KR20130057600A - Method for preparing glutaric acid using recombinant microorganism - Google Patents

Method for preparing glutaric acid using recombinant microorganism Download PDF

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KR20130057600A
KR20130057600A KR1020110123396A KR20110123396A KR20130057600A KR 20130057600 A KR20130057600 A KR 20130057600A KR 1020110123396 A KR1020110123396 A KR 1020110123396A KR 20110123396 A KR20110123396 A KR 20110123396A KR 20130057600 A KR20130057600 A KR 20130057600A
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glutaric acid
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박시재
이승환
송봉근
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한국화학연구원
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Abstract

PURPOSE: A method for preparing glutaric acid from lysine using a recombinant microorganism is provided to ensure significant productivity of the glutaric acid from the lysine and to be used in technology for inexpensively producing a large amount of glutaric acid. CONSTITUTION: A recombinant microorganism for preparing glutaric acid comprises: a step of preparing an expression vector containing one or more genes among genes encoding lysine 2-monooxygenase(DavB), delta-aminovaleramidase(DavA), 5-aminovalerate aminotransferase(DavT), and glutarate semialdehyde dehydrogenase(DavD); a step of transforming one or more expression vectors into a microorganism and preparing a recombinant microorganism containing DavB, DavA, DavT, and DavD genes. The microorganism is E. coli. The expression vector includes: a recombinant vector which is prepared by inserting genes encoding DavB and DavA into an expression vector; and a recombinant vector which is prepared by inserting the genes encoding DavT and DavD into the expression vector.

Description

재조합 미생물을 이용한 글루타릭산의 제조 방법{Method for Preparing glutaric acid using recombinant microorganism}Method for preparing glutaric acid using recombinant microorganism

본 발명은 재조합 미생물을 이용한 라이신(lysine)으로부터 글루타릭산(glutaric acid)의 제조 방법에 관한 것이다.
The present invention relates to a method for preparing glutaric acid from lysine using recombinant microorganisms.

최근 전 세계적인 석유수급 불안과 석유자원 고갈에 대한 위기의식으로 최근에 산업 바이오 기술을 이용하여 바이오매스에서 유래한 대체 생산 방법이나 대체 화합물을 생산하기 위한 전 인류적인 노력이 바이오에너지, 바이오플라스틱, 바이오화합물 등의 다양한 분야에서 가시화되고 있다. 바이오매스를 활용하여 생산되는 바이오 플라스틱 시장의 경우 2002년 Natureworks사에 의해 상업화된 폴리유산 (Poly Lactic acid)이 연 14만 톤 규모로 생산되어 최근 시장이 급속히 확대되고 있다. PHA계 바이오플라스틱인 폴리-(3-하이드록시부틸레이트-코-3-하이드록시발레레이트{poly-(3-hydroxybutyrate-co-3-hydroxyvalarate)}(P(3HB-co-3HV))도 Metabolix와 ADM의 합작회사인 Telles에 의해 5만 톤 규모의 공장이 2010년 완공되어 제품이 시판되고 있다. 또한, 듀폰사가 생산하고 있는 바이오매스 기반의 1,3-프로판디올을 이용하여 PTT 고분자 제품이 현재 상용화되어 있다. 이외에도 숙신산 기반의 PBS 등도 활발히 개발되고 있다.
In recent years, in response to the global oil supply instability and the crisis of depletion of petroleum resources, all human efforts to produce alternative production methods or alternative compounds derived from biomass using industrial biotechnology have recently been developed. It is visualized in various fields, such as a compound. The bioplastics market, which utilizes biomass, is produced in 140,000 tonnes of polylactic acid, commercialized by Natureworks in 2002, and is expanding rapidly. Poly- (3-hydroxybutyrate-co-3-hydroxyvalarate)} (P (3HB-co-3HV)), a PHA-based bioplastic, is also metabolix. The product is being marketed by the Telles, a joint venture between ADM and ADM, with a production capacity of 50,000 tonnes completed in 2010. In addition, PTT polymer products are manufactured using biomass-based 1,3-propanediol produced by DuPont. In addition, SBS-based PBS is being actively developed.

나일론 4의 원료물질인 2-피롤리돈은 감마 부티로락톤(gamma butyrolactone)으로부터 생산될 수 있으며 4-아미노부틸산의 탈수 고리반응에 의해 생산될 수 있다. 4-아미노부틸산의 경우 감마-아미노부틸산(gamma-aminobutyric acid, GABA)이라고도 하는데, 체내에서는 신경전달물질 억제효과에 의한 저혈압 및 진통효과가 있다고 알려져, 현재 식품 및 의약품 소재로 활용되고 있다(Erlander et al., Neurochem Res. 16: 215-226, 1991). 이러한 GABA는 글루타메이트를 원료로 글루타메이트 디카르복실라아제(Glutamate decarboxylase)에 의해 제조될 수 있는데, 현재까지 다양한 글루타메이트 디카르복실라아제가 보고되어 있으며(Proc. Natl. Acad. Sci. USA, 87: 8491-8495, 1990; Protein Expression and Purification 8: 430-438, 1996; Biosci. Biotechnol. Biochem. 66(12): 2600-2605, 2002), 글루타메이트 디카르복실라아제를 이용하여 GABA를 생산하는 공정이 개발된바 있다. 나일론 4 이외에 최근에 나일론 5에 관한 관심 또한 높아지고 있다. 나일론 5의 모노머는 현재 라이신으로부터 유래된 다이아민인 카다베린(cadaverine)이 사용되고 있다.
2-pyrrolidone, a raw material of nylon 4, can be produced from gamma butyrolactone and can be produced by the dehydration ring reaction of 4-aminobutyl acid. 4-aminobutyric acid is also called gamma-aminobutyric acid (GABA), which is known to have hypotension and analgesic effects by neurotransmitter inhibitory effects, and is currently used as a food and pharmaceutical material ( Erlander et al., Neurochem Res. 16: 215-226, 1991). Such GABA can be prepared by glutamate decarboxylase based on glutamate, and various glutamate decarboxylases have been reported to date (Proc. Natl. Acad. Sci. USA, 87: 8491-8495, 1990; Protein Expression and Purification 8: 430-438, 1996; Biosci.Biotechnol.Biochem. 66 (12): 2600-2605, 2002), a process for producing GABA using glutamate decarboxylase This has been developed. In addition to nylon 4, interest in nylon 5 has also increased recently. The monomer of nylon 5 is currently used cadaverine, a diamine derived from lysine.

본 발명자들은 라이신(lysine)으로부터 유래될 수 있는 나일론 5의 또 다른 모노머로서 5-아미노발레르산(5-aminovaleric acid)을 생산하는 공정을 개발하기 위하여 연구한 결과, 대장균에 L-라이신(L-lysine)을 5-아미노발레르아미드(5-aminovaleramide)로 전환하는 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB) 효소와 5-아미노발레르아미드를 5-아미노발레르산(5-aminovaleric acid)으로 전환하는 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소를 증폭함으로써 라이신으로부터 5-아미노발레르산이 생산됨을 확인하였으므로, 상기 재조합 균주는 5-아미노발레르산을 저가로 대량 생산하는데 유용하게 사용할 수 있음을 밝힌바 있다. 또한, 대한민국 등록특허 10-0828625-0000에서는 재조합 대장균을 이용한 의학적 용도의 알파-케토-글루타메이트 및 만티톨의 제조방법이 기재되어 있고, Jouranl of Molecular Catalysis B: Enzymatic 65(2010) 58-21에서는 글루타릭산의 중간 물질인 5-아미노발레르산을 효과적으로 생산하는 방법이 기재되어 있으나, 재조합 미생물을 이용한 글루타릭산을 생산방법은 알려진바 없다.
The present inventors have studied to develop a process for producing 5-aminovaleric acid as another monomer of nylon 5 which may be derived from lysine, and thus L-lysine (L- lysine 2-monooxygenase (DabB) enzyme and 5-aminovaleramide to 5-aminovaleric acid to convert lysine to 5-aminovaleramide It was confirmed that 5-aminovaleric acid is produced from lysine by amplifying the delta-aminovaleramidase (DV-A) enzyme, which can be useful for mass production of low-cost 5-aminovaleric acid. It has been said. In addition, Korean Patent No. 10-0828625-0000 discloses a method for preparing alpha-keto-glutamate and mantitol for medical use using recombinant Escherichia coli, and described in Jouranl of Molecular Catalysis B: Enzymatic 65 (2010) 58-21. Although a method for effectively producing 5-amino valeric acid, which is an intermediate of rutaric acid, has been described, a method for producing glutaric acid using recombinant microorganisms is not known.

폴리머의 모노머로 사용될 수 있는 케미칼은 앞에 예를 든 것과 같이 C3의1,3-프로판디올, 말릭산, C4의 숙신산, 1,4-부탄디올, C5의 1,5-펜탄디올, 글루타릭산등의 다이올과 다이에시드이다.
Chemicals that can be used as monomers in polymers include C3 1,3-propanediol, malic acid, C4 succinic acid, 1,4-butanediol, C5 1,5-pentanediol, glutaric acid, etc. Diols and diesides.

이에 본 발명자들은 라이신(lysine)으로부터 유래될 수 있는 C5의 글루타릭산을 생산하는 공정을 개발하기 위하여 연구한 결과, 대장균에 L-라이신(L-lysine)을 5-아미노발레르아미드(5-aminovaleramide)로 전환하는 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB) 효소와 5-아미노발레르아미드를 5-아미노발레르산(5-aminovaleric acid)으로 전환하는 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레르산을 글루타레이트 세미알데하이드(glutarate semialdehyde)로 전환하는 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT 효소), 글루타레이트 세미알데하이드(glutarate semialdehyde)를 글루타레이트(glutarate)로 전환하는 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 증폭한 결과, 라이신으로부터 5-아미노발레르산을 거쳐 글루타릭산이 생산됨을 확인하여, 상기 재조합 균주는 글루타릭산을 저가의 라이신으로부터 대량 생산하는데 유용하게 사용할 수 있음을 밝힘으로써 본 발명을 완성하였다.
Therefore, the present inventors have studied to develop a process for producing glutathic acid of C5 which may be derived from lysine (lysine). Lysine 2-monooxygenase (DaB) enzyme and delta-aminovaleramidase to convert 5-aminovalericamide to 5-aminovaleric acid. DavA) enzyme, 5-aminovalerate aminotransferase (DaviT enzyme), which converts 5-aminovaleric acid to glutarate semialdehyde, glutarate semialdehyde Amplification of the glutarate semialdehyde dehydrogenase (DVD) enzyme, which converts c) to glutarate, By confirming that glutaric acid is produced via 5-amino valeric acid, the recombinant strain has completed the present invention by revealing that glutaric acid can be usefully used for mass production of low-cost lysine.

본 발명의 목적은, SUMMARY OF THE INVENTION [0006]

라이신 2-단일산화효소(lysine 2-monooxygenase; DavB), 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT), 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD)를 코딩하는 유전자를 포함하는 재조합 벡터를 미생물에 형질전환하여 제조된 글루타릭산(glutaric acid) 생산용 재조합 미생물을 제공하기 위한 것이다.Lysine 2-monooxygenase (DaB), delta-aminovaleramidase (Dava A) enzyme, 5-aminovalerate aminotransferase (DaT), gluta The present invention provides a recombinant microorganism for producing glutaric acid produced by transforming a microorganism into a recombinant vector containing a gene encoding a glutarate semialdehyde dehydrogenase (DVD).

본 발명의 또 다른 목적은, A further object of the present invention is to provide

1) 라이신 2-단일산화효소(DavB), 델타-아미노발레라미다아제(DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;1) Lysine 2-monooxidase (DavB), delta-aminovaleramidase (DavA) enzyme, 5-aminovalate aminotransferase (DavT) enzyme and glutarate semialdehyde dehydrogenase (DavD) enzyme Preparing an expression vector comprising one or more genes of the gene encoding the;

2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 대장균에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 글루타릭산(glutaric acid) 생산용 재조합 대장균 균주를 제조하는 방법을 제공하기 위한 것이다.2) provides a method for producing a recombinant E. coli strain for the production of glutaric acid (glutaric acid) containing all of the DavB, DavA, DavT and DavD gene by transforming E. coli with one or more expression vectors prepared in step 1) It is to.

본 발명의 또 다른 목적은 DavB 효소, DavA 효소, DavT 효소 및 DavD 효소를 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환하여 제조된 재조합 대장균 균주를 이용하여 글루코스 및 라이신이 포함된 배지로부터 글루타릭산을 생산하는 방법을 제공하기 위한 것이다.
Another object of the present invention is glutaric acid from the medium containing glucose and lysine using a recombinant E. coli strain prepared by transforming with a recombinant vector comprising a gene encoding DavB enzyme, DavA enzyme, DavT enzyme and DavD enzyme To provide a way to produce.

상기 목적을 달성하기 위하여, 본 발명은In order to achieve the above object,

1) 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB) 효소, 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;1) Lysine 2-monooxygenase (DaB) enzyme, delta-aminovaleramidase (Dava A) enzyme, 5-aminovalerate aminotransferase (DaT) Preparing an expression vector comprising one or more genes encoding an enzyme and a glutarate semialdehyde dehydrogenase (DVD) enzyme;

2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 미생물에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 재조합 미생물을 제조하는 단계를 포함하는 제조방법으로 제조된 글루타릭산(glutaric acid) 생산용 재조합 미생물을 제공한다. 2) Glutaric acid prepared by the production method comprising the step of transforming the microorganisms of one or more expression vectors prepared in step 1) to produce a recombinant microorganism containing all of the DavB, DavA, DavT and DavD gene ( It provides a recombinant microorganism for the production of glutaric acid).

또한, 본 발명은In addition,

1) 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB), 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;1) Lysine 2-monooxygenase (DaB), delta-aminovaleramidase (Dava A) enzyme, 5-aminovalerate aminotransferase (DaT) enzyme And preparing an expression vector comprising one or more genes of a gene encoding a glutarate semialdehyde dehydrogenase (DVD) enzyme;

2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 대장균에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 글루타릭산(glutaric acid) 생산용 재조합 대장균 균주를 제조하는 방법을 제공한다.2) provides a method for producing a recombinant E. coli strain for the production of glutaric acid (glutaric acid) containing all of the DavB, DavA, DavT and DavD gene by transforming E. coli with one or more expression vectors prepared in step 1) do.

아울러, 본 발명은 In addition,

1) 라이신 2-단일산화효소(DavB), 델타-아미노발레라미다아제(DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;1) Lysine 2-monooxidase (DavB), delta-aminovaleramidase (DavA) enzyme, 5-aminovalate aminotransferase (DavT) enzyme and glutarate semialdehyde dehydrogenase (DavD) enzyme Preparing an expression vector comprising one or more genes of the gene encoding the;

2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 대장균 균주에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 재조합 대장균 균주를 제조하는 단계;2) transforming the E. coli strain with one or more expression vectors prepared in step 1) to prepare a recombinant E. coli strain comprising all of the DavB, DavA, DavT and DavD genes;

3) 단계 2)의 재조합 대장균 균주를 글루코스(glucose) 및 라이신(lysine)을 포함하는 배지에 배양하는 단계; 및3) culturing the recombinant E. coli strain of step 2) in a medium containing glucose and lysine; And

4) 단계 3)의 배양액으로부터 글루타릭산을 수득하는 단계를 포함하는 글루타릭산 생산방법을 제공한다.
4) It provides a glutaric acid production method comprising the step of obtaining glutaric acid from the culture medium of step 3).

본 발명은 L-라이신(L-lysine)을 5-아미노발레르아미드(5-aminovaleramide)로 전환하는 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB)와 5-아미노발레르아미드를 5-아미노발레르산(5-aminovaleric acid)으로 전환하는 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레르산을 글루타레이트 세미알데하이드(glutarate semialdehyde)로 전환하는 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT), 글루타레이트 세미알데하이드를 글루타레이트(glutarate)로 전환하는 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소가 증폭된 재조합 미생물에 관한 것으로, 상기 재조합 미생물을 라이신이 함유된 배지에서 배양할 경우 글루타릭산을 대량생산 할 수 있으므로 글루타릭산 대량생산 산업화기술개발에 유용하게 이용될 수 있다.
The present invention is a 5-aminovaleryl lysine 2-monooxygenase (DabB) and 5-aminovaleramide that converts L-lysine to 5-aminovaleramide. Delta-aminovaleramidase (DavaA) enzyme, which converts to 5-aminovaleric acid, 5-aminovalate aminotransform, which converts 5-aminovaleric acid to glutarate semialdehyde 5-aminovalerate aminotransferase (DavT), a glutarate semialdehyde dehydrogenase (DVD) enzyme that converts glutarate semialdehyde to glutarate When the recombinant microorganism is cultured in a lysine-containing medium, glutaric acid can be mass-produced, which is useful for the development of glutaric acid mass production industrialization technology. Can be used.

이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.

본 발명은,According to the present invention,

1) 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB) 효소, 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;1) Lysine 2-monooxygenase (DaB) enzyme, delta-aminovaleramidase (Dava A) enzyme, 5-aminovalerate aminotransferase (DaT) Preparing an expression vector comprising one or more genes encoding an enzyme and a glutarate semialdehyde dehydrogenase (DVD) enzyme;

2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 미생물에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 재조합 미생물을 제조하는 단계를 포함하는 제조방법으로 제조된 글루타릭산(glutaric acid) 생산용 재조합 미생물을 제공한다.2) Glutaric acid prepared by the production method comprising the step of transforming the microorganisms of one or more expression vectors prepared in step 1) to produce a recombinant microorganism containing all of the DavB, DavA, DavT and DavD gene ( It provides a recombinant microorganism for the production of glutaric acid).

상기 미생물은 대장균인 것이 바람직하고, BL21 codon plus인 것이 보다 바람직하나 이에 한정하지 않는다.
The microorganism is preferably E. coli, and more preferably BL21 codon plus, but is not limited thereto.

또한, 본 발명은 In addition,

1) 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB), 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;1) Lysine 2-monooxygenase (DaB), delta-aminovaleramidase (Dava A) enzyme, 5-aminovalerate aminotransferase (DaT) enzyme And preparing an expression vector comprising one or more genes of a gene encoding a glutarate semialdehyde dehydrogenase (DVD) enzyme;

2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 대장균에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 글루타릭산(glutaric acid) 생산용 재조합 대장균 균주를 제조하는 방법을 제공한다.2) provides a method for producing a recombinant E. coli strain for the production of glutaric acid (glutaric acid) containing all of the DavB, DavA, DavT and DavD gene by transforming E. coli with one or more expression vectors prepared in step 1) do.

상기 단계 1)의 발현 벡터는, The expression vector of step 1),

라이신 2-단일산화효소(DavB) 및 델타-아미노발레라미다아제(DavA) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터; 및A recombinant vector incorporating a gene encoding a lysine 2-monooxidase (DavB) and delta-aminovaleramidase (DavA) enzyme into an expression vector; And

5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터인 것이 바람직하나 이에 한정하지 않는다.Preferably, the recombinant vector is a recombinant vector in which a gene encoding a 5-aminovalate aminotransferase (DavT) enzyme and a glutarate semialdehyde dehydrogenase (DavD) enzyme are inserted into an expression vector.

상기 DavB는 서열번호 1로 기재되는 염기서열을 갖는 것이 바람직하나 이에 한정하지 않는다.DavB preferably has a nucleotide sequence as set forth in SEQ ID NO: 1, but is not limited thereto.

상기 DavA는 서열번호 2로 기재되는 염기서열을 갖는 것이 바람직하나 이에 한정하지 않는다.DavA preferably has a nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

상기 DavT는 서열번호 3으로 기재되는 염기서열을 갖는 것이 바람직하나 이에 한정하지 않는다.The DavT preferably has a nucleotide sequence set forth in SEQ ID NO: 3, but is not limited thereto.

상기 DavD는 서열번호 4로 기재되는 염기서열을 갖는 것이 바람직하나 이에 한정하지 않는다.DavD preferably has a nucleotide sequence set forth in SEQ ID NO: 4, but is not limited thereto.

상기 대장균 균주는 모든 종류의 대장균 균주가 적용될 수 있으나, BL21 codon plus인 것이 보다 바람직하나 이에 한정하지 않는다.
The E. coli strains may be applied to all kinds of E. coli strains, more preferably BL21 codon plus, but is not limited thereto.

또한, 본 발명은, Further, according to the present invention,

1) 라이신 2-단일산화효소(DavB), 델타-아미노발레라미다아제(DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;1) Lysine 2-monooxidase (DavB), delta-aminovaleramidase (DavA) enzyme, 5-aminovalate aminotransferase (DavT) enzyme and glutarate semialdehyde dehydrogenase (DavD) enzyme Preparing an expression vector comprising one or more genes of the gene encoding the;

2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 대장균 균주에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 재조합 대장균 균주를 제조하는 단계;2) transforming the E. coli strain with one or more expression vectors prepared in step 1) to prepare a recombinant E. coli strain comprising all of the DavB, DavA, DavT and DavD genes;

3) 단계 2)의 재조합 대장균 균주를 글루코스(glucose) 및 라이신(lysine)을 포함하는 배지에 배양하는 단계; 및3) culturing the recombinant E. coli strain of step 2) in a medium containing glucose and lysine; And

4) 단계 3)의 배양액으로부터 글루타릭산을 수득하는 단계를 포함하는 글루타릭산 생산방법을 제공한다.4) It provides a glutaric acid production method comprising the step of obtaining glutaric acid from the culture medium of step 3).

상기 단계 1)의 발현 벡터는, The expression vector of step 1),

라이신 2-단일산화효소 및 델타-아미노발레라미다아제(DavA) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터; 및A recombinant vector incorporating a gene encoding a lysine 2-monooxidase and delta-aminovaleramidase (DavA) enzyme into an expression vector; And

5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터인 것이 바람직하나 이에 한정하지 않는다.Preferably, the recombinant vector is a recombinant vector in which a gene encoding a 5-aminovalate aminotransferase (DavT) enzyme and a glutarate semialdehyde dehydrogenase (DavD) enzyme are inserted into an expression vector.

상기 단계 3)의 배지는 대장균 균주의 배양에 이용되는 어떠한 배지도 사용가능하나, LB배지가 보다 바람직하나 이에 한정하지 않는다.As the medium of step 3), any medium used for the culture of E. coli strains may be used, but the LB medium is more preferably but not limited thereto.

상기 단계 3)의 배양은 48 내지 96 시간인 것이 바람직하고 96 시간인 것이 보다 바람직하며, 배양온도는 20 내지 40℃인 것이 바람직하고, 37℃인 것이 보다 바람직하나 이에 한정하지 않는다. The culture of step 3) is preferably 48 to 96 hours, more preferably 96 hours, the culture temperature is preferably 20 to 40 ℃, more preferably 37 ℃ but not limited thereto.

상기 단계 4)의 글루타릭산 수득은 배양액을 분무 건조하여 저농도의 글루타릭산-함유 분말을 대량으로 얻을 수 있고, 배양액을 감압 증발기를 이용하여 농축한 후 동결건조를 진행하여 고농도의 글루타릭산 수득도 가능하며, 또한 이온교환수지를 이용한 정제방법을 사용한 후 농축 및 건조 공정을 수행하여 고순도의 글루타릭산의 수득할 수 있으나 이에 한정하지 않으며, 상기 글루타릭산은 온도 및 pH의 변화에 크게 영향을 받지 않고, 공정 안정성이 있기 때문에 회수 공정상의 제한이 없다. Glutaric acid obtained in step 4) can be obtained by spray-drying the culture solution to obtain a large amount of low concentration glutaric acid-containing powder. It is also possible to obtain a high purity glutaric acid by using a purification method using an ion exchange resin followed by a concentration and drying process, but is not limited thereto. Unaffected and there is no limitation on recovery process because of process stability.

본 발명의 구체적인 실시예에 있어서, 대장균 균주를 라이신 2-단일산화효소(DavB), 5-아미노발레르아미드(5-aminovaleramide)를 5-아미노발레르산(5-aminovaleric acid)으로 전환하는 델타-아미노발레라미다아제(DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD)를 코딩하는 유전자를 함유한 재조합 벡터로 형질전환하여 형질전환된 대장균 균주를 라이신이 포함된 배지에서 배양한 후 글루타릭산 전환 생산능을 확인한 결과, 글루타릭산의 유의적인 전환 생산능을 확인하였다(표 1 참조).In a specific embodiment of the present invention, E. coli strain delta-amino to convert lysine 2-monooxidase (DavB), 5-aminovaleramide to 5-aminovaleric acid Transformed by transformation with a recombinant vector containing genes encoding valeramidase (DavA) enzyme, 5-aminovalate aminotransferase (DavT) enzyme, and glutarate semialdehyde dehydrogenase (DavD) E. coli strains were cultured in a medium containing lysine, and then glutaric acid conversion production capacity was confirmed.

따라서, 본 발명의 재조합 대장균 균주는 글루타릭산을 생산하는데 유용하게 사용될 수 있다.
Therefore, the recombinant E. coli strain of the present invention can be usefully used to produce glutaric acid.

이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.

단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.
However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

<< 실시예Example 1>  1> DavADavA 및  And DavBDavB , 및 , And DavTDavT 및  And DavDDavD 유전자 발현을 위한 발현 벡터의 제조 Preparation of Expression Vectors for Gene Expression

L-라이신(L-lysine)을 5-아미노발레르아미드(5-aminovaleramide)로 전환하는 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB)와 5-아미노발레르아미드를 5-아미노발레르산(5-aminovaleric acid)으로 전환하는 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레르산을 글루타레이트 세미알데하이드(glutarate semialdehyde)로 전환하는 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT), 글루타레이트 세미알데하이드를 글루타레이트(glutarate)로 전환하는 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 발현하는 재조합 E. coli BL21 codon plus를 제조하여, 상기 재조합 대장균 내에서 라이신으로부터 글루타릭산 생산능을 확인하기 위하여, DavA, DavB, DavT 및 DavD 유전자를 발현하는 벡터를 제작하였다. Lysine 2-monooxygenase (DabB) and 5-aminovaleramide convert L-lysine to 5-aminovaleramide to 5-aminovaleric acid (5 delta-aminovaleramidase (DavaA) enzyme, which converts to -aminovaleric acid, and 5-aminovalate aminotransferase, which converts 5-aminovaleric acid to glutarate semialdehyde. 5-aminovalerate aminotransferase; DavT), a recombinant E. coli BL21 codon plus that expresses the glutarate semialdehyde dehydrogenase (DVD) enzyme, which converts glutarate semialdehyde to glutarate. To prepare a vector expressing the DavA, DavB, DavT and DavD gene in order to confirm the production of glutaric acid from lysine in the recombinant E. coli.

구체적으로, 발현용 벡터 pKE12-MCS와 pKA32-MCS는 LacIO promoter를 보유한 발현벡터로서(Park et al. (2011) Appl. Microbiol. Biotechnol. DOI: 10.1007/s00253-011-3530-x), pKE12-MCS에 슈도모나스 푸티다(Pseudomonas putida)의 DavA 유전자를 EcoRI/KpnI으로 절단하고 삽입하여, pKE12DavA를 제조하였고, pKE12DavA에 슈도모나스 푸티다의 DavB 유전자를 KpnI/BamHI으로 절단하고 삽입하여, pKE12DavAB를 제조하였다. 또한 pKA32-MCS에 슈도모나스 푸티다의 DavT 유전자를 EcoRI/KpnI으로 절단하고 삽입하여, pKA12DavT를 제조하였고, pKA32DavT에 슈도모나스 푸티다의 DavD 유전자를 KpnI/BamHI으로 절단하고 삽입하여, pKA32DavTD를 제조하였다. 그런 다음, 상기 제조된 벡터를 대장균에 형질전환 하여 E. coli BL21 codon plus(pKE12DavAB + pKA32DavTD)를 제작하였다.
Specifically, expression vectors pKE12-MCS and pKA32-MCS are expression vectors having a LacIO promoter (Park et al. (2011) Appl. Microbiol. Biotechnol. DOI: 10.1007 / s00253-011-3530-x), pKE12- PKE12DavA was prepared by cleaving and inserting the DavA gene of Pseudomonas putida with EcoRI / KpnI in MCS, and preparing pKE12DavA, and cutting and inserting the DavB gene of Pseudomonas putida in pKE12DavA with KpnI / BamHI to prepare pKE12DavAB. In addition, pKA32DavT was cut and inserted into PKA32-MCS with Pseudomonas putida with EcoRI / KpnI to prepare pKA12DavT, and pKA32DavT was cut and inserted into PKA32DavT with PDP32 and DavD of Pseudomonas putida into pKA32DavTD. Then, E. coli BL21 codon plus (pKE12DavAB + pKA32DavTD) was produced by transforming the prepared vector into E. coli .

<< 실시예Example 2>  2> 글루타릭산Glutaric acid 생산능Production capacity 확인 Confirm

상기 <실시예 1>에서 제작한 E. coli BL21 codon plus(pKE12DavAB + pKA32DavTD)를 이용하여 글루타릭산 생산능을 확인하기 위하여, E. coli BL21 codon plus를 LB배지 및 글루코스 10 g/L와 라이신 10 g/L가 함유된 LB 및 MR 배지에서 배양하였다. 단백질의 발현을 위하여 OD가 0.4일 때 IPTG 1 mM를 첨가하였다. 상기 MR 배지는(pH 6.9) 1리터당 하기와 같은 조성으로 구성하였다: 6.67 g KH2PO4, 4 g(NH4)2HPO4,0.8 g MgSO47H2O, 0.8g 시트르산(citric acid), 5 ml 트레이스 금속 용액(trace metal solution). 상기 트레이스 금속 용액는 1리터당 하기와 같은 조성으로 구성하였다(0.5 M HCl): 10 g FeSO47H2O, 2 g CaCl2, 2.2gZnSO47H2O, 0.5 g MnSO44H2O, 1 g CuSO45H2O, 0.1 g(NH4)6Mo7O244H2O, 0.02 g Na2B4O710H2O. E. coli BL21 codon plus using the E. coli BL21 codon plus (pKE12DavAB + pKA32DavTD) prepared in <Example 1>, E. coli BL21 codon plus LB medium and glucose 10 g / L and lysine Cultured in LB and MR medium containing 10 g / L. 1 mM of IPTG was added when the OD was 0.4 for the expression of the protein. The MR medium (pH 6.9) consisted of the following composition per liter: 6.67 g KH 2 PO 4 , 4 g (NH 4 ) 2 HPO 4 , 0.8 g MgSO 4 7H 2 O, 0.8 g citric acid , 5 ml trace metal solution. The trace metal solution consisted of the following composition per liter (0.5 M HCl): 10 g FeSO 4 7H 2 O, 2 g CaCl 2 , 2.2 gZnSO 4 7H 2 O, 0.5 g MnSO 4 4H 2 O, 1 g CuSO 4 5H 2 O, 0.1 g (NH 4 ) 6 Mo 7 O 24 4H 2 O, 0.02 g Na 2 B 4 O 7 10H 2 O.

그 결과, 표 1에 나타낸 바와 같이 LB배지에서 E. coli BL21 codon plus (pKE12DavAB + pKA32DavTD)를 배양했을 때 라이신으로부터 글루타릭산이 유의적으로 생산되는 것을 확인하였다(표 1). 또한, MR배지에서 배양하였을 때에는 글루타릭산의 생성은 확인되지 않았다.
As a result, as shown in Table 1, it was confirmed that glutaric acid was significantly produced from lysine when E. coli BL21 codon plus (pKE12DavAB + pKA32DavTD) was cultured in LB medium (Table 1). In addition, the production of glutaric acid was not confirmed when cultured in MR medium.

시간time 글루타릭산 (g/L)Glutaric Acid (g / L) 10 초10 seconds 00 24 시간24 hours 0.10.1 48 시간48 hours 0.20.2 72 시간72 hours 0.10.1 96 시간96 hours 0.50.5

<110> Korea Research Institute of Chemical Technology <120> Method for Preparing glutaric acid using recombinant microorganism <130> 11p-10-43 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 560 <212> PRT <213> Unknown <220> <223> lysine 2-monooxygenase sequence <400> 1 Met Asn Lys Lys Asn Arg His Pro Ala Asp Gly Lys Lys Pro Ile Thr 1 5 10 15 Ile Phe Gly Pro Asp Phe Pro Phe Ala Phe Asp Asp Trp Leu Glu His 20 25 30 Pro Ala Gly Leu Gly Ser Ile Pro Ala Glu Arg His Gly Glu Glu Val 35 40 45 Ala Ile Val Gly Ala Gly Ile Ala Gly Leu Val Ala Ala Tyr Glu Leu 50 55 60 Met Lys Leu Gly Leu Lys Pro Val Val Tyr Glu Ala Ser Lys Leu Gly 65 70 75 80 Gly Arg Leu Arg Ser Gln Ala Phe Asn Gly Thr Asp Gly Ile Val Ala 85 90 95 Glu Leu Gly Gly Met Arg Phe Pro Val Ser Ser Thr Ala Phe Tyr His 100 105 110 Tyr Val Asp Lys Leu Gly Leu Glu Thr Lys Pro Phe Pro Asn Pro Leu 115 120 125 Thr Pro Ala Ser Gly Ser Thr Val Ile Asp Leu Glu Gly Gln Thr Tyr 130 135 140 Tyr Ala Glu Lys Pro Thr Asp Leu Pro Gln Leu Phe His Glu Val Ala 145 150 155 160 Asp Ala Trp Ala Asp Ala Leu Glu Ser Gly Ala Gln Phe Ala Asp Ile 165 170 175 Gln Gln Ala Ile Arg Asp Arg Asp Val Pro Arg Leu Lys Glu Leu Trp 180 185 190 Asn Lys Leu Val Pro Leu Trp Asp Asp Arg Thr Phe Tyr Asp Phe Val 195 200 205 Ala Thr Ser Arg Ser Phe Ala Lys Leu Ser Phe Gln His Arg Glu Val 210 215 220 Phe Gly Gln Val Gly Phe Gly Thr Gly Gly Trp Asp Ser Asp Phe Pro 225 230 235 240 Asn Ser Met Leu Glu Ile Phe Arg Val Val Met Thr Asn Cys Asp Asp 245 250 255 His Gln His Leu Val Val Gly Gly Val Glu Gln Val Pro Gln Gly Ile 260 265 270 Trp Arg His Val Pro Glu Arg Cys Val His Trp Pro Glu Gly Thr Ser 275 280 285 Leu Ser Thr Leu His Gly Gly Ala Pro Arg Thr Gly Val Lys Arg Ile 290 295 300 Ala Arg Ala Ser Asp Gly Arg Leu Ala Val Thr Asp Asn Trp Gly Asp 305 310 315 320 Thr Arg His Tyr Ser Ala Val Leu Ala Thr Cys Gln Thr Trp Leu Leu 325 330 335 Thr Thr Gln Ile Asp Cys Glu Glu Ser Leu Phe Ser Gln Lys Met Trp 340 345 350 Met Ala Leu Asp Arg Thr Arg Tyr Met Gln Ser Ser Lys Thr Phe Val 355 360 365 Met Val Asp Arg Pro Phe Trp Lys Asp Lys Asp Pro Glu Thr Gly Arg 370 375 380 Asp Leu Leu Ser Met Thr Leu Thr Asp Arg Leu Thr Arg Gly Thr Tyr 385 390 395 400 Leu Phe Asp Asn Gly Asn Asp Lys Pro Gly Val Ile Cys Leu Ser Tyr 405 410 415 Ser Trp Met Ser Asp Ala Leu Lys Met Leu Pro His Pro Val Glu Lys 420 425 430 Arg Val Gln Leu Ala Leu Asp Ala Leu Lys Lys Ile Tyr Pro Lys Thr 435 440 445 Asp Ile Ala Gly His Ile Ile Gly Asp Pro Ile Thr Val Ser Trp Glu 450 455 460 Ala Asp Pro Tyr Phe Leu Gly Ala Phe Lys Gly Ala Leu Pro Gly His 465 470 475 480 Tyr Arg Tyr Asn Gln Arg Met Tyr Ala His Phe Met Gln Gln Asp Met 485 490 495 Pro Ala Glu Gln Arg Gly Ile Phe Ile Ala Gly Asp Asp Val Ser Trp 500 505 510 Thr Pro Ala Trp Val Glu Gly Ala Val Gln Thr Ser Leu Asn Ala Val 515 520 525 Trp Gly Ile Met Asn His Phe Gly Gly His Thr His Pro Asp Asn Pro 530 535 540 Gly Pro Gly Asp Val Phe Asn Glu Ile Gly Pro Ile Ala Leu Ala Asp 545 550 555 560 <210> 2 <211> 264 <212> PRT <213> Unknown <220> <223> delta-aminovaleramidase sequence <400> 2 Met Arg Ile Ala Leu Tyr Gln Gly Ala Pro Lys Pro Leu Asp Val Pro 1 5 10 15 Gly Asn Leu Gln Arg Leu Arg His Gln Ala Gln Leu Ala Ala Glu Arg 20 25 30 Gly Ala Gln Leu Leu Val Cys Pro Glu Met Phe Leu Thr Gly Tyr Asn 35 40 45 Ile Gly Leu Ala Gln Val Glu Arg Leu Ala Glu Ala Ala Asp Gly Pro 50 55 60 Ala Ala Met Thr Val Val Glu Ile Ala Gln Ala His Arg Ile Ala Ile 65 70 75 80 Val Tyr Gly Tyr Pro Glu Arg Gly Asp Asp Gly Ala Ile Tyr Asn Ser 85 90 95 Val Gln Leu Ile Asp Ala His Gly Arg Ser Leu Ser Asn Tyr Arg Lys 100 105 110 Thr His Leu Phe Gly Glu Leu Asp Arg Ser Met Phe Ser Pro Gly Ala 115 120 125 Asp His Phe Pro Val Val Glu Leu Glu Gly Trp Lys Val Gly Leu Leu 130 135 140 Ile Cys Tyr Asp Ile Glu Phe Pro Glu Asn Ala Arg Arg Leu Ala Leu 145 150 155 160 Asp Gly Ala Glu Leu Ile Leu Val Pro Thr Ala Asn Met Thr Pro Tyr 165 170 175 Asp Phe Thr Cys Gln Val Thr Val Arg Ala Arg Ala Gln Glu Asn Gln 180 185 190 Cys Tyr Leu Val Tyr Ala Asn Tyr Cys Gly Ala Glu Asp Glu Ile Glu 195 200 205 Tyr Cys Gly Gln Ser Ser Ile Ile Gly Pro Asp Gly Ser Leu Leu Ala 210 215 220 Met Ala Gly Arg Asp Glu Cys Gln Leu Leu Ala Glu Leu Glu His Glu 225 230 235 240 Arg Val Val Gln Gly Arg Thr Ala Phe Pro Tyr Leu Thr Asp Leu Arg 245 250 255 Gln Glu Leu His Leu Arg Lys Gly 260 <210> 3 <211> 425 <212> PRT <213> Unknown <220> <223> 5-aminovalerate aminotransferase sequence <400> 3 Met Ser Lys Thr Asn Glu Ser Leu Met Gln Arg Arg Val Ala Ala Val 1 5 10 15 Pro Arg Gly Val Gly Gln Ile His Pro Ile Phe Val Asp Thr Ala Lys 20 25 30 Asn Ser Thr Val Ile Asp Val Glu Gly Arg Glu Leu Ile Asp Phe Ala 35 40 45 Gly Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Lys Val Val 50 55 60 Ala Ala Val Gln Glu Gln Leu Thr Lys Val Ser His Thr Cys Phe Gln 65 70 75 80 Val Leu Ala Tyr Glu Pro Tyr Val Glu Leu Cys Glu Lys Ile Asn Lys 85 90 95 Leu Val Pro Gly Asp Phe Asp Lys Lys Thr Leu Leu Val Thr Thr Gly 100 105 110 Ser Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Gly 115 120 125 Arg Ala Gly Val Ile Ala Phe Thr Gly Gly Tyr His Gly Arg Thr Met 130 135 140 Met Thr Leu Gly Leu Thr Gly Lys Val Val Pro Tyr Ser Ala Gly Met 145 150 155 160 Gly Leu Met Pro Gly Gly Ile Phe Arg Ala Leu Phe Pro Ser Glu Leu 165 170 175 His Gly Ile Ser Val Asp Asp Ala Ile Ala Ser Val Glu Arg Ile Phe 180 185 190 Lys Asn Asp Ala Glu Pro Arg Asp Ile Ala Ala Ile Ile Leu Glu Pro 195 200 205 Val Gln Gly Glu Gly Gly Phe Leu Pro Ala Pro Lys Glu Leu Met Lys 210 215 220 Arg Leu Arg Ala Leu Cys Asp Gln His Gly Ile Leu Leu Ile Ala Asp 225 230 235 240 Glu Val Gln Thr Gly Ala Gly Arg Thr Gly Thr Phe Phe Ala Met Glu 245 250 255 Gln Met Gly Val Ala Pro Asp Leu Thr Thr Phe Ala Lys Ser Ile Ala 260 265 270 Gly Gly Phe Pro Leu Ala Gly Val Cys Gly Lys Ala Glu Tyr Met Asp 275 280 285 Ala Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Ser Pro Ile 290 295 300 Ala Cys Ala Ala Ala Leu Ala Val Ile Glu Val Phe Glu Glu Glu Lys 305 310 315 320 Leu Leu Asp Arg Ser Lys Ala Val Gly Glu Arg Leu Thr Ala Gly Leu 325 330 335 Arg Glu Ile Gln Lys Lys Tyr Pro Ile Ile Gly Asp Val Arg Gly Leu 340 345 350 Gly Ser Met Ile Ala Val Glu Val Phe Glu Lys Gly Thr His Thr Pro 355 360 365 Asn Ala Ala Ala Val Gly Gln Val Val Ala Lys Ala Arg Glu Lys Gly 370 375 380 Leu Ile Leu Leu Ser Cys Gly Thr Tyr Gly Asn Val Leu Arg Ile Leu 385 390 395 400 Val Pro Leu Thr Ala Glu Asp Ala Leu Leu Asp Lys Gly Leu Ala Ile 405 410 415 Ile Glu Glu Cys Phe Ala Glu Ile Ala 420 425 <210> 4 <211> 480 <212> PRT <213> Unknown <220> <223> glutarate semialdehyde dehydrogenase sequence <400> 4 Met Gln Leu Lys Asp Ala Gln Leu Phe Arg Gln Gln Ala Tyr Ile Asn 1 5 10 15 Gly Glu Trp Leu Asp Ala Asp Asn Gly Gln Thr Ile Lys Val Thr Asn 20 25 30 Pro Ala Thr Gly Glu Val Ile Gly Thr Val Pro Lys Met Gly Thr Ala 35 40 45 Glu Thr Arg Arg Ala Ile Glu Ala Ala Asp Lys Ala Leu Pro Ala Trp 50 55 60 Arg Ala Leu Thr Ala Lys Glu Arg Ser Ala Lys Leu Arg Arg Trp Phe 65 70 75 80 Glu Leu Met Ile Glu Asn Gln Asp Asp Leu Ala Arg Leu Met Thr Thr 85 90 95 Glu Gln Gly Lys Pro Leu Ala Glu Ala Lys Gly Glu Ile Ala Tyr Ala 100 105 110 Ala Ser Phe Ile Glu Trp Phe Ala Glu Glu Ala Lys Arg Ile Tyr Gly 115 120 125 Asp Thr Ile Pro Gly His Gln Pro Asp Lys Arg Leu Ile Val Ile Lys 130 135 140 Gln Pro Ile Gly Val Thr Ala Ala Ile Thr Pro Trp Asn Phe Pro Ala 145 150 155 160 Ala Met Ile Thr Arg Lys Ala Gly Pro Ala Leu Ala Ala Gly Cys Thr 165 170 175 Met Val Leu Lys Pro Ala Ser Gln Thr Pro Tyr Ser Ala Leu Ala Leu 180 185 190 Val Glu Leu Ala His Arg Ala Gly Ile Pro Ala Gly Val Leu Ser Val 195 200 205 Val Thr Gly Ser Ala Gly Glu Val Gly Gly Glu Leu Thr Gly Asn Ser 210 215 220 Leu Val Arg Lys Leu Ser Phe Thr Gly Ser Thr Glu Ile Gly Arg Gln 225 230 235 240 Leu Met Glu Glu Cys Ala Lys Asp Ile Lys Lys Val Ser Leu Glu Leu 245 250 255 Gly Gly Asn Ala Pro Phe Ile Val Phe Asp Asp Ala Asp Leu Asp Lys 260 265 270 Ala Val Glu Gly Ala Ile Ile Ser Lys Tyr Arg Asn Asn Gly Gln Thr 275 280 285 Cys Val Cys Ala Asn Arg Ile Tyr Val Gln Asp Gly Val Tyr Asp Ala 290 295 300 Phe Ala Glu Lys Leu Ala Ala Ala Val Ala Lys Leu Lys Ile Gly Asn 305 310 315 320 Gly Leu Glu Glu Gly Thr Thr Thr Gly Pro Leu Ile Asp Gly Lys Ala 325 330 335 Val Ala Lys Val Gln Glu His Ile Glu Asp Ala Val Ser Lys Gly Ala 340 345 350 Lys Val Leu Ser Gly Gly Lys Leu Ile Glu Gly Asn Phe Phe Glu Pro 355 360 365 Thr Ile Leu Val Asp Val Pro Lys Thr Ala Ala Val Ala Lys Glu Glu 370 375 380 Thr Phe Gly Pro Leu Ala Pro Leu Phe Arg Phe Lys Asp Glu Ala Glu 385 390 395 400 Val Ile Ala Met Ser Asn Asp Thr Glu Phe Gly Leu Ala Ser Tyr Phe 405 410 415 Tyr Ala Arg Asp Met Ser Arg Val Phe Arg Val Ala Glu Ala Leu Glu 420 425 430 Tyr Gly Met Val Gly Ile Asn Thr Gly Leu Ile Ser Asn Glu Val Ala 435 440 445 Pro Phe Gly Gly Ile Lys Ala Ser Gly Leu Gly Arg Glu Gly Ser Lys 450 455 460 Tyr Gly Ile Glu Asp Tyr Leu Glu Ile Lys Tyr Leu Cys Ile Ser Val 465 470 475 480 <110> Korea Research Institute of Chemical Technology <120> Method for Preparing glutaric acid using recombinant          microorganism <130> 11p-10-43 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 560 <212> PRT <213> Unknown <220> <223> lysine 2-monooxygenase sequence <400> 1 Met Asn Lys Lys Asn Arg His Pro Ala Asp Gly Lys Lys Pro Ile Thr   1 5 10 15 Ile Phe Gly Pro Asp Phe Pro Phe Ala Phe Asp Asp Trp Leu Glu His              20 25 30 Pro Ala Gly Leu Gly Ser Ile Pro Ala Glu Arg His Gly Glu Glu Val          35 40 45 Ala Ile Val Gly Ala Gly Ale Gly Leu Val Ala Ala Tyr Glu Leu      50 55 60 Met Lys Leu Gly Leu Lys Pro Val Val Tyr Glu Ala Ser Lys Leu Gly  65 70 75 80 Gly Arg Leu Arg Ser Gln Ala Phe Asn Gly Thr Asp Gly Ile Val Ala                  85 90 95 Glu Leu Gly Gly Met Arg Phe Pro Val Ser Ser Thr Ala Phe Tyr His             100 105 110 Tyr Val Asp Lys Leu Gly Leu Glu Thr Lys Pro Phe Pro Asn Pro Leu         115 120 125 Thr Pro Ala Ser Gly Ser Thr Val Ile Asp Leu Glu Gly Gln Thr Tyr     130 135 140 Tyr Ala Glu Lys Pro Thr Asp Leu Pro Gln Leu Phe His Glu Val Ala 145 150 155 160 Asp Ala Trp Ala Asp Ala Leu Glu Ser Gly Ala Gln Phe Ala Asp Ile                 165 170 175 Gln Gln Ala Ile Arg Asp Arg Asp Val Pro Arg Leu Lys Glu Leu Trp             180 185 190 Asn Lys Leu Val Pro Leu Trp Asp Asp Arg Thr Phe Tyr Asp Phe Val         195 200 205 Ala Thr Ser Arg Phe Ala Lys Leu Ser Phe Gln His Arg Glu Val     210 215 220 Phe Gly Gln Val Gly Phe Gly Thr Gly Gly Trp Asp Ser Asp Phe Pro 225 230 235 240 Asn Ser Met Leu Glu Ile Phe Arg Val Val Met Thr Asn Cys Asp Asp                 245 250 255 His Gln His Leu Val Val Gly Gly Val Glu Gln Val Pro Gln Gly Ile             260 265 270 Trp Arg His Val Pro Glu Arg Cys Val His Trp Pro Glu Gly Thr Ser         275 280 285 Leu Ser Thr Leu His Gly Gly Ala Pro Arg Thr Gly Val Lys Arg Ile     290 295 300 Ala Arg Ala Ser Asp Gly Arg Leu Ala Val Thr Asp Asn Trp Gly Asp 305 310 315 320 Thr Arg His Tyr Ser Ala Val Leu Ala Thr Cys Gln Thr Trp Leu Leu                 325 330 335 Thr Gln Ile Asp Cys Glu Glu Ser Leu Phe Ser Gln Lys Met Trp             340 345 350 Met Ala Leu Asp Arg Thr Arg Tyr Met Gln Ser Ser Lys Thr Phe Val         355 360 365 Met Val Asp Arg Pro Phe Trp Lys Asp Lys Asp Pro Glu Thr Gly Arg     370 375 380 Asp Leu Leu Ser Met Thr Leu Thr Asp Arg Leu Thr Arg Gly Thr Tyr 385 390 395 400 Leu Phe Asp Asn Gly Asn Asp Lys Pro Gly Val Ile Cys Leu Ser Tyr                 405 410 415 Ser Trp Met Ser Asp Ala Leu Lys Met Leu Pro His Pro Val Glu Lys             420 425 430 Arg Val Gln Leu Ala Leu Asp Ala Leu Lys Lys Ile Tyr Pro Lys Thr         435 440 445 Asp Ile Ala Gly His Ile Ile Gly Asp Pro Ile Thr Val Ser Trp Glu     450 455 460 Ala Asp Pro Tyr Phe Leu Gly Ala Phe Lys Gly Ala Leu Pro Gly His 465 470 475 480 Tyr Arg Tyr Asn Gln Arg Met Tyr Ala His Phe Met Gln Gln Asp Met                 485 490 495 Pro Ala Glu Gln Arg Gly Ile Phe Ile Ala Gly Asp Asp Val Ser Trp             500 505 510 Thr Pro Ala Trp Val Glu Gly Ala Val Gln Thr Ser Leu Asn Ala Val         515 520 525 Trp Gly Ile Met Asn His Phe Gly Gly His Thr His Pro Asp Asn Pro     530 535 540 Gly Pro Gly Asp Val Phe Asn Glu Ile Gly Pro Ile Ala Leu Ala Asp 545 550 555 560 <210> 2 <211> 264 <212> PRT <213> Unknown <220> <223> delta-aminovaleramidase sequence <400> 2 Met Arg Ile Ala Leu Tyr Gln Gly Ala Pro Lys Pro Leu Asp Val Pro   1 5 10 15 Gly Asn Leu Gln Arg Leu Arg His Gln Ala Gln Leu Ala Ala Glu Arg              20 25 30 Gly Ala Gln Leu Leu Val Cys Pro Glu Met Phe Leu Thr Gly Tyr Asn          35 40 45 Ile Gly Leu Ala Gln Val Glu Arg Leu Ala Glu Ala Ala Asp Gly Pro      50 55 60 Ala Ala Met Thr Val Val Glu Ile Ala Gln Ala His Arg Ile Ala Ile  65 70 75 80 Val Tyr Gly Tyr Pro Glu Arg Gly Asp Asp Gly Ala Ile Tyr Asn Ser                  85 90 95 Val Gln Leu Ile Asp Ala His Gly Arg Ser Leu Ser Asn Tyr Arg Lys             100 105 110 Thr His Leu Phe Gly Glu Leu Asp Arg Ser Met Phe Ser Pro Gly Ala         115 120 125 Asp His Phe Pro Val Val Glu Leu Glu Gly Trp Lys Val Gly Leu Leu     130 135 140 Ile Cys Tyr Asp Ile Glu Phe Pro Glu Asn Ala Arg Arg Leu Ala Leu 145 150 155 160 Asp Gly Ala Glu Leu Ile Leu Val Pro Thr Ala Asn Met Thr Pro Tyr                 165 170 175 Asp Phe Thr Cys Gln Val Thr Val Arg Ala Arg Ala Gln Glu Asn Gln             180 185 190 Cys Tyr Leu Val Tyr Ala Asn Tyr Cys Gly Ala Glu Asp Glu Ile Glu         195 200 205 Tyr Cys Gly Gln Ser Ser Ile Gly Pro Asp Gly Ser Leu Leu Ala     210 215 220 Met Ala Gly Arg Asp Glu Cys Gln Leu Leu Ala Glu Leu Glu His Glu 225 230 235 240 Arg Val Val Gln Gly Arg Thr Ala Phe Pro Tyr Leu Thr Asp Leu Arg                 245 250 255 Gln Glu Leu His Leu Arg Lys Gly             260 <210> 3 <211> 425 <212> PRT <213> Unknown <220> <223> 5-aminovalerate aminotransferase sequence <400> 3 Met Ser Lys Thr Asn Glu Ser Leu Met Gln Arg Arg Val Ala Ala Val   1 5 10 15 Pro Arg Gly Val Gly Gln Ile His Pro Ile Phe Val Asp Thr Ala Lys              20 25 30 Asn Ser Thr Val Ile Asp Val Glu Gly Arg Glu Leu Ile Asp Phe Ala          35 40 45 Gly Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Lys Val Val      50 55 60 Ala Ala Val Gln Glu Gln Leu Thr Lys Val Ser His Thr Cys Phe Gln  65 70 75 80 Val Leu Ala Tyr Glu Pro Tyr Val Glu Leu Cys Glu Lys Ile Asn Lys                  85 90 95 Leu Val Pro Gly Asp Phe Asp Lys Lys Thr Leu Leu Val Thr Thr Gly             100 105 110 Ser Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Gly         115 120 125 Arg Ala Gly Val Ile Ala Phe Thr Gly Gly Tyr His Gly Arg Thr Met     130 135 140 Met Thr Leu Gly Leu Thr Gly Lys Val Val Pro Tyr Ser Ala Gly Met 145 150 155 160 Gly Leu Met Pro Gly Gly Ile Phe Arg Ala Leu Phe Pro Ser Glu Leu                 165 170 175 His Gly Ile Ser Val Asp Asp Ala Ile Ala Ser Val Glu Arg Ile Phe             180 185 190 Lys Asn Asp Ala Glu Pro Arg Asp Ile Ala Ala Ile Ile Leu Glu Pro         195 200 205 Val Gln Gly Glu Gly Gly Phe Leu Pro Ala Pro Lys Glu Leu Met Lys     210 215 220 Arg Leu Arg Ala Leu Cys Asp Gln His Gly Ile Leu Leu Ile Ala Asp 225 230 235 240 Glu Val Gln Thr Gly Ala Gly Arg Thr Gly Thr Phe Phe Ala Met Glu                 245 250 255 Gln Met Gly Val Ala Pro Asp Leu Thr Thr Phe Ala Lys Ser Ile Ala             260 265 270 Gly Gly Phe Pro Leu Ala Gly Val Cys Gly Lys Ala Glu Tyr Met Asp         275 280 285 Ala Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Ser Pro Ile     290 295 300 Ala Cys Ala Ala Ala Leu Ala Val Ile Glu Val Phe Glu Glu Glu Lys 305 310 315 320 Leu Leu Asp Arg Ser Lys Ala Val Gly Glu Arg Leu Thr Ala Gly Leu                 325 330 335 Arg Glu Ile Gln Lys Lys Tyr Pro Ile Ile Gly Asp Val Arg Gly Leu             340 345 350 Gly Ser Met Ile Ala Val Glu Val Phe Glu Lys Gly Thr His Thr Pro         355 360 365 Asn Ala Ala Ala Val Gly Gln Val Val Ala Lys Ala Arg Glu Lys Gly     370 375 380 Leu Ile Leu Leu Ser Cys Gly Thr Tyr Gly Asn Val Leu Arg Ile Leu 385 390 395 400 Val Pro Leu Thr Ala Glu Asp Ala Leu Leu Asp Lys Gly Leu Ala Ile                 405 410 415 Ile Glu Glu Cys Phe Ala Glu Ile Ala             420 425 <210> 4 <211> 480 <212> PRT <213> Unknown <220> <223> glutarate semialdehyde dehydrogenase sequence <400> 4 Met Gln Leu Lys Asp Ala Gln Leu Phe Arg Gln Gln Ala Tyr Ile Asn   1 5 10 15 Gly Glu Trp Leu Asp Ala Asp Asn Gly Gln Thr Ile Lys Val Thr Asn              20 25 30 Pro Ala Thr Gly Glu Val Ile Gly Thr Val Pro Lys Met Gly Thr Ala          35 40 45 Glu Thr Arg Arg Ala Ile Glu Ala Ala Asp Lys Ala Leu Pro Ala Trp      50 55 60 Arg Ala Leu Thr Ala Lys Glu Arg Ser Ala Lys Leu Arg Arg Trp Phe  65 70 75 80 Glu Leu Met Ile Glu Asn Gln Asp Asp Leu Ala Arg Leu Met Thr Thr                  85 90 95 Glu Gln Gly Lys Pro Leu Ala Glu Ala Lys Gly Glu Ile Ala Tyr Ala             100 105 110 Ala Ser Phe Ile Glu Trp Phe Ala Glu Glu Ala Lys Arg Ile Tyr Gly         115 120 125 Asp Thr Ile Pro Gly His Gln Pro Asp Lys Arg Leu Ile Val Ile Lys     130 135 140 Gln Pro Ile Gly Val Thr Ala Ala Ile Thr Pro Trp Asn Phe Pro Ala 145 150 155 160 Ala Met Ile Thr Arg Lys Ala Gly Pro Ala Leu Ala Ala Gly Cys Thr                 165 170 175 Met Val Leu Lys Pro Ala Ser Gln Thr Pro Tyr Ser Ala Leu Ala Leu             180 185 190 Val Glu Leu Ala His Arg Ala Gly Ile Pro Ala Gly Val Leu Ser Val         195 200 205 Val Thr Gly Ser Ala Gly Glu Val Gly Gly Glu Leu Thr Gly Asn Ser     210 215 220 Leu Val Arg Lys Leu Ser Phe Thr Gly Ser Thr Glu Ile Gly Arg Gln 225 230 235 240 Leu Met Glu Glu Cys Ala Lys Asp Ile Lys Lys Val Ser Leu Glu Leu                 245 250 255 Gly Gly Asn Ala Pro Phe Ile Val Phe Asp Asp Ala Asp Leu Asp Lys             260 265 270 Ala Val Glu Gly Ala Ile Ile Ser Lys Tyr Arg Asn Asn Gly Gln Thr         275 280 285 Cys Val Cys Ala Asn Arg Ile Tyr Val Gln Asp Gly Val Tyr Asp Ala     290 295 300 Phe Ala Glu Lys Leu Ala Ala Ala Val Ala Lys Leu Lys Ile Gly Asn 305 310 315 320 Gly Leu Glu Glu Gly Thr Thr Thr Gly Pro Leu Ile Asp Gly Lys Ala                 325 330 335 Val Ala Lys Val Gln Glu His Ile Glu Asp Ala Val Ser Lys Gly Ala             340 345 350 Lys Val Leu Ser Gly Gly Lys Leu Ile Glu Gly Asn Phe Phe Glu Pro         355 360 365 Thr Ile Leu Val Asp Val Pro Lys Thr Ala Ala Val Ala Lys Glu Glu     370 375 380 Thr Phe Gly Pro Leu Ala Pro Leu Phe Arg Phe Lys Asp Glu Ala Glu 385 390 395 400 Val Ile Ala Met Ser Asn Asp Thr Glu Phe Gly Leu Ala Ser Tyr Phe                 405 410 415 Tyr Ala Arg Asp Met Ser Arg Val Phe Arg Val Ala Glu Ala Leu Glu             420 425 430 Tyr Gly Met Val Gly Ile Asn Thr Gly Leu Ile Ser Asn Glu Val Ala         435 440 445 Pro Phe Gly Gly Ile Lys Ala Ser Gly Leu Gly Arg Glu Gly Ser Lys     450 455 460 Tyr Gly Ile Glu Asp Tyr Leu Glu Ile Lys Tyr Leu Cys Ile Ser Val 465 470 475 480

Claims (11)

1) 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB) 효소, 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;
2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 미생물에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 재조합 미생물을 제조하는 단계를 포함하는 제조방법으로 제조된 글루타릭산(glutaric acid) 생산용 재조합 미생물.
1) Lysine 2-monooxygenase (DaB) enzyme, delta-aminovaleramidase (Dava A) enzyme, 5-aminovalerate aminotransferase (DaT) Preparing an expression vector comprising one or more genes encoding an enzyme and a glutarate semialdehyde dehydrogenase (DVD) enzyme;
2) Glutaric acid prepared by the production method comprising the step of transforming the microorganisms of one or more expression vectors prepared in step 1) to produce a recombinant microorganism containing all of the DavB, DavA, DavT and DavD gene ( glutaric acid) Recombinant microorganism for production.
제 1항에 있어서, 상기 미생물은 대장균은 것을 특징으로 하는 글루타릭산 생산용 재조합 미생물.
The recombinant microorganism for producing glutaric acid according to claim 1, wherein the microorganism is Escherichia coli.
제 1항에 있어서, 상기 단계 1)의 발현 벡터는
라이신 2-단일산화효소(DavB) 효소 및 델타-아미노발레라미다아제(DavA) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터; 및
5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터인 것을 특징으로 하는 글루타릭산(glutaric acid) 생산용 재조합 미생물.
According to claim 1, wherein the expression vector of step 1)
A recombinant vector incorporating a gene encoding a lysine 2-monooxidase (DavB) enzyme and a delta-aminovaleramidase (DavA) enzyme into an expression vector; And
Glutaric acid, characterized in that it is a recombinant vector inserted into the expression vector gene encoding the 5-aminovalate aminotransferase (DavT) enzyme and glutarate semialdehyde dehydrogenase (DavD) enzyme ) Recombinant microorganisms for production.
제 1항 또는 제 3항에 있어서, 상기 DavB는 서열번호 1로 기재되는 염기서열을 갖는 것을 특징으로 하는 글루타릭산 생산용 재조합 미생물.
The recombinant microorganism for producing glutaric acid according to claim 1 or 3, wherein the DavB has a nucleotide sequence represented by SEQ ID NO: 1.
제 1항 또는 제 3항에 있어서, 상기 DavA는 서열번호 2로 기재되는 염기서열을 갖는 것을 특징으로 하는 글루타릭산 생산용 재조합 미생물.
The recombinant microorganism for producing glutaric acid according to claim 1 or 3, wherein DavA has a nucleotide sequence as set forth in SEQ ID NO: 2.
제 1항 또는 제 3항에 있어서, 상기 DavT는 서열번호 3으로 기재되는 염기서열을 갖는 것을 특징으로 하는 글루타릭산 생산용 재조합 미생물.
The recombinant microorganism for producing glutaric acid according to claim 1 or 3, wherein the DavT has a nucleotide sequence represented by SEQ ID NO: 3.
제 1항 또는 제 3항에 있어서, 상기 DavD는 서열번호 4로 기재되는 염기서열을 갖는 것을 특징으로 하는 글루타릭산 생산용 재조합 미생물.
The recombinant microorganism for producing glutaric acid according to claim 1 or 3, wherein the DavD has a nucleotide sequence represented by SEQ ID NO: 4.
1) 라이신 2-단일산화효소(lysine 2-monooxygenase; DavB) 효소, 델타-아미노발레라미다아제(delta-aminovaleramidase; DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(5-aminovalerate aminotransferase; DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(glutarate semialdehyde dehydrogenase; DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;
2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 대장균에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 글루타릭산(glutaric acid) 생산용 재조합 대장균 균주를 제조하는 방법.
1) Lysine 2-monooxygenase (DaB) enzyme, delta-aminovaleramidase (Dava A) enzyme, 5-aminovalerate aminotransferase (DaT) Preparing an expression vector comprising one or more genes encoding an enzyme and a glutarate semialdehyde dehydrogenase (DVD) enzyme;
2) A method for preparing a recombinant E. coli strain for producing glutaric acid (glutaric acid) containing all of the DavB, DavA, DavT and DavD gene by transforming E. coli with one or more expression vectors prepared in step 1).
1) 라이신 2-단일산화효소(DavB) 효소, 델타-아미노발레라미다아제(DavA) 효소, 5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자 중 1개 또는 그 이상의 유전자를 포함하는 발현 벡터를 제조하는 단계;
2) 상기 단계 1)에서 제조한 1개 이상의 발현 벡터를 대장균 균주에 형질전환시켜 DavB, DavA, DavT 및 DavD 유전자 모두가 포함된 재조합 대장균 균주를 제조하는 단계;
3) 단계 2)의 재조합 대장균 균주를 글루코스(glucose) 및 라이신(lysine)을 포함하는 배지에 배양하는 단계; 및
4) 단계 3)의 배양액으로부터 글루타릭산을 수득하는 단계를 포함하는 글루타릭산 생산방법.
1) Lysine 2-monooxidase (DavB) enzyme, delta-aminovaleramidase (DavA) enzyme, 5-aminovalate aminotransferase (DavT) enzyme and glutarate semialdehyde dehydrogenase (DavD) Preparing an expression vector comprising one or more genes of a gene encoding an enzyme;
2) transforming the E. coli strain with one or more expression vectors prepared in step 1) to prepare a recombinant E. coli strain comprising all of the DavB, DavA, DavT and DavD genes;
3) culturing the recombinant E. coli strain of step 2) in a medium containing glucose and lysine; And
4) Glutaric acid production method comprising the step of obtaining glutaric acid from the culture medium of step 3).
제 9항에 있어서, 상기 단계 1)의 발현 벡터는
라이신 2-단일산화효소(DavB) 효소 및 델타-아미노발레라미다아제(DavA) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터; 및
5-아미노발레이트 아미노트렌스퍼라제(DavT) 효소 및 글루타레이트 세미알데하이드 디하이드로게나아제(DavD) 효소를 코딩하는 유전자를 발현벡터 내로 삽입한 재조합 벡터인 것을 특징으로 하는 글루타릭산 생산방법.
The method of claim 9, wherein the expression vector of step 1)
A recombinant vector incorporating a gene encoding a lysine 2-monooxidase (DavB) enzyme and a delta-aminovaleramidase (DavA) enzyme into an expression vector; And
A method for producing glutaric acid, characterized in that it is a recombinant vector in which a gene encoding a 5-aminovalate aminotransferase (DavT) enzyme and a glutarate semialdehyde dehydrogenase (DavD) enzyme are inserted into an expression vector.
제 9항에 있어서, 상기 단계 3)의 배양은 48 내지 96 시간 동안 20 내지 40℃에서 실시하는 것을 특징으로 하는 글루타릭산 생산방법.



10. The method for producing glutaric acid according to claim 9, wherein the culturing of step 3) is performed at 20 to 40 ° C for 48 to 96 hours.



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WO2019228937A1 (en) * 2018-05-28 2019-12-05 Universitaet Des Saarlandes Means and methods for the production of glutarate
KR20200022605A (en) * 2018-08-23 2020-03-04 한국화학연구원 Recombinant Corynebacterium glutamicum strain for producing glutaric acid and a method of producing glutaric acid using the same
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