KR100426808B1 - L-threonine producing microorganism and method for producing L-threonine using the same microorganism - Google Patents
L-threonine producing microorganism and method for producing L-threonine using the same microorganism Download PDFInfo
- Publication number
- KR100426808B1 KR100426808B1 KR10-2001-0034560A KR20010034560A KR100426808B1 KR 100426808 B1 KR100426808 B1 KR 100426808B1 KR 20010034560 A KR20010034560 A KR 20010034560A KR 100426808 B1 KR100426808 B1 KR 100426808B1
- Authority
- KR
- South Korea
- Prior art keywords
- threonine
- ppc
- coli
- producing
- pykf
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
본 발명은 L-쓰레오닌 생산 미생물 및 이를 이용한 L-쓰레오닌 제조방법에 관한 것이다. 피루베이트 카이네이즈 이소자임(pyruvate kinase isozyme)을 인코딩하는 pykA 또는 pykF 유전자 발현활성이 억제된 본 발명의 E. coli ABA/ppc-pykF 12 (KCCM-10268)를 이용하면 종래의 방법에 비하여 L-쓰레오닌을 보다 높은 수율로 생산할 수 있으며, 이렇게 제조된 L-쓰레오닌은 사료 및 식품 첨가제와 의약용으로 수액제, 의약품의 합성 원료로 널리 사용될 것이다.The present invention relates to an L-threonine producing microorganism and a method for producing L-threonine using the same. L.-L compared to the conventional method using the E. coli ABA / ppc-pykF 12 (KCCM-10268) of the present invention, which inhibits the expression of pykA or pykF gene, which encodes pyruvate kinase isozyme. It is possible to produce leonin in higher yields, and thus L-threonine will be widely used as a feedstock for food and food additives and as a synthetic raw material for fluids and medicines.
Description
본 발명은 L-쓰레오닌 생산 미생물 및 이를 이용한 L-쓰레오닌 제조방법에 관한 것이다. 보다 상세하게는 2개의 포스포에놀 피루베이트 카르복실레이즈(phosphoenol pyruvate carboxylase; ppc) 코딩 유전자를 보유하여 포스포에놀 피루베이트(PEP)로부터 쓰레오닌 생합성의 전구체인 옥살로아세테이트(oxaloacetate)로 전환해주는 효소인 ppc 발현량이 증대되고, 포도당 해당경로 효소인 피루베이트 카이네이즈(pyruvate kinase) 효소를 인코딩하는 pykA 또는 pykF 유전자의 발현활성을 억제시켜 피루베이트 카이네이즈에 의한 포스포에놀 피루베이트(phosphoenol pyruvate; PEP)로부터 피루베이트로의 전환속도를 감소시킴으로서 균체내에 쓰레오닌 대사의 전구체인 PEP를 축적시켜 L-쓰레오닌의 생산이 향상된 것을 특징으로 하는 E. coli ABA/ppc-pykF 12와 이를 배양하여 L-쓰레오닌을 생산하는 방법에 관한 것이다.The present invention relates to an L-threonine producing microorganism and a method for producing L-threonine using the same. More specifically, oxaloacetate, a precursor of threonine biosynthesis from phosphoenol pyruvate (PEP), possessing two phosphoenol pyruvate carboxylase (ppc) coding genes Increasing the amount of ppc, an enzyme that converts to, and inhibiting the expression activity of the pykA or pykF gene, which encodes the glucose glycolytic pathway enzyme, pyruvate kinase, phosphoenol pyruvate (phosphoenol) by pyruvate kinase E. coli ABA / ppc-pykF 12 and E. coli, characterized by an increase in the production of L-threonine by accumulating PEP, a precursor of threonine metabolism, in the cells by reducing the conversion rate from pyruvate (PEP) to pyruvate. It relates to a method for producing L-threonine by culturing it.
L-쓰레오닌은 필수 아미노산의 일종으로 사료 및 식품 첨가제로 널리 사용되며 의약용으로 수액제, 의약품의 합성 원료로도 사용된다.L-Threonine is an essential amino acid, widely used in feed and food additives, and as a synthetic raw material for fluids and pharmaceuticals.
L-쓰레오닌은 발효법으로 제조하는데 대장균, 코리네형 세균, 세라티아속 세균, 프로덴시아속 균주의 야생주로부터 유도된 인공변이주를 사용하고 있다. 이러한 변이 균주들로는 아미노산 유사체 및 약제 변이 내성주 또는 이들 내성주에 디아미노피메릭산, 메티오닌, 라이신, 이소루이신 영양요구성을 부여한 인공변이주가 공지되어 있다 (일본국 공개특허출원 제평2-219582호, Appl. Microbiol. Biotechnol., 29. 550-553(1988), 한국특허공고 제92-8365호).L-threonine is produced by fermentation method using artificial mutants derived from wild strains of E. coli, Coryneform bacteria, Serratia bacteria, Prodencia strains. Such mutant strains are known amino acid analogs and drug mutant strains or artificial mutants that give these amino acids diaminopimeric acid, methionine, lysine, and isoleucine nutritional composition (Japanese Patent Application Laid-Open No. 2-219582). , Appl. Microbiol.Biotechnol., 29.550-553 (1988), Korean Patent Publication No. 92-8365).
대장균에서 포도당 해당경로의 중간물질중 하나인 포스포에놀 피루베이트(PEP)는 쓰레오닌의 전구체이며, 대장균의 대사 경로 중에서 포스포에놀 피루베이트(PEP)의 주된 사용경로는 PEP:글루코오스 포스포트랜스퍼라제 시스템(glucose phosphotransferase system; PTS)과 피루베이트 카이네이즈 이소자임(pyruvate kinase isozyme)들이다. pykA 유전자와 pykF 유전자에 의해 인코딩되는 피루베이트 카이네이즈 이소자임(pyruvate kinase isozyme)들은 1분자의 포스포에놀 피루베이트(PEP)를 피루베이트로 전환시키면서 1 분자의 ATP를 생성시킨다.Phosphoenol pyruvate (PEP), an intermediate in glucose glycolysis in E. coli, is a precursor to threonine, and the main route of use of phosphoenol pyruvate (PEP) in E. coli metabolic pathways is PEP: glucose. Glucose phosphotransferase system (PTS) and pyruvate kinase isozymes. The pyruvate kinase isozymes encoded by the pykA and pykF genes produce one molecule of ATP by converting one molecule of phosphoenol pyruvate (PEP) into pyruvate.
또한, 포도당 해당경로의 중간물질중 하나인 포스포에놀 피루베이트(PEP)는 쓰레오닌의 전구체인 동시에 트립토판을 포함하는 방향족 화합물의 전구체이다.In addition, phosphoenol pyruvate (PEP), one of the intermediates in glucose glycolysis, is a precursor of threonine and a precursor of aromatic compounds, including tryptophan.
트립토판 생산 균주에서 포스포에놀 피루베이트(PEP)를 트립토판으로 전환시키는 효소인 피루베이트 카이네이즈(pyruvate kinase)의 효소 활성을 약화시킴으로서 트립토판의 수율 향상을 시도한 보고가 있다 (참고문헌: Gosset et al, A direct comparison of approaches for increasing carbon flow to aromaticbisynthesis in Escherichia coli, Journal of industrial microbiology, 1996, 47-52). 하지만 이를 L-쓰레오닌 생산 수율 향상에 적용한 사례는 보고되고 있지 않다.There have been reports of attempts to improve the yield of tryptophan by attenuating the enzyme activity of pyruvate kinase, an enzyme that converts phosphoenol pyruvate (PEP) to tryptophan in tryptophan producing strains (Ref .: Gosset et al, A direct comparison of approaches for increasing carbon flow to aromaticbisynthesis in Escherichia coli, Journal of industrial microbiology, 1996, 47-52). However, there have been no reports of applying this to improving L-threonine production yield.
본 균주개발 방법에서는 위 참고문헌의 선행기술의 균주개발 방법과 비교해 볼 때 유사한 점은 피루베이트 카이네이즈(pyruvate kinase) 효소 활성을 약화시킴으로 PEP 축적을 이루었다는 점이지만, 최종 목표물질이 L-쓰레오닌과 트립토판으로 전혀 다르며, 본 기술을 적용한 균주인 당 연구소 균주의 경우는 쓰레오닌 생합성 관련 효소들이 상당히 강화되어 있고 선행기술에 사용된 모균주는 트립토판을 포함한 방향족화합물 생합성 관련 효소들이 상당히 강화 되어 있어 피루베이트 카이네이즈(pyruvate kinase) 효소 활성을 약화 시켰을 때 전자는 쓰레오닌의 생산이 현저히 증가된 반면 후자는 트립토판의 수율 향상을 일부 보고하였다.In this strain development method, compared with the strain development method of the prior art, the similarity is that PEP accumulation is achieved by attenuating pyruvate kinase enzyme activity, but the final target material is L-Threo It is completely different from nin and tryptophan, and our laboratory strain, which is the strain to which the present technology is applied, has significantly enhanced the enzymes related to threonine biosynthesis, and the parent strain used in the prior art has significantly enhanced the enzymes related to aromatic compound biosynthesis including tryptophan. The former reported a significant increase in threonine production while the latter reported some improvement in tryptophan yield when attenuated pyruvate kinase enzyme activity.
따라서 본 발명에서는 이 피루베이트 카이네이즈 이소자임(pyruvate kinase isozyme)들의 발현활성을 억제시켜 L-쓰레오닌의 전구체로 사용되는 PEP를 축적함으로써 L-쓰레오닌의 수율 향상을 기대할 수 있다.Therefore, the present invention can be expected to improve the yield of L-threonine by inhibiting the expression activity of the pyruvate kinase isozymes to accumulate PEP used as a precursor of L-threonine.
본 발명의 목적은 2개의 포스포에놀 피루베이트 카르복실레이즈(phosphoenol pyruvate carboxylase; ppc) 코딩 유전자를 보유한 L-쓰레오닌 생산 미생물로부터 pykA 또는 pykF 유전자의 발현활성이 억제되어 균체내에 PEP가 보다 많이 축적됨으로서 L-쓰레오닌의 생산성이 증대된 E. coli ABA/ppc-pykF 12(KCCM-10268)과 이를 배양하여 L-쓰레오닌을 고수율로 생산하는 방법을 제공하는 데 있다.An object of the present invention is to inhibit the expression of the pykA or pykF gene from L-threonine producing microorganisms having two phosphoenol pyruvate carboxylase (ppc) coding genes, thereby inhibiting PEP in cells. By accumulating a lot of E. coli ABA / ppc-pykF 12 (KCCM-10268) which has increased the productivity of L- threonine and provides a method for producing L- threonine in high yield.
본 발명은 L-쓰레오닌 생산 미생물 및 이를 이용한 L-쓰레오닌 제조방법에 관한 것이다.The present invention relates to an L-threonine producing microorganism and a method for producing L-threonine using the same.
본 발명에서 사용한 L-쓰레오닌의 생산 균주는 E. coli ABA/ppc-pykF 12로서, E. coli TF4076 (KFCC10718, 대한민국 특허출원 제90-22965)에서 유래한 것으로, E. coli TF4076 (KFCC10718, 대한민국 특허출원 제90-22965)의 염색체로부터 폴리머레이즈 체인 반응(polymerase chain reaction: 이하 PCR)을 통해 얻은 포스포에놀 파이루베이트 카르복실레이즈 유전자 (phosphoenol pyruvate carboxylase gene: 이하 ppc 유전자)를 다시 모균주인 E. coli TF4076의 염색체에 삽입시킴으로써 염색체 DNA 중에 ppc 유전자 수를 2개로 늘림으로써 PEP로부터 쓰레오닌 생합성의 전구체인 옥살로아세테이트(oxaloacetate)로 전환해주는 효소인 ppc 유전자의 발현량을 증가시킴으로써 L-쓰레오닌의 생산량을 향상시킨 E. coli pGm-PPC 16 (KFCC-11229, 대한민국 특허출원 제2001-2373호)를 개발하였으며, 다시 E. coli pGm-PPC 16 (KFCC-11229) 균주에서 포도당의 해당경로 중의 피루베이트 카이네이즈 이소자임(pyruvate kinase isozyme)을 인코딩하는 pykA 혹은 pykF 유전자를 무력화시켜 PEP로부터 피루베이트로 전환되는 속도를 줄임으로써 균체내에 쓰레오닌 대사의 전구체인 PEP를 축적시키고 강화된 포스포에놀 피루베이트 카르복실레이즈(phosphoenol pyruvate carboxylase, ppc)의 작용으로 L-쓰레오닌의 생산량이 향상된 E. coli ABA/ppc-pykF 12 균주이다.The production strain of L-threonine used in the present invention is E. coli ABA / ppc-pykF 12, derived from E. coli TF4076 (KFCC10718, Korean Patent Application No. 90-22965), E. coli TF4076 (KFCC10718 , Phosphoenol pyruvate carboxylase gene (ppc gene) obtained through a polymerase chain reaction (PCR) from the chromosome of Korea Patent Application No. 90-22965) By inserting into the chromosome of the parental strain E. coli TF4076, the number of ppc genes in the chromosomal DNA is increased to two to increase the expression level of the ppc gene, an enzyme that converts PEP into oxaloacetate, a precursor of threonine biosynthesis. E. coli pGm-PPC 16 (KFCC-11229, Republic of Korea Patent Application No. 2001-2373) to improve the production of L- threonine by the development of E. coli pGm-PPC 16 (KFCC-11229) strain In Accumulate and enhance PEP, a precursor of threonine metabolism in the cells by reducing the rate of conversion from PEP to pyruvate by neutralizing the pykA or pykF gene encoding pyruvate kinase isozyme in the glucose pathway. It is a strain of E. coli ABA / ppc-pykF 12, in which the production of L-threonine is improved by the action of phosphoenol pyruvate carboxylase (ppc).
기출원된 당 연구소의 L-쓰레오닌 생산균주인 E. coli TF4076 (KFCC10718,대한민국 특허출원 제90-22965)는 메티오닌 요구성, 쓰레오닌 유사체(AHV: α-아미노-β-하이드록시 발레릭산)에 대한 내성, 라이신 유사체(AEC: S-(2-아미노에틸)-L-시스테인)에 대한 내성, 이소루이신 유사체(α-아미노부티릭에시드) 대한 내성, 메티오닌의 유사체(에티오닌)에 대한 내성 등의 특성을 가지고 있다.E. coli TF4076 (KFCC10718, Republic of Korea Patent Application No. 90-22965), an L-threonine producing strain from the Institute, is a methionine-required, threonine analogue (AHV: α-amino-β-hydroxy valerian Resistance to lysine), resistance to lysine analogs (AEC: S- (2-aminoethyl) -L-cysteine), resistance to isoleucine analogs (α-aminobutyric acid), analogs of methionine (ethionine Resistance to).
따라서, 본 발명의 상기 L-쓰레오닌의 생산균주인 E. coli ABA/ppc-pykF 12를 2001년 5월 9 일자로 서울 서대문구 홍제동 소재 사단법인 한국종균협회에 기탁번호 제 KCCM-10268호로 기탁하였다.Therefore, E. coli ABA / ppc-pykF 12, the production strain of L-threonine of the present invention, was deposited with No. KCCM-10268 to the Korean spawn association of Hong Kong-dong, Seodaemun-gu, Seoul on May 9, 2001. It was.
하기 실시예를 통하여 본 발명의 미생물분리 및 획득방법을 좀더 구체적으로 기술한다.Through the following examples will be described in more detail the microbial separation and acquisition method of the present invention.
실시예 1: 피루베이트 카이네이즈 이소자임의 발현활성이 억제된 균주 개발Example 1 Development of Strains with Inhibitory Expression of Pyruvate Kinase Isozyme
피루베이트 카이네이즈 이소자임의 발현활성이 억제된 균주를 개발하기위하여 피루베이트 카이네이즈 이소자임(pyruvate kinase isozyme)의 발현활성을 억제시키 방법은 위의 참고문헌 1 (Gosset et al, A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli, Journal of industrial microbiology , 1996, 47-52)의 방법을 응용하였고, 또 다른 참고문헌 2 (Ponce et al, Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli : the relative roles of these enzymes in pyruvate biosynthesis, Journal of Bacteriology, 1995, 5719-22)의 균주를 이용하였다.In order to develop a strain in which pyruvate kinase isozyme is inhibited, a method of inhibiting the expression activity of pyruvate kinase isozyme is described in Gosset et al, A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli, Journal of industrial microbiology, 1996, 47-52, and another reference 2 (Ponce et al, Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis, Journal of Bacteriology, 1995, 5719-22).
전체적으로는 박테리오파지 P1-매개 형질도입법 (bacteriophage P1-mediatedtransduction)을 이용하였다. 좀더 상세하게 설명하면 자연형 대장균 W3110을 4개의 삼각 플라스크에 신선배지 25 mL씩을 넣고 배양하면서 박테리오파지 P1 분해물(lysate)를 각각 100 ㎕, 50 ㎕, 10 ㎕, 0 ㎕ 넣고 250 rpm으로 3-4시간 배양한 다음 세포의 분해(lysis)를 확인하였고, 적절한 용해수준에 도달하였을 때 용액 1 mL 을 취한 다음 클로로포름 50 ㎕을 넣고 1분간 흔들어주면서 잘 섞은 다음 12000 rpm에서 2분간 원심분리한 후 상층액 800 ㎕ 을 취하였다. 이렇게 얻은 박테리오파지 P1 분해물로 참고문헌 2 (Ponce et al, Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli : the relative roles of these enzymes in pyruvate biosynthesis, Journal of Bacteriology, 1995, 5719-22)의 균주 E. coli PB25에 대하여 위와 동일한 방법으로 박테리오파지 P1-매개 형질도입법을 수행하여 박테리오파지 P1 분해물(pPB25)을 얻었다.Bacteriophage P1-mediated transduction was used as a whole. In more detail, incubate 25 mL of fresh medium into four Erlenmeyer flasks and incubate 100 μl, 50 μl, 10 μl, 0 μl of bacteriophage P1 lysate, and incubate 3-4 hours at 250 rpm. After culturing, the cells were analyzed for lysis. When the appropriate lysis level was reached, 1 mL of the solution was taken, 50 μl of chloroform was added thereto, shaken for 1 minute, mixed well, centrifuged at 12000 rpm for 2 minutes, and the supernatant 800 Μl was taken. The bacteriophage P1 digest thus obtained was strain E of Reference 2 (Ponce et al, Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis, Journal of Bacteriology, 1995, 5719-22). Bacteriophage P1-mediated transduction was performed on coli PB25 in the same manner to obtain bacteriophage P1 digest (pPB25).
L-쓰레오닌 생산균주인 pGm-PPC 16 (KFCC-11229)을 밤새 배양한 배양액 5 mL을 5000rpm에서 5 분간 원심분리하여 회수한 후 상층액을 버리고 펠릿(pellet)을 MC buffer (0.1 M MgSO4, 5 mM CaCl2) 2.5 mL로 재현탁시켰다. 그 후 위에서 얻은 박테리오파지 P1 분해물(pPB25)을 이용하여 표 1과 같은 조건으로 박테리오파지 P1-매개 형질도입을 수행하였다.5 mL of the culture medium cultured with L-threonine-producing strain pGm-PPC 16 (KFCC-11229) overnight was collected by centrifugation at 5000 rpm for 5 minutes, then the supernatant was discarded and the pellet was transferred to MC buffer (0.1 M MgSO). 4 , 5 mM CaCl 2 ) was resuspended in 2.5 mL. Thereafter, bacteriophage P1-mediated transduction was performed using the bacteriophage P1 digest (pPB25) obtained above under the conditions shown in Table 1.
이후 30 ℃ 에서 교반하지 않고 30분간 배양한 후 1 M 소듐 사이트레이트(sodium citrate)를 100 ㎕ 넣고 잘 섞은 후, 1 mL의 LB 배지를 각 튜브에 넣고 잘 섞어 준 다음 30 ℃ 에서 교반하지 않고 60분간 배양한 후 12000 rpm에서 2분간 원심분리하였다. 이어 상층액 일부분을 버린 후 남은 300 ㎕을 다시 재현탁시킨 다음 적절한 항생제가 들어있는 고체 아가배지에 50 ㎕, 250 ㎕씩 도말한 다음 30 ℃ 배양기에서 밤새 배양하였다.After incubation for 30 minutes without stirring at 30 ℃ 100 ml of 1 M sodium citrate and mix well, 1 mL of LB medium in each tube and mix well and then stirred at 60 ℃ without stirring After culturing for 2 minutes, the mixture was centrifuged at 12000 rpm for 2 minutes. Subsequently, after discarding a portion of the supernatant, the remaining 300 μl was resuspended, and then 50 μl and 250 μl were plated in a solid agar medium containing appropriate antibiotics, and then incubated overnight at 30 ° C. incubator.
생성된 콜로니중에서 pykA 발현이 억제된 돌연변이주는 카나마이신 고체배지(kanamycin plate)에서 pykF 발현이 억제된 돌연변이주는 클로람페니콜 고체배지(chloramphenicol plate)에서 선별하였다.Mutant strains with suppressed pykA expression in the resulting colonies were selected from chloramphenicol plate with mutant strains with pykF expression suppressed in kanamycin plate.
실시예 2: 선별 군주에 대한 삼각 플라스크에서 쓰레오닌 생산 역가 비교 실험Example 2: Comparison of Threonine Production Titers in Erlenmeyer Flasks to Selected Monarchs
카나마이신 고체배지에서 선별한 pykA 발현이 억제된 돌연변이주와 클로람페니콜 고체배지에서 선별한 pykF 발현이 억제된 돌연변이주를 각 15주씩 총 30주를 선별하여 표 2에 나타낸 쓰레오닌 역가배지를 사용하여 삼각 플라스크에서 쓰레오닌 생산성을 비교하였다.A total of 30 strains were selected for the mutant strains with suppressed pykA expression in kanamycin solid medium and the mutant strains with suppressed pykF expression in chloramphenicol solid medium, each of which was selected for triangulation using the threonine titer shown in Table 2. Threonine productivity in the flasks was compared.
32 ℃의 배양기에서 LB 고체 배지중에 밤새 배양한 단일 콜로니들을 25 ml의 역가배지에 1루프씩 접종하여 32 ℃에서 250 rpm으로 48시간 동안 배양하였고 이의 분석 결과를 표 3에 나타내었다. 대조균주인 E. coli TF4076의 역가는 20.0 g/L인 반면 선별한 30주 모두 대조 균주에 비해 우수한 역가를 나타내었으며 특히 25.0 g/L 이상의 역가를 보인 균주도 4주나 되었으며 가장 높은 성적을 보인 균주의 경우 25.5 g/L 의 쓰레오닌을 생산함으로써 수율이 모균주 대비 약 27.5 % 정도 향상되었음을 관찰하고 ABA/ppc-pykF 12 (KCCM-10268)로 명명하였다.Single colonies cultured overnight in LB solid medium in a 32 ° C. incubator were inoculated in a loop of 25 ml titer medium and incubated for 48 hours at 32 ° C. at 250 rpm. The results of the analysis are shown in Table 3. The titer of the control strain E. coli TF4076 was 20.0 g / L, whereas all 30 strains showed superior titers compared to the control strains. Especially, strains showing a titer of 25.0 g / L or more were 4 weeks and showed the highest results. In the case of 25.5 g / L of threonine production yield was observed about 27.5% improvement compared to the parent strain and named as ABA / ppc-pykF 12 (KCCM-10268).
실시예 3: 발효조를 이용한 쓰레오닌 비교시험Example 3: Comparison of threonine using fermenter
실시예 2에서 선별된 우수 균주 E. coli ABA/ppc-pykF 12 (KCCM-10268)와 E. coli TF4076 대조균주를 사용하여 5 L 발효조에서 쓰레오닌 생산성을 비교하여 보았다. 초기 배지 조성은 표 4에 나타내었다. 종균 배양은 LB 배지에 포도당 10 g/L와 L-메티오닌을 0.1 g/L 되게 첨가해 준 배지를 사용하였고 발효조의 초기 접종 부피는 초기 배양 부피의 3-5 %에서 조정하였다. 추가당은 6회에 걸쳐 첨가하였으며 추가한 후의 포도당 농도가 5 % 되게 하였으며 추가시점은 포도당이 고갈된 시점이다. 또한 포도당을 추가할 때 제일인산칼륨(KH2PO4)을 중량기준으로 1 % 되게 같이 첨가해주었다. 초기 배양 부피는 1.5 L이고 최종 부피는 3.0 L이며 발효 종료 후 투입된 총 포도당의 농도는 250 g/L이다. 교반 속도는 700-1000 rpm으로 해주고 pH와 온도는 각각 7.0과 32 ℃로 맞추어 주었다. 배양 중 pH의 조절은 25 내지 28 %의 암모니아수를 사용하였다. 또한 통기량은 0.5 vvm으로 조정하였다. 실험 결과를 표 5에 나타내었다. 대조 균주는 75.3 g/L의 쓰레오닌을 생산하여 소비한 포도당에 대해 30.1 %의 수율을 보인 반면, 신개발 재조합 균주 ABA/ppc-pykF 12 (KCCM-10268)는 98.5 g/L의 쓰레오닌을 생산하여 39.4 %의 수율을 보임으로써 농도와 수율 면에서 대조 균주에 비해 30.8 %의 월등한 성적 향상을 보여주었다. 또한 신개발 재조합 균주에 흔히 나타나는 생육저해 현상에 따른 발효시간 당소모 속도 지연 현상도 전혀 나타나지 않고 대조 균주와 유사한 양상을 보였다.The superior strain E. coli ABA / ppc-pykF 12 (KCCM-10268) and E. coli TF4076 control strains selected in Example 2 were used to compare threonine productivity in a 5 L fermenter. Initial medium compositions are shown in Table 4. The seed culture was a medium in which 10 g / L of glucose and 0.1 g / L of L-methionine were added to the LB medium, and the initial inoculation volume of the fermenter was adjusted at 3-5% of the initial culture volume. Additional sugars were added six times and the glucose concentration was 5% after the addition. The additional time point was when glucose was depleted. In addition, when adding glucose, potassium phosphate (KH 2 PO 4 ) was added together to 1% by weight. The initial culture volume is 1.5 L, the final volume is 3.0 L and the concentration of total glucose added after completion of fermentation is 250 g / L. Stirring speed was 700-1000 rpm and pH and temperature were set at 7.0 and 32 ° C, respectively. The pH of the culture was adjusted to 25 to 28% ammonia water. In addition, the air flow rate was adjusted to 0.5 vvm. The experimental results are shown in Table 5. The control strain produced 35.3% yield on glucose consumed by producing 75.3 g / L of threonine, while the newly developed recombinant strain ABA / ppc-pykF 12 (KCCM-10268) yielded 98.5 g / L of threonine. The yield of 39.4% yielded an excellent performance improvement of 30.8% compared to the control strain in terms of concentration and yield. In addition, there was no delay in fermentation time and sugar consumption due to growth inhibition which is common in newly developed recombinant strains.
본 발명은 L-쓰레오닌 생산 미생물 및 이를 이용한 L-쓰레오닌 제조방법에 관한 것이다. 본 발명의 피루베이트 카이네이즈 이소자임(pyruvate kinase isozyme)을 인코딩한 pykA 또는 pykF 유전자 발현활성을 억제된 본 발명의 E. coli ABA/ppc-pykF 12(KCCM-10268)를 이용하면 종래의 방법에 비하여 L-쓰레오닌을 보다 높은 수율로 생산할 수 있으며, 이렇게 제조된 L-쓰레오닌은 사료 및 식품 첨가제와 의약용으로 수액제, 의약품의 합성 원료로 널리 사용될 것이다.The present invention relates to an L-threonine producing microorganism and a method for producing L-threonine using the same. Compared to the conventional method using the E. coli ABA / ppc-pykF 12 (KCCM-10268) of the present invention suppressed pykA or pykF gene expression activity encoding the pyruvate kinase isozyme of the present invention. L-Threonine can be produced in higher yield, and thus prepared L-Threonine will be widely used as a synthetic raw material for infusions and medicines for feed and food additives and medicine.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0034560A KR100426808B1 (en) | 2001-06-19 | 2001-06-19 | L-threonine producing microorganism and method for producing L-threonine using the same microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0034560A KR100426808B1 (en) | 2001-06-19 | 2001-06-19 | L-threonine producing microorganism and method for producing L-threonine using the same microorganism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20020096233A KR20020096233A (en) | 2002-12-31 |
| KR100426808B1 true KR100426808B1 (en) | 2004-04-13 |
Family
ID=27709821
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-2001-0034560A KR100426808B1 (en) | 2001-06-19 | 2001-06-19 | L-threonine producing microorganism and method for producing L-threonine using the same microorganism |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100426808B1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5931691A (en) * | 1982-08-16 | 1984-02-20 | Ajinomoto Co Inc | Preparation of l-threonine by fermentation |
| US5939307A (en) * | 1996-07-30 | 1999-08-17 | The Archer-Daniels-Midland Company | Strains of Escherichia coli, methods of preparing the same and use thereof in fermentation processes for l-threonine production |
| KR20020061348A (en) * | 2001-01-16 | 2002-07-24 | 제일제당주식회사 | Process for producing l-threonine |
| WO2002064808A1 (en) * | 2001-02-13 | 2002-08-22 | Cheil Jedang Corporation | Method for l-threonine production |
-
2001
- 2001-06-19 KR KR10-2001-0034560A patent/KR100426808B1/en not_active IP Right Cessation
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5931691A (en) * | 1982-08-16 | 1984-02-20 | Ajinomoto Co Inc | Preparation of l-threonine by fermentation |
| US5939307A (en) * | 1996-07-30 | 1999-08-17 | The Archer-Daniels-Midland Company | Strains of Escherichia coli, methods of preparing the same and use thereof in fermentation processes for l-threonine production |
| KR20020061348A (en) * | 2001-01-16 | 2002-07-24 | 제일제당주식회사 | Process for producing l-threonine |
| WO2002064808A1 (en) * | 2001-02-13 | 2002-08-22 | Cheil Jedang Corporation | Method for l-threonine production |
Non-Patent Citations (2)
| Title |
|---|
| J Bacteriol. 1995 Oct;177(19):5719-22 * |
| J Ind Microbiol. 1996 Jul;17(1):47-52 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20020096233A (en) | 2002-12-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100397423B1 (en) | Process for producing L-threonine | |
| US8048650B2 (en) | Microorganism of Corynebacterium genus having enhanced L-lysine productivity and a method of producing L-lysine using the same | |
| EP2115135B1 (en) | A microorganism of corynebacterium genus having enhanced l-lysine productivity and a method of producing l-lysine using the same | |
| US7229794B2 (en) | Microorganism producing L-threonine having inactivated galR gene, method of producing the same and method of producing L-threonine using the microorganism | |
| US20090093030A1 (en) | fadR KNOCK-OUT MICROORGANISM AND METHODS FOR PRODUCING L-THREONINE | |
| KR20000013852A (en) | Preparation method of l-threonine from e.coli dsm 9806(kccm-10133) | |
| Komatsubara et al. | Transductional construction of a threonine-producing strain of Serratia marcescens | |
| KR100426808B1 (en) | L-threonine producing microorganism and method for producing L-threonine using the same microorganism | |
| KR100426807B1 (en) | L-threonine producing microorganism and method for producing L-threonine using the same microorganism | |
| Kase et al. | L-Isoleucine production by analog-resistant mutants derived from threonine-producing strain of Corynebacterium glutamicum | |
| CN101072865A (en) | A microorganism producing l-threonine having an inactivated lysr gene, method for producing the same and method for producing l-threonine using the microorganism | |
| SK16692000A3 (en) | Process for the fermentative preparation of l-lysine by using coryneform bacteria | |
| KR100427480B1 (en) | Process for producing L-threonine | |
| KR100427479B1 (en) | Process for producing L-threonine | |
| KR100608084B1 (en) | A microorganism having an inactivated iclR gene and capable of producing a L-threonine, method for manufacturing the same strain and method for producing L-threonine using the same strain | |
| US7378267B1 (en) | Method for L-threonine production | |
| US7368266B2 (en) | Method for L-threonine production | |
| KR100338373B1 (en) | L-threonine producing microorganism and methods for preparing L-threonine using the same | |
| KR100576344B1 (en) | A nucleic acid encoding glutamyl kinase variant released from feedback inhibition by proline a vector and a host cell containing the same and a method of producing L-threonine using the host cell | |
| KR20100038639A (en) | Microorganism comprising modified purc gene and process for production method of inosine using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20080103 Year of fee payment: 5 |
|
| LAPS | Lapse due to unpaid annual fee |