WO2014182016A1 - Synthèse biologique de l'acide 6-aminocaproïque et micro-organisme transgénique associé - Google Patents

Synthèse biologique de l'acide 6-aminocaproïque et micro-organisme transgénique associé Download PDF

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WO2014182016A1
WO2014182016A1 PCT/KR2014/003933 KR2014003933W WO2014182016A1 WO 2014182016 A1 WO2014182016 A1 WO 2014182016A1 KR 2014003933 W KR2014003933 W KR 2014003933W WO 2014182016 A1 WO2014182016 A1 WO 2014182016A1
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aminocaproic acid
gene
expression vector
producing
encoding
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WO2014182016A9 (fr
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이홍원
안정오
정준기
고희주
박선주
김천석
이혁원
이은교
이주환
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한국생명공학연구원
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Priority to US14/889,543 priority Critical patent/US10087472B2/en
Priority claimed from KR1020140053249A external-priority patent/KR101609448B1/ko
Publication of WO2014182016A1 publication Critical patent/WO2014182016A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom

Definitions

  • the present invention relates to a recombinant microorganism for biologically synthesizing 6-aminocaproic acid in a microorganism for synthesizing caprolactam.
  • Caprolactam is an organic compound that is a lactam of 6-aminohexanoic acid ( ⁇ -aminohexanoic acid, 6-aminocaproic acid). This may alternatively be considered a cyclic amide of caproic acid.
  • One use of caprolactam is as monomer in the production of nylon-6.
  • the most widely used raw materials for the production of caprolactam are aromatic compounds such as benzene, phenol, and toluene.
  • Caprolactam is an oximation reaction between cyclohexanone and hydroxylamine obtained from raw materials of aromatic compounds to prepare an oxime compound, and finally a Beckman displacement reaction using a sulfuric acid catalyst (Beckmann Through rearrangement.
  • caprolactam through this process makes it difficult to avoid the production of by-product ammonium sulfate. Since the yield of caprolactam decreases as more ammonium sulfate is produced in the caprolactam manufacturing process, caprolactam can be obtained with high yield only by suppressing the production of ammonium sulfate.
  • caprolactam manufacturing technology is divided into developing a process for reducing or eliminating the production of ammonium sulfate, a by-product of caprolactam manufacturing process, and developing alternative raw materials.
  • An example of process development to reduce the production of ammonium sulphate is the caprolactam production facility recently built by Sumitomo Japan. It uses gaseous Beckman displacement using a fluid bed gas-phase zeolite-catalyst and an ammoximation reaction with hydrogen peroxide catalyst from EniChem.
  • raw materials developed as substitutes for caprolactam include hexamethylene diamine (HMDA) and tetramethylenediamine (TMDA).
  • HMDA can be prepared from adiponitrile, propylene, acrylonitrile.
  • the manufacturing process of HMDA using adiponitril can be used only by BASF, Solutia, Butachimie, and DuPont.
  • Adiponitril is prepared by reacting butadiene with hydrogen cyanide.
  • Butadiene is also used as a raw material for adipic acid, a raw material of nylon 4,6.
  • the intermediates used in nylon production have their origin in butadiene, and this trend is becoming increasingly widespread.
  • the present inventors produced transgenic microorganisms capable of biosynthesizing 6-aminocaproic acid in microorganisms by expressing genes of enzymes used in the precursor 6-aminocaproic acid biosynthetic pathway for caprolactam.
  • HpaI 4-hydroxy-2-oxo-heptane-1,7-dioate aldolase ) -HpaH ( 2-oxo-hept-3-ene-1,7-dioate dehydratase) gene, nemA (N-ethylmaleimide reductase) Gene, KIVD (alpha-ketoisovalerate decarboxylase) gene; 6-aminocaproic acid biosynthesis, including and including any one or more of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononaoate Aminotransferase) genes; It is to provide an expression vector for.
  • Another object of the present invention is to provide a transformant transformed with the expression vector.
  • another object of the present invention is to provide a caprolactam production method further comprising the step of converting the 6-aminocaproic acid produced by the 6-aminocaproic acid production method to caprolactam.
  • the present invention provides a 6-aminocaproic acid production method.
  • the present invention relates to HpaI (4-hydroxy-2-oxo-heptane-1,7-dioate aldolase ) -HpaH (2-oxo-hept-3-ene-1,7-dioate dehydratase) gene, nemA (N- ethylmaleimide reductase) Gene, KIVD (alpha-ketoisovalerate decarboxylase) gene; 6-aminocaproic acid biosynthesis, including and including any one or more of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononaoate Aminotransferase) genes; Provide an expression vector.
  • the present invention also provides a transformant transformed with the expression vector.
  • the present invention provides a caprolactam production method further comprising converting the 6-aminocaproic acid produced by the 6-aminocaproic acid production method into caprolactam.
  • FIG. 1 is a schematic diagram showing the biosynthetic pathway of 6-aminocaproic acid and enzymes used in the biosynthetic pathway and genes encoding the same.
  • Figure 2 is Hpa I (4-hydroxy-2-oxo-heptane-1,7-dioate aldolase), HpaH (2-oxo-hept-3-ene-1,7-dioate dehydratase), nemA (N-ethylmaleimide reductase) , PIVYCWG vector containing KIVD (alpha-ketoisovalerate decarboxylase), PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononaoate Aminotransferase) genes.
  • HpaI-HpaH aldolase-dehydratase
  • Figure 4 is a standard curve confirming the concentration change of the product NADH in the reverse reaction to confirm the enzymatic activity of reductase ( nemA ).
  • FIG. 5 is a diagram confirming the production of 2-ketopimelic acid by Lc-ms / ms by coupling reaction of aldolase-dehydratase (HpaI-HpaHpaH) and reductase ( nemA );
  • FIG. 6 shows TLC conversion of 6-aminocaproic acid from 2-ketopimelic acid by coupling reaction of decarboxylase ( KIVD ) and transaminase ( PdAT and / or BcAT );
  • FIG. 7 shows LC-ms / ms conversion of 6-aminocaproic acid from 2-ketopimelic acid by coupling reaction of decarboxylase ( KIVD ) and transaminase ( PdAT and / or BcAT );
  • FIG. 8 is a diagram confirming 6-aminocaproic acid biosynthesis activity in E. coli transformed with a pACYCWG vector containing all the genes of the present invention
  • FIG. 9 is a diagram confirming 6-aminocaproic acid biosynthetic activity in Escherichia coli transformed with a pACYCWG vector containing all the genes of the present invention in LC-ms / ms;
  • L1 pACYCWG total protein
  • L2 pACYCWG soluble protein
  • L4 pACYCWG purified protein
  • aldolase-dehydratase HpaI - HpaH ) -58KD;
  • FIG. 11 is a diagram confirming 6-aminocaproic acid biosynthesis activity in Escherichia coli transformed with a pACYCWG vector containing all the genes of the present invention by LC-MS.
  • FIG. 12 is a fermentation graph of E. coli transformed with a pACYCWG vector in a fermentation medium experiment according to an embodiment of the present invention.
  • Figure 13 is a result of confirming 6-aminocaproic acid biosynthetic activity by LC-MS to obtain a supernatant after E. coli culture in the fermentation broth experiment according to an embodiment of the present invention.
  • FIG. 14 is a diagram confirming the expression of all the enzymes cloned in the vector in the fermentation broth experiment according to an embodiment of the present invention by Western blot.
  • 15 is a fermentation graph of E. coli transformed with a pACYCWG-BcAT vector in a strain improvement experiment according to an embodiment of the present invention.
  • 16 is a result of confirming 6-aminocaproic acid biosynthetic activity by LC-MS to obtain a supernatant after E. coli culture in the strain improvement experiment according to an embodiment of the present invention.
  • polynucleotide refers to nucleotide polymers of any length. This term also includes “oligonucleotide derivatives" or “polynucleotide derivatives".
  • An "oligonucleotide derivative” or “polynucleotide derivative” refers to an oligonucleotide or polynucleotide comprising a nucleotide derivative or having a lineage different from the general lineage between the nucleotides, which are used interchangeably.
  • polynucleotide as used herein is used interchangeably with nucleic acids, oligonucleotides and polynucleotides and includes cDNA, mRNA, genomic DNA and the like. As used herein, polynucleotides are encompassed by the term "gene”. Polynucleotides encoding gene sequences include “splicing variants”. Similarly, certain proteins encoded by nucleic acids include proteins encoded by splicing variants encoded thereby. As the name indicates, the term “splicing variant” refers to the product of an alternative splicing variant.
  • the first nucleic acid transcript after transcription is spliced to encode other polypeptides, such as other (obvious) nucleic acid splicing products. Although it is the generation mechanism of splicing variants, this includes exon selective splicing. Other polypeptides derived from the same nucleic acid by incorrect transcription include this definition.
  • the products of splicing reactions are also included in the definition.
  • the term "expression" of a gene product indicates that the gene is affected by a predetermined in vivo action and changed into another form.
  • the term “expression” indicates that the gene, polynucleotide, and the like are transcribed and translated into the polypeptide.
  • the gene is transcribed into mRNA. More preferably these polypeptides have post-translational processing modifications.
  • the "reduction" of the "expression” of the genes, polynucleotides, and polypeptides used herein indicates that the amount of expression when the agent of the invention acts is significantly reduced compared to when the agent is not active.
  • the decrease in expression comprises a decrease in the amount of polypeptide expression. More specifically, the reduction in the amount of expression is at least about 10% or more, preferably at least about 20% or more, more preferably at least about 30% or more, even more preferred when comparing the post-action and pre-action of the agent Preferably at least about 40% or more, even more preferably at least about 50% or more, even more preferably at least about 75% or more, even more preferably at least about 90% or more, even more preferably at least about 100% Abnormal expression decreases.
  • "increase" in the gene, polynucleotide, polypeptide "expression” indicates that the amount of expression when an agent of the present invention acts is significantly increased as compared to when the agent is not active.
  • the increase in expression comprises an increase in the amount of polypeptide expression. More specifically, the increase in the amount of expression is at least about 10% or more, preferably at least about 20% or more, more preferably at least about 30% or more, even more preferred when comparing the post-action and pre-action of the agent Preferably at least about 40% or more, even more preferably at least about 50% or more, even more preferably at least about 75% or more, even more preferably at least about 90% or more, even more preferably at least about 100% As such, even more preferably, a decrease in expression of 200% or an expression not expressed before the action of the agent occurs.
  • the present invention relates to 1) HpaI (4-hydroxy-2-oxo-heptane-1,7-dioate aldolase ) -HpaH ( 2-oxo-hept-3-ene-1,7-dioate dehydratase) gene, nemA (N- ethylmaleimide reductase) Gene, KIVD (alpha-ketoisovalerate decarboxylase) gene; Producing an expression vector comprising; and any one or more of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononaoate Aminotransferase) gene; And
  • step 2) provides a 6-aminocaproic acid (6-aminocaproic acid) production method comprising the step of transforming the expression vector of step 1) to the microorganism.
  • the HpaI-HpaH gene converts aldolase-dehydratase to convert pyruvate and / or succinic semialdehyde (SSA) to 2-oxohept-3-enedioic acid. It is preferred to include a polynucleotide represented by SEQ ID NO: 3 encoding, but is not limited thereto.
  • the nemA gene preferably includes a polynucleotide represented by SEQ ID NO: 4 encoding a reductase for converting 2-oxohept-3-enedioic acid to 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 the enzyme decarboxylase, which converts 2-ketopimelic acid to 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 an enzyme transaminase that converts adipate semialdehyde to 6-aminocaproic acid, but is not limited thereto.
  • the BcAT (adenosylmethionine-8-amino-7-oxononaoate Aminotransferase) gene preferably includes a polynucleotide represented by SEQ ID NO: 7 encoding an enzyme transaminase that converts adipate semialdehyde to 6-aminocaproic acid, but is not limited thereto. .
  • the expression vector of step 1) is a nucleic acid encoding GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, 6HIS, STREP, CBP, CBD, or S-tag affinity tag. It is preferred to further include a sequence, but is not limited thereto.
  • the expression vector of step 1) further comprises a nucleic acid sequence encoding a recognition sequence of kex2p of yeast, purine of mammal, Factor Xa, enterokinase, subtilisin, tobacco etching virus protease, thrombin or ubiquitin hydrolase Preferably, but not limited thereto.
  • the microorganism of step 2) is preferably bacteria, yeast or fungi, more preferably Escherichia coli, but is not limited thereto.
  • step 2) it is preferable to further include the step of producing and secreting 6-aminocaproic acid by fed-batch fermentation of the transformed microorganism of step 2), but is not limited thereto.
  • the polynucleotides of the present invention may comprise base sequences each having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with each base sequence. Can be.
  • the "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
  • the present invention is HpaI-HpaH gene, nemA gene, KIVD (alpha-ketoisovalerate decarboxylase) gene; 6-aminocaproic acid biosynthesis, including and including any one or more of PdAT (beta-alanine-pyruvate transaminase) and BcAT (adenosylmethionine-8-amino-7-oxononaoate Aminotransferase) genes; Provide an expression vector.
  • the expression vector is preferably pACYCWG shown in FIG. 2, but is not limited thereto.
  • Recombinant vectors of the present invention are conventional cloning methods for the genes or fragments thereof for expression vectors (Sambrook et al, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.) Can be obtained by inserting according to In particular, an appropriate adapter may be linked to the gene construct prior to insertion to facilitate cloning of the gene construct.
  • vector expression vector or “recombinant vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell.
  • Vectors can replicate DNA and be reproduced independently in host cells.
  • vector is meant a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express the coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in microbial cells are known.
  • Vectors of the invention can typically be constructed as vectors for cloning or expression.
  • the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.
  • a strong promoter for example, a pL ⁇ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.
  • ribosomal binding sites and transcription / detox termination sequences for initiation of translation.
  • vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series, pACYC184 and pUC19, etc.) often used in the art, phage (e.g. Can be produced by manipulating ⁇ B, ⁇ -Charon, ⁇ z1 and M13, etc.) or viruses (eg SV40, etc.).
  • plasmids eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series, pACYC184 and pUC19, etc.
  • phage e.g. Can be produced by manipulating ⁇ B, ⁇ -Charon, ⁇ z1 and M13, etc.
  • viruses eg SV40, etc.
  • the expression vector will preferably comprise one or more selectable markers.
  • the marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, kanamycin, G418, bleomycin, hygromycin ), But is not limited to antibiotic resistance genes such as chloramphenicol.
  • the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter.
  • the term "promoter” refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.
  • a "constitutive promoter” is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.
  • conventional terminators can be used, for example nopalin synthase (NOS), rice ⁇ -amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens Terminator of the octopine gene, etc., but is not limited thereto.
  • NOS nopalin synthase
  • rice ⁇ -amylase RAmy1 A terminator for example, rice ⁇ -amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens Terminator of the octopine gene, etc., but is not limited thereto.
  • the present invention provides a transformant with the recombinant vector.
  • the transformant is preferably selected from the group consisting of bacteria, yeast and fungi, more preferably bacteria, most preferably E. coli, but is not limited thereto.
  • the transformant preferably converts pyruvate and / or succinic semialdehyde (SSA) to 6-aminocaproic acid, but is not limited thereto.
  • SSA succinic semialdehyde
  • Methods for carrying vectors of the present invention into host cells include microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, Agrobacterium-mediated transfection, DEAE-dextran treatment, and gene balm.
  • the vector can be injected into a host cell by a body or the like.
  • the present invention provides a caprolactam production method further comprising the step of converting the 6-aminocaproic acid produced by the 6-aminocaproic acid production method according to the invention into caprolactam.
  • the inventors encode a gene HpaI , a gene encoding dehydratase, HpaH , a reductase, which encodes aldolase, an enzyme involved in the biosynthetic pathway (see FIG. 1) of 6-aminocaproic acid, a precursor of caprolactam.
  • Genes nemA , gene KIVD encoding decarboxylase and genes BcAT and PdAT encoding transaminase were isolated and introduced into the vector.
  • all of the genes from the vectors were linked and introduced into one vector (FIG. 2).
  • 6-aminocaproic acid which is a precursor of caprolactam
  • a transformant transformed with a vector into which the genes of the present invention are introduced and thus it can be used for biosynthesis of 6-aminocaproic acid.
  • Example 1 Gene cloning and vector construction encoding enzymes of 6-aminocaproic acid biosynthetic pathway
  • the gene HpaI (SEQ ID NO: 1) encoding the enzyme aldolase for converting pyruvate and / or succinic semialdehyde into 4-hydroxy-2-oxoheptanedioic acid from the E.
  • the gene HpaH (SEQ ID NO: 2) encoding the enzyme dehydratase, which converts 4-hydroxy-2-oxoheptanedioic acid to 2-oxohept-3-enedioic acid;
  • the gene nemA (SEQ ID NO: 4) encoding the enzyme reductase that converts 2-oxohept-3-enedioic acid to 2-ketopimelic acid;
  • the gene KIVD alpha-ketoisovalerate decarboxylase) (SEQ ID NO: 5), encoding the enzyme decarboxylase, which converts 2-ketopimelic acid to adipate semialdehyde;
  • PdAT beta-alanine-pyruvate transaminase
  • BcAT adenosylmethionine-8-amino-7-oxononaoate Aminotransferase
  • the amplified PCR product with the linker was introduced into a PET28 (b +) vector digested with restriction enzymes ( Nde I and BamH I) and the expression vectors introduced were named pETHpaI, pETHpaH, pETnemA, pETKIVD, pETBcAT and pETPdAT (Table) 4).
  • a vector was constructed that contains all of the genes Hpa I , HpaH , nemA , KIVD , PdAT and BcAT , which encode enzymes of the 6-aminocaproic acid biosynthesis pathway from Pyruvate.
  • HpaI encoding aldolase and HpaH encoding hydratase were linked to be expressed together using a linker, which was named pETHpaI-HpaH.
  • a linker which was named pETHpaI-HpaH.
  • each of the expression vectors prepared in Example ⁇ 1-1> was used as a template, and each of the primers (SEQ ID NOs 27 to 36) shown in Table 3 was used.
  • PCR was performed (95 ° C. 30 sec, [95 ° C. 30 sec, TM value for each primer 30 sec and 72 ° C. 60 sec, total 30 cycles], 72 degrees 5 min), and the underlined portion of the primer was in-fusion.
  • TM Advantage PCR cloning kit (Clontech, USA).
  • BcAT a linker-containing PCR product
  • pACYC184 digested with restriction enzyme sph I.
  • the vector into which the BcAT was introduced was digested with Hind III and a PdAT PCR product was introduced.
  • the vector introduced with BcAT and PdAT was digested with Sal I and a KIVD PCR product was introduced.
  • the vector introduced with BcAT , PdAT and KIVD was digested with BamH I and nemA PCR products were introduced.
  • the vector into which the enzymes were introduced was digested with Ahd I and then HpaI-HpaH PCR products were introduced.
  • 6-aminocaproic acid gene encoding the enzyme in the biosynthetic pathway HpaI-HpaH (connection HpaI and HpaH), nemA, KIVD, expression was the vector is produced that contains both the PdAT and BcAT, and named it as pACYCWG (Table 4 And FIG. 2).
  • Table 4 Plasmid Explanation source pETI-H PT7, His-tag, kanr; E. coli expression vector carrying aldolase pETnemA PT7, His-tag, kanr; E. coli expression vector carrying reductase pETKIVD PT7, His-tag, kanr; E. coli expression vector carrying decarboxylase pETBcAT PT7, His-tag, kanr; E. coli expression vector carrying transaminase pETPdAT PT7, His-tag, kanr; E. coli expression vector carrying transaminase pACYC184 E. coli cloning vector Mo-bi tec company pACYCWG E. coli cloning vector carrying whole genes contained PT7
  • the plasmids pETHpaI, pETHpaH, pETnemA, pETKIVD, pETBcAT, pETPdAT, pETHpaI-HpaH and pACYCWG prepared in ⁇ Example 1> were transformed into E. coli BL21 (DE3) by thermal shock.
  • HpaI SEQ ID NO: 8
  • HpaH SEQ ID NO: 9
  • Ni-NTA agarose Qiage, Germany
  • Econo Pac Choromatograpy Column Bio-Rad, USA
  • HpaI-HpaH SEQ ID NO: 10
  • nemA SEQ ID NO: 11
  • KIVD SEQ ID NO: 12
  • PdAT SEQ ID NO: 13
  • BcAT SEQ ID NO: 14
  • 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
  • protein purity was as shown in Table 7 below.
  • Example ⁇ 2-1> Aldolase and aldolase-dehydratase expressed by connecting HpaI and HpaH were mixed, adjusted to 100 mM HEPES buffer (pH8.0) for pH balance, and the reaction was induced at 30 ° C. overnight. After the reaction, the absorbance was measured in A570 using a Pyruvate assay kit (Sigma, USA) to confirm the concentration change of the substrate pyruvate (Table 5).
  • TIC is a total separation of 50-300 m / z
  • SIM is a method to monitor peaks that are invisible in full scan (only 155 here)
  • SRM splits molecular ions at high energy. It is a method of generating ions.
  • the reaction since it is difficult to purchase adipate semialdehyde as a substrate, the reaction also proceeds in reverse reaction, so that 20 mM 6-aminocaproic acid is added as a substrate and 10 mM sodium alpha keto glutarate as an amino group is 0.2 mM PLP as a cofactor.
  • 20 mM 6-aminocaproic acid is added as a substrate and 10 mM sodium alpha keto glutarate as an amino group is 0.2 mM PLP as a cofactor.
  • the purified transaminase was added to adjust the volume with 100mM potassium phosphate buffer (pH7.0) for pH balance.
  • specific activity was calculated after measuring the production and concentration change of glutamate, a product of glutamate, in order to confirm the reaction.
  • 2-ketopimelic acid as a substrate, 20 mM glutamate as an amino group donor, 5 mM MgSO 4 and 0.1 mM PLP as cofactor, and KIVD -expressed and purified decarboxylase and BcAT and PdAT to confirm whether the two reactions occur together
  • KIVD -expressed and purified decarboxylase and BcAT and PdAT was added to adjust the volume with 100 mM potassium phosphate buffer (pH 7.0) for pH balance.
  • the product was obtained after inducing an enzymatic reaction at 30 ° C. overnight.
  • the bottom and bottom 1cm portions of both sides of the silica gel plate were lined with a pencil and marked with a pencil with a small dot on the bottom of the sample.
  • the plate was well dried and placed in a tank to seal the inlet and then developed for 1 hour.
  • the developing reagent was mixed well with 5: 1: 5 n-butanol: acetic acid: D.W. and only supernatant was used.
  • the plate was taken out, sprayed with 1% ninhydrin solution, baked at 80 ° C. for 5 minutes, and the color of the spot was checked.
  • Example ⁇ 1-2> biosynthesis of glucose and SSA to 6-aminocaproic acid by expression of a fusion protein (aldolase-dehydratase-reductase-decarboxylase-transaminase) in Escherichia coli transformed with pACYCWG was confirmed.
  • a fusion protein aldolase-dehydratase-reductase-decarboxylase-transaminase
  • Escherichia coli transformed with pACYCWG and 2 Escherichia coli transformed with an empty vector as a control in 2 L of the medium of the composition of Table 9 were incubated at 37 ° C. for 24 hours. After incubation, the cells were centrifuged to obtain pellets. The pellets were washed five times with distilled water and then suspended again in 50 ml of distilled water. 10 g glucose, 4 g / L succinic semialdehyde and 1 ml / L trace elements were added to the suspension, and the in vivo reaction was induced for 16 hours at 37 ° C.
  • TIC is a total separation of 50-300 m / z
  • SIM is a method to monitor peaks that are invisible in full scan (only 155 here)
  • SRM splits molecular ions at high energy. It is a method of generating ions.
  • 6-aminocaproic acid was formed because spots of the same line as 6-aminocaproic acid were seen in lane 3 inducing biosynthetic conversion with the cells cultured for 20 hours (FIG. 8).
  • the biosynthetic pathway into in vivo was confirmed by confirming the 6-aminocaproic acid peak in the results measured at Lc-ms and Lc-ms / ms (FIG. 9).
  • the amount of glucose used for the conversion of glucose to 6-aminocaproic acid (6-ACA) was measured using a glucose analyser, and the amount of 6-aminocaproic acid produced was determined from LC-MS.
  • the amount actually used by the strain was obtained by subtracting last glucose from initial glucose, and 6-ACA was formed as a quantitative value (No plasmid, Pacyc184, HpaIH-nemA, PdAT-Kivd: Negative control).
  • the conversion yield of glucose to 6-ACA was about 2.5% (Table 10).
  • Example ⁇ 4-1> The recombinant protein fused in cells cultured in Example ⁇ 4-1> was subjected to Western blot analysis using SDS-PAGE according to Laemmli's method (Laemmli, UK 1970, Nature 227: 680-685).
  • proteins separated by 10% SDS-PAGE gels were transferred to nitrocellulose membrane.
  • the nitrocellulose membrane was blocked with PBS (phosphate buffered saline) containing 5% skim milk powder, and washed three times with PBST (0.1% Tween20 in PBS).
  • the washed membrane was reacted with His-probe monoclonal antibody (Santa Cruz Biotechnology, USA) for 1 hour at room temperature.
  • Antigens that specifically react with IgG AP (alkaline phosphatase) antibodies were shown in the AP Binding Substrate Kit (Bio-rad, USA) (FIG. 10).
  • 6-aminocaproic acid was formed from the culture supernatant of Escherichia coli transformed with pACYCWG, the culture was analyzed by flask culture.
  • E. coli transformed with pACYCWG in 100 mL of the composition of Table 11 and E. coli transformed with the empty vector as a control were incubated at 28 ° C. for 24 hours. After the culture of E. coli, the supernatant was obtained and analyzed by LC-ms.
  • E. coli transformed with pACYCWG in 2.5L of the medium of the composition of Table 12 was incubated for 24 hours. After incubation, 15 g / L of lactose was added to induce the expression of enzymes at the time of initial sugar consumption and then fed 4 g / L of sugar per hour (FIG. 12). After E. coli culture, the supernatant was obtained and analyzed by LC-MS. As a result, it was confirmed that 6-aminocaproic acid was produced (FIG. 13). In addition, it was confirmed that the desired enzyme was expressed by fermentation culture through Western blot (FIG. 14).
  • a vector was constructed using only PdAT having high specific activity among PdAT and BcAT, and this vector was named pPKNI (pACYCWG-BcAT).
  • pPKNI was transformed into strain HMS174 (DE3) and fermentation induced 6-ACA production. Specifically, E. coli was incubated for 24 hours in a medium of 2.5L of Table 12. After 3 hours of incubation, 0.4 mM IPTG was added to induce the expression of enzymes and 5 g / L of sugar was fed per hour after initial sugar exhaustion (FIG. 15). After E. coli culture, the supernatant was obtained and analyzed by LC-MS / MS. As a result, it was confirmed that 6-aminocaproic acid was produced (FIG. 16).

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Abstract

Cette invention concerne un procédé de préparation d'un micro-organisme recombiné comprenant simultanément les gènes codant pour les enzymes utilisées pour une voie de biosynthèse de l'acide 6-aminocaproïque qui est un précurseur du caprolactame, un procédé de biosynthèse de l'acide 6-aminocaproïque à partir du micro-organisme, et un procédé de production de celui-ci pour synthétiser le caprolactame.
PCT/KR2014/003933 2013-05-06 2014-05-02 Synthèse biologique de l'acide 6-aminocaproïque et micro-organisme transgénique associé WO2014182016A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011515111A (ja) * 2008-03-27 2011-05-19 ジェノマティカ， インコーポレイテッド アジピン酸および他の化合物を生成するための微生物
US20110171699A1 (en) * 2008-03-11 2011-07-14 Petronella Catharina Raemakers-Franken Preparation of 6-aminocaproic acid from 5 -formyl valeri c acid
KR20120034640A (ko) * 2009-05-07 2012-04-12 게노마티카 인코포레이티드 아디페이트, 헥사메틸렌디아민 및 6-아미노카프로산의 생합성을 위한 미생물 및 방법
WO2012177721A1 (fr) * 2011-06-22 2012-12-27 Genomatica, Inc. Microorganismes pour la production d'acide 6-aminocaproïque
US20130017593A1 (en) * 2009-03-11 2013-01-17 Celexion, Llc Biological Synthesis of Difunctional Hexanes and Pentanes from Carbohydrate Feedstocks
JP2013515050A (ja) * 2009-12-22 2013-05-02 ディーエスエム アイピー アセッツ ビー．ブイ． 発酵プロセスで得られた６−アミノカプロン酸からのカプロラクタムの調製

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110171699A1 (en) * 2008-03-11 2011-07-14 Petronella Catharina Raemakers-Franken Preparation of 6-aminocaproic acid from 5 -formyl valeri c acid
JP2011515111A (ja) * 2008-03-27 2011-05-19 ジェノマティカ， インコーポレイテッド アジピン酸および他の化合物を生成するための微生物
US20130017593A1 (en) * 2009-03-11 2013-01-17 Celexion, Llc Biological Synthesis of Difunctional Hexanes and Pentanes from Carbohydrate Feedstocks
KR20120034640A (ko) * 2009-05-07 2012-04-12 게노마티카 인코포레이티드 아디페이트, 헥사메틸렌디아민 및 6-아미노카프로산의 생합성을 위한 미생물 및 방법
JP2013515050A (ja) * 2009-12-22 2013-05-02 ディーエスエム アイピー アセッツ ビー．ブイ． 発酵プロセスで得られた６−アミノカプロン酸からのカプロラクタムの調製
WO2012177721A1 (fr) * 2011-06-22 2012-12-27 Genomatica, Inc. Microorganismes pour la production d'acide 6-aminocaproïque

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