KR20220148694A - Mutant of Corynebacterium glutamicum with enhanced L-lysine productivity and method for preparing L-lysine using the same - Google Patents
Mutant of Corynebacterium glutamicum with enhanced L-lysine productivity and method for preparing L-lysine using the same Download PDFInfo
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- KR20220148694A KR20220148694A KR1020210066151A KR20210066151A KR20220148694A KR 20220148694 A KR20220148694 A KR 20220148694A KR 1020210066151 A KR1020210066151 A KR 1020210066151A KR 20210066151 A KR20210066151 A KR 20210066151A KR 20220148694 A KR20220148694 A KR 20220148694A
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- lysine
- mutant
- corynebacterium
- corynebacterium glutamicum
- strain
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Abstract
Description
본 발명은 L-라이신 생산능이 향상된 코리네박테리움 글루타미쿰 변이주 및 이를 이용한 L-라이신의 생산 방법에 관한 것이다.The present invention relates to a Corynebacterium glutamicum mutant having improved L-lysine-producing ability and a method for producing L-lysine using the same.
L-라이신은 사람이나 동물 체내에서 합성되지 않는 필수아미노산으로서 외부에서 공급되어야 하며, 일반적으로 세균이나 효모와 같은 미생물을 이용한 발효에 의해 생산된다. L-라이신 생산은 자연상태에서 수득된 야생형 균주나 이의 L-라이신 생산능이 향상되도록 변형된 변이주를 이용할 수 있다. 최근에는 L-라이신의 생산 효율을 개선시키기 위해 L-아미노산 및 기타 유용물질 생산에 많이 이용되는 대장균, 코리네박테리움 등의 미생물을 대상으로 유전자 재조합 기술을 적용하여 우수한 L-라이신 생산능을 갖는 다양한 재조합 균주 또는 변이주 및 이를 이용한 L-라이신 생산 방법이 개발되고 있다. L-lysine is an essential amino acid that is not synthesized in humans or animals and must be supplied from the outside, and is generally produced by fermentation using microorganisms such as bacteria or yeast. L- lysine production may use a wild-type strain obtained in the natural state or a mutant strain modified to improve its L- lysine production ability. Recently, in order to improve the production efficiency of L-lysine, genetic recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are often used for the production of L-amino acids and other useful substances, and has excellent L-lysine production ability. Various recombinant strains or mutants and L-lysine production methods using the same are being developed.
한국등록특허 제10-0838038호 및 제10-2139806호에 따르면, L-라이신 생산과 관련된 효소를 포함한 단백질을 암호화하는 유전자의 염기서열 또는 아미노산 서열을 변경하여 그 유전자의 발현을 증가시키거나 불필요한 유전자를 제거함으로써 L-라이신 생산능을 향상시킬 수 있다. 또한, 한국공개특허 제10-2020-0026881호에는 L-라이신 생산에 관여하는 효소를 암호화하는 유전자의 발현을 증가시키기 위하여 유전자의 기존 프로모터를 강한 활성을 가지는 프로모터로 변경하는 방법을 개시하고 있다. According to Korea Patent Registration Nos. 10-0838038 and 10-2139806, the nucleotide sequence or amino acid sequence of a gene encoding a protein including an enzyme related to L-lysine production is changed to increase the expression of the gene or an unnecessary gene. By removing the L- lysine production capacity can be improved. In addition, Korean Patent Application Laid-Open No. 10-2020-0026881 discloses a method of changing an existing promoter of a gene to a promoter having strong activity in order to increase the expression of a gene encoding an enzyme involved in L-lysine production.
이와 같이 L-라이신 생산능을 증가시키는 다양한 방법이 개발되고 있으나, L-라이신 생산에 직간접적으로 연관된 효소, 전사인자, 수송 단백질 등 단백질의 종류가 수십여 종에 이르기 때문에 이러한 단백질의 활성 변화에 따른 L-라이신 생산능 증가 여부에 관해 여전히 많은 연구가 필요한 실정이다.As described above, various methods for increasing L-lysine production capacity have been developed, but since there are dozens of proteins such as enzymes, transcription factors, and transport proteins directly or indirectly related to L-lysine production, it is difficult to change the activity of these proteins. There is still a need for a lot of research on whether the L-lysine production capacity is increased.
본 발명은 L-라이신 생산능이 향상된 코리네박테리움 글루타미쿰(Corynebacterium glutamicum) 변이주를 제공하는 것을 목적으로 한다.An object of the present invention is to provide a mutant Corynebacterium glutamicum having improved L-lysine production ability.
또한, 본 발명은 상기 변이주를 이용한 L-라이신의 생산 방법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a method for producing L-lysine using the mutant.
본 발명자들은 코리네박테리움 글루타미쿰 균주를 이용하여 L-라이신 생산능이 향상된 새로운 변이주를 개발하기 위해 연구한 결과, L-라이신 생합성 경로에서 라이신 전구체인 아스파르테이트의 공급에 관여하는 아스파르테이트 아미노트랜스퍼라제를 암호화하는 aspB 유전자의 프로모터 내 특정 위치의 염기서열을 치환한 경우 L-라이신 생산량이 증가하는 것을 확인함으로써 본 발명을 완성하였다.The present inventors studied to develop a new mutant with improved L-lysine production ability using a Corynebacterium glutamicum strain. As a result, aspartate involved in the supply of aspartate, a lysine precursor, in the L-lysine biosynthesis pathway The present invention was completed by confirming that the production of L-lysine was increased when the nucleotide sequence at a specific position in the promoter of the aspB gene encoding the aminotransferase was substituted.
본 발명의 일 양상은 아스파르테이트 아미노트랜스퍼라제의 활성이 강화되어 L-라이신 생산능이 향상된 코리네박테리움 글루타미쿰 변이주를 제공한다.One aspect of the present invention provides a Corynebacterium glutamicum mutant with enhanced L-lysine production ability by enhancing the activity of aspartate aminotransferase.
본 발명에서 사용된 “아스파르테이트 아미노트랜스퍼라제(aspartate aminotransferase 또는 aspartate transaminase)”는 L-라이신 생합성 경로에서 옥살로아세테이트의 아스파르테이트 전환 반응을 촉매하는 효소를 의미한다.As used herein, "aspartate aminotransferase (aspartate aminotransferase or aspartate transaminase)" refers to an enzyme that catalyzes the conversion of oxaloacetate to aspartate in the L-lysine biosynthetic pathway.
본 발명의 일 구체예에 따르면, 상기 아스파르테이트 아미노트랜스퍼라제는 코리네박테리움(Corynebacterium) 속 균주에서 유래된 것일 수 있다. 구체적으로, 상기 코리네박테리움 속 균주는 코리네박테리움 글루타미쿰(Corynebacterium glutamicum), 코리네박테리움 크루디락티스(Corynebacterium crudilactis), 코리네박테리움 데저티(Corynebacterium deserti), 코리네박테리움 칼루나에(Corynebacterium callunae), 코리네박테리움 수라나래에(Corynebacterium suranareeae), 코리네박테리움 루브리칸티스(Corynebacterium lubricantis), 코리네박테리움 두사넨세(Corynebacterium doosanense), 코리네박테리움 이피시엔스(Corynebacterium efficiens), 코리네박테리움 우테레키(Corynebacterium uterequi), 코리네박테리움 스테셔니스(Corynebacterium stationis), 코리네박테리움 파캔세(Corynebacterium pacaense), 코리네박테리움 싱굴라레(Corynebacterium singulare), 코리네박테리움 휴미레듀센스(Corynebacterium humireducens), 코리네박테리움 마리눔(Corynebacterium marinum), 코리네박테리움 할로톨레란스(Corynebacterium halotolerans), 코리네박테리움 스페니스코룸(Corynebacterium spheniscorum), 코리네박테리움 프레이부르겐세(Corynebacterium freiburgense), 코리네박테리움 스트리아툼(Corynebacterium striatum), 코리네박테리움 카니스(Corynebacterium canis), 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes), 코리네박테리움 레날레(Corynebacterium renale), 코리네박테리움 폴루티솔리(Corynebacterium pollutisoli), 코리네박테리움 이미탄스(Corynebacterium imitans), 코리네박테리움 카스피움(Corynebacterium caspium), 코리네박테리움 테스투디노리스(Corynebacterium testudinoris), 코리네박테리움 슈도펠라지(Corynebacaterium pseudopelargi) 또는 코리네박테리움 플라베스센스(Corynebacterium flavescens)일 수 있으며, 이에 한정되는 것은 아니다.According to one embodiment of the present invention, the aspartate aminotransferase may be derived from a genus strain of Corynebacterium . Specifically, the Corynebacterium sp. strain is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium Corynebacterium callunae , Corynebacterium suranareeae , Corynebacterium lubricantis , Corynebacterium doosanense, Corynebacterium doosanense , Corynebacterium ipish Ens ( Corynebacterium efficiens ), Corynebacterium utereki ( Corynebacterium uterequi ), Corynebacterium stationis ( Corynebacterium stationis ), Corynebacterium pacaense ( Corynebacterium pacaense ), Corynebacterium singulare ( Corynebacterium singulare ) ), Corynebacterium humireducens ( Corynebacterium humireducens ), Corynebacterium marinum ), Corynebacterium halotolerans ( Corynebacterium halotolerans ), Corynebacterium spheniscorum ( Corynebacterium spheniscorum ), Corynebacterium freiburgense ( Corynebacterium freiburgense ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium canis ( Corynebacterium canis ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium Renale ( Corynebacterium renale ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Coryne Bacterium imitans ( Corynebacterium imitans ), Corynebacterium caspium ( Corynebacterium caspium ), Corynebacterium testudinoris ( Corynebacterium testudinoris ), Corynebacterium pseudopelargi ( Corynebacaterium pseudopelargi ) or Corynebacterium fla Bassens ( Corynebacterium flavescens ) It may be, but is not limited thereto.
본 발명에서 사용된 “활성이 강화”는 목적하는 효소, 전사 인자, 수송 단백질 등의 단백질을 암호화하는 유전자의 발현이 새로 도입되거나 증대되어 야생형 균주 또는 변형 전의 균주에 비하여 발현량이 증가되는 것을 의미한다. 이러한 활성의 강화는 유전자를 암호화하는 뉴클레오티드 치환, 삽입, 결실 또는 이들의 조합을 통하여 단백질 자체의 활성이 본래 미생물이 가지고 있는 단백질의 활성에 비해 증가한 경우와, 이를 암호화하는 유전자의 발현 증가 또는 번역 증가 등으로 세포 내에서 전체적인 효소 활성 정도가 야생형 균주 또는 변형 전의 균주에 비하여 높은 경우, 이들의 조합 역시 포함한다.As used in the present invention, "enhanced activity" means that the expression of a gene encoding a protein, such as a desired enzyme, transcription factor, or transport protein, is newly introduced or increased, so that the expression level is increased compared to the wild-type strain or strain before modification. . The enhancement of this activity is when the activity of the protein itself is increased compared to the activity of the protein possessed by the original microorganism through nucleotide substitution, insertion, deletion, or a combination thereof, and the expression or translation increase of the gene encoding it For example, when the overall degree of enzymatic activity in the cell is higher than that of the wild-type strain or the strain before modification, a combination thereof is also included.
본 발명의 일 구체예에 따르면, 상기 아스파르테이트 아미노트랜스퍼라제의 활성 강화는 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자의 프로모터에 위치 특이적 변이를 유발하는 것일 수 있다.According to one embodiment of the present invention, the enhancement of the activity of the aspartate aminotransferase may be to induce a site-specific mutation in the promoter of the gene encoding the aspartate aminotransferase.
본 발명의 일 구체예에 따르면, 상기 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자의 프로모터는 서열번호 1의 염기서열로 표시되는 것일 수 있다.According to one embodiment of the present invention, the promoter of the gene encoding the aspartate aminotransferase may be represented by the nucleotide sequence of SEQ ID NO: 1.
본 발명에서 사용된 “프로모터”는 목적하는 유전자의 mRNA 전사를 개시하는 RNA 중합효소(polymerase)에 대한 결합부위를 포함하여 유전자의 전사를 조절하는 DNA의 특정 부위를 의미하며, 일반적으로 전사 개시점을 기준으로 상위(upstream)에 위치한다. 원핵생물에서의 프로모터는 RNA 중합효소가 결합하는 전사 개시점 주변의 부위로 정의되며, 일반적으로 전사 개시점으로부터 앞쪽으로 -10 영역과 -35 영역의 염기쌍이 떨어져 있는 두 개의 짧은 염기서열로 구성된다. 본 발명에서의 프로모터 변이는 야생형 프로모터에 비해 높은 활성을 가지도록 개량하는 것으로, 전사 개시점의 상위에 위치한 프로모터 영역 안에서 변이를 유발함으로써 하위(downstream)에 위치한 유전자의 발현을 증가시킬 수 있다.As used herein, the term “promoter” refers to a specific region of DNA that regulates transcription of a gene, including a binding site for RNA polymerase that initiates mRNA transcription of a desired gene, and generally refers to a transcription initiation point. It is located upstream based on . In prokaryotes, a promoter is defined as a region around the transcriptional initiation point to which RNA polymerase binds, and is generally composed of two short nucleotide sequences -10 and -35 base pairs away from the transcription initiation point. . The promoter mutation in the present invention is improved to have higher activity than the wild-type promoter, and by inducing mutation in the promoter region located above the transcription start point, it is possible to increase the expression of the gene located downstream.
본 발명의 일 구체예에 따르면, 상기 아스파르테이트 아미노트랜스퍼라제의 활성 강화는 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자의 프로모터 서열 내 전사 개시점으로부터 앞쪽으로 -100 내지 -10 영역 중 하나 이상의 염기가 치환된 것일 수 있다.According to one embodiment of the present invention, the enhancement of the activity of the aspartate aminotransferase is one or more bases in the region of -100 to -10 forward from the transcription start point in the promoter sequence of the gene encoding the aspartate aminotransferase. may be substituted.
보다 구체적으로, 본 발명에서의 프로모터 변이는 -100 내지 -10 영역 중 1개 이상의 염기, 바람직하게는 -95 내지 -15 영역, -95 내지 -55 영역, -55 내지 -40 영역, 또는 -30 내지 -15 영역에서 1개, 2개, 3개, 4개, 또는 5개의 염기가 연속적으로 또는 비연속적으로 치환된 것일 수 있다. More specifically, the promoter mutation in the present invention is one or more bases in the -100 to -10 region, preferably -95 to -15 region, -95 to -55 region, -55 to -40 region, or -30 1, 2, 3, 4, or 5 bases may be continuously or non-contiguously substituted in the to -15 region.
본 발명의 일 실시예에 따르면, 코리네박테리움 글루타미쿰 균주의 아스파르테이트 아미노트랜스퍼라제를 암호화하는 aspB 유전자의 프로모터 서열에서 -22 영역의 염기서열을 C에서 G로 치환하여 aspB 유전자의 새로운 프로모터 서열을 가지는 코리네박테리움 글루타미쿰 변이주를 획득하였다. 이러한 코리네박테리움 글루타미쿰 변이주는 서열번호 2의 염기서열로 표시되는 aspB 유전자의 변이된 프로모터를 포함하는 것일 수 있다.According to an embodiment of the present invention, the nucleotide sequence of the -22 region in the promoter sequence of the aspB gene encoding the aspartate aminotransferase of the Corynebacterium glutamicum strain is substituted from C to G to create a new aspB gene. A Corynebacterium glutamicum mutant having a promoter sequence was obtained. Such a Corynebacterium glutamicum mutant may include a mutated promoter of the aspB gene represented by the nucleotide sequence of SEQ ID NO: 2.
또한, 본 발명의 일 실시예에 따르면, 코리네박테리움 글루타미쿰 균주의 아스파르테이트 아미노트랜스퍼라제를 암호화하는 aspB 유전자의 프로모터 서열에서 -45 영역의 염기서열을 T에서 A로 치환하여 aspB 유전자의 새로운 프로모터 서열을 가지는 코리네박테리움 글루타미쿰 변이주를 획득하였다. 이러한 코리네박테리움 글루타미쿰 변이주는 서열번호 3의 염기서열로 표시되는 aspB 유전자의 변이된 프로모터를 포함하는 것일 수 있다.In addition, according to an embodiment of the present invention, the nucleotide sequence of the -45 region in the promoter sequence of the aspB gene encoding the aspartate aminotransferase of the Corynebacterium glutamicum strain is substituted from T to A, and the aspB gene Corynebacterium glutamicum mutant having a new promoter sequence was obtained. Such a Corynebacterium glutamicum mutant may include a mutated promoter of the aspB gene represented by the nucleotide sequence of SEQ ID NO: 3.
본 발명의 일 실시예에 따르면, 코리네박테리움 글루타미쿰 균주의 아스파르테이트 아미노트랜스퍼라제를 암호화하는 aspB 유전자의 프로모터 서열에서 -88 영역의 염기서열을 T에서 A로 치환하여 aspB 유전자의 새로운 프로모터 서열을 가지는 코리네박테리움 글루타미쿰 변이주를 획득하였다. 이러한 코리네박테리움 글루타미쿰 변이주는 서열번호 4의 염기서열로 표시되는 aspB 유전자의 변이된 프로모터를 포함하는 것일 수 있다.According to an embodiment of the present invention, the nucleotide sequence of the -88 region in the promoter sequence of the aspB gene encoding the aspartate aminotransferase of the Corynebacterium glutamicum strain is substituted from T to A to create a new aspB gene. A Corynebacterium glutamicum mutant having a promoter sequence was obtained. Such a Corynebacterium glutamicum mutant may include a mutated promoter of the aspB gene represented by the nucleotide sequence of SEQ ID NO: 4.
본 발명에서 사용된 “생산능이 향상된”은 모균주에 비해 L-라이신의 생산성이 증가된 것을 의미한다. 상기 모균주는 변이의 대상이 되는 야생형 또는 변이주를 의미하며, 직접 변이의 대상이 되거나 재조합된 벡터 등으로 형질전환되는 대상을 포함한다. 본 발명에 있어서, 모균주는 야생형 코리네박테리움 글루타미쿰 균주 또는 야생형으로부터 변이된 균주일 수 있다. As used in the present invention, "improved productivity" means that the productivity of L-lysine is increased compared to the parent strain. The parent strain means a wild-type or mutant strain to be mutated, and includes a target to be directly mutated or transformed with a recombinant vector. In the present invention, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type.
본 발명의 일 구체예에 따르면, 상기 모균주는 라이신 생산에 관여하는 유전자 (예컨대, lysC, zwf 및 hom 유전자)의 서열에 변이가 유발된 변이주로서 한국미생물보존센터(Korean Culture Center of Microorganisms)에 2021년 4월 2일자 수탁번호 KCCM12969P로 기탁된 코리네박테리움 글루타미쿰 균주 (이하 '코리네박테리움 글루타미쿰 DS1 균주'라 함)인 것일 수 있다.According to one embodiment of the present invention, the parent strain is a mutant strain induced in the sequence of a gene (eg, lysC, zwf and hom genes) involved in lysine production, and the Korean Culture Center of Microorganisms. It may be a Corynebacterium glutamicum strain deposited with accession number KCCM12969P on April 2, 2021 (hereinafter referred to as 'Corynebacterium glutamicum DS1 strain').
이와 같은 본 발명의 L-라이신 생산능이 향상된 코리네박테리움 글루타미쿰 변이주는 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자의 변이된 프로모터 서열을 포함하는 것일 수 있다.As described above, the Corynebacterium glutamicum mutant with improved L-lysine-producing ability of the present invention may include a mutated promoter sequence of a gene encoding aspartate aminotransferase.
본 발명의 일 구체예에 따르면, 상기 변이주는 서열번호 2 내지 4로 표시되는 염기서열 중 어느 하나를 아스파테이트 아미노트랜스퍼라제 유전자의 프로모터 서열로 포함하는 것일 수 있다.According to one embodiment of the present invention, the mutant strain may include any one of the nucleotide sequences shown in SEQ ID NOs: 2 to 4 as the promoter sequence of the aspartate aminotransferase gene.
본 발명의 일 실시예에 따르면, 상기 변이주는 아스파르테이트 아미노트랜스퍼라제를 암호화하는 aspB 유전자의 프로모터 변이를 포함함으로써 모균주에 비해 증가된 L-라이신 생산능을 나타내며, 특히 모균주에 비해 L-라이신 생산량이 3% 이상, 구체적으로는 3 내지 40%, 더욱 구체적으로는 5 내지 30% 증가되어 균주 배양액 1 ℓ 당 65 ~ 90 g의 L-라이신을 생산할 수 있으며, 바람직하게는 70 ~ 80 g의 L-라이신을 생산할 수 있다.According to an embodiment of the present invention, the mutant shows an increased L-lysine-producing ability compared to the parent strain by including a mutation in the promoter of the aspB gene encoding aspartate aminotransferase, and in particular, compared to the parent strain, L- Lysine production is increased by 3% or more, specifically 3 to 40%, more specifically by 5 to 30% to produce 65 to 90 g of L-lysine per 1 liter of strain culture, preferably 70 to 80 g of L-lysine can be produced.
본 발명의 일 구체예에 따른 코리네박테리움 글루타미쿰 변이주는 모균주에 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자의 프로모터 서열이 일부 치환된 변이체를 포함하는 재조합 벡터를 통해 구현될 수 있다.Corynebacterium glutamicum mutant according to an embodiment of the present invention can be implemented through a recombinant vector including a mutant in which the promoter sequence of the gene encoding aspartate aminotransferase in the parent strain is partially substituted.
본 발명에서 사용된 “일부”는 염기서열 또는 폴리뉴클레오티드 서열의 전부가 아닌 것을 의미하며, 1 내지 300개, 바람직하게는 1 내지 100개, 보다 바람직하게는 1 내지 50개일 수 있으나, 이에 한정되는 것은 아니다.As used in the present invention, “some” means not all of the base sequence or polynucleotide sequence, and may be 1 to 300, preferably 1 to 100, more preferably 1 to 50, but is limited thereto. it is not
본 발명에서 사용된 “변이체”는 L-라이신의 생합성에 관여하는 아스파르테이트 아미노트랜스퍼라제 유전자의 프로모터 서열 내 -100 내지 -10 영역 중 하나 이상의 염기가 치환된 프로모터 변이체를 의미한다.As used herein, the term “variant” refers to a promoter variant in which one or more bases in the region of -100 to -10 in the promoter sequence of the aspartate aminotransferase gene involved in the biosynthesis of L-lysine are substituted.
본 발명의 일 구체예에 따르면, 아스파르테이트 아미노트랜스퍼라제 유전자의 프로모터 서열 내 -22 영역의 염기서열이 G로 치환된 변이체는 서열번호 2의 염기서열을, -45 영역의 염기서열이 A로 치환된 변이체는 서열번호 3의 염기서열을, -88 영역의 염기서열이 A로 치환된 변이체는 서열번호 4의 염기서열을 가지는 것일 수 있다.According to one embodiment of the present invention, the mutant in which the nucleotide sequence of the -22 region in the promoter sequence of the aspartate aminotransferase gene is substituted with G is the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence of the -45 region is A The substituted variant may have the nucleotide sequence of SEQ ID NO: 3, and the mutant in which the nucleotide sequence of the -88 region is substituted with A may have the nucleotide sequence of SEQ ID NO: 4.
본 발명에서 사용된 “벡터”는 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 발현 벡터로서 유전자 삽입물이 발현되도록 작동 가능하게 연결된(operably linked) 필수적인 조절요소를 포함하는 유전자 제조물을 의미한다. 여기서, “작동 가능하게 연결된”은 발현이 필요한 유전자와 이의 조절 서열이 서로 기능적으로 결합되어 유전자 발현을 가능케 하는 방식으로 연결된 것을 의미하고, “조절요소”는 전사를 수행하기 위한 프로모터, 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 암호화하는 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함한다. 이러한 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터, 바이러스 벡터 등을 포함하나, 이에 한정되는 것은 아니다.As used herein, the term “vector” refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a gene preparation comprising essential regulatory elements operably linked to express a gene insert. Here, “operably linked” means that a gene requiring expression and its regulatory sequence are functionally linked to each other and linked in a way that enables gene expression, and “regulatory element” refers to a promoter for performing transcription, regulating transcription including any operator sequences for sequencing, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like.
본 발명에서 사용된 “재조합 벡터”는 적합한 숙주세포 내로 형질전환된 후, 숙주세포의 게놈과 무관하게 복제 가능하거나 게놈 그 자체에 봉합될 수 있다. 이때, 상기 "적합한 숙주세포"는 벡터가 복제 가능한 것으로서 복제가 개시되는 특정 염기서열인 복제 원점을 포함할 수 있다. The "recombinant vector" used in the present invention can be replicated independently of the genome of the host cell after transformation into a suitable host cell, or can be sutured into the genome itself. In this case, the "suitable host cell" may include a replication origin, which is a specific nucleotide sequence from which replication is initiated as a vector capable of replication.
상기 형질전환은 숙주세포에 따라 적합한 벡터 도입 기술이 선택되어 목적하는 유전자를 숙주세포 내에서 발현시킬 수 있다. 예를 들면, 벡터 도입은 전기천공법(electroporation), 열 충격(heat-shock), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌글리콜(PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 초산 리튬-DMSO법, 또는 이들의 조합에 의해 수행될 수 있다. 형질전환된 유전자는 숙주세포 내에서 발현될 수 있으면 숙주세포의 염색체 내 삽입 또는 염색체 외에 위치하고 있는 것이든 제한하지 않고 포함될 수 있다.For the transformation, an appropriate vector introduction technique is selected according to the host cell, and the desired gene can be expressed in the host cell. For example, vector introduction can be performed by electroporation, heat-shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE- Dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof can be carried out. As long as the transformed gene can be expressed in the host cell, it may be included without limitation whether it is inserted into the chromosome of the host cell or located outside the chromosome.
상기 숙주세포는 생체내 또는 시험관내에서 본 발명의 재조합 벡터 또는 폴리뉴클레오티드로 형질감염, 형질전환, 또는 감염된 세포를 포함한다. 본 발명의 재조합 벡터를 포함하는 숙주 세포는 재조합 숙주 세포, 재조합 세포 또는 재조합 미생물이다.The host cells include cells transfected, transformed, or infected with the recombinant vector or polynucleotide of the present invention in vivo or in vitro. A host cell comprising the recombinant vector of the present invention is a recombinant host cell, a recombinant cell or a recombinant microorganism.
또한, 본 발명에 의한 재조합 벡터는 선택 마커(selection marker)를 포함할 수 있는데, 상기 선택 마커는 벡터로 형질전환된 형질전환체 (숙주세포)를 선별하기 위한 것으로서 상기 선택 마커가 처리된 배지에서 선택 마커를 발현하는 세포만 생존할 수 있기 때문에, 형질전환 된 세포의 선별이 가능하다. 상기 선택 마커는 대표적인 예로 카나마이신, 스트렙토마이신, 클로람페니콜 등이 있으나, 이에 한정되는 것은 아니다.In addition, the recombinant vector according to the present invention may include a selection marker, the selection marker is for selecting transformants (host cells) transformed with the vector, in the medium treated with the selection marker. Since only cells expressing the selection marker can survive, selection of transformed cells is possible. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, chloramphenicol, and the like.
본 발명의 형질전환용 재조합 벡터 내에 삽입된 유전자들은 상동성 재조합 교차로 인하여 코리네박테리움 속 미생물과 같은 숙주세포 내로 치환될 수 있다.Genes inserted into the recombinant vector for transformation of the present invention can be substituted into a host cell such as a microorganism of the genus Corynebacterium due to homologous recombination crossover.
본 발명의 일 구체예에 따르면, 상기 숙주세포는 코리네박테리움 속 균주일 수 있으며, 예를 들면 코리네박테리움 글루타미쿰(Corynebacterium glutamicum) 균주일 수 있다.According to one embodiment of the present invention, the host cell may be a Corynebacterium sp. strain, for example, Corynebacterium glutamicum ( Corynebacterium glutamicum ) It may be a strain.
또한, 본 발명의 다른 일 양상은 a) 상기 코리네박테리움 글루타미쿰 변이주를 배지에서 배양하는 단계; 및 b) 상기 변이주 또는 변이주가 배양된 배지로부터 L-라이신을 회수하는 단계를 포함하는 L-라이신의 생산 방법을 제공한다.In addition, another aspect of the present invention comprises: a) culturing the Corynebacterium glutamicum mutant in a medium; And b) provides a method for producing L- lysine comprising the step of recovering L-lysine from the culture medium of the mutant or mutant strain.
상기 배양은 당업계에 알려진 적절한 배지와 배양 조건에 따라 이루어질 수 있으며, 통상의 기술자라면 배지 및 배양 조건을 용이하게 조정하여 사용할 수 있다. 구체적으로, 상기 배지는 액체 배지일 수 있으나, 이에 한정되는 것은 아니다. 배양 방법은 예를 들면, 회분식 배양(batch culture), 연속식 배양(continuous culture), 유가식 배양(fed-batch culture) 또는 이들의 조합 배양을 포함할 수 있으나, 이에 한정되는 것은 아니다.The culture may be performed according to an appropriate medium and culture conditions known in the art, and those skilled in the art may easily adjust the medium and culture conditions for use. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, for example, batch culture, continuous culture, fed-batch culture, or a combination culture thereof, but is not limited thereto.
본 발명의 일 구체예에 따르면, 상기 배지는 적절한 방식으로 특정 균주의 요건을 충족해야 하며, 통상의 기술자에 의해 적절하게 변형될 수 있다. 코리네박테리움 속 균주에 대한 배양 배지는 공지된 문헌 (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981)을 참조할 수 있으나, 이에 한정되는 것은 아니다.According to one embodiment of the present invention, the medium should meet the requirements of a specific strain in an appropriate manner, and may be appropriately modified by a person skilled in the art. The culture medium for the Corynebacterium sp. strain may refer to the known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but is not limited thereto.
본 발명의 일 구체예에 따르면, 배지에 다양한 탄소원, 질소원 및 미량원소 성분을 포함할 수 있다. 사용될 수 있는 탄소원으로는 글루코스, 수크로스, 락토스, 프락토스, 말토스, 전분, 셀룰로스와 같은 당 및 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유 등과 같은 오일 및 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤, 에탄올과 같은 알코올, 아세트산과 같은 유기산이 포함된다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있으나, 이에 한정되는 것은 아니다. 사용될 수 있는 질소원으로는 펩톤, 효모 추출물, 육즙, 맥아 추출물, 옥수수 침지액, 대두밀 및 요소 또는 무기 화합물, 예를 들면 황산 암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄이 포함될 수 있다. 질소원 또한 개별적으로 또는 혼합물로서 사용할 수 있으나 이에 한정되는 것은 아니다. 사용될 수 있는 인의 공급원으로는 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨-함유 염이 포함될 수 있으며, 이에 한정되는 것은 아니다. 또한, 배양 배지는 성장에 필요한 황산마그네슘 또는 황산철과 같은 금속염을 함유할 수 있으며, 이에 한정되는 것은 아니다. 그 외에, 아미노산 및 비타민과 같은 필수 성장 물질이 포함될 수 있다. 또한 배양 배지에 적절한 전구체들이 사용될 수 있다. 상기 배지 또는 개별 성분은 배양과정에서 배양액에 적절한 방식에 의해 회분식으로 또는 연속식으로 첨가될 수 있으나, 이에 한정되는 것은 아니다.According to one embodiment of the present invention, the medium may include various carbon sources, nitrogen sources and trace element components. Carbon sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto. Nitrogen sources that may be used include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture, but is not limited thereto. Sources of phosphorus that may be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. In addition, precursors suitable for the culture medium may be used. The medium or individual components may be added batchwise or continuously by an appropriate method to the culture medium during the culturing process, but is not limited thereto.
본 발명의 일 구체예에 따르면, 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산 및 황산과 같은 화합물을 미생물 배양액에 적절한 방식으로 첨가하여 배양액의 pH를 조정할 수 있다. 또한, 배양 중에 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 추가적으로, 배양액의 호기 상태를 유지하기 위하여, 배양액 내로 산소 또는 산소-함유 기체 (예, 공기)를 주입할 수 있다. 배양액의 온도는 통상 20℃ 내지 45℃, 예를 들면 25℃ 내지 40℃일 수 있다. 배양기간은 유용물질이 원하는 생산량으로 수득될 때까지 계속될 수 있으며, 예를 들면 10 내지 160 시간일 수 있다.According to one embodiment of the present invention, it is possible to adjust the pH of the culture medium by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microorganism culture medium in an appropriate manner during culture. In addition, the use of an antifoaming agent such as fatty acid polyglycol ester during culture can suppress the formation of bubbles. Additionally, in order to maintain the aerobic state of the culture medium, oxygen or oxygen-containing gas (eg, air) may be injected into the culture medium. The temperature of the culture medium may be usually 20 °C to 45 °C, for example, 25 °C to 40 °C. The incubation period may be continued until a useful substance is obtained in a desired production amount, for example, it may be 10 to 160 hours.
본 발명의 일 구체예에 따르면, 상기 배양된 변이주 및 변이주가 배양된 배지에서 L-라이신을 회수하는 단계는 배양 방법에 따라 당해 분야에 공지된 적합한 방법을 이용하여 배지로부터 생산된 L-라이신을 수집 또는 회수할 수 있다. 예를 들면 원심분리, 여과, 추출, 분무, 건조, 증발, 침전, 결정화, 전기영동, 분별용해 (예를 들면, 암모늄 설페이트 침전), 크로마토그래피 (예를 들면, 이온 교환, 친화성, 소수성 및 크기배제) 등의 방법을 사용할 수 있으나, 이에 한정되는 것은 않는다.According to one embodiment of the present invention, the step of recovering L-lysine from the cultured medium and the cultured mutant strain is L-lysine produced from the medium using a suitable method known in the art according to the culture method. can be collected or retrieved. e.g. centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractionation (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobicity and size exclusion) may be used, but the present invention is not limited thereto.
본 발명의 일 구체예에 따르면, 라이신을 회수하는 단계는 배양 배지를 저속 원심분리하여 바이오매스를 제거하고 얻어진 상등액을 이온교환 크로마토그래피를 통하여 분리할 수 있다.According to one embodiment of the present invention, in the step of recovering lysine, the biomass is removed by centrifuging the culture medium at low speed, and the obtained supernatant can be separated through ion exchange chromatography.
본 발명의 일 구체예에 따르면, 상기 L-라이신을 회수하는 단계는 L-라이신을 정제하는 공정을 포함할 수 있다.According to one embodiment of the present invention, the step of recovering L-lysine may include a step of purifying L-lysine.
본 발명에 따른 코리네박테리움 글루타미쿰 변이주는 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자의 발현을 증가 또는 강화시킴으로써 모균주에 비해 L-라이신의 생산 수율을 향상시킬 수 있다.The Corynebacterium glutamicum mutant according to the present invention can improve the production yield of L-lysine compared to the parent strain by increasing or enhancing the expression of the gene encoding aspartate aminotransferase.
도 1은 본 발명의 일 실시예에 따라 프로모터 부위의 22번째 서열을 C에서 G로 치환하기 위한 재조합 벡터 pCGI(DS7-2)의 구조를 나타낸 것이다.
도 2는 본 발명의 일 실시예에 따라 프로모터 부위의 45번째 서열을 T에서 A로 치환하기 위한 재조합 벡터 pCGI(DS7-1)의 구조를 나타낸 것이다.
도 3은 본 발명의 일 실시예에 따라 프로모터 부위의 88번째 서열을 T에서 A로 치환하기 위한 재조합 벡터 pCGI(DS7)의 구조를 나타낸 것이다.1 shows the structure of a recombinant vector pCGI (DS7-2) for substituting C to G for the 22nd sequence of the promoter region according to an embodiment of the present invention.
Figure 2 shows the structure of a recombinant vector pCGI (DS7-1) for substituting the 45th sequence of the promoter region from T to A according to an embodiment of the present invention.
3 shows the structure of a recombinant vector pCGI (DS7) for substituting T to A for the 88th sequence of the promoter region according to an embodiment of the present invention.
이하, 본 발명을 보다 상세하게 설명한다. 그러나, 이러한 설명은 본 발명의 이해를 돕기 위하여 예시적으로 제시된 것일 뿐, 본 발명의 범위가 이러한 예시적인 설명에 의하여 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.
실시예 1. 코리네박테리움 글루타미쿰 변이주의 제조Example 1. Preparation of Corynebacterium glutamicum mutants
아스파르테이트 아미노트랜스퍼라제의 활성이 강화된 코리네박테리움 글루타미쿰 변이주를 제조하기 위하여, 코리네박테리움 글루타미쿰 DS1 균주를 사용하여 무작위 돌연변이를 유발하였다.In order to prepare a Corynebacterium glutamicum mutant with enhanced activity of aspartate aminotransferase, random mutagenesis was induced using a Corynebacterium glutamicum DS1 strain.
1-1. 돌연변이 유발1-1. Mutagenesis
코리네박테리움 글루타미쿰 DS1 균주를 CM 액체배지 (글루코오스 5 g, NaCl 2.5 g, 효모 추출물 5.0 g, 유레아(urea) 1.0 g, 폴리펩톤 10.0 g 및 비프(beef) 추출물 5.0 g 함유, pH 6.8) 50 ㎖가 담긴 플라스크에 접종하고 돌연변이 유발물질인 N-메틸-N`-니트로-N-니트로소구아니딘(N-methyl-N`-nitro-N-nitrosoguanidine, NTG)을 최종농도 300 ㎍/㎖로 첨가한 후 30℃에서 200 rpm으로 20시간 동안 진탕 배양한 후 추가적인 변이를 유도하기 위하여 20분간 UV에 노출하였다. 배양을 종료한 후 배양액을 12,000 rpm에서 10분 동안 원심분리하여 상층액을 제거하고, 식염수(saline)로 1회 세척하고 인산 완충액(phosphate buffer)으로 3회 더 세척하였다. 이를 인산 완충액 5 ㎖에 현탁한 후 CM 고체배지 (CM 액체배지에 한천 15 g/ℓ 및 라이신 8%를 더 포함함)에 도말하고 30℃에서 30시간 동안 배양하여 콜로니(colony) 100개를 분리하였다.Corynebacterium glutamicum DS1 strain containing CM broth (glucose 5 g, NaCl 2.5 g, yeast extract 5.0 g, urea 1.0 g, polypeptone 10.0 g and beef extract 5.0 g, pH 6.8 ) inoculated into a flask containing 50 ㎖, and the mutagenic N-methyl-N`-nitro-N-nitrosoguanidine (N-methyl-N`-nitro-N-nitrosoguanidine, NTG) was added to a final concentration of 300 ㎍/㎖ After incubation with shaking at 30° C. at 200 rpm for 20 hours, it was exposed to UV for 20 minutes to induce additional mutation. After completion of the culture, the culture solution was centrifuged at 12,000 rpm for 10 minutes to remove the supernatant, washed once with saline, and washed three more times with phosphate buffer. After suspending this in 5 ml of phosphate buffer, spread it on CM solid medium (CM liquid medium further containing 15 g/L of agar and 8% lysine) and culture at 30° C. for 30 hours to isolate 100 colonies. did.
1-2. L-라이신 생산능이 향상된 변이주 선별1-2. Selection of mutants with improved L-lysine production capacity
분리된 콜로니 100개를 하기 표 1의 생산 액체배지 10 ㎖가 담긴 플라스크에 각각 5%씩 접종하고 30℃에서 200 rpm으로 30시간 동안 진탕 배양하였다. 각 배양액을 OD 610 nm에서 흡광도를 측정하고 L-라이신 생산량을 비교하여 돌연변이가 유발되지 않은 코리네박테리움 글루타미쿰 DS1 균주에 비해 L-라이신을 75.0 g/ℓ 이상 생산하는 콜로니 10개를 선별하였고, 이들의 염기서열 분석을 수행하여 aspB 유전자의 프로모터 변이 위치를 확인하였다. 이들 돌연변이가 유발된 코리네박테리움 글루타미쿰 DS1 변이주의 염기서열 확인 결과, -22 위치 C > G, -45 위치 T > A, -88 위치 T > A의 3가지 유형의 변이를 확인하였다.100 isolated colonies were inoculated 5% each in a flask containing 10 ml of the production broth shown in Table 1, and incubated with shaking at 30° C. at 200 rpm for 30 hours. By measuring the absorbance of each culture medium at OD 610 nm and comparing the L-lysine production, 10 colonies that produce more than 75.0 g / ℓ of L-lysine compared to the Corynebacterium glutamicum DS1 strain that are not mutated are selected and nucleotide sequence analysis was performed to confirm the promoter mutation position of the aspB gene. As a result of confirming the nucleotide sequence of the Corynebacterium glutamicum DS1 mutant in which these mutations were induced, three types of mutations were identified: -22 position C > G, -45 position T > A, and -88 position T > A.
이후에서는 3가지 유형의 aspB 유전자 프로모터 변이에 의한 L-라이신 생산성 증가를 검증하기 위한 실험을 수행하였다.Thereafter, an experiment was performed to verify the increase in L-lysine productivity by mutations in the three types of aspB gene promoters.
실시예 2. 코리네박테리움 글루타미쿰 변이주의 제조Example 2. Preparation of Corynebacterium glutamicum mutants
아스파르테이트 아미노트랜스퍼라제의 활성이 강화된 코리네박테리움 글루타미쿰 변이주를 제조하기 위하여, 코리네박테리움 글루타미쿰 DS1 균주 및 E. coli DH5a (HIT Competent cells™, Cat No. RH618)를 사용하였다.In order to prepare a Corynebacterium glutamicum mutant with enhanced activity of aspartate aminotransferase, Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells™, Cat No. RH618) was used.
상기 코리네박테리움 글루타미쿰 DS1 균주는 증류수 1 L에 글루코오스 5 g, NaCl 2.5 g, 효모 추출물 5.0 g, 유레아(urea) 1.0 g, 폴리펩톤 10.0 g 및 비프(beef) 추출물 5.0 g 조성의 CM 액체배지 또는 고체배지 (필요에 따라 한천 15 g/L 첨가) (pH 6.8)에서 30℃의 온도로 배양하였다.The Corynebacterium glutamicum DS1 strain is a CM of 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone and 5.0 g of beef extract in 1 L of distilled water. It was cultured at a temperature of 30°C in a liquid medium or a solid medium (add 15 g/L of agar if necessary) (pH 6.8).
상기 E. coli DH5a는 증류수 1 L에 트립톤 10.0 g, NaCl 10.0 g 및 효모 추출물 5.0 g 조성의 LB 배지 상에서 37℃의 온도로 배양하였다.The E. coli DH5a was cultured at a temperature of 37° C. on LB medium having a composition of 10.0 g of tryptone, 10.0 g of NaCl and 5.0 g of yeast extract in 1 L of distilled water.
항생제 카나마이신(kanamycin) 및 스트렙토마이신(streptomycine)은 시그마(Sigma)사의 제품을 사용하였고, DNA 시퀀싱 분석은 마크로젠(주)에 의뢰하여 분석하였다.Antibiotics kanamycin and streptomycin were used by Sigma, and DNA sequencing analysis was requested by Macrogen.
2-1. 재조합 벡터의 제작2-1. Construction of Recombinant Vector
균주에 라이신 전구체인 아스파르테이트의 공급을 강화하여 라이신 생산성을 증가시키기 위해 아스파르테이트 아미노트랜스퍼라제의 활성을 강화하고자 하였다. 본 실시예에서 이용한 방법은 아스파르테이트 아미노트랜스퍼라제를 암호화하는 aspB 유전자의 발현을 증가하기 위하여, aspB 유전자의 프로모터에 특이적인 변이를 유발하였다. aspB 유전자의 프로모터 -22 위치의 염기서열을 G로 치환하였고, 상기 실시예1 에서 얻은 각 변이주의 게놈 상에서 aspB 유전자 주변의 1256bp 단편을 PCR로 증폭하여, 재조합 벡터인 pCGI (문헌 [Kim et al., Journal of Microbiological Methods 84 (2011) 128-130] 참조)에 클로닝하였다. 상기 플라스미드를 pCGI(DS7-2)라고 명명하였다 (도 1 참조). 상기 플라스미드를 제작하기 위하여 유전자 단편을 증폭하는데 하기 표 2의 프라이머를 사용하였다.It was attempted to enhance the activity of aspartate aminotransferase in order to increase the productivity of lysine by enhancing the supply of aspartate, a lysine precursor, to the strain. The method used in this Example induced specific mutations in the promoter of the aspB gene in order to increase the expression of the aspB gene encoding aspartate aminotransferase. The nucleotide sequence at the position -22 of the promoter of the aspB gene was substituted with G, and the 1256 bp fragment around the aspB gene was amplified by PCR on the genome of each mutant obtained in Example 1 above, and the recombinant vector pCGI (see Kim et al. , Journal of Microbiological Methods 84 (2011) 128-130]). This plasmid was named pCGI (DS7-2) (see FIG. 1). To construct the plasmid, the primers in Table 2 below were used to amplify the gene fragment.
이상의 프라이머를 이용하여 아래의 조건 하에 PCR을 수행하였다. Thermocycler (TP600, TAKARA BIO Inc., 일본)를 이용하여 각각의 데옥시뉴클레오티드 트리포스페이트 (dATP, dCTP, dGTP, dTTP) 100 μM가 첨가된 반응액에 올리고뉴클레오티드 1 pM, 실시예 1에서 선별된 돌연변이 코리네박테리움 글루타미쿰 DS1 변이주 (프로모터 -22 위치에 변이 발생)의 염색체 DNA 10 ng을 주형(template)으로 이용하여, pfu-X DNA 폴리머라제 혼합물 (Solgent) 1 유닛의 존재 하에서 25 ~ 30 주기(cycle)를 수행하였다. PCR 수행 조건은 (i) 변성(denaturation) 단계: 94℃에서 30초, (ii) 결합(annealing) 단계: 58℃에서 30초, 및 (iii) 확장(extension) 단계: 72℃에서 1 ~ 2분 (1 kb 당 2분의 중합시간 부여)의 조건에서 실시하였다.PCR was performed under the following conditions using the above primers. Using a Thermocycler (TP600, TAKARA BIO Inc., Japan), 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) was added to a reaction solution containing 1 pM of oligonucleotides, mutants selected in Example 1 Using 10 ng of the chromosomal DNA of the Corynebacterium glutamicum DS1 mutant (the mutation occurred at the promoter -22 position) as a template, 25 to 30 in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent) A cycle was performed. PCR conditions were (i) denaturation step: 94°C for 30 seconds, (ii) annealing step: 58°C for 30 seconds, and (iii) extension step: 1-2 at 72°C. It was carried out under the conditions of minutes (providing a polymerization time of 2 minutes per 1 kb).
이와 같이 제조된 유전자 단편을 self-assembly cloning을 이용하여, pCGI 벡터에 클로닝하였다. 상기 벡터를 대장균(E. coli) DH5a에 형질전환시키고, 50 ㎍/㎖의 카나마이신을 함유하는 LB-한천 플레이트 상에 도말하여, 37℃에서 24시간 배양하였다. 최종 형성되는 콜로니를 분리하여 삽입물(insert)이 정확히 벡터에 존재하는지 확인한 후, 이 벡터를 분리하여 코리네박테리움 글루타미쿰 균주의 재조합에 사용하였다.The gene fragment thus prepared was cloned into the pCGI vector using self-assembly cloning. The vector was transformed into E. coli DH5a, spread on an LB-agar plate containing 50 μg/ml kanamycin, and cultured at 37° C. for 24 hours. After isolating the finally formed colony and confirming that the insert is exactly present in the vector, this vector was isolated and used for recombination of the Corynebacterium glutamicum strain.
2-2. 변이주의 제조2-2. manufacturing of mutants
상기 pCGI(DS7-2) 벡터를 이용하여 변이 균주인 DS7-2 균주를 제조하였다. 상기 벡터의 최종 농도가 1 ㎍/㎕ 이상 되도록 준비하여 코리네박테리움 글루타미쿰 DS1 균주에 전기천공법 (문헌 [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347] 참조)을 사용하여 1차 재조합을 유도하였다. 이때, 전기 천공한 균주를 카나마이신이 20 ㎍/㎕ 포함되는 CM 고체배지에 도말하여 콜로니를 분리한 후 게놈상의 유도한 위치에 적절히 삽입되었는지 PCR 및 염기서열 분석을 통해 확인하였다. 이렇게 분리된 균주를 다시 2차 재조합을 유도하기 위하여 CM 액체배지에 접종하고, 하룻밤 이상 배양하여 동일 40 mg/L 스트렙토마이신을 함유한 한천배지에 도말하여 콜로니를 분리하였다. 최종 분리한 콜로니 중에서 카나마이신에 대한 내성 여부를 확인한 후, 항생제 내성이 없는 균주들 중 aspB 유전자의 프로모터에 변이가 도입되었는지 염기서열 분석을 통해 확인하였다 (문헌 [Schafer et al., Gene 145 (1994) 69-73] 참조). 최종적으로 변이 aspB 프로모터가 도입된 코리네박테리움 글루타미쿰 변이주 (DS7-2)를 획득하였다.A mutant strain DS7-2 was prepared using the pCGI (DS7-2) vector. Electroporation (see Tauch et al., FEMS Microbiology letters 123 (1994) 343-347) was used to prepare the vector to have a final concentration of 1 μg/μl or more, and to Corynebacterium glutamicum DS1 strain. to induce primary recombination. At this time, the electroporated strain was spread on a CM solid medium containing 20 μg/μl of kanamycin to isolate colonies, and then it was confirmed through PCR and sequencing whether it was properly inserted into the induced position on the genome. In order to induce secondary recombination of the thus-separated strain again, it was inoculated into a CM broth, cultured overnight or more, and spread on an agar medium containing the same 40 mg/L streptomycin to separate colonies. After confirming the resistance to kanamycin among the finally isolated colonies, it was confirmed through nucleotide sequence analysis whether a mutation was introduced into the promoter of the aspB gene among strains without antibiotic resistance (Schafer et al., Gene 145 (1994)) 69-73]). Finally, a Corynebacterium glutamicum mutant strain (DS7-2) into which the mutated aspB promoter was introduced was obtained.
실시예 3. 코리네박테리움 글루타미쿰 변이주의 제조 Example 3. Preparation of Corynebacterium glutamicum mutants
aspB 유전자의 프로모터 -45 위치의 염기서열을 T에서 A로 치환하고, DNA 주형으로 실시예 1에서 선별된 돌연변이 코리네박테리움 글루타미쿰 DS1 변이주 (프로모터 -45 위치에 변이 발생)를 사용한 것을 제외하고는, 실시예 2와 동일한 방법으로 코리네박테리움 글루타미쿰 변이주를 제조하였다.The nucleotide sequence at the -45 position of the promoter of the aspB gene was substituted from T to A, and the mutant Corynebacterium glutamicum DS1 mutant strain selected in Example 1 (mutation occurred at the promoter -45 position) was used as a DNA template. And, a Corynebacterium glutamicum mutant was prepared in the same manner as in Example 2.
여기서 플라스미드를 제작하기 위하여 유전자 단편을 증폭하는데 상기 표 2의 프라이머를 사용하였고, 제작된 플라스미드 pCGI(DS7-1) 벡터 (도 2 참조)를 이용하여 변이 균주인 DS7-1 균주를 제조하였다. 최종적으로 변이 aspB 유전자가 도입된 코리네박테리움 글루타미쿰 변이주 (DS7-1)를 획득하였다.Here, the primers in Table 2 were used to amplify the gene fragment to construct the plasmid, and the plasmid pCGI (DS7-1) vector (see FIG. 2) was used to prepare a mutant strain DS7-1. Finally, a mutant Corynebacterium glutamicum strain (DS7-1) into which the mutated aspB gene was introduced was obtained.
실시예 4. 코리네박테리움 글루타미쿰 변이주의 제조 Example 4. Preparation of Corynebacterium glutamicum mutants
aspB 유전자의 프로모터 -88 위치의 염기서열을 T에서 A로 치환하고, DNA 주형으로 실시예 1에서 선별된 돌연변이 코리네박테리움 글루타미쿰 DS1 변이주 (프로모터 -88 위치에 변이 발생)를 사용한 것을 제외하고는, 실시예 2와 동일한 방법으로 코리네박테리움 글루타미쿰 변이주를 제조하였다.The nucleotide sequence at the -88 position of the promoter of the aspB gene was substituted from T to A, and the mutant Corynebacterium glutamicum DS1 mutant strain selected in Example 1 (mutation occurred at the promoter -88 position) was used as a DNA template. And, a Corynebacterium glutamicum mutant was prepared in the same manner as in Example 2.
여기서 플라스미드를 제작하기 위하여 유전자 단편을 증폭하는데 상기 표 2의 프라이머를 사용하였고, 제작된 플라스미드 pCGI(DS7) 벡터 (도 3 참조)를 이용하여 변이 균주인 DS7 균주를 제조하였다. 최종적으로 변이 aspB 유전자가 도입된 코리네박테리움 글루타미쿰 변이주 (DS7)를 획득하였다.Here, the primers in Table 2 were used to amplify the gene fragment to construct the plasmid, and a mutated DS7 strain was prepared using the prepared plasmid pCGI (DS7) vector (see FIG. 3). Finally, a Corynebacterium glutamicum mutant strain (DS7) into which the mutated aspB gene was introduced was obtained.
실험예 1. 변이주의 L-라이신 생산성 비교Experimental Example 1. Comparison of L-Lysine Productivity of Mutants
모균주 코리네박테리움 글루타미쿰 DS1 균주와 실시예 2 내지 4에서 제조된 라이신 생산 변이주인 DS7-2, DS7-1 및 DS7 균주의 L-라이신 생산성을 비교하였다.L-lysine productivity of the parent strain Corynebacterium glutamicum DS1 strain and the lysine-producing mutants DS7-2, DS7-1 and DS7 strains prepared in Examples 2 to 4 was compared.
상기 표 1과 같은 조성을 갖는 라이신 배지 10 ml를 함유한 100 ml 플라스크에 각각의 균주를 각각 접종하고, 30℃에서 28시간, 180 rpm의 조건으로 진탕 배양하였다. 배양 종료 후 라이신 분석은 HPLC (Shimazu, 일본)로 L-라이신의 생산량을 측정하였고, 그 결과를 표 3에 나타내었다.Each strain was inoculated into a 100 ml flask containing 10 ml of lysine medium having the composition shown in Table 1, respectively, and cultured with shaking at 30° C. for 28 hours and 180 rpm. Lysine analysis after the end of culture was measured by the production of L-lysine by HPLC (Shimazu, Japan), the results are shown in Table 3.
상기 표 3에 나타낸 바와 같이, 코리네박테리움 글루타미쿰 변이주 DS7-2, DS7-1 및 DS7 균주는 라이신 생합성 경로의 강화를 위해 aspB 유전자의 프로모터 서열의 특정 위치 (-22, -45, 또는 -88 영역)가 최적의 염기서열로 치환됨으로써 모균주 코리네박테리움 글루타미쿰 DS1 균주에 비해 L-라이신의 생산성이 각각 약 3.7%, 7.7%, 16.9% 증가하였으며, 특히 DS7 균주는 다른 변이주들에 비해 생산성이 가장 높은 것으로 확인되었다. 이러한 결과를 통해, aspB 유전자의 발현 강화가 라이신 전구체의 공급을 강화함으로써 균주의 L-라이신 생산능을 향상시킨다는 것을 알 수 있었다.As shown in Table 3 above, Corynebacterium glutamicum mutants DS7-2, DS7-1 and DS7 strains are specific positions (-22, -45, or -88 region) was replaced with the optimal nucleotide sequence, and the productivity of L-lysine was increased by about 3.7%, 7.7%, and 16.9%, respectively, compared to the parent strain Corynebacterium glutamicum DS1 strain. was found to have the highest productivity compared to Through these results, it was found that enhancing the expression of the aspB gene enhances the L-lysine production ability of the strain by enhancing the supply of lysine precursors.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.
<110> DAESANG CORPORATION <120> Mutant of Corynebacterium glutamicum with enhanced L-lysine productivity and method for preparing L-lysine using the same <130> PN210024P1 <150> KR 10-2021-0055536 <151> 2021-04-29 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> promoter sequence of aspB gene <400> 1 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgtctagag tagtggcttg 60 aggtcactgc tctttttttg tgcccttttt tggtccgtct attttgccac cacatgcgga 120 ggtacgcagt tatg 134 <210> 2 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> mutant promoter sequence of aspB gene <400> 2 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgtctagag tagtggcttg 60 aggtcactgc tctttttttg tgcccttttt tggtccgtct attttgccag cacatgcgga 120 ggtacgcagt tatg 134 <210> 3 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> mutant promoter sequence of aspB gene <400> 3 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgtctagag tagtggcttg 60 aggtcactgc tctttttttg tgccctattt tggtccgtct attttgccac cacatgcgga 120 ggtacgcagt tatg 134 <210> 4 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> mutant promoter sequence of aspB gene <400> 4 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgactagag tagtggcttg 60 aggtcactgc tctttttttg tgcccttttt tggtccgtct attttgccac cacatgcgga 120 ggtacgcagt tatg 134 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DS7-F <400> 5 cgagaattac cggcgatg 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DS7-R <400> 6 tcaccgtgaa accagtcg 18 <110> DAESANG CORPORATION <120> Mutant of Corynebacterium glutamicum with enhanced L-lysine productivity and method for preparing L-lysine using the same <130> PN210024P1 <150> KR 10-2021-0055536 <151> 2021-04-29 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> promoter sequence of aspB gene <400> 1 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgtctagag tagtggcttg 60 aggtcactgc tctttttttg tgcccttttt tggtccgtct attttgccac cacatgcgga 120 ggtacgcagt tatg 134 <210> 2 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> mutant promoter sequence of aspB gene <400> 2 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgtctagag tagtggcttg 60 aggtcactgc tctttttttg tgcccttttt tggtccgtct attttgccag cacatgcgga 120 ggtacgcagt tatg 134 <210> 3 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> mutant promoter sequence of aspB gene <400> 3 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgtctagag tagtggcttg 60 aggtcactgc tctttttttg tgccctattt tggtccgtct attttgccac cacatgcgga 120 ggtacgcagt tatg 134 <210> 4 <211> 134 <212> DNA <213> Artificial Sequence <220> <223> mutant promoter sequence of aspB gene <400> 4 agggtcagcg agctctgacg ggtgcgcggg atcccctaaa acgactagag tagtggcttg 60 aggtcactgc tctttttttg tgcccttttt tggtccgtct attttgccac cacatgcgga 120 ggtacgcagt tatg 134 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DS7-F <400> 5 cgagaattac cggcgatg 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DS7-R <400> 6 tcaccgtgaa accagtcg 18
Claims (5)
Corynebacterium glutamicum mutant with enhanced L-lysine production ability by enhancing the activity of aspartate aminotransferase.
상기 아스파르테이트 아미노트랜스퍼라제의 활성 강화는 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자의 프로모터에 위치 특이적 변이를 유발하는 것인 코리네박테리움 글루타미쿰 변이주.
The method according to claim 1,
The enhancement of the activity of the aspartate aminotransferase is a Corynebacterium glutamicum mutant that induces a site-specific mutation in the promoter of the gene encoding the aspartate aminotransferase.
상기 아스파르테이트 아미노트랜스퍼라제를 암호화하는 유전자는 서열번호 1의 염기서열로 표시되는 것인 코리네박테리움 글루타미쿰 변이주.
3. The method according to claim 2,
The gene encoding the aspartate aminotransferase is a Corynebacterium glutamicum mutant represented by the nucleotide sequence of SEQ ID NO: 1.
상기 변이주는 서열번호 2 내지 4로 표시되는 염기서열 중 어느 하나를 포함하는 것인 코리네박테리움 글루타미쿰 변이주.
The method according to claim 1,
The mutant Corynebacterium glutamicum mutant comprising any one of the nucleotide sequences shown in SEQ ID NOs: 2 to 4.
b) 상기 변이주 또는 변이주가 배양된 배지로부터 L-라이신을 회수하는 단계를 포함하는 L-라이신의 생산 방법.a) culturing the mutant of claim 1 in a medium; and
b) a method for producing L-lysine comprising the step of recovering L-lysine from the culture medium of the mutant or mutant strain.
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| PCT/KR2021/006499 WO2022231054A1 (en) | 2021-04-29 | 2021-05-25 | Corynebacterium glutamicum variant having improved l-lysine production ability, and method for producing l-lysine using same |
| EP21939436.8A EP4332229A1 (en) | 2021-04-29 | 2021-05-25 | Corynebacterium glutamicum variant having improved l-lysine production ability, and method for producing l-lysine using same |
| BR112023022523A BR112023022523A2 (en) | 2021-04-29 | 2021-05-25 | PROMOTER, CORYNEBACTERIUM GLUTAMICUM VARIANT AND METHOD FOR L-LYSINE PRODUCTION |
| AU2021442398A AU2021442398A1 (en) | 2021-04-29 | 2021-05-25 | Corynebacterium glutamicum variant having improved l-lysine production ability, and method for producing l-lysine using same |
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| KR20200026881A (en) | 2017-06-07 | 2020-03-11 | 지머젠 인코포레이티드 | Uses thereof to modulate promoter and accessory gene expression from Corynebacterium glutamicum |
| KR102139806B1 (en) | 2020-02-13 | 2020-07-30 | 씨제이제일제당 (주) | Microorganism Comprising Mutated LysE and Method of L-Amino Acid Production Using the Same |
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| KR20200026881A (en) | 2017-06-07 | 2020-03-11 | 지머젠 인코포레이티드 | Uses thereof to modulate promoter and accessory gene expression from Corynebacterium glutamicum |
| KR102139806B1 (en) | 2020-02-13 | 2020-07-30 | 씨제이제일제당 (주) | Microorganism Comprising Mutated LysE and Method of L-Amino Acid Production Using the Same |
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