WO2023216685A1 - 一种以葡萄糖为碳源合成2'-岩藻糖基乳糖的菌株及其构建方法和应用 - Google Patents

一种以葡萄糖为碳源合成2'-岩藻糖基乳糖的菌株及其构建方法和应用 Download PDF

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WO2023216685A1
WO2023216685A1 PCT/CN2023/079163 CN2023079163W WO2023216685A1 WO 2023216685 A1 WO2023216685 A1 WO 2023216685A1 CN 2023079163 W CN2023079163 W CN 2023079163W WO 2023216685 A1 WO2023216685 A1 WO 2023216685A1
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plasmid
lacy
ptrc99a
gene
fermentation
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French (fr)
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夏洪志
徐铮
储呈慧
李古月
牛堃
李江波
孙怡
杨陈亮
张建鸿
朱宇雷
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南通励成生物工程有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of bioengineering technology, and in particular relates to a strain that uses glucose as a carbon source to synthesize 2’-fucosyllactose and its construction method and application.
  • Human milk oligosaccharides are unique substances in human milk. More than 200 types have been discovered so far, most of which are composed of 3 to 6 sugar groups. Most human milk oligosaccharides contain L-fucose, such as 2'-fucosyllactose (referred to as 2'-FL), 6'-sialyllactose (6'-SL), lactose-N-neo sugar (LNnT), etc.; among them, the trisaccharide 2'-FL accounts for a high proportion, reaching 30% of the total amount of human milk oligosaccharides.
  • L-fucose such as 2'-fucosyllactose (referred to as 2'-FL), 6'-sialyllactose (6'-SL), lactose-N-neo sugar (LNnT), etc.
  • the trisaccharide 2'-FL accounts for a high proportion, reaching 30% of the total amount of human milk oligosacchari
  • 2'-FL has been proven to play a key role in promoting the maturation of the immune system of infants and young children, significantly reducing the incidence of illness in infants and young children; it can also improve the relationship between intestinal flora and promote cognitive development of the brain, which is a hot issue in the health industry. .
  • 2'-FL can be used by intestinal probiotics after consumption to effectively regulate intestinal flora; it can also inhibit the combination of Campylobacter and human intestinal mucosa to reduce diarrhea in infants and young children.
  • 2'-FL can indirectly inhibit the growth of pathogenic bacteria by improving the competitive advantage of non-pathogenic bacterial symbionts, and can directly act as an anti-adhesion antibacterial agent to reduce microbial infections, making infants who ingest 2'-FL less susceptible to disease. Otitis media caused by Streptococcus pneumoniae and Pseudomonas aeruginosa.
  • 2'-FL also plays a role in brain development, neuronal transmission and synapse formation, and can stimulate brain development, so adding 2'-FL to the diet can promote brain development and improve learning and memory abilities.
  • 2'-FL Due to the excellent physiological functions of 2'-FL for infants and young children, as well as the year-by-year decrease in breastfeeding rates in my country, 2'-FL has become an urgently needed new product in the food additive market.
  • chemical synthesis of 2'-FL requires many steps and low yield, which cannot meet the needs of large-scale use. Microbial fermentation has been proven to be highly feasible. Therefore, how to increase the production scale of 2′-FL and meet the cost requirements of industrial production of food additives is an urgent problem to be solved.
  • Reported production methods generally use glycerol as a fermentation carbon source to obtain higher 2′-FL yields through high-density fermentation and synthesis.
  • glycerol as a fermentation carbon source
  • it is inconvenient to measure the content of glycerol in real time during the fermentation process and it is also difficult to completely separate the residual glycerol from the product.
  • the production process using glucose as the carbon source is more convenient and the cost is lower than that of glycerol.
  • the glucose effect will cause the ⁇ -galactoside permease LacY gene on the lactose operon in the strain to be unable to be transcribed and expressed, which will block the entry of lactose into the cell. , causing 2′-FL to be unable to be synthesized.
  • This problem can be solved by overexpressing LacY from an independently operating promoter in the strain.
  • LacY is a membrane-bound protein, and overexpression will have a toxic effect on the strain, causing it to grow slowly or even die
  • the object of the present invention is to provide a strain that uses glucose as a carbon source to synthesize 2'-fucosyllactose and its construction method and application.
  • the strain provided by the invention is used to synthesize 2'-fucosyllactose using glucose as a carbon source.
  • -Fucosyllactose yield 3.2g/L.
  • the invention provides a strain that uses glucose as a carbon source to synthesize 2'-fucosyllactose, which is obtained by co-transforming the recombinant plasmids pACYCDuet-J23115-LacY and ptrc99a-ManCB-Gmdfcl-FucT into E. coli;
  • the recombinant plasmid pACYCDuet-J23115-LacY is composed of plasmid pACYCDuet-1 and LacY gene, and the T7 promoter in plasmid pACYCDuet-1 is replaced with the J23115 promoter;
  • the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT is obtained by connecting plasmid ptrc99a to the ManCB gene cluster, Gmdfcl gene cluster, FucT gene, the trc promoter of plasmid ptrc99a, and the terminator of plasmid ptrc99a.
  • nucleotide sequence of the LacY gene is shown in SEQ ID No. 1.
  • nucleotide sequence of the ManCB gene cluster is shown in SEQ ID No. 2;
  • the nucleotide sequence of the Gmdfcl gene cluster is shown in SEQ ID No. 3;
  • the nucleotide sequence of the FucT gene is shown in SEQ ID No. 4.
  • the nucleotide sequence of the trc promoter of the plasmid ptrc99a is shown in SEQ ID No. 5;
  • the nucleotide sequence of the terminator of the plasmid ptrc99a is shown in SEQ ID No. 6.
  • the mass ratio of the recombinant plasmid pACYCDuet-J23115-LacY and ptrc99a-ManCB-Gmdfcl-FucT is 1:1.
  • the Escherichia coli includes Escherichia coli JM109(DE3).
  • the present invention also provides a method for constructing the bacterial strain described in the above technical solution, which includes the following steps:
  • step 2) Connect the linear plasmid obtained in step 1) to the LacY gene to obtain the recombinant plasmid pACYCDuet-LacY.
  • step 2) Combine the gene sequence that does not contain the T7 promoter in step 2) with the J23115 promoter and catalyze it with BsaI and T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-J23115-LacY;
  • the plasmid ptrc99a, the ManCB gene cluster, the Gmdfcl gene cluster, the FucT gene, the trc promoter of the plasmid ptrc99a, and the terminator of the plasmid ptrc99a are catalyzed by BsaI and T4 DNA ligase to obtain the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT;
  • the present invention also provides the application of the bacterial strain described in the above technical solution in the synthesis of 2'-fucosyllactose using glucose as a carbon source.
  • the application includes: inoculating the strain into a fermentation medium for fermentation.
  • the OD 600 of the fermentation broth is 1, adding a final concentration of 0.05-0.2mM IPTG and 5g/L lactose, and continuing the fermentation to obtain 2'-Fucosyllactose.
  • the glucose content in the fermentation medium is 15g/L.
  • the fermentation conditions include: the fermentation temperature is 37°C, and the fermentation speed is 220 rpm;
  • the conditions for continued fermentation include: the temperature for continued fermentation is 25°C, the rotation speed for continued fermentation is 220 rpm, and the time for continued fermentation is 60 hours.
  • the mechanism of the present invention to synthesize 2’-fucosyllactose using glucose as the carbon source is:
  • the present invention overexpresses the key enzyme genes ManCB, Gmdfcl, and FucT for the synthesis of 2'-fucosyllactose.
  • glucose is ingested by the strain, it is successively converted into glucose-6-phosphate and fructose-6-phosphate through the strain's own metabolic system.
  • mannose-6-phosphate mannose-6-phosphate is converted into GDP-mannose through the action of ManCB enzyme system
  • GDP-mannose is converted into GDP-L-fucose through the action of Gmdfcl enzyme system.
  • lactose transporter using a constitutive promoter Protein LacY, lactose can be transported into the strain in the presence of glucose, react with GDP-L-fucose under the action of FucT enzyme, and finally generate 2'-fucosyllactose.
  • the JM109(DE3)J23115 strain can ferment high-yield 2'-FL under the condition of glucose as carbon source, with a yield of 3.2g/L, which is only slightly lower than that of glycerol as a carbon source (yield 3.5g/L).
  • the control strain JM109(DE3)V0 which does not overexpress the LacY gene, cannot ferment and synthesize 2'-FL under the condition of glucose as a carbon source.
  • Figure 1 is a schematic diagram of the recombinant plasmids pACYCDuet-J23115-LacY and ptrc99a-ManCB-Gmdfcl-FucT.
  • the invention provides a strain that uses glucose as a carbon source to synthesize 2'-fucosyllactose, which is obtained by co-transforming the recombinant plasmids pACYCDuet-J23115-LacY and ptrc99a-ManCB-Gmdfcl-FucT into E. coli;
  • the recombinant plasmid pACYCDuet-J23115-LacY is composed of plasmid pACYCDuet-1 and LacY gene, and the T7 promoter in plasmid pACYCDuet-1 is replaced with the J23115 promoter;
  • the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT is obtained by connecting plasmid ptrc99a to the ManCB gene cluster, Gmdfcl gene cluster, FucT gene, the trc promoter of plasmid ptrc99a, and the terminator of plasmid ptrc99a.
  • the plasmid pACYCDuet-1 is commercially available, Addgene order number: 71147.
  • nucleotide sequence of the LacY gene is shown in SEQ ID No. 1, specifically as follows:
  • the nucleotide sequence of the ManCB gene cluster is shown in SEQ ID No. 2.
  • the function of the ManCB gene cluster is to encode the ManCB enzyme system and catalyze the conversion of mannose-6-phosphate into GDP-mannose.
  • the specific nucleotide sequence of the ManCB gene cluster is as follows:
  • the nucleotide sequence of the Gmdfcl gene cluster is shown in SEQ ID No. 3.
  • the function of the Gmdfcl gene cluster is to encode the Gmdfcl enzyme system and catalyze GDP-mannose to GDP-L-fucose.
  • the nucleotide sequence of the Gmdfcl gene cluster is as follows:
  • the nucleotide sequence of the FucT gene is shown in SEQ ID No. 4.
  • the function of the FucT gene is to encode FucT enzyme, which uses GDP-L-fucose and lactose as substrates to synthesize 2' -Fucosyllactose.
  • the nucleotide sequence of the FucT gene is as follows:
  • nucleotide sequence of the trc promoter of the plasmid ptrc99a is shown in SEQ ID No. 5, specifically as follows:
  • nucleotide sequence of the terminator of plasmid ptrc99a is shown in SEQ ID No. 6, specifically as follows:
  • nucleotide sequence of the plasmid ptrc99a is shown in SEQ ID No. 48, specifically as follows:
  • the nucleotide sequence of the J23115 promoter is shown in SEQ ID No. 45.
  • the function of the J23115 promoter is to transcribe the lactose transporter LacY with appropriate intensity.
  • the specific nucleotide sequence of the J23115 promoter is as follows:
  • the mass ratio of the recombinant plasmid pACYCDuet-J23115-LacY and ptrc99a-ManCB-Gmdfcl-FucT is preferably 1:1.
  • the Escherichia coli preferably includes Escherichia coli JM109 (DE3).
  • the invention has no special limitation on the source of Escherichia coli JM109 (DE3), and commercially available products can be used.
  • the present invention also provides a method for constructing the bacterial strain described in the above technical solution, which includes the following steps:
  • step 2) Connect the linear plasmid obtained in step 1) to the LacY gene to obtain the recombinant plasmid pACYCDuet-LacY.
  • step 2) Combine the gene sequence that does not contain the T7 promoter in step 2) with the J23115 promoter and catalyze it with BsaI and T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-J23115-LacY;
  • the plasmid ptrc99a, the ManCB gene cluster, the Gmdfcl gene cluster, the FucT gene, the trc promoter of the plasmid ptrc99a, and the terminator of the plasmid ptrc99a are catalyzed by BsaI and T4 DNA ligase to obtain the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT;
  • plasmid pACYCDuet-1 is double digested by restriction endonuclease NdeI and EcoRV to obtain linear plasmid.
  • the double enzyme digestion reaction system is preferably 100 ⁇ l: NdeI 3 ⁇ l, EcoRV 3 ⁇ l, pACYCDuet-1 vector 500 ng, 10 ⁇ buffer buffer 10 ⁇ l, and the rest is made up with double distilled water.
  • the reaction conditions are preferably 37°C water bath reaction for 8 hours.
  • the obtained linear plasmid is connected to the LacY gene to obtain the recombinant plasmid pACYCDuet-LacY.
  • the recombinant plasmid pACYCDuet-LacY is used as a template and is amplified by PCR to obtain a gene sequence that does not contain the T7 promoter.
  • the present invention has no special limitation on the acquisition method of the LacY gene, and those skilled in the art can obtain it according to routine operations.
  • the present invention has no special limitations on the connection conditions, and those skilled in the art can perform routine operations.
  • the present invention has no special limitations on PCR amplification of gene sequences that do not contain the T7 promoter, and those skilled in the art can follow routine operations.
  • the gene sequence that does not contain the T7 promoter and the J23115 promoter are catalyzed by BsaI and T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-J23115-LacY.
  • the conditions of the catalytic reaction preferably include: 37°C for 1 min, 16°C for 1 min; a total of 30 cycles; and then 60°C for 5 min.
  • 20 ⁇ L of the catalyzed system preferably includes 100 ng of the aforementioned amplified pACYCDuet-LacY DNA, 50 ng of J23115 promoter DNA fragment, 2 ⁇ L of 10 ⁇ Tango buffer, 10 mM ATP, 0.5 ⁇ L of T4 DNA ligase, and double distilled water. Bring to 20 ⁇ L.
  • plasmid ptrc99a, ManCB gene cluster, Gmdfcl gene cluster, FucT gene, trc promoter of plasmid ptrc99a, and terminator of plasmid ptrc99a are catalyzed by BsaI and T4 DNA ligase to obtain the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT.
  • the present invention has no special limitation on the acquisition method of the above genes, and those skilled in the art can obtain them according to routine.
  • the conditions of the catalytic reaction preferably include: 37°C for 1 min, 16°C for 1 min; a total of 30 cycles; and then 60°C for 5 min.
  • the catalyzed system preferably includes 100 ng of the amplified ptrc99a DNA, 100 ng of the ManCB gene cluster fragment, 100 ng of the Gmdfcl gene cluster fragment, 100 ng of the FucT gene fragment, 50 ng of the trc promoter fragment of the plasmid ptrc99a, and 50 ng of the trc promoter fragment of the plasmid ptrc99a.
  • the obtained recombinant plasmid pACYCDuet-J23115-LacY and the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT obtained in step 4) are co-transformed into E. coli to obtain a strain.
  • the mass ratio of the recombinant plasmid pACYCDuet-J23115-LacY and the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT is preferably 1:1.
  • the present invention has no special restrictions on other conditions for transferring the recombinant plasmid pACYCDuet-J23115-LacY and the recombinant plasmid ptrc99a-ManCB-Gmdfcl-FucT into E. coli, and those skilled in the art can follow routine operations.
  • the present invention also provides the application of the bacterial strain described in the above technical solution in the synthesis of 2'-fucosyllactose using glucose as a carbon source.
  • the application preferably includes: inoculating the bacterial strain into a fermentation medium for fermentation.
  • the OD 600 of the fermentation broth is 1, add IPTG with a final concentration of 0.05 to 0.2mM and 5g/L lactose, and continue fermentation to obtain 2'-fucosyllactose.
  • the content of glucose in the fermentation medium is preferably 15 g/L.
  • the fermentation medium preferably includes: 15g/L glucose, 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 25mg/L streptomycin and 34mg/L chloramphenicol.
  • the inoculum amount of the strain is preferably 1%.
  • the fermentation conditions preferably include: the fermentation temperature is 37°C, and the fermentation speed is 220 rpm.
  • the IPTG functions as an inducer, inducing the overexpression of related genes, and the lactose functions as a substrate for synthesizing 2'-fucosyllactose.
  • the conditions for continued fermentation preferably include: the temperature for continued fermentation is 25°C, the rotation speed for continued fermentation is 220 rpm, and the time for continued fermentation is 60 hours.
  • the pACYCDuet-1 vector was double-digested by the restriction enzymes NdeI (Takara Company, Catalog No.: 1161A) and EcoRV (Takara Company, Catalog No.: 1042A).
  • the reaction system was 100 ⁇ l: NdeI 3 ⁇ l, EcoRV 3 ⁇ l, 500 nanograms of pACYCDuet-1 vector, 10 microliters of 10 ⁇ buffer, and the rest are made up with water; the reaction conditions are 37°C water bath reaction for 8 hours, and the enzyme digestion product is purified and recovered using a gel recovery kit (Takara Company's MiniBEST Agarose Gel DNA Extraction Kit, item number: 9762).
  • the LacY gene was obtained by PCR.
  • the template was the genome of E.coli DH5 ⁇ strain (Nanjing Novezan Biotechnology Co., Ltd., Cat. No.: C502-03).
  • the primers were LacY-S and LacY-A.
  • the PCR system was 50 ⁇ l: 0.5 ⁇ l of Ex Taq enzyme (Takara Company, Cat. No.: RR001A), 5 ⁇ l of 10 ⁇ buffer, 2 ⁇ l of forward primer, 2 ⁇ l of reverse primer, and 1 ⁇ l of E. coli DH5 ⁇ strain genomic DNA.
  • the PCR amplification program was: pre-denaturation at 95°C for 1 minute, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute and 30 seconds, and final extension at 72°C for 1 minute.
  • the PCR product was purified and recovered using a gel recovery kit (Takara Company, Cat. No.: 9762).
  • Nanjing Novozan Biotechnology Co., Ltd. II One Step Cloning Kit (Cat. No.: C112-01) recombinantly connects the double-digested linearized pACYCDuet vector with the LacY gene DNA (see the product manual for the connection system and reaction conditions), and transforms E. coli DH5 ⁇ competent cells (Nanjing Novozan Biotechnology Co., Ltd., product number: C502-03), LB plate (containing 34 mg/L chloramphenicol) after coating at 37°C Incubate overnight. The clones obtained were picked and cultured in LB liquid medium (containing 34 mg/L chloramphenicol) for 12 hours.
  • LB liquid medium containing 34 mg/L chloramphenicol
  • the bacterial liquid was verified by PCR (the PCR procedure was the same as above, and the primers were LacY-S and LacY-A) to see if it was a positive clone. Positive clones can obtain amplified DNA bands with a size of approximately 1200 bp.
  • the recombinant plasmid pACYCDuet-LacY of the positive clone was extracted using a plasmid extraction kit (Nanjing Novozan Biotechnology Co., Ltd., Cat. No.: DC201-01).
  • a plasmid extraction kit Najing Novozan Biotechnology Co., Ltd., Cat. No.: DC201-01.
  • Using the pACYCDuet-LacY recombinant plasmid as a template obtain a linear DNA fragment without the T7 promoter through PCR and install an Overhang adapter suitable for Golden gate ligation (the PCR procedure is the same as above, and the primers are shown in Table 1).
  • the PCR system is 50 ⁇ l: Ex Taq enzyme (Takara Company, Cat.
  • RR001A 0.5 ⁇ l, 10 ⁇ buffer buffer 5 ⁇ l, primer pACYCDuet-LacY-S 2 ⁇ l, primer pACYCDuet-LacY-A 2 ⁇ l , pACYCDuet-LacY recombinant plasmid DNA 1 ⁇ l.
  • the PCR amplification program was: pre-denaturation at 95°C for 1 minute, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 5 minutes, and final extension at 72°C for 1 minute.
  • a variety of constitutive promoters were obtained through annealing and polymerization, including J23100, J23101, J23106, J23105, J23115, J23109, and J23113, in order to select the most suitable promoter.
  • Mix the promoter fragment with the pACYCDuet-LacY linearized DNA fragment add BsaI enzyme (New England Biolabs Company, Catalog No.: R3733L) and T4 DNA ligase (New England Biolabs Company, Catalog No.: M0202T) for catalysis; 20 ⁇ L of the catalyzed system includes 100ng of the amplified pACYCDuet-LacY DNA, 50ng of promoter DNA fragment, 2 ⁇ L of 10 ⁇ Tango buffer, 10mM ATP, 0.5 ⁇ L of T4 DNA ligase, and double-distilled water were added to 20 ⁇ L.
  • a PCR instrument can be used for catalysis.
  • the reaction conditions are: (37°C for 1 minute, 16°C for 1 minute) ⁇ 30 cycles, and 60°C for another 5 minutes. Then take 10 microliters of the reaction solution to transform Escherichia coli DH5 ⁇ competent cells (Nanjing Novezan Biotechnology Co., Ltd., Cat. No.: C502-03), spread it on an LB plate (containing 34 mg/L chloramphenicol) and culture it at 37°C overnight. . Whether the obtained clone is correct is determined by the corresponding promoter fragment by PCR. If a DNA fragment of approximately 40 bp is obtained by PCR, it is a positive clone.
  • the linearized fragment of ptrc99a plasmid was obtained by PCR (primers were ptrc99a-S and ptrc99a-A) and installed with an Overhang adapter suitable for Golden gate ligation.
  • the ManCB gene cluster was obtained and installed by PCR (primers were ManCB-S and ManCB-A).
  • PCR primers are Gmdfcl-S and Gmdfcl-A
  • Gmdfcl-S and Gmdfcl-A to obtain the Gmdfcl gene cluster
  • PCR primers are FucT-S and FucT-A
  • FucT gene is synthesized by Universal Biosystems (Anhui) Co., Ltd.
  • the reaction system is: 100ng of ptrc99a linearized fragment, 100ng of ManCB gene cluster fragment, and Gmdfcl gene cluster fragment. 100ng, FucT gene fragment 100ng, trc promoter fragment of plasmid ptrc99a 50ng, terminator fragment of plasmid ptrc99a 50ng, 10 ⁇ Tango buffer 2 ⁇ L, ATP 10mM, T4 DNA ligase 0.5 ⁇ L, double distilled water to make up to 20 ⁇ L
  • the reaction conditions were: (37°C for 1 minute, 16°C for 1 minute) ⁇ 30 cycles, and then 60°C for 5 minutes.
  • the reaction product was transformed into Escherichia coli DH5 ⁇ competent cells (Nanjing Novozan Biotechnology Co., Ltd., Cat. No.: C502-03), spread on LB plates containing 50 mg/L ampicillin, and culture at 37°C overnight. After extracting the plasmid from the clone, verify whether it is correct by enzyme digestion: Use EcoNI enzyme (New England Biolabs, Cat. No.: #R0521S) for 1 hour at 37°C and run on a nucleic acid electrophoresis gel.
  • the enzyme digestion reaction system is 10 ⁇ l: EcoNI enzyme 0.5 ⁇ l, 5 ⁇ l of plasmid, 1 ⁇ l of 10 ⁇ buffer, and the rest of the water.
  • the size of the digested product is approximately 10,000 to 11,000 bp, which is considered correct, and the corresponding clone is a positive clone.
  • the specific method for HPLC determination of 2'-FL concentration in fermentation broth is as follows: liquid phase equipment: Agilent 1260 Infinity II; differential detector detection, model: G7162A-1260 RId; liquid phase column model: Sepax HP-Amino, 4.6*250mm, 5 micron particle size (or equivalent amino column); flow rate: 0.8ml/min, mobile phase: 80% pure acetonitrile: 20% water (v/v), system temperature: 35°C, injection volume 10 ⁇ 20 ⁇ l .
  • the 2'-FL standard used to calculate the concentration was purchased from Shanghai Huicheng Biotechnology Co., Ltd. with a purity of 98% (Cat. No.: GY1141).
  • the sequence number of the J23100 promoter is SEQ ID No.
  • sequence number of the J23101 promoter is SEQ ID No. 42
  • sequence number of the J23106 promoter is SEQ ID No. 43
  • sequence number of the J23105 promoter is SEQ ID No. 44.
  • sequence number of the J23115 promoter is SEQ ID No. 45
  • sequence number of the J23109 promoter is SEQ ID No. 46
  • sequence number of the J23113 promoter is SEQ ID No. 47.
  • JM109(DE3)J23115 dual plasmid strain has an OD 600 value of 15.8 when using glycerol as the carbon source, but the yield of 2′-FL is only 0.8g/L, and the OD 600 value is 14.2 when using glucose as the carbon source. But no product (0g/L) was detected.

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Abstract

提供了一种以葡萄糖为碳源合成2'-岩藻糖基乳糖的菌株及其构建方法和应用，属于生物工程技术领域。所述菌株将重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌获得。采用提供的菌株以葡萄糖为碳源来合成2'-岩藻糖基乳糖，产量为3.2g/L。

Description

一种以葡萄糖为碳源合成2’-岩藻糖基乳糖的菌株及其构建方法和应用 技术领域
本发明属于生物工程技术领域，尤其涉及一种以葡萄糖为碳源合成2’-岩藻糖基乳糖的菌株及其构建方法和应用。
背景技术
人乳寡糖(human milk oligosaccharides，简称HMO)是人类乳汁中独有的物质，迄今已发现了200多种，多由3～6个糖基组成。大部分人乳寡糖都含有L-岩藻糖，例如2'-岩藻糖基乳糖(简称2'-FL)、6'-唾液酸乳糖(6'-SL)、乳糖-N-新四糖(LNnT)等；其中三糖2'-FL的占比很高，达人乳寡糖总量的30％。2'-FL已被证明对婴幼儿免疫系统成熟起到关键的促进作用，显著降低婴幼儿得病率；还能改善肠道菌群关系、促进大脑认知发育，是大健康产业关注的热点问题。
近期研究表明，2'-FL食用后可被肠道益生菌利用，有效调节肠道菌群；还可抑制弯曲杆菌与人肠粘膜的结合来减少婴幼儿腹泻，数据表明：2'-FL可使空肠弯曲杆菌对人体的侵袭能力降低80％，从而抑制肠道粘膜促炎因子和信号的释放，并减少婴儿由空肠弯曲杆菌引发的腹泻；2'-FL还可以通过调节人肠道上皮细胞CD14的表达来减轻炎症。所以在奶粉中添加适量2'-FL可以增强新生婴儿的免疫力，有效增强婴儿体质。2'-FL可以通过提高非致病菌共生体的竞争优势来间接抑制致病菌的生长，并能直接充当抗黏附抗菌剂减少微生物感染，使摄入2'-FL的婴儿不容易患由肺炎链球菌、绿脓杆菌引起的中耳炎。此外，2'-FL在大脑发育、神经元传递和突触的形成中也起作用，能够刺激大脑发育，所以在饮食中添加2'-FL可以促进大脑发育并且能够改善学习和记忆能力。由于2'-FL针对婴幼儿的卓越生理功能，以及我国母乳喂养率的逐年下降，导致2'-FL成为食品添加剂市场上急需的新产品。然而牛奶、羊奶中都不含有这种物质，所以2'-FL只能通过人工合成来大量获得。在技术手段上，化学合成2'-FL的步骤多、产量低，不能满足规模化使用的需求。微生物发酵法已被证明具有很强的可行性，因而如何提高2′-FL的生产规模、满足工业化生产食品添加剂的成本要求，是亟待解决的问题。
已报道的生产方法一般使用甘油作为发酵碳源，通过高密度发酵及合成来获得较高的2′-FL产量。然而甘油在发酵过程中不便于实时测定其含量，残留的甘油也较难与产物完全分离。使用葡萄糖为碳源生产工艺更为便利，成本也低于甘油，但葡萄糖效应会导致菌株体内乳糖操纵子上的β-半乳糖苷透性酶LacY基因无法转录表达，这将阻断乳糖进入细胞，导致2′-FL无法合成。将LacY在菌株体内由独立运作的启动子来过表达可以解决这一问题，然而LacY属于膜结合蛋白，过量表达会对菌株产生毒害作用，造成其生长缓慢甚至死亡。
发明内容
有鉴于此，本发明的目的在于提供一种以葡萄糖为碳源合成2’-岩藻糖基乳糖的菌株及其构建方法和应用，采用本发明提供的菌株以葡萄糖为碳源来合成2’-岩藻糖基乳糖，产量为3.2g/L。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种以葡萄糖为碳源合成2’-岩藻糖基乳糖的菌株，将重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌获得；
所述重组质粒pACYCDuet-J23115-LacY是将质粒pACYCDuet-1与LacY基因连接，并将质粒pACYCDuet-1中的T7启动子替换为J23115启动子；
所述重组质粒ptrc99a-ManCB-Gmdfcl-FucT是将质粒ptrc99a与ManCB基因簇、Gmdfcl基因簇、FucT基因、质粒ptrc99a的trc启动子、质粒ptrc99a的终止子连接后得到。
优选的，所述LacY基因的核苷酸序列如SEQ ID No.1所示。
优选的，所述ManCB基因簇的核苷酸序列如SEQ ID No.2所示；
所述Gmdfcl基因簇的核苷酸序列如SEQ ID No.3所示；
所述FucT基因的核苷酸序列如SEQ ID No.4所示；
所述质粒ptrc99a的trc启动子的核苷酸序列如SEQ ID No.5所示；
所述质粒ptrc99a的终止子的核苷酸序列如SEQ ID No.6所示。
优选的，所述重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT的质量比为1:1。
优选的，所述大肠杆菌包括大肠杆菌JM109(DE3)。
本发明还提供了上述技术方案所述的菌株的构建方法，包括以下步骤：
1)将质粒pACYCDuet-1经限制性内切酶NdeI和EcoRV双酶切，得到线性质粒；
2)将所述步骤1)得到的线性质粒与LacY基因连接，得到重组质粒pACYCDuet-LacY，以所述重组质粒pACYCDuet-LacY为模板，经PCR扩增，得到不含有T7启动子的基因序列；
3)将所述步骤2)不含有T7启动子的基因序列与J23115启动子经BsaI和T4DNA连接酶催化，得到重组质粒pACYCDuet-J23115-LacY；
4)将质粒ptrc99a与ManCB基因簇、Gmdfcl基因簇、FucT基因、质粒ptrc99a的trc启动子、质粒ptrc99a的终止子经BsaI和T4DNA连接酶催化，得到重组质粒ptrc99a-ManCB-Gmdfcl-FucT；
5)将所述步骤3)得到的重组质粒pACYCDuet-J23115-LacY与步骤4)得到的重组质粒ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌中，得到菌株。
本发明还提供了上述技术方案所述的菌株在以葡萄糖为碳源合成2’-岩藻糖基乳糖中的应用。
优选的，所述应用包括：将所述菌株接种到发酵培养基中进行发酵，当发酵液的OD600为1时，加入终浓度为0.05～0.2mM IPTG和5g/L乳糖，继续发酵，得到2’-岩藻糖基乳糖。
优选的，所述发酵培养基中葡萄糖的含量为15g/L。
优选的，所述发酵的条件包括：发酵的温度为37℃，发酵的转速为220rpm；
所述继续发酵的条件包括：继续发酵的温度为25℃，继续发酵的转速为220rpm，继续发酵的时间为60h。
本发明以葡萄糖为碳源合成2’-岩藻糖基乳糖的机理为：
本发明过表达了2’-岩藻糖基乳糖合成的关键酶基因ManCB、Gmdfcl、以及FucT，葡萄糖被菌株摄入后经过菌株自身代谢系统相继转化为葡萄糖-6-磷酸、果糖-6-磷酸、和甘露糖-6-磷酸，甘露糖-6-磷酸通过ManCB酶系作用转化为GDP-甘露糖，GDP-甘露糖经过Gmdfcl酶系作用转化为GDP-L-岩藻糖。由于使用组成型启动子过表达了乳糖转运 蛋白LacY，乳糖能够在葡萄糖存在的环境下被转运进菌株体内，在FucT酶的作用下和GDP-L-岩藻糖发生反应，最终生成2’-岩藻糖基乳糖。
本发明的有益效果：
JM109(DE3)J23115菌株可以在葡萄糖为碳源的条件下发酵高产2′-FL，产量为3.2g/L，产量仅略低于以甘油为碳源的条件(产量3.5g/L)。而没有过表达LacY基因的对照菌株JM109(DE3)V0，则无法在葡萄糖为碳源的条件下发酵合成2′-FL。
附图说明
图1为重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT示意图。
具体实施方式
本发明提供了一种以葡萄糖为碳源合成2’-岩藻糖基乳糖的菌株，将重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌获得；
所述重组质粒pACYCDuet-J23115-LacY是将质粒pACYCDuet-1与LacY基因连接，并将质粒pACYCDuet-1中的T7启动子替换为J23115启动子；
所述重组质粒ptrc99a-ManCB-Gmdfcl-FucT是将质粒ptrc99a与ManCB基因簇、Gmdfcl基因簇、FucT基因、质粒ptrc99a的trc启动子、质粒ptrc99a的终止子连接后得到。
在本发明中，所述质粒pACYCDuet-1为市售，Addgene订购号：71147。
在本发明中，所述LacY基因的核苷酸序列如SEQ ID No.1所示，具体如下：

在本发明中，所述ManCB基因簇的核苷酸序列如SEQ ID No.2所示，所述ManCB基因簇的作用是编码ManCB酶系，催化甘露糖-6-磷酸为GDP-甘露糖。所述ManCB基因簇的的核苷酸序列具体如下：

在本发明中，所述Gmdfcl基因簇的核苷酸序列如SEQ ID No.3所示，所述Gmdfcl基因簇的作用是编码Gmdfcl酶系，催化GDP-甘露糖为GDP-L-岩藻糖。在本发明中，所述Gmdfcl基因簇的核苷酸序列具体如下：

在本发明中，所述FucT基因的核苷酸序列如SEQ ID No.4所示，所述FucT基因的作用是编码FucT酶，以GDP-L-岩藻糖和乳糖为底物合成2’-岩藻糖基乳糖。在本发明中，所述FucT基因的核苷酸序列具体如下：

在本发明中，所述质粒ptrc99a的trc启动子的核苷酸序列如SEQ ID No.5所示，具体如下：
在本发明中，所述质粒ptrc99a的终止子的核苷酸序列如SEQ ID No.6所示，具体如下：
在本发明中，所述质粒ptrc99a的核苷酸序列如SEQ ID No.48所示，具体如下：


在本发明中，所述J23115启动子的核苷酸序列如SEQ ID No.45所示，所述J23115启动子的作用是以合适的强度转录乳糖转运蛋白LacY。所述J23115启动子的核苷酸序列具体如下：
在本发明中，所述重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT的质量比优选为1:1。在本发明中，所述大肠杆菌优选包括大肠杆菌JM109(DE3)，本发明对所述大肠杆菌JM109(DE3)的来源没有特殊限定，采用市售商品即可。
本发明还提供了上述技术方案所述的菌株的构建方法，包括以下步骤：
1)将质粒pACYCDuet-1经限制性内切酶NdeI和EcoRV双酶切，得到线性质粒；
2)将所述步骤1)得到的线性质粒与LacY基因连接，得到重组质粒pACYCDuet-LacY，以所述重组质粒pACYCDuet-LacY为模板，经PCR扩增，得到不含有T7启动子的基因序列；
3)将所述步骤2)不含有T7启动子的基因序列与J23115启动子经BsaI和T4DNA连接酶催化，得到重组质粒pACYCDuet-J23115-LacY；
4)将质粒ptrc99a与ManCB基因簇、Gmdfcl基因簇、FucT基因、质粒ptrc99a的trc启动子、质粒ptrc99a的终止子经BsaI和T4DNA连接酶催化，得到重组质粒ptrc99a-ManCB-Gmdfcl-FucT；
5)将所述步骤3)得到的重组质粒pACYCDuet-J23115-LacY与步骤4)得到的重组质粒ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌中，得到菌株。
本发明将质粒pACYCDuet-1经限制性内切酶NdeI和EcoRV双酶切，得到线性质粒。在本发明中，所述双酶切的反应体系优选为100μl：NdeI 3μl、EcoRV 3μl、pACYCDuet-1载体500ng、10×缓冲液buffer 10μl、其余用双蒸水补齐。在本发明中，反应条件优选为37℃水浴反应8h。
本发明将得到的线性质粒与LacY基因连接，得到重组质粒pACYCDuet-LacY，以所述重组质粒pACYCDuet-LacY为模板，经PCR扩增，得到不含有T7启动子的基因序列。本发明对所述LacY基因的获取方式没有特殊限定，本领域技术人员根据常规操作获取即可。本发明对所述连接的条件没有特殊限定，本领域技术人员依据常规操作即可。本发明对PCR扩增不含有T7启动子的基因序列没有特殊限定，本领域技术人员依据常规操作即可。
本发明将不含有T7启动子的基因序列与J23115启动子经BsaI和T4DNA连接酶催化，得到重组质粒pACYCDuet-J23115-LacY。在本发明中，所述催化反应的条件优选包括：37℃ 1min，16℃ 1min；共30个循环；再60℃ 5min。在本发明中，所述催化的体系20μL优选包括前述扩增出的pACYCDuet-LacY DNA 100ng，J23115启动子DNA片段50ng，10×Tango buffer 2μL，ATP 10mM，T4DNA连接酶0.5μL，双蒸水补齐至20μL。
本发明将质粒ptrc99a与ManCB基因簇、Gmdfcl基因簇、FucT基因、质粒ptrc99a的trc启动子、质粒ptrc99a的终止子经BsaI和T4DNA连接酶催化，得到重组质粒ptrc99a-ManCB-Gmdfcl-FucT。本发明对上述基因的获取方式没有特殊限定，本领域技术人员根据常规获取即可。在本发明中，所述催化反应的条件优选包括：37℃1min，16℃1min；共30个循环；再60℃5min。在本发明中，所述催化的体系优选包括前述扩增出的ptrc99a DNA 100ng，ManCB基因簇片段100ng，Gmdfcl基因簇片段100ng，FucT基因片段100ng，质粒ptrc99a的trc启动子片段50ng，质粒ptrc99a的终止子片段50ng，10×Tango buffer 2μL，ATP 10mM，T4DNA连接酶0.5μL，双蒸水补齐至20μL。
本发明将得到的重组质粒pACYCDuet-J23115-LacY与步骤4)得到的重组质粒ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌中，得到菌株。在本发明中，所述重组质粒pACYCDuet-J23115-LacY与重组质粒ptrc99a-ManCB-Gmdfcl-FucT的质量比优选为1:1。本发明对将重组质粒pACYCDuet-J23115-LacY与重组质粒ptrc99a-ManCB-Gmdfcl-FucT转入大肠杆菌的其它条件没有特殊限定，本领域技术人员根据常规操作即可。
本发明还提供了上述技术方案所述的菌株在以葡萄糖为碳源合成2’-岩藻糖基乳糖中的应用。
在本发明中，所述应用优选包括：将所述菌株接种到发酵培养基中进行发酵，当发 酵液的OD600为1时，加入终浓度为0.05～0.2mM IPTG和5g/L乳糖，继续发酵，得到2’-岩藻糖基乳糖。
在本发明中，所述发酵培养基中葡萄糖的含量优选为15g/L。在本发明中，所述发酵培养基优选包括：15g/L葡萄糖、10g/L蛋白胨、5g/L酵母粉、10g/L氯化钠、链霉素25mg/L和氯霉素34mg/L。在本发明中，所述菌株的接种量优选为1％。在本发明中，所述发酵的条件优选包括：发酵的温度为37℃，发酵的转速为220rpm。
在本发明中，所述IPTG的作用是诱导剂，诱导相关基因过表达，所述乳糖的作用是合成2’-岩藻糖基乳糖的底物。在本发明中，所述所述继续发酵的条件优选包括：继续发酵的温度为25℃，继续发酵的转速为220rpm，继续发酵的时间为60h。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
1)构建携带组成型启动子的pACYCDuet-LacY重组质粒
通过限制性内切酶NdeI(Takara公司，货号：1161A)和EcoRV(Takara公司，货号：1042A)双酶切pACYCDuet-1载体，反应体系为100微升：NdeI 3微升、EcoRV 3微升、pACYCDuet-1载体500纳克、10×缓冲液buffer 10微升、其余用水补齐；反应条件为37℃水浴反应8小时，酶切产物使用胶回收试剂盒纯化回收(Takara公司MiniBEST Agarose Gel DNA Extraction Kit，货号：9762)。
通过PCR获得LacY基因，模板为E.coli DH5ɑ菌株(南京诺唯赞生物科技股份有限公司，货号：C502-03)的基因组，引物为LacY-S和LacY-A，PCR体系为50微升：Ex Taq酶(Takara公司，货号：RR001A)0.5微升、10×缓冲液buffer 5微升、正向引物2微升、反向引物2微升、大肠杆菌DH5ɑ菌株基因组DNA 1微升。PCR扩增程序为：95℃预变性1分钟，30个循环的95℃变性30秒、55℃退火30秒、72℃延伸1分30秒，72℃终延伸1分钟。PCR产物使用胶回收试剂盒纯化回收(Takara公司，货号：9762)。
然后使用南京诺唯赞生物科技股份有限公司的II One Step Cloning Kit试剂盒(货号：C112-01)将双酶切线性化的pACYCDuet载体与LacY基因DNA进行重组连接(连接体系和反应条件见产品说明书)，转化大肠杆菌DH5ɑ感受态细胞(南京诺唯赞生物科技股份有限公司，货号：C502-03)，LB平板(含34mg/L氯霉素)涂布后37℃ 过夜培养。所得克隆挑取后在LB液体培养基(含34mg/L氯霉素)中培养12小时，将菌液通过PCR验证(PCR程序同上，引物为LacY-S和LacY-A)是否为阳性克隆，阳性克隆可以得到大小约1200bp的扩增DNA条带。
使用质粒提取试剂盒(南京诺唯赞生物科技股份有限公司，货号：DC201-01)提取阳性克隆的重组质粒pACYCDuet-LacY。以pACYCDuet-LacY重组质粒为模板，通过PCR获得不含T7启动子的线性DNA片段并装上适合于Golden gate连接的Overhang接头(PCR程序同上，引物见表1)。PCR体系为50微升：Ex Taq酶(Takara公司，货号：RR001A)0.5微升、10×缓冲液buffer 5微升、引物pACYCDuet-LacY-S 2微升、引物pACYCDuet-LacY-A 2微升、pACYCDuet-LacY重组质粒DNA 1微升。PCR扩增程序为：95℃预变性1分钟，30个循环的95℃变性30秒、55℃退火30秒、72℃延伸5分钟，72℃终延伸1分钟。
将多种组成型启动子通过退火聚合分别获得，包括J23100、J23101、J23106、J23105、J23115、J23109、J23113，以便于从中挑选出最适合的启动子。将启动子片段与pACYCDuet-LacY线性化DNA片段混合，加入BsaI酶(New England Biolabs公司，货号：R3733L)和T4DNA连接酶(New England Biolabs公司，货号：M0202T)催化；所述催化的体系20μL包括前述扩增出的pACYCDuet-LacY DNA 100ng，启动子DNA片段50ng，10×Tango buffer 2μL，ATP 10mM，T4DNA连接酶0.5μL，双蒸水补齐至20μL。催化可使用PCR仪，反应条件为：(37℃1分钟、16℃1分钟)×30个循环，60℃再反应5分钟。然后取10微升反应液转化大肠杆菌DH5ɑ感受态细胞(南京诺唯赞生物科技股份有限公司，货号：C502-03)，LB平板(含34mg/L氯霉素)涂布后37℃过夜培养。所得克隆是否正确通过PCR对应的启动子片段确定，PCR得到约40bp DNA片段的即为阳性克隆。
表1引物列表


2)构建重组质粒ptrc99a-ManCB-Gmdfcl-FucT
通过PCR(引物为ptrc99a-S和ptrc99a-A)获得ptrc99a质粒的线性化片段并装上适合于Golden gate连接的Overhang接头，PCR(引物为ManCB-S和ManCB-A)获得ManCB基因簇并装上适合于Golden gate连接的Overhang接头，PCR(引物为Gmdfcl-S和Gmdfcl-A)获得Gmdfcl基因簇并装上适合于Golden gate连接的Overhang接头，PCR(引物为FucT-S和FucT-A)获得FucT基因(由通用生物系统(安徽)有限公司经密码子优化后合成，序列见序列表)并装上适合于Golden gate连接的Overhang接头，PCR ptrc99a质粒上的trc启动子和终止子序列(序列见序列表)，并装上适合于Golden gate连接的Overhang接头(引物分别为P-S1/P-A1、P-S2/P-A2，Ter-S1/Ter-A1、Ter-S2/Ter-A2)。
使用BsaI酶(New England Biolabs公司，货号：R3733L)和T4DNA连接酶(New England Biolabs公司，货号：M0202T)共同催化，反应体系为：ptrc99a线性化片段100ng，ManCB基因簇片段100ng，Gmdfcl基因簇片段100ng，FucT基因片段100ng，质粒ptrc99a的trc启动子片段50ng，质粒ptrc99a的终止子片段50ng，10×Tango buffer 2μL，ATP 10mM，T4DNA连接酶0.5μL，双蒸水补齐至20μL
反应条件为：(37℃ 1分钟、16℃ 1分钟)×30个循环，60℃再反应5分钟。反应产物转化大肠杆菌DH5ɑ感受态细胞(南京诺唯赞生物科技股份有限公司，货号： C502-03)，涂布含50mg/L氨苄青霉素的LB平板，37℃过夜培养。所得克隆提取质粒后通过酶切验证是否正确：使用EcoNI酶(New England Biolabs公司，货号：#R0521S)37℃酶切1小时后跑核酸电泳胶，酶切反应体系为10微升：EcoNI酶0.5微升，质粒5微升，10×缓冲液1微升，其余水补齐。酶切产物大小约为10000～11000bp之间即为正确，对应克隆即为阳性克隆。
表2引物列表

3)构建携带组成型启动子的pACYCDuet-LacY重组质粒和重组质粒ptrc99a-ManCB-Gmdfcl-FucT的共转化
通过质粒转化方法将步骤1)和步骤2)制备得到的pACYCDuet-组成型启动子-LacY重组质粒和重组质粒ptrc99a-ManCB-Gmdfcl-FucT共转化到JM109(DE3)菌株中，即获得发酵用的JM109(DE3)双质粒菌株。
具体步骤如下：
(1)制备含有50mg/L氨苄青霉素和34mg/L氯霉素的LB琼脂平板(配方为10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，20g/L琼脂)；
(2)取一个1.5ml离心管，加入100μL JM109(DE3)感受态细胞悬液并置于冰上；加入1μL重组质粒pACYCDuet-组成型启动子-LacY(100ng/μL浓度)、1μL重组质粒ptrc99a-ManCB-Gmdfcl-FucT(100ng/μL浓度)，用移液器轻柔混匀，冰上静置20min；
(3)42℃水浴中热激90秒，然后迅速置冰上3～5min，整个过程不要振荡菌液；
(4)加入1mL LB液体培养基(不含抗生素)，混匀后37℃振荡培养(100rpm)1小时，使细菌恢复正常生长状态，并表达质粒编码的抗生素抗性基因；
(5)取100μL菌液至含有50mg/L氨苄青霉素和34mg/L氯霉素的LB琼脂平板上，涂布均匀；
(6)待菌液被培养基吸收后，37℃倒置培养12～16小时，出现单菌落后挑取JM109(DE3)V1单菌落到含有50mg/L氨苄青霉素和34mg/L氯霉素的LB液体培养基中37℃培养至浑浊，吸取500μL菌液至灭过菌的EP管，再加入500μL 40％(w/w)浓度甘油，混匀后-80℃保藏待用，记为实验组；
(7)以相同的转化方法，将重组质粒ptrc99a-ManCB-Gmdfcl-FucT转化到JM109(DE3)感受态细胞，即获得用做对照实验的JM109(DE3)V0单质粒菌株。该菌株没有过表达乳糖转运蛋白LacY，只通过单质粒ptrc99a-ManCB-Gmdfcl-FucT过表达了2’-岩藻糖基乳糖合成所需的关键酶基因ManCB、Gmdfcl、和FucT。
实施例2
菌株的摇瓶发酵合成2′-FL
使用三角摇瓶发酵，摇瓶规格500mL，装液量100mL，将实施例1制备的JM109(DE3)V0(单质粒对照组)和JM109(DE3)V1(双质粒实验组)分别以1％(v/v)接种量分别接种到摇瓶中，内有含葡萄糖或甘油的LB培养基(配方为15g/L葡萄糖(或甘油)，10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，链霉素25mg/L，氯霉素34mg/L)，37℃，220rpm培养至OD600＝1.0，冷却至25℃，加入终浓度0.2mM IPTG和5g/L乳糖，继续25℃、220rpm培养发酵60小时。将摇瓶发酵液通过12000rpm离心5分钟后，取上清液95℃加热10分钟灭活可溶性蛋白质，再次12000rpm离心5分钟后，吸入注射器并流过0.22微米滤头，滤出液即可进行高效液相色谱法(HPLC)分析。HPLC测定发酵液中2’-FL浓度的具体方法如下：液相设备：Agilent 1260 Infinity II；示差检测器检测，型号：G7162A-1260 RId；液相柱型号：Sepax HP-Amino，4.6*250mm、5微米粒径(或同等规格氨基柱)；流速：0.8ml/min，流动相：80％纯乙腈：20％水(v/v)，系统温度：35℃，进样量10～20微升。用于计算浓度的2’-FL标准品购自上海惠诚生物科技有限公司，纯度98％(货号：GY1141)。J23100启动子的序列号为SEQ ID No.41，J23101启动子的序列号为SEQ ID No.42，J23106启动子的序列号为SEQ ID No.43，J23105启动子的序列号为SEQ ID No.44，J23115启动子的序列号为SEQ ID No.45，J23109启动子的序列号为SEQ ID No.46，J23113启动子的序列号为SEQ ID No.47。
2′-FL产量结果如表3所示：
表3不同组成型启动子的双质粒菌株发酵合成2′-FL产量情况
HPLC结果表明：J23115启动子对应双质粒菌株的2′-FL发酵产量最高，而反映菌株生长情况的OD600值也较高，故其对应的JM109(DE3)J23115双质粒菌株即为本发明要求保护的高产2’-FL的工程菌。而当以甘油为碳源时，JM109(DE3)J23115菌株的2′-FL产量为3.5g/L，OD600值为13.4。比较JM109(DE3)J23115双质粒菌株和JM109(DE3)V0 单质粒菌株，JM109(DE3)V0在使用甘油为碳源时OD600值为15.8，但2′-FL的产量仅为0.8g/L，而在使用葡萄糖为碳源时OD600值为14.2，但未测得产物(0g/L)。
以上结果表明：JM109(DE3)J23115菌株可以在葡萄糖为碳源的条件下发酵高产2′-FL，产量仅略低于以甘油为碳源的条件。而没有过表达LacY基因的对照菌株JM109(DE3)V0，则无法在葡萄糖为碳源的条件下发酵合成2′-FL。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种以葡萄糖为碳源合成2’-岩藻糖基乳糖的菌株，其特征在于，将重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌获得；
    所述重组质粒pACYCDuet-J23115-LacY是将质粒pACYCDuet-1与LacY基因连接，并将质粒pACYCDuet-1中的T7启动子替换为J23115启动子；
    所述重组质粒ptrc99a-ManCB-Gmdfcl-FucT是将质粒ptrc99a与ManCB基因簇、Gmdfcl基因簇、FucT基因、质粒ptrc99a的trc启动子、质粒ptrc99a的终止子连接后得到。
  2. 根据权利要求1所述的菌株，其特征在于，所述LacY基因的核苷酸序列如SEQ ID No.1所示。
  3. 根据权利要求1所述的菌株，其特征在于，所述ManCB基因簇的核苷酸序列如SEQ ID No.2所示；
    所述Gmdfcl基因簇的核苷酸序列如SEQ ID No.3所示；
    所述FucT基因的核苷酸序列如SEQ ID No.4所示；
    所述质粒ptrc99a的trc启动子的核苷酸序列如SEQ ID No.5所示；
    所述质粒ptrc99a的终止子的核苷酸序列如SEQ ID No.6所示；
    所述J23115启动子的核苷酸序列如SEQ ID No.45所示。
  4. 根据权利要求1所述的菌株，其特征在于，所述重组质粒pACYCDuet-J23115-LacY和ptrc99a-ManCB-Gmdfcl-FucT的质量比为1:1。
  5. 根据权利要求1所述的菌株，其特征在于，所述大肠杆菌包括大肠杆菌JM109(DE3)。
  6. 权利要求1-5任一项所述的菌株的构建方法，其特征在于，包括以下步骤：
    1)将质粒pACYCDuet-1经限制性内切酶NdeI和EcoRV双酶切，得到线性质粒；
    2)将所述步骤1)得到的线性质粒与LacY基因连接，得到重组质粒pACYCDuet-LacY，以所述重组质粒pACYCDuet-LacY为模板，经PCR扩增，得到不含有T7启动子的基因序列；
    3)将所述步骤2)不含有T7启动子的基因序列与J23115启动子经BsaI和T4DNA连接酶催化，得到重组质粒pACYCDuet-J23115-LacY；
    4)将质粒ptrc99a与ManCB基因簇、Gmdfcl基因簇、FucT基因、质粒ptrc99a的trc启动子、质粒ptrc99a的终止子经BsaI和T4DNA连接酶催化，得到重组质粒ptrc99a-ManCB-Gmdfcl-FucT；
    5)将所述步骤3)得到的重组质粒pACYCDuet-J23115-LacY与步骤4)得到的重组质粒ptrc99a-ManCB-Gmdfcl-FucT共转化到大肠杆菌中，得到菌株。
  7. 权利要求1-5任一项所述的菌株在以葡萄糖为碳源合成2’-岩藻糖基乳糖中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述应用包括：将所述菌株接种到发酵培养基中进行发酵，当发酵液的OD600为1时，加入终浓度为0.05-0.2mM IPTG和5g/L乳糖，继续发酵，得到2’-岩藻糖基乳糖。
  9. 根据权利要求7所述的应用，其特征在于，所述发酵培养基中葡萄糖的含量为15g/L。
  10. 根据权利要求7所述的应用，其特征在于，所述发酵的条件包括：发酵的温度为37℃，发酵的转速为220rpm；
    所述继续发酵的条件包括：继续发酵的温度为25℃，继续发酵的转速为220rpm，继续发酵的时间为60h。
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