KR101049023B1 - Corynebacterium ammoniagenes with 5'-inosinic acid production capacity and method for producing 5'-inosine acid using the same - Google Patents

Corynebacterium ammoniagenes with 5'-inosinic acid production capacity and method for producing 5'-inosine acid using the same Download PDF

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KR101049023B1
KR101049023B1 KR1020080020284A KR20080020284A KR101049023B1 KR 101049023 B1 KR101049023 B1 KR 101049023B1 KR 1020080020284 A KR1020080020284 A KR 1020080020284A KR 20080020284 A KR20080020284 A KR 20080020284A KR 101049023 B1 KR101049023 B1 KR 101049023B1
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김철하
최종수
김정환
김형석
백민지
최혜진
황수연
권나라
심재익
권중근
안태민
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Abstract

본 발명은 글루코오스-6-포스페이트 아이소머라아제(Glucose-6-phosphate isomerase)의 활성이 강화된5'-이노신산의 생산능을 가지는 코리네박테리움 암모니아게네스 및 이를 이용한 5'-이노신산의 생산방법에 관한 것이다.The present invention is a corynebacterium ammonia genes having a production capacity of 5'-inosinic acid enhanced by the activity of glucose-6-phosphate isomerase (Glucose-6-phosphate isomerase) and a method for producing 5'-inosine acid using the same It is about.

코리네박테리움 암모니아게네스, 5'-이노신산, 글루코오스-6-포스페이트 아이소머라아제, pgi Corynebacterium ammonia genes, 5'-inosinic acid, glucose-6-phosphate isomerase, pgi

Description

5'-이노신산 생산능이 향상된 코리네박테리움 암모니아게네스 및 그를 이용한 5'-이노신산의 생산방법{CORYNEBACTERIUM AMMONIAGENES HAVING ENHANCED 5'-INOSINIC ACID PRODUCTIVITY AND METHOD OF PRODUCING 5'-INOSINIC ACID USING THE SAME}5'-INOSINIC ACID PRODUCTIVITY AND METHOD OF PRODUCING 5'-INOSINIC ACID USING THE SAME} Corynebacterium ammonia genes with improved 5'-inosinic acid production

본 발명은 글루코오스-6-포스페이트 아이소머라아제(glucose-6-phosphate isomerase)의 활성을 강화시킴으로써 5'-이노신산의 생산능이 강화된 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes) 및 이를 이용한 5'-이노신산을 생산하는 방법에 관한 것이다.The present invention is enhanced by the activity of glucose-6-phosphate isomerase (glucose-6-phosphate isomerase) Corynebacterium ammonia gene (Coynebacterium) is enhanced the production capacity of 5'-inosinic acid ammoniagenes ) and a method for producing 5'-inosinic acid using the same.

5'-이노신산(5'-inosinic acid)은 핵산 생합성 대사계의 중간물질로서, 동식물의 체내에서 생리적으로 중요한 역할을 수행할 뿐 아니라, 식품, 의약품 및 각종 의료적 이용 등 다방면에 이용되고 있으며, 특히 글루타민산 나트륨과 같이 사용하면 맛의 상승효과가 커서 정미성 조미료로 각광을 받고 있는 핵산계 조미료 중 하나이다.5'-inosinic acid (5'-inosinic acid) is an intermediate of the nucleic acid biosynthetic metabolic system, and plays a physiologically important role in the body of animals and plants, and is used in various fields such as food, medicine, and various medical uses. In particular, when used together with sodium glutamate, it is one of nucleic acid-based seasonings that has been spotlighted as a seasoning seasoning because of its synergistic effect.

5'-이노신산을 제조하는 방법으로는, 효모 세포로부터 추출한 리보핵산을 효소적으로 분해하는 방법(일본 특허공고 제1614/1957호 등), 발효에 의해 생산된 이 노신을 화학적으로 인산화하는 방법 (Agri. Biol. Chem., 36, 1511(1972) 등) 및 5'-이노신산을 생산할 수 있는 미생물을 배양하고 배지에 축적된 5'-이노신산을 회수하는 방법이 있다. 이러한 방법 중에서 현재 가장 많이 사용되고 있는 방법이 미생물을 이용하여 5'-이노신산을 생산하는 방법이다. 5'-이노신산을 생산하는데 이용하는 미생물 중 코리네박테리움 (Corynebacterium) 속 균주, 예를 들어 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes)를 발효시킴으로써 제조하는 방법(대한민국 특허공개 제2003-42972호 등) 등이 공지되어 있다.As a method for producing 5'-inosinic acid, a method of enzymatically decomposing ribonucleic acid extracted from yeast cells (Japanese Patent Publication No. 1614/1957, etc.), a method of chemically phosphorylating this nosine produced by fermentation ( Agri. Biol. Chem., 36, 1511 (1972) and the like, and a method of culturing microorganisms capable of producing 5'-inosine acid and recovering 5'-inosine acid accumulated in the medium. The most widely used method among these methods is a method of producing 5'-inosinic acid using microorganisms. Among the microorganisms used to produce 5'-inosinic acid, a strain of the genus Corynebacterium , for example Corynebacterium Method of manufacturing by fermentation of ammoniagenes) there is a known light (including Republic of Korea Patent Publication No. 2003-42972 call).

글루코오스-6-포스페이트 아이소머라아제는 글루코네오제네시스(gluconeogenesis) 경로에서 프룩토오스-6-포스페이트(fructose 6-phosphate)와 글루코오스-6-포스페이트(glucose 6-phosphate)의 가역반응에 관여하는 효소이다.Glucose-6-phosphate isomerase is an enzyme involved in the reversible reaction of fructose-6-phosphate and glucose-6-phosphate in the gluconeogenesis pathway. .

이전 연구에서는 이노신을 생산하는 대장균(E.coli)에서 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 유전자(이하 pgi유전자라 칭한다)를 파쇄하였을 경우, 이노신 농도 향상의 효과가 보고된 적이 있었고(Biosci. Biotechnol. Niochem, 69 (7), 1248-1255, 2005), 코리네박테리움 글루타미쿰에서도 pgi 유전자 변이에 의하여 라이신 생산능이 증가하였다는 보고가 있었다(Journal of Biotechnology 104 (2003) 185-197). 또한, 이외 다른 일부 미생물에서도 pgi 유전자의 서열 및 특성이 보고된 바 있으나, 코리네박테리움 암모니아게네스에서의 pgi 유전자의 서열 및 특성에 대해 보고된 바는 아직 없다.Previous studies have reported the effect of improving inosine concentrations when the gene encoding glucose-6-phosphate isomerase (hereinafter referred to as pgi gene ) in inosine-producing E. coli has been reported (Biosci). Biotechnol.Niochem, 69 (7), 1248-1255, 2005), also reported that lysine production was increased by pgi gene mutation in Corynebacterium glutamicum (Journal of Biotechnology 104 (2003) 185-197 ). Also, other than Even with some other microorganisms pgi Although the sequence and properties of genes have been reported, pgi in Corynebacterium ammonia genes There is no report on the sequence and properties of genes.

이에 본 발명자들은 코리네박테리움 속 균주로부터 5'-이노신산을 고농도로 생산할 수 있는 균주를 개발하고자 5'-이노신산 생합성 경로와 그 전구체 합성 경로인 펜토스 포스페이트 경로를 연구하던 중, 코리네박테리움 암모니아게네스에서 글루코오스-6-포스페이트 아이소머라아제의 활성을 증가시킴으로써 궁극적으로 이노신산 생합성 전구체 경로인 펜토스 포스페이트 경로가 강화되어 5'-이노신산이 고농도로 생산된다는 것을 발견함으로써 본 발명을 완성하게 되었다.Therefore, the present inventors are studying the 5'-inosinic acid biosynthesis pathway and the precursor synthesis pathway, the pentose phosphate pathway, to develop a strain capable of producing a high concentration of 5'-inosinic acid from a strain of Corynebacterium. By increasing the activity of glucose-6-phosphate isomerase in ammonia genes, the present invention was completed by discovering that 5'-inosinic acid is produced in high concentration, thereby enhancing the pentose phosphate pathway, which is the inosinic acid biosynthetic precursor pathway.

따라서, 본 발명의 목적은 글루코오스-6-포스페이트 아이소머라아제의 활성을 강화시킴으로써 5'-이노신산의 생산능이 향상된 코리네박테리움 암모니아게네스를 제공하는 것이다.Accordingly, it is an object of the present invention to provide Corynebacterium ammonia genes with enhanced production of 5'-inosinic acid by enhancing the activity of glucose-6-phosphate isomerase.

본 발명의 또 다른 목적은 상기 미생물을 배양하여 그 배양액으로부터 5'-이노신산을 생산하는 방법을 제공하는 것이다.Still another object of the present invention is to provide a method of culturing the microorganism and producing 5'-inosinic acid from the culture solution.

상기와 같은 목적을 달성하기 위해, 본 발명은 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자를 포함하는 재조합 벡터로 형질전환시키거나 또는 내재적 pgi 유전자에 더하여 1 카피 이상의 pgi 유전자를 새로 도입하여 글루코오스-6-포스페이트 아이소머라아제의 활성을 강화시킴으로써 5'-이노신산의 생산능이 향상된 코리네박테리움 암모니아게네스를 제공한다.In order to achieve the above object, the present invention is a pgi encoding glucose-6-phosphate isomerase Ability of a 5'-inosinate produced by newly introducing a transgenic reduce or pgi gene or more than one copy in addition to the intrinsic pgi gene with the recombinant vector containing the gene to enhance the activity of the glucose-6-phosphate epimerase enhanced iso Corynebacterium Provides ammonia genes.

본 발명은 또한 상기 미생물을 배양하여 그 배양액으로부터 5'-이노신산을 생산하는 방법을 제공한다.The present invention also provides a method of culturing the microorganism to produce 5'-inosinic acid from the culture.

본 발명에 따른 코리네박테리움 암모니아게네스는 글루코스-6-포스페이트 아이소머라아제의 활성이 코리네박테리움 속 미생물이 가지고 있는 고유의 활성보다 증가되어 있어 5'-이노신산을 고농도로 생산할 수 있기 때문에, 수율 향상에 의한 생산 원가 절감을 이룰 수 있다.Corynebacterium ammonia gene according to the present invention is because the activity of glucose-6-phosphate isomerase is increased than the intrinsic activity of the microorganisms of Corynebacterium can produce high concentration of 5'-inosinic acid As a result, production costs can be reduced by improving yield.

본 발명은 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자를 포함하는 재조합 벡터로 형질전환시키거나, 내재적 pgi 유전자에 더하여 1 카피 이상의 pgi 유전자를 새로 도입하여 글루코오스-6-포스페이트 아이소머라아제의 활성을 강화시킴으로써 5'-이노신산의 생산능이 향상된 코리네박테리움 암모니아게네스를 제공한다.The present invention relates to pgi encoding glucose-6-phosphate isomerase Either transformed with a recombinant vector containing the gene, or improved Corynebacterium ability of 5'-inosinate production by enhancing one or more copies pgi glucose-6-phosphate isomerase activity of iso-kinase by introducing new genes in addition to the intrinsic pgi gene Provides ammonia genes.

글루코오스-6-포스페이트 아이소머라아제는 글루코오스-6-포스페이트와 프룩토오스-6-포스페이트의 가역 반응을 담당하는 효소이며, 글라이코라이시스 경로와 글루코네오제네시스 경로의 부분을 담당하고 있다.Glucose-6-phosphate isomerase is an enzyme responsible for the reversible reaction of glucose-6-phosphate and fructose-6-phosphate, and is responsible for the glycosylation and part of the gluconeogenic pathway.

상기 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자의 코리네박테리움 암모니아게네스에서의 서열 및 특성에 대해서는 아직 보고된 바가 없다. Pgi encoding the glucose-6-phosphate isomerase The sequence and properties of the gene in Corynebacterium ammonia genes have not been reported yet.

본 발명의 구체적 실시예에서는 코리네박테리움 암모니아게네스 ATCC 6872의 염색체 서열분석을 통하여, 상기 pgi 유전자가 약 1635bp 크기로 존재함을 확인하였다. 본 발명의 상기 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자의 뉴클레오티드 서열은 바람직하게는 서열번호 1의 뉴클레오티드 서열을 갖 는다.In a specific embodiment of the present invention through the chromosome sequencing of Corynebacterium ammonia genes ATCC 6872, the pgi It was confirmed that the gene is present in the size of about 1635bp. The nucleotide sequence of the pgi gene encoding the glucose-6-phosphate isomerase of the present invention preferably has a nucleotide sequence of SEQ ID NO: 1.

본 발명에 있어서, 상기 효소의 활성을 강화시키는 것은 당해 효소를 암호화하는 유전자의 발현을 증가시킴으로써 달성된다. 당해 유전자의 발현 증가는 당해 효소의 유전자의 카피수를 증가시킴으로써 달성된다. 예를 들면, 상기 효소를 암호화하는 유전자 단편을 벡터에 연결하여 코리네박테리움 속 미생물에 도입하여 형질전환시킴으로써 달성된다.In the present invention, enhancing the activity of the enzyme is achieved by increasing the expression of the gene encoding the enzyme. Increased expression of the gene is achieved by increasing the copy number of the gene of the enzyme. For example, a gene fragment encoding the enzyme may be linked to a vector and introduced into a microorganism of the genus Corynebacterium to achieve transformation.

본 발명의 바람직한 양태에서, 상기 pgi 유전자를 포함한 재조합 벡터는 중합효소 연쇄반응을 통하여 얻은 pgi 유전자를 DNA 제한효소, 예를 들어 BamHI 및 Xbal 등으로 절단하고, 동일한 DNA 제한효소로 절단한 벡터에 DNA T4 리가아제 등을 사용하여 삽입시켜 제조할 수 있다. 사용 가능한 벡터는 특별히 제한되는 것은 아니며, 공지된 발현벡터를 사용할 수 있다. 바람직하게는 pECCG117 벡터(Biotechnology letters vol 13, No.10, p.721-726(1991) 또는 대한민국 특허공고 제92-7401호)를 사용할 수 있으며, 더욱 바람직하게는 상기 pECCG117 벡터에 pgi 유전자를 도입시켜 제조한 pECCG117-pgi 벡터를 사용할 수 있다.In a preferred embodiment of the present invention, the recombinant vector comprising the pgi gene is a pgi gene obtained through a polymerase chain reaction DNA restriction enzymes, for example BamHI And Xbal and the like , and inserted into a vector cut with the same DNA restriction enzyme using DNA T4 ligase or the like. The vector which can be used is not specifically limited, A well-known expression vector can be used. Preferably, the pECCG117 vector (Biotechnology letters vol 13, No. 10, p.721-726 (1991) or Korean Patent Publication No. 92-7401) can be used, and more preferably, the pgi gene is introduced into the pECCG117 vector. PECCG117- pgi vector prepared by the above method can be used.

본 발명의 구체적 실시예에서, 상기 재조합 벡터, 예를 들어 pECCG117-pgi를 사용하여 선형 DNA단편을 통상적인 일렉트로포레이션(electroporation) 방법을 이용하여 미생물 균주(예를 들어, 코리네박테리움 암모니아게네스 (Corynebacterium ammoniagenes) KCCM-10610, 대한민국 특허공고 제10-2006-0060404호)에 전달하고, 선별마커인 항생제 카나마이신(kanamycin)을 포함하는 배지에서 배양하여 선별할 수 있다. 선별된 변이주 내의 pECCG117-pgi 벡터의 삽입은 중합효소 연쇄 반응을 통하여 확인할 수 있다.In a specific embodiment of the present invention, a microbial strain (eg, Corynebacterium ammonia crab) is prepared using a conventional electroporation method for a linear DNA fragment using the recombinant vector, eg, pECCG117- pgi . Ness ( Corynebacterium ammoniagenes ) KCCM-10610, Republic of Korea Patent Publication No. 10-2006-0060404) and can be selected by culturing in a medium containing the antibiotic kanamycin (kanamycin), a selection marker. Insertion of pECCG117- pgi vector in selected mutant strains can be confirmed by polymerase chain reaction.

또한, 본 발명에서는 글루코스-6-포스페이트 아이소머라아제의 활성을 강화시키기 위하여, 코리네박테리움 속 미생물에 내재적으로 존재하는 pgi 유전자에 더하여 1카피 이상의 pgi 유전자를 도입함으로써 활성을 강화시킬 수 있다. 이와 같이, 염색체 DNA에 목적 유전자를 삽입하기 위한 벡터로는 염색체 삽입용 pDZ벡터를 사용할 수 있다.In the present invention, it is possible to enhance the glucose-6-phosphate activity by isopropyl in order to enhance the activity of the epimerase, introducing the implicit presence pgi gene in addition to the at least one copy of the pgi gene into Corynebacterium spp. As such, as a vector for inserting a target gene into chromosomal DNA, a pDZ vector for chromosomal insertion can be used.

본 발명의 구체적 실시예에서, 염색체 내로 pgi 유전자를 삽입하기 위한 염색체 삽입용 벡터는 중합효소 연쇄반응을 통하여 얻은 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 유전자 단편 2개를 DNA 제한효소, 예를 들어 BamHI 및 Xbal 등으로 절단하고, 동일한 DNA 제한효소로 절단한 벡터에 DNA T4 리가아제 등을 사용하여 제조할 수 있다. 사용 가능한 벡터는 pDZ벡터이다. 상기 염색체 내 2카피 삽입용 벡터는 pDZ벡터에 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 유전자 2개를 삽입시켜 제조한 pDZ-2pgi 벡터가 바람직하다(도1 또는 도3 참고). 상기 유전자의 염색체 내로의 삽입은 당업계에 알려진 임의의 방법, 예를 들면, 상동 재조합에 의하여 이루어 질 수 있다.In a specific embodiment of the present invention, the chromosomal insertion vector for inserting the pgi gene into the chromosome comprises two DNA fragments encoding glucose-6-phosphate isomerase obtained through a polymerase chain reaction. For example, DNA T4 ligase or the like can be prepared by using a vector cleaved with BamHI , Xbal, or the like, and digested with the same DNA restriction enzyme. The vector that can be used is a pDZ vector. Preferably, the vector for inserting two copies of the chromosome is a pDZ-2 pgi vector prepared by inserting two genes encoding glucose-6-phosphate isomerase into the pDZ vector (see FIG. 1 or 3). Insertion of the gene into the chromosome can be by any method known in the art, for example homologous recombination.

본 발명의 구체적 실시예에서, 상기 재조합 벡터, 예를 들어 pDZ-2pgi를 사용하여 선형 DNA단편을 통상적인 일렉트로포레이션(electroporation) 방법을 이용하여 미생물 균주(예를 들어, 코리네박테리움 암모니아게네스 (Corynebacterium ammoniagenes) KCCM-10610, 대한민국 특허공고 제10-2006-0060404호)에 전달하고, 선별마커인 항생제 카나마이신(kanamycin)을 포함하는 배지 및 X-gal(5-브로모-4- 클로로-3-인돌릴-β-D-갈락토시드)을 포함하는 배지에서 배양하여 선별할 수 있다. 선별된 균주 내의 2 카피 삽입은 중합효소 연쇄 반응을 통하여 확인할 수 있다.In a specific embodiment of the present invention, the microbial strain (eg, Corynebacterium ammonia) by using a conventional electroporation method of a linear DNA fragment using the recombinant vector, for example pDZ-2 pgi It is delivered to Genes ( Corynebacterium ammoniagenes ) KCCM-10610, Korean Patent Publication No. 10-2006-0060404), and the medium containing the selection marker antibiotic kanamycin and X-gal (5-bromo-4-chloro) 3-indolyl-β-D-galactoside) may be selected by culturing in a medium containing. Insertion of two copies in selected strains can be confirmed through polymerase chain reaction.

본 발명의 가장 바람직한 양태는 글루코오스-6-포스페이트 아이소머라아제 활성을 증가시켜 5'-이노신산을 고농도로 생산하도록 형질전환된 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes) CN01-0207 (수탁번호:KCCM-10918P) 일 수 있다.The most preferred embodiment of the present invention is Corynebacterium transformed to increase glucose-6-phosphate isomerase activity to produce high concentrations of 5'-inosinic acid ( Corynebacterium) ammoniagenes ) CN01-0207 (Accession Number: KCCM-10918P).

또한, 본 발명은 상기의 형질전환된 코리네박테리움 암모니아게네스를 배양하여 그 배양액으로부터 5'-이노신산을 생산하는 방법에 관한 것이다.The present invention also relates to a method of culturing the transformed Corynebacterium ammonia genes to produce 5'-inosinic acid from the culture.

더욱 상세하게는, 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자를 포함하는 재조합 벡터 또는 염색체내 pgi 유전자 2카피 삽입용 벡터로 형질전환 시켜 5'-이노신산의 생산성이 향상된 코리네박테리움 암모니아게네스 CN01-0206 및 CN01-0207(KCCM-10918P)에 의한 직접 발효법으로 배양액 중에 5'-이노신산을 축적시켜 생산하는 방법에 관한 것이다.More specifically, Corynebacterium ammonia with improved productivity of 5'-inosinic acid is transformed with a recombinant vector comprising a pgi gene encoding glucose-6-phosphate isomerase or a vector for inserting two copies of pgi gene into a chromosome. The present invention relates to a method of accumulating 5'-inosinic acid in a culture by direct fermentation with Gennes CN01-0206 and CN01-0207 (KCCM-10918P).

상기의 형질전환된 코리네박테리움 암모니아게네스를 적당한 탄소원, 질소원, 아미노산, 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.The transformed Corynebacterium ammonia genes may be cultured under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins, and the like while controlling temperature, pH, and the like.

탄소원으로는 글루코오스와 프룩토오스가 사용될 수 있고, 질소원으로는 암모니아, 염화암모늄, 황산암모늄과 같은 각종 무기질소원 및 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크, 또는 그의 분해생성물 등 유기질소원이 사용될 수 있다. 무기화합물로는 인산1수소칼륨, 인산2수소칼륨, 황산마그네슘, 황산철, 황산망간, 탄산칼슘 등이 사용될 수 있으며, 이외에 필요에 따라 비타민 및 영양요구성 염기 등이 첨가될 수 있다.As a carbon source, glucose and fructose may be used, and as a nitrogen source, various inorganic nitrogen sources such as ammonia, ammonium chloride, and ammonium sulfate and peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, and fish Or organic nitrogen sources such as degradation products thereof, degreasing soy cakes, or degradation products thereof. As the inorganic compound, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate and the like may be used. In addition, vitamins and nutrient-containing bases may be added as necessary.

배양은 호기적 조건 하에서 예를 들면, 진탕 배양 또는 통기 교반 배양에 의해, 바람직하게는 20 내지 40℃의 온도에서 수행될 수 있다. 배지의 pH는 배양하는 동안 중성 근처에서 유지하는 것이 바람직하다. 배양은 5 내지 6일 동안 수행할 수 있으며, 직접 발효에 의해 축적된 5'-이노신산을 통상의 방법으로 회수할 수 있다.The culturing may be carried out under aerobic conditions, for example by shaking culture or aeration stirred culture, preferably at a temperature of 20 to 40 ° C. The pH of the medium is preferably maintained near neutral during the culture. Cultivation can be performed for 5 to 6 days, and the 5'-inosinic acid accumulated by direct fermentation can be recovered by a conventional method.

본 발명의 구체적 실시예에서, 본 발명에 의하여 형질전환된 코리네박테리움 암모니아게네스는 형질전환하지 않은 코리네박테리움 속 미생물에 비하여 높은 수율로 5'-이노신산을 생산하였다.In a specific embodiment of the present invention, Corynebacterium ammonia genes transformed by the present invention produced 5'-inosinic acid in high yield compared to the non-transfected Corynebacterium microorganism.

이하, 본 발명을 실시예를 통하여 더욱 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시하기 위한 것으로, 본 발명이 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the present invention is not limited by these examples.

실시예Example 1 :  One : pgipgi 유전자  gene 클로닝Cloning 및 플라스미드 증폭용 벡터 제작 Vector production for amplification and plasmid

코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes) ATCC 6872의 염색체 유전자를 분리하고, 이를 주형으로 하여 서열번호 2와 서열번호 3의 프라이머를 이용하여 중합효소 연쇄반응을 통하여 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자(서열번호1)를 얻었다.Corynebacterium ammoniagenes's Ness (Corynebacterium ammoniagenes ) A pgi gene (SEQ ID NO: 1) that isolates the chromosomal gene of ATCC 6872 and encodes glucose-6-phosphate isomerase through polymerase chain reaction using primers of SEQ ID NO: 2 and SEQ ID NO: 3 as a template. )

얻어진 pgi 유전자의 단편을 제한효소 BamHI (New England Biolabs, Beverly, MA)과 Xbal (New England Biolabs, Beverly, MA)으로 절단하였다. 리가아제(New England Biolabs, Beverly, MA)를 이용하여, 상기 유전자 단편을 BamHI 와 XalI 제한효소로 절단시킨 선상의 pECCG117 벡터에 접합하였다. 제조된 벡터를 pECCG117-pgi로 명명하였다.A fragment of the obtained pgi gene was digested with restriction enzymes BamH I (New England Biolabs, Beverly, MA) and Xbal (New England Biolabs, Beverly, MA). Using the ligase (New England Biolabs, Beverly, Mass.), The gene fragment was BamHI. And the linear pECCG117 vector digested with XalI restriction enzyme. The produced vector was named pECCG117- pgi .

상기 pECCG117-pgi 벡터를 코리네박테리움 암모니아게네스 KCCM-10610에 일렉트로포레이션(electroporation)을 통하여 형질전환하고, 1리터 당 카나마이신 (kanamycin) 25㎎을 포함하는 CM 고체배지 (육즙 10g/L, 효모엑기스 10g/L, 박토펩톤 10g/L, 염화나트륨 2.5g/L, 박토아가 1.7%, pH 7.0)에서 배양하여 단일 콜로니들을 회수하였다. 회수된 콜로니들을 동일한 항생제가 첨가된 CM 액체배지에서 키운 다음, 플라스미드를 분리하여 그 크기를 일차로 확인하고, 2차로 pECCG117의 다중클로닝 부위의 양끝단을 포함하는 프라이머를 이용하여 콜로니 PCR을 수행함으로써, 삽입된 DNA의 크기를 통해 형질전환 여부를 확인하였다. 상기 클로닝 과정 및 클로닝된 pECCG117- pgi 벡터는 도1과 같다.PECCG117- pgi Vectors were transformed into Corynebacterium ammonia genes KCCM-10610 via electroporation, and CM solid medium containing 25 mg of kanamycin (l gravy 10g / L, yeast extract 10g /) L, bactopeptone 10g / L, sodium chloride 2.5g / L, bactoa 1.7%, pH 7.0) to recover single colonies. The recovered colonies were grown in a CM liquid medium to which the same antibiotic was added, the plasmids were isolated and their size was determined first, and secondly, colony PCR was performed using primers containing both ends of the multicloning site of pECCG117. The presence or absence of transformation was confirmed through the size of the inserted DNA. The cloning procedure and cloned pECCG117- The pgi vector is shown in FIG.

pgi 유전자를 증폭하기 위하여 사용된 프라이머들의 서열은 각각 다음과 같다.The sequences of the primers used to amplify the pgi gene are as follows.

프라이머 pgi-1F (서열번호 2):Primer pgi -1F (SEQ ID NO: 2):

5'- CGC GGA TCC GGG GAT TGG CTC TTC CTT CTC -3'5'- CGC GGA TCC GGG GAT TGG CTC TTC CTT CTC -3 '

프라이머 pgi -R (서열번호 3):Primer pgi -R (SEQ ID NO: 3):

5'- TGC TCT AGA CCA CGC GCC AAT GCC GTA CAG-3'5'- TGC TCT AGA CCA CGC GCC AAT GCC GTA CAG-3 '

이 과정을 통해 선별된 pgi 유전자를 포함하는 재조합 벡터로 형질전환된 균 주를 코리네박테리움 암모니아게네스 (Corynebacterium ammoniagenes) CN01-0206로 명명하였다.The strain transformed with the recombinant vector containing the pgi gene selected through this process was identified as Corynebacterium. ammoniagenes ) CN01-0206.

실시예Example 2 : 염색체 삽입용 벡터 ( 2: vector for chromosome insertion ( pDZpDZ )의 제작 및 이를 이용한 유전자의 삽입 방법) Construction and insertion of gene using the same

본 실시예에서는 대장균 클로닝용 벡터 pACYC177 (New England Biolab, GenBank accetion # X06402)를 기본 벡터로 사용하여, 코리네박테리움 암모니아게네스 내로의 염색체 삽입용 벡터 pDZ를 제작하였으며, 제작 과정은 다음과 같다.In the present example, using the vector pACYC177 (New England Biolab, GenBank accetion # X06402) for cloning the E. coli, a vector pDZ for chromosome insertion into Corynebacterium ammonia genes was prepared. .

상기 pACYC177 벡터를 AcuI 및 BanI 제한 효소로 처리한 후, 클레나우 효소처리를 통하여 평활 말단화하였다. 선별 마커로 사용될 대장균 유래의 lacZ 유전자는 대장균 K12 W3110의 게놈 DNA로부터 PCR을 통하여 자체 프로모터를 포함하도록 증폭한 후, T4 DNA 폴리머라제 및 폴리뉴클레오티드 키나아제 처리를 통하여 5' 말단 인산화 및 평활화함으로써 준비하였다.The pACYC177 vector was treated with AcuI and BanI restriction enzymes, and then blunt ended via Klenow enzyme treatment. The lacZ gene derived from Escherichia coli to be used as a selection marker was prepared by amplifying the genomic DNA of Escherichia coli K12 W3110 to include its own promoter through PCR, and then phosphorylating and smoothing 5 'terminal through T4 DNA polymerase and polynucleotide kinase treatment.

이상과 같이 준비한 2종의 DNA 단편을 접합하였으며, 접합된 환상 DNA 분자의 제한효소 BamHI 부위로 인위적으로 합성한 다수의 제한효소 인식 부위를 포함하고 있는 아답터(adaptor) 서열을 삽입하여, 염색체 삽입용 벡터 pDZ를 완성하였다. 도 2는 코리네박테리움 염색체 삽입용 벡터 pDZ를 나타내는 도면이다.Two DNA fragments prepared as described above were conjugated, and an adapter sequence including a plurality of restriction enzyme recognition sites artificially synthesized into the restriction enzyme BamHI site of the conjugated circular DNA molecule was inserted for chromosome insertion. Vector pDZ was completed. 2 is a diagram showing a vector pDZ for corynebacterium chromosome insertion.

이하의 실시예에서는 코리네박테리움 암모니아게네스의 염색체 내로 여분의 pgi 유전자를 삽입하기 위하여, 상기 pgi 유전자를 연속적으로 2 카피 포함하고 있는 pDZ 벡터를 5'-이노신산 생산능을 가진 균주인 코리네박테리움 암모니아게네스 KCCM-10610에 일렉트로포레이션법으로 형질전환한 후, 카나마이신 25mg/L를 함유한 선별 배지에서 염색체상의 동 유전자와의 상동성에 의해 삽입된 균주를 선별하였다. In the following example, in order to insert an extra pgi gene into the chromosome of Corynebacterium ammonia genes, Coryne , a strain having 5'-inosinic acid production, is a pDZ vector containing two copies of the pgi gene in succession. After transforming bacterium ammonia genes KCCM-10610 by the electroporation method, the inserted strain was selected by homology with the same gene on the chromosome in a selection medium containing 25 mg / L of kanamycin.

벡터의 성공적인 염색체 삽입은 X-gal (5-브로모-4-클로로-3-인돌릴-β-D-갈락토시드)을 포함한 고체배지에서 푸른색을 나타내는가 여부를 확인함으로써 가능하였다. 1차 염색체 삽입된 균주를 영양 배지에서 진탕배양 (30℃, 8시간)한 후, 각각 10-4으로부터 10-10까지 희석하여, X-gal을 포함하고 있는 고체배지에 도말하였다. 대부분의 콜로니가 푸른색을 띄는데 반하여 낮은 비율로 나타나는 백색의 콜로니를 선별함으로써, 2차 교차 (crossover)에 의해 삽입된 염색체상의 벡터 서열이 제거된 균주를 선별하였다. 이상과 같이 선별된 균주는 최종적으로 항생제 카나마이신에 대한 감수성 여부의 확인 및 PCR을 통하여 유전자 구조 확인 과정을 거쳐 최종 선정되었다.Successful chromosomal insertion of the vector was made possible by checking whether it was blue in solid media, including X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactosid). The primary chromosome-inserted strains were shaken in nutrient medium (30 ° C., 8 hours), diluted from 10 −4 to 10 −10 , respectively, and plated on solid medium containing X-gal. While most colonies were blue, white colonies appearing at a low rate, and strains from which the vector sequence on the chromosome inserted by the second crossover were removed were selected. The strains selected as described above were finally selected through the gene structure confirmation process through PCR and confirmation of susceptibility to the antibiotic kanamycin.

실시예Example 3 : 염색체 내 2  3: in chromosome 2 copycopy 삽입용 벡터 제작 Create vector for insertion

코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes) ATCC 6872의 염색체 유전자를 분리하고, 이를 주형으로 하여 중합효소는 PfuUltra TM 고-신뢰 DNA 폴리머라제 (Stratagene)를 사용하였으며, PCR 조건은 변성 96℃, 30초; 어닐링 53℃, 30초; 및 중합반응 72℃, 1분을 30회 반복하여 증폭하였다.Corynebacterium ammoniagenes's Ness (Corynebacterium ammoniagenes ) The chromosomal gene of ATCC 6872 was isolated and used as a template for the polymerase as PfuUltra ™ high-trust DNA polymerase (Stratagene), and PCR conditions were denatured 96 ° C., 30 seconds; Annealing 53 ° C., 30 seconds; And amplification by repeating the polymerization reaction 72 DEG C for 1 minute 30 times.

그 결과, 프로모터 부위를 포함한 pgi 유전자 두 쌍 (pgi-A, pgi-B)을 얻었 다. pgi-A 는 서열번호 2와 3을 프라이머로 사용하여 증폭된 것이며, pgi-B는 서열번호 3과 4를 프라이머로 사용하여 증폭된 것이다. 상기 증폭 산물을 TOPO 클로닝 키트(Invitrogen)를 이용하여 대장균 벡터 pCR2.1에 클로닝하여 pCR-pgi-A와 pCR-pgi-B 벡터를 얻었다. 상기 pCR 벡터에 pgi-A와 pgi-B의 각 말단에 포함된 제한효소(pgi-A: BamHI + Xbal, pgi-B: Xbal)를 처리하여, 상기 pCR 벡터로부터 각각의 pgi 유전자를 분리하였다. 다음으로, 제한효소 BamHI과 Xbal가 처리된 pDZ 벡터에 3 조각 접합 (3-piece ligation)을 통하여 클로닝하여, 최종적으로 pgi 2 카피가 연속적으로 클로닝된 pDZ-2pgi 재조합 벡터를 제작하였다. 도 3는 코리네박테리움 염색체 삽입용 벡터 pDZ-2pgi를 나타내는 도면이다.As a result, the pgi containing the promoter site Two pairs of genes ( pgi -A, pgi -B) were obtained. p gi -A Is amplified using SEQ ID NO: 2 and 3 as a primer, pgi -B is amplified using SEQ ID NO: 3 and 4 as a primer. The amplification product was cloned into E. coli vector pCR2.1 using the TOPO cloning kit (Invitrogen) to obtain pCR- pgi -A and pCR- pgi -B vectors. Restriction enzymes contained in each end of pgi -A and pgi -B in the pCR vector ( pgi -A: BamHI + Xbal , pgi -B: Xbal ) to separate each pgi gene from the pCR vector. Next, the pDZ vector treated with the restriction enzymes BamHI and Xbal was cloned through 3-piece ligation to finally prepare a pDZ-2 pgi recombinant vector in which pgi 2 copies were continuously cloned. Fig. 3 shows the vector pDZ-2 pgi for corynebacterium chromosome insertion.

상기 pDZ-2pgi 벡터를 코리네박테리움 암모니아게네스 KCCM-10610에 일렉트로포레이션 (electroporation)을 통하여 형질전환하고, 2차 교차 과정을 거쳐 염색체 상에서 pgi 유전자의 바로 옆에 1 카피의 pgi 유전자를 추가로 삽입하여 카피 수를 2개로 증가시킨 균주를 얻었다. 연속적으로 삽입된 pgi 유전자는 2 카피의 pgi 연결부위를 증폭할 수 있는 서열번호 5와 6의 프라이머를 이용한 PCR을 통하여 최종 확인하였다.The pDZ-2 pgi vector was transformed by electroporation to Corynebacterium ammonia genes KCCM-10610, and a second copy of the pgi gene was located next to the pgi gene on the chromosome. Further insertion yielded a strain in which the copy number was increased to two. The continuously inserted pgi gene was finally confirmed by PCR using primers of SEQ ID NOs: 5 and 6 capable of amplifying 2 copies of the pgi linkage.

pDZ-2pgi 제작을 위한 유전자 증폭 및 상기 벡터로 형질전환되어 2 카피의 pgi 유전자가 도입된 균주를 확인하기 위하여 사용된 프라이머들의 서열은 각각 다음과 같다.The sequences of the primers used to identify the gene amplification for constructing pDZ-2 pgi and the strain transformed with the vector into which two copies of the pgi gene were introduced are as follows.

프라이머 pgi -R (서열번호 3):Primer pgi -R (SEQ ID NO: 3):

5'- TGC TCT AGA CCA CGC GCC AAT GCC GTA CAG-3'5'- TGC TCT AGA CCA CGC GCC AAT GCC GTA CAG-3 '

프라이머 pgi-2F (서열번호 4):Primer pgi -2F (SEQ ID NO: 4):

5'- GCC CTC TAG AGG GAT TGG CTC TTC CTT CTC TAC C-3'5'- GCC CTC TAG AGG GAT TGG CTC TTC CTT CTC TAC C-3 '

프라이머 2pgi-1F (서열번호 5):Primer 2 pgi -1F (SEQ ID NO: 5):

5'- GGA ATT AGG TAA GCA ACA AGC CAA CG-3'5'- GGA ATT AGG TAA GCA ACA AGC CAA CG-3 '

프라이머 2pgi-1R (서열번호 6):Primer 2 pgi -1R (SEQ ID NO: 6):

5'- GCT TTG CCC ATG CAG GTT GTT GCG-3'5'- GCT TTG CCC ATG CAG GTT GTT GCG-3 '

이 과정을 통해 선별된 염색체 내에 2 카피의 pgi 유전자가 삽입된 균주를 코리네박테리움 암모니아게네스 (Corynebacterium ammoniagenes) CN01-0207로 명명하였고, 그를 2008년 1월 8일자로 대한민국 서울특별시 서대문구 홍제 1동 361-221번지에 소재하는 국제기탁기관인 한국종균협회 부설 한국미생물보존센터에 수탁번호 KCCM-10918P로 기탁하였다.In this process, two copies of the pgi gene were inserted into the selected chromosome. Corynebacterium ammoniagenes ) CN01-0207 and deposited on January 8, 2008 with the Korea Microbiological Conservation Center affiliated with the Korea Spawn Association, an international depository located at 361-221 Hongje 1-dong, Seodaemun-gu, Seoul, Korea. It was.

실시예Example 4 : 삼각 플라스크  4: Erlenmeyer flask 발효역가Fermentation potency 시험 exam

종배지 3ml를 지름 18mm 시험관에 분주하고 가압 살균한 후, 코리네박테리움 암모니아게네스 (Corynebacterium ammoniagenes) CN01-0206와 CN01-0207 (KCCM-10918P)를 접종하고, 30℃에서 24시간 진탕 배양하여 종 배양액으로 사용하였다. 발효배지 27ml를 500ml 진탕용 삼각플라스크에 분주하고, 120℃에서 10분간 가압 살균한 후, 종배양액 3ml을 접종하여 5 내지 6일간 배양하였다. 배양 조건은 회전 수 200rpm, 온도 32℃, pH 7.2로 조절하였다.After dispensing 3 ml of seed medium into an 18 mm diameter test tube and autoclaving it, inoculated with Corynebacterium ammoniagenes CN01-0206 and CN01-0207 (KCCM-10918P), and incubated shaking at 30 ° C for 24 hours. Used as species culture. 27 ml of the fermentation broth was dispensed into a 500 ml shake flask, sterilized under pressure at 120 ° C. for 10 minutes, and then inoculated with 3 ml of the seed culture solution and incubated for 5 to 6 days. Culture conditions were adjusted to 200 rpm, temperature 32 ℃, pH 7.2.

이때, 배지 내 5'-이노신산 축적량을 모균주인 코리네박테리움 암모니아게네스KCCM-10610 과 비교한 결과는 하기 표1과 같으며, 이는 코리네박테리움 암모니아게네스 CN01-0206과 CN01-0207(KCCM-10918P) 균주가 동일한 조건하에서 모균주인 코리네박테리움 암모니아게네스 KCCM-10610 대비 단위 시간당 생산하는 5'-이노신산의 생산성이 3.6 ~ 6.6% 증가함을 나타낸다.At this time, the result of comparing the 5'-inosinic acid accumulation in the medium with the parent strain Corynebacterium ammonia genes KCCM-10610 is shown in Table 1 below, which is corynebacterium ammonia genes CN01-0206 and CN01-0207. (KCCM-10918P) shows that the productivity of 5'-inosinic acid produced per unit time is 3.6-6.6% higher than the parent strain Corynebacterium ammonia gene KCCM-10610 under the same conditions.

상기 종배지 및 발효배지의 조성은 다음과 같다.The composition of the seed medium and fermentation medium is as follows.

종배지 : 포도당 1%, 펩톤1%, 육즙 1%, 효모엑기스 1%, 염화나트륨 0.25%, 아데닌 100mg/l, 구아닌100mg/l, pH 7.2 Species medium : glucose 1%, peptone 1%, gravy 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100mg / l, guanine 100mg / l, pH 7.2

플라스크 발효배지 : 글루타민산 나트륨 0.1%, 암모늄클로라이드 1%, 황산마그네슘 1 .2%, 염화칼슘 0.01%, 황산철 20mg/l, 황산망간 20mg/l, 황산아연 20mg/l, 황산구리 5mg/l, L-시스테인 23mg/l, 알라닌 24mg/l, 니코틴산 8mg/l, 비오틴 45㎍/l, 티아민염산 5mg/l, 아데닌 30mg/l, 인산(85%) 1.9%, 포도당 4.2%, 원당 2.4% 되게 첨가하여 사용 Flask fermentation medium : 0.1% sodium glutamate, 1% ammonium chloride, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg / l, manganese sulfate 20mg / l, zinc sulfate 20mg / l, copper sulfate 5mg / l, L- Cysteine 23mg / l, alanine 24mg / l, nicotinic acid 8mg / l, biotin 45㎍ / l, thiamine hydrochloride 5mg / l, adenine 30mg / l, phosphoric acid (85%) 1.9%, glucose 4.2%, raw sugar 2.4% use

균주명Strain name Cell OD
(배양 5일후)Cell od
(5 days after culture) 생산성(g/l/hr)
(배양 5일후)Productivity (g / l / hr)
(5 days after culture) 대조군(KCCM-10610)Control Group (KCCM-10610) 30.230.2 0.1360.136 CN01-0206CN01-0206 33.533.5 0.1410.141 CN01-0207(KCCM-10918P)CN01-0207 (KCCM-10918P) 31.931.9 0.1450.145

도 1은 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자의 클로닝 과정 및 클로닝된 pECCG117-pgi 벡터를 나타낸다.1 shows the cloning process of the pgi gene encoding glucose-6-phosphate isomerase and the cloned pECCG117- pgi vector.

도 2는 염색체내 유전자 삽입을 위한 pDZ 벡터를 나타낸다.2 shows a pDZ vector for intrachromosomal gene insertion.

도 3은 글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자를 염색체내에 2 copy 삽입하기 위한 pDZ-2pgi 벡터를 나타낸다.Figure 3 shows a pDZ-2 pgi vector for inserting two copies of the pgi gene encoding glucose-6-phosphate isomerase into the chromosome.

<110> CJ Cheiljedang Corporation <120> A microorganism of corynebacterium genus having enhanced 5'-inosinic acid productivity and method of producing 5'-inosinic acid using the same <130> PA07-0414 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1635 <212> DNA <213> Corynebacterium ammoniagenes ATCC 6872 <400> 1 atggaaataa cgcaacaacc tgcatgggca aagcttcagc agcttttcga agctaagaag 60 cacacaacgc ttcgacagct ttttgctgcc gatgccaccc gcgcttcggc tttaaccttc 120 gatgctgcgg ggctgcatgt ggacctttcc aagaatctta ttgactccga agtggttgca 180 gcgctggtag aactcgctga gcaagcaggg gttgaagcac gccgcgcaga catgtttagc 240 ggcaagcaca tcaataccac tgaagaccgc gctgtactgc ataccgcatt gcgtcttccg 300 gtcgaagcta acctagtggt cgatgagcaa gatgtcgcag ccgatgttca cgaagtcctc 360 ggtcgcatgc gtgatttcgc aactgcattg cgttcgggca agtggctggg gcataccggc 420 cataccatca agaaggtggt caacgtcggc atcggtggtt ctgatttggg gcctgcgatg 480 gcagcccagg cactgcgcag ctatgaagtc gcgggaatct ccgccgagtt tgtctccaat 540 gtggacccag ccgatttggc aaagactttg gatgggctcg atgccggctc cactcttttc 600 atcattgctt ccaagacttt taccacgcag gaaactttgt ctaacgccca cgctgctcgt 660 cgttggatgc tggagcaatt cgatggtgat gagtcttcca tcgccaagca ctttgttgcg 720 gtatcgacca acgccgaaaa ggtcgcggaa ttcggcattg atactgaaaa catgtttggg 780 ttttggaact gggtcggcgg acgttactct gtcgatgcag ccattggctt gtctctgatg 840 tgcaccattg gcccgctaga tttcatgcgt ttcctcgaag gcttccacgc catggatgag 900 cacttccgta ccacgccgct ggaatctaat gttccaacac tcatggcgct gctgagcatc 960 tggtacacca acttctatgg cgcacagtct cacgctgtac ttccctactc tgaggatctc 1020 ggccgtttcc ctgcttacct acagcagctg acgatggaat ctaacggcaa gtccgtgcac 1080 cacgacggtt ctgcagtgtc aaccaccacc agtcccattt actggggtga gccaggaacc 1140 aatggccagc acgctttctt ccagctactg caccagggga cgcacttggt tccggctgat 1200 ttcattggat ttgcccgccc gaaggaagat ttccccactg ccgatggcac aggctccatg 1260 catgatctac tcatgggtaa tctcttcgca caaaccaagg tgctggcctt tggcaaaacc 1320 gcagaagaaa tagccaggga aggcatcgca gaagaactcg tggctcacaa ggtcatgccg 1380 ggcaatcgcc ccaccaccac tatcttggcc gaagagctca cccctgcagc cttgggcagc 1440 ttgattgcgc tttacgaaca catcgtgttt gtcgaaggcg tcatctggga tatcaactcc 1500 tttgaccaat ggggcgtgga attaggtaag caacaagcca acgacctagc gccggcggtc 1560 tctggcaagg cggatatcga taccggtgat gaatcgaccg acgcgctgat ctcctggttt 1620 cttaaacacc gttaa 1635 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer-pgi-1F <400> 2 cgcggatccg gggattggct cttccttctc 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer-pgi-R <400> 3 tgctctagac cacgcgccaa tgccgtacag 30 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer-pgi-2F <400> 4 gccctctaga gggattggct cttccttctc tacc 34 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer-2pgi-1F <400> 5 ggaattaggt aagcaacaag ccaacg 26 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer-2pgi-1R <400> 6 gctttgccca tgcaggttgt tgcg 24 <110> CJ Cheiljedang Corporation <120> A microorganism of corynebacterium genus having enhanced          5'-inosinic acid productivity and method of producing 5'-inosinic          acid using the same <130> PA07-0414 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1635 <212> DNA <213> Corynebacterium ammoniagenes ATCC 6872 <400> 1 atggaaataa cgcaacaacc tgcatgggca aagcttcagc agcttttcga agctaagaag 60 cacacaacgc ttcgacagct ttttgctgcc gatgccaccc gcgcttcggc tttaaccttc 120 gatgctgcgg ggctgcatgt ggacctttcc aagaatctta ttgactccga agtggttgca 180 gcgctggtag aactcgctga gcaagcaggg gttgaagcac gccgcgcaga catgtttagc 240 ggcaagcaca tcaataccac tgaagaccgc gctgtactgc ataccgcatt gcgtcttccg 300 gtcgaagcta acctagtggt cgatgagcaa gatgtcgcag ccgatgttca cgaagtcctc 360 ggtcgcatgc gtgatttcgc aactgcattg cgttcgggca agtggctggg gcataccggc 420 cataccatca agaaggtggt caacgtcggc atcggtggtt ctgatttggg gcctgcgatg 480 gcagcccagg cactgcgcag ctatgaagtc gcgggaatct ccgccgagtt tgtctccaat 540 gtggacccag ccgatttggc aaagactttg gatgggctcg atgccggctc cactcttttc 600 atcattgctt ccaagacttt taccacgcag gaaactttgt ctaacgccca cgctgctcgt 660 cgttggatgc tggagcaatt cgatggtgat gagtcttcca tcgccaagca ctttgttgcg 720 gtatcgacca acgccgaaaa ggtcgcggaa ttcggcattg atactgaaaa catgtttggg 780 ttttggaact gggtcggcgg acgttactct gtcgatgcag ccattggctt gtctctgatg 840 tgcaccattg gcccgctaga tttcatgcgt ttcctcgaag gcttccacgc catggatgag 900 cacttccgta ccacgccgct ggaatctaat gttccaacac tcatggcgct gctgagcatc 960 tggtacacca acttctatgg cgcacagtct cacgctgtac ttccctactc tgaggatctc 1020 ggccgtttcc ctgcttacct acagcagctg acgatggaat ctaacggcaa gtccgtgcac 1080 cacgacggtt ctgcagtgtc aaccaccacc agtcccattt actggggtga gccaggaacc 1140 aatggccagc acgctttctt ccagctactg caccagggga cgcacttggt tccggctgat 1200 ttcattggat ttgcccgccc gaaggaagat ttccccactg ccgatggcac aggctccatg 1260 catgatctac tcatgggtaa tctcttcgca caaaccaagg tgctggcctt tggcaaaacc 1320 gcagaagaaa tagccaggga aggcatcgca gaagaactcg tggctcacaa ggtcatgccg 1380 ggcaatcgcc ccaccaccac tatcttggcc gaagagctca cccctgcagc cttgggcagc 1440 ttgattgcgc tttacgaaca catcgtgttt gtcgaaggcg tcatctggga tatcaactcc 1500 tttgaccaat ggggcgtgga attaggtaag caacaagcca acgacctagc gccggcggtc 1560 tctggcaagg cggatatcga taccggtgat gaatcgaccg acgcgctgat ctcctggttt 1620 cttaaacacc gttaa 1635 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer-pgi-1F <400> 2 cgcggatccg gggattggct cttccttctc 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer-pgi-R <400> 3 tgctctagac cacgcgccaa tgccgtacag 30 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer-pgi-2F <400> 4 gccctctaga gggattggct cttccttctc tacc 34 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer-2pgi-1F <400> 5 ggaattaggt aagcaacaag ccaacg 26 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer-2pgi-1R <400> 6 gctttgccca tgcaggttgt tgcg 24  

Claims (7)

글루코오스-6-포스페이트 아이소머라아제를 암호화하는 pgi 유전자를 포함하는 재조합 벡터로 형질전환시키거나, 또는 내재적 pgi 유전자에 더하여 1 카피 이상의 pgi 유전자를 새로 도입함으로써 글루코오스-6-포스페이트 아이소머라아제(glucose-6-phosphate isomerase)의 활성이 강화된 것을 특징으로 하는, 5'-이노신산 생산능을 가지는 코리네박테리움 암모니아게네스.Glucose-6-phosphate to iso transformed with a recombinant vector containing the pgi gene coding for epimerase, or in addition to the intrinsic pgi gene introduced by the pgi gene least one copy of glucose-6-phosphate epimerase iso (glucose- 6-phosphate isomerase) Corynebacterium ammonia gene having a 5'-inosinic acid production capacity, characterized in that enhanced activity. 삭제delete 제1항에 있어서, The method of claim 1, 상기 pgi 유전자는 서열번호 1의 염기서열을 가지는 것을 특징으로 하는, 5'-이노신산 생산능을 가지는 코리네박테리움 암모니아게네스.The pgi gene is characterized in that having the nucleotide sequence of SEQ ID NO: 1, Corynebacterium ammonia genes having a 5'-inosinic acid production capacity. 제1항에 있어서, The method of claim 1, 상기 재조합 백터는 도1의 개열지도를 가지는 벡터 pECCG117-pgi인 것을 특징으로 하는, 5'-이노신산 생산능을 가지는 코리네박테리움 암모니아게네스.The recombinant vector is a vector pECCG117- pgi having a cleavage map of Figure 1, Corynebacterium ammonia gene having a 5'-inosinic acid production capacity. 제1항에 있어서, The method of claim 1, 상기 도입은 코리네박테리움 속 미생물을 도3의 개열지도를 가지는 pDZ-2pgi 벡터로 형질전환시킴으로써 달성되는 것을 특징으로 하는, 5'-이노신산 생산능을 가지는 코리네박테리움 암모니아게네스.The introduction of the Corynebacterium ammonia gene having a 5'-inosinic acid, characterized in that the transformation is achieved by transforming a microorganism of the genus Corynebacterium with pDZ-2 pgi vector having a cleavage map of FIG. 제1항에 있어서,The method of claim 1, 상기 코리네박테리움 암모니아게네스는 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes) CN01-0207(수탁번호:KCCM-10918P)인 것을 특징으로 하는, 5'-이노신산 생산능을 가지는 코리네박테리움 암모니아게네스.The Corynebacterium ammonia genes is Corynebacterium ammonia genes ( Corynebacterium ammoniagenes ) CN01-0207 (Accession Number: KCCM-10918P), characterized in that, Corynebacterium ammonia having a 5'-inosinic acid production capacity Gennes. 제1항, 제3항 내지 제6항 중 어느 한 항에 따른 미생물을 배양하고, 그 배양액으로부터 5'-이노신산을 생산하는 방법.A method for culturing a microorganism according to any one of claims 1 to 3, and producing 5'-inosinic acid from the culture solution.
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WO2019147078A1 (en) 2018-01-25 2019-08-01 씨제이제일제당 (주) Corynebacterium sp. microorganism producing purine nucleotide and method for producing purine nucleotide by using same

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US6586214B1 (en) 1999-09-15 2003-07-01 Degussa Ag Method for increasing the metabolic flux through the pentose phosphate cycle in coryneform bacteria by regulation of the phosphoglucose isomerase (pgi gene)
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US6830903B1 (en) 1999-07-23 2004-12-14 Archer-Daniels-Midland Company Methods for producing L-amino acids using a corynebacterium glutamicum with a disrupted pgi gene
US6586214B1 (en) 1999-09-15 2003-07-01 Degussa Ag Method for increasing the metabolic flux through the pentose phosphate cycle in coryneform bacteria by regulation of the phosphoglucose isomerase (pgi gene)
US20030092137A1 (en) 2001-03-17 2003-05-15 Mike Farwick Process for the preparation of L-amino acids by using coryneform bacteria

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019147078A1 (en) 2018-01-25 2019-08-01 씨제이제일제당 (주) Corynebacterium sp. microorganism producing purine nucleotide and method for producing purine nucleotide by using same
US11421200B2 (en) 2018-01-25 2022-08-23 Cj Cheiljedang Corporation Microorganism of the genus Corynebacterium producing purine nucleotide and a method for producing purine nucleotide by using the same

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