lactis
GGTGGTGGTGCTCGAGTGATTTATTTTGTTC
Lactococcus
lactis
CGCGCGGCAGCCATATGATGATCTATTTTGA
Bacillus
cereus
GGTGGTGGTGCTCGAGCCTCATCACTTCATA
Bacillus
cereus
CGCGCGGCAGCCATATGATGAACCAACCGCA
Paracoccus
denificans
GGTGGTGGTGCTCGAGGGCCACCTCGGCAAA
Paracoccus
denificans
1F: forward primer, R: reverse primer
<1-2> Construction of Vector in which Genes Encoding Each Enzyme are Ligated
The vector including all HpaI, HpaH, nemA, KIVD, PdAT, and BcAT genes encoding the enzymes in the pathway biosynthesizing 6-aminocaproic acid from pyruvate was constructed.
Specifically, HpaI encoding aldolase and HpaH encoding hydratase were ligated to be expressed together using a ligase, and were assigned as pETHpaI-HpaH. Further, in order to insert the genes encoding the enzymes above into a vector, the expression vector prepared in Example <1-1> was used as a template, and PCR (95° C., 30 seconds, [95° C., 30 seconds, TM value for each primer: 30 seconds and 72° C., 60 seconds, total 30 cycles], 72° C., 5 minutes) was conducted using each primer in Table 3 (SEQ ID NOs: 27 to 36). The parts of primers that were introduced into an In-fusion™ Advantage PCR cloning kit (Clontech, USA) are underlined. BcAT, which is a PCR product having a first ligase, was introduced into pACYC184 decomposed by restriction enzyme sphI. The vector in which BcAT was introduced was then decomposed by HindIII, and PdAT, a PCR product, was introduced thereto. Furthermore, the vector in which BcAT and PdAT were introduced was decomposed by SalI, and KIVD, a PCR product, was introduced. Furthermore, the vector in which BcAT, PdAT, and KIVD were introduced was decomposed by BamHI, and nemA, a PCR product, was introduced. Lastly, the vector in which all the enzymes above introduced was decomposed by AhdI, and HpaI-HpaH, a PCR product, was introduced.
As a result, the expression vector including all genes HpaI-HpaH (ligation of HpaI and HpaH), nemA, KIVD, PdAT, and BcAT encoding enzymes in the biosynthesis pathway of 6-aminocaproic acid was prepared, and was assigned as pACYCWG (Table 4 and
CGATACTATGACTGATAATACGACTCACTA
CATGGCGTTGACTCTCAAAAAACCCCTCAA
CCCGTCCTGTGGATGTAATACGACTCACTA
CCGGCGTAGAGGATCCAAAAAACCCCTCAA
AAGGGAGAGCGTCGATAATACGACTCACTA
AAGGGCATCGGTCGACAAAAAACCCCTCAA
CCATCTCCTTGCATGTAATACGACTCACTA
AAGGAATGGTGCATGCAAAAAACCCCTCAA
TATCATCGATAAGCTTAATACGACTCACTA
TACCGCATTAAAGCTCAAAAAACCCCTCAA
E. coli cloning vector
E. coli cloning vector carrying whole genes
<2-1> Expression and Purification of Each Enzyme and Fusion Protein
The plasmids, namely, pETHpaI, pETHpaH, pETnemA, pETKIVD, pETBcAT, pETPdAT, pETHpaI-HpaH, and pACYCWG prepared in Example 1, were transformed into E. coli BL21(DE3) via thermal shock. The transformant was cultured in an LB medium containing 50 μg/mL of antibiotics at 37° C. When the culture solution of the transformant reached a concentration of A600=0.5, 0.5 mM of IPTG (isopropyl-β-thio-D-galactopyranoside) was added, and the resultant was further cultured for 3 hours at 37° C. The cultured cells were collected by centrifugation and the pellet produced was disrupted by sonication. After the disruption of pellet, HpaI (SEQ ID NO: 8), HpaH (SEQ ID NO: 9), HpaI-HpaH (SEQ ID NO: 10), nemA (SEQ ID NO: 11), KIVD (SEQ ID NO: 12), PdAT (SEQ ID NO: 13), and BcAT (SEQ ID NO: 14) were purified from the supernatant using Ni-NTA agarose (Qiage, Germany) and an Econo Pac Chromatography Column (Bio-Rad, USA) according to the manufacturer's description. The concentrations of purified proteins were measured by a BCA protein assay kit Pierce (USA), and as a result, the concentrations were HpaI-H: 0.216 mg/mL; Kivd: 0.523 mg/mL; PdAT: 0.176 mg/mL; BcAT: 0.632 mg/mL; nemA: 0.659 mg/mL, and protein purity was measured as shown in
<3-1> Confirmation of Activity of Purified Aldolase-Dehydratase (HpaI-HpaH)
An enzymatic reaction was carried out to confirm the conversion of pyruvate and/or succinic semialdehyde to 2-oxohept-3-enedioic acid by aldolase and aldolase-dehydratase (HpaI-HpaH) enzymes (purified using E. coli transformed into pETHpaI-HpaH) purified in Example 2-1.
Specifically, 4 g/L of pyruvate, 4 g/L of succinic semialdehyde (SSA), which are substrates, and 50 mM of MnCl2, serving as a cofactor, were mixed together with aldolase, which is a protein expressing purified HpaI, and aldolase-dehydratase expressed by ligating HpaI and HpaH, both of which were purified in Example 2-1 above, respectively. The volume was then adjusted by 100 mM HEPES buffer (pH 8.0), and the reaction was carried out overnight at 30° C. After the reaction, the change in concentration of pyruvate, a substrate, was confirmed by measuring absorbance at A570 using a pyruvate assay kit (Sigma, USA).
As a result, it was confirmed that the concentration of pyruvate, a substrate for aldolase-hydratase (HpaI-HpaH), was reduced, implying that the conversion of pyruvate and/or succinic semialdehyde to 2-oxohept-3-enedioic acid was carried out (
<3-2> Confirmation of Activity of Purified Reductase (nemA)
An enzymatic reaction was carried out to confirm the conversion of 2-oxohept-3-enedioic acid to 2-ketopimelic acid by reductase (nemA) purified in Example 2-1.
Specifically, as it is difficult to purchase 2-oxohept-3-enedioic acid, a substrate, 2-ketopimelic acid was used as a substrate using the fact that the reaction is also carried out in the reverse direction. 4 mM of NAD, 0.4 mM of FeSO4, which are cofactors, and reductase, in which nemA is expressed, were added to the substrate. The volume was then adjusted by 1 M potassium phosphate buffer (pH 5.3) to balance the pH, and the reaction was induced at 30° C. After the reaction, the amount of NADH produced during the conversion of NAD to NADH was measured as absorbance at A450 using an NADH assay kit (abcam. USA), confirming the conversion effect (Table 6).
As a result, it was confirmed that the reductase, which was expressed and purified by pETnemA, produced NADH, confirming the conversion effect of 2-ketopimelic acid to 2-oxohept-3-enedioic acid (
<3-3> Analysis of Coupling Reaction of Aldose-Dehydratase with Reductase
The conversion of pyruvate and/or succinic semialdehyde to 2-oxohept-3-enedioic acid was confirmed in Example 3-1, and the conversion of 2-oxohept-3-enedioic acid to 2-ketopimelic acid was confirmed in Example 3-2 via the reverse reaction. Herein, it was confirmed whether two reactions occur simultaneously by the two enzymes.
Specifically, 4 g/L of pyruvate, 4 g/L of SSA, and 200 mM of NADH were added as substrates, and 50 mM of MnCl2 was added thereto as a cofactor. Aldolase-hydratase, in which HpaI-HpaH is expressed, and reductase, in which nemA is expressed, were then added thereto, and the volume thereof was adjusted by 100 mM potassium phosphate buffer to balance the pH. Subsequently, an enzymatic reaction was induced to confirm 2-ketopimelic acid, which is a product, using LC-MS/MS. TIC indicates the overall isolation from 50 m/z to 300 m/z, SIM is a method for separately monitoring peaks of molecular ions (herein, 155) invisible in full scan, and SRM is a method for producing daughter ions from the molecular ions by splitting the same at high energy.
As a result, peaks were formed at similar times with 2-ketopimelic acid (Sigma Aldrich), which is a control group, and the mass of peaks was identical (
<3-4> Confirmation of Activity of Purified Transaminase (BcAT or PdAT)
An enzymatic reaction was carried out to confirm the conversion of adipate semialdehyde to 6-aminocaproic acid by the transaminase purified in Example 2-1.
Specifically, as it was difficult to purchase adipate semialdehyde, which is a substrate, 20 mM of 6-aminocaproic acid was added as a substrate using the fact that the reaction also occurs in the reverse direction. 10 mM of sodium alpha-ketoglutarate was then added thereto as an amino group donor, 0.2 mM of PLP was added thereto as a cofactor, and subsequently, transaminase, in which BcAT and PdAT were expressed and purified in Example 2-1, was added thereto. The volume thereof was adjusted by 100 mM potassium phosphate buffer (pH 7.0) to balance the pH. The enzymatic reaction was carried out overnight at 30° C., and the formation of glutamate, a product, and a change in concentrations was measured via glutamate analyzer to confirm the reaction, and a specific activity was then calculated.
As a result, it was confirmed that glutamate was formed in both PdAT and BcAT, and the activity of glutamate was higher in PdAT (Table 8).
<3-5> Analysis of Coupling Reaction of Transaminase (BcAT and/or PdAT) with Decarboxylase (KIVD)
Herein, it was confirmed whether 2-ketopimelic acid was converted to 6-aminocaproic acid by transaminase (BcAT and/or PdAT), confirmed in Example 3-4, and decarboxylase (KIVD) of the present invention.
Specifically, 2-ketopimelic acid as a substrate, 20 mM of glutamate as an amino group donor, 5 mM of MgSO4 as a cofactor, and 0.1 mM of PLP were added to confirm whether the two reactions occur simultaneously. Decarboxylase, in which KIVD was expressed and purified, and transaminase, in which BcAT and PdAT were expressed and purified, were then added thereto, and the volume of the resultant was adjusted by 100 mM potassium phosphate buffer (pH 7.0) to balance the pH. The enzymatic reaction was induced overnight at 30° C., and a product was obtained.
To confirm the product by TLC, the underside of a silica gel plate was marked by pencil with a line at the 1 cm point and with a dot where the sample was to be loaded. The thus-obtained product was loaded onto the plate by 1 μL drops a total of 5 times, the plate was inserted into a tank after drying, and the entrance was sealed to develop the plate for 1 hour. The developing reagent was mixed at a ratio of 5:1:5 between n-butanol:acetic acid:D.W., and only the supernatant was used. After the development of the plate, the plate was removed, sprayed with a 1% ninhydrin solution, and placed back into the tank for 5 minutes at 80° C. to confirm the color of the spots.
Also, the product was confirmed by LC-MS/MS (column: 250 mm×4.6 mm OptimaPak C18 (RS Tech, Korea); mobile phase: A—20% acetonitrile, B—80% distilled water; velocity: 300 μL/min; load amount: 1 μL; ionization: ESI(+)-MS, positive scan mode; source voltage=2.5 kV; capillary temperature=350° C.; m/z=50 to 150; step size=0.1 m/z; nebulizer pressure=100 psi).
As a result, it was confirmed by TLC and LC-MS/MS that 6-aminocaproic acid was produced (
<4-1> Confirmation of Biosynthesis of 6-Aminocaproic Acid
Herein, the biosynthesis of 6-aminocaproic acid from glucose and SSA was confirmed by the expression of fusion protein (aldolase-dehydratase-reductase-decarboxylase-transaminase) transformed into pACYCWG in E. coli in Example 1-2 above.
Specifically, in 2 L of medium containing the components shown in Table 9, E. coli transformed into pACYCWG and E. coli transformed into an empty vector, which is a control group, were cultured in a fed-batch culture for 24 hours at 37° C.
After culturing, cells were subjected to centrifugation to obtain a pellet, and the pellet was washed with distilled water five times and suspended in 50 mL of distilled water. 10 g of glucose, 4 g/L of succinic semialdehyde, and 1 mL/L of trace elements were added to the suspension, and an in vivo reaction was induced for 16 hours at 37° C. After the reaction, 6-aminocaproic acid was confirmed by TLC and LC-MS (column: 250 mm×4.6 mm OptimaPak C18 (RS Tech, Korea); mobile phase: A—20% acetonitrile, B—80% distilled water; velocity: 300 μL/min; load amount: 1 μL; ionization: ESI(+)-MS, positive scan mode; source voltage=2.5 kV; capillary temperature=350° C.; m/z=50 to 150; step size=0.1 m/z; nebulizer pressure=100 psi) by merely obtaining the supernatant. TIC indicates the overall isolation from 50 m/z to 300 m/z, SIM is a method for separately monitoring peaks of molecular ions (herein, 155) invisible in full scan, and SRM is a method for producing daughter ions from the molecular ions by splitting the same at high energy.
As a result, lane #3, in which the biosynthetic conversion was induced with cells cultured for 20 hours, showed a spot on the same line as 6-aminocaproic acid, confirming that 6-aminocaproic acid was produced (
Also, to calculate a conversion yield from glucose to 6-aminocaproic acid (6-ACA), the amount of glucose used was measured by a glucose analyzer and the amount of 6-aminocaproic acid produced was calculated from LC-MS. During an actual reaction, 10 g of glucose was added, however, the actual amount of glucose used by the strain was calculated by the initial amount of glucose minus the final amount of glucose, and the amount of 6-ACA formed was measured with a quantitative value (no plasmid, Pacycl84, HpaIH-nemA, PdAT-Kivd: negative control group). As a result, it was confirmed that the conversion yield from glucose to 6-ACA was shown to be about 2.5% (Table. 10).
<4-2> Confirmation of Enzyme Expression in Cells
The fusion recombinant proteins in cells cultured in Example 4-1 were subjected to western blot analysis using SDS-PAGE according to Laemmli procedures (Laemmli, U. K. 1970, Nature 227:680-685).
Specifically, proteins isolated by 10% SDS-PAGE gel were transferred onto a nitrocellulose membrane. After the transfer, the nitrocellulose membrane was blocked with 5% PBS (phosphate buffered saline) containing dried skim milk and washed with PBST (0.1% Tween20 in PBS) three times. The thus-washed membrane was reacted with His-probe monoclonal antibodies (Santa Cruz Biotechnology, USA) for 1 hour. An antigen that specifically reacts with an IgG AP (alkaline phosphatase) antibody was then shown in an AP conjugate substrate kit (Bio-rad, USA) (
<5-1> Culturing of Flask
A flask was cultured to analyze its culture medium in order to confirm whether 6-aminocaproic acid was produced from a culture supernatant of E. coli transformed into pACYCWG.
Specifically, in 100 mL of medium containing the components shown in Table 11, E. coli transformed into pACYCWG and E. coli transformed into an empty vector, which is a control group, were cultured for 24 hours at 28° C. After culturing of E. coli, the supernatant was obtained and analyzed by LC-MS.
As a result, it was confirmed through an LC-MS spectrum that the biosynthetic conversion was induced (
<5-2> Fermentation Culture
In 2.5 L of medium containing the components shown in Table 12, E. coli transformed into pACYCWG was cultured for 24 hours for mass culturing of 6-aminocaproic acid. After culturing, 15 g/L of lactose was added at the point when all of the initial glucose was consumed to induce the expression of enzymes, and then 4 g/L of glucose was fed each hour (
<5-3> Strain Improvement
Until now, two types of transaminase were used. However, as poor expression levels were observed due to many enzymes, only one type of transaminase was used herein. PdAT having a high specific activity was used rather than BcAT to construct a vector, and the vector was assigned as pPKNI (pACYCWG-BcAT).
A strain HMS174(DE3), in which RecA gene encoding recombinase was mutated, was selected and used because of the possibility that recombination might occur in the strain, as the size of the vector is huge.
pPKNI was transformed into the strain HMS174(DE3), and the production of 6-ACA was induced by fermentation. Specifically, in 2.5 L of medium containing the components shown in Table 12, E. coli was cultured for 24 hours. 3 hours after the culturing, 0.4 mM of IPTG was added to induce the expression of enzymes, and 5 g/L of glucose was fed after all of the initial glucose was consumed (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2013-0050676 | May 2013 | KR | national |
| 10-2014-0053249 | May 2014 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2014/003933 | 5/2/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/182016 | 11/13/2014 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20110171699 | Raemakers-Franken et al. | Jul 2011 | A1 |
| 20130017593 | Baynes et al. | Jan 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 2011-515111 | May 2011 | JP |
| 2013-515050 | May 2013 | JP |
| 10-2012-0034640 | Apr 2012 | KR |
| 2012177721 | Dec 2012 | WO |
| Entry |
|---|
| International Search Report issued in corresponding International Patent Application No. PCT/KR2014/003933 dated Aug. 26, 2014. |
| Number | Date | Country | |
|---|---|---|---|
| 20160257976 A1 | Sep 2016 | US |
