KR20230090947A - Carbon source-inducible expression vector expressable in methylorubrum strain and gene expression system using same - Google Patents
Carbon source-inducible expression vector expressable in methylorubrum strain and gene expression system using same Download PDFInfo
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- KR20230090947A KR20230090947A KR1020210180175A KR20210180175A KR20230090947A KR 20230090947 A KR20230090947 A KR 20230090947A KR 1020210180175 A KR1020210180175 A KR 1020210180175A KR 20210180175 A KR20210180175 A KR 20210180175A KR 20230090947 A KR20230090947 A KR 20230090947A
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- methylorubrum
- gene expression
- hpdr
- strain
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Abstract
Description
본 발명은 메틸로루브룸속 균주에서 탄소원 유도 발현이 가능한 발현 벡터 및 이를 이용한 유전자 발현 시스템에 관한 것이다.The present invention relates to an expression vector capable of carbon source-induced expression in strains of the genus Methylolrubrum and a gene expression system using the same.
연료, 화학 물질 및 기타 부가가치 제품을 생산하기 위한 공급원료로서 단일 탄소(C1) 화합물과 미생물을 이용하는 것은 화석 연료의 고갈 및 환경 문제를 해결하기 위한 이상적인 전략으로 평가된다. 그 중 포름산염(formate) 및 메탄올(methanol)과 같은 환원된 단일 탄소(C1) 화합물을 비교적 저렴한 비용으로 합성할 수 있으며 다양한 산업에서 발생하는 풍부하므로, 지속 가능하고 친환경적인 미생물의 공급원료로 평가된다(Belkhelfa et al., Front Microbiol. 2019 Jun 20;10:1313). Using single carbon (C1) compounds and microorganisms as feedstocks to produce fuels, chemicals and other value-added products is considered an ideal strategy to address fossil fuel depletion and environmental challenges. Among them, reduced single carbon (C1) compounds such as formate and methanol can be synthesized at a relatively low cost and are abundant in various industries, so they are evaluated as sustainable and environmentally friendly microbial feedstocks. (Belkhelfa et al ., Front Microbiol. 2019 Jun 20;10:1313).
한편, 메틸영양미생물 중 메틸영양 알파-프로테오박테리움인 Methylorubrum extorquens AM1은 포르메이트(C1) 및 메탄올(C1)과 같은 단일 탄소뿐만 아니라 아세테이트(C2), 피루브산(C3) 및 석시네이트(C4)와 같은 다중 탄소 화합물을 모두 사용할 수 있다. 또한, M. extorquens은 다양한 대사 모델 및 과발현, 녹아웃 및 트랜스포존 돌연변이 유발 등이 용이한 유전자 세트를 가지고 있어 생명공학적 적용을 위한 매력적인 숙주로 평가받고 있다.On the other hand, among methylotrophic microorganisms, Methylorubrum extorquens AM1, a methylotrophic alpha-proteobacterium, contains not only single carbons such as formate (C1) and methanol (C1), but also acetate (C2), pyruvic acid (C3) and succinate (C4). Any multi-carbon compound such as can be used. In addition, M. extorquens is evaluated as an attractive host for biotechnology applications because it has various metabolic models and gene sets that are easy to overexpress, knockout, and transposon mutagenesis.
그러나, 상기 M. extorquens의 유전자를 과발현 시키는데 사용되는 λPL 및 λPR 프로모터는 비활성 상태인 경우가 많으며, Plac 프로모터는 M. extorquens에서 약한 발현을 나타내므로 적합하지 않은 것으로 알려져 있다. 또한, M. extorquens의 유전자 발현에 사용된다 보고된 PmxaF, PcoxB, PfumC 및 Ptuf 프로모터 세트는 발현 시스템의 통제가 어려우며 이로인해 구조적 불안정한 표적 단백질을 생산하므로, 세포에 유해하다는 문제가 있었다(Schada Von Borzyskowski et al., ACS Synth Biol. 2015 Apr 17;4(4):430-43).However, the λPL and λPR promoters used to overexpress the M. extorquens gene are often inactive, and the Plac promoter is known to be unsuitable because it shows weak expression in M. extorquens . In addition, the PmxaF, PcoxB, PfumC, and Ptuf promoter sets reported to be used for gene expression of M. extorquens are difficult to control in the expression system and produce structurally unstable target proteins, which is harmful to cells (Schada Von Borzyskowski et al ., ACS Synth Biol. 2015 Apr 17;4(4):430-43).
따라서, M. extorquens에서도 화학적 유도제 없이 안정적으로 유전자를 발현시킬 수 있는 시스템 또는 방법에 대한 개발이 필요한 실정이다. 또한, M. extorquens을 이용한 생합성 과정에서 이용되는 기질과 이를 통해 생산된 생산물에 대한 안정적인 유전자 발현 시스템은 미생물의 생합성을 이용하는 폭 넓은 분야의 산업에서 이용 가능하므로 지속적인 개발이 필요하다. Therefore, it is necessary to develop a system or method capable of stably expressing genes in M. extorquens without chemical inducers. In addition, a stable gene expression system for the substrate used in the biosynthesis process using M. extorquens and the product produced therethrough is available in a wide range of industries using microbial biosynthesis, so continuous development is required.
이에 본 발명자들은 메틸로루브룸속 균주에서 안정적으로 유전자를 발현하는 방법에 대해 연구한 결과, 탄소원 기질을 유도제로 하는 유전자 발현 벡터 및 이를 메틸로루브룸속 균주에 도입시켜 유전자 발현이 안정적으로 증가하는 것을 확인하여 본 발명을 완성하였다.Accordingly, the present inventors studied a method for stably expressing genes in strains belonging to the genus Methylolubrum. As a result, a gene expression vector using a carbon source substrate as an inducer and a gene expression vector stably increased by introducing the same into strains belonging to the genus Methylolubrum It was confirmed that the present invention was completed.
상기 목적을 달성하기 위하여, 본 발명의 일 측면은, HpdR 전사인자를 코딩하는 유전자 및 P hpdH 프로모터를 포함하는 3-하이드록시프로피온산 또는 레불린산의 존재시 메틸로루브룸속 균주에서 발현 가능한 벡터를 제공하는 것이다. In order to achieve the above object, an aspect of the present invention is a vector that can be expressed in Methylolorubrum strains in the presence of 3-hydroxypropionic acid or levulinic acid containing a gene encoding an HpdR transcription factor and a PhpdH promoter is to provide
본 발명의 다른 측면은, 상기 발현 벡터를 포함하는 형질전환체를 제공하는 것이다.Another aspect of the present invention is to provide a transformant comprising the expression vector.
본 발명의 또 다른 측면은, HpdR 전사인자를 코딩하는 유전자 및 P hpdH 프로모터를 포함하는 발현 벡터를 포함하는 메틸로루브룸속 균주를 3-하이드록시프로피온산 또는 레불린산의 존재 하에 배양하는 단계를 포함하는 유전자 발현 방법을 제공하는 것이다. Another aspect of the present invention includes the steps of culturing a strain of the genus Methylolorubrum containing an expression vector containing a gene encoding an HpdR transcription factor and a P hpdH promoter in the presence of 3-hydroxypropionic acid or levulinic acid. It is to provide a gene expression method comprising.
본 발명의 발현 벡터 및 이를 포함하는 형질전환체를 이용함으로써, 저비용의 탄소원 기질을 유도제로 하여 안정적이고 효율적으로 유전자를 발현시킬 수 있으며 상기 형질전환체를 배양하는 데 있어서 탄소원 기질의 농도를 조절함으로써 유전자 발현을 조절할 수 있다. By using the expression vector of the present invention and a transformant containing the same, stable and efficient gene expression can be achieved using a low-cost carbon source substrate as an inducer, and by controlling the concentration of the carbon source substrate in culturing the transformant Can regulate gene expression.
따라서, 본 발명의 발현 벡터 및 이를 포함하는 형질전환체는 미생물을 이용한 생화학 내지 생물학적 물질 생산에 유용하게 활용할 수 있다.Therefore, the expression vector of the present invention and a transformant containing the same can be usefully used for biochemical or biological material production using microorganisms.
도 1은 본 발명의 일 실시예에서 사용한 탄소원 기질 유도성 유전자 발현 카세트를 포함하는 발현 벡터의 모식도이다.
도 2는 메틸로루브룸속 균주의 탄소원 기질 유도성 유전자 발현 시스템과 IPTG 유도성 유전자 발현 시스템의 유전자 발현량을 형광 강도 측정을 통해 비교한 것이다.
도 3은 HpdR/P hpdH 유전자 발현 시스템과 LacI/P L/O4 유전자 발현 시스템의 유전자 발현량을 유세포 분석을 통해 비교한 것이다.
도 4는 레불린산(LA) 첨가 시간별 HpdR/P hpdH 유전자 발현 시스템의 발현량을 형광 강도 측정을 통해 확인한 것이다.
도 5는 레불린산 첨가 농도별 HpdR/P hpdH 유전자 발현 시스템의 발현량을 형광 강도 측정을 통해 확인한 것이다.
도 6은 레불린산 첨가 농도별 HpdR/P hpdH 유전자 발현 시스템의 발현량을 MHRH01의 세포 성장을 통해 확인한 것이다.
도 7은 첨가 농도별 HpdR/P hpdH 유전자 발현 시스템의 발현량을 유세포 분석을 통해 확인한 것이다.
도 8은 pMHRH-eGFP+ 벡터를 도입한 여러 Methylorubrum 균주(MHRH01, CM4, DM4, PA1, TK0001 및 ATCC55366)의 유전자 발현량을 형광 강도 측정을 통해 확인한 것이다.
도 9는 형질전환체 MHRH01의 레불린산(LA) 첨가 직후, 24 시간 후 및 48시간 후 LA 잔류량을 확인한 것이다.
도 10은 형질전환체 MHRH01 및 MPLO4의 eGFP+ 발현량을 SDS-PAGE를 통해 확인한 것이다.
도 11은 형질전환체 MHRH01 및 MPLO4이 생성한 GFP 단백질을 정량 분석한 것이다. 1 is a schematic diagram of an expression vector including a carbon source substrate inducible gene expression cassette used in an embodiment of the present invention.
Figure 2 compares the gene expression levels of the carbon source substrate inducible gene expression system and the IPTG inducible gene expression system of strains of the genus Methylolrubrum through fluorescence intensity measurement.
3 is HpdR / P hpdH The gene expression levels of the gene expression system and the LacI/P L/O4 gene expression system were compared through flow cytometry.
Figure 4 is HpdR / P hpdH by levulinic acid (LA) addition time The expression level of the gene expression system was confirmed by measuring the fluorescence intensity.
5 is HpdR / P hpdH by levulinic acid addition concentration The expression level of the gene expression system was confirmed by measuring the fluorescence intensity.
6 is HpdR / P hpdH by levulinic acid addition concentration The expression level of the gene expression system was confirmed through cell growth of MHRH01.
7 is HpdR / P hpdH by addition concentration The expression level of the gene expression system was confirmed through flow cytometry.
Figure 8 confirms the gene expression level of several Methylorubrum strains (MHRH01, CM4, DM4, PA1, TK0001 and ATCC55366) introduced with pMHRH-eGFP + vector through fluorescence intensity measurement.
Figure 9 confirms the amount of LA remaining immediately after addition of levulinic acid (LA) in the transformant MHRH01, after 24 hours, and after 48 hours.
10 shows the eGFP + expression level of transformants MHRH01 and MPLO4 through SDS-PAGE.
11 is a quantitative analysis of GFP proteins produced by transformants MHRH01 and MPLO4.
이하, 본 발명을 구체적으로 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 측면은 HpdR 전사인자를 코딩하는 유전자 및 P hpdH 프로모터를 포함하는 3-하이드록시프로피온산 또는 레불린산의 존재시 메틸로루브룸속 균주에서 발현 가능한 벡터를 제공한다.One aspect of the present invention provides a vector capable of being expressed in Methylolorubrum strains in the presence of 3-hydroxypropionic acid or levulinic acid, including a gene encoding an HpdR transcription factor and a PhpdH promoter.
상기 HpdR 전사인자는 LysR 타입의 전사활성인자로서, 3-하이드록시프로피온산(3-HP)의 이화 작용을 조절할 수 있으며 3-HP가 HpdR에 결합함으로써, 하위 유전자(downstream gene)의 프로모터 서열과 RNA 중합효소의 결합을 유도할 수 있다. 본 발명의 하나의 태양에 따르면, 상기 HpdR 전사인자는 서열번호 1로 표시되는 아미노산 서열을 포함할 수 있으며, 상기 아미노산 서열은 서열번호 2로 표시되는 염기서열로 코딩될 수 있다.The HpdR transcription factor is a LysR-type transcriptional activator, which can regulate the catabolic action of 3-hydroxypropionic acid (3-HP), and when 3-HP binds to HpdR, the promoter sequence and RNA of a downstream gene binding of the polymerase may be induced. According to one aspect of the present invention, the HpdR transcription factor may include the amino acid sequence represented by SEQ ID NO: 1, and the amino acid sequence may be encoded by the nucleotide sequence represented by SEQ ID NO: 2.
상기 P hpdH 프로모터는 HpdR 전사인자와 결합하는 염기서열이며, 구체적으로 HpdR 전사인자를 코딩하는 유전자와 HpdR 전사인자가 조절하는 hpdH 유전자 사이에 존재할 수 있다. 본 발명의 하나의 태양에 따르면, 상기 P hpdH 프로모터는 서열번호 3으로 표시되는 염기서열을 포함할 수 있다.The P hpdH promoter is a nucleotide sequence that binds to the HpdR transcription factor, and may specifically exist between a gene encoding the HpdR transcription factor and the hpdH gene regulated by the HpdR transcription factor. According to one aspect of the present invention, the P hpdH promoter may include the nucleotide sequence represented by SEQ ID NO: 3.
본 발명의 용어 "전사인자"는 DNA의 특정 부위에 결합하여 유전자의 발현을 촉진하거나 억제하는 단백질을 의미한다. 특히, 전사인자는 DNA-결합 영역(DNA-binding domain, DBD)과 트랜스크립션 조절 영역(transcriptional regulation domain)을 갖는다.The term "transcription factor" of the present invention refers to a protein that binds to a specific site of DNA and promotes or inhibits gene expression. In particular, transcription factors have a DNA-binding domain (DBD) and a transcriptional regulation domain.
본 발명의 용어 "프로모터"는 유전자의 기본 전사 및/또는 조절 전사에 필요한, 일반적으로 코딩 서열 바로 상류의 DNA 서열을 의미한다. 특히, 프로모터는 전사의 개시를 가능하게 하는 정보를 가지며, 일반적으로 전사 개시 시작 부위 및 중합효소 복합체에 대한 결합 부위를 갖는다.As used herein, the term "promoter" refers to a DNA sequence necessary for basic and/or regulated transcription of a gene, usually immediately upstream of a coding sequence. In particular, a promoter carries information enabling the initiation of transcription and usually has a transcription initiation start site and a binding site for the polymerase complex.
본 발명에서 사용되는 염기서열은, 서열 목록에 기재된 서열과 실질적인 동일성을 나타내는 서열을 포함하는 것으로 이해된다. 여기서, "실질적인 동일성"은 본 발명의 서열과 임의의 다른 서열로서 70% 이상의 상동성, 구체적으로 80% 이상의 상동성, 보다 더 구체적으로 90% 이상의 상동성을 갖는 서열이며, 이들 상동성 범위에 있는 염기서열도 본 발명의 권리범위에 포함되는 것으로 해석된다.The nucleotide sequence used in the present invention is understood to include sequences showing substantial identity to the sequences described in the sequence listing. Here, "substantially identical" is a sequence having at least 70% homology, specifically at least 80% homology, and more specifically at least 90% homology with any other sequence with the sequence of the present invention, within these homology ranges. It is also construed that the nucleotide sequence in the present invention is included in the scope of the present invention.
본 발명에 따르면, 상기 메틸로루브룸속 균주는 Methylorubrum extorquens, Methylorubrum aminovorans, Methylorubrum podarium, Methylorubrum pseudosasae, Methylorubrum rhodesianum, Methylorubrum rhodinum, Methylorubrum salsuginis 또는 Methylorubrum suomiense일 수 있으며, 구체적으로, Methylorubrum extorquens일 수 있다. 더욱 구체적으로, 메틸로루브룸속 균주는 Methylorubrum extorquens M1, Methylorubrum extorquens CM4, Methylorubrum extorquens DM4, Methylorubrum extorquens PA1, Methylorubrum extorquens TK0001 및 Methylorubrum extorquens ATCC55366 균주일 수 있으며, 본 발명의 일 실시예에서는 Methylorubrum extorquens AM1을 이용하였다.According to the present invention, the strain of the genus Methylorubrum may be Methylorubrum extorquens , Methylorubrum aminovorans , Methylorubrum podarium , Methylorubrum pseudosasae , Methylorubrum rhodesianum , Methylorubrum rhodinum , Methylorubrum salsuginis or Methylorubrum suomiense , specifically, Methylorubrum extorquens . More specifically, the genus Methylorubrum strains may be Methylorubrum extorquens M1, Methylorubrum extorquens CM4, Methylorubrum extorquens DM4, Methylorubrum extorquens PA1, Methylorubrum extorquens TK0001 and Methylorubrum extorquens ATCC55366 strains, and in one embodiment of the present invention, Methylorubrum extorquens AM1 used
본 발명의 용어 "발현 가능한 벡터(이하, 발현 벡터)"는 숙주세포에서 목적으로 하는 외래 유전자를 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동 가능하게 연결된 필수적인 조절 요소를 포함하는 벡터를 의미한다. The term "expressible vector (hereinafter, expression vector)" as used herein refers to a vector capable of expressing a foreign gene of interest in a host cell, and includes essential regulatory elements operably linked to express a gene insert. do.
상기 발현 벡터는 형질전환체에 의해서 발현되는 유전자 및 조절 서열로 구성되는 DNA 벡터로서, 오픈 리딩 프레임, 폴리아데닐화 부위를 포함하는 3' 비번역 영역을 추가로 포함할 수 있다. 또한, 공지의 방법에 의하여 인위적으로 디자인된 재조합 발현 벡터일 수 있다. The expression vector is a DNA vector composed of genes and regulatory sequences expressed by a transformant, and may further include an open reading frame and a 3' untranslated region including a polyadenylation site. In addition, it may be a recombinant expression vector artificially designed by a known method.
상기 발현 벡터는 공지된 다양한 벡터를 사용하여 제조할 수 있으며, 예를 들어, 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터 또는 바이러스 벡터 등을 사용할 수 있다. 본 발명의 일 실시예에서는, 상기 발현 벡터로 pCM110_P mxaF _Fdh1을 사용하였다.The expression vector can be prepared using various known vectors, and for example, a plasmid vector, cosmid vector, bacteriophage vector, or viral vector can be used. In one embodiment of the present invention, pCM110_P mxaF _Fdh1 was used as the expression vector.
본 발명의 다른 측면은, 상기 발현 벡터를 포함하는 형질전환체를 제공한다.Another aspect of the present invention provides a transformant comprising the expression vector.
본 발명의 용어 "형질전환체"는 외래의 유전물질을 포함하여 유전형질이 변화된 미생물과 같은 생물체를 의미한다. 본 발명에 따른 형질전환체는 유전자 조작이 용이하며 다양한 발현 시스템에 적용될 수 있으며, 대량 배양에 이용될 수 있다. As used herein, the term "transformant" refers to organisms such as microorganisms in which genetic traits have been changed by including foreign genetic material. The transformant according to the present invention is easy to genetically manipulate, can be applied to various expression systems, and can be used for mass culture.
본 발명에 따르면, 상기 형질전환체는 Methylorubrum extorquens, Methylorubrum aminovorans, Methylorubrum podarium, Methylorubrum pseudosasae, Methylorubrum rhodesianum, Methylorubrum rhodinum, Methylorubrum salsuginis 또는 Methylorubrum suomiense일 수 있으며, 구체적으로, Methylorubrum extorquens일 수 있다. 더욱 구체적으로, 상기 형질전환체는 Methylorubrum extorquens M1, Methylorubrum extorquens CM4, Methylorubrum extorquens DM4, Methylorubrum extorquens PA1, Methylorubrum extorquens TK0001 및 Methylorubrum extorquens ATCC55366 균주일 수 있으며, 본 발명의 일 실시예에서는 Methylorubrum extorquens AM1을 이용하였다.According to the present invention, the transformant may be Methylorubrum extorquens , Methylorubrum aminovorans , Methylorubrum podarium , Methylorubrum pseudosasae , Methylorubrum rhodesianum , Methylorubrum rhodinum , Methylorubrum salsuginis or Methylorubrum suomiense , specifically, Methylorubrum extorquens . More specifically, the transformant may be Methylorubrum extorquens M1, Methylorubrum extorquens CM4, Methylorubrum extorquens DM4, Methylorubrum extorquens PA1, Methylorubrum extorquens TK0001 and Methylorubrum extorquens ATCC55366 strains, and in one embodiment of the present invention, Methylorubrum extorquens AM1 was used. .
상기 발현 벡터는 형질전환 방법을 통해 미생물, 균주 내로 도입시킬 수 있다.The expression vector can be introduced into a microorganism or strain through a transformation method.
상기 형질전환시키는 방법으로는, 당해 분야에서 공지된 방법을 이용할 수 있으며, 예를 들면, 일시적 형질감염(transient transfection), 미세주사, 형질도입(transduction), 세포융합, 칼슘 포스페이트 침전법, 리포좀 매개된 형질감염(liposome-mediated transfection), DEAE 덱스트란-매개된 형질감염(DEAE Dextran-mediated transfection), 폴리브렌-매개된 형질감염(polybrene-mediated transfection), 전기침공법(electroporation), 유전자 총(gene gun) 또는 세포 내로 핵산을 유입시키기 위한 다른 공지의 방법에 의해 세포 내로 도입할 수 있지만, 이에 제한되는 것은 아니다. 본 발명의 일 실시예에서는 전기천공법을 사용하였다.As the method for the transformation, methods known in the art may be used, and for example, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, and liposome mediation. Liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun ( gene gun) or other known methods for introducing nucleic acids into cells, but are not limited thereto. In one embodiment of the present invention, electroporation was used.
본 발명에 따르면 HpdR 전사인자를 코딩하는 유전자 및 P hpdH 프로모터를 포함하는 메틸로루브룸속 균주를 3-하이드록시프로피온산 또는 레불린산 존재 하에 배양하는 단계를 포함할 수 있다.According to the present invention, a step of culturing a strain of the genus Methylorubrum containing a gene encoding an HpdR transcription factor and a P hpdH promoter in the presence of 3-hydroxypropionic acid or levulinic acid may be included.
상기 메틸로루브룸속 균주를 배양하는 단계는 균주를 MSM 배지에서 전-배양(pre-cultures)하는 단계를 포함할 수 있다. 본 발명의 일 실시예에서는, 균주를 리터당 1.62 g NH4Cl, 0.2 g MgSO4, 2.21 g K2HPO4, 1.25 g NaH2PO42H2O, 15 mg Na2EDTA2H2O, 4.5 mg ZnSO47H2O, 0.3 mg CoCl26H2O, 1 mg MnCl24H2O, 1 mg H3BO3, 2.5 mg CaCl2, 0.4 mg of Na2MoO42H2O, 3 mg FeSO47H2O, 0.3 mg CuSO45H2O, and 4 g succinate를 포함하는 MSM 배지에서 전-배양하였다.The step of culturing the strain of the genus Methylorubrum may include pre-culturing the strain in MSM medium. In one embodiment of the present invention, the strain is 1.62 g NH 4 Cl, 0.2 g MgSO 4 , 2.21 g K 2 HPO 4 , 1.25 g NaH 2 PO 4 2H2O, 15 mg Na 2 EDTA2H 2 O, 4.5 mg ZnSO 4 7H2O per liter , 0.3 mg CoCl 2 6H 2 O, 1 mg MnCl 2 4H 2 O, 1 mg H 3 BO 3 , 2.5 mg CaCl 2 , 0.4 mg of Na 2 MoO 4 2H 2 O, 3 mg FeSO 4 7H 2 O, 0.3 mg CuSO 4 They were pre-cultured in MSM medium containing 5H 2 O, and 4 g succinate.
본 발명의 또 다른 측면은, HpdR 전사인자를 코딩하는 유전자 및 P hpdH 프로모터를 포함하는 발현 벡터를 포함하는 메틸로루브룸속 균주를 3-하이드록시프로피온산 또는 레불린산의 존재 하에 배양하는 단계를 포함하는, 유전자 발현 방법을 제공한다.Another aspect of the present invention includes the steps of culturing a strain of the genus Methylolorubrum containing an expression vector containing a gene encoding an HpdR transcription factor and a P hpdH promoter in the presence of 3-hydroxypropionic acid or levulinic acid. Including, it provides a gene expression method.
상기 메틸로루브룸속 균주를 레불린산의 존재하에 배양하는 경우 레불린산은 0.01 mM 내지 50 mM, 0.01 mM 내지 45 mM, 0.01 mM 내지 40 mM, 0.01 mM 내지 35 mM 0.01 mM 내지 30 mM, 0.05mM 내지 25mM, 0.1mM 내지 20mM의 농도로 포함될 수 있으며, 구체적으로, 0.1 mM 내지 20 mM의 농도로 포함될 수 있다. 상기 농도 범위일 때, 최소한의 탄소원 기질을 사용하여 유전자를 최대로 발현시키는 것이 가능할 수 있다.When the Methylolubrum strain is cultured in the presence of levulinic acid, levulinic acid is 0.01 mM to 50 mM, 0.01 mM to 45 mM, 0.01 mM to 40 mM, 0.01 mM to 35 mM 0.01 mM to 30 mM, 0.05 mM It may be included at a concentration of mM to 25 mM, 0.1 mM to 20 mM, and specifically, may be included at a concentration of 0.1 mM to 20 mM. In the above concentration range, it may be possible to maximize gene expression using a minimum amount of carbon source substrate.
본 발명의 일실시예에서는, 레불린산 농도에 따른 메틸로루브룸속 균주의 유전자 발현을 확인하였다. 구체적으로, 레불린산을 0.1 에서 20 mM까지 농도를 증가시킴에 따라 유전자 발현이 증가하는 것을 확인하였다. 반면, 레불린산을 20 mM 이상으로 농도를 증가시킴에 따라 형광 강도가 감소하는 것을 확인하였으며, 특히, 40, 50, 및 100 mM의 레불린산을 첨가할 때는 20 mM의 레불린산을 첨가할 때에 비해 각각 약10%, 28%, 및 68% 감소된 효율이 나타내는 것을 확인하였다. 또한, 0.1 내지 20 mM의 농도롤 첨가한 샘플들의 경우, 유전자 발현이 균일하게 유지됨을 확인하였다. In one embodiment of the present invention, the gene expression of strains belonging to the genus Methylolorubrum according to the concentration of levulinic acid was confirmed. Specifically, it was confirmed that gene expression increased as the concentration of levulinic acid increased from 0.1 to 20 mM. On the other hand, it was confirmed that the fluorescence intensity decreased as the concentration of levulinic acid increased to 20 mM or higher. In particular, when 40, 50, and 100 mM levulinic acid was added, 20 mM levulinic acid was added. It was confirmed that the efficiency decreased by about 10%, 28%, and 68%, respectively, compared to when In addition, in the case of samples added at a concentration of 0.1 to 20 mM, it was confirmed that gene expression was maintained uniformly.
상기 유전자 발현 방법에 있어 배양 시간은 1시간 내지 58시간, 2시간 내지 56시간, 3시간 내시 54시간, 4시간 내지 52시간, 5시간 내지 50시간, 또는 6시간 내지 48시간일 수 있으며, 바람직하게, 6시간 내지 48시간일 수 있다.In the gene expression method, the culture time may be 1 hour to 58 hours, 2 hours to 56 hours, 3 hours, 54 hours, 4 hours to 52 hours, 5 hours to 50 hours, or 6 hours to 48 hours, preferably. Preferably, it may be 6 to 48 hours.
이하, 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.
참고예: 유전자 발현 시스템 Reference Example: Gene Expression System
유전자 발현 시스템의 세부사항을 하기 표 1에 나타낸다. 또한, 표 2에 전사인자 및 프로모터의 유전자 서열정보를 나타낸다.Details of the gene expression system are shown in Table 1 below. In addition, Table 2 shows gene sequence information of transcription factors and promoters.
HpdR
제조예 1. HpdR/PPreparation Example 1. HpdR/P hpdHhpdH 유전자 발현 카세트를 포함하는 발현 벡터 제조 Preparation of expression vectors containing gene expression cassettes
HpdR/P hpdH 유전자 발현 카세트를 포함하는 발현 벡터를 제조하기 위해서, 먼저, P. putida KT2440 균주의 게놈 DNA를 주형으로 하며 HpdR/P hpdH 유전자 발현 카세트를 포함하는 pPROBE-eGFP+ 벡터(이하, pHRH_eGFP+)를 제조하였다(Sathesh-Prabu et al., Scientifc Reports, 2021, 11:18079).In order to prepare an expression vector containing the HpdR/P hpdH gene expression cassette, first, pPROBE-eGFP + vector (hereinafter referred to as pHRH_eGFP) containing the HpdR/P hpdH gene expression cassette using the genomic DNA of the P. putida KT2440 strain as a template was prepared. + ) was prepared (Sathesh-Prabu et al ., Scientific Reports, 2021, 11:18079).
이후, 상기 pHRH_eGFP+를 주형으로 하여 Q5TM DNA 중합효소(New England Biolabs, USA)를 이용해서 하기 표 3의 조건으로 PCR을 진행하였다. 이때, PCR에 사용한 프라이머는 하기 표 4에 나타내었다. Subsequently, PCR was performed under the conditions shown in Table 3 below using Q5 TM DNA polymerase (New England Biolabs, USA) using the pHRH_eGFP + as a template. At this time, the primers used for PCR are shown in Table 4 below.
상기 PCR을 통해 얻은 유전자 단편을 인서트 DNA로 사용하고, pCM110_P mxaF _Fdh1 벡터를 XbaI/BamHI 제한효소로 절단하여 백본 벡터로 사용하였다. 이때, 인서트 DNA와 벡본 벡터의 라이게이션은 Gibson assembly cloning kit(New England Biolabs, USA)를 이용하여 수행하였다. 상기의 과정을 통해 제조된 HpdR/P hpdH 유전자 발현 카세트를 포함하는 발현 벡터를 pMHRH_eGFP+로 명명하였다.The gene fragment obtained through the PCR was used as an insert DNA, and the pCM110_P mxaF _Fdh1 vector was digested with Xba I/ BamH I restriction enzymes and used as a backbone vector. At this time, ligation of the insert DNA and the backbone vector was performed using the Gibson assembly cloning kit (New England Biolabs, USA). The expression vector containing the HpdR/P hpdH gene expression cassette prepared through the above process was named pMHRH_eGFP + .
제조예 2. HexR/PPreparation Example 2. HexR/P zwf1zwf1 ,, LvaR/PLvaR/P lvaAlvaA 또는 MmsR/P or MmsR/P mmsAmmsA 유전자 발현 카세트를 포함하는 발현 벡터 제조 Preparation of expression vectors containing gene expression cassettes
HexR/P zwf1 , LvaR/P lvaA 또는 MmsR/P mmsA 유전자 발현 카세트를 포함하는 발현 벡터를 제조하기 위해서, 먼저, P. putida KT2440 균주의 게놈 DNA를 주형으로 하며 HexR/P zwf1 , LvaR/P lvaA 또는 MmsR/P mmsA 유전자 발현 카세트를 포함하는 pPROBE-eGFP+ 벡터를 각각 제조하였다(Sathesh-Prabu et al., Scientifc Reports, 2021, 11:18079).To prepare an expression vector containing HexR/P zwf1 , LvaR/P lvaA or MmsR/P mmsA gene expression cassettes, first, genomic DNA of P. putida KT2440 strain is used as a template and HexR/P zwf1 , LvaR/P lvaA Alternatively, a pPROBE-eGFP + vector containing the MmsR/P mmsA gene expression cassette was prepared (Sathesh-Prabu et al ., Scientifc Reports, 2021, 11:18079).
이후, 상기 제조예 1과 동일한 방법 및 조건으로 PCR을 수행하였으며, 얻어진 DNA 단편을 인서트로 하여 pCM110_P mxaF _Fdh1 벡터에 각각 클로닝하였다. 상기 과정을 통해 제조된 HexR/P zwf1 유전자 발현 카세트를 포함하는 발현 벡터는 pMHRZ_eGFP+, LvaR/P lvaA 유전자 발현 카세트를 포함하는 발현 벡터는 pMLRL_eGFP+, 그리고 MmsR/P mmsA 유전자 발현 카세트를 포함하는 발현 벡터는 pMMRM_eGFP+로 각각 명명하였다.Thereafter, PCR was performed in the same manner and conditions as in Preparation Example 1, and the obtained DNA fragments were cloned into pCM110_P mxaF _Fdh1 vectors as inserts, respectively. The expression vector containing the HexR / P zwf1 gene expression cassette prepared through the above process was pMHRZ_eGFP + , the expression vector containing the LvaR / P lvaA gene expression cassette was pMLRL_eGFP + , and the expression vector containing the MmsR / P mmsA gene expression cassette The vectors were each named pMMRM_eGFP + .
제조예 3. XutR/PPreparation Example 3. XutR/P xutAxutA 유전자 발현 카세트를 포함하는 발현 벡터 제조 Preparation of expression vectors containing gene expression cassettes
XutR/P xutA 유전자 발현 카세트를 포함하는 발현 벡터를 제조하기 위해서, 먼저, P. fluorescens SBW25 균주의 게놈 DNA를 주형으로 하며 XutR/P xutA 유전자 발현 카세트를 포함하는 pPROBE-eGFP+ 벡터를 제조하였다(Sathesh-Prabu et al., Scientifc Reports, 2021, 11:18079).In order to prepare an expression vector containing the XutR / P xutA gene expression cassette, first, pPROBE-eGFP + vector containing the XutR / P xutA gene expression cassette was prepared using the genomic DNA of the P. fluorescens SBW25 strain as a template ( Sathesh-Prabu et al ., Scientifc Reports, 2021, 11:18079).
이후, 상기 제조예 1과 동일한 방법 및 조건으로 PCR을 수행하였으며, 얻어진 DNA 단편을 인서트로 하여 pCM110_P mxaF _Fdh1 벡터에 클로닝하였다. 상기 과정을 통해 제조된 XutR/P xutA 유전자 발현 카세트를 포함하는 발현 벡터를 pMXRX_eGFP+로 명명하였다.Thereafter, PCR was performed in the same manner and conditions as in Preparation Example 1, and the obtained DNA fragment was used as an insert and cloned into the pCM110_P mxaF _Fdh1 vector. The expression vector containing the XutR/P xutA gene expression cassette prepared through the above process was named pMXRX_eGFP + .
제조예 4. LacI/PPreparation Example 4. LacI/P L/O4L/O4 유전자 발현 카세트를 포함하는 발현 벡터 제조 Preparation of expression vectors containing gene expression cassettes
E.coli MG1655균주의 게놈 DNA를 주형으로 하며 LacI/P L/O4 유전자 발현 카세트를 포함하는 pCM110 플라스미드의 제조는 ACS Synth. Biol. 8, 2451-2456. doi:10.1021/acssynbio.9b00220.를 참고하였다. Preparation of the pCM110 plasmid containing the LacI/P L/O4 gene expression cassette using the genomic DNA of E.coli strain MG1655 as a template was performed by ACS Synth. Biol. 8, 2451-2456. See doi:10.1021/acssynbio.9b00220.
구체적으로, E.coli MG1655균주의 게놈 DNA를 주형으로 하여, LacI 전사인자를 코딩하는 유전자 및 이에 결합하는 P L/O4 프로모터를 포함하는 영역을 Q5TM DNA 중합효소(New England Biolabs, USA), PL04-FP 및 PL04-RP 프라이머를 이용하여 하기 표 5의 조건으로 1차 PCR을 수행하였다. 이후, 제조된 1차 PCR 단편을 주형으로 PL04-FP 및 PL04-RP2 프라이머를 이용하여 하기 표 5의 조건으로 2차 PCR을 수행하였으며, PL04 promoter 단편을 제작하였다. 이때, PCR에 사용한 프라이머는 하기 표 6에 나타내었다. Specifically, using the genomic DNA of E.coli strain MG1655 as a template, the region including the gene encoding the LacI transcription factor and the P L/O4 promoter coupled thereto was converted into Q5 TM DNA polymerase (New England Biolabs, USA), Primary PCR was performed using the PL04-FP and PL04-RP primers under the conditions shown in Table 5 below. Then, using the prepared primary PCR fragment as a template and using the PL04-FP and PL04-RP2 primers, secondary PCR was performed under the conditions shown in Table 5 below, and a PL04 promoter fragment was prepared. At this time, the primers used for PCR are shown in Table 6 below.
상기 PCR을 통해 얻은 유전자 단편을 인서트 DNA로 사용하고, pCM110 벡터를 제한효소로 절단하여 백본 벡터로 사용하였다. 이때, 인서트 DNA와 벡본 벡터의 라이게이션은 Gibson assembly cloning kit(New England Biolabs, USA)를 이용하여 수행하였다. 상기의 과정을 통해 제조된 LacI/P L/O4 유전자 발현 카세트를 포함하는 pCM110 벡터를 pCM110_P L/O4 로 명명하였다.The gene fragment obtained through the PCR was used as an insert DNA, and the pCM110 vector was digested with a restriction enzyme and used as a backbone vector. At this time, ligation of the insert DNA and the backbone vector was performed using a Gibson assembly cloning kit (New England Biolabs, USA). The pCM110 vector containing the LacI/P L/O4 gene expression cassette prepared through the above process was named pCM110_P L/O4 .
이후, 상기 pCM110_P L/O4 를 주형으로 하여 Q5TM DNA 중합효소(New England Biolabs, USA)를 이용해서 하기 표 7의 조건으로 PCR을 진행하였다. 이때, PCR에 사용한 프라이머는 하기 표 8에 나타내었다. Thereafter, PCR was performed under the conditions shown in Table 7 below using Q5 TM DNA polymerase (New England Biolabs, USA) using the pCM110_P L/O4 as a template. At this time, the primers used for PCR are shown in Table 8 below.
상기 PCR을 통해 얻은 유전자 단편을 인서트 DNA로 사용하고, pCM110_P mxaF _Fdh1 벡터를 제한효소로 절단하여 백본 벡터로 사용하였다. 이때, 인서트 DNA와 벡본 벡터의 라이게이션은 Gibson assembly cloning kit(New England Biolabs, USA)를 이용하여 수행하였다. 상기의 과정을 통해 제조된 LacI/P L/O4 유전자 발현 카세트를 포함하는 pCM110_P mxaF _Fdh1 벡터를 pMPLO4_eGFP+로 명명하였다.The gene fragment obtained through the PCR was used as an insert DNA, and the pCM110_P mxaF _Fdh1 vector was digested with a restriction enzyme and used as a backbone vector. At this time, ligation of the insert DNA and the backbone vector was performed using a Gibson assembly cloning kit (New England Biolabs, USA). The pCM110_P mxaF _Fdh1 vector containing the LacI/P L/O4 gene expression cassette prepared through the above process was named pMPLO4_eGFP + .
제조예 5. 유전자 발현 카세트를 포함하는 형질전환체(MHRH01, MRHZ01, MLRL01, MMRM01, MXRX01 및 MPLO4) 제조Preparation Example 5. Preparation of Transformants Containing Gene Expression Cassettes (MHRH01, MRHZ01, MLRL01, MMRM01, MXRX01 and MPLO4)
상기 제조예 1 내지 4에서 제조된 유전자 발현 카세트를 각각 포함하는 발현 벡터를 Methylorubrum extorquens AM1(이하, M. extorquens AM1)에 도입하여 형질전환체를 제조하였다.Transformants were prepared by introducing the expression vectors each containing the gene expression cassettes prepared in Preparation Examples 1 to 4 into Methylorubrum extorquens AM1 (hereinafter referred to as M. extorquens AM1).
구체적으로, M. extorquens AM1를 MSM(minimal salt medium)배지에 분주한 후, 인큐베이터 진탕기에서 넣어 200 rpm, 30℃ 조건에서 교반하면서 배양하였다. 이때, MSM배지는 리터당 1.62 g NH4Cl, 0.2 g MgSO4, 2.21 g K2HPO4, 1.25 g NaH2PO42H2O, 15 mg Na2EDTA2H2O, 4.5 mg ZnSO47H2O, 0.3 mg CoCl26H2O, 1 mg MnCl24H2O, 1 mg H3BO3, 2.5 mg CaCl2, 0.4 mg of Na2MoO42H2O, 3 mg FeSO47H2O, 0.3 mg CuSO45H2O, and 4 g succinate를 포함하였다.Specifically, after dispensing M. extorquens AM1 into MSM (minimal salt medium) medium, it was put in an incubator shaker and cultured while stirring at 200 rpm and 30°C. At this time, the MSM medium contained 1.62 g NH 4 Cl, 0.2 g MgSO 4 , 2.21 g K 2 HPO 4 , 1.25 g NaH 2 PO 4 2H2O, 15 mg Na 2 EDTA2H 2 O, 4.5 mg ZnSO 4 7H2O, and 0.3 mg CoCl 2 per liter. 6H2O, 1 mg MnCl 2 4H 2 O, 1 mg H 3 BO 3 , 2.5 mg CaCl 2 , 0.4 mg of Na 2 MoO 4 2H 2 O, 3 mg FeSO 4 7H 2 O, 0.3 mg CuSO 4 5H 2 O, and 4 g succinate was included.
이후, MicroPulser 전기천공기(Bio-Rad, USA)를 사용하여 1.8 kV, 0.1 cm 큐벳 간격(cuvette gap)의 조건에서 배양된 M. extorquens AM1를 발현 벡터로 각각 형질전환시켰다. Thereafter, M. extorquens AM1 cultured under conditions of 1.8 kV and 0.1 cm cuvette gap were transformed with the expression vectors using a MicroPulser electroporator (Bio-Rad, USA).
M. extorquens AM1 균주에 HpdR/P hpdH , HexR/P zwf1 , LvaR/P lvaA , MmsR/P mmsA , XutR/P xutA 또는 LacI/P L/O4 를 포함하는 발현 벡터가 도입된 형질전환체를 MHRH01, MRHZ01, MLRL01, MMRM01, MXRX01 및 MPLO4로 각각 명명하였다.A transformant into which an expression vector containing HpdR/P hpdH , HexR/P zwf1 , LvaR/P lvaA , MmsR/P mmsA , XutR/P xutA or LacI/P L/O4 was introduced into M. extorquens AM1 strain was MHRH01 , MRHZ01, MLRL01, MMRM01, MXRX01 and MPLO4, respectively.
실시예 1. 형질전환체를 이용한 각 유전자 발현 시스템의 유전자 발현량 확인Example 1. Confirmation of gene expression level of each gene expression system using transformants
상기 제조예 5에서 제조한 각각의 형질전환체를 이용하여 각 유전자 발현 시스템별 유전자 발현량을 비교 분석하였다.Gene expression levels for each gene expression system were comparatively analyzed using each transformant prepared in Preparation Example 5.
먼저, 상기 제조예 5에서 제조한 각각의 형질전환체를 MSM 배지에 넣어 late exponential phase에 도달할 때까지 전-배양(pre-cultures) 한 후, 5 ㎖의 MSM 배지에 다시 계대배양하여 12시간 동안 배양하였다. 이후, OD600 값이 0.5 내지 0.7에 도달하면 각 유전자 발현 시스템의 유도체인 포도당(MRHZ01), 자일로스(MXRX01), 레불린산 또는 3-하이드록시프로피온산(MHRH01, MLRL01 및 MMRM01)를 10 mM 농도로 처리하였으며, IPTG(MPLO4)를 1 mM의 농도로 처리하였다. 그 후, 각 유도체가 처리된 형질전환체를 24시간 더 배양한 후, 180 ㎕씩 수집하여 바닥 Corning 96-웰 플레이트(bottom Corning 96-well plate)에 접종하였으며, Infinite F200 PRO 형광 판독기(Tecan, Austria)를 사용하여 GFP 형광 강도를 측정하였다.First, each transformant prepared in Preparation Example 5 was put in MSM medium and pre-cultured until reaching the late exponential phase, and then subcultured again in 5 ml of MSM medium for 12 hours cultured for a while. Subsequently, when the OD 600 value reached 0.5 to 0.7, glucose (MRHZ01), xylose (MXRX01), levulinic acid or 3-hydroxypropionic acid (MHRH01, MLRL01 and MMRM01), which are derivatives of each gene expression system, were added at 10 mM concentration. , and IPTG (MPLO4) was treated at a concentration of 1 mM. Thereafter, the transformants treated with each derivative were cultured for 24 hours more, and then 180 μl of each was collected and inoculated into a bottom Corning 96-well plate, and Infinite F200 PRO fluorescence reader (Tecan, Austria) was used to measure GFP fluorescence intensity.
그 결과, 도 2에 나타난 바와 같이, HpdR/P hpdH 유전자 발현 시스템들이 대조군에 비해 3HP(3-하이드록시프로피온산) 또는 LA(레불린산)으로 유도된 경우 유의적으로 증가된 형광 강도(P < 0.05) 나타낸다는 것을 확인하였다. 또한, LA로 유도된 HpdR/P hpdH 의 형광 강도는 HpdR/P hpdH 의 공지된 유도제인 3HP로 유도된 HpdR/P hpdH 의 형광 강도와 유사한 수준이었으며, LA로 유도된 HpdR/P hpdH (MHRH01)는 IPTG(1 mM)로 유도된 LacI/P L/O4 대비 94% 효율을 달성하였음을 확인하였다.As a result, as shown in FIG. 2, the fluorescence intensity (P < 0.05) was confirmed. In addition, the fluorescence intensity of HpdR/ PhpdH induced by LA was similar to that of HpdR/ PhpdH induced by 3HP , a known inducer of HpdR/PhpdH, and HpdR/PhpdH induced by LA (MHRH01) confirmed that 94% efficiency was achieved compared to LacI/P L/O4 induced by IPTG (1 mM).
이어서, HpdR/P hpdH 유전자 발현 시스템과 LacI/P L/O4 유전자 발현 시스템을 FACS를 이용하여 비교 분석하였다.Then, the HpdR/P hpdH gene expression system and the LacI/P L/O4 gene expression system were comparatively analyzed using FACS.
그 결과, 도 3에 나타난 바와 같이, HpdR/P hpdH 유전자 발현 시스템의 효율이 LacI/ PL/O4 유전자 발현 시스템의 효율과 유사하다는 것을 확인하였다. 또한, 비-유도 조건에서는 HpdR/P hpdH 유전자 발현 시스템이 LacI/ PL/O4 유전자 발현 시스템 비해 적어도 3배는 적게 누설되는 것을 확인하였다.As a result, as shown in FIG. 3 , it was confirmed that the efficiency of the HpdR/ PhpdH gene expression system was similar to that of the LacI/ PL/O4 gene expression system. In addition, it was confirmed that the HpdR/P hpdH gene expression system leaked at least three times less than the LacI/ PL/O4 gene expression system under non-inducing conditions.
이를 통해, 3HP 또는 LA을 유도제로 하는 HpdR/P hpdH 유전자 발현 시스템은 M. extorquens AM1 균주내에서 안정적으로 발현하며, LacI/ PL/O4 유전자 발현 시스템과 유사한 수준의 효율을 가진다는 것을 확인하였다.Through this, it was confirmed that the HpdR/P hpdH gene expression system using 3HP or LA as an inducer was stably expressed in the M. extorquens AM1 strain and had a similar level of efficiency to the LacI/ PL/O4 gene expression system.
실시예 2. HpdR/PExample 2. HpdR/P hpdHhpdH 유전자 발현 시스템의 레불린산 첨가 시간 최적화 Optimizing Levulinic Acid Addition Time for Gene Expression Systems
HpdR/P hpdH 유전자 발현 시스템의 LA 유도를 위한 최적 시간을 측정하기 위해, 상기 형질전환체 MHRH01를 실시예 1와 같은 방법으로 배양하여 배양물을 제조한 후에, 초기 접종(OD600 
그 결과, 도 4에 나타난 바와 같이, HpdR/P hpdH 유전자 발현 시스템은 다양한 LA 첨가 시점에서 eGFP+의 발현에 유의적인 차이 (P > 0.05)를 나타내지 않는 것을 확인하였다. As a result, as shown in FIG. 4 , it was confirmed that the HpdR/P hpdH gene expression system did not show a significant difference (P > 0.05) in the expression of eGFP + at various time points of LA addition.
이를 통해, HpdR/P hpdH 유전자 발현 시스템의 발현에 있어 LA 첨가 시점은 크게 상관이 없다는 것을 확인하였으며, 이는 초기 성장기 또는 지수 성장기 동안에도 LA의 첨가가 가능하여 유도제가 최적 세포 밀도에서 첨가되도록 하기 위해 세포 성장을 계속 모니터링할 필요가 없다는 것을 나타낸다.Through this, it was confirmed that the time point of LA addition was not significantly related to the expression of the HpdR/ PhpdH gene expression system, and it was possible to add LA even during the early growth phase or the exponential growth phase, so that the inducer was added at the optimal cell density. Indicates that there is no need to continuously monitor cell growth.
실시예 3. HpdR/PExample 3. HpdR/P hpdHhpdH 유전자 발현 시스템의 레불린산 첨가 농도 최적화 Optimization of levulinic acid addition concentration of gene expression system
HpdR/P hpdH 유전자 발현 시스템의 LA 유도를 위한 최적 농도를 측정하기 위해, 상기 형질전환체 MHRH01을 실시예 1와 같은 방법으로 배양하여 배양물을 제조한 후에, 다양한 농도(0.1, 0.25, 0.5, 1, 2, 4, 8, 10, 20, 40, 50, 및 100 mM)의 LA를 배양 시작 시에(0 h) 단일 용량으로 첨가하였다. 이후, LA 첨가후 12 내지 24 시간후에 각 샘플들을 수거하여 GFP 형광 강도를 측정하였다.In order to determine the optimal concentration for LA induction of the HpdR / P hpdH gene expression system, after culturing the transformant MHRH01 in the same manner as in Example 1 to prepare a culture, various concentrations (0.1, 0.25, 0.5, 1, 2, 4, 8, 10, 20, 40, 50, and 100 mM) of LA was added in a single dose at the beginning of the culture (0 h). Then, each sample was collected 12 to 24 hours after LA addition and the GFP fluorescence intensity was measured.
그 결과, 도 5에 나타난 바와 같이, 형광 강도는 LA를 20 mM까지 농도를 증가시킴에 따라 증가하는 것을 확인하였으며, 저농도의 LA(0.1 mM)에 의해서도 형광이 발현함을 확인하였다. 다만, LA의 농도가 20 mM를 초과하여 사용하는 경우 형광 강도가 오히려 감소하는 것을 확인하였으며, 40, 50, 및 100 mM의 LA에서는 20 mM의 LA에 비해 각각 약10%, 28%, 및 68% 감소된 효율이 나타내는 것을 확인하였다. As a result, as shown in FIG. 5, it was confirmed that the fluorescence intensity increased as the concentration of LA increased up to 20 mM, and it was confirmed that fluorescence was expressed even at a low concentration of LA (0.1 mM). However, it was confirmed that the fluorescence intensity rather decreased when the concentration of LA exceeded 20 mM, and at 40, 50, and 100 mM LA, compared to 20 mM LA, respectively, about 10%, 28%, and 68 It was confirmed that the % reduced efficiency was indicated.
이어서, 각 샘플들을 수거하여 세포 성장을 측정하였으며, 이때, 바이오크롬 라이브라S22 분광광도계(Biochrom, 영국)를 사용하여 세포 밀도를 모니터링하였다.Subsequently, each sample was collected and cell growth was measured, and at this time, cell density was monitored using a Biochrom Libra S22 spectrophotometer (Biochrom, UK).
그 결과, 도 6에 나타난 바와 같이, LA의 농도를 20 mM를 초과하여 사용함에 따라 세포 성장이 감소하는 것을 확인하였으며, 50 mM의 LA에서는 세포 성장이 최대 1.5배 감소된다는 것을 확인하였다. As a result, as shown in FIG. 6, it was confirmed that cell growth decreased as LA concentration exceeded 20 mM, and it was confirmed that cell growth was reduced up to 1.5 times at 50 mM LA.
또한, 각 샘플들을 수거하여 유세포 분석하였다. In addition, each sample was collected and analyzed by flow cytometry.
그 결과, 도 7에 나타난 바와 같이, LA를 0.1 내지 20 mM의 농도롤 첨가한 샘플들의 경우, 모두 유사한 수준의 발현을 나타냄을 확인하였다. As a result, as shown in FIG. 7 , it was confirmed that the samples in which LA was added at a concentration of 0.1 to 20 mM showed similar levels of expression.
이를 통해, HpdR/P hpdH 유전자 발현 시스템은 용량-의존적으로 발현하며, 유도체인 LA의 농도가 0.1 내지 20 mM 범위일 때, 최적의 효율을 나타내는 것을 확인하였다. Through this, it was confirmed that the HpdR/ PhpdH gene expression system expresses dose-dependently and exhibits optimal efficiency when the concentration of the derivative, LA, is in the range of 0.1 to 20 mM.
실시예 4. HpdR/PExample 4. HpdR/P hpdHhpdH 유전자 발현 시스템의 균주 무관성 확인 Verification of strain indifference of gene expression system
HpdR/P hpdH 유전자 발현 시스템의 균주 무관성을 확인하기 위해 HpdR/P hpdH 유전자 발현 시스템를 포함하는 발현 벡터 pMHRH_eGFP+를 CM4(KCTC 32005), DM4(DSM 6343), PA1(DSM 23939), TK0001(DSM1337) 및 ATCC55366과 같은 메틸로루브룸(Methylorubrum extorquens) 균주에 형질전환시켰으며, 이를 CM4HRH01, DM4HRH01, PA1HRH01, TK01HRH01 및 AT66HRH01로 각각 명명하였다.In order to confirm the strain indifference of the HpdR/P hpdH gene expression system, expression vectors pMHRH_eGFP + containing the HpdR/P hpdH gene expression system were selected from CM4 (KCTC 32005), DM4 (DSM 6343), PA1 (DSM 23939), and TK0001 (DSM1337). ) And methylorubrum such as ATCC55366 ( Methylorubrum extorquens ) Transformed into strains, which were named CM4HRH01, DM4HRH01, PA1HRH01, TK01HRH01 and AT66HRH01, respectively.
각 형질전환체를 상기 실시예 1와 같은 방법으로 배양하여 배양물을 제조한 후에, 10 mM의 LA를 배양 시작 시에(0 h) 첨가하였다. 이후, 각 샘플들을 수거하여 GFP 형광 강도를 측정하였다.After culturing each transformant in the same manner as in Example 1 to prepare a culture, 10 mM LA was added at the start of the culture (0 h). Then, each sample was collected and the GFP fluorescence intensity was measured.
그 결과, 도 8에 나타난 바와 같이, 이들 균주에서도 MHRH01에서 관찰된 수준과 유사한 발현을 나타내는 것을 확인하였으며, 이를 통해 HpdR/P hpdH 유전자 발현 시스템이 메틸로루브룸속의 다양한 균주에서 균등한 발현 수준을 나타냈고 이러한 균주들에 광범위하게 적용될 수 있음을 확인하였다.As a result, as shown in FIG. 8, it was confirmed that these strains also exhibited similar expression levels to those observed in MHRH01, and through this, the HpdR/ PhpdH gene expression system showed equal expression levels in various strains of the genus Methylolrubrum. and confirmed that it can be widely applied to these strains.
실시예 5. 형질전환체 MHRH01의 레불린산 잔류 농도 확인Example 5. Confirmation of the residual concentration of levulinic acid in the transformant MHRH01
형질전환체 MHRH01의 LA 잔류 농도를 측정하기 위해, 형질전환체 MHRH01를 실시예 1과 같은 방법으로 배양하여 배양물을 제조한 후에, 10 mM의 LA를 배양 시작 시에(0 h) 첨가하였다. 이후, LA 첨가 직후, 24 시간후 및 48 시간후에 각 샘플들을 수거하여 LA 잔류 농도를 측정하였다.In order to measure the LA residual concentration of the transformant MHRH01, after culturing the transformant MHRH01 in the same manner as in Example 1 to prepare a culture, 10 mM LA was added at the start of the culture (0 h). Thereafter, samples were collected immediately after addition of LA, after 24 hours and after 48 hours, and the residual concentration of LA was measured.
그 결과, 도 9에 나타난 바와 같이, 세포 성장에 따른 흡광도(OD600)는 LA 첨가 직후(0 h)에 0.125에서 LA 첨가 48 시간후 1.94로 증가하였으나, LA의 잔류 농도는 거의 변함이 없는 것을 확인하였다. As a result, as shown in FIG. 9, the absorbance (OD 600 ) according to cell growth increased from 0.125 immediately after LA addition (0 h) to 1.94 48 hours after LA addition, but the residual concentration of LA was almost unchanged. Confirmed.
이를 통해, 형질전환체 MHRH01는 충분히 성장한 후에도, 유도체인 LA를 과도하게 소비하지 않는다는 것을 확인하였다.Through this, it was confirmed that the transformant MHRH01 does not excessively consume the derivative, LA, even after sufficient growth.
실시예 6. 형질전환체 MHRH01의 eGFPExample 6. eGFP of transformant MHRH01 단백질 생성 효율Protein production efficiency 확인check
GFP 단백질을 생성하기 위해 제작된 HpdR/P hpdH 유전자 발현 시스템의 효율을 확인하기 위하여, 형질전환체 MHRH01를 실시예 1와 같은 방법으로 배양하여 배양물을 제조한 후에, 10 mM의 LA를 첨가하여 eGFP+의 발현을 유도하였으며 24 시간 후에 세포를 수확하였다. 또한, 대조군으로서 형질전환체 MPL04을 MSM 배지에서 배양한 후, 1 mM의 IPTG를 첨가하여 eGFP+의 발현을 유도하였으며 24 시간 후에 세포를 수확하였다. 이후, 수확한 각 세포를 50 mM의 MOPS 및 10 mM의 이미다졸을 포함하는 ice-cold 세포 용해 완충액에 재현탁시키고, VCX130 초음파 처리기(Sonics, 미국)를 사용하여 35% 진폭으로 6분 동안 초음파 처리하여 세포 용해물을 제조하였다. 소혈청 알부민(BSA)을 함유한 브래드포드(Bradford) 분석 시약을 사용하여 단백질 농도를 먼저 측정하였다. 이어서, 세포 용해물을 SDS-PAGE 완충액에 희석시키고, 웰 당 5 ㎍를 15% 폴리아크릴아미드 겔 상에 부과하여 SDS-PAGE를 수행하였다. 생성된 단백질 밴드를 쿠마시 브릴리언트 블루(Coomassie Brilliant Blue) R-250으로 염색하여 가시화하였다. 각 샘플의 eGFP+ 농도는 이미 농도를 알고 있는 정제된 GFP(Abcam, 미국)의 선형 관계에 기반하여 제조사의 프로토콜에 따라 계산하였다.In order to confirm the efficiency of the HpdR / P hpdH gene expression system designed to produce GFP protein, the transformant MHRH01 was cultured in the same manner as in Example 1 to prepare a culture, and then 10 mM LA was added to Expression of eGFP + was induced and cells were harvested after 24 hours. In addition, as a control, after culturing the transformant MPL04 in MSM medium, 1 mM IPTG was added to induce the expression of eGFP + , and the cells were harvested after 24 hours. Then, each harvested cell was resuspended in ice-cold cell lysis buffer containing 50 mM MOPS and 10 mM imidazole, and sonicated for 6 minutes at 35% amplitude using a VCX130 sonicator (Sonics, USA). treatment to prepare cell lysates. Protein concentration was first determined using Bradford assay reagent containing bovine serum albumin (BSA). Cell lysates were then diluted in SDS-PAGE buffer and subjected to SDS-PAGE by applying 5 μg per well onto a 15% polyacrylamide gel. The resulting protein band was visualized by staining with Coomassie Brilliant Blue R-250. The eGFP + concentration of each sample was calculated according to the manufacturer's protocol based on the linear relationship of purified GFP (Abcam, USA) with known concentration.
그 결과, 도 10에 나타난 바와 같이, LA으로 유도된 형질전환체 MHRH01가 IPTG로 유도된 형질전환체 MPL04와 마찬가지로 eGFP+(27 kDa)을 강하게 발현하며(w/LA 레인), LA의 부재하에서는 eGFP+ 단백질을 거의 생성하지 않는 것을 확인하였다(w/o 레인). As a result, as shown in FIG. 10, the transformant MHRH01 induced by LA strongly expresses eGFP + (27 kDa) like the transformant MPL04 induced by IPTG (w/LA lane), and in the absence of LA It was confirmed that almost no eGFP + protein was produced (w/o lanes).
이어서, 각 세포 용해물을 수거하여 각 형질전환체가 생성한 GFP의 농도를 측정하였다.Then, each cell lysate was collected and the concentration of GFP produced by each transformant was measured.
그 결과, 도 11에 나타난 바와 같이, MHRH01는 42 mg/g의 바이오매스(건조 세포 중량)로 총 단백질의 약 10%에 해당하는 GFP를 생성하였으며, 이는 46 mg/g의 바이오매스(건조 세포 중량)로 총 단백질의 약 11%에 해당하는 GFP를 생성한 MPL04와 유사한 수준임을 확인하였다.As a result, as shown in FIG. 11, MHRH01 produced GFP corresponding to about 10% of the total protein with 42 mg/g of biomass (dry cell weight), which was 46 mg/g of biomass (dry cell weight). It was confirmed that the level was similar to that of MPL04, which produced GFP corresponding to about 11% of the total protein by weight).
이를 통해, LA-유도성 HpdR/P hpdH 유전자 발현 시스템의 효율은 IPTG-유도성 LacI/P L/O4 유전자 발현 시스템과 동등한 수준임을 확인하였다(90%) (P > 0.05).Through this, it was confirmed that the efficiency of the LA-inducible HpdR/P hpdH gene expression system was equivalent to that of the IPTG-inducible LacI/P L/O4 gene expression system (90%) (P > 0.05).
<110> UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> CARBON SOURCE-INDUCIBLE EXPRESSION VECTOR EXPRESSABLE IN METHYLORUBRUM STRAIN AND GENE EXPRESSION SYSTEM USING SAME <130> FPD/202110-0038 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 295 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for HpdR <400> 1 Met Phe Asp Trp Asn Asp Leu Arg Phe Phe Leu Glu Leu Gln Arg Ser 1 5 10 15 Gly Arg Leu Leu Thr Ala Ala Lys Arg Leu Asn Thr Thr His Ser Thr 20 25 30 Val Ala Arg His Ile Glu Ser Ile Glu Gln Ser Leu Gly Thr Ala Leu 35 40 45 Phe Val Gln His Ala Gln Gly Tyr Glu Leu Thr Pro Ser Gly Gln Ala 50 55 60 Leu Leu Lys His Ala Glu Ala Met Glu Asn Val Ala Leu Leu Ala Gln 65 70 75 80 Glu Glu Ile Thr Gln Ala Ile Thr Pro Leu Gly Lys Ile Arg Leu Gly 85 90 95 Val Thr Glu Gly Ile Gly Ile Met Phe Phe Thr Pro Arg Met Asn Ala 100 105 110 Leu Phe Glu Arg Tyr Pro Gly Leu Glu Val Glu Leu Val Ala Val Pro 115 120 125 Arg Phe Val Ser Ile Leu Asn Arg Glu Ala Glu Ile Ser Ile His Leu 130 135 140 Glu Arg Pro Asn Ala Asp Leu Leu Ile Thr Arg Lys Leu Thr Asp Tyr 145 150 155 160 Arg Leu Ala Leu Tyr Ala Ser Gln Ala Tyr Leu Asp Arg Ala Pro Pro 165 170 175 Leu Arg His Arg Glu Asp Leu Ala Arg His Ser Trp Ile Gly Tyr Val 180 185 190 Asp Asp Leu Leu Phe Ser Gln Glu Leu Leu Phe Leu Asn Ser Phe Cys 195 200 205 Arg Val Pro Asn Val Val Phe Arg Ser Thr Ser Val Ile Ala Gln Gln 210 215 220 Ala Ala Ala Arg Ala Gly Leu Gly Ile Ala Val Leu Pro Asn Tyr Met 225 230 235 240 Ala Arg His Asp Pro Thr Leu Val Arg Val Leu Pro Gly Glu Thr Ile 245 250 255 Gln Arg Ser Tyr Trp Ile Cys Thr Arg Arg Glu Leu His Lys Ser Val 260 265 270 Arg Leu Arg Val Val Trp Asp Tyr Leu Leu Ala Leu Cys Ala Ala Glu 275 280 285 Gln Ser Glu Leu Leu Ala Glu 290 295 <210> 2 <211> 888 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for HpdR <400> 2 ctactcggct agcaactcgc tttgttcggc cgcgcacaac gccagcaggt agtcccacac 60 cacccgcagg cgcacagact tgtgcagctc gcggcgggtg cagatccagt agctgcgctg 120 gatggtttct cccggcagca cgcgcaccag tgtcgggtcg tggcgggcca tgtagttggg 180 cagcacggcg atgcccagcc cggcgcgggc ggcggcttgc tgggcgatca cgctggtgct 240 tcgaaacacc acgttgggta ctcggcagaa actgttgaga aacagcagtt cctggctgaa 300 cagcaaatcg tcaacgtagc cgatccagct gtgccgggcc aggtcttcgc ggtggcgcag 360 tggcggcgcg cggtccaggt aggcctggct ggcgtatagc gccaggcggt agtcggtgag 420 tttgcgggtg atcagcagat cggcgttggg ccgctccaga tggatgctga tttctgcctc 480 gcggttgagg atgctgacga agcgcggcac cgctaccaat tccacctcca ggccggggta 540 gcgctcgaac aacgcgttca tgcgtggggt gaagaacatg atgccgatgc cttcggttac 600 ccccaggcga atcttgccca gcggggtgat ggcctgggtg atttcctcct gtgccagcag 660 ggcgacgttc tccatggctt cggcatgctt gagcagggct tgacccgagg gggtcagctc 720 gtacccctgg gcatgttgca cgaacagcgc ggtccccagg ctctgctcga tgctctcgat 780 atgccgggcc acggtgctgt gggtagtgtt caggcgcttg gcggcggtca gcaagcggcc 840 gctgcgctgc aactcgagga aaaaccgcag atcattccag tcgaacat 888 <210> 3 <211> 233 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PhpdH <400> 3 gcttgtcctt tatggcagtt cgttccggcc tcttaacggg catgcccaca ggtgcggtga 60 acaccctgaa ggtaacgtga tccctgagct gcgggcgagc ccgtgaagag gtcggtacag 120 gcttgcccct gtgctaaaac gcacagcggc tgcgcgaaat ctcgtgtttc atccacgaaa 180 ttactcacta agatggatcg ggacaagaat aaaaaacagg cgcgaggttg cac 233 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Pro-FP <400> 4 tgcatgcctg caggtcgact ctagacagga attggggatc ggaag 45 <210> 5 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Pro-RP <400> 5 gttagcagcc ctcgagtttg gatccgtcca agctcagcta attaagc 47 <210> 6 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> PLO4-FP <400> 6 cgtttccacc gaattagctt gcatgcctgc aggtcgactc tagatcactg cccgctttcc 60 agtcggg 67 <210> 7 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> PLO4-RP <400> 7 tcctgctgat gtgctcagta tcattgttat ccgctcacat gtcaacaccg ccagagataa 60 tttatcgcat gcaccattcc ttgcggcggc 90 <210> 8 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> PLO4-RP2 <400> 8 caactcagct tcctttcggg ctttgttagc agccggatcc gtcagtgcgt cctgctgatg 60 tgctcagtat cattg 75 <210> 9 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> LacI/PL/O4-FP <400> 9 cgtttccacc gaattagctt gcatgcctgc aggtcgactc tagatcactg cccgctttcc 60 agtcggg 67 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> LacI/PL/O4-RP <400> 10 cgacggatcc atgtatatct ccttcttaaa gttaaacaaa gtcag 45 <110> UNIST (ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> CARBON SOURCE-INDUCIBLE EXPRESSION VECTOR EXPRESSABLE IN METHYLORUBRUM STRAIN AND GENE EXPRESSION SYSTEM USING SAME <130> FPD/202110-0038 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 295 <212> PRT <213> artificial sequence <220> <223> amino acid sequence for HpdR <400> 1 Met Phe Asp Trp Asn Asp Leu Arg Phe Phe Leu Glu Leu Gln Arg Ser 1 5 10 15 Gly Arg Leu Leu Thr Ala Ala Lys Arg Leu Asn Thr Thr His Ser Thr 20 25 30 Val Ala Arg His Ile Glu Ser Ile Glu Gln Ser Leu Gly Thr Ala Leu 35 40 45 Phe Val Gln His Ala Gln Gly Tyr Glu Leu Thr Pro Ser Gly Gln Ala 50 55 60 Leu Leu Lys His Ala Glu Ala Met Glu Asn Val Ala Leu Leu Ala Gln 65 70 75 80 Glu Glu Ile Thr Gln Ala Ile Thr Pro Leu Gly Lys Ile Arg Leu Gly 85 90 95 Val Thr Glu Gly Ile Gly Ile Met Phe Phe Thr Pro Arg Met Asn Ala 100 105 110 Leu Phe Glu Arg Tyr Pro Gly Leu Glu Val Glu Leu Val Ala Val Pro 115 120 125 Arg Phe Val Ser Ile Leu Asn Arg Glu Ala Glu Ile Ser Ile His Leu 130 135 140 Glu Arg Pro Asn Ala Asp Leu Leu Ile Thr Arg Lys Leu Thr Asp Tyr 145 150 155 160 Arg Leu Ala Leu Tyr Ala Ser Gln Ala Tyr Leu Asp Arg Ala Pro Pro 165 170 175 Leu Arg His Arg Glu Asp Leu Ala Arg His Ser Trp Ile Gly Tyr Val 180 185 190 Asp Asp Leu Leu Phe Ser Gln Glu Leu Leu Phe Leu Asn Ser Phe Cys 195 200 205 Arg Val Pro Asn Val Val Phe Arg Ser Thr Ser Val Ile Ala Gln Gln 210 215 220 Ala Ala Ala Arg Ala Gly Leu Gly Ile Ala Val Leu Pro Asn Tyr Met 225 230 235 240 Ala Arg His Asp Pro Thr Leu Val Arg Val Leu Pro Gly Glu Thr Ile 245 250 255 Gln Arg Ser Tyr Trp Ile Cys Thr Arg Arg Glu Leu His Lys Ser Val 260 265 270 Arg Leu Arg Val Val Trp Asp Tyr Leu Leu Ala Leu Cys Ala Ala Glu 275 280 285 Gln Ser Glu Leu Leu Ala Glu 290 295 <210> 2 <211> 888 <212> DNA <213> artificial sequence <220> <223> nucleotide sequence for HpdR <400> 2 ctactcggct agcaactcgc tttgttcggc cgcgcacaac gccagcaggt agtcccacac 60 cacccgcagg cgcacagact tgtgcagctc gcggcgggtg cagatccagt agctgcgctg 120 gatggtttct cccggcagca cgcgcaccag tgtcgggtcg tggcgggcca tgtagttggg 180 cagcacggcg atgcccagcc cggcgcgggc ggcggcttgc tgggcgatca cgctggtgct 240 tcgaaacacc acgttgggta ctcggcagaa actgttgaga aacagcagtt cctggctgaa 300 cagcaaatcg tcaacgtagc cgatccagct gtgccgggcc aggtcttcgc ggtggcgcag 360 tggcggcgcg cggtccaggt aggcctggct ggcgtatagc gccaggcggt agtcggtgag 420 tttgcgggtg atcagcat cggcgttggg ccgctccaga tggatgctga tttctgcctc 480 gcggttgagg atgctgacga agcgcggcac cgctaccaat tccacctcca ggccggggta 540 gcgctcgaac aacgcgttca tgcgtggggt gaagaacatg atgccgatgc cttcggttac 600 ccccaggcga atcttgccca gcggggtgat ggcctgggtg atttcctcct gtgccagcag 660 ggcgacgttc tccatggctt cggcatgctt gagcagggct tgacccgagg gggtcagctc 720 gtacccctgg gcatgttgca cgaacagcgc ggtccccagg ctctgctcga tgctctcgat 780 atgccgggcc acggtgctgt gggtagtgtt caggcgcttg gcggcggtca gcaagcggcc 840 gctgcgctgc aactcgagga aaaaccgcag atcattccag tcgaacat 888 <210> 3 <211> 233 <212> DNA <213> artificial sequence <220> <223> nucleotide sequence for PhpdH <400> 3 gcttgtcctt tatggcagtt cgttccggcc tcttaacggg catgcccaca ggtgcggtga 60 acaccctgaa ggtaacgtga tccctgagct gcgggcgagc ccgtgaagag gtcggtacag 120 gcttgcccct gtgctaaaac gcacagcggc tgcgcgaaat ctcgtgtttc atccacgaaa 180 ttactcacta agatggatcg ggacaagaat aaaaaacagg cgcgaggttg cac 233 <210> 4 <211> 45 <212> DNA <213> artificial sequence <220> <223> Pro-FP <400> 4 tgcatgcctg caggtcgact ctagacagga attggggatc ggaag 45 <210> 5 <211> 47 <212> DNA <213> artificial sequence <220> <223> Pro-RP <400> 5 gttagcagcc ctcgagtttg gatccgtcca agctcagcta attaagc 47 <210> 6 <211> 67 <212> DNA <213> artificial sequence <220> <223> PLO4-FP <400> 6 cgtttccacc gaattagctt gcatgcctgc aggtcgactc tagatcactg cccgctttcc 60 agtcggg 67 <210> 7 <211> 90 <212> DNA <213> artificial sequence <220> <223> PLO4-RP <400> 7 tcctgctgat gtgctcagta tcattgttat ccgctcacat gtcaacaccg ccagagataa 60 tttatcgcat gcaccattcc ttgcggcggc 90 <210> 8 <211> 75 <212> DNA <213> artificial sequence <220> <223> PLO4-RP2 <400> 8 caactcagct tcctttcggg ctttgttagc agccggatcc gtcagtgcgt cctgctgatg 60 tgctcagtat cattg 75 <210> 9 <211> 67 <212> DNA <213> artificial sequence <220> <223> LacI/PL/O4-FP <400> 9 cgtttccacc gaattagctt gcatgcctgc aggtcgactc tagatcactg cccgctttcc 60 agtcggg 67 <210> 10 <211> 45 <212> DNA <213> artificial sequence <220> <223> LacI/PL/O4-RP <400> 10 cgacggatcc atgtatatct ccttcttaaa gttaaacaaa gtcag 45
Claims (10)
상기 HpdR 전사인자는 서열번호 1로 표시되는 아미노산 서열을 포함하는 것인, 메틸로루브룸속 균주에서 발현 가능한 벡터.According to claim 1,
Wherein the HpdR transcription factor comprises the amino acid sequence represented by SEQ ID NO: 1, a vector capable of being expressed in a strain of the genus Methylolorubrum.
상기 아미노산 서열은 서열번호 2로 표시되는 염기서열로 코딩되는 것인, 메틸로루브룸속 균주에서 발현 가능한 벡터.According to claim 2,
The amino acid sequence is encoded by the nucleotide sequence represented by SEQ ID NO: 2, a vector capable of being expressed in strains of the genus Methylolorubrum.
상기 P hpdH 프로모터는 서열번호 3으로 표시되는 염기서열을 포함하는 것인, 메틸로루브룸속 균주에서 발현 가능한 벡터.According to claim 1,
Wherein the P hpdH promoter comprises the nucleotide sequence represented by SEQ ID NO: 3, a vector capable of being expressed in a strain of the genus Methylolorubrum.
상기 메틸로루브룸속 균주는 Methylorubrum extorquens, Methylorubrum aminovorans, Methylorubrum podarium, Methylorubrum pseudosasae, Methylorubrum rhodesianum, Methylorubrum rhodinum, Methylorubrum salsuginis 및 Methylorubrum suomiense에서 선택되는, 메틸로루브룸속 균주에서 발현 가능한 벡터.According to claim 1,
The Methylorubrum strain is selected from Methylorubrum extorquens , Methylorubrum aminovorans , Methylorubrum podarium , Methylorubrum pseudosasae , Methylorubrum rhodesianum , Methylorubrum rhodinum , Methylorubrum salsuginis and Methylorubrum suomiense .
상기 형질전환체는 Methylorubrum extorquens, Methylorubrum aminovorans, Methylorubrum podarium, Methylorubrum pseudosasae, Methylorubrum rhodesianum, Methylorubrum rhodinum, Methylorubrum salsuginis 및 Methylorubrum suomiense에서 선택되는, 형질전환체.According to claim 6,
The transformant is selected from Methylorubrum extorquens , Methylorubrum aminovorans , Methylorubrum podarium , Methylorubrum pseudosasae , Methylorubrum rhodesianum , Methylorubrum rhodinum , Methylorubrum salsuginis and Methylorubrum suomiense .
상기 배양 단계에서 레불린산이 0.01 mM 내지 30 mM의 농도로 배지에 포함되는, 유전자 발현 방법.According to claim 8,
In the culturing step, levulinic acid is contained in the medium at a concentration of 0.01 mM to 30 mM, the gene expression method.
배양 시간은 6시간 내지 48시간인, 유전자 발현 방법.According to claim 8,
The incubation time is 6 hours to 48 hours, the gene expression method.
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