KR102281525B1 - Transformed methanotrophs for producing cadaverine and uses thereof - Google Patents
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Abstract
본 발명은 카다베린 고생성능을 가지는 형질전환 메탄자화균 및 이를 이용한 카다베린의 제조방법에 관한 것으로, 본 발명의 형질전환 메탄자화균은 메탄으로부터 카다베린을 생산할 수 있는바, 친환경적이고 경제적으로 카다베린를 대량 생산하는데 유용하게 사용될 수 있다.The present invention relates to a transgenic methanogen having high cadaverine performance and a method for producing cadaverine using the same. The transgenic methanogenic bacteria of the present invention can produce cadaverine from methane, so it is environmentally friendly and economically cadaverine. It can be usefully used for mass production of berine.
Description
본 발명은 카다베린 고생성능을 가지는 형질전환 메탄자화균 및 이를 이용한 카다베린의 제조방법에 관한 것이다.The present invention relates to a transformed methanogen having high cadaverine performance and a method for producing cadaverine using the same.
메탄은 이산화탄소와 비교하여 강력한 온실 가스보다 24배나 많음에도 불구하고 바이오 매스를 중요한 화학 물질로 생물 전환시키는 탄소 공급 원료이며, 석유계 기반의 탄소원을 대체할 잠재력 있는 탄소원으로 평가받고 있다. 이로 인해 메탄은 화학 합성을 위한 매우 저렴하고 재생 가능한 원천이되고 미래 산업 생명 공학을 위한 차세대 공급 원료가 될 수 있다. 지난 수십 년 동안 인위적 활동으로 인하여 대기 중에 축적되는 메탄의 양을 해결하고 메탄을 값 싸고 재생 가능한 주요 제품 공급원으로 활용하기 위한 노력이 지속되고 있다. Methane is a carbon feedstock that bioconverts biomass into an important chemical, despite being 24 times more potent than a greenhouse gas compared to carbon dioxide, and is evaluated as a potential carbon source to replace petroleum-based carbon sources. This could make methane a very inexpensive and renewable source for chemical synthesis and a next-generation feedstock for future industrial biotechnology. Efforts have been made over the past few decades to address the amount of methane accumulating in the atmosphere due to anthropogenic activity and to utilize methane as a major source of cheap and renewable products.
최근 유전자 조작 기술의 개발을 통해 메탄을 유일한 공급 원료로 사용하여 산업 생물 촉매로서 메타자화균의 잠재력을 확장되고 있다. 유산소, 카로티노이드, 2,3-부탄디올, 숙신산 및 푸트레신과 같은 천연 및 비천연의 여러 화합물의 생합성을 위한 플랫폼으로서 메타자화균를 사용할 수 있을 뿐만 아니라 주요한 메탄 이용 경로들이 밝혀지고 있다. Recent developments in genetically engineered technologies have expanded the potential of metamagnets as industrial biocatalysts using methane as the sole feedstock. As a platform for the biosynthesis of various natural and non-natural compounds such as aerobic, carotenoid, 2,3-butanediol, succinic acid and putrescine, metamagnets can be used as a platform, as well as major methane utilization pathways are being identified.
그러나, 지난 수십년동안 저렴한 메탄자원을 이용하여 고부가가치 산물을 생물 전환하는 다양한 노력들이 시도되어 왔으나, 메탄자화균의 낮은 성장률, 메탄자화균의 생리학에 대한 부족한 이해 및 유전자 도구의 부재는 메탄자화균을 산업용 균주로 발전시키는데 한계를 나타내었다. However, various efforts have been made over the past few decades to bioconvert high value-added products using inexpensive methane resources. It showed a limit in developing the fungus into an industrial strain.
본 발명의 목적은 피루브산 카복실화효소(pyruvate carboxylase), 디아미노피멜린산 카복실화효소(diaminopimelate decarboxylase), 라이신 카복실화효소(lysine decarboxylase) 및 카다베린-라이신 역수용체 단백질(cadaverine-lysine antiporter protein)이 활성화된 카다베린을 생산하는 형질전환 메탄자화균을 제공하는 것이다. An object of the present invention is pyruvate carboxylase (pyruvate carboxylase), diaminopimelate decarboxylase, lysine carboxylase (lysine decarboxylase) and cadaverine-lysine antiporter protein (cadaverine-lysine antiporter protein) It is to provide a transformed methanogen producing this activated cadaverine.
본 발명의 다른 목적은 상기 형질전환 메탄자화균을 이용하여 메탄 기질로부터 카다베린을 고생산하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for high production of cadaverine from a methane substrate using the transformed methanogen.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시 형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는 피루브산 카복실화효소(pyruvate carboxylase), 디아미노피멜린산 카복실화효소(diaminopimelate decarboxylase), 라이신 카복실화효소(lysine decarboxylase) 및 카다베린-라이신 역수용체 단백질(cadaverine-lysine antiporter protein)이 활성화된 카다베린을 생산하는 형질전환 메탄자화균을 제공한다.One aspect of the present invention for achieving the above object is pyruvate carboxylase (pyruvate carboxylase), diaminopimelic acid carboxylase (diaminopimelate decarboxylase), lysine carboxylase (lysine decarboxylase) and cadaverine-lysine inverse receptor Provided is a transgenic methanogen producing cadaverine-lysine antiporter protein-activated cadaverine.
또한, 발명자들은 카다베린을 고생산할 수 있는 방법으로, 메탄 등 바이오매스를 활용한 방법에 대해 예의 연구 노력한 결과, 놀랍게도 본 발명의 형질전환 메탄자화균을 이용할 경우 카다베린을 고생산이 가능함을 확인하였다. Type II methanotrophs,Methylosinus trichosporium OB3b에 카다베린 생합성 경로를 도입함으로서 메탄으로부터 카다베린을 고생산 할 수 있음은 지금까지 전혀 알려지지 않았고, 본 발명을 통해 최초로 개발되었다는 점에서 그 의의가 매우 크다고 할 수 있다.In addition, the inventors made intensive research efforts on a method that utilizes biomass such as methane as a method for high production of cadaverine, and surprisingly, it was confirmed that high production of cadaverine is possible when the transformed methanogen of the present invention is used. did. Type II methanotrophs, Methylosinus trichosporium OB3b, by introducing the cadaverine biosynthetic pathway to high production of cadaverine from methane, was not known at all until now, and it can be said that it is very significant in that it was developed for the first time through the present invention.
본 발명에서 용어 "카다베린(cadaverine)" 은 NH2(CH2)5NH2의 분자식을 가지는 5개의 탄소로 구성된 다이아민(diamine) 유기화합물이고, 5-diaminopentane으로 알려져 있으며 많은 산업적 응용에 있어서 중요한 기반 화학물질이다. 카다베린은 폴리아마이드나 폴리우레탄과 같은 고분자의 구성요소, 킬레이팅제 또는 다른 첨가제로 사용될 수 있다. In the present invention, the term "cadaverine" is a diamine organic compound consisting of five carbons having a molecular formula of NH 2 (CH 2 ) 5 NH 2 , known as 5-diaminopentane, and in many industrial applications It is an important base chemical. Cadarberine can be used as a component of polymers such as polyamides or polyurethanes, as chelating agents or as other additives.
본 발명에서 용어 "메탄자화균(metahnotroph)"은 메탄을 주요 탄소원 또는 에너지원으로 사용하는 세균을 의미한다. 상기 메탄자화균은 본 발명에서 형질전환의 대상이 되는 숙주 균주를 의미할 수 있으며, 본 발명의 목적상 메탄을 탄소원으로 사용하여, 옥살아세트산(OAA)를 출발물질로 L-lysine을 거쳐 최종적으로 카다베린(cadaverine)을 생산할 수 있는 한 특별히 이에 제한되지 않는다. 또한, 메탄, 메탄올, 메틸아민 등 C1 화합물을 에너지원으로 사용하는 메틸자화균(methylotroph) 중에서 메탄을 함께 사용할 수 있는 균주 또한 본 발명의 메탄화균에 포함될 수 있음은 당업자에게 자명하다.In the present invention, the term “metahnotroph” refers to bacteria using methane as a major carbon source or energy source. The methanogen may mean a host strain to be transformed in the present invention, and for the purpose of the present invention, methane is used as a carbon source, oxaloacetic acid (OAA) is used as a starting material, and finally through L-lysine. As long as it can produce cadaverine, it is not particularly limited thereto. In addition, it is apparent to those skilled in the art that a strain capable of using methane together among methylotrophs using C1 compounds such as methane, methanol, and methylamine as an energy source may also be included in the methanating bacteria of the present invention.
상기 메탄자화균은 메탄 등 C1 화합물을 에너지원으로 사용할 수 있는 것인한 특별히 이에 제한되지 않으나, 메틸로모나스 속(Methylomonas), 메틸로박터속(Methylobacter), 메틸로코커스 속(Methylococcus), 메틸로스페라 속(Methylosphaera), 메틸로칼덤 속(Methylocaldum), 메틸로글로버스 속(Methyloglobus), 메틸로사르시나 속(Methylosarcina), 메틸로프로펀더스 속(Methyloprofundus), 메틸로썰머스 속(Methylothermus), 메틸로할로비우스 속(Methylohalobius), 메틸로게아 속(Methylogaea), 메틸로마리넘 속(Methylomarinum), 메틸로벌럼 속(Methylovulum), 메틸로마리노범 속(Methylomarinovum), 메틸로러브럼 속(Methylorubrum), 메틸로파라코커스 속(Methyloparacoccus), 메틸로시스티스 속(Methylocystis), 메틸로셀라 속(Methylocella), 메틸로캡사 속(Methylocapsa), 메틸로퍼룰라 속(Methylofurula), 메틸아시디필럼 속(Methylacidiphilum), 메틸아시디마이크로비움 속(Methylacidimicrobium), 메틸로마이크로비움 속(Methylomicrobium) 또는 메틸로시너스 속(Methylosinus) 균주일 수 있으며, 구체적으로 메틸로시너스 트리코스포륨(Methylosinus trichosporium) OB3b일 수 있다.The methanogenic bacteria is not particularly limited thereto, as long as it can use a C1 compound such as methane as an energy source, but Methylomonas, Methylobacter, Methylococcus, methylose Methylosphaera, Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus ), genus Methylohalobius, genus Methylogaea, genus Methylomarinum, genus Methylovulum, genus Methylomarinovum, genus Methyloloverum Methylorubrum, Methyloparacoccus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylacid It may be a phyllum genus (Methylacidiphilum), a methylacidimicrobium genus (Methylacidimicrobium), a methylomicrobium genus (Methylomicrobium) or a methylosinus genus (Methylosinus) strain, specifically methylosinus trichosporium OB3b can be
이러한 메탄자화균을 이용한 바이오전환 공정의 경우, 비교적 저렴한 메탄을 탄소원으로 사용할 수 있어 경제적으로 유리하며, 대기 중의 메탄을 사용 가능하다는 점에서 온실가스의 방출 예방 등 환경적인 면에서도 장점이 있다. 또한, 기존에 포도당(glucose)을 이용한 공정 대비 산소의 손실이 없어 최종 생산 물질의 수율 또한 높다.In the case of the bioconversion process using such methane magnetizing bacteria, relatively inexpensive methane can be used as a carbon source, which is economically advantageous, and in that methane in the atmosphere can be used, there are environmental advantages such as prevention of greenhouse gas emission. In addition, there is no loss of oxygen compared to the conventional process using glucose, so the yield of the final product is also high.
본 발명에서 용어 "형질전환 메탄자화균"은 상기 메탄자화균의 유전자를 도입하거나 또는 제거하여 형질을 전환시킨 균주를 의미한다.In the present invention, the term "transformed methanogenic bacteria" refers to a strain transformed by introducing or removing the gene of the methanogenic bacteria.
본 발명에서 용어 "피루브산 카복실화효소(pyruvate carboxylase)"는 피루브산을 옥살아세트산(OAA)으로 전환하는 효소이다.As used herein, the term "pyruvate carboxylase" is an enzyme that converts pyruvate into oxalic acid (OAA).
상기 피루브산 카복실화효소(pyruvate carboxylase)를 코딩하는 유전자(Pyc)는 Methylomonas sp. DH-1 유래일 수 있으며, 구체적으로 서열번호 1의 염기서열로 구성된 것일 수 있다. 보다 구체적으로, 상기 서열번호 1의 서열과 70% 이상, 구체적으로는 80% 이상, 보다 구체적으로는 90%이상, 보다 더 구체적으로는 95%이상, 가장 구체적으로는 99% 이상의 상동성을 나타내는 염기서열로서 실질적으로 pyruvate carboxylase 활성을 가진 단백질을 발현 가능한 경우, 제한없이 포함될 수 있다.The gene encoding the pyruvate carboxylase ( Pyc ) is Methylomonas sp. It may be derived from DH-1, and specifically may be composed of the nucleotide sequence of SEQ ID NO: 1. More specifically, 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, most specifically 99% or more homology with the sequence of SEQ ID NO: 1 If a protein having substantially pyruvate carboxylase activity can be expressed as a base sequence, it may be included without limitation.
본 발명의 용어 "디아미노피멜린산 카복실화효소(diaminopimelate decarboxylase)"는 Meso-diaminopimelate를 라이신으로 전환하는 효소이다.The term "diaminopimelate decarboxylase" as used herein is an enzyme that converts meso-diaminopimelate to lysine.
상기 디아미노피멜린산 카복실화효소(diaminopimelate decarboxylase)를 코딩하는 유전자(lysA)는 M. trichosporium OB3b는 서열번호 2의 염기서열로 구성된 것일 수 있다. 보다 구체적으로, 상기 서열번호 2의 서열과 70% 이상, 구체적으로는 80% 이상, 보다 구체적으로는 90%이상, 보다 더 구체적으로는 95%이상, 가장 구체적으로는 99% 이상의 상동성을 나타내는 염기서열로서 실질적으로 디아미노피멜린산 카복실화효소(diaminopimelate decarboxylase) 활성을 가진 단백질을 발현 가능한 경우, 제한없이 포함될 수 있다.The gene encoding the diaminopimelate decarboxylase ( lysA ) may be M. trichosporium OB3b consisting of the nucleotide sequence of SEQ ID NO: 2. More specifically, 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, most specifically 99% or more homology with the sequence of SEQ ID NO: 2 If it is possible to express a protein having substantially diaminopimelate decarboxylase activity as a base sequence, it may be included without limitation.
본 발명의 용어 "라이신 카복실화효소(lysine decarboxylase)"는 라이신을 카다베린으로 전환하는 효소이다.The term "lysine decarboxylase" as used herein is an enzyme that converts lysine to cadaverine.
상기 라이신 카복실화효소(lysine decarboxylase)를 코딩하는 유전자(ldcC)는 서열번호 3의 염기서열로 구성된 것일 수 있다. 보다 구체적으로, 상기 서열번호 3의 서열과 70% 이상, 구체적으로는 80% 이상, 보다 구체적으로는 90%이상, 보다 더 구체적으로는 95% 이상, 가장 구체적으로는 99% 이상의 상동성을 나타내는 염기서열로서 실질적으로 lysine decarboxylase 활성을 가진 단백질을 발현 가능한 경우, 제한없이 포함될 수 있다.The gene encoding the lysine decarboxylase ( ldcC ) may be composed of the nucleotide sequence of SEQ ID NO: 3. More specifically, 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, most specifically 99% or more homology with the sequence of SEQ ID NO: 3 If a protein having substantially lysine decarboxylase activity can be expressed as a base sequence, it may be included without limitation.
본 발명의 용어 "카다베린-라이신 역수용체 단백질(cadaverine-lysine antiporter protein)"을 암호화하는 cadB 유전자는 카다베린을 세포 외로 내보낼뿐만 아니라 기질 라이신을 세포 내로 유입시킨다. 카다베린-라이신 역수용체 단백질(cadaverine-lysine antiporter protein)를 코딩하는 유전자(cadB)는 서열번호 4의 염기서열로 구성된 것일 수 있다.The cadB gene encoding the term "cadaverine-lysine antiporter protein" of the present invention not only exports cadaverine out of the cell, but also introduces a substrate lysine into the cell. The cadaverine-lysine antireceptor protein (cadaverine-lysine antiporter protein) coding gene (cadB ) may be composed of the nucleotide sequence of SEQ ID NO: 4.
전술한 각각의 효소 또는 단백질은 본 발명에서 사용되는 각각의 활성을 나타내는한, 각 효소 또는 단백질을 구성하는 아미노산 서열의 하나 이상의 위치에서의 하나 이상의 아미노산이 치환, 결실, 삽입, 첨가 또는 역위된 아미노산 서열을포함할수 있는데, 상기 각 효소 또는 단백질의 활성을 나타내는 한, 상기 각 효소또는 단백질의 아미노산 서열에 대하여, 80% 이상, 구체적으로는 90% 이상, 보다구체적으로는 95% 이상, 보다 더 구체적으로는 99% 이상의 상동성을 가지는 것으로, 실질적으로 각 효소 또는 단백질과 동일하거나 상응하는 활성을 가지는 아미노산 서열의 경우도 본 발명의 범주에 포함됨은 당업자에게 자명하다.Each of the above-described enzymes or proteins is an amino acid in which one or more amino acids at one or more positions in the amino acid sequence constituting each enzyme or protein are substituted, deleted, inserted, added or inverted, as long as they exhibit the respective activity used in the present invention. It may include a sequence, as long as it shows the activity of each enzyme or protein, with respect to the amino acid sequence of each enzyme or protein, 80% or more, specifically 90% or more, more specifically 95% or more, more specifically It is apparent to those skilled in the art that an amino acid sequence having substantially the same or corresponding activity as each enzyme or protein is included in the scope of the present invention as having a homology of 99% or more.
상기 단백질을 코딩하는 폴리뉴클레오티드의 일부 또는 전체를 결실하는 방법은 세균 내 염색체 삽입용 벡터를 통해 염색체 내 내재적 목적 단백질을 암호화하는 폴리뉴클레오티드를일부 핵산 서열이 결실된 폴리뉴클레오티드 또는 마커 유전자로 교체함으로써 수행될 수 있다. 본 발명에서 용어 "강화"는 효소 또는 단백질의 활성이 야생형 또는그 내재적 활성에 비해 증가되는 것을 의미한다. 상기 효소 또는 단백질의 활성 강화는 당업계에 알려진 임의의 방법에 의해 수행될 수 있다.The method of deleting part or all of the polynucleotide encoding the protein is performed by replacing a polynucleotide encoding an endogenous target protein in a chromosome with a polynucleotide or a marker gene having a partial nucleic acid sequence deleted through a vector for chromosome insertion in bacteria. can be In the present invention, the term "enhancement" means that the activity of an enzyme or protein is increased compared to the wild type or its intrinsic activity. Enhancing the activity of the enzyme or protein may be performed by any method known in the art.
상기 목적을 달성하기 위한 본 발명의 다른 하나의 양태는 상기 카다베린 생산용 형질전환 메탄자화균을 배양하는 단계를 포함하는, 카다베린 제조방법을 제공하는 것이다.Another aspect of the present invention for achieving the above object is to provide a method for producing cadaverine, comprising the step of culturing the transformed methanogenic bacteria for the production of cadaverine.
본 발명에서 용어 "배양"은 목적하는 세포 또는 조직 등을 인공적으로 조절한 환경 조건에서 생육하는 것을 의미한다. 상기 환경 조건은 대표적으로 영양소,온도, 삼투압, pH, 기체 조성, 빛 등이 있으나, 직접적인 영향을 주는 것은 배지이며, 배지는 크게 액체배지와 고체배지로 나뉠 수 있다. 본 발명의 형질전환 메탄자화균의 배양은 당업계에 널리 알려져 있는 방법을 이용하여 수행할 수 있다.In the present invention, the term "culture" refers to growth in environmental conditions artificially regulated, such as a target cell or tissue. The environmental conditions typically include nutrients, temperature, osmotic pressure, pH, gas composition, light, etc., but it is the medium that has a direct influence, and the medium can be largely divided into a liquid medium and a solid medium. Culturing of the transformant methanogenic bacteria of the present invention can be performed using a method well known in the art.
구체적으로, 상기 배양은 상기 형질전환 메탄자화균으로부터 카다베린을 생산할 수 있는 한 특별히 이에 제한되지 않으나, 유가 배양 또는 주입배치 또는 반복 유가 배양 공정(fed batch or repeated fed batch process)에서 연속식으로 배양할 수 있다. 배양에 사용되는 배지는 메탄자화균의 배양에 사용되는것으로 알려진 NMS(nitrate mineral salts) 배지를 사용할 수 있고, 메탄자화균에 따라 상기 배지에 포함된 성분 또는 이의 함량을 적절히 조절한 배지를 사용할 수있으나, 이에 특별히 제한되는 것은 아니다. 본 발명에서 형질전환 메탄자화균의 배양 온도는 15℃ 내지 45℃, 구체적으로 20℃ 내지 40℃, 보다 구체적으로 25℃내지 35℃일 수 있으며, 메탄과 균주의 원활한 접촉을 위해, 150rpm 내지 300rpm,구체적으로 180rpm 내지 270rpm, 보다 구체적으로 200rpm 내지 250rpm으로 교반할수 있으나, 이에 제한되지 않는다.Specifically, the culture is not particularly limited as long as it can produce cadaverine from the transformed methanogen, but continuously cultured in fed batch or fed batch or repeated fed batch process. can do. As the medium used for culture, NMS (nitrate mineral salts) medium known to be used for culturing of methanogenic bacteria can be used, and a medium in which the components contained in the medium or its content are appropriately adjusted according to the methanogenic bacteria can be used. However, it is not particularly limited thereto. In the present invention, the culture temperature of the transformant methanogenic bacteria may be 15 ℃ to 45 ℃, specifically 20 ℃ to 40 ℃, more specifically 25 ℃ to 35 ℃, for smooth contact between the methane and the strain, 150rpm to 300rpm , Specifically, 180 rpm to 270 rpm, more specifically 200 rpm to 250 rpm may be stirred, but is not limited thereto.
본 발명에 있어서, 상기 카다베린의 제조방법은 상기 형질전환 메탄자화균을 메탄을 포함하는 배양액에 배양하는 것일 수 있다.In the present invention, the method for producing cadaverine may be culturing the transformed methanogen in a culture medium containing methane.
본 발명에 있어서, 상기 제조방법은 배양액으로부터 카다베린을 회수하는 단계를 추가로 포함할 수 있다. 상기 카다베린을 회수하는 단계는 배양 방법에 따라 당업계에 공지된 적합한 방법에 의해 수행될 수 있다. 구체적으로, 공지된 카다베린 회수 방법은 특별히 이에 제한되지 않으나, 원심분리, 여과, 추출, 분무, 건조, 증발, 침전, 결정화, 전기영동, 분별 용해(예를 들면 암모늄 설페이트 침전), 크로마토그래피(예를 들면 HPLC, 이온교환, 친화성, 소수성 및 크기배제) 등의 방법이 사용될 수 있다.In the present invention, the manufacturing method may further comprise the step of recovering cadaverine from the culture medium. The step of recovering the cadaverine may be performed by a suitable method known in the art according to the culture method. Specifically, the known cadaverine recovery method is not particularly limited thereto, but centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (eg, ammonium sulfate precipitation), chromatography ( For example, methods such as HPLC, ion exchange, affinity, hydrophobicity and size exclusion) may be used.
본 발명의 형질전환 메탄자화균은 메탄으로부터 카다베린을 생산할 수 있는바, 친환경적이고 경제적으로 카다베린를 대량 생산하는데 유용하게 사용될 수 있다.The transformant methanogenic bacteria of the present invention can produce cadaverine from methane, and can be effectively used for mass production of cadaverine in an environmentally friendly and economical manner.
도 1은 카다베린 생성을 위한 메탄자화균의 대사 경로를 나타낸 모식도이다:
Pyc(pyruvate carboxylase); ppc(Phosphoenolpyruvate carboxylase); lysC(aspartokinase); lysA(diaminopimelate decarboxylase), cadA 또는 ldcC(lysine decarboxylase); cadB(a cadaverine-lysine antiporter protein).
도 2는 20% 메탄(v/v)이 보충된 NMS 배지에 보조인자로서 PLP를 첨가한 M. trichosporium OB3b 형질 전환체의 카다베린의 생성을 나타낸 그래프이다.
도 3은 20% 메탄(v/v)이 보충된 NMS 배지에서 M. trichosporium OB3b 형질 전환체의 성장(A) 및 카다베린 생산(B)의 비교한 것을 나타낸 그래프이다.
도 4는 20% 메탄(v/v)이 보충된 NMS 배지에서 M. trichosporium OB3b 형질 전환체의 성장(A) 및 카다베린 생산(B)을 비교한 그래프이다.
도 5는 재조합 OB3b/cad4 균주를 유가 배양기에서 배양하여 시간 당 생산되는 카다베린의 양을 나타낸 그래프이다.1 is a schematic diagram showing the metabolic pathway of methanogenic bacteria for cadaverine production:
Pyc (pyruvate carboxylase); ppc (Phosphoenolpyruvate carboxylase); lysC (aspartokinase); lysA (diaminopimelate decarboxylase), cadA or ldcC (lysine decarboxylase); cadB (a cadaverine-lysine antiporter protein).
Figure 2 is a graph showing the production of cadaverine in M. trichosporium OB3b transformants added with PLP as a cofactor to NMS medium supplemented with 20% methane (v/v).
3 is a graph showing the comparison of growth (A) and cadaverine production (B) of M. trichosporium OB3b transformants in NMS medium supplemented with 20% methane (v/v).
4 is a graph comparing the growth (A) and cadaverine production (B) of M. trichosporium OB3b transformants in NMS medium supplemented with 20% methane (v/v).
5 is a graph showing the amount of cadaverine produced per hour by culturing a recombinant OB3b/cad4 strain in a fed-batch culture medium.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 메탄에서 카다베린의 생산을 한 유형 II methanotrophs, Methylosinus trichosporium OB3b의 대사 공학적으로 형질전환된 균주를 개발하였다.The present inventors developed a metabolically engineered strain of Methylosinus trichosporium OB3b, a type II methanotrophs that produced cadaverine in methane.
먼저, L-라이신을 카다베린으로 전환시키는 L-lysine decarboxylase는 tac 프로모터하에 대장균으로부터 2개의 유전자 cadA 또는 ldcC의 플라스미드-기반 발현에 의해 M. trichosporium OB3b에 도입되었다. 두 유전자의 효소 활성을 비교한 결과, ldcC는 cadA보다 카다베린 전환 능력이 우수했다. 다음으로, L-라이신 생합성 풀을 증가시키기 위하여, aspartokinase II 또는 meso-diaminopimelate decarboxylase(lysA)를 과발현시켜 재조합 균쥬 OB3b/cad1과 OB3b/cad2를 얻었으며 이를 이용하여 각각 16.8 mg/L 및 30 mg/L의 카다베린을 생성하였다. 또한, Methyomonas sp. DH-1. 유래의 pyruvate carboxylase(pyc)를 OB3b/cad2 균주에 추가로 도입하여 재조합 OB3b/cad3 균주를 얻었으며 이는 초기 균주보다 13배 더 높은 역가의 카다베린(41.94 mg/L에 상응함)을 생성하였다. 카다베린 생산성을 향상시키기 위해, 대장균으로부터의 cadaverine-lysine antiporter CadB를 OB3b/cad3 균주에 도입하였다. 최종 형질전환된 균주 OB3b/cad4는 유가 배양(fed-batch)에 의해 0.153 mmol/g DCW/일의 생산성으로 192.09 m/L의 카다베린을 생산할 수 있었다. 이 결과는 형질전환된 메탄자화균을 사용하여 유일한 탄소원으로서 메탄으로부터 카다베린의 생산됨을 확인하였으므로 본 발명을 완성하였다.First, L-lysine decarboxylase, which converts L-lysine to cadaverine, was introduced into M. trichosporium OB3b by plasmid-based expression of two genes cadA or ldcC from E. coli under the tac promoter. As a result of comparing the enzymatic activity of the two genes, ldcC had better cadaverine conversion ability than cadA. Next, in order to increase the L-lysine biosynthesis pool, recombinant strains OB3b/cad1 and OB3b/cad2 were obtained by overexpressing aspartokinase II or meso-diaminopimelate decarboxylase (lysA), and using them, 16.8 mg/L and 30 mg/L, respectively L of cadaverine was produced. In addition, Methyomonas sp. DH-1. Derived pyruvate carboxylase (pyc) was additionally introduced into the OB3b/cad2 strain to obtain a recombinant OB3b/cad3 strain, which produced 13-fold higher titer of cadaverine (corresponding to 41.94 mg/L) than the initial strain. To improve cadaverine productivity, the cadaverine-lysine antiporter CadB from E. coli was introduced into the OB3b/cad3 strain. The final transformed strain OB3b/cad4 was able to produce 192.09 m/L of cadaverine with a productivity of 0.153 mmol/g DCW/day by fed-batch. This result confirmed the production of cadaverine from methane as the only carbon source using the transformed methanogen, thus completing the present invention.
이하, 본 발명은 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.
<실시예 1> 실험 재료 및 방법<Example 1> Experimental materials and methods
<1-1> 시약 및 올리고뉴클레오티드<1-1> Reagents and oligonucleotides
Lamp Pfu 중합 효소 및 제한 효소는 BioFACT (한국)에서 구입하였다. Snap gene을 사용하여 설계되었고 Macrogen (서울, 한국)에 의해 합성된 프라이머는 하기 표 1에 나타내었다. 일부 유전자의 합성 리보솜 결합 부위 (RBS)는 리보솜 결합 부위 계산기에 기초하여 설계되었다. Kit of Wizard® Genomic DNA Purification Kit (Promega, USA)를 사용하여 게놈 DNA를 추출하였다. PCR 정제 키트 및 플라스미드 추출 키트는 GeneAll Biotechnology(한국)에서 구입하였다. NE (England)에서 구입한 모든 플라스미드는 Gibson 어셈블리를 사용하여 제작하였다. Lamp Pfu polymerase and restriction enzyme were purchased from BioFACT (Korea). Primers designed using Snap gene and synthesized by Macrogen (Seoul, Korea) are shown in Table 1 below. The synthetic ribosome binding site (RBS) of some genes was designed based on the ribosome binding site calculator. Genomic DNA was extracted using the Kit of Wizard® Genomic DNA Purification Kit (Promega, USA). PCR purification kit and plasmid extraction kit were purchased from GeneAll Biotechnology (Korea). All plasmids purchased from NE (England) were constructed using Gibson assembly.
<1-2> 균주 및 플라스미드의 구성<1-2> Composition of strains and plasmids
프라이머 pAWP89-Tac-For/pAWP89-Tac-Rev(표 1)를 사용하여 벡터 pAWP89를 선형화하기 위해 역 PCR을 사용하였다. 라이신 디카복실레이즈 유전자 ldcC 및 cadA를 게놈 DNA로부터 증폭시키고 Gibson 어셈블리(표 2)에 의해 pAWP89 벡터로 구축하였다. 2가지 형태의 라이신 디카복실레이즈 ldcC 및 cadA가 둘다 tac 프로모터하에 M. trichosporium OB3b에서 발현시켰다.Reverse PCR was used to linearize the vector pAWP89 using the primers pAWP89-Tac-For/pAWP89-Tac-Rev (Table 1). Lysine decarboxylase genes ldcC and cadA were amplified from genomic DNA and constructed into pAWP89 vector by Gibson assembly (Table 2). Both forms of the lysine decarboxylase ldcC and cadA were expressed in M. trichosporium OB3b under the tac promoter.
MG1655 Escherichia coli
MG1655
<1-3> Methylosinus trichosporium OB3b의 컨쥬게이션<1-3> Conjugation of Methylosinus trichosporium OB3b
상기 실시예 1-3을 통하여 얻어진 재조합 발현용 벡터를 야생형 메탄자화균 M. trichosporium OB3b에 형질전환시키기 위하여, 컨주게이션(conjugation)을 실시하였다. OD600에서 약 0.2까지 자란 M.trichosporium OB3b를 사용하였다 M. trichosporium OB3b 50ml 및 도입될 플라스미드를 포함하는 공여자 E. coli S17-1 10ml를 NMS로 세척하였다. 이후, 이들을 0.2 μm 멸균 니트로셀룰로스 필터에 혼합된 세포를 도포하였다. 필터를 0.02%(w/v) 프로테오스-펩톤(proteose-peptone)을 포함한 NMS 한천 배지에 놓고, 메탄(대기 중 50 %)의 존재하에 30℃에서 24시간 동안 배양하였다. 0.02%(w/v) 프로테오스-펩톤(proteose-peptone)을 포함한 NMS 한천 플레이트로부터의 세포를 10mL NMS 배지에 재현탁시키고, 7,000 g에서 5분 동안 원심분리하여 농축시켰다. 세포 펠렛은 카나마이신이 포함된 NMS 한천에 도말하고 단일한 콜로니가 나타날 때까지 메탄/공기(1:1, v/v)와 함께 2-3주 동안 배양하였다. In order to transform the recombinant expression vector obtained in Examples 1-3 into M. trichosporium OB3b, a wild-type methanogen, conjugation was performed. M. trichosporium OB3b grown at OD600 to about 0.2 was used. 50 ml of M. trichosporium OB3b and 10 ml of donor E. coli S17-1 containing the plasmid to be introduced were washed with NMS. Thereafter, the mixed cells were applied to a 0.2 μm sterile nitrocellulose filter. The filter was placed on NMS agar medium containing 0.02% (w/v) proteose-peptone, and incubated at 30° C. for 24 hours in the presence of methane (50% in air). Cells from NMS agar plates containing 0.02% (w/v) proteose-peptone were resuspended in 10 mL NMS medium, and concentrated by centrifugation at 7,000 g for 5 minutes. Cell pellets were plated on NMS agar containing kanamycin and incubated with methane/air (1:1, v/v) for 2-3 weeks until single colonies appeared.
<1-4> 배지 및 배양 조건<1-4> Medium and culture conditions
M. trichosporium OB3b는 이전 연구(Hwang et al., 2015)에서 기술된 바와 같이, 5μM의 CuSO4를 포함한 NMS(nitrate mineral salt) 배지에서 배양하였다. M. trichosporium OB3b 세포를 50ml의 NMS 배지가 들어있고 스크류 캡으로 밀봉된 500ml 배플 플라스크(baffled flask)에서 30℃로 230 rpm으로 교반하며 배양하였다. 기밀식 주사기(gas-tight syringe)를 사용한 가스 교환에 의해 최종 농도 30%(v/v) 메탄을 공급하였으며, 헤드 스페이스를 매일 환기한 후, 30%(v/v) 메탄을 공급하였다. 세포 배양의 광학 밀도는 분광 광도계에서 측정하였다. 50μg/ml의 최종 농도를 갖는 카나마이신(Km)을 재조합 플라스미드를 함유하는 M. trichosporium OB3b 및 대장균 모두의 선별하는 데에 사용하였다. M. trichosporium OB3b was cultured in NMS (nitrate mineral salt) medium containing 5 μM CuSO 4 as described in a previous study (Hwang et al., 2015). M. trichosporium OB3b cells were cultured in a 500 ml baffled flask containing 50 ml of NMS medium and sealed with a screw cap at 30° C. and 230 rpm with agitation. A final concentration of 30% (v/v) methane was supplied by gas exchange using a gas-tight syringe, and after daily ventilation of the headspace, 30% (v/v) methane was supplied. The optical density of the cell culture was measured on a spectrophotometer. Kanamycin (Km) with a final concentration of 50 μg/ml was used for selection of both M. trichosporium OB3b and E. coli containing recombinant plasmids.
<1-5> 효소 분석<1-5> Enzyme analysis
M. trichosporium OB3b 형질전환체에서 라이신 디카복실레이즈 효소의 활성을 확인하기 위해, 정지상(stationary phase) 세포를 수집하였다. 효소 활성은 재조합 균주를 추출하고 초음파 처리한 조추출물을 사용하여 측정하였다. 단백질의 농도는 브래드 포드(Bradford) 방법에 의해 측정하었다. 라이신 디카복실레이즈 (cadA 및 ldcC) 활성은 라이신을 카다베린으로 변환한 것을 HPLC 분석을 사용하여 측정하였다. 500mM 아세트산 나트륨 완충액(pH 6.0)에서 보조 인자로서 1mM L-리신, 0.1mM 피리독살-5-포스페이 (PLP)와의 반응, 무세포 추출물 20μL를 Thermomixer로 37℃에서 30분 혼합하였다. 100℃에서 5분 동안 가열하여 반응을 종료하였다. 라이신 디카복실레이즈 활성의 1 유닛(unit)은 37℃에서 분당 1μmol의 카다베린을 전환시키는데 필요한 효소의 양으로 결정되었다. 이어서, 반응의 50μL를 라이신 및 카다베린 농도 둘 모두를 결정하기 위해 유도체화에 사용하였다. 음성 대조군은 조추출물 또는 L-라이신을 제거하는 것을 사용하였다.To confirm the activity of lysine decarboxylase enzyme in M. trichosporium OB3b transformants, stationary phase cells were collected. Enzyme activity was measured using the crude extract extracted from the recombinant strain and treated with sonication. The protein concentration was determined by the Bradford method. Lysine decarboxylase (cadA and ldcC) activity was determined using HPLC analysis of the conversion of lysine to cadaverine. Reaction with 1 mM L-lysine, 0.1 mM pyridoxal-5-phosphate (PLP) as a cofactor in 500 mM sodium acetate buffer (pH 6.0), and 20 μL of cell-free extract were mixed in a Thermomixer at 37° C. for 30 minutes. The reaction was terminated by heating at 100° C. for 5 minutes. One unit of lysine decarboxylase activity was determined as the amount of enzyme required to convert 1 μmol of cadaverine per minute at 37°C. 50 μL of the reaction was then used for derivatization to determine both lysine and cadaverine concentrations. A negative control was used to remove the crude extract or L-lysine.
<1-6> 생물 반응기 배양<1-6> Bioreactor Cultivation
생물 반응기에서 발효는 3L의 NMS 배지를 함유하는 5L 유리 생물 반응기에서 수행되었다. 메탄 및 공기를 3:7 비율로 혼합한 가스는 0.2 vvm의 속도로 지속적으로 공급하였다. 가스 조성 및 유량은 mass flow controller (Brooks, Pennsylvania, USA)에 의해 제어되었다. 발효는 30℃에서 수행되었고 임펠러의 속도는 200rpm였다. 세포 성장 동안 pH 증가를 방지하기 위해 2N HCl 용액을 공급함으로써 pH 값을 7.0 미만으로 제어하였다. 배양이 진행되는 동안 표시된 시간마다 액체 샘플을 수집하였다. 세포 배양(OD600)의 광학 밀도는 UV-가시 광선 분광 광도계로 측정하였다. 출구 가스 조성은 카복센 1000 (Carboxen 1000, Supelco, Bellefonte, PA) 컬럼이 장착된 GC에 의해 분석되었다.Fermentation in the bioreactor was performed in a 5 L glass bioreactor containing 3 L of NMS medium. A gas mixture of methane and air in a ratio of 3:7 was continuously supplied at a rate of 0.2 vvm. Gas composition and flow rates were controlled by a mass flow controller (Brooks, Pennsylvania, USA). Fermentation was carried out at 30 °C and the speed of It was 200 rpm. The pH value was controlled below 7.0 by supplying 2N HCl solution to prevent the pH increase during cell growth. Liquid samples were collected at indicated times during incubation. The optical density of the cell culture (OD600) was measured with a UV-visible light spectrophotometer. The outlet gas composition was analyzed by GC equipped with a Carboxen 1000 (
<1-7> 분석방법<1-7> Analysis method
배양액 중의 라이신 및 카다베린의 양을 분석하기 위하여 배양시간별로 상층액을 수집하였다. 라이신 및 카다베린의 디아민 유도체는 300μL의 50mM 붕산 나트륨 완충액(pH 9), 100μL의 메탄올, 47μL의 증류수, 3μL의 200mM diethylethoxymethylenemalonate(DEEMM), 및 상층액 50μL을 함유하는 500μL 반응 혼합물에서 수득하였다. DEEMM는 라이신 또는 카다베린을 유도체화를 위해 사용되었고 유도체화 반응을 70℃에서 2시간 동안 수행하였다(Alaiz et al., 1992). 반응액은 0.2μm 필터를 이용하여 여과한 후, reverse-phase Symmetry C18 칼럼(4.6×250 mm HPLC Column)이 장착된 HPLC(일본, Jasco Co.)에서 UV 검출기(284 nm)에서 측정하였다. 컬럼의 온도를 35℃로 유지하였고, 이동상은 아세토 니트릴(A) 및 25 mM 아세트산 나트륨 완충액(pH 4.8)(B), 유속은 1.0 mL/분이었다. 이동산은 다음과 같이 구배법(Gradient Method)으로 사용하였다: 0-2분, 20-25% A; 2-32분, 25-60% A; 32-37분, 60-20%; 37-40분, 20-20% (Kim et al., 2015).In order to analyze the amount of lysine and cadaverine in the culture medium, the supernatant was collected for each culture time. Diamine derivatives of lysine and cadaverine were obtained in 500 μL reaction mixture containing 300 μL of 50 mM sodium borate buffer (pH 9), 100 μL of methanol, 47 μL of distilled water, 3 μL of 200 mM diethylethoxymethylenemalonate (DEEMM), and 50 μL of the supernatant. DEEMM was used to derivatize lysine or cadaverine, and the derivatization reaction was performed at 70° C. for 2 hours (Alaiz et al., 1992). After filtration using a 0.2 μm filter, the reaction solution was measured with a UV detector (284 nm) in HPLC (Jasco Co., Japan) equipped with a reverse-phase Symmetry C 18 column (4.6×250 mm HPLC Column). The temperature of the column was maintained at 35° C., the mobile phases were acetonitrile (A) and 25 mM sodium acetate buffer (pH 4.8) (B), and the flow rate was 1.0 mL/min. Mobile acids were used in the Gradient Method as follows: 0-2 min, 20-25% A; 2-32 min, 25-60% A; 32-37 min, 60-20%; 37-40 min, 20-20% (Kim et al., 2015).
<실시예 2> 실험결과<Example 2> Experimental results
<2-1> M. trichosporium OB3b에서 라이신 디카복실레이즈의 발현에 의한 카다베린 생산 경로의 구축<2-1> Construction of cadaverine production pathway by expression of lysine decarboxylase in M. trichosporium OB3b
M. trichosporium OB3b를 메탄으로부터 카다베린 생성 플랫폼 균주를 만들기위한 모델 균주로 사용하였다. 카다베린 생산, 활용 및 분해 경로가 카다베린 역가에 영향을 미칠 가능성이 가장 높기 때문에, 우선 M. trichosporium OB3b의 게놈에서 관련 유전자의 존재를 조사하였다. 일반적으로, 카다베린은 미생물에서 라이신 디카복실레이즈를 사용하여 라이신의 탈카르복실화에 의해 생물 기반 물질을 생산할 수 있다. 게놈에서 라이신 디카복실레이즈로 추정되는 유전자는 확인되지 않았다. E. coli 또는 코리네박테리움과는 달리 M. trichosporium OB3b는 putrescine/cadaverine aminopropyltransferase (speE), spermidine acetyltransferase (speG), glutamate-putrescine/glutamate-cadaverine ligase (puuA) 및 putrescine/cadaverine aminotransferase (ygjG)와 같은 카다베린 이용 경로와 관련된 주요 효소 중 putrescine/cadaverine aminopropyltransferase (speE)만을 갖는 것을 확인하였다. M. trichosporium OB3bb에는 라이신 디카복실레이즈 유전자가 없기 때문에, 대장균 MG1655의 라이신 디카복실레이즈 유전자 ldcC 또는 cadA를 각각 tac 프로모터하에 발현 플라스미드에 클로닝하였다. M. trichosporium OB3b에서 발현된 2종의 라이신 디카복실레이즈의 효소 활성을 평가하기 위해 시험 관내 효소 분석을 수행하였다. 이들 발현 유전자의 효소 활성은 재조합 균주의 조추출물로부터 측정하였다.M. trichosporium OB3b was used as a model strain for making a cadaverine-producing platform strain from methane. Since cadaverine production, utilization and degradation pathways are most likely to affect cadaverine titer, we first investigated the presence of the relevant genes in the genome of M. trichosporium OB3b. In general, cadaverine can produce biological-based substances by decarboxylation of lysine using lysine decarboxylase in microorganisms. A gene presumed to be lysine decarboxylase in the genome was not identified. Unlike E. coli or Corynebacterium, M. trichosporium OB3b contains putrescine/cadaverine aminopropyltransferase (speE), spermidine acetyltransferase (speG), glutamate-putrescine/glutamate-cadaverine ligase (puuA) and putrescine/cadaverine aminotransferase ( Among the major enzymes related to the same cadaverine utilization pathway, only putrescine/cadaverine aminopropyltransferase (speE) was confirmed. Since there is no lysine decarboxylase gene in M. trichosporium OB3bb, the lysine decarboxylase genes ldcC or cadA of E. coli MG1655 were cloned into expression plasmids under the tac promoter, respectively. An in vitro enzymatic assay was performed to evaluate the enzymatic activity of two lysine decarboxylases expressed in M. trichosporium OB3b. The enzymatic activity of these expressed genes was measured from crude extracts of recombinant strains.
그 결과, 야생형(WT) 균주에서는 라이신 디카복실레이즈의 활성이 나타나지 않았으며 재조합 균주에서만 관찰되었다. ldcC(5.815 ± 0.028 U/mg/min)의 비활성 (specific activity)은 cadA(4.299 ± 0.018 U/mg/min)보다 1.35배 높았다(표 3). ldcC가 M. trichosporium에서 cadA에 비해 우수한 카다베린 전환 능력을 가지고 있음을 확인하였다.As a result, the activity of lysine decarboxylase did not appear in the wild-type (WT) strain and was observed only in the recombinant strain. The specific activity of ldcC (5.815 ± 0.028 U/mg/min) was 1.35 times higher than that of cadA (4.299 ± 0.018 U/mg/min) (Table 3). It was confirmed that ldcC had superior cadaverine conversion ability compared to cadA in M. trichosporium.
또한, 플라스크에서 탄소원으로서 메탄을 사용하여 카다베린을 생산하기 위해 재조합 균주, OB3b/LdcC 및 OB3b/CadA를 배양하였다. OB3b/LdcC를 사용하여 6 일 발효 후 3.253±0.042 mg/L 카다베린을 얻었으며, OB3b/CadA(1.66±0.034 mg/L)를 사용하여 얻은 것보다 1.95배 높았다(도 3B).In addition, recombinant strains, OB3b/LdcC and OB3b/CadA, were cultured to produce cadaverine using methane as a carbon source in flasks. After 6 days of fermentation using OB3b/LdcC, 3.253±0.042 mg/L cadaverine was obtained, which was 1.95 times higher than that obtained using OB3b/CadA (1.66±0.034 mg/L) ( FIG. 3B ).
라이신 디카복실레이즈 활성이 보조 인자로서 pyridoxal-5-phosphate (PLP)에 의존하는 것으로 알려져 있으므로, pydoxal-5-phosphate hydrate을 NMS 배지에 첨가한 다음 카다베린 역가를 비교하였다. Since it is known that lysine decarboxylase activity depends on pyridoxal-5-phosphate (PLP) as a cofactor, pydoxal-5-phosphate hydrate was added to NMS medium and then cadaverine titers were compared.
그 결과, PLP의 첨가는 카다베린을 생성하는데 영향을 미치지 않았으며(도 2), M. trichosporium OB3b의 대사를 통해 생성된 PLP의 양이 카다베린 생산에 충분하였다. 이러한 결과로, E.coli로부터의 ldcC 유전자를 보유하는 재조합 OB3b/LdcC 균주는 PLP-free NMS 배지를 사용하여 메탄으로부터 카다베린을 생산하기에 적합한 균주로 확인되었다. 그러나 L-라이신 풀이 낮기 때문에 생산량이 적으므로 L-라이신의 이용 가능성을 증가시키기 위해 유전자를 추가로 형질전환하였다. As a result, the addition of PLP did not affect the production of cadaverine ( FIG. 2 ), and the amount of PLP produced through the metabolism of M. trichosporium OB3b was sufficient for the production of cadaverine. As a result, the recombinant OB3b/LdcC strain carrying the ldcC gene from E. coli was identified as a suitable strain for producing cadaverine from methane using PLP-free NMS medium. However, since the L-lysine pool is low, the production is low, and the gene was further transformed to increase the availability of L-lysine.
<2-2> 전구체 공급을 증가시키기 위한 라이신 합성 경로 재설계<2-2> Redesign of lysine synthesis pathway to increase precursor supply
카다베린의 직접 전구체인 L-라이신의 대사 플럭스를 증가시켜 카다베린의 생산을 향상시키고자 하였다. E.coli, Corynebacterium 및 Bacillus methanolicus에서 L-라이신 생합성은 전사 억제 및 피드백 억제를 통한 다양한 단계(코딩된 lyC, asd, dapB, dapD 및 lysA 유전자에 의해)를 통하여 라이신에 의해 조절되는 것으로 알려져 있다. 대장균과 마찬가지로 M. trichosporium OB3b의 L-라이신 생합성은 L-aspartate의 9개의 연속적인 효소 반응이 요구되므로 메탄자화균의 라이신 생합성에서 아미노산에 의한 라이신 생합성 경로의 전사 조절 및 피드백 억제에 대해서는 아직보고 된 바가 없다. Aspartokinase III(lysC) 또는 diaminopimelate decarboxylase(lysA)는 tac 프로모터하에 발현용 플라스미드 pAWP89-lysC 또는 pAWP89-lysA를 구축하고 (표 2), 2종의 플라스미드를 야생형 M. trichosporium OB3b로 형질전환시켜, 재조합 균주 OB3b/lysC 및 OB3b/lysA를 얻었다. 진탕 플라스크에서 L-라이신 생성에 대해 재조합 균주(OB3b/lysC 및 OB3b/lysA)를 시험하였고, 동일한 조건하에서 야생형 균주는 5.4±0.018 mg/L L-라이신을 생산하는 반면, OB3b/lysC(7.33±0.012 mg/L) 및 OB3b/lysA(14.87±0.021 mg/L)는 1.35배 및 2.75배 증가한 L-라이신을 생산하였다(표 4). It was attempted to improve the production of cadaverine by increasing the metabolic flux of L-lysine, a direct precursor of cadaverine. It is known that L-lysine biosynthesis in E. coli, Corynebacterium and Bacillus methanolicus is regulated by lysine through various steps (by the encoded lyC, asd, dapB, dapD and lysA genes) through transcriptional repression and feedback repression. As in E. coli, L-lysine biosynthesis of M. trichosporium OB3b requires nine consecutive enzymatic reactions of L-aspartate, so the transcriptional regulation and feedback inhibition of lysine biosynthetic pathways by amino acids in lysine biosynthesis of methanobacteria have not yet been reported. there is no bar Aspartokinase III (lysC) or diaminopimelate decarboxylase (lysA) was constructed by constructing a plasmid pAWP89-lysC or pAWP89-lysA for expression under the tac promoter (Table 2), and transforming the two plasmids into wild-type M. trichosporium OB3b, resulting in a recombinant strain OB3b/lysC and OB3b/lysA were obtained. Recombinant strains (OB3b/lysC and OB3b/lysA) were tested for L-lysine production in shake flasks, and under the same conditions the wild-type strain produced 5.4±0.018 mg/L L-lysine, while OB3b/lysC (7.33± 0.012 mg/L) and OB3b/lysA (14.87±0.021 mg/L) produced 1.35-fold and 2.75-fold increase in L-lysine (Table 4) .
이들 결과는 lysC 및 lysA 유전자 모두 L-라이신에 의해 억제되고, 라이신 생합성 경로의 피드백-억제된 효소의 과발현은 M. trichosporium OB3b에서 L-라이신 생산에 대해 우수한 효과를 가질 수 있음을 입증한다.These results demonstrate that both lysC and lysA genes are inhibited by L-lysine, and overexpression of feedback-repressed enzymes of the lysine biosynthetic pathway can have a superior effect on L-lysine production in M. trichosporium OB3b.
또한, ldcC 단독의 발현은 소량의 카다베린을 생성(3.253±0.042 mg/L)하는 반면에, lysC 및 lysA와의 공동발현은 카다베린의 생성(16.867 ± 0.019 mg/L 및 30.575±0.045 mg/L)을 향상시킴을 확인하였다(도 3 및 표 4). In addition, expression of ldcC alone produced small amounts of cadaverine (3.253 ± 0.042 mg/L), whereas co-expression with lysC and lysA produced cadaverine (16.867 ± 0.019 mg/L and 30.575 ± 0.045 mg/L). ) was confirmed to improve (Fig. 3 and Table 4) .
<2-3> CO<2-3> CO 22 동화 효율 개선에 따른 라이신 및 카다베린의 생산성 향상 분석 Analysis of productivity improvement of lysine and cadaverine according to the improvement of assimilation efficiency
상류 전구체인 oxaloacetate(OAA)의 부족은 카다베린 생합성 경로의 생산성을 제한할 수 있다. M. trichosporium OB3b의 카다베린 생합성 경로는 OAA로부터 출발하여 tricarboxylic acid(TCA) 사이클에서 나누어진다. 카다베린 생산을 향상시키는 다른 연구에 따르면 phosphoenolpyruvate carboxylase(PEPC) 및 pyruvate carboxylase(PYC)의 발현뿐만 아니라 phosphoenolpyruvate carboxykinase(PEPCK)의 발현을 억제하여 옥살로아세테이트(oxaloacetate)를 향상시키는 것으로 알려져 있다. M. trichosporium OB3b는 PEPC를 암호화하는 유전자를 가지고 있으며서, PEPCK를 암호화하는 유전자를 가지고 있지 않아 대장균 및 C. glutamincum에 비해 라이신 및 카다베린 생산을 위한 합성 경로 재설계를 위한 노력을 절감할 수 있다.The lack of an upstream precursor, oxaloacetate (OAA), may limit the productivity of the cadaverine biosynthetic pathway. The cadaverine biosynthetic pathway in M. trichosporium OB3b is divided in the tricarboxylic acid (TCA) cycle starting from OAA. Another study that enhances cadaverine production has been known to enhance oxaloacetate by inhibiting the expression of phosphoenolpyruvate carboxylase (PEPCK), as well as the expression of phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PYC). M. trichosporium OB3b has a gene encoding PEPC and does not have a gene encoding PEPCK, so it can reduce the effort for redesigning the synthetic pathway for lysine and cadaverine production compared to E. coli and C. glutamincum. .
M. trichosporium Ob3b에는 pyruvate carboxylase를 코딩하는 pyc 유전자가 없기 때문에 Methylomonas sp. DH-1 유래의 pyc 유전자를 선택하여 피루베이트에서 옥살로 아세테이트로의 플럭스를 재설계하고자 하였다. pyc 유전자를 lysA를 함유하는 발현 벡터에 클로닝하여 플라스미드 pAWP89-lysA-pyc를 구축하고 이를 야생형 균주에 형질전환시켜 재조합 OB3b/lysA-pyc 균주를 얻었다.Since M. trichosporium Ob3b lacks the pyc gene encoding pyruvate carboxylase, Methylomonas sp. We tried to redesign the flux from pyruvate to oxaloacetate by selecting the pyc gene derived from DH-1. The pyc gene was cloned into an expression vector containing lysA to construct a plasmid pAWP89-lysA-pyc, which was transformed into a wild-type strain to obtain a recombinant OB3b/lysA-pyc strain.
그 결과, OB3b/lysA-pyc 균주의 L-라이신 역가(21.66±0.018 mg/L)는 OB3b/lysA 균주(14.87±0.021 mg/L)보다 1.45배 더 높았다(표 4). 카다베린 생산에 대한 lysA 및 pyc 유전자의 공동발현 효과를 평가하기 위하여, 플라스미드 pAWP89-lysA-pyc를 OB3b/LdcC 균주로 형질 전환한 후, 재조합 OB3b/cad3 균주를 얻었다. OB3b/cad3를 이용하여 6일간 배양한 결과, 41.937±0.278 mg/L의 카다베린을 생산하였고, 이는 초기 균주 OB3b/LdcC보다 생산성이 12.9배 증가한 결과이다(도 4B).As a result, the L-lysine titer (21.66±0.018 mg/L) of the OB3b/lysA-pyc strain was 1.45 times higher than that of the OB3b/lysA strain (14.87±0.021 mg/L) (Table 4). To evaluate the effect of co-expression of lysA and pyc genes on cadaverine production, the plasmid pAWP89-lysA-pyc was transformed into an OB3b/LdcC strain, and then a recombinant OB3b/cad3 strain was obtained. As a result of culturing for 6 days using OB3b/cad3, cadaverine of 41.937±0.278 mg/L was produced, which is a result of a 12.9-fold increase in productivity compared to the initial strain OB3b/LdcC ( FIG. 4B ).
<2-4> 카다베린-라이신 역수송체에 의해 M. trichosporium OB3에서 카다베린의 생성 분석 <2-4> Analysis of cadaverine production in M. trichosporium OB3 by cadaverine-lysine reverse transporter
cadB 유전자는 카다베린-라이신 역수용체를 암호화하는 것으로 알려져있다. cadB의 기능은 생성물 카다베린을 세포 외로 내보낼뿐만 아니라 기질 라이신을 세포 내로 유입시킨다(Ma et al., 2017). M. trichosporium OB3b 내 cadB의 과발현은 카다베린 생산성을 향상시킬 것으로 예상되어 형질전환 균주 OB3b/cad4를 제작하였다. 형질전환된 균주 OB3b/cad4의 카다베린의 최대 역가는 약 53.56±0.155 mg/L로 관찰되었고 카다베린의 생산성은 약 0.201±0.002 mmol/g DCW/일로, 이는 OB3b/LdC 및 OB3b/cad3 균주와 비교하여 각각 카다베린 생산성이 16.5배 및 1.3배 향상된 결과이다(표 4 및 도 4B). 카다베린의 분비 효율은 균주 OB3b/cad3에 비해 21.7% 향상되었으며, M. trichosporium OB3b 내 cadB의 과발현은 카다베린 생산성을 향상시킴을 확인하였다.The cadB gene is known to encode a cadaverine-lysine inverse receptor. The function of cadB is not only to export the product cadaverine out of the cell, but also to bring the substrate lysine into the cell (Ma et al., 2017). The overexpression of cadB in M. trichosporium OB3b was expected to improve cadaverine productivity, so a transformed strain OB3b/cad4 was prepared. The maximum titer of cadaverine in the transformed strain OB3b/cad4 was observed to be about 53.56±0.155 mg/L, and the productivity of cadaverine was about 0.201±0.002 mmol/g DCW/day, which was comparable to that of OB3b/LdC and OB3b/cad3 strains. In comparison, cadaverine productivity was improved by 16.5 times and 1.3 times, respectively (Table 4 and FIG. 4B). The secretion efficiency of cadaverine was improved by 21.7% compared to strain OB3b/cad3, and it was confirmed that overexpression of cadB in M. trichosporium OB3b improves cadaverine productivity.
<2-5> 생물 반응기에서 재조합 M. trichosporium OB3b 균주의 생산성 분석<2-5> Productivity analysis of recombinant M. trichosporium OB3b strain in bioreactor
1L 유가 배양기에서 카다베린의 생성을 확인하였다. OB3b/cad4 균주를 유가 배양기에서 배양하였다. 유가 배양의 경우, 10% CH4 및 40% 공기를 연속적으로 제공하였다. 접종에 사용된 OB3b/cad4 균주를 플라스크 배양에서 동일한 조건에서 배양하였다. Production of cadaverine was confirmed in a 1L fed-batch incubator. The OB3b/cad4 strain was cultured in a fed-batch incubator. For fed-batch culture, 10% CH 4 and 40% air were continuously provided. The OB3b/cad4 strain used for inoculation was cultured under the same conditions in flask culture.
결과, 플라스크 배양에서 최대 OD600은 6일 후 약 2.797인 반면, 생물반응기의 최대 세포 밀도는 OD600은 약 5.726(8일 배양)로 생물 반응기에서의 세포 성장이 플라스크 배양보다 빨랐으며, 유가 배양 8일 후 카다베린의 역가는 192.09 mg/L이었다(도 5). Result, while from about 2.797 after the maximum OD 600 for 6 days in the flask cultures, the maximum cell density of the bioreactor is OD 600 were about 5.726 the growth of cells in a bioreactor (8 day culture) faster than flask culture, Batch Culture After 8 days, the titer of cadaverine was 192.09 mg/L ( FIG. 5 ).
<110> University-Industry Cooperation Group of Kyung Hee University <120> Transformed methanotrophs for producing cadaverine and uses thereof <130> PN1910-474 <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 3258 <212> DNA <213> Unknown <220> <223> pyc gene from Methylomonas sp. DH-1 <400> 1 atgttacgca aaatcctgat cgccaatcgc ggcgagattg ccgtccgcat cattcgcgcc 60 tgcgccgaaa tgggcatccg ttccgcagcg atctattccg aagccgaccg ctttgccctg 120 cacgtcaaaa aggccgacga gtcttattac atcggcagcg agccgttggc gggttacctg 180 aatatctacg gcttggtcga cttggctctg cgcaccggtt gcgatgcgat tcatccgggc 240 tacggcttcc tgtcggaaaa cgcccaattt gccaaagcct gtcaggagcg cggattagtc 300 tttatcgggc cgtcggcgga agtcatccag cgcatgggcg acaagactga agcgcgggcg 360 gcgatgaaag ctgccggcct gccggtgacg cccggttccg acggcaacct cgaaaatgtc 420 gagcaggcct tgcaggtggc cgagcagatc ggctacccga tcatgttgaa agccacctcc 480 ggcggcggcg gccgtggcat ccgccgttgc gataaccccg acgagttgcg gcaaaattat 540 caacgcgtca tttccgaagc gaccaaggcc ttcggcagtg ccgacgtgtt tctggaaaaa 600 tgcgtcgtca atccgattca tattgaggtg caagtgttgg ccgaccaatt cggcaacact 660 atccatttgt acgaacgcga ttgctcggta cagcgccgca accaaaaact gatcgaaatc 720 gcgccgtcgc cgcaactcga cgaggcccaa cgccaatact tgggcggttt ggcggtgatg 780 gcggctaaag ccgtcggcta caccaacgcc ggtactgtcg agtttttatt ggacagcaat 840 ggccgtttct atttcatgga aatgaatacc cgcgtgcagg tcgaacacac gatcaccgaa 900 actattaccg gtgtcgacat cgtcgaggaa cagattcggg tcgccgccgg tctgccgcta 960 cgtttcaagc aggaagaaat cgtcagacgc ggttatgcaa tccagtttcg ggtcaacgcc 1020 gaggacccga aaaacaattt cctgcccagc ttcggccaca tttcccgtta ctacgcgccc 1080 ggcggccccg gcgtgcgcac cgacaccgcg atttataccg gctacgaagt gccgccgttt 1140 tacgattcga tgctggccaa agtcatcgtc agcgcgatga cctgggaaga tgccatcaat 1200 cgcggcgagc gggccttgaa ggacatgggt ttgttcggca tcaaaaccac gattccctat 1260 tacctgcgga tactggccca ccccgatttt cgccgcggcc gcttcaacac cggtttcgtc 1320 gaagccaatc ccgatttaat caattattcc aacaaaccgc gcccggaaat tctggccagc 1380 gtgctggcgg ccgttgtcgc ttcccatacc ggcctctaag ccaaggaaac tctcatgagc 1440 aaagtgtatg taaccgacgt tattctgcgc gacgcccacc agtcgctgat cgccacccgg 1500 atgcgcaccg aagacatgtt gccggcttgc acgatgttgg acgacatcgg ctattggtcg 1560 ttggaatgct ggggcggcgc cacctttgat gcctgcttgc gcttcttgaa agaagaccct 1620 tgggagcgct tgagcaaact gaaagcagcc ctgccaaata ccaaattgca gatgctgctg 1680 cgtggccaaa atctgctcgg ttatcgtcac tattccgacg acgtggtgcg taagttcgtc 1740 gagaccgccg cccgcaacgg catggacgta ttccgcatct tcgacgcgct gaacgatatt 1800 cgtaacttgg aaaccgccat cgaagccacc cgcgaggccg gcaaacatgc ccagggcacg 1860 atttgctaca ccaccagtcc ggtacacgat atcgccagtt tcatcaaact gggtaaaggt 1920 ttggccaacc tgggctgtaa ctcgattgcg atcaaggaca tggccggctt gttgacgcca 1980 tacatcgccg gcgaaatggt caaagctctg aaagacgccg tcgatttgcc gttgcatttg 2040 cactgccatg ctacggccgg cttagcggaa atgtgccaga tcaaggccat cgaagccggc 2100 tgcgagcacg tcgataccgc gctgtcgtcc tgggccggcg gcaccagcca tccaccgacc 2160 gaaagcctgg tcaccgcgtt gcgcggcacc gattacgaca cggggctgga tttggataaa 2220 ctgcaagcgg tgaacgacta tttcgccgaa gtccgcaaaa aataccgccg cttcgaaagc 2280 gaatttaccg gcatcgacac ccgcgtccac atcttccaag tcccgggcgg catgatctcc 2340 aacctggcca accaattgaa agaacgcaac gcactggacc ggatcgacga ggtgtataaa 2400 gaaatcccgg aagtccgcaa agacctgggc tatccgccat tggtgacacc aacctcgcaa 2460 atcgtcggca cccaggcggt gttgaacgtg ctgatcggca agcgttacga gaccatcagc 2520 aacgaggtta aacgctacct gcacggcggc tacggcaagg cgccggcgcc ggtcaatccg 2580 caattgctgg ccaaggcggt cggtaaagag gaaatcatcg aatgccggcc ggccgatttg 2640 ctcaagcccg agttcgagca tcttcgtggc gaaatcgccc atctggcgtt gaacgacgag 2700 gacgtactga gttacgcgat gtttccggaa atcggcaaac aattcctgga attgcgttcg 2760 aacgacaacc tagtgccgga gccgttggaa ttggaggctg cgcctaaacc gggcgaggtc 2820 aaaaaagccc cgaccgaatt caacgtggcg ttgcacggcg agcaatacca cgtcaaagtt 2880 accggcgccg gcccgaaaaa ccaaagcctg cggcattttt acttcaccgt ggacggcatg 2940 ccggaagaaa tcgtcatcga gaccttggac gaaatcgtac tggacggcgg cgcccatggc 3000 gcggtgcaaa gcgcaatcgc cagcaagcgc cgccggccca gcgagccggg cgatgtggtg 3060 accagcatgc cgtgcaacat catcgacgtg ctggtcaagg agggacagaa agtcaacgcc 3120 ggccaggcct tgctggtcac cgaagcgatg aaaatggaga ccgaaatcac ggcaccgatt 3180 gccggcgtgg ttaaagccat ttacgtcgcc aaaggcgatg cggttaaccc caacgaggtg 3240 ttggtggaaa taaattga 3258 <210> 2 <211> 1269 <212> DNA <213> Unknown <220> <223> lysA gene from M. trichosporium OB3b <400> 2 atgcatcatt tcgactatcg cgccggcgtc ctccacgccg aggacgtggc cctgccgaag 60 atcgccgagg acgtcggcgc gcccttctac tgctatagcg cggcgacgat ccggcggcac 120 ttcaccgtct tttccgccgc tttcgcgggg ctcgacgcgc tcgtctgcta tgcggtgaag 180 gccaattcca atcaggccgt gctcgccctg ctcgccggac tcggcgccgg catggacgtc 240 gtctccggcg gcgagctgcg ccgcgcccgc gccgccggcg ttcccgcgag caagatcacc 300 ttctccggcg tcggcaagac ggcgcaggag atcgcgctcg cgatcgacga aggcatcttc 360 tgcttcaatg tggagtccga gccggagctc gaggcgatcg ctgcgatcgc tgcggcgaag 420 gggagacgcg cccatatctc tctgcgcgtc aatccggacg tcgacgccaa gacccacgcc 480 aagatctcga ccggcctcgc cgagaataaa ttcggcgtgc ccttgtcgcg cgcccgcgac 540 atttacgcgc aggcggcgcg gctgccggga ctcgacatcg tcggcgtcga catgcacatc 600 ggctcgcaga tcactgatct tcgccccttc gacgcggcct tttccctgct cgccgagctc 660 gtcgtcggcc tgcgcgccga cggacacgcg atctcccatg tcgatctcgg cggcggcctc 720 ggcattccct atcacgccgg cgaggacccg gcctcctatc atccagaccg ctatgcggag 780 atcgtgcgcc gccatatggg cgcgctcggc tgcaagctcc tgttcgagcc cggccggctc 840 atcgtcggca atgccggcgt gctggtgacg cgcgtcctct atgtgaagca gggcgaggcc 900 aagaccttcg tcatcgtcga cgccggcatg aacgatctcg tccgtccgac cctctacgac 960 gcctggcacg agatcattcc ggtgaccgag cccgcgcggg ggcggaacga gattctcgcc 1020 gatgtcgtcg ggccggtgtg cgagaccggc gattacattg cgctcggccg ctcgatcccg 1080 gcgccggcgc agggcgatct gctcgcggtt ctgacctccg gggcctatgg cgccgtgcag 1140 gcgggaacct ataattcgcg gccgctcatt cccgaggtgc tggtcgacgg cgcgcgctgg 1200 gcggtggtgc gcgcgcgtcc gagcatagag agcctcatcg cgctcgacag cgttcccgat 1260 tggctgtga 1269 <210> 3 <211> 2142 <212> DNA <213> Unknown <220> <223> ldcC gene from E.coli <400> 3 atgaacatca ttgccattat gggaccgcat ggcgtctttt ataaagatga gcccatcaaa 60 gaactggagt cggcgctggt ggcgcaaggc tttcagatta tctggccaca aaacagcgtt 120 gatttgctga aatttatcga gcataaccct cgaatttgcg gcgtgatttt tgactgggat 180 gagtacagtc tcgatttatg tagcgatatc aatcagctta atgaatatct cccgctttat 240 gccttcatca acacccactc gacgatggat gtcagcgtgc aggatatgcg gatggcgctc 300 tggttttttg aatatgcgct ggggcaggcg gaagatatcg ccattcgtat gcgtcagtac 360 accgacgaat atcttgataa cattacaccg ccgttcacga aagccttgtt tacctacgtc 420 aaagagcgga agtacacctt ttgtacgccg gggcatatgg gcggcaccgc atatcaaaaa 480 agcccggttg gctgtctgtt ttatgatttt ttcggcggga atactcttaa ggctgatgtc 540 tctatttcgg tcaccgagct tggttcgttg ctcgaccaca ccgggccaca cctggaagcg 600 gaagagtaca tcgcgcggac ttttggcgcg gaacagagtt atatcgttac caacggaaca 660 tcgacgtcga acaaaattgt gggtatgtac gccgcgccat ccggcagtac gctgttgatc 720 gaccgcaatt gtcataaatc gctggcgcat ctgttgatga tgaacgatgt agtgccagtc 780 tggctgaaac cgacgcgtaa tgcgttgggg attcttggtg ggatcccgcg ccgtgaattt 840 actcgcgaca gcatcgaaga gaaagtcgct gctaccacgc aagcacaatg gccggttcat 900 gcggtgatca ccaactccac ctatgatggc ttgctctaca acaccgactg gatcaaacag 960 acgctggatg tcccgtcgat tcacttcgat tctgcctggg tgccgtacac ccattttcat 1020 ccgatctacc agggtaaaag tggtatgagc ggcgagcgtg ttgcgggaaa agtgatcttc 1080 gaaacgcaat cgacccacaa aatgctggcg gcgttatcgc aggcttcgct gatccacatt 1140 aaaggcgagt atgacgaaga ggcctttaac gaagccttta tgatgcatac caccacctcg 1200 cccagttatc ccattgttgc ttcggttgag acggcggcgg cgatgctgcg tggtaatccg 1260 ggcaaacggc tgattaaccg ttcagtagaa cgagctctgc attttcgcaa agaggtccag 1320 cggctgcggg aagagtctga cggttggttt ttcgatatct ggcaaccgcc gcaggtggat 1380 gaagccgaat gctggcccgt tgcgcctggc gaacagtggc acggctttaa cgatgcggat 1440 gccgatcata tgtttctcga tccggttaaa gtcactattt tgacaccggg gatggacgag 1500 cagggcaata tgagcgagga ggggatcccg gcggcgctgg tagcaaaatt cctcgacgaa 1560 cgtgggatcg tagtagagaa aaccggccct tataacctgc tgtttctctt tagtattggc 1620 atcgataaaa ccaaagcaat gggattattg cgtgggttga cggaattcaa acgctcttac 1680 gatctcaacc tgcggatcaa aaatatgcta cccgatctct atgcagaaga tcccgatttc 1740 taccgcaata tgcgtattca ggatctggca caagggatcc ataagctgat tcgtaaacac 1800 gatcttcccg gtttgatgtt gcgggcattc gatactttgc cggagatgat catgacgcca 1860 catcaggcat ggcaacgaca aattaaaggc gaagtagaaa ccattgcgct ggaacaactg 1920 gtcggtagag tatcggcaaa tatgatcctg ccttatccac cgggcgtacc gctgttgatg 1980 cctggagaaa tgctgaccaa agagagccgc acagtactcg attttctact gatgctttgt 2040 tccgtcgggc aacattaccc cggttttgaa acggatattc acggcgcgaa acaggacgaa 2100 gacggcgttt accgcgtacg agtcctaaaa atggcgggat aa 2142 <210> 4 <211> 1335 <212> DNA <213> Unknown <220> <223> cadB gene from E.coli <400> 4 atgagttctg ccaagaagat cgggctattt gcctgtaccg gtgttgttgc cggtaatatg 60 atggggagcg gtattgcatt attacctgcg aacctagcaa gtatcggtgg tattgctatc 120 tggggttgga ttatctctat tattggtgca atgtcgctgg cgtatgtata tgcccgactg 180 gcaacaaaaa acccgcaaca aggtggccca attgcttatg ccggagaaat ttcccctgca 240 tttggttttc agacaggtgt tctttattac catgctaact ggattggtaa cctggcgatt 300 ggtattaccg ctgtatctta tctttccacc ttcttcccag tattaaatga tcctgttccg 360 gcgggtatcg cctgtattgc tatcgtctgg gtatttacct ttgtaaatat gctcggcggt 420 acttgggtaa gccgtttaac cactattggt ctggtgctgg ttcttattcc tgtggtgatg 480 actgctattg ttggctggca ttggtttgat gcggcaactt atgcagctaa ctggaatact 540 gcggatacca ctgatggtca tgcgatcatt aaaagtattc tgctctgcct gtgggccttc 600 gtgggtgttg aatccgcagc tgtaagtact ggtatggtta aaaacccgaa acgtaccgtt 660 ccgctggcaa ccatgctggg tactggttta gcaggtattg tttacatcgc tgcgactcag 720 gtgctttccg gtatgtatcc gtcttctgta atggcggctt ccggtgctcc gtttgcaatc 780 agtgcttcaa ctatcctcgg taactgggct gcgccgctgg tttctgcatt caccgccttt 840 gcgtgcctga cttctctggg ctcctggatg atgttggtag gccaggcagg tgtacgtgcc 900 gctaacgacg gtaacttccc gaaagtttat ggtgaagtcg acagcaacgg tattccgaaa 960 aaaggtctgc tgctggctgc agtgaaaatg actgccctga tgatccttat cactctgatg 1020 aactctgccg gtggtaaagc atctgacctg ttcggtgaac tgaccggtat cgcagtactg 1080 ctgactatgc tgccgtattt ctactcttgc gttgacctga ttcgttttga aggcgttaac 1140 atccgcaact ttgtcagcct gatctgctct gtactgggtt gcgtgttctg cttcatcgcg 1200 ctgatgggcg caagctcctt cgagctggca ggtaccttca tcgtcagcct gattatcctg 1260 atgttctacg ctcgcaaaat gcacgagcgc cagagccact caatggataa ccacaccgcg 1320 tctaacgcac attaa 1335 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pAWP89-Tac-For <400> 5 tagttgtcgg gaagatgcgt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pAWP89-Tac-Rev <400> 6 agctgtttcc tgtgtgaata 20 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ldcC-For <400> 7 tattcacaca ggaaacagct atgaacatca ttgccattat gggac 45 <210> 8 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> ldcC-Rev <400> 8 gcatcttccc gacaactatt atcccgccat ttttaggact c 41 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> cadA-K12-For <400> 9 tattcacaca ggaaacagct atgaacgtta ttgcaatatt 40 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> cadA-K12-Rev <400> 10 gcatcttccc gacaactatt attttttgct ttctt 35 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> lysA-For <400> 11 gggaacaaaa gctgggtaca tgcatcattt cgactatcg 39 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> LysA-Rev <400> 12 cgaggggggg cccggtactc acagccaatc gggaacgc 38 <210> 13 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> lysC-For <400> 13 gggaacaaaa gctgggtaca tggctcgtct ggtgatgaa 39 <210> 14 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> LysC-Rev <400> 14 cgaggggggg cccggtactc aggccgcgtc cagcccat 38 <210> 15 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Pyc-For <400> 15 ggtatcgata agcttgatat cgtcacggcg cagctgcgcc ctaccgctgt taaaggagac 60 attttatgtt acgcaaaatc ctgat 85 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Pyc-Rev <400> 16 gatcccccgg gctgcaggtc aatttatttc caccaaca 38 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Ptac-ldc-BamHI-For <400> 17 ttgacctgcc agcccggggt tgacaattaa tcatcggct 39 <210> 18 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Ptac-ldc-BamHI-Rev <400> 18 gccgctctag aactagtgtt atcccgccat ttttagga 38 <210> 19 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> cadB-XbaI-For <400> 19 ggcgggataa cactagtttt aaccaacagt ttttctacct aaaggaggcg ttttatgagt 60 tctgccaaga agat 74 <110> University-Industry Cooperation Group of Kyung Hee University <120> Transformed methanotrophs for producing cadaverine and uses it <130> PN1910-474 <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 3258 <212> DNA <213> Unknown <220> <223> pyc gene from Methylomonas sp. DH-1 <400> 1 atgttacgca aaatcctgat cgccaatcgc ggcgagattg ccgtccgcat cattcgcgcc 60 tgcgccgaaa tgggcatccg ttccgcagcg atctattccg aagccgaccg ctttgccctg 120 cacgtcaaaa aggccgacga gtcttattac atcggcagcg agccgttggc gggttacctg 180 aatatctacg gcttggtcga cttggctctg cgcaccggtt gcgatgcgat tcatccgggc 240 tacggcttcc tgtcggaaaa cgcccaattt gccaaagcct gtcaggagcg cggattagtc 300 tttatcgggc cgtcggcgga agtcatccag cgcatgggcg acaagactga agcgcgggcg 360 gcgatgaaag ctgccggcct gccggtgacg cccggttccg acggcaacct cgaaaatgtc 420 gagcaggcct tgcaggtggc cgagcagatc ggctacccga tcatgttgaa agccacctcc 480 ggcggcggcg gccgtggcat ccgccgttgc gataaccccg acgagttgcg gcaaaattat 540 caacgcgtca tttccgaagc gaccaaggcc ttcggcagtg ccgacgtgtt tctggaaaaa 600 tgcgtcgtca atccgattca tattgaggtg caagtgttgg ccgaccaatt cggcaacact 660 atccatttgt acgaacgcga ttgctcggta cagcgccgca accaaaaact gatcgaaatc 720 gcgccgtcgc cgcaactcga cgaggcccaa cgccaatact tgggcggttt ggcggtgatg 780 gcggctaaag ccgtcggcta caccaacgcc ggtactgtcg agtttttatt ggacagcaat 840 ggccgtttct atttcatgga aatgaatacc cgcgtgcagg tcgaacacac gatcaccgaa 900 actattaccg gtgtcgacat cgtcgaggaa cagattcggg tcgccgccgg tctgccgcta 960 cgtttcaagc aggaagaaat cgtcagacgc ggttatgcaa tccagtttcg ggtcaacgcc 1020 gaggacccga aaaacaattt cctgcccagc ttcggccaca tttcccgtta ctacgcgccc 1080 ggcggccccg gcgtgcgcac cgacaccgcg atttataccg gctacgaagt gccgccgttt 1140 tacgattcga tgctggccaa agtcatcgtc agcgcgatga cctgggaaga tgccatcaat 1200 cgcggcgagc gggccttgaa ggacatgggt ttgttcggca tcaaaaccac gattccctat 1260 tacctgcgga tactggccca ccccgatttt cgccgcggcc gcttcaacac cggtttcgtc 1320 gaagccaatc ccgatttaat caattattcc aacaaaccgc gcccggaaat tctggccagc 1380 gtgctggcgg ccgttgtcgc ttcccatacc ggcctctaag ccaaggaaac tctcatgagc 1440 aaagtgtatg taaccgacgt tattctgcgc gacgcccacc agtcgctgat cgccacccgg 1500 atgcgcaccg aagacatgtt gccggcttgc acgatgttgg acgacatcgg ctattggtcg 1560 ttggaatgct ggggcggcgc cacctttgat gcctgcttgc gcttcttgaa agaagaccct 1620 tgggagcgct tgagcaaact gaaagcagcc ctgccaaata ccaaattgca gatgctgctg 1680 cgtggccaaa atctgctcgg ttatcgtcac tattccgacg acgtggtgcg taagttcgtc 1740 gagaccgccg cccgcaacgg catggacgta ttccgcatct tcgacgcgct gaacgatatt 1800 cgtaacttgg aaaccgccat cgaagccacc cgcgaggccg gcaaacatgc ccagggcacg 1860 atttgctaca ccaccagtcc ggtacacgat atcgccagtt tcatcaaact gggtaaaggt 1920 ttggccaacc tgggctgtaa ctcgattgcg atcaaggaca tggccggctt gttgacgcca 1980 tacatcgccg gcgaaatggt caaagctctg aaagacgccg tcgatttgcc gttgcatttg 2040 cactgccatg ctacggccgg cttagcggaa atgtgccaga tcaaggccat cgaagccggc 2100 tgcgagcacg tcgataccgc gctgtcgtcc tgggccggcg gcaccagcca tccaccgacc 2160 gaaagcctgg tcaccgcgtt gcgcggcacc gattacgaca cggggctgga tttggataaa 2220 ctgcaagcgg tgaacgacta tttcgccgaa gtccgcaaaa aataccgccg cttcgaaagc 2280 gaatttaccg gcatcgacac ccgcgtccac atcttccaag tcccgggcgg catgatctcc 2340 aacctggcca accaattgaa agaacgcaac gcactggacc ggatcgacga ggtgtataaa 2400 gaaatcccgg aagtccgcaa agacctgggc tatccgccat tggtgacacc aacctcgcaa 2460 atcgtcggca cccaggcggt gttgaacgtg ctgatcggca agcgttacga gaccatcagc 2520 aacgaggtta aacgctacct gcacggcggc tacggcaagg cgccggcgcc ggtcaatccg 2580 caattgctgg ccaaggcggt cggtaaagag gaaatcatcg aatgccggcc ggccgatttg 2640 ctcaagcccg agttcgagca tcttcgtggc gaaatcgccc atctggcgtt gaacgacgag 2700 gacgtactga gttacgcgat gtttccggaa atcggcaaac aattcctgga attgcgttcg 2760 aacgacaacc tagtgccgga gccgttggaa ttggaggctg cgcctaaacc gggcgaggtc 2820 aaaaaagccc cgaccgaatt caacgtggcg ttgcacggcg agcaatacca cgtcaaagtt 2880 accggcgccg gcccgaaaaa ccaaagcctg cggcattttt acttcaccgt ggacggcatg 2940 ccggaagaaa tcgtcatcga gaccttggac gaaatcgtac tggacggcgg cgcccatggc 3000 gcggtgcaaa gcgcaatcgc cagcaagcgc cgccggccca gcgagccggg cgatgtggtg 3060 accagcatgc cgtgcaacat catcgacgtg ctggtcaagg agggacagaa agtcaacgcc 3120 ggccaggcct tgctggtcac cgaagcgatg aaaatggaga ccgaaatcac ggcaccgatt 3180 gccggcgtgg ttaaagccat ttacgtcgcc aaaggcgatg cggttaaccc caacgaggtg 3240 ttggtggaaa taaattga 3258 <210> 2 <211> 1269 <212> DNA <213> Unknown <220> <223> lysA gene from M. trichosporium OB3b <400> 2 atgcatcatt tcgactatcg cgccggcgtc ctccacgccg aggacgtggc cctgccgaag 60 atcgccgagg acgtcggcgc gcccttctac tgctatagcg cggcgacgat ccggcggcac 120 ttcaccgtct tttccgccgc tttcgcgggg ctcgacgcgc tcgtctgcta tgcggtgaag 180 gccaattcca atcaggccgt gctcgccctg ctcgccggac tcggcgccgg catggacgtc 240 gtctccggcg gcgagctgcg ccgcgcccgc gccgccggcg ttcccgcgag caagatcacc 300 ttctccggcg tcggcaagac ggcgcaggag atcgcgctcg cgatcgacga aggcatcttc 360 tgcttcaatg tggagtccga gccggagctc gaggcgatcg ctgcgatcgc tgcggcgaag 420 gggagacgcg cccatatctc tctgcgcgtc aatccggacg tcgacgccaa gacccacgcc 480 aagatctcga ccggcctcgc cgagaataaa ttcggcgtgc ccttgtcgcg cgcccgcgac 540 atttacgcgc aggcggcgcg gctgccggga ctcgacatcg tcggcgtcga catgcacatc 600 ggctcgcaga tcactgatct tcgccccttc gacgcggcct tttccctgct cgccgagctc 660 gtcgtcggcc tgcgcgccga cggacacgcg atctcccatg tcgatctcgg cggcggcctc 720 ggcattccct atcacgccgg cgaggacccg gcctcctatc atccagaccg ctatgcggag 780 atcgtgcgcc gccatatggg cgcgctcggc tgcaagctcc tgttcgagcc cggccggctc 840 atcgtcggca atgccggcgt gctggtgacg cgcgtcctct atgtgaagca gggcgaggcc 900 aagaccttcg tcatcgtcga cgccggcatg aacgatctcg tccgtccgac cctctacgac 960 gcctggcacg agatcattcc ggtgaccgag cccgcgcggg ggcggaacga gattctcgcc 1020 gatgtcgtcg ggccggtgtg cgagaccggc gattacattg cgctcggccg ctcgatcccg 1080 gcgccggcgc agggcgatct gctcgcggtt ctgacctccg gggcctatgg cgccgtgcag 1140 gcgggaacct ataattcgcg gccgctcatt cccgaggtgc tggtcgacgg cgcgcgctgg 1200 gcggtggtgc gcgcgcgtcc gagcatagag agcctcatcg cgctcgacag cgttcccgat 1260 tggctgtga 1269 <210> 3 <211> 2142 <212> DNA <213> Unknown <220> <223> ldcC gene from E. coli <400> 3 atgaacatca ttgccattat gggaccgcat ggcgtctttt ataaagatga gcccatcaaa 60 gaactggagt cggcgctggt ggcgcaaggc tttcagatta tctggccaca aaacagcgtt 120 gatttgctga aatttatcga gcataaccct cgaatttgcg gcgtgatttt tgactgggat 180 gagtacagtc tcgatttatg tagcgatatc aatcagctta atgaatatct cccgctttat 240 gccttcatca acacccactc gacgatggat gtcagcgtgc aggatatgcg gatggcgctc 300 tggttttttg aatatgcgct ggggcaggcg gaagatatcg ccattcgtat gcgtcagtac 360 accgacgaat atcttgataa cattacaccg ccgttcacga aagccttgtt tacctacgtc 420 aaagagcgga agtacacctt ttgtacgccg gggcatatgg gcggcaccgc atatcaaaaa 480 agcccggttg gctgtctgtt ttatgatttt ttcggcggga atactcttaa ggctgatgtc 540 tctatttcgg tcaccgagct tggttcgttg ctcgaccaca ccgggccaca cctggaagcg 600 gaagagtaca tcgcgcggac ttttggcgcg gaacagagtt atatcgttac caacggaaca 660 tcgacgtcga acaaaattgt gggtatgtac gccgcgccat ccggcagtac gctgttgatc 720 gaccgcaatt gtcataaatc gctggcgcat ctgttgatga tgaacgatgt agtgccagtc 780 tggctgaaac cgacgcgtaa tgcgttgggg attcttggtg ggatcccgcg ccgtgaattt 840 actcgcgaca gcatcgaaga gaaagtcgct gctaccacgc aagcacaatg gccggttcat 900 gcggtgatca ccaactccac ctatgatggc ttgctctaca acaccgactg gatcaaacag 960 acgctggatg tcccgtcgat tcacttcgat tctgcctggg tgccgtacac ccattttcat 1020 ccgatctacc agggtaaaag tggtatgagc ggcgagcgtg ttgcgggaaa agtgatcttc 1080 gaaacgcaat cgacccacaa aatgctggcg gcgttatcgc aggcttcgct gatccacatt 1140 aaaggcgagt atgacgaaga ggcctttaac gaagccttta tgatgcatac caccacctcg 1200 cccagttatc ccattgttgc ttcggttgag acggcggcgg cgatgctgcg tggtaatccg 1260 ggcaaacggc tgattaaccg ttcagtagaa cgagctctgc attttcgcaa agaggtccag 1320 cggctgcggg aagagtctga cggttggttt ttcgatatct ggcaaccgcc gcaggtggat 1380 gaagccgaat gctggcccgt tgcgcctggc gaacagtggc acggctttaa cgatgcggat 1440 gccgatcata tgtttctcga tccggttaaa gtcactattt tgacaccggg gatggacgag 1500 cagggcaata tgagcgagga ggggatcccg gcggcgctgg tagcaaaatt cctcgacgaa 1560 cgtgggatcg tagtagagaa aaccggccct tataacctgc tgtttctctt tagtattggc 1620 atcgataaaa ccaaagcaat gggattattg cgtgggttga cggaattcaa acgctcttac 1680 gatctcaacc tgcggatcaa aaatatgcta cccgatctct atgcagaaga tcccgatttc 1740 taccgcaata tgcgtattca ggatctggca caagggatcc ataagctgat tcgtaaacac 1800 gatcttcccg gtttgatgtt gcgggcattc gatactttgc cggagatgat catgacgcca 1860 catcaggcat ggcaacgaca aattaaaggc gaagtagaaa ccattgcgct ggaacaactg 1920 gtcggtagag tatcggcaaa tatgatcctg ccttatccac cgggcgtacc gctgttgatg 1980 cctggagaaa tgctgaccaa agagagccgc acagtactcg attttctact gatgctttgt 2040 tccgtcgggc aacattaccc cggttttgaa acggatattc acggcgcgaa acaggacgaa 2100 gacggcgttt accgcgtacg agtcctaaaa atggcgggat aa 2142 <210> 4 <211> 1335 <212> DNA <213> Unknown <220> <223> cadB gene from E. coli <400> 4 atgagttctg ccaagaagat cgggctattt gcctgtaccg gtgttgttgc cggtaatatg 60 atggggagcg gtattgcatt attacctgcg aacctagcaa gtatcggtgg tattgctatc 120 tggggttgga ttatctctat tattggtgca atgtcgctgg cgtatgtata tgcccgactg 180 gcaacaaaaa acccgcaaca aggtggccca attgcttatg ccggagaaat ttcccctgca 240 tttggttttc agacaggtgt tctttattac catgctaact ggattggtaa cctggcgatt 300 ggtattaccg ctgtatctta tctttccacc ttcttcccag tattaaatga tcctgttccg 360 gcgggtatcg cctgtattgc tatcgtctgg gtatttacct ttgtaaatat gctcggcggt 420 acttgggtaa gccgtttaac cactattggt ctggtgctgg ttcttattcc tgtggtgatg 480 actgctattg ttggctggca ttggtttgat gcggcaactt atgcagctaa ctggaatact 540 gcggatacca ctgatggtca tgcgatcatt aaaagtattc tgctctgcct gtgggccttc 600 gtgggtgttg aatccgcagc tgtaagtact ggtatggtta aaaacccgaa acgtaccgtt 660 ccgctggcaa ccatgctggg tactggttta gcaggtattg tttacatcgc tgcgactcag 720 gtgctttccg gtatgtatcc gtcttctgta atggcggctt ccggtgctcc gtttgcaatc 780 agtgcttcaa ctatcctcgg taactgggct gcgccgctgg tttctgcatt caccgccttt 840 gcgtgcctga cttctctggg ctcctggatg atgttggtag gccaggcagg tgtacgtgcc 900 gctaacgacg gtaacttccc gaaagtttat ggtgaagtcg acagcaacgg tattccgaaa 960 aaaggtctgc tgctggctgc agtgaaaatg actgccctga tgatccttat cactctgatg 1020 aactctgccg gtggtaaagc atctgacctg ttcggtgaac tgaccggtat cgcagtactg 1080 ctgactatgc tgccgtattt ctactcttgc gttgacctga ttcgttttga aggcgttaac 1140 atccgcaact ttgtcagcct gatctgctct gtactgggtt gcgtgttctg cttcatcgcg 1200 ctgatgggcg caagctcctt cgagctggca ggtaccttca tcgtcagcct gattatcctg 1260 atgttctacg ctcgcaaaat gcacgagcgc cagagccact caatggataa ccacaccgcg 1320 tctaacgcac attaa 1335 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pAWP89-Tac-For <400> 5 tagttgtcgg gaagatgcgt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pAWP89-Tac-Rev <400> 6 agctgtttcc tgtgtgaata 20 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ldcC-For <400> 7 tattcacaca ggaaacagct atgaacatca ttgccattat gggac 45 <210> 8 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> ldcC-Rev <400> 8 gcatcttccc gacaactatt atcccgccat ttttaggact c 41 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> cadA-K12-For <400> 9 tattcacaca ggaaacagct atgaacgtta ttgcaatatt 40 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> cadA-K12-Rev <400> 10 gcatcttccc gacaactatt attttttgct ttctt 35 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> lysA-For <400> 11 gggaacaaaa gctgggtaca tgcatcattt cgactatcg 39 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> LysA-Rev <400> 12 cgaggggggg cccggtactc acagccaatc gggaacgc 38 <210> 13 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> lysC-For <400> 13 gggaacaaaa gctgggtaca tggctcgtct ggtgatgaa 39 <210> 14 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> LysC-Rev <400> 14 cgaggggggg cccggtactc aggccgcgtc cagcccat 38 <210> 15 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Pyc-For <400> 15 ggtatcgata agcttgatat cgtcacggcg cagctgcgcc ctaccgctgt taaaggagac 60 attttatgtt acgcaaaatc ctgat 85 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Pyc-Rev <400> 16 gatcccccgg gctgcaggtc aatttatttc caccaaca 38 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Ptac-ldc-BamHI-For <400> 17 ttgacctgcc agcccggggt tgacaattaa tcatcggct 39 <210> 18 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Ptac-ldc-BamHI-Rev <400> 18 gccgctctag aactagtgtt atcccgccat ttttagga 38 <210> 19 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> cadB-XbaI-For <400> 19 ggcgggataa cactagtttt aaccaacagt ttttctacct aaaggaggcg ttttatgagt 60 tctgccaaga agat 74
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