KR20240000147A - Development of lysine decarboxylase mutants with increased soluble expression and their applications - Google Patents
Development of lysine decarboxylase mutants with increased soluble expression and their applications Download PDFInfo
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- KR20240000147A KR20240000147A KR1020220076728A KR20220076728A KR20240000147A KR 20240000147 A KR20240000147 A KR 20240000147A KR 1020220076728 A KR1020220076728 A KR 1020220076728A KR 20220076728 A KR20220076728 A KR 20220076728A KR 20240000147 A KR20240000147 A KR 20240000147A
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- South Korea
- Prior art keywords
- leu
- gly
- ala
- ile
- glu
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- 108010048581 Lysine decarboxylase Proteins 0.000 title claims abstract description 47
- 230000001965 increasing effect Effects 0.000 title description 37
- 230000014509 gene expression Effects 0.000 title description 14
- 238000011161 development Methods 0.000 title description 2
- 238000004519 manufacturing process Methods 0.000 claims abstract description 28
- 230000035772 mutation Effects 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 12
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 7
- 102200151056 rs397514647 Human genes 0.000 claims description 6
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 51
- 102000004169 proteins and genes Human genes 0.000 abstract description 49
- 150000003839 salts Chemical class 0.000 abstract description 10
- 241000588724 Escherichia coli Species 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 3
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 description 44
- 235000018102 proteins Nutrition 0.000 description 44
- 230000000694 effects Effects 0.000 description 39
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 37
- 238000006243 chemical reaction Methods 0.000 description 32
- 108010038633 aspartylglutamate Proteins 0.000 description 28
- 150000001413 amino acids Chemical group 0.000 description 27
- 108091035707 Consensus sequence Proteins 0.000 description 23
- 239000004472 Lysine Substances 0.000 description 22
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- BFYHIHGIHGROAT-HTUGSXCWSA-N Phe-Glu-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BFYHIHGIHGROAT-HTUGSXCWSA-N 0.000 description 14
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 14
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 14
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 14
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 14
- 108010005942 methionylglycine Proteins 0.000 description 14
- 108010020532 tyrosyl-proline Proteins 0.000 description 14
- PARSHQDZROHERM-NHCYSSNCSA-N Ile-Lys-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)O)N PARSHQDZROHERM-NHCYSSNCSA-N 0.000 description 13
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 12
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- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 10
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Classifications
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01018—Lysine decarboxylase (4.1.1.18)
Abstract
본 발명은 대장균 유래 항시성(constitutive) 발현의 라이신 디카르복실라아제 (LdcC)의 용해도 증대를 위한 단백질 공학 기술과 이를 통한 새로운 변이주의 제조 및 라이신 디카르복실라아제의 용해도를 결정하는 중요한 염결합(salt bridge)을 갖는 변이주의 제조에 관한 것이다. The present invention provides protein engineering technology to increase the solubility of constitutively expressed lysine decarboxylase (LdcC) derived from Escherichia coli, production of new mutant strains using the same, and important salts that determine the solubility of lysine decarboxylase. It relates to the production of mutant strains with salt bridges.
Description
본 발명은 단백질 공학기술을 이용하여 LdcC의 용해도를 증가시키고 이를 통해 제대로 접힌(folding) LdcC의 생산량 증대 및 카다베린 생산 증대를 달성하는 라이신 디카르복실라아제의 변이주 및 이의 제조 방법에 관한 발명이다. The present invention relates to a mutant strain of lysine decarboxylase and a method for producing the same that increase the solubility of LdcC using protein engineering technology, thereby increasing the production of properly folded LdcC and increasing cadaverine production. .
라이신 디카르복실라아제는 양자를 소모하여 라이신을 탈탄산하는 효소로 카다베린을 생성한다. 특히 대장균에는 두가지 라이신 디카르복실라아제가 존재하는데 하나는 지속적으로(constitutive) 발현되면서 반응 최적 pH가 7.4인 항시성(constitutive) 라이신 디카르복실라아제(LdcC), 다른 하나는 유도성으로(inducible) 발현되며 반응 최적 pH가 5.6인 유도성(inducible) 라이신 디카르복실라아제(CadA)이다.Lysine decarboxylase is an enzyme that decarboxylates lysine by consuming protons to produce cadaverine. In particular, there are two types of lysine decarboxylase in E. coli: one is constitutive lysine decarboxylase (LdcC), which is expressed constitutively and has an optimal reaction pH of 7.4, and the other is inducible ( It is an inducible lysine decarboxylase (CadA) that is expressed and has an optimal reaction pH of 5.6.
대장균에서 라이신 디카르복실라아제는 외부 혹은 내부의 pH에 대응하여 세포의 내부 pH를 조절해주는 역할을 하는 효소로 반응에서 양자를 사용하기 때문에 산성 조건(acidic)에서 빠르게 세포 내부를 염기성(basic)으로 만든다. 비록 효소의 1차적 역할은 세포 pH 조절이지만 이를 통해 생산되는 카다베린은 다양한 분야에 활용될 수 있어 카다베린의 효율적인 생산은 바이오산업에서 많은 관심을 받아왔다.In E. coli, lysine decarboxylase is an enzyme that controls the internal pH of the cell in response to external or internal pH. Because it uses protons in the reaction, it quickly changes the inside of the cell from acidic to basic. It is made with Although the primary role of the enzyme is to regulate cell pH, the cadaverine produced through it can be used in a variety of fields, so the efficient production of cadaverine has received much attention in the bio industry.
라이신 디카르복실라아제 반응은 도 1 과 같은 반응을 진행한다. 해당 반응은 양자를 소모하여 라이신을 탈탄산시켜 카다베린을 만들게 된다. 따라서 반응이 진행될수록 반응액은 알칼리성으로 변하게 되며 이런 반응의 특성상 약산성 조건에서 활성이 뛰어난 유도성(inducible) 라이신 디카르복실라아제(CadA)는 활성을 빨리 잃게 된다. 이를 해결하기 위해서는 반응 최적 pH가 중성인 항시성(constitutive) 라이신 디카르복실라아제(LdcC)를 사용할 수 있으나 LdcC의 경우 용해도가 낮아 산업적 적용이 어렵다. The lysine decarboxylase reaction proceeds as shown in Figure 1. This reaction consumes protons to decarboxylate lysine to create cadaverine. Therefore, as the reaction progresses, the reaction solution becomes more alkaline, and due to the nature of this reaction, inducible lysine decarboxylase (CadA), which is highly active under mildly acidic conditions, quickly loses its activity. To solve this problem, constitutive lysine decarboxylase (LdcC), which has a neutral reaction optimal pH, can be used. However, LdcC has low solubility, making industrial application difficult.
카다베린은 식물의 성장을 돕거나 부정맥, 이질 등의 질병의 치료에 도움을 주며 대표적으로 나일론의 단량체로 사용되는 고부가물질 중 하나이다. 특히 최근 환경보호에 대해 세계적인 관심이 높아지면서 친환경적으로 폴리머를 생산할 수 있는 녹색화학(green chemistry)에 대한 관심이 높다. 따라서 카다베린의 생합성은 산업적인 가치가 높으며 장기적으로 환경보호에 있어서도 중요하다. Cadaverine helps plants grow and treat diseases such as arrhythmia and dysentery. It is one of the high value-added substances typically used as a monomer for nylon. In particular, as global interest in environmental protection has recently increased, interest in green chemistry, which can produce polymers in an environmentally friendly manner, is high. Therefore, the biosynthesis of cadaverine has high industrial value and is important for long-term environmental protection.
현재 산업에서 카다베린 생산에 있어 가장 많이 활용되는 효소는 CadA이다. CadA는 대장균 유래의 효소이기 때문에 바이오산업에서의 생변환에 있어 가장 많이 사용되는 대장균내 발현에서 외래 유전자들에 비해 이점이 있다. 특히 CadA의 경우 다른 유래의 라이신 디카르복실라아제와 비교하였을 때 활성과 열안정성이 뛰어난 편에 속하기에 생변환에 있어 적합한 효소로 인식되고 있다. 때문에 CadA를 이용한 카다베린 생산에 대한 다양한 방법들이 개발되었으며 CadA의 열안정성을 단백질 공학적 변이로 증가시키거나 CadA의 활성을 더욱 증가시키는 연구 등이 진행되었다. Currently, the enzyme most widely used in industry to produce cadaverine is CadA. Because CadA is an enzyme derived from E. coli, it has an advantage over foreign genes in expression in E. coli, which is the most commonly used enzyme for biotransformation in the bio industry. In particular, CadA is recognized as a suitable enzyme for biotransformation because it has excellent activity and thermal stability compared to lysine decarboxylase of other origins. Therefore, various methods for producing cadaverine using CadA have been developed, and research has been conducted to increase the thermal stability of CadA through protein engineering mutations or to further increase the activity of CadA.
카다베린 생산을 위한 종래기술들은 대부분 CadA에 집중하였고 LdcC에 대해서 단백질 공학을 진행한 발명은 거의 없다.Most of the prior technologies for producing cadaverine focused on CadA, and there were few inventions that carried out protein engineering for LdcC.
CadA는 비록 활성과 열안정성이 뛰어나지만 반응 최적 pH가 5.6으로 반응 메커니즘상 반응이 진행될수록 반응액의 수소이온이 소모되어 반응액이 점점 염기성으로 변하는 해당 반응에서는 효소의 활성을 빨리 잃게 만드는 원인이 된다. 따라서 산업에서 고농도 장시간 반응을 진행할 경우 효소의 활성 유지를 위해 반응 pH를 지속적으로 낮춰주는 작업이 필요하고 이러한 추가 작업은 카다베린 생산의 경제성을 떨어뜨린다.Although CadA has excellent activity and thermal stability, the optimal reaction pH is 5.6. As the reaction progresses, hydrogen ions in the reaction solution are consumed as the reaction progresses, causing the enzyme to quickly lose activity in the reaction in which the reaction solution becomes more basic. do. Therefore, when high concentration and long-time reactions are carried out in industry, it is necessary to continuously lower the reaction pH to maintain enzyme activity, and this additional work reduces the economic feasibility of cadaverine production.
이러한 문제를 해결하기 위해 반응 최적 pH가 7.4인 LdcC를 사용하면 알칼리성 반응 환경에서 CadA에 비해 상대적으로 오랜 기간 활성을 유지할 수 있다. 하지만 LdcC는 대장균내에서 과발현시 전체 발현량이 CadA만큼 발현되는 것으로 관찰되었으나 용해도는 CadA에 비해 상당히 부족함이 확인되었다. 따라서 LdcC의 용해도를 높이는 작업이 LdcC의 산업적 적용에 있어 중요하다. 그러나 아직까지 LdcC의 용해도를 증가시키는 연구는 보고되지 않았다. 특히, LdcC의 단백질 변이를 통해 용해도를 증가시키려는 시도는 없었다.To solve this problem, using LdcC, which has an optimal reaction pH of 7.4, can maintain activity for a relatively long period of time compared to CadA in an alkaline reaction environment. However, when LdcC was overexpressed in E. coli, it was observed that the total expression amount was as high as CadA, but its solubility was confirmed to be significantly insufficient compared to CadA. Therefore, increasing the solubility of LdcC is important for the industrial application of LdcC. However, no studies on increasing the solubility of LdcC have been reported yet. In particular, there have been no attempts to increase the solubility of LdcC through protein modification.
이러한 상황 하에서, 본 발명자들은, 공통서열(consensus sequence)을 토대로 단백질 라이브러리를 제작하여 LdcC의 용해도를 높이는 변이주들을 선별하고 해당 변이주들의 특성 분석을 진행하였다. 선별한 변이주들은 기존 야생형 대비 훨씬 뛰어난 용해도를 보이면서도 더 높은 활성을 갖는 것을 확인하였다. 뿐만 아니라 라이신 디카르복실라아제의 용해도를 결정하는 중요한 염결합(salt bridge)의 존재를 확인하였다. Under these circumstances, the present inventors created a protein library based on the consensus sequence, selected mutant strains that increase the solubility of LdcC, and analyzed the characteristics of the mutant strains. The selected mutants were confirmed to have much better solubility and higher activity compared to the existing wild type. In addition, the presence of an important salt bridge that determines the solubility of lysine decarboxylase was confirmed.
본 발명에서는 LdcC의 용해도 증가를 위해 단백질 공학적 변이를 통해 용해도가 증가된 변이주를 탐색하였다. 이러한 단백질공학적 변이를 위해서는 단백질 변이주 라이브러리 구축과 이를 빠르게 탐색할 수 있는 스크리닝 시스템의 개발이 필수이다. 따라서 본 발명에서는 단백질 공통서열을 통해 변이주 라이브러리를 구축하고 녹색 형광 단백질(green fluorescent protein)을 이용한 용해도 스크리닝 시스템을 개발하여 용해도 증가 변이주들을 선별하였다. In the present invention, in order to increase the solubility of LdcC, mutant strains with increased solubility were searched through protein engineering mutations. For these protein engineering mutations, it is essential to build a library of protein mutant strains and develop a screening system that can quickly search for them. Therefore, in the present invention, a mutant library was constructed using the protein consensus sequence and a solubility screening system using green fluorescent protein was developed to select mutant strains with increased solubility.
본 발명은 야생형보다 라이신 디카르복실라아제(LdcC)의 용해도가 증가한 변이주를 제공한다. 구체적으로, 본 발명은 야생형 대비 훨씬 뛰어난 용해도를 보이면서도 더 높은 활성을 갖는 라이신 디카르복실라아제(LdcC)의 변이주를 제공한다.The present invention provides a mutant strain with increased solubility of lysine decarboxylase (LdcC) than the wild type. Specifically, the present invention provides a mutant strain of lysine decarboxylase (LdcC) that exhibits much better solubility and higher activity compared to the wild type.
또한, 본 발명은 라이신 디카르복실라아제(LdcC)의 용해도가 증가한 변이주를 제조하는 방법을 제공한다.Additionally, the present invention provides a method for producing a mutant strain with increased solubility of lysine decarboxylase (LdcC).
또한, 본 발명은 라이신 디카르복실라아제의 용해도를 결정하는 중요한 염결합(salt bridge)을 갖는 변이주 및 이의 제조 방법을 제공한다. Additionally, the present invention provides a mutant strain having an important salt bridge that determines the solubility of lysine decarboxylase and a method for producing the same.
구체적으로, 본 발명은 하기를 제공한다.Specifically, the present invention provides:
1) 서열번호 1 (LdcC) 의 아미노산 서열에서 M711E 또는 M711D 의 변이를 포함하는, 라이신 디카르복실라아제의 변이주.1) A mutant strain of lysine decarboxylase containing the mutation M711E or M711D in the amino acid sequence of SEQ ID NO: 1 (LdcC).
2) 1) 에 있어서, V467L/T671A/V682I/M711E 의 변이를 포함하는, 라이신 디카르복실라아제의 변이주.2) The mutant strain of lysine decarboxylase according to 1), comprising the mutation V467L/T671A/V682I/M711E.
3) 1) 에 있어서, 3) In 1),
L27Q/Q109T/R143G/V467L/T671A/V682I/M711E, L27Q/Q109T/R143G/V467L/T671A/V682I/M711E,
A388R/V467L/T671A/V682I/M711E, 또는 A388R/V467L/T671A/V682I/M711E, or
L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711EL27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E
의 변이를 포함하는, 라이신 디카르복실라아제의 변이주.A mutant strain of lysine decarboxylase containing a mutation of .
4) 1) 에 있어서, 서열번호 2 내지 7 중 어느 하나의 서열을 갖는, 라이신 디카르복실라아제의 변이주. 4) The mutant strain of lysine decarboxylase according to 1), which has the sequence of any one of SEQ ID NOs: 2 to 7.
5) 1) 항에 있어서, 711 위치의 잔기와 643 위치의 아르기닌 잔기가 염결합을 이루는, 라이신 디카르복실라아제의 변이주.5) The mutant strain of lysine decarboxylase according to item 1), wherein the residue at position 711 and the arginine residue at position 643 form a salt bond.
6) 1) 내지 5) 중 어느 하나에 있어서, 야생형 라이신 디카르복실라아제(LdcC)에 비해 향상된 용해도를 갖는, 라이신 디카르복실라아제의 변이주.6) The mutant strain of lysine decarboxylase according to any one of 1) to 5), which has improved solubility compared to wild-type lysine decarboxylase (LdcC).
7) 서열번호 1 (LdcC) 의 아미노산 서열에서, 643 위치의 아르기닌 잔기와 염결합을 형성할 수 있도록 711 위치의 메티오닌 잔기를 치환하는 것을 포함하는, 라이신 디카르복실라아제 변이주의 제조 방법.7) In the amino acid sequence of SEQ ID NO: 1 (LdcC), a method for producing a lysine decarboxylase mutant, comprising substituting the methionine residue at position 711 so as to form a salt bond with the arginine residue at position 643.
8) 7) 에 있어서, 상기 치환은 M711E 또는 M711D 인, 라이신 디카르복실라아제 변이주의 제조 방법.8) The method of producing a lysine decarboxylase mutant according to 7), wherein the substitution is M711E or M711D.
본 발명은 용해도가 증가된 라이신 디카르복실라아제(LdcC) 변이주를 통해 제대로 접힌(folding) LdcC의 생산량을 증대시키고 카다베린 생산을 증대시킬 수 있다. The present invention can increase the production of properly folded LdcC and increase cadaverine production through a lysine decarboxylase (LdcC) mutant strain with increased solubility.
구체적으로, 단백질의 용해도 증가를 통해 단백질 접힘(folding)에 있어 기능성 단백질(functional protein)의 양을 늘리는 중요한 역할을 하여, 궁극적으로 단백질을 더 오랜 기간 보관하고 사용할 수 있게 해준다.Specifically, it plays an important role in increasing the amount of functional proteins in protein folding by increasing the solubility of proteins, ultimately allowing proteins to be stored and used for a longer period of time.
본 발명의 변이주들은 용해도 증가의 영향으로 용해성 추출물(soluble extract) 형태에서 야생형에 비해 더 높은 활성을 보여 카다베린 생산량 증대에 활용될 수 있다.The mutant strains of the present invention show higher activity than the wild type in the form of soluble extract due to increased solubility and can be used to increase cadaverine production.
본 발명에서 밝혀낸 중요 염결합은 추후 다른 유래의 다양한 라이신 디카르복실라아제에의 용해도 증가에도 적용될 수 있다.The important salt binding discovered in the present invention can later be applied to increase the solubility of various lysine decarboxylases of other origins.
최적 pH가 높은 LdcC의 적용은 CadA에 비해 더 오랜기간 활성을 잃지 않고 알칼리성에서 반응하게 하여 산업적으로 경제성을 확보할 수 있다.The application of LdcC, which has a high optimal pH, can ensure industrial economic feasibility by reacting in alkaline conditions without losing activity for a longer period of time compared to CadA.
도 1 은 라이신 디카르복실라아제의 반응 도식도를 나타낸다.
도 2 는 용해도 증가 변이주 스크리닝을 위한 split GFP 리포터 시스템의 모식도를 나타낸다.
도 3 은 (a) LdcC에 대한 단백질 공통서열 분석 결과 및 (b) 라이브러리 변이 분포 결과를 나타낸다.
도 4 는 (a) 순차적 유세포분리 결과 및 (b) 이를 통한 변이주 선별을 나타낸다.
도 5 는 LdcC 변이주 평가를 위한 T7 발현 벡터 시스템을 나타낸다.
도 6 은 (a) 변이주 1, 2, 3 의 SDS-PAGE 발현 양상 및 (b) 정제 후 수용성 단백질 양 분석을 나타낸다. 도 6(a) 에서, T 는 LdcC 의 전체 발현량을 확인할 수 있는 전체 분획(total fraction), S 는 수용성 단백질의 양만을 확인할 수 있는 용해성 분획(soluble fraction) 을 나타낸다.
도 7 은 (a) 변이주 4와 M711E 의 SDS-PAGE 발현 양상 및 (b) 정제 후 수용성 단백질 양 분석을 나타낸다. 도 7(a) 에서, T 는 LdcC 의 전체 발현량을 확인할 수 있는 total fraction, S 는 수용성 단백질의 양만을 확인할 수 있는 용해성 분획(soluble fraction) 을 나타낸다.
도 8 은 (a) 변이주들의 비활성도 측정 및 (b) 용해성 분획(soluble fraction)의 전체 활성(total activity) 측정을 나타낸다.
도 9 는 저농도 및 고농도에서의 변이주 활성 확인을 나타낸다. 도 9(A) 는 50mM 라이신 반응. 도 9(B) 는 1M 라이신 반응을 나타낸다. 야생형은 회색(□), 변이주1은 빨강(○), 변이주2는 파랑(△), 변이주3 은 초록(▽), 변이주4 는 보라(◇), 변이주 M711E 는 금색(◁)을 나타낸다.
도 10 은 (a) 야생형 LdcC 711번 잔기 구조 및 (b) M711E 변이주 구조를 나타낸다.
도 11 은 R643A, M711A, M711D 변이주의 SDS-PAGE 발현 양상을 나타낸다. T 는 LdcC 의 전체 발현량을 확인할 수 있는 전체 분획(total fraction), S 는 수용성 단백질의 양만을 확인할 수 있는 용해성 분획(soluble fraction) 을 나타낸다.
도 12 는 LdcC와 CadA, M711E 변이주의 염결합 위치 아미노산 서열의 비교를 나타낸다.Figure 1 shows a schematic diagram of the reaction of lysine decarboxylase.
Figure 2 shows a schematic diagram of the split GFP reporter system for screening mutant strains with increased solubility.
Figure 3 shows (a) the protein consensus sequence analysis results for LdcC and (b) the library mutation distribution results.
Figure 4 shows (a) sequential flow cytometry results and (b) mutant strain selection through this.
Figure 5 shows the T7 expression vector system for evaluating LdcC mutant strains.
Figure 6 shows (a) the SDS-PAGE expression pattern of mutant strains 1, 2, and 3 and (b) analysis of the amount of soluble protein after purification. In Figure 6(a), T represents the total fraction that can confirm the total expression level of LdcC, and S represents the soluble fraction that can confirm only the amount of soluble protein.
Figure 7 shows (a) the SDS-PAGE expression pattern of mutant strains 4 and M711E and (b) analysis of the amount of soluble protein after purification. In Figure 7(a), T represents the total fraction that can confirm the total expression level of LdcC, and S represents the soluble fraction that can confirm only the amount of water-soluble protein.
Figure 8 shows (a) measurement of specific activity of mutant strains and (b) measurement of total activity of soluble fraction.
Figure 9 shows confirmation of mutant strain activity at low and high concentrations. Figure 9(A) shows 50mM lysine reaction. Figure 9(B) shows the 1M lysine reaction. The wild type is gray (□), mutant strain 1 is red (○), mutant strain 2 is blue (△), mutant strain 3 is green (▽), mutant strain 4 is purple (◇), and mutant strain M711E is gold (◁).
Figure 10 shows (a) the structure of residue 711 of wild-type LdcC and (b) the structure of the M711E mutant strain.
Figure 11 shows the SDS-PAGE expression patterns of the R643A, M711A, and M711D mutant strains. T represents the total fraction that can confirm the total expression level of LdcC, and S represents the soluble fraction that can confirm only the amount of soluble protein.
Figure 12 shows a comparison of the salt binding site amino acid sequences of LdcC, CadA, and the M711E mutant strain.
이하, 본 발명의 내용을 상세하게 서술한다. 또한, 이하에 기재하는 발명 구성 요건의 설명은, 본 발명의 실시양태의 일례 (대표예) 이고, 본 발명은 그 요지를 초과하지 않는 한, 이들 형태에 특정되는 것은 아니다.Hereinafter, the contents of the present invention will be described in detail. In addition, the description of the invention constituent elements described below is an example (representative example) of the embodiment of the present invention, and the present invention is not specific to these forms as long as it does not exceed the gist thereof.
하나의 양태에서, 본 발명은, 야생형 LdcC (서열번호 1) 의 아미노산 서열에서, M711E 또는 M711D 의 변이를 포함하는, 라이신 디카르복실라아제의 변이주를 제공한다.In one embodiment, the present invention provides a mutant strain of lysine decarboxylase, comprising the mutation M711E or M711D in the amino acid sequence of wild-type LdcC (SEQ ID NO: 1).
본 발명의 변이주는, 야생형 LdcC (서열번호 1) 의 아미노산 서열에서 V467L/T671A/V682I/M711E 의 변이를 포함할 수 있다.The mutant strain of the present invention may include the mutations V467L/T671A/V682I/M711E in the amino acid sequence of wild-type LdcC (SEQ ID NO: 1).
본 발명의 변이주는, 야생형 LdcC (서열번호 1) 의 아미노산 서열에서 L27Q/Q109T/R143G/V467L/T671A/V682I/M711E 의 변이를 포함할 수 있다.The mutant strain of the present invention may include the mutations L27Q/Q109T/R143G/V467L/T671A/V682I/M711E in the amino acid sequence of wild-type LdcC (SEQ ID NO: 1).
본 발명의 변이주는, 야생형 LdcC (서열번호 1) 의 아미노산 서열에서 A388R/V467L/T671A/V682I/M711E 의 변이를 포함할 수 있다.The mutant strain of the present invention may include the mutations A388R/V467L/T671A/V682I/M711E in the amino acid sequence of wild-type LdcC (SEQ ID NO: 1).
본 발명의 변이주는, 야생형 LdcC (서열번호 1) 의 아미노산 서열에서 L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E 의 변이를 포함할 수 있다.The mutant strain of the present invention may include the mutations L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E in the amino acid sequence of wild-type LdcC (SEQ ID NO: 1).
본 발명의 변이주는, 하기 중 어느 하나의 아미노산 서열을 가질 수 있다: The mutant strain of the present invention may have any of the following amino acid sequences:
서열번호 1 (LdcC)의 아미노산 서열에서 L27Q/Q109T/R143G/V467L/T671A/V682I/M711E 의 변이를 갖는, 서열번호 2 의 아미노산 서열;The amino acid sequence of SEQ ID NO: 2, which has the mutations L27Q/Q109T/R143G/V467L/T671A/V682I/M711E in the amino acid sequence of SEQ ID NO: 1 (LdcC);
서열번호 1 (LdcC)의 아미노산 서열에서 A388R/V467L/T671A/V682I/M711E 의 변이를 갖는, 서열번호 3 의 아미노산 서열;The amino acid sequence of SEQ ID NO: 3, having the mutation A388R/V467L/T671A/V682I/M711E in the amino acid sequence of SEQ ID NO: 1 (LdcC);
서열번호 1 (LdcC)의 아미노산 서열에서, L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E 의 변이를 갖는, 서열번호 4 의 아미노산 서열;In the amino acid sequence of SEQ ID NO: 1 (LdcC), the amino acid sequence of SEQ ID NO: 4, having the mutations L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E;
서열번호 1 (LdcC)의 아미노산 서열에서, V467L/T671A/V682I/M711E 의 변이를 갖는, 서열번호 5 의 아미노산 서열;The amino acid sequence of SEQ ID NO: 5, having the mutations V467L/T671A/V682I/M711E in the amino acid sequence of SEQ ID NO: 1 (LdcC);
서열번호 1 (LdcC)의 아미노산 서열에서, M711E 의 변이를 갖는, 서열번호 6 의 아미노산 서열;The amino acid sequence of SEQ ID NO: 6, with the mutation M711E in the amino acid sequence of SEQ ID NO: 1 (LdcC);
서열번호 1 (LdcC)의 아미노산 서열에서, M711D 의 변이를 갖는, 서열번호 7 의 아미노산 서열.The amino acid sequence of SEQ ID NO: 7, with the mutation of M711D in the amino acid sequence of SEQ ID NO: 1 (LdcC).
본 발명의 변이주는, 711 위치의 잔기와 643 위치의 아르기닌 잔기가 염결합을 이룰 수 있다.In the mutant strain of the present invention, the residue at position 711 and the arginine residue at position 643 can form a salt bond.
본 발명에서 염결합(salt bridge)은 라이신 디카르복실라아제의 용해도를 결정하는 중요한 역할을 한다. 711 위치의 잔기가 643 위치의 아르기닌 잔기와 염결합을 형성하는 경우, 야생형에 비해 용해도가 크게 증가한다. 바람직하게는, 711 위치의 메티오닌 잔기가 글루탐산 또는 아스파르트산으로 치환되는 경우, 염결합을 형성한다. 염결합을 제거하는 경우, 수용성 LdcC 가 크게 감소한다.In the present invention, salt bridge plays an important role in determining the solubility of lysine decarboxylase. When the residue at position 711 forms a salt bond with the arginine residue at position 643, solubility increases significantly compared to the wild type. Preferably, when the methionine residue at position 711 is replaced with glutamic acid or aspartic acid, a salt bond is formed. When salt bonds are removed, water-soluble LdcC is greatly reduced.
본 발명의 변이주는, 야생형 라이신 디카르복실라아제(LdcC) 에 비해 향상된 용해도를 갖는다. 본 발명에서 용해도는 물에 대한 용해도를 의미하며, 용해도는 수용성 단백질의 양을 비교함으로써 평가할 수 있다.The mutant strain of the present invention has improved solubility compared to wild-type lysine decarboxylase (LdcC). In the present invention, solubility refers to solubility in water, and solubility can be evaluated by comparing the amount of water-soluble protein.
본 발명의 변이주는, 야생형 라이신 디카르복실라아제(LdcC) 에 비해 향상된 활성을 나타낸다. 본 발명에서 활성은 용해성 분획(soluble fraction)에 대한 전체 활성(total activity)을 측정함으로써 평가할 수 있다. 본 발명에서 용해도의 증가가 카다베린 생산량 증대에 직접적인 영향을 준다. 본 발명의 변이주는, 바람직하게는, 고농도의 라이신에서 높은 활성을 나타낼 수 있다. 상기 고농도의 라이신은, 바람직하게는 1M 이상일 수 있다.The mutant strain of the present invention exhibits improved activity compared to wild-type lysine decarboxylase (LdcC). In the present invention, activity can be evaluated by measuring the total activity of the soluble fraction. In the present invention, the increase in solubility directly affects the increase in cadaverine production. The mutant strain of the present invention can preferably exhibit high activity at high concentrations of lysine. The high concentration of lysine may preferably be 1M or more.
또 다른 양태에서, 본 발명은, 야생형 LdcC (서열번호 1) 의 아미노산 서열에서, 643 위치의 아르기닌 잔기와 염결합을 형성할 수 있도록 711 위치의 메티오닌 잔기를 치환하는 것을 포함하는, 라이신 디카르복실라아제 변이주의 제조 방법을 제공한다.In another aspect, the present invention provides a lysine decarboxylic acid comprising substituting the methionine residue at position 711 so as to form a salt bond with the arginine residue at position 643 in the amino acid sequence of wild-type LdcC (SEQ ID NO: 1). A method for producing a rase mutant strain is provided.
본 발명의 제조 방법에서, 상기 치환은 M711E 또는 M711D 일 수 있다.In the production method of the present invention, the substitution may be M711E or M711D.
본 발명의 제조 방법은, 야생형 라이신 디카르복실라아제(LdcC) 에 비해 향상된 용해도를 갖는 라이신 디카르복실라아제의 변이주를 제조할 수 있다.The production method of the present invention can produce a mutant strain of lysine decarboxylase with improved solubility compared to wild-type lysine decarboxylase (LdcC).
본 발명의 제조 방법은, 야생형 라이신 디카르복실라아제(LdcC) 에 비해 향상된 활성을 나타내는 라이신 디카르복실라아제의 변이주를 제조할 수 있다.The production method of the present invention can produce a mutant strain of lysine decarboxylase that exhibits improved activity compared to wild-type lysine decarboxylase (LdcC).
본 발명은, 단백질의 공통서열(consensus sequence)을 이용하여 공통적(consensus)이지 않은 잔기를 공통적인 아미노산으로 치환하여 용해도를 증가시킬 수 있다.The present invention can increase solubility by substituting non-consensus residues with common amino acids using the consensus sequence of proteins.
단백질 공통서열이란 다중서열정렬(multiple sequence alignment)을 토대로 정렬된 위치에 가장 많이 분포하는 아미노산 서열을 일컫는다. 단백질 공학분야에서는 공통서열로의 변이를 통해 단백질의 안정성을 증대시키는 연구를 활발히 진행하였고 실제로 많은 단백질들을 이와 같은 방법으로 안정성 증대를 이루어냈다. 또한, 일부 연구에서는 안정성의 증대와 용해도의 증대 간의 상관관계를 밝혔다. 그렇기에 단백질의 공통서열을 이용하여 공통적(consensus)이지 않은 잔기를 공통적인 아미노산으로 치환하여 용해도를 증가시키는 것이 가능하다. Protein consensus sequence refers to the amino acid sequence most distributed in the aligned position based on multiple sequence alignment. In the field of protein engineering, research has been actively conducted to increase protein stability through mutation to a common sequence, and stability has actually been improved for many proteins using this method. Additionally, some studies have revealed a correlation between increased stability and increased solubility. Therefore, it is possible to increase solubility by substituting non-consensus residues with common amino acids using the consensus sequence of the protein.
이러한 단백질공학적 변이를 위해서는 단백질 변이주 라이브러리 구축과 이를 빠르게 탐색할 수 있는 스크리닝 시스템의 개발이 필수이다. 따라서 본 발명에서는 단백질 공통서열을 통해 변이주 라이브러리를 구축하고 녹색 형광 단백질(green fluorescent protein)을 이용한 용해도 스크리닝 시스템을 개발하여 용해도 증가 변이주들을 선별하였다. For these protein engineering mutations, it is essential to build a library of protein mutant strains and develop a screening system that can quickly search for them. Therefore, in the present invention, a mutant library was constructed using the protein consensus sequence and a solubility screening system using green fluorescent protein was developed to select mutant strains with increased solubility.
녹색 형광 단백질은 바이오 분야 전반에서 이미징이나 리포터 유전자로 널리 사용되는 단백질로 발현량이 증가할수록 높아지는 형광의 세기로 유전자의 발현량 등을 분석할 수 있다. 11개의 베타 스트랜드(beta strand)가 하나의 배럴(barrel) 구조를 이루고 있는 녹색 형광 단백질은 11번째 스트랜드(strand)가 분리되면 형광을 띄지 않게 된다. 이를 응용하여 11번 strand(GFP11)를 분리하여 목적단백질의 C 말단에 붙여 용해도의 리포터 태그로써 사용하는 split GFP가 개발되었다. GFP11은 목적단백질의 용해도가 증가하여 제대로 접힌 단백질이 증가할수록 1-10 스트랜드(strand)(GFP1-10)와의 결합이 증가해 형광이 증가하여 용해도가 증가한 목적단백질을 선별하는 것이 가능하다.Green fluorescent protein is a protein widely used as an imaging or reporter gene throughout the biofield. The expression level of a gene can be analyzed through the intensity of fluorescence, which increases as the expression level increases. Green fluorescent protein, which consists of 11 beta strands forming a barrel, loses fluorescence when the 11th strand is separated. By applying this, split GFP was developed in which strand 11 (GFP11) was isolated and attached to the C terminus of the target protein to use it as a solubility reporter tag. GFP11 increases the solubility of the target protein, and as the number of properly folded proteins increases, the binding to strands 1-10 (GFP1-10) increases, resulting in increased fluorescence, making it possible to select target proteins with increased solubility.
본 발명에서 라이신 디카르복실라아제의 공통서열은 하기와 같다:The common sequence of lysine decarboxylase in the present invention is as follows:
LdcC 의 L27 위치의 공통서열 E, Q, K, A, TConsensus sequence E, Q, K, A, T at position L27 of LdcC
LdcC 의 Q109 위치의 공통서열 T, A, S, L, MConsensus sequence T, A, S, L, M at position Q109 of LdcC
LdcC 의 M117 위치의 공통서열 I, VConsensus sequences I and V of the M117 position of LdcC
LdcC 의 R143 위치의 공통서열 G, S, Y, H, AConsensus sequence G, S, Y, H, A at position R143 of LdcC
LdcC 의 L238 위치의 공통서열 V, I, AConsensus sequence V, I, A at position L238 of LdcC
LdcC 의 N255 위치의 공통서열 S, T, VConsensus sequence S, T, V at position N255 of LdcC
LdcC 의 T293 위치의 공통서열 PConsensus sequence P at position T293 of LdcC
LdcC 의 Q294 위치의 공통서열 L, N, GConsensus sequence L, N, G at position Q294 of LdcC
LdcC 의 A388 위치의 공통서열 R, T, LConsensus sequence R, T, L at position A388 of LdcC
LdcC 의 V467 위치의 공통서열 LConsensus sequence L at position V467 of LdcC
LdcC 의 T671 위치의 공통서열 P, A, SConsensus sequence P, A, S at position T671 of LdcC
LdcC 의 V682 위치의 공통서열 I, FConsensus sequence I, F of position V682 of LdcC
LdcC 의 M711 위치의 공통서열 E, Q, N, D, AConsensus sequence E, Q, N, D, A at position M711 of LdcC
[실시예][Example]
본 발명의 구체적인 방법을 실시예로서 상세히 설명하나, 본 발명의 기술적 범위가 이들 실시예에 한정되는 것은 아니다.Specific methods of the present invention will be described in detail as examples, but the technical scope of the present invention is not limited to these examples.
실시예 1: 용해도 증가 LdcC를 선별하기 위한 리포터 시스템 구축Example 1: Construction of a reporter system to select LdcC with increased solubility
단백질 용해도 증가를 빠르게 스크리닝하기 위해서 녹색형광단백질(GFP)을 이용한 스크리닝 시스템 구축을 우선적으로 진행하였다. GFP를 용해도 리포터 시스템으로 응용하기 위해 11번째 스트랜드(strand)를 목적단백질의 태그처럼 사용하는 split GFP 시스템을 구축하였다. 이를 위해 1번부터 10번까지의 스트랜드(strand)(GFP1-10)을 pET28a 벡터에 클로닝하여 T7 프로모터로 발현되도록 하였으며 LdcC와 GFP11을 (GGGS)2 링커로 이어 붙여 pBAD/HisA 벡터에 클로닝하여 아라비노오스(arabinose) 프로모터로 발현되도록 제작하였다 (도 2). 기존의 두 벡터는 호환되지 않는 복제기점을 가지고 있기에 pET28a의 복제기점을 p15A로 교체하는 작업을 진행하였다. 이 시스템을 통해 LdcC의 변이주의 용해도가 높을수록 단백질 접힘이 더 잘 일어나 더 높은 형광을 띄게 되고 이를 통해 용해도가 증가한 변이주를 스크리닝 할 수 있다.In order to quickly screen for increased protein solubility, we first conducted construction of a screening system using green fluorescent protein (GFP). To apply GFP as a solubility reporter system, a split GFP system was constructed using the 11th strand as a tag for the target protein. For this purpose, strands 1 to 10 (GFP1-10) were cloned into the pET28a vector to be expressed with the T7 promoter, and LdcC and GFP11 were connected with a (GGGS) 2 linker and cloned into the pBAD/HisA vector. It was designed to be expressed with an arabinose promoter (Figure 2). Since the two existing vectors have incompatible replication origins, we proceeded with replacing the replication origin of pET28a with p15A. Through this system, the higher the solubility of the LdcC mutant strain, the more likely it is to fold the protein, resulting in higher fluorescence. This allows screening for mutant strains with increased solubility.
실시예 2: 단백질 공통서열(consensus sequence) 분석 및 이를 통한 라이브러리 구축Example 2: Protein consensus sequence analysis and library construction through this
단백질 공통서열 분석을 위해 Consensus Finder (문헌 [Biochemistry 2017, 56, 50, 6521-6532] 참조)라는 컴퓨터 프로그램을 이용하였다. 위 프로그램으로 총 219개의 다양한 유래 라이신 디카르복실라아제 서열에 대해 다중서열정렬(multiple sequence alignment)을 진행하고 서열의 각 위치에 대한 단백질 공통서열을 분석하였다. 이를 토대로 LdcC가 총 13개 위치에 대해 공통서열이 아닌 다른 아미노산으로 치환되어 있는 것을 확인하였고 해당 위치들에 대한 공통서열의 정보를 얻었다 (도 3(a)). LdcC에 대한 공통서열 라이브러리 구축을 위해 Darwin assembly라는 방법을 이용하였다 (문헌 [Nucleic Acids Research, Volume 46, Issue 8, 4 May 2018, Page e51] 참조). 예측한 공통서열 전부를 라이브러리로 제작하기에는 크기가 너무 커 현실적으로 제작이 어렵기에 각 위치마다 최대 3개까지 총 13개 위치 33개의 변이로 제한하였다. 제작한 라이브러리는 DH5a에 전기천공법(electroporation)으로 형질전환하여 100μg/mL의 암피실린이 녹아있는 한천 플레이트(agar plate)에 도포하여 37℃에서 하루 배양하였다. 이를 통해 얻은 콜로니 일부를 DNA 시퀀싱을 통해 해당 위치마다 변이가 이루어졌는지 확인작업을 진행하였고 변이가 원하는 위치에만 일어남을 확인하였다 (도 3(b)).For protein consensus sequence analysis, a computer program called Consensus Finder (see literature [Biochemistry 2017, 56, 50, 6521-6532]) was used. Using the above program, multiple sequence alignment was performed on a total of 219 lysine decarboxylase sequences from various sources, and the protein consensus sequence for each position in the sequence was analyzed. Based on this, it was confirmed that a total of 13 positions in LdcC were substituted with amino acids other than the common sequence, and information on the common sequence for those positions was obtained (Figure 3(a)). To construct a common sequence library for LdcC, a method called Darwin assembly was used (see the literature [Nucleic Acids Research, Volume 46, Issue 8, 4 May 2018, Page e51]). Because the size of all predicted common sequences was too large to produce as a library, it was realistically difficult to produce, so it was limited to a total of 33 mutations in 13 positions, up to 3 at each position. The prepared library was transformed into DH5a by electroporation, applied to an agar plate containing 100 μg/mL ampicillin, and cultured at 37°C for one day. DNA sequencing of some of the colonies obtained through this process was performed to confirm whether mutations occurred at each location, and it was confirmed that mutations occurred only at the desired location (Figure 3(b)).
실시예 3: 유세포분석기(FACS)를 이용한 용해도 증가 변이주 스크리닝Example 3: Screening of mutant strains with increased solubility using flow cytometry (FACS)
실시예 2 에서 제작하였던 라이브러리를 GFP1-10벡터와 함께 BL21(DE3) 균주로 형질전환하여 이를 유세포분석기를 통한 형광 스크리닝에 사용하였다. 형질전환한 세포를 100μg/mL 암피실린과 100μg/mL 카나마이신이 녹아있는 액체 LB에 하룻밤동안 37℃에서 배양한 후 250mL 삼각플라스크에 50mL 수준으로 스케일업을 진행하였다. 이후 OD600 ~0.2-0.4에서 아라비노오스(arabinose) 0.2%로 유도(induction)를 37℃에서 4시간 진행하고 IPTG 1mM로 4시간 더 진행하였다. 유도(Induction)가 끝난 배양액은 4000rpm에서 10분간 원심분리하여 세포를 얻었고 50mM PBS(phosphate buffered saline) pH7.4로 세척하였다. 세척한 세포는 107 cells/mL 이하로 희석하여 유세포분석에 사용하였다. The library prepared in Example 2 was transformed into the BL21(DE3) strain along with the GFP1-10 vector, and used for fluorescence screening using a flow cytometer. The transformed cells were cultured overnight at 37°C in liquid LB containing 100 μg/mL ampicillin and 100 μg/mL kanamycin, and then scaled up to 50 mL in a 250 mL Erlenmeyer flask. Afterwards, induction was carried out with arabinose 0.2% at OD 600 ~0.2-0.4 for 4 hours at 37°C, and then with IPTG 1mM for another 4 hours. After induction, the culture medium was centrifuged at 4000 rpm for 10 minutes to obtain cells, and washed with 50mM PBS (phosphate buffered saline) pH 7.4. Washed cells were diluted to 10 7 cells/mL or less and used for flow cytometry.
유세포분석기 (Bio-rad; S3e)에서 FSC-A vs. SSC-A, SSC-H vs. SSC-W, FSC-H vs. FSC-A 게이팅을 통해 최대한 단일세포만 분석할 수 있도록 불순물을 제거하고 분석을 진행하였다. 형광값의 상위 5%를 선별(sorting)하였으며 약 1만개의 세포를 선별하였다. 선별한 세포는 액체 LB에서 37℃에서 1시간 회복(recovery)후 고체 LB배지에 도포하였다. 위 선별 작업을 총 3번 순차적으로 반복하여 형광값이 높아진 변이주들을 선별하였으며 각각의 선별 과정에서 전체적인 형광값이 점차 증가하는 것을 확인하였다 (도 4(a)).FSC-A vs. FSC-A in flow cytometry (Bio-rad; S3e). SSC-A, SSC-H vs. SSC-W, FSC-H vs. Impurities were removed and analysis was performed through FSC-A gating so that only single cells could be analyzed as much as possible. The top 5% of fluorescence values were sorted, and approximately 10,000 cells were selected. The selected cells were recovered in liquid LB at 37°C for 1 hour and then applied to solid LB medium. The above selection process was sequentially repeated a total of three times to select mutants with increased fluorescence values, and it was confirmed that the overall fluorescence value gradually increased during each selection process (Figure 4(a)).
3번의 선별과정 후 얻은 세포들은 고체 LB배지를 이용한 형광 스크리닝을 진행하였다. 100μg/mL 암피실린과 100μg/mL 카나마이신을 녹인 고체 LB배지위에 나이트로셀룰로오스 필터종이를 얹고 세포를 도포하여 37℃에서 하룻밤 키웠다. 나이트로셀룰로오스 필터종이 위에 자란 콜로니들을 필터째로 100μg/mL 암피실린, 100μg/mL 카나마이신, 0.2% 아라비노오스(arabinose)를 첨가한 고체 LB배지로 옮겨 다시 37℃에서 2시간동안 GFP11의 유도(induction)를 진행하였다. 이후 필터종이를 100μg/mL 암피실린, 100μg/mL 카나마이신, 1mM IPTG 고체 LB배지로 옮겨 37℃에서 2시간동안 GFP1-10의 유도(induction)를 진행하였다. 위 작업을 거친 후 형광분광광도계를 사용하여 콜로니의 형광을 측정하였다. 측정한 형광은 ImageJ 프로그램으로 형광세기를 분석하여 세기가 가장 강한 변이주 3개를 선별하였다 (도 4(b)). 선별한 변이주들은 DNA 시퀀싱을 진행하여 각각 다음과 같은 변이를 갖는 것을 확인하였다. Cells obtained after three selection processes were subjected to fluorescence screening using solid LB medium. Nitrocellulose filter paper was placed on solid LB medium containing 100 μg/mL ampicillin and 100 μg/mL kanamycin, and cells were spread and grown overnight at 37°C. Colonies grown on nitrocellulose filter paper were transferred to solid LB medium supplemented with 100 μg/mL ampicillin, 100 μg/mL kanamycin, and 0.2% arabinose with the filter intact, and then incubated at 37°C for 2 hours for induction of GFP11. ) was carried out. Afterwards, the filter paper was transferred to solid LB medium containing 100μg/mL ampicillin, 100μg/mL kanamycin, and 1mM IPTG, and induction of GFP1-10 was performed at 37°C for 2 hours. After completing the above operations, the fluorescence of the colonies was measured using a fluorescence spectrophotometer. The measured fluorescence was analyzed using the ImageJ program, and the three mutants with the strongest intensity were selected (Figure 4(b)). The selected mutant strains underwent DNA sequencing and were confirmed to have the following mutations.
변이주 1: L27Q/Q109T/R143G/V467L/T671A/V682I/M711E,Mutant 1: L27Q/Q109T/R143G/V467L/T671A/V682I/M711E,
변이주 2: A388R/V467L/T671A/V682I/M711E,Mutant 2: A388R/V467L/T671A/V682I/M711E,
변이주 3: L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E.Mutant 3: L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E.
실시예 4: 변이주 평가용 벡터 제작 Example 4: Production of vectors for evaluating mutant strains
변이주 선별에 사용한 벡터는 발현의 세기가 약한 pBAD/HisA 벡터이기에 세계적으로 많이 사용하는 과발현 벡터인 pET28a 벡터로 변이주를 옮겨 평가하고자 하였다. 실시예 3 에서 찾은 3개의 변이주와 이후 제작한 모든 변이주는 pET28a 벡터에서 평가를 진행하였다. 변이주들은 NcoI과 XhoI 제한효소를 처리하여 pET28a 벡터에 라이게이션(ligation)하여 제작하였다 (도 5). Since the vector used to select mutant strains was the pBAD/HisA vector, which has a weak expression intensity, we attempted to transfer and evaluate the mutant strains into the pET28a vector, an overexpression vector widely used worldwide. The three mutant strains found in Example 3 and all mutant strains produced thereafter were evaluated using the pET28a vector. Mutants were produced by treatment with NcoI and XhoI restriction enzymes and ligated into the pET28a vector (Figure 5).
실험예 1: 변이주 용해도 평가Experimental Example 1: Evaluation of solubility of mutant strains
실시예 4 에서 제작한 변이주 벡터를 BL21(DE3)에 형질전환하여 용해도 평가를 진행하였다. 100μg/mL 카나마이신이 녹아있는 액체 LB에 하룻밤동안 37℃에서 배양한 변이주들 및 야생형을 250mL 삼각플라스크에서 50mL 수준으로 스케일업을 진행하여 OD600 ~0.6-0.8까지 키웠다. IPTG 0.1mM을 첨가하여 18℃에서 하룻밤동안 유도(induction)를 진행한 후 4000rpm에서 10분간 원심분리하여 세포를 수확하였다. 수확한 세포는 50mM sodium phosphate buffer pH7.4로 세척한 후 각 변이주들 및 야생형을 OD600 30에 맞춰 5mL에 녹여 초음파 파쇄기로 세포 분쇄를 진행하였다. 세포 파쇄액은 LdcC의 전체 발현량을 알아볼 수 있는 전체 분획(total fraction)으로 일부 사용하고 나머지를 16000rpm으로 30분간 원심분리하여 수용성 단백질의 양만을 확인할 수 있는 용해성 분획(soluble fraction)을 얻었다. 분리한 전체(total) 및 용해성(soluble) 분획은 2x SDS 젤-로딩 염료와 100mM DTT(dithiothreitol)과 함께 튜브에 넣어 10분간 고온에서 끓여 SDS-PAGE분석을 위해 단백질 접힘을 풀었다. 이후 12% SDS-PAGE gel에 5μL씩 넣어 SDS-PAGE 분석을 수행하였다. The mutant vector prepared in Example 4 was transformed into BL21 (DE3) and solubility was evaluated. Mutants and wild types cultured overnight at 37°C in liquid LB containing 100 μg/mL kanamycin were scaled up to 50 mL in a 250 mL Erlenmeyer flask and grown to OD 600 ~0.6-0.8. After induction was performed overnight at 18°C by adding 0.1mM of IPTG, cells were harvested by centrifugation at 4000rpm for 10 minutes. The harvested cells were washed with 50mM sodium phosphate buffer pH 7.4, and then each mutant strain and wild type were dissolved in 5 mL to an OD of 600 30, and the cells were pulverized using an ultrasonicator. A portion of the cell lysate was used as a total fraction to determine the total expression level of LdcC, and the remainder was centrifuged at 16000 rpm for 30 minutes to obtain a soluble fraction to determine only the amount of soluble protein. The separated total and soluble fractions were placed in a tube with 2x SDS gel-loading dye and 100mM DTT (dithiothreitol) and boiled at high temperature for 10 minutes to unfold the protein for SDS-PAGE analysis. Afterwards, SDS-PAGE analysis was performed by adding 5 μL each to a 12% SDS-PAGE gel.
실시예 3 에서 선별한 변이주 1, 2, 3을 SDS-PAGE를 통해 확인한 결과 야생형 대비 모두 용해도가 크게 증가함을 확인하였다 (도 6(a)). 또한 His-tag을 이용한 단백질 정제를 진행한 후 정제된 수용성 단백질의 양을 비교하였다. Ni-NTA 비드(bead) 500μL를 His-tag 컬럼에 넣고 50mM 인산나트륨 버퍼(sodium phosphate buffer) pH7.4에 300mM 염화나트륨과 5mM 이미다졸을 녹인 버퍼(버퍼1)를 5mL씩 두차례 흘려 bead를 활성화시켜주었다. 이후 용해성 분획(soluble fraction)을 비드(bead)에 1시간가량 바인딩 시켜준 후 바인딩 되지 않은 단백질들을 제거해주었다. 버퍼1에서 이미다졸 양만 30mM로 늘린 버퍼2 5mL로 한차례 세척한 후 다시 버퍼2에서 이미다졸양을 50mM로 늘린 버퍼3 5mL로 한차례 더 세척하였다. 50mM 인산나트륨 버퍼(sodium phosphate buffer) pH7.4에 이미다졸 250mM을 녹인 버퍼4 500μL로 분리 정제한 단백질을 얻었다. 정제한 단백질의 양을 브래드퍼드 단백질 정량법으로 3차례 측정하고 세포건조무게를 측정하여 이로 나누어 주었다 (도 6(b)). 단백질양을 정제 후 단순 농도로 비교할 경우 정제 후 수용성 단백질의 절대량이 많으나 부피가 커 농도가 낮은 경우나 반대로 절대량은 적지만 부피가 작아 농도가 높아지는 오류를 범할 수 있어 세포건조무게로 나누는 정규화(normalization) 작업을 진행하였다. 이를 통해 변이주 1, 2, 3은 각각 야생형 대비 10배, 6.1배, 5배 수용성 단백질의 양이 증가하여 6.41mg/g DCW, 3.92mg/g DCW, 3.24mg/g DCW의 정제된 LdcC를 얻을 수 있음을 확인하였다.As a result of confirming the mutant strains 1, 2, and 3 selected in Example 3 through SDS-PAGE, it was confirmed that the solubility of all strains was significantly increased compared to the wild type (Figure 6(a)). In addition, after protein purification using His-tag, the amount of purified water-soluble protein was compared. Put 500 μL of Ni-NTA beads on a His-tag column and activate the beads by flowing 5 mL each of buffer (buffer 1) containing 300mM sodium chloride and 5mM imidazole in 50mM sodium phosphate buffer pH 7.4 twice. I ordered it. Afterwards, the soluble fraction was bound to beads for about 1 hour, and then unbound proteins were removed. It was washed once with 5 mL of buffer 2 in which only the amount of imidazole in buffer 1 was increased to 30mM, and then washed again with 5 mL of buffer 3 in which the amount of imidazole in buffer 2 was increased to 50mM. Proteins were separated and purified with 500 μL of buffer 4 containing 250mM imidazole dissolved in 50mM sodium phosphate buffer pH 7.4. The amount of purified protein was measured three times using the Bradford protein quantification method, and the dry cell weight was measured and divided (Figure 6(b)). When comparing the amount of protein with simple concentration after purification, an error may be made in which the absolute amount of water-soluble protein after purification is large, but the concentration is low due to the large volume, or, conversely, the absolute amount is low, but the concentration is high due to the small volume, so normalization by dividing by cell dry weight is possible. ) Work was carried out. Through this, mutant strains 1, 2, and 3 increased the amount of soluble protein by 10, 6.1, and 5 times, respectively, compared to the wild type, resulting in purified LdcC of 6.41 mg/g DCW, 3.92 mg/g DCW, and 3.24 mg/g DCW. It was confirmed that it was possible.
추가적으로 변이주 4 와 M711E 변이주에 대해서도 같은 방식으로 용해도 평가를 수행하였다. 변이주 4는 변이주 1, 2, 3이 공통적으로 가지고 있는 4개의 변이로 구성된 변이주로 V467L/T671A/V682I/M711E의 변이로 구성된다. 위 변이주 1, 2, 3에 대한 평가 방법과 동일하게 용해도 평가를 수행하였고 변이주 4와 M711E 또한 야생형 대비 용해도가 증가함을 확인하였다 (도 7). 변이주 4와 M711E는 야생형 대비 각각 3.9배와 10.8배 증가한 2.49mg/g DCW와 6.95mg/g DCW의 정제된 수용성 LdcC를 얻을 수 있었다.Additionally, solubility evaluation was performed in the same manner for mutant strain 4 and M711E mutant strain. Mutant strain 4 is a mutant strain composed of four mutations common to mutant strains 1, 2, and 3, and consists of the mutations V467L/T671A/V682I/M711E. Solubility evaluation was performed in the same manner as the evaluation method for mutant strains 1, 2, and 3 above, and it was confirmed that mutant strains 4 and M711E also had increased solubility compared to the wild type (Figure 7). Mutant strains 4 and M711E were able to obtain purified water-soluble LdcC of 2.49 mg/g DCW and 6.95 mg/g DCW, which is a 3.9-fold and 10.8-fold increase, respectively, compared to the wild type.
실험예 2: 변이주 활성 평가Experimental Example 2: Evaluation of mutant strain activity
용해도 증가가 실제 카다베린 생산에 영향을 주는지 알아보기 위해 변이주들의 활성 평가를 진행하였다. LdcC의 비활성도(specific activity)를 측정하기 위해 다음과 같은 반응 조건을 사용하였다. 100nM의 효소에 기질인 라이신 50mM과 조효소인 피리독살인산(PLP) 0.1mM를 넣어 1시간동안 50℃에서 반응을 진행하였다. 버퍼는 50mM 인산나트륨 버퍼(sodium phosphate buffer) pH7.4를 사용하여 pH를 맞추었다. 반응 후 반응액은 10분간 끓인 후 16000rpm으로 10분간 원심분리하여 사용한 효소를 반응액과 분리하였다. 이후 상등액의 유도체화를 진행하였다 (문헌 [Journal of Molecular Catalysis B: Enzymatic 115 (2015) 151-154] 참조). 유도체화 후 고성능 액체 크로마토그래피(HPLC)로 생산한 카다베린의 양을 분석하였다. 변이주들에 대해 비활성도를 분석한 결과 M711E만이 야생형에 비해 높은 비활성도를 보였으며 다른 변이주들은 야생형에 비해 비활성도가 낮음을 확인하였다 (도 8(a)). 야생형이 44.2μmole/min/mg의 비활성도를 보일 때 변이주 1, 2, 3, 4는 절반 수준인 18.5, 22.9, 28.9, 23.6μmole/min/mg의 비활성도를 각각 나타내었으나 M711E는 야생형 대비 2배 수준인 84.1μmole/min/mg의 비활성도를 나타내었다. The activity of mutant strains was evaluated to determine whether increased solubility affects actual cadaverine production. The following reaction conditions were used to measure the specific activity of LdcC. 50mM of lysine, a substrate, and 0.1mM of pyridoxal phosphate (PLP), a coenzyme, were added to 100nM of enzyme, and the reaction was performed at 50°C for 1 hour. The pH of the buffer was adjusted using 50mM sodium phosphate buffer pH 7.4. After reaction, the reaction solution was boiled for 10 minutes and then centrifuged at 16000 rpm for 10 minutes to separate the used enzyme from the reaction solution. Afterwards, derivatization of the supernatant was performed (see document [ Journal of Molecular Catalysis B: Enzymatic 115 (2015) 151-154]). After derivatization, the amount of cadaverine produced was analyzed by high-performance liquid chromatography (HPLC). As a result of analyzing the specific activity of the mutant strains, it was confirmed that only M711E showed higher specific activity than the wild type, and other mutant strains showed lower specific activity than the wild type (Figure 8(a)). When the wild type showed a specific activity of 44.2 μmole/min/mg, mutant strains 1, 2, 3, and 4 showed a specific activity of 18.5, 22.9, 28.9, and 23.6 μmole/min/mg, respectively, which is half the level, but M711E showed a specific activity of 2 μmole/min/mg compared to the wild type. It showed a specific activity of 84.1μmole/min/mg, which is twice the level.
비활성도는 낮지만 용해도의 증가가 실질적인 카다베린의 생산량 증대와 연결될 수 있기에 용해성 분획(soluble fraction) 형태로 전체 활성(total activity)을 측정하였다. 정제하지 않은 용해성 분획(soluble fraction) 5% (v/v)을 라이신 1M과 PLP 0.1mM를 넣어 30분간 50℃에서 50mM 인산나트륨 버퍼(sodium phosphate buffer) pH7.4를 사용하여 반응을 진행하였다. 이후 비활성도 측정 실험과 마찬가지로 유도체화 및 분석을 진행하였다. 분석 결과 모든 변이주들이 야생형에 비해 높은 전체 활성(total activity)을 보이는 것을 확인하였고 특히 가장 생산량이 높은 변이주 3은 야생형 대비 4.9배 높은 카다베린 생산량을 보였다 (도 8(b)). 변이주 1, 2, 3, 4, M711E는 각각 197.8, 389.0, 423.2, 269.3, 122.1U의 전체 활성(total activity)을 보여 야생형에 비해 각각 2.3, 4.5, 4.9, 3.1, 1.4배 높은 카다베린 생산량을 나타내었다. 이 결과를 토대로 용해도의 증가가 카다베린 생산량 증대에 직접적인 영향을 준다는 것을 확인함과 동시에 본 발명에서 개발한 변이주들이 야생형에 비해 궁극적으로 더 높은 카다베린 생산량을 보이는 것을 확인하였다.Although the specific activity is low, the increase in solubility can be linked to a substantial increase in cadaverine production, so the total activity was measured in the form of soluble fraction. 5% (v/v) of the unpurified soluble fraction was added with 1M lysine and 0.1mM PLP, and the reaction was performed using 50mM sodium phosphate buffer, pH 7.4, at 50°C for 30 minutes. Afterwards, derivatization and analysis were performed as in the specific activity measurement experiment. As a result of the analysis, it was confirmed that all mutant strains showed higher total activity than the wild type, and in particular, mutant strain 3, which had the highest production, showed cadaverine production 4.9 times higher than the wild type (Figure 8(b)). Mutants 1, 2, 3, 4, and M711E showed total activity of 197.8, 389.0, 423.2, 269.3, and 122.1U, respectively, producing cadaverine production 2.3, 4.5, 4.9, 3.1, and 1.4 times higher than the wild type, respectively. indicated. Based on these results, it was confirmed that increased solubility has a direct effect on increasing cadaverine production, and at the same time, it was confirmed that the mutant strains developed in the present invention ultimately showed higher cadaverine production compared to the wild type.
실험예 3: 농도에 따른 활성 비교Experimental Example 3: Comparison of activity according to concentration
변이주 1, 2, 3, 4, M711E에 대하여 저농도 및 고농도에서의 변이주 활성을 확인하였다. 저농도 라이신 반응은 라이신 50mM과 피리독살인산(PLP) 0.1mM로 진행하였으며, 50℃에서 5% (v/v) 용해성 분획을 사용하여 진행하였다. 고농도 라이신 반응은 이외 모든 조건은 동일하나 라이신만 1M로 진행하였다. 분석 결과 고농도 (1M) 라이신에서 모든 변이주들이 야생형보다 높은 카다베린 생산량을 나타내었다. M711E 변이주는 저농도 (50mM) 라이신 반응에서는 높은 활성을 보였으나(도 9A), 고농도 (1M) 라이신 반응에서는 다른 변이주(변이주 1, 2, 3, 4)들의 활성이 더 높았다(도 9B).The activity of mutant strains 1, 2, 3, 4, and M711E was confirmed at low and high concentrations. The low-concentration lysine reaction was carried out with 50mM of lysine and 0.1mM of pyridoxal phosphate (PLP), and was carried out using 5% (v/v) soluble fraction at 50°C. All other conditions for the high-concentration lysine reaction were the same, but only lysine was used at 1M. As a result of the analysis, all mutant strains showed higher cadaverine production than the wild type at high concentration (1M) lysine. The M711E mutant showed high activity in low concentration (50mM) lysine reaction (Figure 9A), but other mutants (mutants 1, 2, 3, and 4) showed higher activity in high concentration (1M) lysine reaction (Figure 9B).
실험예 4: 변이주 용해도 증가 원인 규명 및 실험적 증명Experimental Example 4: Identification and experimental proof of the cause of increased solubility of mutant strains
M711E 변이주의 구조를 LdcC 야생형의 PDB 구조를 UCSF Chimera 프로그램의 인실리코 변이 기능을 통해 제작하여 확인하였다. 이를 통해 M711E로의 변이가 구조적으로 근처에 있는 R643 잔기와 염결합(salt bridge)을 할 가능성을 확인하였다 (도 10). The structure of the M711E mutant strain was confirmed by constructing the PDB structure of the LdcC wild type using the in silico mutation function of the UCSF Chimera program. Through this, it was confirmed that the mutation to M711E may form a salt bridge with the structurally nearby R643 residue (FIG. 10).
해당 염결합이 실제 LdcC의 용해도에 영향을 미치는지 알아보기 위해 해당 염결합을 제거하는 R643A와 M711A 변이 및 염결합은 가능하나 아미노산의 길이가 달라 염결합에 영향을 주는 M711D 변이주를 제작하고 SDS-PAGE 분석을 통해 용해도의 변화를 확인하였다 (도 11). 확인결과 예상대로 염결합을 제거한 R643A와 M711A 변이주는 수용성 LdcC가 모두 사라지는 것을 확인하였고 염결합이 가능한 M711D는 M711E보다는 낮지만 용해도가 야생형에 비해 크게 증가하는 것을 확인하였다. To determine whether the salt binding actually affects the solubility of LdcC, the R643A and M711A mutations, which remove the salt binding, and the M711D mutant, which is capable of salt binding but affects salt binding due to a different amino acid length, were created and SDS-PAGE Changes in solubility were confirmed through analysis (Figure 11). As a result, as expected, the R643A and M711A mutants with salt binding removed showed that all water-soluble LdcC disappeared, and the solubility of M711D, which is capable of salt binding, was confirmed to be lower than M711E, but significantly increased compared to the wild type.
특히 용해도가 높은 것으로 알려진 CadA에도 해당 염결합이 동일하게 존재하는 것을 확인하였다 (도 12). 따라서 본 발명은 해당 위치의 염결합이 라이신 디카르복실라아제의 용해도에 영향을 크게 미치는 것을 확인하였다. In particular, it was confirmed that the same salt bond exists in CadA, which is known to have high solubility (Figure 12). Therefore, the present invention confirmed that salt binding at the corresponding position greatly affects the solubility of lysine decarboxylase.
<110> Seoul National University R&DB Foundation <120> DEVELOPMENT OF LYSINE DECARBOXYLASE MUTANTS WITH INCREASED SOLUBLE EXPRESSION AND THEIR APPLICATIONS <130> Y22KP-111 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 713 <212> PRT <213> Escherichia coli <400> 1 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Val Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Thr Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Met Ala Gly 705 710 <210> 2 <211> 713 <212> PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (L27Q/Q109T/R143G/V467L/T671A/V682I/M711E) <400> 2 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Gln Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Thr Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Gly Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 3 <211> 713 <212> PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (A388R/V467L/T671A/V682I/M711E) <400> 3 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Arg Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 4 <211> 713 <212> PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E) <400> 4 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Lys Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Gly Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Arg Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 5 <211> 713 <212> PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (V467L/T671A/V682I/M711E) <400> 5 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 6 <211> 713 <212> PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (M711E) <400> 6 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Val Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Thr Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 7 <211> 713 <212> PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (M711D) <400> 7 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Val Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Thr Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Asp Ala Gly 705 710 <110> Seoul National University R&DB Foundation <120> DEVELOPMENT OF LYSINE DECARBOXYLASE MUTANTS WITH INCREASED SOLUBLE EXPRESSION AND THEIR APPLICATIONS <130> Y22KP-111 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 713 <212> PRT <213> Escherichia coli <400> 1 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Val Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Thr Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Met Ala Gly 705 710 <210> 2 <211> 713 <212 > PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (L27Q/Q109T/R143G/V467L/T671A/V682I/M711E) <400> 2 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Gln Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Thr Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Gly Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 3 <211> 713 <212 > PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (A388R/V467L/T671A/V682I/M711E) <400> 3 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Arg Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 4 <211> 713 <212 > PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E) <400> 4 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Lys Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Gly Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Arg Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 5 <211> 713 <212> PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase (LdcC) mutant (V467L/T671A/V682I/M711E) <400> 5 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Leu Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Ala Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Ile Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 6 <211> 713 <212 > PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase (LdcC) mutant (M711E) <400> 6 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Val Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Thr Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700 Arg Val Arg Val Leu Lys Glu Ala Gly 705 710 <210> 7 <211> 713 <212 > PRT <213> Artificial Sequence <220> <223> Lysine dearboxylase(LdcC) mutant (M711D) <400> 7 Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp 1 5 10 15 Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Leu Ala Gln Gly Phe Gln 20 25 30 Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile Glu His 35 40 45 Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu Tyr Ser Leu 50 55 60 Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr Leu Pro Leu Tyr 65 70 75 80 Ala Phe Ile Asn Thr His Ser Thr Met Asp Val Ser Val Gln Asp Met 85 90 95 Arg Met Ala Leu Trp Phe Phe Glu Tyr Ala Leu Gly Gln Ala Glu Asp 100 105 110 Ile Ala Ile Arg Met Arg Gln Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125 Thr Pro Pro Phe Thr Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140 Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys 145 150 155 160 Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu 165 170 175 Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu Leu Asp 180 185 190 His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe 195 200 205 Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn Gly Thr Ser Thr Ser Asn 210 215 220 Lys Ile Val Gly Met Tyr Ala Ala Pro Ser Gly Ser Thr Leu Leu Ile 225 230 235 240 Asp Arg Asn Cys His Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255 Val Val Pro Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270 Gly Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280 285 Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr 290 295 300 Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile Lys Gln 305 310 315 320 Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335 Thr His Phe His Pro Ile Tyr Gln Gly Lys Ser Gly Met Ser Gly Glu 340 345 350 Arg Val Ala Gly Lys Val Ile Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365 Leu Ala Ala Leu Ser Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380 Asp Glu Glu Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser 385 390 395 400 Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu 405 410 415 Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu Arg Ala 420 425 430 Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu Glu Ser Asp Gly 435 440 445 Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln Val Asp Glu Ala Glu Cys 450 455 460 Trp Pro Val Ala Pro Gly Glu Gln Trp His Gly Phe Asn Asp Ala Asp 465 470 475 480 Ala Asp His Met Phe Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495 Gly Met Asp Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510 Leu Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520 525 Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr 530 535 540 Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg Ser Tyr 545 550 555 560 Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro Asp Leu Tyr Ala Glu 565 570 575 Asp Pro Asp Phe Tyr Arg Asn Met Arg Ile Gln Asp Leu Ala Gln Gly 580 585 590 Ile His Lys Leu Ile Arg Lys His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605 Ala Phe Asp Thr Leu Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620 Gln Arg Gln Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu 625 630 635 640 Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Pro Gly Val 645 650 655 Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg Thr Val 660 665 670 Leu Asp Phe Leu Leu Met Leu Cys Ser Val Gly Gln His Tyr Pro Gly 675 680 685 Phe Glu Thr Asp Ile His Gly Ala Lys Gln Asp Glu Asp Gly Val Tyr 690 695 700Arg Val Arg Val Leu Lys Asp Ala Gly 705 710
Claims (8)
L27Q/Q109T/R143G/V467L/T671A/V682I/M711E,
A388R/V467L/T671A/V682I/M711E, 또는
L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E
의 변이를 포함하는, 라이신 디카르복실라아제의 변이주.According to claim 1,
L27Q/Q109T/R143G/V467L/T671A/V682I/M711E,
A388R/V467L/T671A/V682I/M711E, or
L27K/Q109A/R143G/A388R/V467L/T671A/V682I/M711E
A mutant strain of lysine decarboxylase containing a mutation of .
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| PCT/KR2023/008670 WO2023249426A1 (en) | 2022-06-23 | 2023-06-22 | Development of solubility-enhanced lysine decarboxylase mutant and application thereof |
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| KR101231897B1 (en) * | 2010-08-03 | 2013-02-08 | 한국과학기술원 | Varient Microorganism Having Cadaverine Producing Ability and Method for Preparing Cadaverine Using the Same |
| US9765369B2 (en) * | 2012-01-18 | 2017-09-19 | Mitsui Chemicals, Inc. | Method for producing 1,5-pentamethylenediamine, mutant lysine decarboxylase, method for producing 1,5-pentamethylene diisocyanate and method for producing polyisocyanate composition |
| KR101873541B1 (en) * | 2016-01-28 | 2018-07-04 | 건국대학교 산학협력단 | A Method for continuedly production of large volume cadaverine using immobilized carrier and lysine decarboxylase―overexpressing recombinant E.coli |
| KR20180029139A (en) * | 2016-09-09 | 2018-03-20 | 홍익대학교세종캠퍼스산학협력단 | Methods for preparing high-concentrated cadaverine |
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| KR20170017341A (en) | 2015-08-06 | 2017-02-15 | 서울대학교산학협력단 | Method for producing lysine decarboxylase mutants and their applications |
| KR20170030824A (en) | 2015-09-10 | 2017-03-20 | 대상 주식회사 | Gene encoding lysine decarboxylase derived from H. alvei, recombinant vector, host cell and method for producing cadaverine using the same |
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