WO2021093022A1 - 重组大肠杆菌系统及其构建方法和其在合成α-1,2-岩藻糖基化寡糖中的应用 - Google Patents

重组大肠杆菌系统及其构建方法和其在合成α-1,2-岩藻糖基化寡糖中的应用 Download PDF

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WO2021093022A1
WO2021093022A1 PCT/CN2019/120873 CN2019120873W WO2021093022A1 WO 2021093022 A1 WO2021093022 A1 WO 2021093022A1 CN 2019120873 W CN2019120873 W CN 2019120873W WO 2021093022 A1 WO2021093022 A1 WO 2021093022A1
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gdp
gene
fucosyltransferase
fucose
recombinant
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黄锦
薛晓舟
李玉
秦慧民
佟毅
李义
陈博
李凡
安泰
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中粮营养健康研究院有限公司
吉林中粮生化有限公司
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Priority to CN201980006916.9A priority Critical patent/CN113166770A/zh
Priority to AU2019400151A priority patent/AU2019400151B2/en
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides

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  • the present invention relates to the technical field of genetic engineering, in particular to a recombinant Escherichia coli system, a method for constructing the recombinant Escherichia coli system, the application of the recombinant Escherichia coli system in the synthesis of ⁇ -1,2-fucosylated oligosaccharides and a method for constructing the recombinant Escherichia coli system. Synthesis method of ⁇ -1,2-fucosylated oligosaccharides.
  • oligosaccharides With the rapid development of biotechnology and the food industry, the research and development of functional oligosaccharides has become the forefront of international biotechnology, mainly focusing on polysaccharides derived from natural plants and microorganisms, and low-molecular-weight polysaccharides prepared after degradation by acid and alkali. Oligosaccharides; and, the oligosaccharide industry has become an emerging industry used in food, medicine, agriculture and chemical industries, and has a huge market share. Fucosylated oligosaccharides are a class of functional oligosaccharides, which play an important role in blood transport, selective mediation of leukocyte endothelial adhesion, and ontogeny. According to research, when the content of fucosylated oligosaccharides in the human body is reduced, it will cause some serious diseases, such as leukocyte adhesion defects, and more serious ones will directly lead to the occurrence of cancer and atherosclerosis.
  • Human milk oligosaccharides are free oligosaccharides in human milk. Its content in human milk is second only to fat and lactose. It is the third largest solid substance in human milk and is even more important in human milk. The active factor. The structure of HMOs is complex and diverse, and 70% of HMOs are fucosylated oligosaccharides. Human milk oligosaccharides have physiological functions such as regulating intestinal flora, enhancing immunity, anti-viral infection, reducing inflammation, and promoting infant brain development. As a kind of functional oligosaccharide substance with special structure, it participates in infant's non-immune defense system. The enhancement of infant nutrition and health care will surely play its huge role.
  • the enzymatic synthesis process is a process in which all enzymes involved in catalyzing the synthesis of fucosylated oligosaccharides are separated and purified and catalyzed in vitro.
  • the method can improve the activity of the enzyme by modifying the fucosyltransferase involved in enzyme catalysis, and further increase the production of fucosylated oligosaccharides.
  • ⁇ -1,2-Fucosyltransferase is an enzyme that transfers a fucosyl group to the corresponding oligosaccharide to form an ⁇ -1,2-linkage.
  • ⁇ -1,2-Fucosyltransferase belongs to Glycosyltransferase family11. At present, the gene of ⁇ -1,2-Fucosyltransferase has been found in mammals, viruses, plants, and bacteria. There are also different degrees of research progress on the nature of science. ⁇ -1,2-Fucosyltransferase exhibits a wide range of substrate specificity, which provides great advantages for the synthesis of ⁇ -1,2-Fucosylated oligosaccharides. However, the existing technology uses biotechnology to produce When fucosylated oligosaccharides are used, the yield of fucosylated oligosaccharides needs to be further improved.
  • the technical problem to be solved by the present invention is to provide a method for enzymatically synthesizing ⁇ -1,2-fucosylated oligosaccharides, so as to solve the complex process route and high cost of chemical synthesis in the prior art, as well as the yield of biological method. Need to improve.
  • the present invention provides a recombinant Escherichia coli system having an ⁇ -1,2-fucosyltransferase gene and a GDP-fucose pyrophosphorylase gene.
  • the present invention provides a method for constructing a recombinant E. coli system as described above, the method comprising:
  • step (3) Transform the expression vector obtained in step (1) and step (2) into E. coli cells to obtain the recombinant E. coli system.
  • the present invention provides the application of the recombinant E. coli system described above in the synthesis of ⁇ -1,2-fucosylated oligosaccharides.
  • the present invention provides a method for synthesizing ⁇ -1,2-fucosylated oligosaccharides, the method comprising:
  • step (2) Contact the ⁇ -1,2-fucosyltransferase and GDP-fucose pyrophosphorylase obtained in step (1) with the conversion substrate to synthesize the ⁇ -1,2-fucose Glycated oligosaccharides.
  • the present invention constructs genetic engineering bacteria to express ⁇ -1,2-fucose by transforming ⁇ -1,2-fucose transferase gene and GDP-fucose pyrophosphorylase gene into Escherichia coli Base transferase and GDP-fucose pyrophosphorylase, and the expressed ⁇ -1,2-fucose pyrophosphorylase and GDP-fucose pyrophosphorylase are used to enzymatically synthesize ⁇ -1,2 -Fucosylated oligosaccharides, compared to other sources (for example, Pirkko et al. used recombinant E.
  • Figure 1 is an SDS-PAGE electrophoresis diagram of ⁇ -1,2-fucosyltransferase (HpFutr) (a) and GDP-fucose pyrophosphorylase (FKP) (b).
  • HpFutr ⁇ -1,2-fucosyltransferase
  • FKP GDP-fucose pyrophosphorylase
  • Figure 2 is a schematic diagram of the synthesis process of ⁇ -1,2-fucosylated oligosaccharides provided by the present invention.
  • a recombinant Escherichia coli system which is transformed with an ⁇ -1,2-fucosyltransferase gene and a GDP-fucose pyrophosphorylase gene.
  • the ⁇ -1,2-fucosyltransferase gene can be various existing nucleotide sequences encoding ⁇ -1,2-fucosyltransferase, according to the present invention.
  • the ⁇ -1,2-fucosyltransferase gene is derived from Helicobacter pylori, and more preferably, the ⁇ -1,2-fucosyltransferase gene has SEQ ID NO :1 The nucleotide sequence shown.
  • the GDP-fucose pyrophosphorylase gene may be various existing nucleotide sequences encoding GDP-fucose pyrophosphorylase.
  • the GDP-fucose pyrophosphorylase gene is derived from Bacteroides fragilis. More preferably, the GDP-fucose pyrophosphorylase gene has the nucleotide sequence shown in SEQ ID NO: 2.
  • the ⁇ -1,2-fucosyltransferase gene and GDP-fucose pyrophosphorylase gene can be inserted into a vector to construct and express ⁇ -1,2-fucosyltransferase.
  • the recombinant vector for the transferase and the recombinant vector for expressing GDP-fucose pyrophosphorylase, and then the constructed recombinant expression vector is transformed into the Escherichia coli host cell, thereby obtaining the recombinant Escherichia coli system of the present invention.
  • the construction of the recombinant vector can be carried out in accordance with conventional methods in the art.
  • it can be designed with a primer pair containing the expected restriction site for ⁇ -1,2-fucosyltransferase gene and GDP-fucosyltransferase gene.
  • the cut products are ligated under the action of DNA ligase to obtain the expected recombinant vector.
  • the plasmid used to construct the recombinant vector can be various plasmids that can be expressed in E. coli conventionally in the art. According to a preferred embodiment of the present invention, it is used to construct a plasmid containing ⁇ -1,2-
  • the plasmid of the recombinant vector of the fucosyltransferase gene is the pCold I plasmid; according to another preferred embodiment of the present invention, the plasmid used to construct the recombinant vector of the GDP-fucose pyrophosphorylase gene is the pET28a plasmid.
  • the transformation refers to the treatment of cells by some known methods in molecular biology and genetic engineering, so that the treated cells are in a competent state, and thus contact with exogenous DNA, so that the exogenous DNA can be brought into contact with the exogenous DNA.
  • transformation methods include protoplast transformation, chemical transformation and electroporation transformation.
  • the Escherichia coli may be various conventional Escherichia coli strains used for expression of foreign genes.
  • the Escherichia coli cell is an Escherichia coli C43 cell.
  • the ⁇ -1,2-fucosyltransferase gene and GDP-fucose pyrophosphorylase gene can be respectively transformed into different E. coli cells, and then respectively The Escherichia coli transformed with the ⁇ -1,2-fucosyltransferase gene and the GDP-fucose pyrophosphorylase gene are multiplied and the corresponding gene is expressed, thereby obtaining the target product.
  • the ⁇ -1,2-fucosyltransferase gene and GDP-fucose pyrophosphorylase gene can be respectively transformed into the same E. coli cell, and then The Escherichia coli transformed with ⁇ -1,2-fucosyltransferase gene and GDP-fucose pyrophosphorylase gene is multiplied and the corresponding genes are expressed to obtain the target product.
  • step (3) Transform the expression vectors obtained in step (1) and step (2) into the same or different E. coli cells to obtain the recombinant E. coli system.
  • PCR is performed on the ⁇ -1,2-fucosyltransferase gene through the forward primer shown in SEQ ID NO: 3 and the reverse primer shown in SEQ ID NO: 4 Amplify to obtain the restriction sites of BamHI and Hind III, then double digest the PCR product and the corresponding plasmid with BamHI and Hind III, and then ligate by DNA ligase to obtain the ⁇ -1,2 -An expression vector for the fucosyltransferase gene.
  • the GDP-fucose pyrophosphorylase gene is amplified by PCR through the forward primer shown in SEQ ID NO: 5 and the reverse primer shown in SEQ ID NO: 6 , To obtain the restriction sites of BamHI and XhoI, and then double-enzyme the PCR product and the corresponding plasmid with BamHI and XhoI, and then ligate by DNA ligase to obtain the GDP-fucose pyrophosphorylase Gene expression vector.
  • the method further includes a step of screening the transformed E. coli.
  • the screening step can be carried out according to a conventional method in the art, for example, The transformed E. coli was spread on the LB plate containing Amp and Kan for the cultivation of single colony strains.
  • the E. coli transformed with the recombinant vector can obtain Amp and Kan resistance, so that the transformed recombinant vector can be obtained through the screening of this step A positive clone of E. coli.
  • the obtained recombinant Escherichia coli cells can be stored in a 15% glycerol tube for preservation of strains.
  • the present invention provides the application of the aforementioned recombinant E. coli system in the synthesis of ⁇ -1,2-fucosylated oligosaccharides.
  • the present invention provides a method for synthesizing ⁇ -1,2-fucosylated oligosaccharides, the method comprising:
  • step (2) Contact the ⁇ -1,2-fucosyltransferase and GDP-fucose pyrophosphorylase obtained in step (1) with the conversion substrate to synthesize the ⁇ -1,2-fucose Glycated oligosaccharides.
  • the "conditions under which Escherichia coli can multiply” refers to the conditions known in the art for the growth and multiplication of Escherichia coli, for example, in LB solid or liquid medium at 15-40°C condition.
  • an LB liquid medium containing ampicillin final concentration may be 40-60 ⁇ g/mL
  • kanamycin final concentration may be 40-60 ⁇ g/mL
  • IPTG concentration can be 0.1-0.5mM
  • the recombinant E. coli is induced and cultured at 15-17°C to express the ⁇ -1,2-fucosyltransferase Enzyme gene and the GDP-fucose pyrophosphorylase gene to obtain ⁇ -1,2-fucose pyrophosphorylase and GDP-fucose pyrophosphorylase.
  • the timing of adding the IPTG is a conventional choice in the art, for example, IPTG can be added when the OD value of the culture solution reaches 0.6-0.8.
  • the time of induction culture can be selected in a wide range, for example, it can be 12-20 hours.
  • the method of the present invention when the culture is completed, also includes the separation and purification of proteins from the bacteria obtained by the culture, so as to obtain purified ⁇ -1,2-fucosyltransferase and GDP-fucose Pyrophosphorylase.
  • the method of protein separation and purification can refer to conventional methods in the art.
  • the culture solution can be collected by centrifugation, and the bacteria can be broken in the lysis buffer by a method such as ultrasound, so that the purpose is The protein is dissolved, and then the supernatant is collected by centrifugation. Finally, the resulting supernatant can be separated and mixed by a nickel column.
  • a Ni-NTA superflow nickel column can be used.
  • the transformation substrate in step (2), is known in the art to be able to synthesize ⁇ -1,2-fucosylated oligos under the action of ⁇ -1,2-fucosyltransferase.
  • a sugar substrate for example, the substrate may contain GDP-fucose and lactose.
  • the conversion substrate preferably also contains Tris-HCl, MgSO 4 , ATP and GTP.
  • the concentration of each component can be varied within a wide range. According to a preferred embodiment of the present invention, relative to the conversion substrate per liter, the concentration of GDP-fucose is 5-15 mmol, and the concentration of lactose is 5-15 mmol. The concentration is 5-15 mmol, the concentration of Tris-HCl is 15-25 mmol, the concentration of MgSO 4 is 4-6 mmol, the concentration of ATP is 5-15 mmol, and the concentration of GTP is 5-15 mmol.
  • the dosage of the ⁇ -1,2-fucosyltransferase and GDP-fucose pyrophosphorylase can be selected in a wide range, in order to more effectively improve the synthesis of ⁇ -1 .
  • the efficiency of 2-fucosylated oligosaccharides, preferably, relative to the conversion substrate per liter, the amount of the ⁇ -1,2-fucosyltransferase is 30-50 ⁇ g (for example, 30 ⁇ g, 32 ⁇ g, 34 ⁇ g, 36 ⁇ g, 38 ⁇ g, 40 ⁇ g, 42 ⁇ g, 44 ⁇ g, 46 ⁇ g, 48 ⁇ g, 50 ⁇ g),
  • the GDP-fucose pyrophosphorylase dosage is 30-50 ⁇ g (for example, 30 ⁇ g, 32 ⁇ g, 34 ⁇ g, 36 ⁇ g, 38 ⁇ g , 40 ⁇ g, 42 ⁇ g, 44 ⁇ g, 46 ⁇ g, 48 ⁇ g, 50 ⁇ g).
  • the contact form of the above-mentioned ⁇ -1,2-fucosyltransferase and GDP-fucose pyrophosphorylase with the conversion substrate can be the supernatant obtained after the bacterial cells are broken. Liquid, it can also be purified protein after purification, but no matter what form it is in contact with the conversion substrate, the above-mentioned ⁇ -1,2-fucosyltransferase and GDP-fucose pyrophosphate
  • the dosage of the enzyme is based on the amount of ⁇ -1,2-fucosyltransferase and GDP-fucose pyrophosphorylase itself.
  • the synthesis conditions of the ⁇ -1,2-fucosylated oligosaccharide may be conventional conditions in the art, for example, the temperature may be 36-38°C.
  • SEQ ID NO: 1 ( ⁇ -1,2-fucosyltransferase gene), the nucleotide sequence shown, and the positive sequence shown in SEQ ID NO: 3. And the reverse primer shown in SEQ ID NO: 4.
  • SEQ ID NO:1 ⁇ -1,2-fucosyltransferase gene
  • amplify ⁇ -1,2-rock through the PCR reaction system and amplification program shown in Table 1
  • fucosyltransferase gene agarose gel electrophoresis was used to confirm the amplified DNA fragment (approximately 900 bp, in line with expectations), and the PCR product was recovered with an agarose gel DNA recovery kit (purchased from Tiangen Biochemical).
  • SEQ ID NO: 2 GDP-fucose pyrophosphorylase gene
  • SEQ ID NO: 5 The reverse primer shown in SEQ ID NO: 6.
  • SEQ ID NO: 2 GDP-fucose pyrophosphorylase gene
  • the GDP-fucose pyrophosphorylase gene was amplified through the PCR reaction system and amplification program shown in Table 2
  • Agarose gel electrophoresis was used to confirm the amplified DNA fragment (approximately 2800 bp, in line with expectations), and the PCR product was recovered with an agarose gel DNA recovery kit (purchased from Tiangen Biochemical).
  • This example is used to illustrate the construction of a recombinant E. coli system
  • Example 1 and Example 2 The recombinant plasmids constructed in Example 1 and Example 2 were respectively transformed into different E. coli C43 (DE3) competent cells, cultured overnight at 37°C, and a single clone containing ampicillin and kanamycin was selected. Cultured in the liquid medium of LB, preserved the strain with 15% glycerol, obtained E.coil C43 (DE3) recombinant strain, that is, constructed recombinant strain expressing ⁇ -1,2-fucosyltransferase gene And constructed a recombinant strain of GDP-fucose pyrophosphorylase gene.
  • E.coil C43 (DE3) recombinant strain that is, constructed recombinant strain expressing ⁇ -1,2-fucosyltransferase gene
  • constructed a recombinant strain of GDP-fucose pyrophosphorylase gene constructed a recombinant strain of GDP-fucose pyrophosphorylase gene.
  • This example is used to illustrate the protein expression and purification of ⁇ -1,2-fucosyltransferase and GDP-fucose pyrophosphorylase
  • the obtained recombinant bacteria were respectively inoculated into 10 ml (containing 50 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin) LB liquid medium, and cultured overnight at 37° C. as a seed solution. Transfer 1 vol% inoculum to 100 mL (containing 50 ⁇ g/mL ampicillin) LB liquid medium, culture with shaking at 37°C until OD 600 reaches 0.6-0.8, add IPTG to a final concentration of 0.1-0.5 mM, and shake at 16°C At 16 hours of culture, the cells were collected by centrifugation at 5000r/min and 15min.
  • ⁇ -1,2-fucosyltransferase and GDP-haloside pyrophosphorylase specifically catalyze the production of ⁇ -1,2-fucosylated oligosaccharides, and the specific catalytic process To: transfer fucose from the glycosyl donor GDP-fucose to the corresponding acceptor oligosaccharide to form an ⁇ -1,2-link.

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Abstract

涉及基因工程技术领域，提供了重组大肠杆菌系统及其构建方法和其在合成α-1,2-岩藻糖基化寡糖中的应用。该重组大肠杆菌系统具有α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因。通过将α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因转化到大肠杆菌中，构建基因工程菌，以表达α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶，并将表达的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶用于酶法合成α-1,2-岩藻糖基化寡糖的。

Description

重组大肠杆菌系统及其构建方法和其在合成α-1,2-岩藻糖基化寡糖中的应用 技术领域
本发明涉及基因工程技术领域，具体涉及重组大肠杆菌系统、构建该重组大肠杆菌系统的方法，该重组大肠杆菌系统在合成α-1,2-岩藻糖基化寡糖中的应用以及一种α-1,2-岩藻糖基化寡糖的合成方法。
背景技术
随着生物科技和食品工业的快速发展，功能性寡糖的研究与开发已成为国际生物技术的前沿方向，主要集中于天然植物和微生物来源的多糖，经酸碱等降解后制备的低分子量的寡糖；并且，寡糖产业已成为应用于食品、医药、农业和化工等行业的新兴产业，且市场份额巨大。岩藻糖基化寡糖即是一类功能性寡糖类物质，其在血液运输、选择性调解白细胞内皮粘合以及个体发育过程中都起着重要的作用。据研究，当人体内的岩藻糖基化寡糖含量减少时，则会引发一些严重的疾病，像白细胞粘合缺陷病，更严重者则会直接导致癌症和动脉粥样硬化的发生。
人乳寡糖(HMOs)是一种于人乳中游离的寡糖，其在人乳中的含量仅次于脂肪和乳糖，是人乳中的第三大固体物质，更是人乳中重要的活性因子。HMOs的结构复杂且种类繁多，而且70％的HMOs都属于岩藻糖基化寡糖。人乳寡糖具有调节肠道菌群、增强免疫力、抗病毒感染、减少炎症以及促进婴儿大脑发育等生理作用，作为一类特殊结构的功能性寡糖类物质，参与婴儿的非免疫防御体系的增强，在婴儿营养保健方面必将发挥其巨大的作用。
最初生产岩藻糖基化寡糖的工艺主要是利用化学合成法，但化学合成法一方面存在合成路线复杂、且反应条件控制严格的缺陷，而且合成时还需要注意催化剂的选择，设计合适保护基团以及确定供体的离去基团；而且因为合成寡糖的糖苷供体价格昂贵而导致最终的合成成本昂贵。
相比于化学合成方法，利用生物技术生产岩藻糖基化寡糖具有诸多优点，并且随着岩藻糖基转移酶基因在细菌中被发现，全细胞生物合成法和酶法合成工 艺成为生物技术领域生产岩藻糖基化寡糖的两大主要路线。全细胞生物合成法是利用基因工程和代谢工程技术将相应的基因插入到细胞的基因组内，并利用细胞自身的代谢生成岩藻糖基化寡糖。该方法需要细胞表达过量的岩藻糖基转移酶，并提供大量的供体GDP-岩藻糖(鸟苷酸二磷酸-岩藻糖)以保证受体及产物在细胞内不被分解掉。酶法合成工艺是将所有参与催化合成岩藻糖基化寡糖的酶分离纯化出来并在体外进行催化反应的过程。该方法可通过对参与酶催化的岩藻糖基转移酶的改造，以提高酶的活性，进一步提高岩藻糖基化寡糖的产量。
α-1,2-岩藻糖基转移酶是将岩藻糖基转移到相应寡糖上以形成α-1,2-连接的酶。α-1,2-岩藻糖基转移酶属于Glycosyltransferase family11，目前已在哺乳动物、病毒、植物、细菌中均发现了α-1,2-岩藻糖基转移酶的基因，并对其酶学性质也有不同程度的研究进展。α-1,2-岩藻糖基转移酶表现出广泛的底物特异性，为合成α-1,2-岩藻糖基化寡糖提供了巨大的优势，然而现有技术利用生物技术生产岩藻糖基化寡糖时，岩藻糖基化寡糖的产量还需要进一步提高。
发明内容
本发明所要解决的技术问题在于提供一种酶法合成α-1,2-岩藻糖基化寡糖的方法，以解决现有技术中化学合成法工艺路线复杂及成本较高以及生物法产量需要提高的问题。
为了实现上述目的，第一方面，本发明提供一种重组大肠杆菌系统，该重组大肠杆菌系统具有α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因。
第二方面，本发明提供如上所述的重组大肠杆菌系统的构建方法，该方法包括：
(1)克隆α-1,2-岩藻糖基转移酶基因，并构建所述α-1,2-岩藻糖基转移酶基因的表达载体；
(2)克隆GDP-岩藻糖焦磷酸化酶基因，并构建所述GDP-岩藻糖焦磷酸化酶基因的表达载体；
(3)将步骤(1)和步骤(2)获得的表达载体转化入大肠杆菌细胞中，得到所述重组大肠杆菌系统。
第三方面，本发明提供如上所述的重组大肠杆菌系统在合成α-1,2-岩藻糖基 化寡糖中的应用。
第四方面，本发明提供一种α-1,2-岩藻糖基化寡糖的合成方法，该方法包括：
(1)在大肠杆菌能够繁殖的条件下，使如上所述的重组大肠杆菌系统繁殖并表达所述α-1,2-岩藻糖基转移酶基因和所述GDP-岩藻糖焦磷酸化酶基因，获得α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶；
(2)将步骤(1)获得的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶与转化底物接触，合成所述α-1,2-岩藻糖基化寡糖。
本发明通过将α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因转化到大肠杆菌中，构建基因工程菌，以表达α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶，并将表达的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶用于酶法合成α-1,2-岩藻糖基化寡糖的，相比于他来源的(例如，Pirkko等人利用重组大肠杆菌合成GDP-甘露糖，并进一步利用酵母菌表达系统成功合成GDP-岩藻糖[Mattila P,Jarkko
Figure PCTCN2019120873-appb-000001
Hortling S,et al.Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae[J].Glycobiology,2000,10(10):1041-7.])的α-1,2-岩藻糖基转移酶催化产率提高了145.23％。
附图说明
图1为α-1,2-岩藻糖基转移酶(HpFutr)(a)和GDP-岩藻糖焦磷酸化酶(FKP)(b)的SDS-PAGE电泳图。
图2为本发明提供的α-1,2-岩藻糖基化寡糖的合成过程示意图。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
根据本发明的第一方面，提供一种重组大肠杆菌系统，该重组大肠杆菌系 统转化有α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因。
根据本发明，所述α-1,2-岩藻糖基转移酶基因可以为现有的各种编码α-1,2-岩藻糖基转移酶的核苷酸序列，根据本发明一种优选的实施方式，所述α-1,2-岩藻糖基转移酶基因来源于幽门螺杆菌，更优选的，所述α-1,2-岩藻糖基转移酶基因具有如SEQ ID NO:1所示的核苷酸序列。
根据本发明，所述GDP-岩藻糖焦磷酸化酶基因可以为现有的各种编码GDP-岩藻糖焦磷酸化酶的核苷酸序列，根据本发明一种优选的实施方式，所述GDP-岩藻糖焦磷酸化酶基因来源于脆弱拟杆菌，更优选的，所述GDP-岩藻糖焦磷酸化酶基因具有如SEQ ID NO:2所示的核苷酸序列。
根据本发明，所述α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因可以通过分别插入到载体中从而构建表达α-1,2-岩藻糖基转移酶的重组载体和表达GDP-岩藻糖焦磷酸化酶的重组载体，然后再将构建的重组表达载体转化到大肠杆菌宿主细胞中，从而获得本发明的重组大肠杆菌系统。
其中，所述重组载体的构建可以按照本领域常规的方法进行，例如，可以通过设计有含有预期酶切位点的引物对α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因进行PCR扩增，使得PCR扩增产物带有预期的酶切位点，然后再通过相应的限制性内切酶对获得的PCR产物和目的载体进行酶切，并将酶切产物在DNA连接酶的作用下连接，获得预期的重组载体。
其中，所述用于构建所述重组载体的质粒可以为本领域常规的能够在大肠杆菌中表达的各种质粒，根据本发明一种优选的实施方式，用于构建含有α-1,2-岩藻糖基转移酶基因的重组载体的质粒为pCold I质粒；根据本发明另一种优选的实施方式，用于构建GDP-岩藻糖焦磷酸化酶基因的重组载体的质粒为pET28a质粒。
根据本发明，所述转化是指通过采用分子生物学和基因工程中的一些已知方法处理细胞，使经处理后的细胞处于感受态，并由此与外源DNA接触，从而使外源DNA进入处于感受态的细胞中，常用的转化方法包括原生质体转化法、化学转化法和电穿孔转化法。
根据本发明，所述大肠杆菌可以为常规的各种用于外源基因表达的大肠杆菌菌种，根据本发明一种优选的实施方式，所述大肠杆菌细胞为大肠杆菌C43 细胞。
根据本发明一种具体的实施方式，可以将所述α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因分别转化到不同的大肠杆菌细胞中，进而分别使转化有α-1,2-岩藻糖基转移酶基因和转化有GDP-岩藻糖焦磷酸化酶基因的大肠杆菌繁殖并表达相应基因，从而获得目标产物。
根据本发明另一种具体的实施方式，可以将所述α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因分别转化到同一个大肠杆菌细胞中，进而使转化有α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因的大肠杆菌繁殖并表达相应基因，从而获得目标产物。
根据本发明的第二方面，提供了如上所述的重组大肠杆菌系统的构建方法，该方法包括：
(1)克隆α-1,2-岩藻糖基转移酶基因，并构建所述α-1,2-岩藻糖基转移酶基因的表达载体；
(2)克隆GDP-岩藻糖焦磷酸化酶基因，并构建所述GDP-岩藻糖焦磷酸化酶基因的表达载体；
(3)将步骤(1)和步骤(2)获得的表达载体转化入同一个或不同的大肠杆菌细胞中，得到所述重组大肠杆菌系统。
根据本发明一种优选的实施方式，通过SEQ ID NO:3所示的正向引物和SEQ ID NO:4所示的反向引物对α-1,2-岩藻糖基转移酶基因进行PCR扩增，使其获得BamHI和Hind III的酶切位点，然后用BamHI和Hind III对PCR产物和相应的质粒进行双酶切，之后通过DNA连接酶进行连接，获得所述α-1,2-岩藻糖基转移酶基因的表达载体。
根据本发明另一种优选的实施方式，通过SEQ ID NO:5所示的正向引物和SEQ ID NO:6所示的反向引物对GDP-岩藻糖焦磷酸化酶基因进行PCR扩增，使其获得BamHI和XhoI的酶切位点，然后用BamHI和XhoI对PCR产物和相应的质粒进行双酶切，之后通过DNA连接酶进行连接，获得所述GDP-岩藻糖焦磷酸化酶基因的表达载体。
根据本发明，为了得到稳定表达目的基因的大肠杆菌阳性克隆，所述方法还包括对转化后的大肠杆菌进行筛选的步骤，所述筛选步骤可以按照本领域常规 的方法进行，例如，可以先将转化后的大肠杆菌涂布于含有Amp和Kan的LB平板上进行单菌落菌株的培养，转化有重组载体的大肠杆菌能够获得Amp和Kan抗性，从而通过该步骤的筛选可得到转化有重组载体的大肠杆菌阳性克隆。
根据本发明，可以将获得的重组大肠杆菌细胞保存于15％的甘油管中进行菌种的保存。
第三方面，本发明提供了如上所述的重组大肠杆菌系统在合成α-1,2-岩藻糖基化寡糖中的应用。
第四方面，本发明提供了一种α-1,2-岩藻糖基化寡糖的合成方法，该方法包括：
(1)在大肠杆菌能够繁殖的条件下，使如上所述的重组大肠杆菌系统繁殖并表达所述α-1,2-岩藻糖基转移酶基因和所述GDP-岩藻糖焦磷酸化酶基因，获得α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶；
(2)将步骤(1)获得的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶与转化底物接触，合成所述α-1,2-岩藻糖基化寡糖。
根据本发明，步骤(1)中，所述“大肠杆菌能够繁殖的条件”是指本领域公知的大肠杆菌可生长增殖的条件，例如，在LB固体或液体培养基中，15-40℃的条件。
根据本发明一种优选的实施方式，在含有氨苄霉素(终浓度可以为40-60μg/mL)和卡那霉素(终浓度可以为40-60μg/mL)的LB液体培养基中，并且在添加有IPTG(浓度可以为0.1-0.5mM)的情况下，于15-17℃条件下进行所述重组大肠杆菌的诱导培养，使其表达所述α-1,2-岩藻糖基转移酶基因和所述GDP-岩藻糖焦磷酸化酶基因，获得α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶。
其中，所述IPTG的加入时机为本领域常规的选择，例如，可以当培养液的OD值达到0.6-0.8时加入IPTG。
其中，诱导培养的时间可以在较宽的范围内进行选择，例如，可以为12-20小时。
根据本发明，当培养结束后，本发明的方法还包括对培养获得的菌体进行蛋白质的分离与纯化，从而获得纯化的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦 磷酸化酶。
其中，对于蛋白质的分离和纯化方法可以参照本领域常规的方法，例如，可以先将培养液通过离心收集菌体，并在裂解缓冲液中，通过例如超声的方法对菌体进行破碎，使得目的蛋白溶出，然后离心收集上清，最后可以通过镍柱对所得上清液进行蛋白质的分离和混合，例如，可以使用Ni-NTA superflow(天根生化)的镍柱。
根据本发明，步骤(2)中，所述转化底物为本领域公知的能够在α-1,2-岩藻糖基转移酶的作用下合成α-1,2-岩藻糖基化寡糖的底物，例如，所述底物可以含有GDP-岩藻糖和乳糖。
为了更为有效的提高合成α-1,2-岩藻糖基化寡糖的效率，所述转化底物中还优选含有Tris-HCl、MgSO 4、ATP和GTP。其中，各组分的浓度可在较宽的范围内进行变动，根据本发明一种优选的实施方式，相对于每升的转化底物，GDP-岩藻糖的浓度为5-15mmol，乳糖的浓度为5-15mmol、Tris-HCl的浓度为15-25mmol，MgSO 4的浓度为4-6mmol，ATP的浓度为5-15mmol，GTP的浓度为5-15mmol。
根据本发明，所述α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶的用量可以在较宽的范围内进行选择，为了更为有效的提高合成α-1,2-岩藻糖基化寡糖的效率，优选的，相对于每升的转化底物，所述α-1,2-岩藻糖基转移酶的用量为30-50μg(例如，30μg、32μg、34μg、36μg、38μg、40μg、42μg、44μg、46μg、48μg、50μg)，所述GDP-岩藻糖焦磷酸化酶的用量为30-50μg(例如，30μg、32μg、34μg、36μg、38μg、40μg、42μg、44μg、46μg、48μg、50μg)。
此处需要说明的是，如上所述的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶与转化底物的接触形式可以为菌体破碎后获得的上清液，也可以为经过纯化后的纯化蛋白，然而无论以何种形式与所述转化底物接触，如上所述的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶的用量均是以α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶本身计的用量。
根据本发明，所述α-1,2-岩藻糖基化寡糖的合成条件可以为本领域常规的条件，例如，温度可以为36-38℃。
以下将通过实施例对本发明进行详细描述。
实施例1
本实施例用于说明α-1,2-岩藻糖基转移酶基因重组质粒的构建
(1)委托英潍捷基Invitrogen公司合成如下SEQ ID NO:1(α-1,2-岩藻糖基转移酶基因)、所示的核苷酸序列、SEQ ID NO:3所示的正向引物和SEQ ID NO:4所示的反向引物。
SEQ ID NO1：
Figure PCTCN2019120873-appb-000002
SEQ ID NO:3：
F：5’-CGGGATCCATGGCTTTTAAAGTGGTGC-3’；
SEQ ID NO:4：
R：5’-CCCAAGCTTGTTATACTTTTGGGATTTTACCTCAAAATG-3’。
(2)以SEQ ID NO:1(α-1,2-岩藻糖基转移酶基因)为模板，通过如表1所示的PCR反应体系和扩增程序扩增α-1,2-岩藻糖基转移酶基因，以琼脂糖凝胶电泳检测确认扩增DNA片段(大约900bp，符合预期)，并用琼脂糖凝胶DNA回收试剂盒(购自天根生化)回收PCR产物。
表1
Figure PCTCN2019120873-appb-000003
(3)α-1,2-岩藻糖基转移酶基因DNA的酶切及连接：对回收的PCR产物及质粒pCold I，用相同的限制性内切酶BamHI和Hind III进行双酶切处理，随后用琼脂糖凝胶DNA回收试剂盒回收酶切的片段，用Solution I连接酶(购自takara公司)反应连接，得到重组质粒。
(4)转化和重组质粒的提取：取连接后的重组质粒加入到50μl JM109感受态细胞中，冰浴30min，42℃热击90s，立即冰浴5min，加入500μl LB液体培养基，于37℃振荡培养1h。低速离心菌液，弃掉部分上清，将剩余菌体重悬，涂布于含卡那霉素的LB平板上，于37℃过夜培养。挑去单菌落进行PCR验证，测序确定阳性克隆，用质粒小提试剂盒提取该阳性克隆质粒。
实施例2
本实施例用于说明GDP-岩藻糖焦磷酸化酶基因重组质粒的构建
(1)委托英潍捷基Invitrogen公司合成如下SEQ ID NO:2(GDP-岩藻糖焦磷酸化酶基因)、所示的核苷酸序列、SEQ ID NO:5所示的正向引物和SEQ ID  NO:6所示的反向引物。
SEQ ID NO:2：
Figure PCTCN2019120873-appb-000004
Figure PCTCN2019120873-appb-000005
Figure PCTCN2019120873-appb-000006
SEQ ID NO:5：
F：5’-CGCGGATCCGGATCCATGCAGAAGC-3’；
SEQ ID NO:6：
R：5’-CCGCTCGAGCTCGAGTTAGCTACGGCTAACC-3’；
(2)以SEQ ID NO:2(GDP-岩藻糖焦磷酸化酶基因)为模板，通过如表2所示的PCR反应体系和扩增程序扩增GDP-岩藻糖焦磷酸化酶基因，以琼脂糖凝胶电泳检测确认扩增DNA片段(大约2800bp，符合预期)，并用琼脂糖凝胶DNA回收试剂盒(购自天根生化)回收PCR产物。
表2
Figure PCTCN2019120873-appb-000007
(3)GDP-岩藻糖焦磷酸化酶基因DNA的酶切及连接：对回收的PCR产物及表达载体pET28a，用相同的限制性内切酶BamHI和XhoI进行双酶切处理，随后用琼脂糖凝胶DNA回收试剂盒回收酶切的片段，用Solution I连接酶(购自takara公司)反应连接，得到重组质粒。
(4)转化和重组质粒的提取：取连接后的重组质粒加入到50μl JM109感受态细胞中，冰浴30min，42℃热击90s，立即冰浴5min，加入500μl LB液体培养基，于37℃振荡培养1h。低速离心菌液，弃掉部分上清，将剩余菌体重悬，涂布于含卡那霉素的LB平板上，于37℃过夜培养。挑去单菌落进行PCR验证，测序确定阳性克隆，用质粒小提试剂盒提取该阳性克隆质粒。
实施例3
本实施例用于说明重组大肠杆菌系统的构建
(1)将实施例1和实施例2中构建的重组质粒分别转化不同的大肠杆菌C43(DE3)感受态细胞中，37℃过夜培养，挑取单克隆于含氨苄霉素和卡那霉素的LB的液体培养基中培养，用15％甘油保存菌种，获得E.coil C43(DE3)重组菌，也即，构建的表达α-1,2-岩藻糖基转移酶基因的重组菌株和构建的GDP-岩藻糖焦磷酸化酶基因的重组菌株。
实施例4
本实施例用于说明α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶的蛋白表达与纯化
(1)蛋白表达
将所得重组菌分别接种于10ml(含有50μg/mL氨苄霉素和50μg/mL卡那霉素)LB液体培养基中，37℃过夜培养作为种子液。以1体积％接种量转接到100mL(含有50μg/mL氨苄霉素)LB液体培养基，37℃振荡培养至OD 600达到0.6-0.8，加入IPTG至终浓度为0.1-0.5mM，16℃振荡培养16时，5000r/min、15min下离心收集菌体。
(2)蛋白分离与纯化
向菌体加入20mL破碎缓冲液(50mM Tris(pH 8.0)，100mM NaCl，20％(v/v)甘油，1％(v/v)Triton X-100)并重悬，超声破碎菌体，高速离心，收集上清液与平衡好的Ni-NTA superflow(天根生化)结合1h，使混合的样品流经纯化柱，用洗杂缓冲液洗去杂质蛋白。再用洗脱缓冲液溶出目的蛋白。通过12％非变性聚丙烯酰胺凝胶电泳(SDS-PAGE)，鉴定得到的目的蛋白(α-1,2-岩藻糖基转移酶(a)和GDP-岩藻糖焦磷酸化酶(b))高纯度分别高达95％以上，结果如图1所示。
实施例5
本实施例用于说明α-1,2-岩藻糖基化寡糖的产率测定
如图2所示，α-1,2-岩藻糖基转移酶及GDP-盐藻糖焦磷酸化酶特异性催化生 产α-1,2-岩藻糖基化寡糖，其具体催化过程为：将岩藻糖从糖基供体GDP-岩藻糖上转移到相应受体寡糖上形成α-1,2-连接。
具体的，在EP管中，相对于1L的反应底物，加入20mmol Tris-HCl、5mmol MgSO 4、10mmol ATP、10mmol GTP、10mmol乳糖、10mmol GDP-岩藻糖、40μg实施例4纯化的α-1,2-岩藻糖基转移酶和40μg实施例4纯化的GDP-岩藻糖焦磷酸化酶，于37℃过夜反应。根据乳糖与α-1,2-岩藻糖基化寡糖转化关系的标准曲线及反应体系中乳糖减少量测得α-1,2-岩藻糖基化寡糖的产率，测得所得α-1,2-岩藻糖基化寡糖的产率为82.76％。
而[Mattila P,Jarkko
Figure PCTCN2019120873-appb-000008
Hortling S,et al.Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae[J].Glycobiology,2000,10(10):1041-7.]在最优条件下的α-1,2-岩藻糖基转移酶催化产率提高了145.23％。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (10)

  1. 一种重组大肠杆菌系统，其特征在于，该重组大肠杆菌系统具有α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因。
  2. 根据权利要求1所述的重组大肠杆菌系统，其中，所述α-1,2-岩藻糖基转移酶基因来源于源自幽门螺杆菌，和/或所述GDP-岩藻糖焦磷酸化酶基因来源于脆弱拟杆菌；
    优选的，所述α-1,2-岩藻糖基转移酶基因具有如SEQ ID NO:1所示的核苷酸序列，和/或所述GDP-岩藻糖焦磷酸化酶基因具有如SEQ ID NO:2所示的核苷酸序列。
  3. 根据权利要求1或2所述的重组大肠杆菌系统，其中，用于构建所述大肠杆菌系统的大肠杆菌细胞为大肠杆菌C43细胞；
    优选的，所述α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因包括在同一大肠杆菌细胞中；或者
    所述α-1,2-岩藻糖基转移酶基因和GDP-岩藻糖焦磷酸化酶基因包括在不同大肠杆菌细胞中。
  4. 权利要求1-3中任意一项所述的重组大肠杆菌系统的构建方法，其特征在于，该方法包括：
    (1)克隆α-1,2-岩藻糖基转移酶基因，并构建所述α-1,2-岩藻糖基转移酶基因的表达载体；
    (2)克隆GDP-岩藻糖焦磷酸化酶基因，并构建所述GDP-岩藻糖焦磷酸化酶基因的表达载体；
    (3)将步骤(1)和步骤(2)获得的表达载体转化入大肠杆菌细胞中，得到所述重组大肠杆菌系统。
  5. 根据权利要求4所述的方法，其中，步骤(1)中，使用pCold I质粒构 建所述α-1,2-岩藻糖基转移酶基因的表达载体；
    优选的，所述α-1,2-岩藻糖基转移酶基因的表达载体的构建方法包括：
    (1)以克隆的α-1,2-岩藻糖基转移酶基因为模板，以SEQ ID NO:3所示的正向引物和SEQ ID NO:4所示的反向引物为引物进行PCR扩增；
    (2)将所得PCR扩增产物用BamHI和Hind III进行双酶切处理，并与BamHI和Hind III双酶切后的质粒相连接，得到所述α-1,2-岩藻糖基转移酶基因的表达载体。
  6. 根据权利要求4所述的方法，其中，步骤(2)中，使用pET28a质粒建所述GDP-岩藻糖焦磷酸化酶基因的表达载体；
    优选的，所述GDP-岩藻糖焦磷酸化酶基因的表达载体的构建方法包括：
    (1)以克隆的GDP-岩藻糖焦磷酸化酶基因为模板，以SEQ ID NO:5所示的正向引物和SEQ ID NO:6所示的反向引物为引物进行PCR扩增；
    (2)将所得PCR扩增产物用BamHI和XhoI进行双酶切处理，并与BamHI和XhoI双酶切后的质粒相连接，得到所述GDP-岩藻糖焦磷酸化酶基因的表达载体。
  7. 权利要求1-3中任意一项所述的重组大肠杆菌系统在合成α-1,2-岩藻糖基化寡糖中的应用。
  8. 一种α-1,2-岩藻糖基化寡糖的合成方法，其特征在于，该方法包括：
    (1)在大肠杆菌能够繁殖的条件下，使权利要求1-3中任意一项所述的重组大肠杆菌系统繁殖并表达所述α-1,2-岩藻糖基转移酶基因和所述GDP-岩藻糖焦磷酸化酶基因，获得α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶；
    (2)将步骤(1)获得的α-1,2-岩藻糖基转移酶和GDP-岩藻糖焦磷酸化酶与转化底物接触，合成所述α-1,2-岩藻糖基化寡糖。
  9. 根据权利要求8所述的方法，其中，所述转化底物含有GDP-岩藻糖和乳糖。
  10. 根据权利要求8所述的方法，其中，所述转化底物还含有Tris-HCl、MgSO 4、ATP和GTP；
    优选的，相对于每升的转化底物，GDP-岩藻糖的浓度为5-15mmol，乳糖的浓度为5-15mmol、Tris-HCl的浓度为15-25mmol，MgSO 4的浓度为4-6mmol，ATP的浓度为5-15mmol，GTP的浓度为5-15mmol，所述α-1,2-岩藻糖基转移酶的用量为30-50μg，所述GDP-岩藻糖焦磷酸化酶的用量为30-50μg。
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