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KR102154256B1 - Recombinant Corynebacterium Glutamicum for the production of 3'-fucosyllactose and method for the production of 3'-fucosyllactose therefrom - Google Patents

Recombinant Corynebacterium Glutamicum for the production of 3'-fucosyllactose and method for the production of 3'-fucosyllactose therefrom Download PDF

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KR102154256B1
KR102154256B1 KR1020190119983A KR20190119983A KR102154256B1 KR 102154256 B1 KR102154256 B1 KR 102154256B1 KR 1020190119983 A KR1020190119983 A KR 1020190119983A KR 20190119983 A KR20190119983 A KR 20190119983A KR 102154256 B1 KR102154256 B1 KR 102154256B1
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Abstract

The present invention relates to a production method of 3′-fucosylactose using Corynebacterium glutamicum. According to the production method of 3′-fucosylactose using Corynebacterium glutamicum, 3′-fucosylactose is produced using recombinant Corynebacterium glutamicum. According to the present invention, compared to conventional colon bacillus, 3′-fucosylactose can be safely produced.

Description

코리네박테리움 글루타미쿰을 이용한 3'-푸코실락토오스의 생산방법 {Recombinant Corynebacterium Glutamicum for the production of 3'-fucosyllactose and method for the production of 3'-fucosyllactose therefrom}Recombinant Corynebacterium Glutamicum for the production of 3'-fucosyllactose and method for the production of 3'-fucosyllactose therefrom}

본 발명은 코리네박테리움 글루타미쿰을 이용한 3'-푸코실락토오스의 생산방법에 관한 것으로, 상세하게는 α-1,3-푸코오스 전이효소 (α-1,3- fucosyltransferase)가 발현되도록 형질전환되고, GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase)가 발현되도록 형질전환되며, GDP-L-푸코오스 신타아제 (GDP-L-fucose synthase)가 발현되도록 형질전환되고, 락토오즈 퍼미아제 (lactose permease)가 발현되도록 형질전환되며, ABC 트랜스포터 퍼미아제(transporter permease)가 발현되도록 형질전환되고, 포스포만노뮤타아제 (Phosphomannomutase) 및 GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)를 보유하고 있는 것을 특징으로 하는 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)을 이용하여 3'-푸코실락토오스를 생산하는 방법에 관한 것이다.The present invention relates to a method for producing 3'-fucosyllactose using Corynebacterium glutamicum, and in detail, to express α-1,3-fucosyltransferase (α-1,3-fucosyltransferase). Transformed and transformed to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase), and GDP-L-fucose synthase (GDP- L-fucose synthase) is transformed to be expressed, lactose permease is transformed to be expressed, ABC transporter permease is transformed to be expressed, and phosphomannomutase (Phosphomannomutase) and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase), characterized in that it has a recombinant Corynebacterium glutamicum ( Corynebacterium glutamicum ) relates to a method for producing 3'-fucosyl lactose.

사람의 모유에는 200여 종 이상의 독특한 구조를 가지는 올리고당 (human milk oligosaccharides, HMO)이 다른 포유류의 젖에 비해 상당히 높은 농도 (5~15g/L)로 존재한다. HMO는 D-글루코오스 (Glc), D-갈락토오스 (Gal), N-아세틸글루코사민 (N-aetylglucosamine, GlcNAc), L-푸코오스 (L-fucose, Fuc)와 시알산 (sialc acid) [Sia; N-acetyl neuraminic acid (Neu5Ac)]으로 구성되어 있다.Human milk oligosaccharides (HMO), which have more than 200 species of unique structure, are present in human milk at a significantly higher concentration (5-15g/L) than that of other mammals. HMO includes D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and sialc acid [Sia; It is composed of [N-acetyl neuraminic acid (Neu5Ac)].

HMO의 구조는 매우 다양하고 복잡하기 때문에, 다른 잔기와 글리코실 결합을 가지는 200개 정도의 이성질체가 서로 다른 중합도(DP 3-20)로 존재할 수 있다. 다만, 구조적 복잡성에 불구하고, HMO는 몇 가지 공통적인 구조를 보이는데, 대부분 HMO는 환원 말단에 락토오스 (Galβ1-4Glc) 잔기를 가진다. 락토오스의 Gal은 α-(2,3)-과 α-(2,6)-결합으로 각각 3-시알릴락토오스 (3-sialyllactose) 또는 6-시알릴락토오스 (6-sialyllactose)의 형태로 시알화되거나, α-(1,2)-과 α-(1,3)-결합으로 각각 2-푸코실락토오스 (2'-fucosyllactose, 2'-FL) 또는 3-푸코실락토오스 (3'-fucosyllactose, 3-FL)의 형태로 푸코실화 (fucosylation)될 수 있다.Since the structure of HMO is very diverse and complex, about 200 isomers having different moieties and glycosyl bonds can exist at different degrees of polymerization (DP 3-20). However, despite the structural complexity, HMOs show several common structures, and most HMOs have lactose (Galβ1-4Glc) residues at the reducing end. Gal of lactose is sialylated in the form of 3-sialyllactose or 6-sialyllactose by α-(2,3)- and α-(2,6)-bonds, respectively. Or, in α-(1,2)- and α-(1,3)-bonds, respectively, 2-fucosyllactose (2'-fucosyllactose, 2'-FL) or 3-fucosyllactose (3'-fucosyllactose, 3-FL) can be fucosylated.

한편, 3'-푸코실락토오스는 다양한 생물학적 활성에 관여하는 주요 HMO인 것으로 보고되고 있다. 3'-푸코실락토오스의 생산방법으로는 직접 모유로부터 추출하는 방법과 화학적 또는 효소적 방법으로 합성하는 방법이 있다. 하지만, 직접 추출하는 방법은 모유수급의 한계와 낮은 생산성이란 단점이 있으며, 화학적 합성법은 고가의 기질, 낮은 이성체 선택성 (stereo-selectivity) 및 생산수율, 독성 유기용매 사용 등의 문제가 존재한다. 또한, 효소적 합성법은 푸코오스의 공여체 (donor)로 이용되는 GDP-L-fucose가 매우 고가이고, 푸코오스 전이효소 (fucosyltransferase)의 정제비용이 많이 소요되는 단점이 있다.On the other hand, it is reported that 3'-fucosylactose is a major HMO involved in various biological activities. Methods for producing 3'-fucosyl lactose include direct extraction from breast milk and synthesis by chemical or enzymatic methods. However, the direct extraction method has disadvantages such as limitation of breast milk supply and demand and low productivity, and the chemical synthesis method has problems such as expensive substrates, low stereo-selectivity and production yield, and the use of toxic organic solvents. In addition, the enzymatic synthesis method has disadvantages that GDP-L-fucose, which is used as a donor of fucose, is very expensive, and the purification cost of fucosyltransferase is high.

이와 같이 직접추출, 화학적 또는 효소적 생산법은 푸코실락토오스의 대량생산에 적용이 어려운 실정이다. 그러나 미생물을 이용한 생물공학적 생산방법은, 단순한 공정을 통해 저렴한 기질로부터 푸코실락토오스를 대량으로 생산할 수 있기 때문에, 건강기능성식품 및 의약품 소재로의 개발 가능성을 지닌 3'-푸코실락토오스를 생산하기 위한 방법으로 각광받고 있다.As described above, direct extraction, chemical or enzymatic production methods are difficult to apply to mass production of fucosyl lactose. However, the biotechnological production method using microorganisms can produce a large amount of fucosyl lactose from an inexpensive substrate through a simple process, so it is intended to produce 3'-fucosyl lactose with potential for development as a health functional food and pharmaceutical material. It is in the spotlight as a method

그러나 미생물을 이용한 3'-푸코실락토오스 생산에 관한 종래의 기술은 대부분 재조합 대장균을 이용한 생산기술로, 실험용으로 이용되는 대장균은 실제로는 병원균이 아니지만 소비자들에게는 해로운 균이라는 인식이 강하고, 세포막 성분이 엔도톡신으로 작용할 수 있기 때문에 분리 정제의 비용이 많이 소비되는 단점이 있어, 식품 및 의약품 소재인 3'-푸코실락토오스를 생산하는 숙주세포로써 대장균을 이용하기에는 어려움이 있는 실정이다.However, the conventional techniques for the production of 3'-fucosylactose using microorganisms are mostly production technologies using recombinant E. coli, and E. coli used for experimentation is not actually a pathogen, but consumers are aware that it is a harmful bacteria. Since it can act as an endotoxin, there is a disadvantage that the cost of separation and purification is high, and it is difficult to use E. coli as a host cell that produces 3'-fucosylactose, a material for food and pharmaceuticals.

대한민국공개특허 제10-2018-0118544호(2018.10.31.)에는, 코리네박테리움 글루타미쿰을 이용한 3'-푸코실락토오스의 생산방법에 관하여 기재되어 있다.In Korean Patent Application Publication No. 10-2018-0118544 (October 31, 2018), a method for producing 3'-Fucosyl lactose using Corynebacterium glutamicum is described.

본 발명에서는 식품 및 의약품 소재인 3'-푸코실락토오스를 생산하는 숙주세포로서, 대장균보다 안전한 코리네박테리움 글루타미쿰 (Corynebactrium glutamicum)을 이용하되, 고농도, 고수율, 고생산성으로 3'-푸코실락토오스를 생산하는 방법을 개발하여 제공하고자 한다.In the present invention, as a host cell producing 3'-fucosylactose, which is a food and pharmaceutical material, using Corynebactrium glutamicum , which is safer than E. coli, 3'- with high concentration, high yield, and high productivity. It is intended to develop and provide a method for producing fucosyl lactose.

본 발명은 α-1,3-푸코오스 전이효소 (α-1,3- fucosyltransferase)가 발현되도록 형질전환되고, GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase)가 발현되도록 형질전환되며, GDP-L-푸코오스 신타아제 (GDP-L-fucose synthase)가 발현되도록 형질전환되고, 락토오즈 퍼미아제 (lactose permease)가 발현되도록 형질전환되며, ABC 트랜스포터 퍼미아제(transporter permease)가 발현되도록 형질전환되고, 포스포만노뮤타아제 (Phosphomannomutase) 및 GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)를 보유하고 있는 것을 특징으로 하는 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)을 제공한다.The present invention is transformed to express α-1,3-fucosyltransferase, and GDP-D-mannose-4,6-dehydratase (GDP-D-mannose -4,6-dehydratase), transformed to express GDP-L-fucose synthase, and transformed to express lactose permease. Transformed, and transformed to express ABC transporter permease, Phosphomannomutase and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate) guanylyltransferase) provides a recombinant Corynebacterium glutamicum ( Corynebacterium glutamicum ), characterized in that it has.

본 발명의 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)에 있어, 상기 α-1,3-푸코오스 전이효소 (α-1,3-fucosyltransferase)는, 바람직하게 azoT 유전자로 암호화된 것이고, tac 프로모터에 의해 전사가 조절되는 것이 좋다.The recombinant Corynebacterium glutamicum of the present invention ( Corynebacterium glutamicum ), the α-1,3-fucosyltransferase is preferably encoded by the azoT gene, and transcription is preferably regulated by the tac promoter.

본 발명의 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)에 있어, 상기 GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase)는, 바람직하게 gmd 유전자 또는 noeL 유전자로 암호화된 것일 수 있다.The recombinant Corynebacterium glutamicum of the present invention ( Corynebacterium glutamicum ), the GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase) may be preferably encoded by a gmd gene or a noeL gene.

본 발명의 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)에 있어, 상기 재조합 코리네박테리움 글루타미쿰은, 바람직하게 포스포만노뮤타아제 (Phosphomannomutase)가 과발현되도록 형질전환되고, GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)가 과발현되도록 형질전환된 것이 좋다.In the recombinant Corynebacterium glutamicum of the present invention, the recombinant Corynebacterium glutamicum is preferably transformed to overexpress Phosphomannomutase, and GTP-mannose- It is preferable that it is transformed to overexpress 1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase).

한편, 본 발명은 락토오스가 첨가된 배지에, α-1,3-푸코오스 전이효소 (α-1,3- fucosyltransferase)가 발현되도록 형질전환되고, GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase)가 발현되도록 형질전환되며, GDP-L-푸코오스 신타아제 (GDP-L-fucose synthase)가 발현되도록 형질전환되고, 락토오즈 퍼미아제 (lactose permease)가 발현되도록 형질전환되며, 서열번호 5의 핵산서열로 구성된 ABC 트랜스포터 퍼미아제(transporter permease)가 발현되도록 형질전환되고, 포스포만노뮤타아제 (Phosphomannomutase) 및 GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)를 보유하고 있는 것을 특징으로 하는 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)을 배양하는 것을 특징으로 하는 3'-푸코실락토오스의 생산방법을 제공한다.On the other hand, the present invention is transformed to express α-1,3-fucosyltransferase (α-1,3-fucosyltransferase) in a medium to which lactose is added, and GDP-D-mannose-4,6-dehai Transformed to express dratase (GDP-D-mannose-4,6-dehydratase), transformed to express GDP-L-fucose synthase, and lactose permia Agent (lactose permease) is transformed to express, ABC transporter permease consisting of the nucleic acid sequence of SEQ ID NO: 5 is transformed to be expressed, and phosphomannomutase (Phosphomannomutase) and GTP-mannose- 1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase), characterized in that it has a recombinant Corynebacterium glutamicum ( Corynebacterium glutamicum ) characterized by culturing 3'-Fuco Provides a method for producing silactose.

본 발명의 3'-푸코실락토오스의 생산방법에 있어, 상기 배지는, 바람직하게 글루코오스를 더 포함하는 것이 좋다. 이때, 상기 푸코실락토오스의 생산방법은, 더욱 바람직하게 글루코오스 또는 락토오스를 추가로 공급하는 회분식 배양 또는 유가식 배양인 것일 수 있다.In the method for producing 3'-fucosylactose of the present invention, the medium preferably further contains glucose. At this time, the method for producing the fucosyl lactose may be a batch culture or fed-batch culture that additionally supplies glucose or lactose.

본 발명에 의하면, GRAS 균주인 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum) 균주를 사용하여 3'-푸코실락토오스를 생산할 수 있는데, 종래의 대장균에 비해 안전하게 3'-푸코실락토오스를 생산할 수 있다. 또한, 본 발명의 코리네박테리움 글루타미쿰 균주를 이용할 경우, ABC 트랜스포터 퍼미아제(transporter permease)의 도입에 의해 고농도, 고수율, 고생산성으로 3'-푸코실락토오스를 생산할 수 있다.According to the present invention, the GRAS strain, Corynebacterium glutamicum, can be used to produce 3'-fucosylactose, which can safely produce 3'-fucosylactose compared to conventional E. coli. . In addition, when using the Corynebacterium glutamicum strain of the present invention, 3'-fucosyllactose can be produced with high concentration, high yield, and high productivity by the introduction of ABC transporter permease.

도 1은 코리네박테리움 글루타미쿰 균주에서 GDP-L-fucose 및 3'-푸코실락토오스를 생합성하기 위하여 도입한 대사경로를 도식화한 것이다.
도 2는 코리네박테리움 글루타미쿰 pVBCLE + pENGWTA(CO)에서 생산된 3'-푸코실락토오스를 HPLC를 통해 측정한 결과이다.
도 3은 재조합 코리네박테리움 글루타미쿰 (C. glutamicum) pVBCLE + pENGWTA(CO)을 이용한 플라스크 회분식 배양결과를 나타낸 그래프이다. 광학밀도 (OD600)가 약 0.8에 도달하면, IPTG와 락토오스를 최종 농도가 각각 1.0mM, 10g/L (화살표)이 되도록 첨가하였다. 그래프 중 기호는 다음과 같다: ●건조세포중량, ▲글루코오스, ■락토오스, ▼락테이트, ◆3'-푸코실락토오스.
도 4는 재조합 코리네박테리움 글루타미쿰 (C. glutamicum) pVBCLE + pENGWTA(CO)를 이용한 유가식 배양결과를 나타낸 그래프이다. 초기에 투입한 40g/L 글루코오스가 모두 소모된 후, 글루코오스를 연속식 (continuous feeding)방법으로 공급하기 시작하였고, IPTG와 락토오스를 동시에 첨가하였다 (화살표). 그래프 중 기호는 다음과 같다: ●건조세포중량, ▲ 글루코오스, ■락토오스, ▼락테이트, ◆3'-푸코실락토오스.
1 is a schematic diagram of the metabolic pathways introduced to biosynthesize GDP-L-fucose and 3'-fucosylactose in Corynebacterium glutamicum strains.
FIG. 2 is a result of measuring 3′-fucosyl lactose produced in Corynebacterium glutamicum pVBCLE + pENGWTA (CO) through HPLC.
3 is a graph showing the results of batch culture of flasks using recombinant Corynebacterium glutamicum (C. glutamicum) pVBCLE + pENGWTA (CO). When the optical density (OD 600 ) reached about 0.8, IPTG and lactose were added so that the final concentrations were 1.0mM and 10g/L (arrow), respectively. The symbols in the graph are as follows: ●Dry cell weight, ▲glucose, ■lactose, ▼lactate, ◆3'-fucosylactose.
Figure 4 is a graph showing the fed-batch culture results using recombinant Corynebacterium glutamicum (C. glutamicum) pVBCLE + pENGWTA (CO). After the initial 40g/L glucose was all consumed, glucose was started to be supplied by a continuous feeding method, and IPTG and lactose were simultaneously added (arrow). The symbols in the graph are as follows: ● Dry cell weight, ▲ glucose, ■ lactose, ▼ lactate, ◆ 3'-fucosyl lactose.

본 발명은 α-1,3-푸코오스 전이효소 (α-1,3- fucosyltransferase)가 발현되도록 형질전환되고, GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase)가 발현되도록 형질전환되며, GDP-L-푸코오스 신타아제 (GDP-L-fucose synthase)가 발현되도록 형질전환되고, 락토오즈 퍼미아제 (lactose permease)가 발현되도록 형질전환되며, ABC 트랜스포터 퍼미아제(transporter permease)가 발현되도록 형질전환되고, 포스포만노뮤타아제 (Phosphomannomutase) 및 GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)를 보유하고 있는 것을 특징으로 하는 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)을 제공한다.The present invention is transformed to express α-1,3-fucosyltransferase, and GDP-D-mannose-4,6-dehydratase (GDP-D-mannose -4,6-dehydratase), transformed to express GDP-L-fucose synthase, and transformed to express lactose permease. Is converted, Transformed to express ABC transporter permease, Phosphomannomutase and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase) It provides a recombinant Corynebacterium glutamicum ( Corynebacterium glutamicum ) characterized in that it has.

본 발명의 발명자는 대한민국 특허출원번호 제10-2016-0012803호 (2016.02.02)를 통해, 대장균을 이용한 3'-푸코실락토오스 생산방법을 출원하였다. 하지만, 3'-푸코실락토오스를 기능성 식품첨가물로 이용함에 있어, 이를 대장균을 통해 생산하는 것은, 대장균이 갖는 여러 안전상의 염려로 인해 문제가 될 수 있다는 지적이 많았다. 따라서, 본 발명에서는 식품안전상 문제가 없는 대체 균주를 통해 3'-푸코실락토오스를 생산해보고자 하였다.The inventors of the present invention applied for a method for producing 3'-fucosylactose using E. coli through Korean Patent Application No. 10-2016-0012803 (2016.02.02). However, in the use of 3'-fucosilactose as a functional food additive, it has been pointed out that producing it through E. coli may be a problem due to various safety concerns of E. coli. Therefore, in the present invention, it was attempted to produce 3'-fucosyl lactose through an alternative strain without food safety problems.

본 발명에서는 3'-푸코실락토오스를 생산하는 숙주세포로서 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)을 선정하였는데, 이 균주는 종래에 사용하던 대장균과는 달리 GRAS (generally recognized as safe)로 인정된 균주일 뿐만 아니라, 엔도톡신을 생산하지 않으며, 식품첨가물인 아미노산 및 핵산의 산업적 생산에 널리 이용되고 있는 균주이다. 따라서, 코리네박테리움 글루타미쿰은 식품 및 의약품 소재의 생산을 위해 적합한 균주라 할 수 있고, 안전성 측면에서 소비자의 우려를 불식시킬 수 있는 장점이 있다.In the present invention, as a host cell producing 3'-fucosylactose, Corynebacterium glutamicum ( Corynebacterium glutamicum ) was selected, and this strain is not only a strain recognized as GRAS (generally recognized as safe), unlike E. coli used in the past, but also does not produce endotoxins, and is widely used for industrial production of amino acids and nucleic acids as food additives. It is a strain becoming. Therefore, Corynebacterium glutamicum can be said to be a suitable strain for the production of food and pharmaceutical materials, and has the advantage of eliminating concerns of consumers in terms of safety.

그런데 대장균과 코리네박테리움 글루타미쿰은 균주 자체의 유전적 특성이 다르기 때문에, 대장균에 적용하였던 전략과는 다른 전략을 사용해야 한다. 3'-푸코실락토오스를 생산하기 위해 대장균이든 코리네박테리움 글루타미쿰이든 기본적으로 외래의 α-1,3-푸코오스 전이효소 (α-1,3-fucosyltransferase)를 도입해야 하는 것은 동일하나, 코리네박테리움 글루타미쿰은 그 외에 추가적으로 GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase, Gmd, noeL), GDP-L-푸코오스 신타아제 (GDP-L-fucose synthase, 이 효소는 'GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase'로도 불림. 또한, 약어로는 'WcaG'로 불리는데, 이 효소를 암호화하는 유전자를 특히 'WcaG'로 부름), 락토오즈 퍼미아제 (lactose permease, LacY)를 도입해야 한다. 즉, 대장균에는 GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase, Gmd, noeL), GDP-L-푸코오스 신타아제 (GDP-L-fucose synthase, WcaG), 락토오즈 퍼미아제 (lactose permease, LacY)를 암호화하는 유전자를 가지나, 코리네박테리움 글루타미쿰 균주는 상기 효소들을 암호화하는 유전자를 가지지 않기 때문에, 이를 외부에서 도입시켜 이를 발현시켜 주어야 하는 것이다.However, because E. coli and Corynebacterium glutamicum have different genetic characteristics of the strain itself, a strategy different from the strategy applied to E. coli should be used. In order to produce 3'-fucosyl lactose, whether it is E. coli or Corynebacterium glutamicum, it is basically the same that foreign α-1,3-fucosyltransferase must be introduced. , Corynebacterium glutamicum is in addition to GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase, Gmd , noeL ), GDP-L- fuco Os synthase (GDP-L-fucose synthase, this enzyme is also called'GDP -4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase'. Also,' WcaG ' for abbreviation is The gene encoding this enzyme is specifically called'WcaG '), and lactose permease ( LacY ) must be introduced. That is, in E. coli GDP-D-mannose-4,6-dehydratase ( Gmd , noeL ), GDP-L-fucose synthase (GDP-L- fucose synthase, WcaG ), lactose permease ( LacY ) has a gene encoding the gene, but because Corynebacterium glutamicum strain does not have the gene encoding the enzymes, it is introduced from the outside to It must be revealed.

한편, 본 발명에 있어, 상기 α-1,3-푸코오스 전이효소 (α-1,3-fucosyltransferase)를 코딩하는 유전자는, azoT 유전자로 암호화된 것을 사용하는 것이 좋고, tac 프로모터에 의해 전사가 조절되는 것이 좋은데, 상기 tac_azoT 유전자는 바람직하게 서열번호 7의 핵산서열로 구성된 것일 수 있다. α-1,3-푸코실락토오스를 생산하기 위해서는, GDP-L-푸코오스 (GDP-L-fucose)와 락토오즈 (lactose)를 기질로 하여 α-1,3-푸코실락토오스 생산 반응을 수행하는 α-1,3-푸코오스 전이효소 (α-1,3-fucosyltransferase)가 필요하다 (도 1 참조 요망). On the other hand, in the present invention, the gene encoding the α-1,3-fucosyltransferase (α-1,3-fucosyltransferase) is preferably used to be encoded by the azoT gene, and transcription is performed by the tac promoter. It is good to be regulated, but the tac_ azoT gene may be preferably composed of the nucleic acid sequence of SEQ ID NO: 7. In order to produce α-1,3-fucosyllactose, α-1,3-fucosyllactose production reaction is performed using GDP-L-fucose and lactose as substrates. The α-1,3-fucosyltransferase is required (see Fig. 1).

이 효소는 다양한 미생물에 존재하는데, 본 발명에서는 아조스피릴럼 브라실렌스(Azospirillum brasilense)에서 유래한 유전자(azoT)를 사용한 것이다. 다른 유래의 α-1,3-푸코오스 전이효소를 사용한 경우에는 3'-푸코실락토오스의 생산량이 미미하였으나, azoT 유전자를 사용한 경우, 다른 유래의 것을 사용하는 것에 비해 3'-푸코실락토오스의 생산량이 현저히 높게 나타났다. 이때, Tac 프로모터(promoter)는 스트롱 프로모터(strong promoter)로 강력하게 발현되고, 유도성 프로모터(inducible promoter)로써 조절이 가능한 장점이 있다. 본 발명에서는 tac 프로모터를 azoT 유전자의 앞에 추가하여 azoT 유전자가 좀 더 강하게 발현되도록 유도한 것이다.The enzyme used is a gene (azoT) derived from azo RY rilreom bra chamber lances (Azospirillum brasilense) for the various micro-organisms present in the present invention. When α-1,3-fucose transferase from other sources were used, the production of 3'-fucosylactose was insignificant, but when using the azoT gene, the 3'- fucosyllactose was The output was remarkably high. At this time, the Tac promoter is strongly expressed as a strong promoter, and has an advantage that can be regulated as an inducible promoter. In the present invention, and the addition of the tac promoter in front of the gene to induce azoT azoT the gene such that a more strongly expressed.

또한, GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase, noeL)도 바람직하게 아조스피릴럼 브라실렌스(Azospirillum brasilense)에서 유래한 유전자(noeL)로 암호화된 것이 좋다. 한편, GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase, Gmd), GDP-L-푸코오스 신타아제 (GDP-L-fucose-synthase, WcaG) 및 락토오즈 퍼미아제 (lactose permease, LacY)를 암호화하는 유전자는 대장균에서 유래한 것을 사용하는 것이 좋다.In addition, GDP-D- mannose-4, 6 having a gene derived from other Hydra dehydratase (GDP-D-mannose-4,6 -dehydratase, noeL) also preferably azo RY rilreom bra chamber lances (Azospirillum brasilense) ( noeL ) encrypted. Meanwhile, GDP-D-mannose-4,6-dehydratase ( Gmd ), GDP-L-fucose synthase (GDP-L-fucose-synthase, WcaG ) and lactose permease (lactose permease, LacY ) genes encoding E. coli is preferably used.

한편, 본 발명에 있어서, 상기 GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase)는, 바람직하게 gmd 유전자 또는 noeL 유전자로 암화화되어 있을 수 있는데, 더욱 바람직하게는 이들 두 유전자를 동시에 발현되는 것이 좋다. 즉, gmd 발현 시스템에 noeL을 추가적으로 발현하여 결과적으로 두 가지 유전자를 모두 발현시킨 시스템을 구축할 수 있는 것이다. gmd는 대장균 유래이고, noeL은 아조스피릴럼 브라실렌스(Azospirillum brasilense) 유래인데, 같은 효소를 암호화하는 유전자라 하여도 유래가 다르면 발휘하는 효율도 다를 것이라 생각하여, 본 발명에서는 종래에 알려진 gmd 발현 시스템에 noeL을 추가적으로 발현하여 결과적으로 두 가지 유전자를 모두 발현시킨 시스템을 구축한 것이다.On the other hand, in the present invention, the GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase) is preferably cancerized with a gmd gene or noeL gene. However, more preferably, these two genes are preferably expressed at the same time. In other words, it is possible to construct a system in which noeL is additionally expressed in the gmd expression system and as a result, both genes are expressed. gmd is derived from E. coli, are azo noeL RY rilreom bra chamber lances (Azospirillum brasilense ), but even if the genes encoding the same enzyme are different, the efficiency exerted will be different, so in the present invention, noeL is additionally expressed in the conventionally known gmd expression system, and as a result, both genes are expressed. The system was built.

한편, 본 발명의 ABC 트랜스포터 퍼미아제(transporter permease)는, 본 발명에서 균체 내에서 생산된 3'-푸코실락토오스를 균체 밖으로 배출하는 역할을 수행하는데, 통상적으로 익스포터(exporter)라고도 부른다. ABC 트랜스포터 퍼미아제는 기질결합단백질(substrate binding protein)에 기질이 붙고, ATP가 소모되어 기질을 세포안으로 들여오는 수송체로서, 여기서는 단지 푸코실락토오스가 지나다닐 수 있는 통로를 도입함으로써, 세포내에서 만들어진 프코실락토오스가 세포밖으로 빠져나갈 수 있는 길을 만들어 준 것이다.On the other hand, the ABC transporter permease of the present invention plays a role of discharging 3'-fucosylactose produced in the cells in the present invention, and is generally referred to as an exporter. ABC transporter permase is a transporter in which a substrate is attached to a substrate binding protein and ATP is consumed to bring the substrate into the cell. Here, by introducing a pathway through which fucosyl lactose can only pass, the cell It made a way for the pcosylactose produced inside to escape out of the cell.

본 발명에서는 바람직하게 비피도박테리움 인판티스(Bifidobacterium infantis) 유래의 서열번호 5의 핵산서열로 암호화된 것을 이용하여 실험을 수행하였는데, 균체 내에서 생산되어 축적된 3'-푸코실락토오스이 이 효소의 도입으로 인해 균체 밖으로 잘 배출됨 (균체를 파쇄할 필요가 없어 배지 중으로 3'-푸코실락토오스를 배출하는 것은 분리·정제 공정에 매우 효과적임)을 확인할 수 있었다.In the present invention, preferably Bifidobacterium Infantis ( Bifidobacterium infantis ) derived from the nucleic acid sequence of SEQ ID NO: 5, and the experiment was carried out, and 3'-fucosyl lactose produced and accumulated in the cells is well discharged out of the cells due to the introduction of this enzyme (requires disruption of the cells. It was confirmed that the discharge of 3'-fucosyllactose into the medium is very effective in the separation and purification process).

다만, 본 발명에서는 ABC 트랜스포터 퍼미아제(transporter permease)로 비피도박테리움 인판티스(Bifidobacterium infantis) 유래의 서열번호 5의 핵산서열로 암호화된 것을 사용하였으나, 비피도박테리움의 푸코실락토오스 수송에 관여하는 ABC 트랜스포터(transporter) 중에 트랜스멤브레인 도메인(transmembrane domain)을 가진 ABC 트랜스포터 퍼미아제가 푸코실락토오스가 지나갈 수 있는 통로로써 사용될 수 있기 때문에, 비피도박테리움 인판티스(Bifidobacterium infantis) 유래의 서열번호 5의 암호화된 것 외에 ABC 트랜스포터 퍼미아제(transporter permease) 류에 속하는 것은 어느 것이나 동일한 효과가 발생할 것으로 추론할 수 있었다.However, in the present invention, the one encoded by the nucleic acid sequence of SEQ ID NO: 5 derived from Bifidobacterium infantis was used as ABC transporter permease, but the transport of fucosyl lactose of Bifidobacterium Because ABC transporter permase having a transmembrane domain among the ABC transporters involved in can be used as a pathway through which fucosylactose can pass, Bifidobacterium infantis ( Bifidobacterium) infantis ) It could be inferred that any one belonging to the ABC transporter permease class other than the encoded one of SEQ ID NO: 5 derived from it would have the same effect.

한편, 본 발명의 재조합 코리네박테리움 글루타미쿰은 바람직하게 포스포만노뮤타아제 (Phosphomannomutase)가 과발현되도록 형질전환되고, GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)가 과발현되도록 형질전환되는데, 코리네박테리움 글루타미쿰은 포스포만노뮤타아제 (Phosphomannomutase, ManB), GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase, ManC)를 암호화하는 유전자를 자체적으로 보유하여 발현시킬 수 있기 때문에, 굳이 이 효소를 암호화하는 유전자를 도입시켜줄 필요는 없으나, 대량 생산을 위해서는 이 효소를 과발현시켜줄 필요가 있다. 따라서, 본 발명에서는 이들 두 효소를 과발현할 수 있도록 코리네박테리움 글루타미쿰을 형질전환하는 것이 필요한 것이다.On the other hand, the recombinant Corynebacterium glutamicum of the present invention is preferably transformed to overexpress Phosphomannomutase, GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1 -phosphate guanylyltransferase) is transformed to overexpress, Corynebacterium glutamicum is Phosphomannomutase ( ManB ), GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1- Since the gene encoding phosphate guanylyltransferase, ManC ) can be retained and expressed, it is not necessary to introduce the gene encoding this enzyme, but it is necessary to overexpress this enzyme for mass production. Therefore, in the present invention, it is necessary to transform Corynebacterium glutamicum so that these two enzymes can be overexpressed.

한편, 상기 효소들의 작용은 도 1을 통해서 이해될 수 있으므로, 이에 대한 설명은 생략하기로 한다. 다만, 락토오즈 퍼미아제 (lactose permease, LacY)는 균주 외부에 존재하는 락토오스를 균주 내부로 수송하는데 관여하는 효소임을 특별히 밝혀두는 바이다. 하기 본 발명의 실시예에서는 대장균의 Lac 오페론에서 lacZ가 제거된 lacYA 유전자를 도입하여 실험하였으나, 본 발명에서 Lac 오페론의 도입 이유가 락토오스의 유입에 관한 것이기 때문에, 굳이 lacA 유전자까지는 필요 없고, lacY 유전자만 도입시켜도 충분하다.Meanwhile, since the action of the enzymes can be understood through FIG. 1, a description thereof will be omitted. However, lactose permease (lactose permease, LacY ) is a bar that is specifically identified as an enzyme involved in transporting lactose present outside the strain into the strain. To, but experiments by the embodiment of the present invention introducing the lacYA gene lacZ is removed from the Lac operon of E. coli, because it relates to the invention of the free reason of Lac operon, it lactose coming from, deliberately not required by lacA gene, lacY gene It is enough to introduce only.

한편, 본 발명에서 사용하는 '발현'이라는 용어는, 본 발명의 코리네박테리움 글루타미쿰 균주가 자체적으로 발현시킬 수 없는 효소를, 인위적으로 발현시키기 위해 외부 유래의 유전자를 균주 내로 도입하여 발현시키는 것을 의미하고, '과발현'이라는 용어는 본 발명의 코리네박테리움 글루타미쿰 균주가 자체적으로 해당 효소를 암호화하는 유전자를 가져, 스스로 발현시킬 수 있으나, 대량생산을 위한 목적으로 이의 발현량을 증대시키기 위해 인위적으로 해당 효소의 발현량을 증대시켜 과발현한 것을 의미한다.Meanwhile, the term'expression' used in the present invention is expressed by introducing an external gene into the strain in order to artificially express an enzyme that the Corynebacterium glutamicum strain of the present invention cannot express on its own. The term'overexpression' means that the strain of Corynebacterium glutamicum of the present invention has a gene encoding the enzyme and can express it by itself, but for the purpose of mass production, the expression level thereof In order to increase it, it means overexpression by artificially increasing the expression level of the enzyme.

한편, 본 발명자들은 상기에서 설명한 형질전환 전략을 통해, 코리네박테리움 글루타미쿰 (C. glutamicum)에서 모유올리고당인 3'-푸코실락토오스를 대량 생산할 수 있음을 확인할 수 있었다.On the other hand, the present inventors were able to confirm that through the transformation strategy described above, it was possible to mass-produce 3'-fucosyl lactose, which is a human milk oligosaccharide, from Corynebacterium glutamicum ( C. glutamicum ).

한편, 본 발명은 락토오스가 첨가된 배지에, 본 발명의 재조합 코리네박테리움 글루타미쿰을 배양하는 것을 특징으로 하는 3'-푸코실락토오스의 생산방법을 제공한다. 본 발명의 재조합 코리네박테리움 글루타미쿰 균주를 이용할 경우, 고농도, 고수율, 고생산성으로 3'-푸코실락토오스를 생산할수 있다.On the other hand, the present invention provides a method for producing 3'-fucosylactose, characterized in that the recombinant Corynebacterium glutamicum of the present invention is cultured in a medium to which lactose is added. When using the recombinant Corynebacterium glutamicum strain of the present invention, it is possible to produce 3'-fucosyl lactose with high concentration, high yield, and high productivity.

한편, 상기 본 발명의 3'-푸코실락토오스의 생산방법에 있어, 상기 배지는, 바람직하게 글루코오스를 더 포함하는 것이 좋다. 글루코오스가 배지에 추가됨으로써 균주의 생육이 활발해져 더욱 높은 생산성으로 3'-푸코실락토오스를 생산할 수 있다.On the other hand, in the method for producing 3'-fucosyl lactose of the present invention, the medium preferably further contains glucose. When glucose is added to the medium, the growth of the strain becomes active, so that 3'-fucosyl lactose can be produced with higher productivity.

한편, 상기 본 발명의 3'-푸코실락토오스의 생산방법은, 회분식 또는 락토오스를 추가로 공급하는 유가식 배양을 통해 수행될 수 있다. 회분식 또는 유가식 배양에 관한 세부 지엽적 기술들은 당업계의 공지 기술을 사용할 수 있으므로, 이에 대해서는 그 기재를 생략하기로 한다.On the other hand, the method for producing 3'-fucosyl lactose of the present invention may be carried out through batch or fed-batch culture in which lactose is additionally supplied. Detailed local techniques for batch-type or fed-batch culture may use known techniques in the art, and thus description thereof will be omitted.

한편, 본 발명의 균주 코리네박테리움 글루타미쿰은, 3'-푸코실락토오스 생산의 기질인 락토오스를 균체 내로 유입하기 위하여 락토오즈 퍼미아제 (lactose permease)를 도입하였다. 즉, 코리네박테리움 글루타미쿰을 이용하여 3'-FL을 생산하기 위해서는 락토오즈를 균체 내로 유입시킬 수 있는 락토오즈 퍼미아제로 형질전환시켜 줘야 하는데, 본 발명의 균주는 이 효소로 형질전환된 것이다.On the other hand, the strain Corynebacterium glutamicum of the present invention introduced lactose permease to introduce lactose, which is a substrate for the production of 3'-fucosylactose, into the cells. That is, in order to produce 3'-FL using Corynebacterium glutamicum, it must be transformed with lactose permase, which can introduce lactose into the cells, and the strain of the present invention is transformed with this enzyme. It was done.

그런데 락토오즈 퍼미아제는 통상적으로 포도당 존재하에서 "glucose repression (포도당 저해)"을 받아 그 활성이 저해된다. 그 결과, 포도당 존재하에서 락토오즈의 유입이 일어나지 못하게 되고, 결과적으로 3'-푸코실락토오스를 생산하지 못하게 된다.However, lactose permase is usually inhibited by receiving "glucose repression" in the presence of glucose. As a result, inflow of lactose does not occur in the presence of glucose, and as a result, 3'-fucosyl lactose cannot be produced.

하지만, 3'-푸코실락토오스의 생산을 위한 호스트 균주로 본 발명에서 사용한 코리네박테리움 글루타미쿰에서는 "glucose repression (포도당 저해)" 작용이 일어나지 않아, 포도당의 존재하에서도 락토오스의 유입에 따른 3'-FL을 생산할 수가 있었고, 이를 통해 3'-푸코실락토오스의 생산성을 극대화할 수 있었다.However, in the Corynebacterium glutamicum used in the present invention as a host strain for the production of 3'-fucosyllactose, the "glucose repression (glucose inhibition)" action does not occur, according to the influx of lactose even in the presence of glucose. It was possible to produce 3'-FL, and through this, the productivity of 3'-fucosylactose could be maximized.

한편, 본 발명의 코리네박테리움 글루타미쿰을 이용한 3'-푸코실락토오스의 생산방법은 3'-푸코실락토오스를 'nongrowth-associated product formation'의 생산 패턴으로 생산함이 본 발명의 하기 실험을 통해 확인되었다.On the other hand, the production method of 3'-fucosylactose using Corynebacterium glutamicum of the present invention is to produce 3'-fucosylactose in the production pattern of'nongrowth-associated product formation'. Confirmed through.

대사산물의 생산이 호스트 균주의 생육과 무관하게 생산되는 'nongrowth-associated product formation'은 대사산물의 생산을 위한 호스트 균주의 생장이 같이 요구되지 않기 때문에, 호스트 균주를 대량으로 배양한 후, 기질을 투입하여 단시간에 대사산물을 대량으로 생산할 수 있는 장점이 있다. 또한, 배양에 사용된 호스트 균주를 반복해서 사용할 수도 있기 때문에 생산성을 극대화시킬 수 있는 장점도 있다. 따라서, 본 발명에서 코리네박테리움 글루타미쿰을 사용하여 구축한 3'-푸코실락토오스 생산방법은 3'-푸코실락토오스의 생산을 극대화할 수 있는 방법인 것이다.'Nongrowth-associated product formation', in which the production of metabolites is produced irrespective of the growth of the host strain, does not require the growth of the host strain for the production of metabolites, so after culturing the host strain in large quantities, the substrate is It has the advantage of being able to mass-produce metabolites in a short time by inputting it. In addition, since the host strain used for cultivation may be repeatedly used, there is also an advantage of maximizing productivity. Therefore, the method for producing 3'-fucosylactose constructed using Corynebacterium glutamicum in the present invention is a method capable of maximizing the production of 3'-fucosylactose.

이하, 본 발명의 내용을 하기 실시예를 통해 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the content of the present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited to the following examples, and includes modifications of the technical idea equivalent thereto.

[[ 실시예Example 1: 재조합 균주 및 플라스미드 제작] 1: Recombinant strain and plasmid construction]

플라스미드 제작을 위해 클로닝은 대장균 (Escherichia coli TOP10)을 이용하였고, 3'-푸코실락토오스 (3'-fucosyllactose, 3'-FL)의 생산을 위하여 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum) ATCC 13032를 이용하였다. 선행연구에서 개발된 manBmanC 그리고 lacYA와 exporter 유전자 클러스터를 발현 하는 pVBCLE 발현용 플라스미드를 이용하였다. 또한, gmdwcaG 유전자를 발현하는 pEGW 발현용 플라스미드에 α-1,3-푸코오스 전이효소 (α-1,3- fucosyltransferase(azoT))와 noeL 유전자를 발현시키기 위하여 벡터를 구축하였다. 이때, α-1,3-푸코오스 전이효소(azoT)의 유래는 아조스피릴럼 브라실렌스(Azospirillum brasilense) ATCC 29145이며, 코리네박테리움 글루타미쿰에 맞도록 코돈최적화 된 서열을 사용하였다. pEGW 벡터에 Sac1의 제한효소를 이용하여 tac 프로모터를 앞에 위치시키고 코돈최적화된 α-1,3-푸코오스 전이효소 (tac_azoT(CO)를 삽입하였고, Kpn1의 제한효소를 이용하여 noeL 유전자를 삽입하였다. 상기 azoT(CO)는 azoT 유전자를 코리네박테리움에 맞도록 코돈최적화 시킨 것이다. 한편, 상기에서 사용된 manB , manC , gmd , WcaG , noeL . lacYA 및 코돈최적화된 α-1,3-푸코오스 전이효소 (azoT(CO))의 유전자 서열, 균주, 플라스미드 및 올리고뉴클레오티드 를 하기 표 1 내지 3에 기재하였다.For plasmid production, cloning was performed using Escherichia coli TOP10), and for the production of 3'-fucosyllactose (3'-FL), Corynebacterium glutamicum ( Corynebacterium glutamicum ) ATCC 13032 was used. A manB and manC and a plasmid for expression pVBCLE expressing lacYA and exporter gene cluster developed in previous studies was used. In addition, a vector was constructed to express α-1,3-fucosyltransferase (azoT) and noeL gene in a plasmid for pEGW expression expressing gmd and wcaG genes. At this time, α-1,3- origin of fucose transferase (azoT) azo RY rilreom bra lances and chamber (Azospirillum brasilense) ATCC 29145, was used as the codon optimized sequences to match Corynebacterium glutamicum. It was located in front of the tac promoter by using a restriction enzyme Sac 1 to pEGW vector and insert the codon optimized α-1,3- fucose transferase (tac_azoT (CO), using the restriction enzymes Kpn 1 noeL the gene was inserted. the azoT (CO) which is codon optimized to fit the azoT gene in Corynebacterium. on the other hand, the manB, manC, gmd, WcaG, noeL. lacYA and codon-optimized α-1,3 used in the -The gene sequence, strain, plasmid and oligonucleotide of fucose transferase (azoT(CO)) are shown in Tables 1 to 3 below.

유전자명Gene name 서열번호Sequence number manBmanB 서열번호 1SEQ ID NO: 1 manCmanC 서열번호 2SEQ ID NO: 2 gmdgmd -- WcaGWcaG 서열번호 3SEQ ID NO: 3 lacYAlacYA 서열번호 4SEQ ID NO: 4 ExporterExporter 서열번호 5SEQ ID NO: 5 noeLnoeL 서열번호 6SEQ ID NO: 6 tac_ azoT(CO) tac_ azoT (CO) 서열번호 7SEQ ID NO: 7 tac_Promoter tac_ Promoter 서열번호 8SEQ ID NO: 8 azoT(CO) azoT (CO) 서열번호 9SEQ ID NO: 9

균주Strain 관련된 특징Related features E. coli TOP10 E. coli TOP10 F-, mcrA Δ(mrr - hsdRMS- mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG F -, mcr A Δ (mrr - hsd RMS - mcr BC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ ( ara-leu ) 7 697 galU galK rpsL (Str R ) endA1 nupG C. C. glutamicumglutamicum WW 플라스미드Plasmid 관련된 특징Related features pEKEx2pEKEx2 KmR ;C . glutamicum/E. coli shuttle vector for regulated gene expression (Ptac,lacIq,pBL1, oriVC.g., oriVE.c.)Km R ; C. glutamicum / E. coli shuttle vector for regulated gene expression (P tac ,lacIq,pBL1, oriVC.g., oriVE.c.) pVWEx2pVWEx2 TcR ;C . glutamicum/E. coli shuttle vector for regulated gene expression (Ptac,lacIq,pBL1, oriVC.g., oriVE.c.)Tc R ; C. glutamicum / E. coli shuttle vector for regulated gene expression (P tac ,lacIq,pBL1, oriVC.g., oriVE.c.) pEGWpEGW pEKEx2 + gmd - wcaG pEKEx2 + gmd - wcaG pVBCLEpVBCLE pVWEx2 + manB + manC + lacYA + exporter pVWEx2 + manB + manC + lacYA + exporter pENGWTA(CO)pENGWTA(CO) pEKEx2 + noeL + tac_ azoT (CO) pEKEx2 + noeL + tac_ azoT (CO)

프라이머primer 이름 name 서열(5'→3')Sequence (5'→3') 서열번호Sequence number F_inf_KpnI_noeL(pENGWTA(CO))F_inf_KpnI_ noeL (pENGWTA(CO)) CTAGAGGATCCCCGGGTACC TTGGCGCGGATCGCATTGATCTTCGCTAGAGGATCCCCG GGTACC TTGGCGCGGATCGCATTGATCTTCG 서열번호 10SEQ ID NO: 10 R_inf_KpnI_noeL(pENGWTA(CO))R_inf_KpnI_ noeL (pENGWTA(CO)) TTGTATATCTCCTTGGTACC TCAAGGTTGCGGCAGGGCTTGTATATCTCCTT GGTACC TCAAGGTTGCGGCAGGGC 서열번호 11SEQ ID NO: 11 F_inf_Sac1_Tac_RBS_azoT(pEGWTA(CO))F_inf_Sac1_Tac_RBS_ azoT (pEGWTA(CO)) GCTTTCGGGGGTAAGAGCTC TCAGGCAGCCATCGGAAGGCTTTCGGGGGTAA GAGCTC TCAGGCAGCCATCGGAAG 서열번호 12SEQ ID NO: 12 R_inf_sacI_COazoT(pEGWTA(CO))R_inf_sacI_CO azoT (pEGWTA(CO)) CGGCCAGTGAATTCGAGCTC TTATAAGCGGGATTCGATCCAGTCCGGCCAGTGAATTC GAGCTC TTATAAGCGGGATTCGATCCAGTC 서열번호 13SEQ ID NO: 13

* 굵은체로 표시된 서열은 특정 제한효소의 인지 부위를 나타냄.* Sequences in bold indicate the recognition site of a specific restriction enzyme.

[[ 실시예Example 2: 재조합 2: recombination 코리네박테리움Corynebacterium 글루타미쿰을Glutamicum 이용한 3'- Used 3'- 푸코실락토오스의Fucosylactose 생산] production]

(1) 배양조건 및 방법(1) Culture conditions and methods

종균배양에는 적절한 항생제 (kanamycin 25㎍/㎖, tetracycline 5㎍/㎖)가 포함된 5㎖의 BHI (Brain Heart Infusion) 배지가 담긴 실험관을 이용하였고, 온도는 30℃, 교반 속도를 250 rpm으로 유지하며 12시간 동안 배양하였다.For spawn culture, a test tube containing 5 ml of BHI (Brain Heart Infusion) medium containing appropriate antibiotics (kanamycin 25µg/ml, tetracycline 5µg/ml) was used, and the temperature was 30℃ and the stirring speed was maintained at 250 rpm. And incubated for 12 hours.

회분식 배양은 100㎖ ((NH4)2SO4 20g/L, urea 5g/L, KH2PO4 1g/L, K2HPO4 1g/L, MgSO4 0.25g/L, MOPS 42g/L, CaCl2 10㎎/L, Biotin 0.2㎎/L, Protocatechuic acid 30㎎/L, FeSO47H20 10㎎/L, MnSO4H2O 10㎎/L, ZnSO47H2O 1㎎/L, CuSO4 0.2㎎/L, NiCl26H2O 0.02㎎/L, pH 7.0)가 담긴 250㎖ 플라스크에서 30℃로 수행하였다. 교반속도는 250 rpm으로 유지하며 배양하였다. 회분식 배양시에는 광학밀도 (OD600)가 0.8에 도달한 시점에서 IPTG (isopropyl-β락토오스를 최종 농도가 각각 1.0mM, 10g/L가 되도록 첨가하였다.Batch culture is 100㎖ ((NH 4 ) 2 SO 4 20g/L, urea 5g/L, KH 2 PO 4 1g/L, K 2 HPO 4 1g/L, MgSO 4 0.25g/L, MOPS 42g/L, CaCl 2 10 mg/L, Biotin 0.2 mg/L, Protocatechuic acid 30 mg/L, FeSO 4 7H 2 0 10 mg/L, MnSO 4 H 2 O 10 mg/L, ZnSO 4 7H 2 O 1 mg/L, CuSO 4 0.2 mg/L, NiCl 2 6H 2 O 0.02 mg/L, pH 7.0) in a 250 ml flask containing 30°C. The stirring speed was maintained at 250 rpm and cultured. In batch culture, when the optical density (OD 600 ) reached 0.8, isopropyl-β lactose (IPTG) was added so that the final concentrations were 1.0mM and 10g/L, respectively.

고농도 세포배양을 위한 유가식 배양은 40g/L의 글루코오스 및 적절한 항생제 (kanamycin 25㎍/㎖, tetracycline 5㎍/㎖)를 포함하는 1.0L의 최소배지를 포함하는 2.5L 들이의 생물반응기 (bioreactor, Kobiotech, Incheon, Korea)에서 실시하였다. 초기에 첨가한 글루코오스가 완전히 고갈된 후, 800g/L의 글루코오스를 포함하는 유입용액 (feeding solution)을 5.7g/L/h의 속도로 연속식 (continuous feeding) 방법으로 공급하였다. 이와 동시에, tac 프로모터-매개 유전자 발현을 유도하여 3'-푸코실락토오스를 생산하기 위해 IPTG, 락토오스를 최종 농도가 1.0mM, 10g/L가 되도록 첨가하였다.Fed-batch culture for high-concentration cell culture is a 2.5L bioreactor containing 1.0L minimal medium containing 40g/L glucose and appropriate antibiotics (kanamycin 25㎍/mL, tetracycline 5㎍/mL). Kobiotech, Incheon, Korea). After the initially added glucose was completely depleted, a feeding solution containing 800 g/L of glucose was supplied by a continuous feeding method at a rate of 5.7 g/L/h. At the same time, in order to induce tac promoter-mediated gene expression to produce 3'-fucosylactose, IPTG and lactose were added to a final concentration of 1.0 mM and 10 g/L.

발효 도중 배지의 pH가 설정포인트 (set-point)보다 더 낮아지면 자동으로 28% NH4OH가 공급되고, 높아지면 2N HCl이 첨가되어 pH가 일정범위 내 (pH 6.98~7.02)에서 유지되도록 하였다. 배지의 pH는 pH 전극 (Mettler Toledo, USA)을 사용하여 실시간으로 측정되었다. 교반 속도 및 통기 속도는 산소결핍을 방지하기 위하여 각각 1000 rpm 및 2vvm으로 유지하였다.During fermentation, when the pH of the medium was lower than the set-point, 28% NH 4 OH was automatically supplied, and when it increased, 2N HCl was added to maintain the pH within a certain range (pH 6.98 to 7.02). . The pH of the medium was measured in real time using a pH electrode (Mettler Toledo, USA). The stirring speed and the aeration speed were maintained at 1000 rpm and 2vvm, respectively, to prevent oxygen deficiency.

(2) 세포 및 (2) cells and 대사산물의Metabolite 농도 결정 Concentration determination

건조세포중량은 광학밀도 (optical density, OD)에 미리 측정한 변환 상수 0.3을 곱해 결정하였다. 광학밀도 (OD)는 샘플을 적절하게 희석하여 광학밀도를 0.1-0.5 사이의 범위에 맞춘 후에 분광광도계 (spectrophotometer, Ultrospec 2000, Amersham Pharmacia Biotech, USA)를 사용하여 흡광도 600㎚에서 측정하였다.The dry cell weight was determined by multiplying the optical density (OD) by a previously measured conversion constant of 0.3. Optical density (OD) was measured at 600 nm using a spectrophotometer (Ultrospec 2000, Amersham Pharmacia Biotech, USA) after appropriately diluting the sample and adjusting the optical density to a range of 0.1-0.5.

3'-푸코실락토오스, 락토오스, 락테이트, 글루코오스 및 아세트산의 농도는 'Carbohydrate Analysis column (Rezex ROA-organic acid, Phenomenex, USA)' 및 'RI (refractive index)' 검출기가 장착된 HPLC (high performance liquid chromatography) (Agilent 1100LC, USA)를 이용하여 측정하였다. 60℃에서 가열된 컬럼을 적용하여 10배 희석된 20㎕의 배양 배지를 분석하였다. 0.6㎖/min 유속으로 5mM의 H2SO4 용액을 이동상으로 사용하였다. The concentrations of 3'-fucosylactose, lactose, lactate, glucose and acetic acid are measured by HPLC (high performance) equipped with'Carbohydrate Analysis column (Rezex ROA-organic acid, Phenomenex, USA)'and'RI (refractive index)' detectors. liquid chromatography) (Agilent 1100LC, USA). A column heated at 60° C. was applied to analyze 20 μl of the culture medium diluted 10 times. A 5 mM H 2 SO 4 solution was used as the mobile phase at a flow rate of 0.6 ml/min.

(3) (3) 회분식Batch 배양을 통한 3'- 3'- through culture 푸코실락토오스의Fucosylactose 생산 결과 Production results

3'-푸코실락토오스 생산성능 및 발효특징을 알아보기 위하여 ManB, ManC, Gmd, WcaG, noeL, tac_azoT(CO) 및 lacZ가 제거된 lac 오페론 (lacYA)와 익스포터(exporter)를 도입한 재조합 코리네박테리움 글루타미쿰을 플라스크에서 회분식 배양을 실시하였다. 광학밀도 (OD600)가 0.8에 도달한 시점에서 IPTG, 락토오스를 최종 농도가 각각 1.0mM, 10g/L가 되도록 첨가하였다.Recombinant Corynes introduced with lac operon (lacYA) and exporter from which ManB, ManC, Gmd, WcaG, noeL, tac_azoT(CO) and lacZ have been removed to investigate 3'-fucosylactose productivity and fermentation characteristics. Bacterium glutamicum was batch cultured in a flask. When the optical density (OD 600 ) reached 0.8, IPTG and lactose were added so that the final concentrations were 1.0mM and 10g/L, respectively.

플라스크 회분식 배양의 결과, 1.33g/L의 3'-푸코실락토오스가 생산되었고, 이때의 락토오스 대비 3'-푸코실락토오스의 수율은 0.35 mole 3'-푸코실락토오스 /mole 락토오스, 생산성은 0.02g/L/h였다 (도 3 및 표 4).As a result of the flask batch culture, 1.33 g/L of 3'-fucosyl lactose was produced, and the yield of 3'-fucosyl lactose compared to lactose at this time was 0.35 mole 3'-fucosyl lactose/mole lactose, and the productivity was 0.02 g. /L/h (Fig. 3 and Table 4).

도 2는 코리네박테리움 글루타미쿰 pVBCLE + pENGWTA(CO)에서 생산된 3'-푸코실락토오스를 HPLC를 통해 측정한 결과이다. 상기 회분식 배양의 결과는 하기 표 4에 기재하였으며, 도 3은 재조합 코리네박테리움 글루타미쿰 (C. glutamicum) pVBCLE + pENGWTA(CO)을 이용한 플라스크 회분식 배양결과를 나타낸 그래프이다. 광학밀도 (OD600)가 약 0.8에 도달하면, IPTG와 락토오스를 최종 농도가 각각 1.0 mM, 10g/L (화살표)이 되도록 첨가하였다. 그래프 중 기호는 다음과 같다: ●건조세포중량, ▲글루코오스, ■락토오스, ▼락테이트, ◆3'-푸코실락토오스.FIG. 2 is a result of measuring 3′-fucosyl lactose produced in Corynebacterium glutamicum pVBCLE + pENGWTA (CO) through HPLC. The results of the batch culture are shown in Table 4 below, and FIG. 3 is a graph showing the flask batch culture results using recombinant Corynebacterium glutamicum ( C. glutamicum ) pVBCLE + pENGWTA (CO). When the optical density (OD 600 ) reached about 0.8, IPTG and lactose were added so that the final concentrations were 1.0 mM and 10 g/L (arrow), respectively. The symbols in the graph are as follows: ●Dry cell weight, ▲glucose, ■lactose, ▼lactate, ◆3'-fucosylactose.

재조합 코리네박테리움 글루타미쿰 (C. glutamicum) pVBCLE + pENGWTA(CO)를 이용한 플라스크 회분식 배양 결과Flask batch culture results using recombinant Corynebacterium glutamicum ( C. glutamicum ) pVBCLE + pENGWTA(CO) 최종 건조
세포 중량
(g/L)
Final drying
Cell weight
(g/L)
락토오스 소모량a
(g/L)
Lactose consumption a
(g/L)
최대 3'-푸코실락토오스 농도a
(g/L)
Max. 3'-fucosylactose concentration a
(g/L)
수율 (mole 3'-푸코실락토오스
/mole 락토오스)
Yield (mole 3'-fucosylactose
/mole lactose)
생산성a (g/L/h)Productivity a (g/L/h)
회분식Batch 14.314.3 0.880.88 1.331.33 0.350.35 0.020.02

a락토오스와 3-푸코실락토오스의 농도는 배지에 있는 것만을 정량한 수치임. a The concentrations of lactose and 3-fucosylactose are quantified values only in the medium.

(4) (4) 유가식Federation 배양을 통한 Through culture 3'-3'- 푸코실락토오스의Fucosylactose 생산 결과 Production results

고농도의 세포배양을 통하여 고농도 3'-푸코실락토오스를 생산 하기 위해 pVBCLE, pENGWTA(CO) 플라스미드를 도입한 재조합 코리네박테리움 글루타미쿰 (C. glutamicum)을 이용하여 2.5L 수준의 발효기에서 유가식 배양을 실시하였다.In order to produce high-concentration 3'-fucosylactose through high-concentration cell culture, the recombinant Corynebacterium glutamicum ( C. glutamicum ) introduced with pVBCLE and pENGWTA(CO) plasmids is used in a 2.5L fermentor with a milk price. Food culture was performed.

초기에 첨가해준 40g/L의 글루코오스를 모두 소모한 시점부터 세포생장을 유지하기 위해 피딩용액 (feeding solution)을 연속식 (continuous feeding)방법을 이용하여 5.7g/L/h의 속도로 공급하였다. 이와 동시에 3'-푸코실락토오스의 생산을 유도하기 위해 IPTG와 락토오스를 첨가해주었다.The feeding solution was supplied at a rate of 5.7 g/L/h using a continuous feeding method in order to maintain cell growth from the point at which the initially added 40 g/L of glucose was consumed. At the same time, IPTG and lactose were added to induce the production of 3'-fucosylactose.

실험 결과, 발효가 진행되는 동안 아세트산은 전혀 생성되지 않았으며, 글루코오스의 대사를 통해 세포는 최종적으로 건조세포중량 55.5g/L에 도달하였다. 또한, 최대 3'-푸코실락토오스의 농도는 10.0g/L, 락토오스 대비 생산수율은 0.53 mole 3'-푸코실락토오스/mole 락토오스이고, 생산성은 0.06 g/L/h를 얻을 수 있었다 (도 4 및 표 5).As a result of the experiment, acetic acid was not produced at all during fermentation, and the cells finally reached a dry cell weight of 55.5 g/L through glucose metabolism. In addition, the maximum concentration of 3'-fucosyl lactose was 10.0 g/L, the production yield relative to lactose was 0.53 mole 3'-fucosyl lactose/mole lactose, and the productivity was 0.06 g/L/h (Fig. 4 And Table 5).

3'-푸코실락토오스의 생산을 위한 유가식 배양의 결과는 하기 표 5에 기재하였으며, 도 4는 재조합 코리네박테리움 글루타미쿰 (C. glutamicum) pVBCLE + pENGWTA(CO 를 이용한 유가식 배양결과를 나타낸 그래프이다. 초기에 투입한 40g/L 글루코오스가 모두 소모된 후, 글루코오스를 연속식 (continuous feeding)방법으로 공급하기 시작하였고, IPTG와 락토오스를 동시에 첨가하였다 (화살표). 그래프 중 기호는 다음과 같다: ●건조세포중량, ▲ 글루코오스, ■락토오스, ▼락테이 트, ◆3'-푸코실락토오스.The results of fed-batch culture for the production of 3'-fucosylactose are shown in Table 5 below, and FIG. 4 is a fed-batch culture result using recombinant Corynebacterium glutamicum (C. glutamicum) pVBCLE + pENGWTA (CO) After the initial 40g/L glucose was all consumed, glucose was started to be supplied by a continuous feeding method, and IPTG and lactose were added at the same time (arrow). Same as: Dry cell weight, ▲ Glucose, ■ Lactose, ▼ Lactate, ◆ 3'-Fucault room lactose.

재조합 코리네박테리움 글루타미쿰 (C. glutamicum) pVBCLE + pENGWTA(CO)를 이용한 배양기 유가식 배양 결과Incubator fed-batch culture results using recombinant Corynebacterium glutamicum ( C. glutamicum ) pVBCLE + pENGWTA(CO) 최종 건조
세포 중량
(g/L)
Final drying
Cell weight
(g/L)
락토오스 소모량a
(g/L)
Lactose consumption a
(g/L)
최대 3'-푸코실락토오스 농도a
(g/L)
Max. 3'-fucosylactose concentration a
(g/L)
수율 (mole 3'-푸코실락토오스
/mole 락토오스)
Yield (mole 3'-fucosylactose
/mole lactose)
생산성a (g/L/h)Productivity a (g/L/h)
유가식Federation 55.555.5 14.714.7 10.010.0 0.530.53 0.060.06

a락토오스와 3-푸코실락토오스의 농도는 배지에 있는 것만을 정량한 수치임. a The concentrations of lactose and 3-fucosylactose are quantified values only in the medium.

최대 3'푸코실락토오스 농도 10.0g/L는 본 발명자들이 이전 특허출원 10-2018-0045845(출원일자: 2018년 4월 20일)에서 생산하였던 최대 3'푸코실락토오스 농도 3.6g/L보다 약 3배 정도 더 높은 것이다.The maximum 3'fucosyl lactose concentration of 10.0 g/L is less than the maximum 3'fucosyl lactose concentration of 3.6 g/L produced by the present inventors in the previous patent application 10-2018-0045845 (application date: April 20, 2018). It is about three times higher.

<110> Seoul National University R&DB Foundation <120> Recombinant Corynebacterium Glutamicum for the production of 3'-fucosyllactose and method for the production of 3'-fucosyllactose therefrom <130> YP-19-128 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 1377 <212> DNA <213> Corynebacterium glutamicum <400> 1 atgcgtaccc gtgaatctgt cacggctgta attaaggcgt atgacgtccg tggtgttgtt 60 ggtgtcgata ttgatgctga tttcatttct gagactggcg ctgcctttgg tcggctcatg 120 cgtagtgagg gtgaaaccac cgttgctatt ggccatgaca tgcgtgattc ctcccctgaa 180 ttggccaagg cgtttgccga tggcgtgact gcacagggtt tggatgttgt tcatttggga 240 ctgacttcta ctgatgagct gtactttgcg tccggaacct tgaagtgtgc tggtgcgatg 300 tttactgcgt cgcataaccc cgctgagtac aacggcatca agttgtgtcg tgcgggtgct 360 cgtccggtcg gtcaggattc tggtttggcc aacatcattg atgatctggt tgagggtgtt 420 ccagcgtttg atggtgagtc aggttcggtt tctgagcagg atttgctgag cgcatatgcc 480 gagtacctca atgagcttgt tgatctgaag aacatccgcc cgttgaaggt tgctgtggat 540 gcggcaaacg gcatgggtgg gttcactgtc cctgaggtat tcaagggtct gccacttgat 600 gttgcgccac tgtattttga gcttgacggc aatttcccca accatgaggc caatcctctg 660 gagcctgcca acctggttga tttgcagaag tttaccgtag agaccggatc tgatatcggt 720 ttggcgttcg acggcgatgc ggatcgttgc ttcgtggtcg atgagaaggg ccagccagtc 780 agcccttcgg cgatctgtgc gatcgtagcg gagcgttact tggagaagct tccgggttcc 840 accatcatcc acaacctgat tacctctaag gctgtgcctg aggtgattgc tgaaaacggt 900 ggcactgcgg tgcgtactcg cgtgggtcac tccttcatca aggcgaagat ggcagagacc 960 ggtgcggcct ttggtggcga gcactctgcg cactactact tcactgagtt cttcaatgcg 1020 gactccggca ttttggctgc gatgcacgtg ctggctgcgc tgggaagcca ggaccagcca 1080 ctcagtgaga tgatggctag gtataaccgg tacgttgctt caggcgagtt gaactcccgt 1140 ttggctaatg cagaggcgca gcaagagcgc acccaggctg tgctcgatgc gttcgctgat 1200 cgcaccgagt ccgtggacac ccttgacggc gtgactgtgg aactcaagga cacctccgcg 1260 tggttcaacg tgcgtgcgtc caacaccgag ccgctgcttc gcctcaatgt tgaagctgca 1320 tcgaaggaag aagtcgatgc gttggtagcg gagattctag ggattatccg cgcataa 1377 <210> 2 <211> 1089 <212> DNA <213> Corynebacterium glutamicum <400> 2 atgactttaa ctgacaacag caaaaacgtt gatgctgtca tcttggtcgg tggcaaaggt 60 acccgactgc gccccctgac cgtcaatact ccaaagccaa tgctgccaac tgctggccac 120 ccattcttga cccacctttt ggcccgcatc aaggccgcag gcatcacaca cgtcgtgctg 180 ggaacgtcat tcaaagctga agtcttcgag gaatacttcg gagatggctc cgaaatgggc 240 ttggaaattg aatatgtcgt cgaggatcag cctttgggca ctggtggtgg catccgaaac 300 gtctacgaca agctgcgtca cgatactgcg attgtgttca acggcgatgt gctctccggt 360 gcggatctca acagcattct ggacacccac cgcgaaaagg acgcagatct gaccatgcat 420 ctcgtgcgcg tagctaaccc tcgtgcgttt ggttgcgtcc ccaccgatga ggatggtcgc 480 gtcagcgaat tccttgaaaa gaccgaagat ccaccaaccg atcagatcaa cgccggctgc 540 tacgtgttca agaaggaact catcgagcag atcccggcag gccgagcagt ttccgtcgag 600 cgcgaaacct tccctcagct gttggaagaa ggcaagcgag tcttcggcca cgtcgacgct 660 tcctactggc gcgacatggg caccccaagc gacttcgtcc gcggctcggc tgacctggtc 720 cgcggcattg cgtactcccc attgctcgaa ggcaaaacag gagagtcgct tgtcgacgcc 780 tccgccggcg ttcgcgacgg cgtcctgctg ctcggcggaa ccgtagtcgg ccgcggcact 840 gagatcggtg ccggctgccg cgttgacaac actgttattt tcgacggcgt caccattgaa 900 ccaggtgcgg tcattgaaaa ttccatcatt tcctcgggag cacgcatcgg tgctaatgcg 960 cacatctccg gttgcatcat tggcgagggc gcacaggttg gtgctcggtg tgaactcaac 1020 gcagggatgc gcgtcttccc aggcgttgtg atcccagaca gcggaattcg tttttcgtct 1080 gatcagtag 1089 <210> 3 <211> 2090 <212> DNA <213> Escherichia coli <400> 3 atgtcaaaag tcgctctcat caccggtgta accggacaag acggttctta cctggcagag 60 tttctgctgg aaaaaggtta cgaggtgcat ggtattaagc gtcgcgcatc gtcattcaac 120 accgagcgcg tggatcacat ttatcaggat ccgcacacct gcaacccgaa attccatctg 180 cattatggcg acctgagtga tacctctaac ctgacgcgca ttttgcgtga agtacagccg 240 gatgaagtgt acaacctggg cgcaatgagc cacgttgcgg tctcttttga gtcaccagaa 300 tataccgctg acgtcgacgc gatgggtacg ctgcgcctgc tggaggcgat ccgcttcctc 360 ggtctggaaa agaaaactcg tttctatcag gcttccacct ctgaactgta tggtctggtg 420 caggaaattc cgcagaaaga gaccacgccg ttctacccgc gatctccgta tgcggtcgcc 480 aaactgtacg cctactggat caccgttaac taccgtgaat cctacggcat gtacgcctgt 540 aacggaattc tcttcaacca tgaatccccg cgccgcggcg aaaccttcgt tacccgcaaa 600 atcacccgcg caatcgccaa catcgcccag gggctggagt cgtgcctgta cctcggcaat 660 atggattccc tgcgtgactg gggccacgcc aaagactacg taaaaatgca gtggatgatg 720 ctgcagcagg aacagccgga agatttcgtt atcgcgaccg gcgttcagta ctccgtgcgt 780 cagttcgtgg aaatggcggc agcacagctg ggcatcaaac tgcgctttga aggcacgggc 840 gttgaagaga agggcattgt ggtttccgtc accgggcatg acgcgccggg cgttaaaccg 900 ggtgatgtga ttatcgctgt tgacccgcgt tacttccgtc cggctgaagt tgaaacgctg 960 ctcggcgacc cgaccaaagc gcacgaaaaa ctgggctgga aaccggaaat caccctcaga 1020 gagatggtgt ctgaaatggt ggctaatgac ctcgaagcgg cgaaaaaaca ctctctgctg 1080 aaatctcacg gctacgacgt ggcgatcgcg ctggagtcat aagcatgagt aaacaacgag 1140 tttttattgc tggtcatcgc gggatggtcg gttccgccat caggcggcag ctcgaacagc 1200 gcggtgatgt ggaactggta ttacgcaccc gcgacgagct gaacctgctg gacagccgcg 1260 ccgtgcatga tttctttgcc agcgaacgta ttgaccaggt ctatctggcg gcggcgaaag 1320 tgggcggcat tgttgccaac aacacctatc cggcggattt catctaccag aacatgatga 1380 ttgagagcaa catcattcac gccgcgcatc agaacgacgt gaacaaactg ctgtttctcg 1440 gatcgtcctg catctacccg aaactggcaa aacagccgat ggcagaaagc gagttgttgc 1500 agggcacgct ggagccgact aacgagcctt atgctattgc caaaatcgcc gggatcaaac 1560 tgtgcgaatc atacaaccgc cagtacggac gcgattaccg ctcagtcatg ccgaccaacc 1620 tgtacgggcc acacgacaac ttccacccga gtaattcgca tgtgatccca gcattgctgc 1680 gtcgcttcca cgaggcgacg gcacagaatg cgccggacgt ggtggtatgg ggcagcggta 1740 caccgatgcg cgaatttctg cacgtcgatg atatggcggc ggcgagcatt catgtcatgg 1800 agctggcgca tgaagtctgg ctggagaaca cccagccgat gttgtcgcac attaacgtcg 1860 gcacgggcgt tgactgcact atccgcgagc tggcgcaaac catcgccaaa gtggtgggtt 1920 acaaaggccg ggtggttttt gatgccagca aaccggatgg cacgccgcgc aaactgctgg 1980 atgtgacgcg cctgcatcag cttggctggt atcacgaaat ctcactggaa gcggggcttg 2040 ccagcactta ccagtggttc cttgagaatc aagaccgctt tcgggggtaa 2090 <210> 4 <211> 3335 <212> DNA <213> Escherichia coli <400> 4 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa 60 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc 120 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg 180 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa 240 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg 300 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg 360 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc 420 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct 480 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc 540 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc 600 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt 660 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa 720 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag 780 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc 840 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat 900 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg 960 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc 1020 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa 1080 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 1140 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 1200 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 1260 atttcacaca ggaaacagct atgtactatt taaaaaacac aaacttttgg atgttcggtt 1320 tattcttttt cttttacttt tttatcatgg gagcctactt cccgtttttc ccgatttggc 1380 tacatgacat caaccatatc agcaaaagtg atacgggtat tatttttgcc gctatttctc 1440 tgttctcgct attattccaa ccgctgtttg gtctgctttc tgacaaactc gggctgcgca 1500 aatacctgct gtggattatt accggcatgt tagtgatgtt tgcgccgttc tttattttta 1560 tcttcgggcc actgttacaa tacaacattt tagtaggatc gattgttggt ggtatttatc 1620 taggcttttg ttttaacgcc ggtgcgccag cagtagaggc atttattgag aaagtcagcc 1680 gtcgcagtaa tttcgaattt ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt 1740 gtgcctcgat tgtcggcatc atgttcacca tcaataatca gtttgttttc tggctgggct 1800 ctggctgtgc actcatcctc gccgttttac tctttttcgc caaaacggat gcgccctctt 1860 ctgccacggt tgccaatgcg gtaggtgcca accattcggc atttagcctt aagctggcac 1920 tggaactgtt cagacagcca aaactgtggt ttttgtcact gtatgttatt ggcgtttcct 1980 gcacctacga tgtttttgac caacagtttg ctaatttctt tacttcgttc tttgctaccg 2040 gtgaacaggg tacgcgggta tttggctacg taacgacaat gggcgaatta cttaacgcct 2100 cgattatgtt ctttgcgcca ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc 2160 tggctggcac tattatgtct gtacgtatta ttggctcatc gttcgccacc tcagcgctgg 2220 aagtggttat tctgaaaacg ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct 2280 ttaaatatat taccagccag tttgaagtgc gtttttcagc gacgatttat ctggtctgtt 2340 tctgcttctt taagcaactg gcgatgattt ttatgtctgt actggcgggc aatatgtatg 2400 aaagcatcgg tttccagggc gcttatctgg tgctgggtct ggtggcgctg ggcttcacct 2460 taatttccgt gttcacgctt agcggccccg gcccgctttc cctgctgcgt cgtcaggtga 2520 atgaagtcgc ttaagcaatc aatgtcggat gcggcgcgag cgccttatcc gaccaacata 2580 tcataacgga gtgatcgcat tgaacatgcc aatgaccgaa agaataagag caggcaagct 2640 atttaccgat atgtgcgaag gcttaccgga aaaaagactt cgtgggaaaa cgttaatgta 2700 tgagtttaat cactcgcatc catcagaagt tgaaaaaaga gaaagcctga ttaaagaaat 2760 gtttgccacg gtaggggaaa acgcctgggt agaaccgcct gtctatttct cttacggttc 2820 caacatccat ataggccgca atttttatgc aaatttcaat ttaaccattg tcgatgacta 2880 cacggtaaca atcggtgata acgtactgat tgcacccaac gttactcttt ccgttacggg 2940 acaccctgta caccatgaat tgagaaaaaa cggcgagatg tactcttttc cgataacgat 3000 tggcaataac gtctggatcg gaagtcatgt ggttattaat ccaggcgtca ccatcgggga 3060 taattctgtt attggcgcgg gtagtatcgt cacaaaagac attccaccaa acgtcgtggc 3120 ggctggcgtt ccttgtcggg ttattcgcga aataaacgac cgggataagc actattattt 3180 caaagattat aaagttgaat cgtcagttta aattataaaa attgcctgat acgctgcgct 3240 tatcaggcct acaagttcag cgatctacat tagccgcatc cggcatgaac aaagcgcagg 3300 aacaagcgtc gcatcatgcc tctttgaccc acagc 3335 <210> 5 <211> 1890 <212> DNA <213> Bifidobacterium infantis <400> 5 atgagtgaag caatcgcaag accgcgttcc aagtcgctgc agcgcagaga cgccaaactg 60 gcgctcaagg caagcaagca ttacaagcgc atgcagcagc gtgagccggc tccgaagctg 120 accggcaagc agcgtgtgct caactggctg ctgcacatca ttatggctgt gatggtcatc 180 tactgcctcg tgccgctgct gtgggtcgtc ttctcttcca caaagaccag tgagggcatc 240 ttcagctctt tcggcctgtg gttcgatgac aagaatgtgt tctggcagaa cgtgcaggat 300 acgtttgctt atcagcatgg tgtctacact cgttggctgt tcaatacgat catgtacgca 360 gtggtagcag gtgttggtgc aaccatcatt gccactttcg ctgggtatgc catcgccacc 420 atgcgattcc ctggacgtaa cgccctgctg gccgtcactc tggcattcat gtcaattcct 480 tctacggtga tcaccgtgcc gctgttcctc atgtattcga agatcgggct ggtcggcact 540 ccgtgggccg tgattattcc gcagcttgcc acgccgttcg gcctgtatct gatgatcatc 600 tatgcgcaga cctcgattcc ggtctcgctg atcgaagccg cgaaactgga cggtgcaaac 660 acttggacca tcttctggaa ggtcggtttc ccgctgctgt ctccaggttt cgtcaccgta 720 ctgctgttca cgctggtcgg tgtttggaac aactacttcc tgccgctgat catgcttacg 780 aataccaacg actacccgtt gacggtcggt ctgaacatgt ggctgaagat gggtgcccag 840 ggaacttcag acggtcaggt tccgaacaac ctcatcatca cgggctcgct tatcgcagtt 900 gttccgttga tcatcgcatt catgttcctg caaaagtact ggcagtctgg cctcgcggcc 960 ggctctgtca agcagtgaat gacaaatgca acggcgcaac cggatacttc cgtgatgcgc 1020 aagccgaagc gccaatacat aggcatattg tattgtcttc catacgtggt ggtcttcctg 1080 ttcggcatga tcgtcccgat gttctacgcg ctttatctca gcttcttcaa gcagtccctt 1140 ctcggtggca ccacattcgc gggcttcgac aacttcatcc gagcgttcaa ggatgaggct 1200 ttgtggggcg gctttaggaa cgtgctgatt tacgcggcga tccagattcc gatgaatctg 1260 attctgtccc tcgtggcggc gctcgtcttg gactcccaga ggatccgcca tatcgcggtg 1320 ccgcgaatcc tgctgttcct gccttatgcc gtcccaggcg tcatcgcggc gctgatgtgg 1380 ggatacattt acggcgataa gtatggtctt tttggacaaa tcgccggtat gtttggcgta 1440 gcggctccta atatgttgtc gaagcagctc atgttgttcg ccatcgcgaa catctgcact 1500 tggtgcttcc ttggctataa catgctgatt tattattctg cactgatcgg cattccgaac 1560 gacctgtacg aatctgcccg tattgacggc gcttcggagc tgcgcatcgc ttggtccgtg 1620 aagatccccc agattaagag caccatcgtg atgacggtcc tcttctctgt gatcggcacg 1680 ctccagctgt tcaacgaacc gaacatcttg cgtacttccg ccccggacgt gatcaacagc 1740 agctatacgc cgaacatcta cacctacaac ctcgcattca atggccagaa cgtcaactat 1800 gccgctgccg tctctctggt catcggcatc atcgtcatgg ccttggtcgc cgtcgttaag 1860 atcatcggca acaagtggga gaacaagtga 1890 <210> 6 <211> 969 <212> DNA <213> Azospirillum brasilense <400> 6 ttggcgcgga tcgcattgat cttcggaatc tccgggcagg atggaagcta tcttgcccgg 60 cacctgctcg accgcggcta caccgtccat ggcacgtcgc gggactgcga gatcgcatcg 120 ttccgcaacc tgacgattct gggcatccgc gaccgggtaa acgtccattc ggcgaccctg 180 catgatttcc gcagcatcgt gcaggtgatc cgggcggtga agcccgacga gatctacaac 240 ctctccgccc agtcggcggt gggtctgtcc ttcgagcagc cggtcgagac gatcgacagc 300 atcctgaacg gcacgctgaa cattctggag gccgtccgct tcctgggggt ggactcgcgc 360 ttctaccacg cctcctccag cgagagcttc ggcaacaccc tggacgagcc ggccagcgag 420 acgacggcct ttcgtccctg cagcccttat ggcgtcgcca agtcggcagc ccactggatg 480 gtcgccaact atcggcaggc ctacgggctc tatgcctgct cgggcattct gttcaatcat 540 gaatcgccgc tgcgcccggc gcgtttcgtc acccgcaaga ttgtccgcgg cgccatcgcg 600 gtggcgcagg ggcgcctgaa gacgctggaa ttgggcaacc tcgccgtggc gcgggattgg 660 ggctgggcgc cggaatatgt ggacgccatg tggcgcatgc tccagcagga cgagccggac 720 gatttcgtca tcgccaccgg ccggatgcat cccctggagg acttcgtccg gctggccttt 780 gcgcatttcg gcctagactg gcgggagcat gtggtccagg gggcgacgca tcggcgaccg 840 atggacatcc attgcagcgt cggcaacccg gccagggccg aagcccgcct gggctggaag 900 gccgaaaccc tgatgccgga tctcgtcgga cgtctggtcg aggcggagtt ggccctgccg 960 caaccttga 969 <210> 7 <211> 1257 <212> DNA <213> Artificial Sequence <220> <223> tac_azoT(CO) <400> 7 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 60 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 120 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 180 attgtgagcg gataacaatt tcacacagga aacagaatta aaagatatga ccatgattac 240 gccaagcttg catgcctgca ggtcgactct agaggatcca tgctcgatca acgtacgagc 300 gcattcttgg aagaatttct ggcgaaaccg ggaggtgatc ccgagcgact cgaccgcttc 360 ttacttcacg gcccgtaccg tggccggcgg ggcggcaagc cgcgattgaa acttgcgttc 420 cacgatttct ggccagagtt tgacaaggga accaattttt tcatcgaaat tctgtcctcc 480 cgcttcgatc tgtcggtagt ggaagacgat tctgacctcg ccattgtgtc tgtcttcggc 540 gggcggcacc gcgaggctcg ctcacgccgc accctcttct tcaccggaga gaacgttcgc 600 ccaccgcttg acggtttcga tatggctgtg tccttcgacc gcgttgacga tccacgccat 660 tacaggctgc cactctacgt catgcacgcc tacgagcaca tgcgagaggg cgcggtgcca 720 catttttgtt cacctgtcct gccaccagtg cctccgacaa gagctgcttt tgcagaacgt 780 ggattttgcg cctttttgta caagaatcct aacggtgaaa ggcgtaaccg ctttttcccg 840 gtgctggacg gtcgtcgacg tgttgattct gtgggctggc atctaaacaa taccggttcc 900 gtcgtgaaaa tgggatggtt gtcgaagatc cgtgtcttcg aacgctaccg tttcgccttc 960 gcattcgaga acgctagcca tcccggttat cttactgaaa agatcctgga cgtcttccag 1020 gccggcgcgg tgcctttgta ttggggtgat cccgatctag aacgcgaagt tgcagtcggc 1080 agctttatcg acgtgagtcg cttcgcaact gatgaggaag ctgtggacca cattcttgca 1140 gtggatgatg attacgacgc ctactgcgca caccgcgcag ttgcgccttt tctggggacc 1200 gaggaatttt atttcgatgc ctaccgcctc gctgactgga tcgaatcccg cttataa 1257 <210> 8 <211> 279 <212> DNA <213> Escherichia coli <400> 8 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 60 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 120 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 180 attgtgagcg gataacaatt tcacacagga aacagaatta aaagatatga ccatgattac 240 gccaagcttg catgcctgca ggtcgactct agaggatcc 279 <210> 9 <211> 978 <212> DNA <213> Artificial Sequence <220> <223> azoT(CO) <400> 9 atgctcgatc aacgtacgag cgcattcttg gaagaatttc tggcgaaacc gggaggtgat 60 cccgagcgac tcgaccgctt cttacttcac ggcccgtacc gtggccggcg gggcggcaag 120 ccgcgattga aacttgcgtt ccacgatttc tggccagagt ttgacaaggg aaccaatttt 180 ttcatcgaaa ttctgtcctc ccgcttcgat ctgtcggtag tggaagacga ttctgacctc 240 gccattgtgt ctgtcttcgg cgggcggcac cgcgaggctc gctcacgccg caccctcttc 300 ttcaccggag agaacgttcg cccaccgctt gacggtttcg atatggctgt gtccttcgac 360 cgcgttgacg atccacgcca ttacaggctg ccactctacg tcatgcacgc ctacgagcac 420 atgcgagagg gcgcggtgcc acatttttgt tcacctgtcc tgccaccagt gcctccgaca 480 agagctgctt ttgcagaacg tggattttgc gcctttttgt acaagaatcc taacggtgaa 540 aggcgtaacc gctttttccc ggtgctggac ggtcgtcgac gtgttgattc tgtgggctgg 600 catctaaaca ataccggttc cgtcgtgaaa atgggatggt tgtcgaagat ccgtgtcttc 660 gaacgctacc gtttcgcctt cgcattcgag aacgctagcc atcccggtta tcttactgaa 720 aagatcctgg acgtcttcca ggccggcgcg gtgcctttgt attggggtga tcccgatcta 780 gaacgcgaag ttgcagtcgg cagctttatc gacgtgagtc gcttcgcaac tgatgaggaa 840 gctgtggacc acattcttgc agtggatgat gattacgacg cctactgcgc acaccgcgca 900 gttgcgcctt ttctggggac cgaggaattt tatttcgatg cctaccgcct cgctgactgg 960 atcgaatccc gcttataa 978 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> F_inf_KpnI_noeL(pENGWTA(CO)) <400> 10 ctagaggatc cccgggtacc ttggcgcgga tcgcattgat cttcg 45 <210> 11 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> R_inf_KpnI_noeL(pENGWTA(CO)) <400> 11 ttgtatatct ccttggtacc tcaaggttgc ggcagggc 38 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> F_inf_Sac1_Tac_RBS_azoT(pEGWTA(CO)) <400> 12 gctttcgggg gtaagagctc tcaggcagcc atcggaag 38 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> R_inf_sacI_COazoT(pEGWTA(CO)) <400> 13 cggccagtga attcgagctc ttataagcgg gattcgatcc agtc 44 <110> Seoul National University R&DB Foundation <120> Recombinant Corynebacterium Glutamicum for the production of 3'-fucosyllactose and method for the production of 3'-fucosyllactose therefrom <130> YP-19-128 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 1377 <212> DNA <213> Corynebacterium glutamicum <400> 1 atgcgtaccc gtgaatctgt cacggctgta attaaggcgt atgacgtccg tggtgttgtt 60 ggtgtcgata ttgatgctga tttcatttct gagactggcg ctgcctttgg tcggctcatg 120 cgtagtgagg gtgaaaccac cgttgctatt ggccatgaca tgcgtgattc ctcccctgaa 180 ttggccaagg cgtttgccga tggcgtgact gcacagggtt tggatgttgt tcatttggga 240 ctgacttcta ctgatgagct gtactttgcg tccggaacct tgaagtgtgc tggtgcgatg 300 tttactgcgt cgcataaccc cgctgagtac aacggcatca agttgtgtcg tgcgggtgct 360 cgtccggtcg gtcaggattc tggtttggcc aacatcattg atgatctggt tgagggtgtt 420 ccagcgtttg atggtgagtc aggttcggtt tctgagcagg atttgctgag cgcatatgcc 480 gagtacctca atgagcttgt tgatctgaag aacatccgcc cgttgaaggt tgctgtggat 540 gcggcaaacg gcatgggtgg gttcactgtc cctgaggtat tcaagggtct gccacttgat 600 gttgcgccac tgtattttga gcttgacggc aatttcccca accatgaggc caatcctctg 660 gagcctgcca acctggttga tttgcagaag tttaccgtag agaccggatc tgatatcggt 720 ttggcgttcg acggcgatgc ggatcgttgc ttcgtggtcg atgagaaggg ccagccagtc 780 agcccttcgg cgatctgtgc gatcgtagcg gagcgttact tggagaagct tccgggttcc 840 accatcatcc acaacctgat tacctctaag gctgtgcctg aggtgattgc tgaaaacggt 900 ggcactgcgg tgcgtactcg cgtgggtcac tccttcatca aggcgaagat ggcagagacc 960 ggtgcggcct ttggtggcga gcactctgcg cactactact tcactgagtt cttcaatgcg 1020 gactccggca ttttggctgc gatgcacgtg ctggctgcgc tgggaagcca ggaccagcca 1080 ctcagtgaga tgatggctag gtataaccgg tacgttgctt caggcgagtt gaactcccgt 1140 ttggctaatg cagaggcgca gcaagagcgc acccaggctg tgctcgatgc gttcgctgat 1200 cgcaccgagt ccgtggacac ccttgacggc gtgactgtgg aactcaagga cacctccgcg 1260 tggttcaacg tgcgtgcgtc caacaccgag ccgctgcttc gcctcaatgt tgaagctgca 1320 tcgaaggaag aagtcgatgc gttggtagcg gagattctag ggattatccg cgcataa 1377 <210> 2 <211> 1089 <212> DNA <213> Corynebacterium glutamicum <400> 2 atgactttaa ctgacaacag caaaaacgtt gatgctgtca tcttggtcgg tggcaaaggt 60 acccgactgc gccccctgac cgtcaatact ccaaagccaa tgctgccaac tgctggccac 120 ccattcttga cccacctttt ggcccgcatc aaggccgcag gcatcacaca cgtcgtgctg 180 ggaacgtcat tcaaagctga agtcttcgag gaatacttcg gagatggctc cgaaatgggc 240 ttggaaattg aatatgtcgt cgaggatcag cctttgggca ctggtggtgg catccgaaac 300 gtctacgaca agctgcgtca cgatactgcg attgtgttca acggcgatgt gctctccggt 360 gcggatctca acagcattct ggacacccac cgcgaaaagg acgcagatct gaccatgcat 420 ctcgtgcgcg tagctaaccc tcgtgcgttt ggttgcgtcc ccaccgatga ggatggtcgc 480 gtcagcgaat tccttgaaaa gaccgaagat ccaccaaccg atcagatcaa cgccggctgc 540 tacgtgttca agaaggaact catcgagcag atcccggcag gccgagcagt ttccgtcgag 600 cgcgaaacct tccctcagct gttggaagaa ggcaagcgag tcttcggcca cgtcgacgct 660 tcctactggc gcgacatggg caccccaagc gacttcgtcc gcggctcggc tgacctggtc 720 cgcggcattg cgtactcccc attgctcgaa ggcaaaacag gagagtcgct tgtcgacgcc 780 tccgccggcg ttcgcgacgg cgtcctgctg ctcggcggaa ccgtagtcgg ccgcggcact 840 gagatcggtg ccggctgccg cgttgacaac actgttattt tcgacggcgt caccattgaa 900 ccaggtgcgg tcattgaaaa ttccatcatt tcctcgggag cacgcatcgg tgctaatgcg 960 cacatctccg gttgcatcat tggcgagggc gcacaggttg gtgctcggtg tgaactcaac 1020 gcagggatgc gcgtcttccc aggcgttgtg atcccagaca gcggaattcg tttttcgtct 1080 gatcagtag 1089 <210> 3 <211> 2090 <212> DNA <213> Escherichia coli <400> 3 atgtcaaaag tcgctctcat caccggtgta accggacaag acggttctta cctggcagag 60 tttctgctgg aaaaaggtta cgaggtgcat ggtattaagc gtcgcgcatc gtcattcaac 120 accgagcgcg tggatcacat ttatcaggat ccgcacacct gcaacccgaa attccatctg 180 cattatggcg acctgagtga tacctctaac ctgacgcgca ttttgcgtga agtacagccg 240 gatgaagtgt acaacctggg cgcaatgagc cacgttgcgg tctcttttga gtcaccagaa 300 tataccgctg acgtcgacgc gatgggtacg ctgcgcctgc tggaggcgat ccgcttcctc 360 ggtctggaaa agaaaactcg tttctatcag gcttccacct ctgaactgta tggtctggtg 420 caggaaattc cgcagaaaga gaccacgccg ttctacccgc gatctccgta tgcggtcgcc 480 aaactgtacg cctactggat caccgttaac taccgtgaat cctacggcat gtacgcctgt 540 aacggaattc tcttcaacca tgaatccccg cgccgcggcg aaaccttcgt tacccgcaaa 600 atcacccgcg caatcgccaa catcgcccag gggctggagt cgtgcctgta cctcggcaat 660 atggattccc tgcgtgactg gggccacgcc aaagactacg taaaaatgca gtggatgatg 720 ctgcagcagg aacagccgga agatttcgtt atcgcgaccg gcgttcagta ctccgtgcgt 780 cagttcgtgg aaatggcggc agcacagctg ggcatcaaac tgcgctttga aggcacgggc 840 gttgaagaga agggcattgt ggtttccgtc accgggcatg acgcgccggg cgttaaaccg 900 ggtgatgtga ttatcgctgt tgacccgcgt tacttccgtc cggctgaagt tgaaacgctg 960 ctcggcgacc cgaccaaagc gcacgaaaaa ctgggctgga aaccggaaat caccctcaga 1020 gagatggtgt ctgaaatggt ggctaatgac ctcgaagcgg cgaaaaaaca ctctctgctg 1080 aaatctcacg gctacgacgt ggcgatcgcg ctggagtcat aagcatgagt aaacaacgag 1140 tttttattgc tggtcatcgc gggatggtcg gttccgccat caggcggcag ctcgaacagc 1200 gcggtgatgt ggaactggta ttacgcaccc gcgacgagct gaacctgctg gacagccgcg 1260 ccgtgcatga tttctttgcc agcgaacgta ttgaccaggt ctatctggcg gcggcgaaag 1320 tgggcggcat tgttgccaac aacacctatc cggcggattt catctaccag aacatgatga 1380 ttgagagcaa catcattcac gccgcgcatc agaacgacgt gaacaaactg ctgtttctcg 1440 gatcgtcctg catctacccg aaactggcaa aacagccgat ggcagaaagc gagttgttgc 1500 agggcacgct ggagccgact aacgagcctt atgctattgc caaaatcgcc gggatcaaac 1560 tgtgcgaatc atacaaccgc cagtacggac gcgattaccg ctcagtcatg ccgaccaacc 1620 tgtacgggcc acacgacaac ttccacccga gtaattcgca tgtgatccca gcattgctgc 1680 gtcgcttcca cgaggcgacg gcacagaatg cgccggacgt ggtggtatgg ggcagcggta 1740 caccgatgcg cgaatttctg cacgtcgatg atatggcggc ggcgagcatt catgtcatgg 1800 agctggcgca tgaagtctgg ctggagaaca cccagccgat gttgtcgcac attaacgtcg 1860 gcacgggcgt tgactgcact atccgcgagc tggcgcaaac catcgccaaa gtggtgggtt 1920 acaaaggccg ggtggttttt gatgccagca aaccggatgg cacgccgcgc aaactgctgg 1980 atgtgacgcg cctgcatcag cttggctggt atcacgaaat ctcactggaa gcggggcttg 2040 ccagcactta ccagtggttc cttgagaatc aagaccgctt tcgggggtaa 2090 <210> 4 <211> 3335 <212> DNA <213> Escherichia coli <400> 4 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa 60 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc 120 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg 180 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa 240 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg 300 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg 360 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc 420 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct 480 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc 540 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc 600 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt 660 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa 720 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag 780 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc 840 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat 900 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg 960 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc 1020 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa 1080 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 1140 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 1200 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 1260 atttcacaca ggaaacagct atgtactatt taaaaaacac aaacttttgg atgttcggtt 1320 tattcttttt cttttacttt tttatcatgg gagcctactt cccgtttttc ccgatttggc 1380 tacatgacat caaccatatc agcaaaagtg atacgggtat tatttttgcc gctatttctc 1440 tgttctcgct attattccaa ccgctgtttg gtctgctttc tgacaaactc gggctgcgca 1500 aatacctgct gtggattatt accggcatgt tagtgatgtt tgcgccgttc tttattttta 1560 tcttcgggcc actgttacaa tacaacattt tagtaggatc gattgttggt ggtatttatc 1620 taggcttttg ttttaacgcc ggtgcgccag cagtagaggc atttattgag aaagtcagcc 1680 gtcgcagtaa tttcgaattt ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt 1740 gtgcctcgat tgtcggcatc atgttcacca tcaataatca gtttgttttc tggctgggct 1800 ctggctgtgc actcatcctc gccgttttac tctttttcgc caaaacggat gcgccctctt 1860 ctgccacggt tgccaatgcg gtaggtgcca accattcggc atttagcctt aagctggcac 1920 tggaactgtt cagacagcca aaactgtggt ttttgtcact gtatgttatt ggcgtttcct 1980 gcacctacga tgtttttgac caacagtttg ctaatttctt tacttcgttc tttgctaccg 2040 gtgaacaggg tacgcgggta tttggctacg taacgacaat gggcgaatta cttaacgcct 2100 cgattatgtt ctttgcgcca ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc 2160 tggctggcac tattatgtct gtacgtatta ttggctcatc gttcgccacc tcagcgctgg 2220 aagtggttat tctgaaaacg ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct 2280 ttaaatatat taccagccag tttgaagtgc gtttttcagc gacgatttat ctggtctgtt 2340 tctgcttctt taagcaactg gcgatgattt ttatgtctgt actggcgggc aatatgtatg 2400 aaagcatcgg tttccagggc gcttatctgg tgctgggtct ggtggcgctg ggcttcacct 2460 taatttccgt gttcacgctt agcggccccg gcccgctttc cctgctgcgt cgtcaggtga 2520 atgaagtcgc ttaagcaatc aatgtcggat gcggcgcgag cgccttatcc gaccaacata 2580 tcataacgga gtgatcgcat tgaacatgcc aatgaccgaa agaataagag caggcaagct 2640 atttaccgat atgtgcgaag gcttaccgga aaaaagactt cgtgggaaaa cgttaatgta 2700 tgagtttaat cactcgcatc catcagaagt tgaaaaaaga gaaagcctga ttaaagaaat 2760 gtttgccacg gtaggggaaa acgcctgggt agaaccgcct gtctatttct cttacggttc 2820 caacatccat ataggccgca atttttatgc aaatttcaat ttaaccattg tcgatgacta 2880 cacggtaaca atcggtgata acgtactgat tgcacccaac gttactcttt ccgttacggg 2940 acaccctgta caccatgaat tgagaaaaaa cggcgagatg tactcttttc cgataacgat 3000 tggcaataac gtctggatcg gaagtcatgt ggttattaat ccaggcgtca ccatcgggga 3060 taattctgtt attggcgcgg gtagtatcgt cacaaaagac attccaccaa acgtcgtggc 3120 ggctggcgtt ccttgtcggg ttattcgcga aataaacgac cgggataagc actattattt 3180 caaagattat aaagttgaat cgtcagttta aattataaaa attgcctgat acgctgcgct 3240 tatcaggcct acaagttcag cgatctacat tagccgcatc cggcatgaac aaagcgcagg 3300 aacaagcgtc gcatcatgcc tctttgaccc acagc 3335 <210> 5 <211> 1890 <212> DNA <213> Bifidobacterium infantis <400> 5 atgagtgaag caatcgcaag accgcgttcc aagtcgctgc agcgcagaga cgccaaactg 60 gcgctcaagg caagcaagca ttacaagcgc atgcagcagc gtgagccggc tccgaagctg 120 accggcaagc agcgtgtgct caactggctg ctgcacatca ttatggctgt gatggtcatc 180 tactgcctcg tgccgctgct gtgggtcgtc ttctcttcca caaagaccag tgagggcatc 240 ttcagctctt tcggcctgtg gttcgatgac aagaatgtgt tctggcagaa cgtgcaggat 300 acgtttgctt atcagcatgg tgtctacact cgttggctgt tcaatacgat catgtacgca 360 gtggtagcag gtgttggtgc aaccatcatt gccactttcg ctgggtatgc catcgccacc 420 atgcgattcc ctggacgtaa cgccctgctg gccgtcactc tggcattcat gtcaattcct 480 tctacggtga tcaccgtgcc gctgttcctc atgtattcga agatcgggct ggtcggcact 540 ccgtgggccg tgattattcc gcagcttgcc acgccgttcg gcctgtatct gatgatcatc 600 tatgcgcaga cctcgattcc ggtctcgctg atcgaagccg cgaaactgga cggtgcaaac 660 acttggacca tcttctggaa ggtcggtttc ccgctgctgt ctccaggttt cgtcaccgta 720 ctgctgttca cgctggtcgg tgtttggaac aactacttcc tgccgctgat catgcttacg 780 aataccaacg actacccgtt gacggtcggt ctgaacatgt ggctgaagat gggtgcccag 840 ggaacttcag acggtcaggt tccgaacaac ctcatcatca cgggctcgct tatcgcagtt 900 gttccgttga tcatcgcatt catgttcctg caaaagtact ggcagtctgg cctcgcggcc 960 ggctctgtca agcagtgaat gacaaatgca acggcgcaac cggatacttc cgtgatgcgc 1020 aagccgaagc gccaatacat aggcatattg tattgtcttc catacgtggt ggtcttcctg 1080 ttcggcatga tcgtcccgat gttctacgcg ctttatctca gcttcttcaa gcagtccctt 1140 ctcggtggca ccacattcgc gggcttcgac aacttcatcc gagcgttcaa ggatgaggct 1200 ttgtggggcg gctttaggaa cgtgctgatt tacgcggcga tccagattcc gatgaatctg 1260 attctgtccc tcgtggcggc gctcgtcttg gactcccaga ggatccgcca tatcgcggtg 1320 ccgcgaatcc tgctgttcct gccttatgcc gtcccaggcg tcatcgcggc gctgatgtgg 1380 ggatacattt acggcgataa gtatggtctt tttggacaaa tcgccggtat gtttggcgta 1440 gcggctccta atatgttgtc gaagcagctc atgttgttcg ccatcgcgaa catctgcact 1500 tggtgcttcc ttggctataa catgctgatt tattattctg cactgatcgg cattccgaac 1560 gacctgtacg aatctgcccg tattgacggc gcttcggagc tgcgcatcgc ttggtccgtg 1620 aagatccccc agattaagag caccatcgtg atgacggtcc tcttctctgt gatcggcacg 1680 ctccagctgt tcaacgaacc gaacatcttg cgtacttccg ccccggacgt gatcaacagc 1740 agctatacgc cgaacatcta cacctacaac ctcgcattca atggccagaa cgtcaactat 1800 gccgctgccg tctctctggt catcggcatc atcgtcatgg ccttggtcgc cgtcgttaag 1860 atcatcggca acaagtggga gaacaagtga 1890 <210> 6 <211> 969 <212> DNA <213> Azospirillum brasilense <400> 6 ttggcgcgga tcgcattgat cttcggaatc tccgggcagg atggaagcta tcttgcccgg 60 cacctgctcg accgcggcta caccgtccat ggcacgtcgc gggactgcga gatcgcatcg 120 ttccgcaacc tgacgattct gggcatccgc gaccgggtaa acgtccattc ggcgaccctg 180 catgatttcc gcagcatcgt gcaggtgatc cgggcggtga agcccgacga gatctacaac 240 ctctccgccc agtcggcggt gggtctgtcc ttcgagcagc cggtcgagac gatcgacagc 300 atcctgaacg gcacgctgaa cattctggag gccgtccgct tcctgggggt ggactcgcgc 360 ttctaccacg cctcctccag cgagagcttc ggcaacaccc tggacgagcc ggccagcgag 420 acgacggcct ttcgtccctg cagcccttat ggcgtcgcca agtcggcagc ccactggatg 480 gtcgccaact atcggcaggc ctacgggctc tatgcctgct cgggcattct gttcaatcat 540 gaatcgccgc tgcgcccggc gcgtttcgtc acccgcaaga ttgtccgcgg cgccatcgcg 600 gtggcgcagg ggcgcctgaa gacgctggaa ttgggcaacc tcgccgtggc gcgggattgg 660 ggctgggcgc cggaatatgt ggacgccatg tggcgcatgc tccagcagga cgagccggac 720 gatttcgtca tcgccaccgg ccggatgcat cccctggagg acttcgtccg gctggccttt 780 gcgcatttcg gcctagactg gcgggagcat gtggtccagg gggcgacgca tcggcgaccg 840 atggacatcc attgcagcgt cggcaacccg gccagggccg aagcccgcct gggctggaag 900 gccgaaaccc tgatgccgga tctcgtcgga cgtctggtcg aggcggagtt ggccctgccg 960 caaccttga 969 <210> 7 <211> 1257 <212> DNA <213> Artificial Sequence <220> <223> tac_azoT(CO) <400> 7 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 60 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 120 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 180 attgtgagcg gataacaatt tcacacagga aacagaatta aaagatatga ccatgattac 240 gccaagcttg catgcctgca ggtcgactct agaggatcca tgctcgatca acgtacgagc 300 gcattcttgg aagaatttct ggcgaaaccg ggaggtgatc ccgagcgact cgaccgcttc 360 ttacttcacg gcccgtaccg tggccggcgg ggcggcaagc cgcgattgaa acttgcgttc 420 cacgatttct ggccagagtt tgacaaggga accaattttt tcatcgaaat tctgtcctcc 480 cgcttcgatc tgtcggtagt ggaagacgat tctgacctcg ccattgtgtc tgtcttcggc 540 gggcggcacc gcgaggctcg ctcacgccgc accctcttct tcaccggaga gaacgttcgc 600 ccaccgcttg acggtttcga tatggctgtg tccttcgacc gcgttgacga tccacgccat 660 tacaggctgc cactctacgt catgcacgcc tacgagcaca tgcgagaggg cgcggtgcca 720 catttttgtt cacctgtcct gccaccagtg cctccgacaa gagctgcttt tgcagaacgt 780 ggattttgcg cctttttgta caagaatcct aacggtgaaa ggcgtaaccg ctttttcccg 840 gtgctggacg gtcgtcgacg tgttgattct gtgggctggc atctaaacaa taccggttcc 900 gtcgtgaaaa tgggatggtt gtcgaagatc cgtgtcttcg aacgctaccg tttcgccttc 960 gcattcgaga acgctagcca tcccggttat cttactgaaa agatcctgga cgtcttccag 1020 gccggcgcgg tgcctttgta ttggggtgat cccgatctag aacgcgaagt tgcagtcggc 1080 agctttatcg acgtgagtcg cttcgcaact gatgaggaag ctgtggacca cattcttgca 1140 gtggatgatg attacgacgc ctactgcgca caccgcgcag ttgcgccttt tctggggacc 1200 gaggaatttt atttcgatgc ctaccgcctc gctgactgga tcgaatcccg cttataa 1257 <210> 8 <211> 279 <212> DNA <213> Escherichia coli <400> 8 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 60 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 120 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 180 attgtgagcg gataacaatt tcacacagga aacagaatta aaagatatga ccatgattac 240 gccaagcttg catgcctgca ggtcgactct agaggatcc 279 <210> 9 <211> 978 <212> DNA <213> Artificial Sequence <220> <223> azoT(CO) <400> 9 atgctcgatc aacgtacgag cgcattcttg gaagaatttc tggcgaaacc gggaggtgat 60 cccgagcgac tcgaccgctt cttacttcac ggcccgtacc gtggccggcg gggcggcaag 120 ccgcgattga aacttgcgtt ccacgatttc tggccagagt ttgacaaggg aaccaatttt 180 ttcatcgaaa ttctgtcctc ccgcttcgat ctgtcggtag tggaagacga ttctgacctc 240 gccattgtgt ctgtcttcgg cgggcggcac cgcgaggctc gctcacgccg caccctcttc 300 ttcaccggag agaacgttcg cccaccgctt gacggtttcg atatggctgt gtccttcgac 360 cgcgttgacg atccacgcca ttacaggctg ccactctacg tcatgcacgc ctacgagcac 420 atgcgagagg gcgcggtgcc acatttttgt tcacctgtcc tgccaccagt gcctccgaca 480 agagctgctt ttgcagaacg tggattttgc gcctttttgt acaagaatcc taacggtgaa 540 aggcgtaacc gctttttccc ggtgctggac ggtcgtcgac gtgttgattc tgtgggctgg 600 catctaaaca ataccggttc cgtcgtgaaa atgggatggt tgtcgaagat ccgtgtcttc 660 gaacgctacc gtttcgcctt cgcattcgag aacgctagcc atcccggtta tcttactgaa 720 aagatcctgg acgtcttcca ggccggcgcg gtgcctttgt attggggtga tcccgatcta 780 gaacgcgaag ttgcagtcgg cagctttatc gacgtgagtc gcttcgcaac tgatgaggaa 840 gctgtggacc acattcttgc agtggatgat gattacgacg cctactgcgc acaccgcgca 900 gttgcgcctt ttctggggac cgaggaattt tatttcgatg cctaccgcct cgctgactgg 960 atcgaatccc gcttataa 978 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> F_inf_KpnI_noeL(pENGWTA(CO)) <400> 10 ctagaggatc cccgggtacc ttggcgcgga tcgcattgat cttcg 45 <210> 11 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> R_inf_KpnI_noeL(pENGWTA(CO)) <400> 11 ttgtatatct ccttggtacc tcaaggttgc ggcagggc 38 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> F_inf_Sac1_Tac_RBS_azoT(pEGWTA(CO)) <400> 12 gctttcgggg gtaagagctc tcaggcagcc atcggaag 38 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> R_inf_sacI_COazoT(pEGWTA(CO)) <400> 13 cggccagtga attcgagctc ttataagcgg gattcgatcc agtc 44

Claims (7)

서열번호 9에 기재된 핵산서열을 가지는 azoT 유전자로 암호화된 것이고, tac 프로모터에 의해 전사가 조절되는 α-1,3-푸코오스 전이효소 (α-1,3- fucosyltransferase)가 발현되도록 형질전환되고,
gmd 유전자 및 noeL 유전자로 암호화된 GDP-D-만노오스-4,6-데하이드라타아제 (GDP-D-mannose-4,6-dehydratase)가 발현되도록 형질전환되며,
GDP-L-푸코오스 신타아제 (GDP-L-fucose synthase)가 발현되도록 형질전환되고,
락토오즈 퍼미아제 (lactose permease)가 발현되도록 형질전환되고,
서열번호 5에 기재된 핵산서열로 암호화된 ABC 트랜스포터 퍼미아제(transporter permease)가 발현되도록 형질전환되며,
포스포만노뮤타아제 (Phosphomannomutase) 및 GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)를 보유하고 있는 것을 특징으로 하는 3'-푸코실락토오스 생산용 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum).
It is encoded by the azoT gene having the nucleic acid sequence set forth in SEQ ID NO: 9, and is transformed to express α-1,3-fucosyltransferase, which transcription is regulated by the tac promoter,
It is transformed to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase) encoded by the gmd gene and the noeL gene,
Transformed to express GDP-L-fucose synthase,
Transformed to express lactose permease,
It is transformed to express ABC transporter permease encoded with the nucleic acid sequence set forth in SEQ ID NO: 5,
Recombinant Corey for the production of 3'-fucosylactose, characterized by having Phosphomannomutase and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase) Nebacterium glutamicum ( Corynebacterium glutamicum ).
삭제delete 삭제delete 제1항에 있어서,
상기 재조합 코리네박테리움 글루타미쿰은,
포스포만노뮤타아제 (Phosphomannomutase)가 과발현되도록 형질전환되고,
GTP-만노오스-1-포스페이트 구아닐릴트랜스퍼라아제 (GTP-mannose-1-phosphate guanylyltransferase)가 과발현되도록 형질전환된 것을 특징으로 하는 3'-푸코실락토오스 생산용 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum).
The method of claim 1,
The recombinant Corynebacterium glutamicum,
Phosphomannomutase is transformed to overexpress,
Recombinant Corynebacterium glutamicum for the production of 3'-fucosylactose, characterized in that it has been transformed to overexpress GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase) ( Corynebacterium glutamicum ).
락토오스가 첨가된 배지에, 제1항의 재조합 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum)을 배양하는 것을 특징으로 하는 3'-푸코실락토오스의 생산방법.
A method for producing 3'-fucosyl lactose, characterized in that the recombinant Corynebacterium glutamicum of claim 1 is cultured in a medium to which lactose is added.
제5항에 있어서,
상기 배지는,
글루코오스를 더 포함하는 것을 특징으로 하는 3'-푸코실락토오스의 생산방법.
The method of claim 5,
The medium,
3'-Fucosyl lactose production method, characterized in that it further comprises glucose.
제5항에 있어서,
상기 푸코실락토오스의 생산방법은,
글루코오스 또는 락토오스를 추가로 공급하는 회분식 배양 또는 유가식 배양인 것을 특징으로 하는 3'-푸코실락토오스의 생산방법.
The method of claim 5,
The method for producing the fucosyl lactose,
A method for producing 3'-fucosyl lactose, characterized in that it is a batch culture or fed-batch culture that additionally supplies glucose or lactose.
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