WO2023169200A1 - 重组酵母菌及其应用 - Google Patents

重组酵母菌及其应用 Download PDF

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WO2023169200A1
WO2023169200A1 PCT/CN2023/077280 CN2023077280W WO2023169200A1 WO 2023169200 A1 WO2023169200 A1 WO 2023169200A1 CN 2023077280 W CN2023077280 W CN 2023077280W WO 2023169200 A1 WO2023169200 A1 WO 2023169200A1
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acid sequence
lactose
amino acid
seq
recombinant
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PCT/CN2023/077280
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French (fr)
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方诩
李凤仪
韩丽娟
殷文成
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山东恒鲁生物科技有限公司
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Publication of WO2023169200A1 publication Critical patent/WO2023169200A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01056—Lipopolysaccharide N-acetylglucosaminyltransferase (2.4.1.56)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • 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 present invention relates to recombinant yeast for producing lactose-N-trisaccharide and its application, and belongs to the field of bioengineering.
  • HMOs Human milk oligosaccharides
  • N-tetraose (LNT) and lactose-N-neotetraose (LNnT) are both produced by microbial fermentation.
  • HMOs acidic and neutral oligosaccharides
  • the concentration of total HMOs (acidic and neutral oligosaccharides) in breast milk decreases as the lactation period prolongs; therefore, timely and sufficient supplementation of HMOs will help maintain the healthy growth of infants and young children.
  • the synthesis of HMOs has always been a huge challenge faced by synthetic biology/chemists. From the perspective of the synthesis of sugar chains in vivo, the synthesis of such molecules is not the replication of a single template, but requires multiple glycosyltransferases and Glycosidases are co-regulated, which determines the complexity, diversity and microscopic heterogeneity of the sugar chain structure.
  • Lactose-N-triose (English name Lacto-N-trioseII, abbreviated as LNTII, also known as GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc, CAS: 75645-27-1, chemical structural formula is as formula 1).
  • LNTII is not only an important oligosaccharide of HMOs, but also an important precursor for the synthesis of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). body.
  • LNT lacto-N-tetraose
  • LNnT lacto-N-neotetraose
  • LNTII can be synthesized by enzyme-catalyzed formation of ⁇ -1,3-glycosidic bond between N-acetylglucosamine (GlcNAc) and ⁇ -lactose.
  • GlcNAc N-acetylglucosamine
  • ⁇ -lactose N-acetylglucosamine
  • Biosynthetic technology has received widespread attention in recent years due to its advantages such as high specificity and scalable mass production.
  • the enzyme catalysis method has the advantages of less reaction by-products and strong controllability, its catalytic efficiency is low.
  • patent CN202010386045.2 discloses the use of ⁇ -N-acetylhexosaminidase derived from Haloferula sp. in the synthesis of LNTII.
  • Application which uses the substrate chitin as an amino sugar donor, has a conversion rate of only less than 14%.
  • ⁇ -cell catalysis directly utilizes high-energy activated substrate forms in the body, such as UDP-GlcNAc, which is an easier method to achieve high conversion efficiency.
  • Chinese patent CN201680052611.8 discloses a genetically modified microbial host cell that produces A method that requires oligosaccharides
  • CN202010834657.3 discloses a genetically engineered bacterium that improves LNTII and a production method.
  • LNTII-producing hosts are Escherichia coli, but the removal of endotoxins from Escherichia coli is a major challenge in large-scale industrial production. Therefore, finding a host with stable yield, high production efficiency and good safety to synthesize LNTII is of great value for the industrial application and promotion of human milk oligosaccharides.
  • microbial strains containing modifications related to the acetylglucosaminyl transferase gene.
  • the invention provides enzymes for catalyzing the production of lactose-N-triose (LNTII).
  • the enzyme used to catalyze the production of lactose-N-trisaccharide (LNTII) belongs to the glycoside hydrolase 28 family according to the classification of Carbohydrate-Active-Enzymes, that is, the CAZy database.
  • the enzyme of the glycoside hydrolase 28 family is acetylglucosaminyl transferase ⁇ -N-acetylglucosaminyl transferase.
  • the enzyme that catalyzes the production of lactose-N-trisaccharide is preferably from Neisseria meningitidis (Neisseria meningitidis), EC No: 2.4.1.56; Genebank number AAC44084.1, and its amino acid sequence is such as SEQ ID NO:1; or contain the amino acid sequence shown in SEQ ID NO:1, or have at least 70% amino acid sequence identity with SEQ ID NO:1, or contain at least 70% amino acid sequence with SEQ ID NO:1 Sequence consistency.
  • lactose-N-trisaccharide is preferably derived from Helicobacter pylori, EC No: 2.4.1.129; Genebank number WP000199766.1; its amino acid sequence is as shown in SEQ ID NO: 2 shown; or have at least 70% amino acid sequence identity with SEQ ID NO:2, or contain the amino acid sequence shown in SEQ ID NO:2, or contain at least 70% amino acid sequence identity with SEQ ID NO:2.
  • the enzyme that catalyzes the production of lactose-N-trisaccharide is preferably derived from Salmonella enterica enterica), EC No: 2.4.1.155; Genebank number WP_140239824.1, which has the amino acid sequence shown in SEQ ID NO: 3; or has at least 70% amino acid sequence identity with SEQ ID NO: 3, or contains SEQ ID NO :3, or comprise at least 70% amino acid sequence identity with SEQ ID NO:3.
  • the enzyme that generates lactose-N-trisaccharide includes an amino acid sequence that is at least 70%, at least 71%, at least 72%, or at least 73% identical to SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3.
  • the invention describes a recombinant cell comprising one or more (eg, two, several) heterologous polynucleotides.
  • the heterologous polynucleotide is selected from heterologous polynucleotides encoding acetylglucosaminyltransferase, which has the ability to generate LNTII.
  • the recombinant cells preferably, comprise the amino acid sequence of the enzyme shown in the above SEQ ID NO: 1 and/or SEQ ID NO: 2 and/or SEQ ID NO: 3 for generating lactose-N-trisaccharide and its coding nucleic acid sequence
  • the present invention describes using yeast cells as hosts to efficiently synthesize lactose-N-trisaccharide ( Lac-N-Triose II, LNTII).
  • the recombinant cells are yeast cells, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells ; Further preferred ones are Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxinus, Yarrowia lipolytica, and Rhodotorula graminis ), Saccha romyces pastorianus and other cells; Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxinus, Yarrowia lipolytica are all Yeast cells that have passed the GRAS food safety certification of the US Food and Drug Administration are more likely to be accepted in terms of food or food additive safety management.
  • yeast cells such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells
  • the present invention provides a new production process of lactose-N-trisaccharide (LNTII), which can easily synthesize lactose-N-trisaccharide (LNTII) through fermentation, which not only simplifies the synthesis steps, but also improves the safety of the target product.
  • LNTII lactose-N-trisaccharide
  • the recombinant yeast comprises (1) an acetylglucosaminyltransferase capable of producing lactose-N-triose and (2) Destruction of marker genes.
  • disruption means that the coding region and/or control sequences of the reference gene are partially or completely modified (eg by deletion, insertion and/or substitution of one or more nucleotides) such that expression of the encoded polypeptide is absent ( Inactivation) or reduced, and/or the enzymatic activity of the encoded polypeptide is absent or reduced.
  • the disruptive effect can be measured using techniques known in the art, such as detecting the absence or reduction of lactose consumption using measurements from cell-free extracts referenced herein.
  • the marker gene includes ⁇ -galactosidase or a partial amino acid sequence of ⁇ -galactosidase, which is at least 50%, at least 51%, and at least 52% consistent with SEQ ID NO:4 and/or SEQ ID NO:5.
  • the recombinant yeast is a Kluyveromyces lactis recombinant yeast cell; further preferably, it contains at least one marker gene that has been destroyed, and contains the amino acid sequence of the marker, and the nucleic acid sequence encoding the marker.
  • the nucleic acid sequence of the marker gene is Genebank number M84410.1; its amino acid sequence is Genebank number AAA35265.1.
  • the recombinant yeast is a Kluyveromyces marxianus recombinant yeast cell; further preferably, it contains at least one marker gene that has been destroyed, and includes the amino acid sequence of the marker, and the nucleic acid sequence encoding the marker.
  • the nucleic acid sequence of the marker gene is Genebank number XM_022818497.1; its amino acid sequence Genebank number is XP_022675157.1.
  • the marker gene is the ⁇ -galactosidase gene lac4.
  • the disruption of the marker gene preferably refers to the disruption of the lac4 gene.
  • the disruption of the lac4 gene preferably refers to the disruption of part of the marker gene.
  • the recombinant Kluyveromyces lactis strain is preferably a recombinant Kluyveromyces lactis strain KL-NMLgtA and/or KL-SELgtA and/or KL-HPLgtA.
  • the recombinant Kluyveromyces marxianus strain is preferably the recombinant Kluyveromyces marxianus strain KM-NMLgtA and/or KM-SELgtA and/or KM-HPLgtA.
  • the recombinant Kluyveromyces lactis strain StrainKL-2-02 of the present invention is classified and named: Kluyveromyces lactis, and the deposit number is: CCTCC NO:M 2022118.
  • the recombinant yeast of the present invention can be genetically modified through ultraviolet mutagenesis to further increase the production of LNTII.
  • the invention also provides the expression method of the recombinant yeast cell, which includes: (a) cultivating the parent strain; (b) destroying the marker gene in the parent strain of (a); (c) introducing the ability to produce lactose-N -Trisaccharide enzyme; preferably, it also includes the step of (d) isolating the recombinant strain.
  • parent strain refers to the strain that was disrupted to produce the recombinant strains described herein.
  • the parent may be a naturally occurring (wild type) or pre-modified strain.
  • the yeast strain is a Kluyveromyces sp. strain; more preferably, it is a Kluyveromyces lactis strain, K.lactic and/or Kluyveromyces marxianus; further preferably, it is a Kluyveromyces sp. strain.
  • the application of the Kluyveromyces lactis recombinant yeast strain and the Kluyveromyces marxianus recombinant yeast strain of the present invention in the preparation of lactose-N-trisaccharide LNTII, the UDP-acetylglucosamine (UDP-GlcNAc) and lactose synthesized by their own metabolism are The reaction donor generates lactose-N-trisaccharide (LNTII) under the catalysis of the heterologously introduced 28 family glycoside hydrolase.
  • the present invention also provides the application of the Kluyveromyces lactis recombinant yeast and/or Kluyveromyces marxianus recombinant yeast in producing lactose-N-trisaccharide.
  • the medium used is an aqueous solution; more preferably, the medium used is an aqueous solution containing lactose and/or glucose and/or fructose and/or sucrose and/or galactose.
  • the aqueous solution needs to add the Kluyveromyces lactis recombinant yeast strain and/or the Kluyveromyces marxianus recombinant yeast strain.
  • the applied culture temperature is 15-60°C; further preferably, the applied culture temperature is 28-32°C.
  • the medium also contains inorganic salts and/or trace elements; preferably, the inorganic salts and/or trace elements are selected from dipotassium hydrogen phosphate. , magnesium sulfate, ammonium sulfate, manganese sulfate, potassium sulfate or any combination thereof; preferably, the final concentration of the inorganic salt or trace element is 3-7mM.
  • the present invention also provides a method for producing the lactose-N-trisaccharide (LNTII), which method includes: (a) cultivating the recombinant yeast cells in a fermentable medium under suitable conditions to produce lactose -N-trisaccharide (LNTII); and (b) recovering the lactose-N-trisaccharide (LNTII).
  • lactose-N-trisaccharide is preferably LNTII obtained after separation and purification by adding activated carbon or other methods.
  • lactose-N-trisaccharide preferably utilizes the recombinant yeast to perform mixed fermentation to obtain LNTII.
  • sequence identity refers to the correlation between two amino acid sequences or between two nucleotide sequences, described by the parameter “sequence identity”, also known as “sequence identity”.
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector.
  • host cell covers a parent cell that differs from the parent cell due to mutations that occur during replication. any descendants.
  • the term "recombinant cell” is defined as a non-naturally occurring host cell that includes one or more (eg, two, several) heterologous polynucleotides.
  • the host cell can be a mutant strain described herein, or be further disrupted to provide a mutant strain described herein.
  • the invention provides a new method for manufacturing lactose-N-trisaccharide (LNTII), which uses recombinant Kluyveromyces lactis (Kluyveromyces lactis and Kluyveromyces marxianus) as a cell factory to synthesize human milk oligos.
  • LNTII in sugar.
  • the entire manufacturing process is simple and controllable, has high biological safety, is easy to expand in scale, has a wide range of raw material sources, has low product preparation costs and has high practical value.
  • It uses lactose as the substrate to synthesize lactose-N-trisaccharide (LNTII). Conversion rates are higher.
  • the present invention proves that recombinant yeast cells have great potential in realizing efficient production of human milk oligosaccharides, lays the foundation for large-scale industrial production of lactose-N-trisaccharide (LNTII) and its analogs, and has important economic value and social benefits.
  • LNTII lactose-N-trisaccharide
  • the raw materials used in the present invention are easily available, the method is simple and easy to implement, is suitable for industrial production, and has excellent application prospects.
  • Figure 2 is an HPLC chromatogram of the product synthesized by the original strain of Kluyveromyces lactis and the control strain StrainKL-1 in Example 3.
  • Figure 3 shows the HPLC chromatograms of the synthetic products and LNTII standard of the Kluyveromyces lactis strain StrainKL-2-01 in Example 4 and the fermentation broth of the recombinant strain StrainKL-2-02 in Example 6.
  • Figure 4 is an HPLC chromatogram of the purified synthetic product and LNTII standard of the recombinant Kluyveromyces lactis strain StrainKL-2-01 fermentation broth in Example 4.
  • Figure 5 is an HPLC chromatogram of the purified synthetic product and LNTII standard of the Kluyveromyces lactis recombinant strain StrainKL-2-02 fermentation broth in Example 6.
  • Figure 7 is an LC-MS analysis chart of the product with a peak elution time of 12.10 minutes in the fermentation broth of the recombinant Kluyveromyces lactis strain StrainKL-2-02 in Figure 5.
  • Figure 8 is a 1 H-NMR analysis spectrum of the product of the fermentation broth of the recombinant Kluyveromyces lactis strain StrainKL-2-02 in Figure 5 with a peak time of 12.10 minutes.
  • Figure 9 is a 13 C-NMR analysis spectrum of the product of the fermentation broth of the recombinant Kluyveromyces lactis strain StrainKL-2-02 in Figure 5 with a peak time of 12.10 minutes.
  • Kluyveromyces lactis and/or Kluyveromyces marxianus which have good biological safety and are widely used in industry, are used as hosts. Knock out the ⁇ -galactosidase gene lac4 of Kluyveromyces lactis and/or Kluyveromyces marxianus to prevent lactose from being consumed in the yeast strain; and insert the gene NMLgtA from Neisseria meningitidis encoding acetyl glycosyltransferase and/or the gene encoding acetyl glycosaminotransferase (SELgtA) from Salmonella enterica and/or the gene encoding acetyl glycosaminotransferase (HPLgtA) from Helicobacter pylori was integrated into Kluyveromyces lactis or M. The genome of Luyveromyces luyveromyces was used to obtain a strain that synthe.
  • the first stage is the bacterial growth phase: the cells are cultured with glucose as the carbon source to accumulate biomass and enzymes until the cell growth enters the late logarithmic phase or stationary phase.
  • the second stage is the product synthesis period: first, culture at 30°C and 200rpm with shaking for 40 hours, then add 2g/L lactose, 20g/L glucose and metal ions with a final concentration of 3-7mM to the culture medium.
  • the final product is lactose-N-trisaccharide (LNTII).
  • the protein produced by the host cell can be obtained by any known process for isolating and purifying proteins. These processes include, but are not limited to, salting out and solvent precipitation, ultrafiltration, gel electrophoresis, ion exchange chromatography, affinity chromatography, reversed phase high performance liquid chromatography, hydrophobic interaction chromatography, mixed mode chromatography, hydroxyapatite chromatography, and others. Electric focusing.
  • Specific embodiments of the present invention also provide a method for synthesizing lactose-N-trisaccharide (LNTII).
  • the production method includes: respectively amplifying genes encoding acetylglucosaminyltransferase derived from Neisseria meningitidis, Salmonella enterica and Helicobacter pylori. And construct an expression cassette suitable for Kluyveromyces lactis and/or Kluyveromyces marxianus; introduce the expression cassette into Kluyveromyces lactis DSM70799 and/or Kluyveromyces marxianus DMKU3-1042, and integrate it into the bacteria from the genome of the strain; screen out positive yeast transformants; ferment and culture the genetically engineered bacteria to obtain lactose-N-trisaccharide (LNTII); use high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance chromatography to identify the fermentation broth and its purified products. .
  • LNTII lactose-N-trisaccharide
  • E. coli BL21 and expression vector pET-28a were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. Experimental methods without specifying specific conditions in the following examples were carried out according to conventional conditions, such as the conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer of the corresponding biological reagents.
  • the reaction procedure of the PCR amplification reaction can be a conventional PCR amplification reaction procedure.
  • the preservation number of Saccharomyces cerevisiae in the ATCC Culture Collection Center is CICC1964.
  • Instrument 126-quadrupole time-of-flight high-resolution mass spectrometer (liquid mass spectrometry).
  • HPLC High performance liquid chromatography
  • the LNTII standard was purchased from the French ELICITYL company.
  • Example 1 Construction of ⁇ LAC4 marker KL and/or KM knockout cassette and KL and/or KM expression cassette of enzyme genes of glycoside hydrolase 28 family.
  • PCR amplification components Phanta Super-Fidelity DNA Polymerase 1 ⁇ L, 2 ⁇ Phanta Max Buffer 25 ⁇ L, dNTPs (10mM) 1 ⁇ L, primer F 2 ⁇ L, primer R 2 ⁇ L, template DNA 1 ⁇ L, ddH 2 O 18 ⁇ L, total volume 50 ⁇ L.
  • PCR amplification steps (1) Pre-denaturation at 95°C, 3min; (2) Denaturation at 95°C, 0.25min; (3) Annealing at 54°C, 0.5min; (4) Extension at 72°C, 0.5min; step (2)- (4) Cycle 30 times; (5) Final extension at 72°C for 5 minutes; (6) Store at 4°C.
  • PUG6 plasmid as template, PUG6-Kpn1 and PUG6-Bamh1 as primers (PUG6 does not have enzyme cutting sites for Kpn1 and Bamh1, and requires PCR amplification to introduce the required enzyme cutting sites) for PCR amplification.
  • PCR amplification components Phanta Super-Fidelity DNA Polymerase 1 ⁇ L, 2 ⁇ Phanta Max Buffer 25 ⁇ L, dNTPs (10mM) 1 ⁇ L, primer F 2 ⁇ L, primer R 2 ⁇ L, template DNA 1 ⁇ L, ddH 2 O 18 ⁇ L, total volume 50 ⁇ L.
  • PCR amplification steps (1) Pre-denaturation at 95°C, 3min; (2) Denaturation at 95°C, 0.25min; (3) Annealing at 54°C, 0.5min; (4) Extension at 72°C, 3min; steps (2)-( 4) Cycle 30 times; (5) Final extension at 72°C for 5 minutes; (6) Store at 4°C.
  • agarose gel electrophoresis is performed for identification, and the bands that meet the expected size are purified and recovered using a gel recovery kit.
  • LAC4 promoter region and terminator sequence region were connected into the PUG6 vector through two enzyme digestion ligations to construct KL- ⁇ LAC4 and KM- ⁇ LAC4 knockout cassettes.
  • the first enzyme digestion ligation use Kpn1 and Bamh1 to connect the termination sequence obtained in step 1.2 and the PUG6 vector respectively.
  • the enzyme digestion temperature is 37°C and the time is about 2 hours.
  • Enzyme digestion system DNA fragment X ⁇ L (total DNA amount is about 2-3ng), Kpn1 2 ⁇ L, Bamh1 2 ⁇ L, Buffer 5 ⁇ L, ddH 2 O 41-X ⁇ L, total volume 50 ⁇ L.
  • E. coli transformation is carried out promptly after the ligation is completed. Select the correct transformant for plasmid extraction, and use this plasmid as a new vector for the second enzyme digestion and ligation.
  • the second enzyme cutting and ligation double-digest the PUG6 vector with the termination sequence (the vector itself carries Nde1 and Not1 enzyme cutting sites) and the promoter region with Nde1 and Not1 respectively.
  • the enzyme cutting temperature is 37°C and the time About 2h.
  • agarose gel electrophoresis was performed for identification, and the bands that met the expected size were purified and recovered using a gel recovery kit.
  • the purified fragments were ligated using T4 DNA ligase. E. coli transformation is carried out promptly after the ligation is completed. Select the correct transformants for plasmid extraction to obtain the knockout vector carrying the ⁇ LAC4 knockout cassette.
  • the lgtA KL expression cassette includes its KL- ⁇ LAC4::NMLgtA, KL- ⁇ LAC4::HPLgtA, KL- ⁇ LAC4::SELgtA, KL-18S::NMLgtA, KL-18S::HPLgt A, and KL-18S:: SELgt A expression cassette.
  • the lgtA KM expression cassette includes its KM- ⁇ LAC4::NMLgtA, KM- ⁇ LAC4::HPLgtA, KM- ⁇ LAC4::SELgtA, KM-18S::NMLgtA, KM-18S::HPLgt A, and KM-18S:: SELgt A expression cassette.
  • Promoter region sequence As shown in SEQ ID NO:6, it is derived from the Kluyveromyces lactis LAC4 promoter region sequence; as shown in SEQ ID NO:7, it is derived from the Kluyveromyces marxianus LAC4 promoter region sequence.
  • the ADH1 terminator sequence is derived from Saccharomyces cerevisiae
  • PCR amplification was performed using NMLgtA/HPLgtA/SELgtA gene, Kluyveromyces lactis genome, Kluyveromyces marxianus genome, and Saccharomyces cerevisiae genome as templates respectively.
  • PCR amplification components Phanta Super-Fidelity DNA Polymerase 1 ⁇ L, 2 ⁇ Phanta Max Buffer 25 ⁇ L, dNTPs (10mM) 1 ⁇ L, primer F 2 ⁇ L, primer R 2 ⁇ L, template DNA 1 ⁇ L, ddH 2 O 18 ⁇ L, total volume 50 ⁇ L.
  • PCR amplification steps (1) Pre-denaturation 95°C, 3min; (2) Denaturation 95°C, 0.25min; (3) Annealing 54°C, 0.5min; (4) Extension at 72°C, 1.5min; cycle steps (2)-(4) 30 times; (5) Final extension at 72°C, 5min; (6) Store at 4°C.
  • agarose gel electrophoresis is performed for identification, and the bands that meet the expected size are purified and recovered using a gel recovery kit.
  • the DNA concentration of the recovered product was measured using a micrometer, and its concentration was diluted to 1ng/ ⁇ L using ddH2O. Fusion PCR amplification was performed using the diluted DNA fragment as a template and ADH1-Not1 and LAC4Pro-Nde1/LAC4Pro-S as primers.
  • PCR amplification components Phanta Super-Fidelity DNA Polymerase 1 ⁇ L, 2 ⁇ Phanta Max Buffer 25 ⁇ L, dNTPs (10mM) 1 ⁇ L, primer F 2 ⁇ L, primer R 2 ⁇ L, template DNA 1 ⁇ L, ddH 2 O 18 ⁇ L, total volume 50 ⁇ L.
  • PCR amplification was performed using Kluyveromyces lactis genome, Kluyveromyces marxianus genome and PUG6 vector as templates.
  • PCR amplification components Phanta Super-Fidelity DNA Polymerase 1 ⁇ L, 2 ⁇ Phanta Max Buffer 25 ⁇ L, dNTPs (10mM) 1 ⁇ L, primer F 2 ⁇ L, primer R 2 ⁇ L, template DNA 1 ⁇ L, ddH 2 O 18 ⁇ L, total volume 50 ⁇ L.
  • agarose gel electrophoresis is performed for identification, and the bands that meet the expected size are purified and recovered using a gel recovery kit.
  • the fusion sequence obtained in step 2.1 and the ⁇ LAC4 knockout vector were double digested with Nde1 and Not1 respectively.
  • the digestion temperature was 37°C and the time was about 2 hours.
  • agarose gel electrophoresis was performed for identification, and the bands that met the expected size were purified and recovered using a gel recovery kit.
  • the purified fragments were ligated using T4 DNA ligase. Immediately after the connection is completed Perform E. coli transformation.
  • step 2.1 Mix the fusion sequence obtained in step 2.1 with the upstream and downstream homology arm sequences, backbone sequences, and resistance sequences in step 2.2 in a total volume of 5 ⁇ L and a molar ratio of 1:1:1:1:1, and then add 5 ⁇ L of seamless cloning MIX Make a connection.
  • the connection temperature is 50°C and the time is 30-60min.
  • transform E. coli in time select the correct transformant for plasmid extraction, and obtain the expression vector KL-18S::NMLgtA and/or KL-18S::SELgtA carrying the NMLgtA and/or SELgtA and/or HPLgtA expression cassette.
  • 50 ⁇ TAE solution Prepared with ddH 2 O, containing 2M Tris, 100mM Na 2 EDTA ⁇ H 2 O, 2% SDS, and adjust the pH to 8.5.
  • Genome extraction buffer Prepared with ddH 2 O, containing 200mM Tris-HCl, 250mM NaCl, 2% SDS, 25mM EDTA, and adjust the pH to 8.0.
  • Escherichia coli medium prepared from 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and water. An additional 20g/L agar powder is required for solid culture medium. Use high-temperature steam sterilization at 121°C for 20 minutes before use.
  • Yeast culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, and water. An additional 20g/L agar powder is required for solid culture media. Use high-temperature steam sterilization at 115°C for 30 minutes before use.
  • E. coli was cultured on solid plates in a constant-temperature incubator set at 37°C, and in shake flasks on a shaker with a temperature set at 37°C and a rotation speed of 200 rpm.
  • the yeast was cultured on a solid plate in a constant temperature incubator with a temperature set to 30°C, and in a shaker flask with a temperature set to 30°C and a rotational speed set to 200 rpm.
  • Example 2 Construction of recombinant strains of Kluyveromyces lactis and Kluyveromyces marxianus:
  • yeast transformation is performed to obtain control strains StrainKL-1 and StrainKM-1 respectively.
  • the yeast transformation method is shown in this example.
  • KM-18S::NMLgtA and/or KM-18S::SELgt A and/or KM-18S ::HPLgt A expression cassette was successfully constructed and transformed into yeast respectively to obtain the recombinant strain StrainKM-2-01/StrainKM-2-05/StrainKM-2-02 and the recombinant strain StrainKM-2-03/StrainKM-2-06/StrainKM. -2-04.
  • the yeast transformation method is as follows:
  • yeast competent cells take a small amount of frozen yeast strain and streak it on a flat solid medium, and incubate it upside down at 30°C for 2 days. Pick a single yeast colony and culture it in 50 mL liquid culture medium at 30°C and 220 rpm until the OD 600 is between 0.8-1.5. Collect the bacterial cells and wash them with 25 mL of sterile water, centrifuge at 1500 ⁇ g for 10 min at room temperature, and discard the supernatant. Add 1 mL of 100 mM lithium chloride buffer, resuspend the pellet, centrifuge at 12,000 rpm for 30 s, and discard the supernatant. Add 400 ⁇ L 100mM again of lithium chloride buffer, resuspend the pellet to obtain competent yeast cells, and aliquot into 50 ⁇ L/tube for transformation.
  • Transformation Centrifuge the competent yeast prepared above, and use Tips to remove the residual lithium chloride solution. For each transformation, add in the following order: 50% PEG3350 (240 ⁇ L); 1 M LiCl (36 ⁇ L); 2 mg/mL single-stranded Salmon sperm DNA (25 ⁇ L); 5 to 10 ⁇ g/50 ⁇ L H 2 O plasmid DNA (50 ⁇ L), vigorously Vortex and mix until the precipitated cells are completely evenly distributed; incubate in a 30°C water bath for 30 minutes; heat shock in a 42°C water bath for 20 to 25 minutes; collect the yeast cells after centrifugation at 8000 rpm for 10 minutes; then, resuspend the yeast in 500 ⁇ L liquid culture medium, 30°C Incubate on a shaking table; after 1 to 4 hours, take 25 to 100 ⁇ L of bacterial liquid and spread it on the selective medium plate, and incubate it upside down at 30°C.
  • the KL- ⁇ LAC4::NMLgtA and/or KL- ⁇ LAC4::SELgtA and/or KL- ⁇ LAC4::HPLgtA expression cassette is introduced into the original Kluyveromyces lactis strain StrainKL-0; KM-18S ::NMLgtA and/or KM-18S::SELgt A and/or KM-18S::HPLgtA expression cassette is introduced into Max Kluyver’s original starting strain StrainKM-0.
  • the plasmid DNA is the KL- ⁇ LAC4 knockout cassette constructed in Example 1, and/or KL- ⁇ LAC4::NMLgtA and/or KL- ⁇ LAC4::SELgtA and/or KL- ⁇ LAC4::HPLgtA, and KL- 18S::NMLgtA and/or KL-18S::SELgtA and/or KL-18S::HPLgtA expression cassette.
  • the plasmid DNA is the KM- ⁇ LAC4 knockout cassette constructed in Example 1, and/or KM- ⁇ LAC4::NMLgtA and/or KM- ⁇ LAC4::SELgtA and/or KM- ⁇ LAC4::HPLgtA, and KM- 18S::NMLgtA and/or KM-18S::SELgtA and/or KM-18S::HPLgtA expression cassette.
  • the plate is cultured statically for 2-3 days, and the single colony of the transformed transformant is verified.
  • the verification method is as follows:
  • the specific conditions of the culture medium in this example are as follows:
  • Solid culture medium 1 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 20g/L agar powder is prepared by adding water, and is sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Liquid culture medium 1 20g/L tryptone, 10g/L yeast extract, 20g/L glucose is prepared by adding water, and is sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Solid culture medium 2 20g/L tryptone, 10g/L yeast extract, 20g/L lactose, 20g/L agar powder is prepared by adding water, and is sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Liquid culture medium 2 20g/L tryptone, 10g/L yeast extract, 20g/L lactose is prepared by adding water, and is sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Example 3 Fermentation experiments of the original strain of Kluyveromyces lactis cells and its control strain, and the original strain of Kluyveromyces marxianus cells and its control strain.
  • the synthesis verification experiment of LNTII was conducted using two-stage culture on the original strain of Kluyveromyces lactis cells and its control strain, and the original strain of Kluyveromyces marxianus cells and its control strain respectively.
  • the first stage is the cell growth phase: cells are cultured with glucose as the carbon source, and the amount of cells and enzymes are accumulated respectively until the cell growth enters the late logarithmic phase or the stationary phase. Streak the strain and culture it in solid medium. After culturing for 2-3 days at 30°C, pick a single colony and inoculate it into 1.5mL liquid medium. Culture it overnight at 30°C and 200rpm with shaking.
  • inoculate 2% of the inoculum into a 50mL liquid medium shake flask, culture at 30°C and 200rpm until OD600 1, then inoculate 2% of the inoculum into 5L liquid medium (10L volume fermenter), 30°C, 200rpm Shake culture at 200 rpm to accumulate bacterial cells.
  • the second stage is the product synthesis period: when the culture is shaken at 30°C and 200 rpm for 40 hours, feeding begins (record the flow Add the amount of feed), the total fermentation time is 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and a high-pressure homogenizer was used to disrupt the bacterial cells. The proteins were removed by centrifugation. After the supernatant was filtered through a 0.22 ⁇ m filter to remove impurities, the LNTII content was measured using HPLC and the Klutinol lactate was measured using a biosensor. The ethanol content in the fermentation broth of StrainKL-0, the original starting strain of Kluyveri and Max Kluyve, is recorded in Table 5.
  • Kluyveromyces lactis original starting strain and control strain Kluyveromyx maximus original starting strain and control strain do not have the ability to produce LNTII.
  • the specific conditions of the culture medium in this example are as follows:
  • Solid culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 20g/L agar powder, sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Liquid culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 8g/L lactose, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate and manganese sulfate with a final concentration of 5mM respectively, after 115°C Sterilize with high temperature steam for 30 minutes before use.
  • Feeding materials 2g/L lactose, 20g/L glucose, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate and manganese sulfate with a final concentration of 5mM respectively.
  • Example 4 The recombinant Kluyveromyces lactis strain StrainKL-2-01 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-01 catalyze the synthesis of LNTII.
  • Two-stage culture was also used for the preparation of LNTII for the recombinant strains StrainKL-2-01 and/or StrainKM-2-01.
  • the second stage is the product synthesis stage: except for the recombinant strain, the others are the same as the second stage of Example 3.
  • the LNTII content in StrainKL-2-01 or StrainKM-2-01 fermentation broth measured by HPLC method was 8.53g/L and 8.75g/L respectively, and the LNTII conversion rates of lactose synthesis were 51.53% and 53.53% respectively.
  • Recombinant strain StrainKL-2-01 original fermentation The HPLC pattern of the liquid is shown in Figure 2, curve B, and the HPLC pattern of the purified fermentation broth is shown in Figure 3.
  • HPLC chromatogram of the product synthesized by the recombinant strain StrainKM-2-01 is the same as that of the product synthesized by StrainKL-2-01.
  • the ethanol content in the fermentation broth of StrainKL-2-01 and/or StrainKM-2-01 was measured using a biosensor to be approximately 25.1g/L and 25.7g/L respectively.
  • the solid culture medium, liquid culture medium, and feeding materials are the same as those in Example 3.
  • Example 5 The recombinant Kluyveromyces lactis strain StrainKL-2-03 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-03 catalyze the synthesis of LNTII.
  • the first stage is the growth stage of bacterial cells: except for the bacteria, everything else is the same as the first stage of Example 4.
  • the second stage is the product synthesis stage: except for the recombinant strain, the others are the same as the second stage of Example 4.
  • the LNTII content in the fermentation broth of StrainKL-2-03 and/or StrainKM-2-03 was measured by HPLC method to be 7.21g/L and 7.15g/L respectively, and the LNTII conversion rates of lactose synthesis were 45.24% and 44.21% respectively.
  • the ethanol contents measured using biosensors were approximately 25g/L and 24.3g/L respectively.
  • the solid culture medium, liquid culture medium and feeding materials are the same as those in Example 3.
  • Example 6 The recombinant Kluyveromyces lactis strain StrainKL-2-02 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-02 catalyze the synthesis of LNTII.
  • the first stage is the growth stage of bacterial cells: except for the bacteria, everything else is the same as the first stage of Example 4.
  • the second stage is the product synthesis stage: except for the recombinant strain, the others are the same as the second stage of Example 4.
  • the LNTII content in the fermentation broth of StrainKL-2-02 and/or StrainKM-2-02 was measured by HPLC method to be 15.23g/l and 15.27g/l respectively, and the LNTII conversion rates of lactose synthesis were 95.57% and 95.71% respectively.
  • the HPLC profile of the original fermentation broth of the recombinant strain StrainKL-2-02 is shown in Figure 2, curve C, and the HPLC profile of the purified fermentation broth is shown in Figure 4.
  • HPLC chromatogram of the product synthesized by the recombinant strain StrainKM-2-02 is the same as that of the product synthesized by StrainKL-2-02.
  • ethanol content in StrainKL-2-02 and/or StrainKM-2-02 fermentation broth was measured using a biosensor to be approximately 25.2g/l and 25.6g/l respectively.
  • the solid culture medium, liquid culture medium, and feeding materials are the same as those in Example 3.
  • Example 7 The recombinant Kluyveromyces lactis strain StrainKL-2-04 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-04 catalyze the synthesis of LNTII.
  • Two-stage culture was also used for the preparation of LNTII for the recombinant strains StrainKL-2-04 and/or StrainKM-2-04.
  • the first stage is the growth stage of bacterial cells: except for the bacteria, everything else is the same as the first stage of Example 4.
  • the second stage is the product synthesis stage: except for the recombinant strain, the others are the same as the second stage of Example 4.
  • the LNTII content in StrainKL-2-04 and/or StrainKM-2-04 fermentation broth measured by HPLC method was 14.57g/L and 14.71g/L respectively, and the LNTII conversion rates of lactose synthesis were 91.42% and 92.02% respectively.
  • the ethanol content measured using biosensors was approximately 24.6g/L and 24.9g/L respectively.
  • the solid culture medium, liquid culture medium, and feeding materials are the same as those in Example 3.
  • Example 8 The recombinant Kluyveromyces lactis strain StrainKL-2-05 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-05 catalyze the synthesis of LNTII.
  • the first stage is the growth stage of bacterial cells: except for the bacteria, everything else is the same as the first stage of Example 4.
  • the second stage is the product synthesis stage: except for the recombinant strain, the others are the same as the second stage of Example 4.
  • the LNTII content in the fermentation broth of StrainKL-2-05 and/or StrainKM-2-05 was measured by HPLC method to be 3.23g/L and 3.27g/L respectively, and the LNTII conversion rates of lactose synthesis were 20.27% and 20.52% respectively.
  • HPLC chromatogram of the product synthesized by the recombinant strain StrainKM-2-05 is the same as that of the product synthesized by StrainKL-2-05.
  • the ethanol content in the fermentation broth of StrainKL-2-05 and/or StrainKM-2-05 was measured using a biosensor to be approximately 26.1g/L and 26.3g/L respectively.
  • the solid culture medium, liquid culture medium, and feeding materials are the same as those in Example 3.
  • Example 9 The recombinant Kluyveromyces lactis strain StrainKL-2-06 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-06 catalyze the synthesis of LNTII.
  • the first stage is the growth stage of bacterial cells: except for the bacteria, everything else is the same as the first stage of Example 4.
  • the second stage is the product synthesis stage: except for the recombinant strain, the others are the same as the second stage of Example 4.
  • the LNTII content in the fermentation broth of StrainKL-2-06 and/or StrainKM-2-06 was measured by HPLC method to be 2.45g/L and 2.17g/L respectively, and the LNTII conversion rates of lactose synthesis were 15.37% and 13.61% respectively.
  • the ethanol contents measured using biosensors were approximately 26.3g/L and 27.1g/L respectively.
  • the solid culture medium, liquid culture medium, and feeding materials are the same as those in Example 3.
  • Example 10 The recombinant Kluyveromyces lactis strain StrainKL-2-01 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-01 catalyze the synthesis of LNTII.
  • the first stage is the same as the first stage of Example 4 except for the bacteria.
  • the second stage is the product synthesis period: when the culture is shaken at 15°C and 200rpm for 40 hours, fed feeding begins, and the total amount of The fermentation time is 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the protein was removed by centrifugation using a high-pressure homogenizer. The supernatant was passed through a 0.22 ⁇ m filter to remove impurities and the LNTII content was measured using HPLC.
  • the LNTII contents in the fermentation broth of StrainKL-2-01 and/or StrainKM-2-01 were measured by HPLC to be 4.73g/L and 4.67g/L respectively, and the conversion rates of lactose into LNTII were 29.81% and 29.68% respectively.
  • the ethanol content measured using biosensors was approximately 21.4g/L and 21.5g/L respectively.
  • the specific conditions of the culture medium in this example are as follows:
  • Liquid culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 8g/L lactose, ammonium sulfate and manganese sulfate with final concentrations of 3mM respectively, sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Feeding materials 2g/L lactose, 20g/L glucose, ammonium sulfate and manganese sulfate with final concentrations of 3mM respectively.
  • Example 11 The recombinant Kluyveromyces lactis strain StrainKL-2-01 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-01 catalyze the synthesis of LNTII.
  • the first stage is the same as the first stage of Example 4 except for the bacteria.
  • the second stage is the product synthesis period: when the culture is shaken at 60°C and 200 rpm for 40 hours, feed feeding begins for LNTII fermentation, with a total fermentation time of 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the protein was removed by centrifugation using a high-pressure homogenizer. The supernatant was passed through a 0.22 ⁇ m filter to remove impurities and the LNTII content was measured using HPLC.
  • the LNTII content in the fermentation broth of StrainKL-2-01 and/or StrainKM-2-01 was measured by HPLC method to be 1.12g/L and 1.15g/L respectively, and the conversion rates of LNTII synthesized from lactose were 7.02% and 7.07% respectively.
  • the ethanol contents measured using biosensors were approximately 18.0g/L and 18.2g/L respectively.
  • the specific conditions of the culture medium in this example are as follows:
  • Liquid culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 8g/L lactose, dipotassium hydrogen phosphate and magnesium sulfate with a final concentration of 7mM respectively, sterilized by high-temperature steam at 115°C for 30 minutes before use. use.
  • Feeding materials 2g/L lactose, 20g/L glucose, dipotassium hydrogen phosphate and magnesium sulfate with final concentrations of 7mM respectively.
  • Example 12 LNTII is catalyzed by Kluyveri lactis recombinant yeast strain StrainKL-2-01 and Kluyverom marxianus recombinant yeast strain StrainKM-2-01.
  • the first stage is the same as the first stage of Example 4 except for the bacteria.
  • the second stage is the product synthesis period: when the culture is shaken at 28°C and 200 rpm for 40 hours, feed feeding begins for LNTII fermentation, with a total fermentation time of 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the protein was removed by centrifugation using a high-pressure homogenizer. The supernatant was passed through a 0.22 ⁇ m filter to remove impurities and the LNTII content was measured using HPLC.
  • the LNTII content in StrainKL-2-01 and/or StrainKM-2-01 fermentation broth measured by HPLC method was 7.89g/L respectively. and 7.87g/L, the conversion rates of lactose synthesis LNTII are 49.51% and 49.49% respectively.
  • the ethanol content measured using biosensors was approximately 24.8g/L and 24.7g/L respectively.
  • the specific conditions of the culture medium in this example are as follows:
  • Liquid culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 8g/L lactose, magnesium sulfate with a final concentration of 6mM, sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Feeding materials 2g/L lactose, 20g/L glucose, and magnesium sulfate with a final concentration of 6mM.
  • Example 13 The recombinant Kluyveromyces lactis strain StrainKL-2-01 and the Kluyveromyces marxianus recombinant yeast strain StrainKM-2-01 catalyze the synthesis of LNTII.
  • the first stage is the same as the first stage of Example 4 except for the bacteria.
  • the second stage is the product synthesis stage: except for the liquid culture medium and feed materials, everything else is the same as the second stage of Example 10.
  • the LNTII content in the fermentation broth of StrainKL-2-01 and/or StrainKM-2-01 was measured by HPLC method to be 7.17g/L and 7.15g/L respectively, and the LNTII conversion rates of lactose synthesis were 44.99% and 44.97% respectively.
  • the ethanol contents measured using biosensors were approximately 24.3 mg/L and 24.5 mg/L respectively.
  • the specific conditions of the culture medium in this example are as follows:
  • Liquid culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 8g/L lactose, potassium sulfate with a final concentration of 6mM, sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Feeding materials 2g/L lactose, 20g/L glucose, and potassium sulfate with a final concentration of 6mM.
  • Example 14 Purification of the synthetic product in Example 6.
  • Example 15 The mutations caused by ultraviolet mutagenesis are very random, and the effects of mutations are also random. In order to obtain effective positive mutations, multiple rounds of ultraviolet mutagenesis are usually required for screening, which requires a large workload and cannot be achieved. Possibility of obtaining effective positive mutations. However, because UV mutagenesis requires simple equipment, low cost, and can obtain a large number of mutants in a short time, it is a commonly used mutation breeding method.
  • the applicant used the recombinant strains StrainKL-2-01, StrainKL-2-02 and StrainKM-2-01, StrainKM-2-02 as starting strains respectively, and genetically modified them through ultraviolet mutagenesis to further improve their LNTII properties. Yield.
  • the recombinant strains StrainKL-2-01, StrainKL-2-02 and StrainKM-2-01, StrainKM-2-02 were cultured in cells using glucose as the carbon source, and the amount of bacteria and enzymes were accumulated until the cells grew into the correct state. terminal or stable period. Streak the strain and culture it in solid medium. After culturing for 2-3 days at 30°C, pick a single colony and inoculate it into 1.5mL liquid medium. Culture it overnight at 30°C and 200rpm with shaking. Use sterile water to wash the bacterial cells to make a suspension, dilute it to 1 ⁇ 10 6 cells/mL, and irradiate it with a UV lamp (40W) for 2-10 minutes at a distance of about 22cm. The lethality rate reaches more than 90%. Coat the plate and incubate at 30°C for 48 hours.
  • the mutant strains StrainKL-2-07/StrainKL-2-08 and StrainKM-2-07/StrainKM-2-08 obtained through the above screening were transferred to liquid culture medium 1, cultured with shaking at 30°C and 200rpm for 24 hours, and then centrifuged. Remove the upper culture medium, then transfer it to liquid culture medium 2, and culture it with shaking at 30°C and 200 rpm for 40 hours. Then start feeding and the total fermentation time is 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and a high-pressure homogenizer was used to disrupt the bacterial cells. The protein was removed by centrifugation. The supernatant was passed through a 0.22 ⁇ m filter to remove impurities and the LNTII content was measured using HPLC.
  • the concentrations of LNTII in the StrainKL-2-07/StrainKL-2-08 fermentation broth are 10.32g/L and 20.44g/L respectively, and the LNTII conversion rates of lactose synthesis are 64.76% and 98.76% respectively;
  • StrainKM-2-07 The yields of LNTII in /StrainKM-2-08 fermentation broth were 10.81g/L and 20.67g/L respectively, and the LNTII conversion rates of lactose synthesis were 67.84% and 99.06% respectively, both of which were significantly higher than the starting strain.
  • Solid culture medium 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 20g/L agar powder, sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Liquid culture medium 1 20g/L tryptone, 10g/L yeast extract, 20g/L glucose is prepared by adding water, and is sterilized by high-temperature steam at 115°C for 30 minutes before use.
  • Liquid culture medium 2 20g/L tryptone, 10g/L yeast extract, 20g/L glucose, 8g/L lactose, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate and manganese sulfate with a final concentration of 5mM respectively, after 115 °C high temperature steam sterilization for 30 minutes before use.
  • Feeding materials 5g/L lactose, 20g/L glucose, dipotassium hydrogen phosphate, magnesium sulfate, and sulfur at a final concentration of 5mM.
  • Ammonium acid and manganese sulfate 5g/L lactose, 20g/L glucose, dipotassium hydrogen phosphate, magnesium sulfate, and sulfur at a final concentration of 5mM.
  • Example 16 Recombinant strains StrainKL-2-01 and/or StrainKL-2-02 and StrainKM-2-01 and/or StrainKM-2-02 catalyze the synthesis of LNTII.
  • the recombinant strains StrainKL-2-01 and/or StrainKL-2-02 and StrainKM-2-01 and/or StrainKM-2-02 were cultured in two stages to prepare LNTII.
  • the second stage is the product synthesis period: the same as in Example 3. .
  • the LNTII contents in the recombinant strains StrainKL-2-01 and/or StrainKM-2-01/fermentation broth were 4.21 mg/L and 4.25 mg/L respectively, and the LNTII conversion rates of lactose synthesis were 26.41% and 26.42% respectively, measured using biosensors.
  • the ethanol contents in the fermentation broth of the original starting strain StrainKM-0 were approximately 22.0g/L and 22.2g/L respectively.
  • the LNTII contents in the recombinant strains StrainKL-2-02 and/or StrainKM-2-02/fermentation broth were 7.18g/L and 7.28g/L respectively, and the LNTII conversion rates of lactose synthesis were 45.68% and 45.88% respectively, measured using biosensors.
  • the ethanol contents in the fermentation broth of the original starting strain StrainKM-0 were approximately 24.3g/L and 24.1g/L respectively.
  • the specific conditions of the culture medium in this example are as follows:
  • Liquid culture medium same as Example 3.
  • Feeding materials 2g/L lactose, 20g/L glucose.
  • Feeding materials 2g/L lactose, 20g/L glucose, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate and manganese sulfate with a final concentration of 5mM respectively.
  • HPLC High-performance liquid phase analysis
  • the peak time of LNTII standard is 12.10min.
  • the HPLC chart in Figure 2 shows that the fermentation products of the recombinant strains StrainKL-2-01 and/or StrainKM-2-01, StrainKL-2-02 and/or StrainKM-2-02 in Examples 4 and 6 have relatively strong activity at 12.10 min. The peak appears, which is consistent with the peak time of the LNTII standard.
  • Figure 3-4 HPLC chart clearly shows that the purified fermentation broth of the recombinant strains StrainKL-2-01 and/or StrainKM-2-01, StrainKL-2-02 and/or StrainKM-2-02 in Examples 4 and 6 A strong absorption peak appeared at 12.10 min, which was consistent with the peak time of the LNTII standard. It was initially determined that the absorption peak at 12.10 min in Examples 4 and 6 may be lactose-N-triose (LNTII), and LNTII was produced during the fermentation process. .
  • LNTII lactose-N-triose
  • HPLC patterns of the purified fermentation broth of the recombinant strains StrainKL-2-03 and StrainKM-2-03 in Example 5 and the recombinant strains StrainKL-2-04 and StrainKM-2-04 in Example 7 and the recombinant strains in Example 4 and 6 The HPLC patterns of the fermentation broth were consistent.
  • the molecular formula of LNTII is C 20 H 35 NO 16 and the molecular weight is 545.495.
  • the molecular weight of LNTII+H is 546.2029.
  • the molecular weight of LNTII+Na is 568.485.
  • the molecular weight of LNTII+K is 584.593.
  • Figure 5 shows that the molecular weight of LNTII standard LNTII+H is 546.2029, the molecular weight of LNTII+Na is 568.1851, and the molecular weight of LNTII+K is 584.1552.
  • Figure 6 shows that in the synthetic product obtained from the fermentation of StrainKL-2-02 and/or StrainKM-2-02 in Example 6, there is a material component with an absorption peak at 12.10 min.
  • the peak with a molecular weight of 546.2039 in the mass spectrometry analysis spectrum is the same as that of the standard substance.
  • LNTII+H is similar
  • the peak with a molecular weight of 568.1859 is similar to the LNTII+Na of the standard product
  • the peak with a molecular weight of 584.1559 is similar to the LNTII+K of the standard product.
  • strains StrainKL-2-01, StrainKL-2-03, StrainKL-2-04, StrainKL-2-05, StrainKL-2-06, and StrainKM-2-01 also have absorption peaks at 12.10 min.
  • StrainKM-2-03, StrainKM-2-04, StrainKM-2-05, and StrainKM-2-06 also synthesized the same substances as the recombinant strains StrainKL-2-02 and StrainKM-2-02, that is, the recombinant strain StrainKL- 2-01, StrainKL-2-02, StrainKL-2-03, StrainKL-2-04, StrainKL-2-05, StrainKL-2-06, StrainKM-2-01, StrainKM-2-02, StrainKM-2- 03. StrainKM-2-04, StrainKM-2-05, and StrainKM-2-06 catalytically synthesized LNTII.
  • Example 18 Comparison of the growth curves of the control strain StrainKL-1 and the original strain StrainKL-0.
  • the present invention uses recombinant yeast of acetylglucosaminyl transferase ⁇ -N-acetylglucosaminyl transferase to synthesize LNTII for the first time.
  • the recombinant yeast containing acetylglucosaminyl transferase ⁇ -N-acetylglucosaminyl transferase has very excellent ability to synthesize LNTII.
  • recombinant yeast containing acetylglucosaminyl transferase derived from Helicobacter pylori which synthesizes lactose
  • the conversion rate of LNTII is much higher than that of recombinant yeast containing acetylglucosaminyl transferase derived from Neisseria meningitidis and acetylglucosaminyl transferase derived from Salmonella enterica Recombinant yeast enzyme ⁇ -N-acetylglucosaminyl transferase, and the conversion rate of lactose synthesis LNTII is higher than that in E. coli.

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Abstract

提供一种用于生成乳糖-N-三糖的重组酵母及其应用。该重组酵母含有乙酰氨基葡萄糖转移酶β-N-acetylglucosaminyl transferase的氨基酸序列及其编码核酸序列，和/或以及标志物基因的破坏。

Description

重组酵母菌及其应用
本申请要求于2022年03月11日提交中国专利局、申请号为202210234497.8、发明名称为“重组酵母菌及其应用”以及于2022年11月21日提交中国专利局、申请号为202211453172.5、发明名称为“重组酵母菌及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及用于生成乳糖-N-三糖的重组酵母及其应用，属于生物工程领域。
背景技术
母乳低聚糖(Human milk oligosaccharides，HMOs)是一类复合低聚糖，由单糖及衍生物、唾液酸等结构单元通过糖苷键连接而成。
HMOs添加的婴幼儿配方奶粉以及膳食补充剂等，已经在美国、欧盟、澳大利亚和新西兰等国家市场上销售。
目前，欧盟EFSA、美国FDA均批准了乳糖-N-四糖(Lacto-N-tetraose，LNT)、乳糖-N-新四糖(Lacto-N-neotetraose，LNnT)的上市，批准上市的乳糖-N-四糖(LNT)、乳糖-N-新四糖(LNnT)均采用微生物发酵法生产。
21世纪以来，随着研究方法的进步，科学家发现母乳总HMOs(酸性和中性低聚糖)浓度随泌乳期延长而下降；因此，及时足量的补充HMOs将有利于维护婴幼儿的健康成长。然而，HMOs的合成一直是合成生物学/化学工作者面临的巨大挑战，从生物体内糖链的合成来看，这类分子的合成不是单一模板的复制，而是需要多种糖基转移酶和糖苷酶共同调控，这也就决定了糖链结构的复杂性、多样性和微观不均一性。
乳糖-N-三糖(英文名称Lacto-N-trioseII，简称LNTII，又称GlcNAc-β1,3-Gal-β1,4-Glc，CAS：75645-27-1，化学结构式如式1)。
LNTII不仅是HMOs的一种重要的寡糖，而且也是合成乳糖-N-四糖(Lacto-N-tetraose，LNT)、乳糖-N-新四糖(Lacto-N-neotetraose，LNnT)的重要前体。虽然有文献公开了LNTII制备技术或制备策略，但因其结构相对复杂，收率、立体和区域选择性控制是困扰寡糖合成的难题。因此，安全性好、特异性高的可持续的LNTII生产技术对于HMOs的产业化显得尤为重要。LNTII可由酶催化N-乙酰氨基葡萄糖(GlcNAc)和β-乳糖形成β-1,3-糖苷键合成 (Nyffenegger等，Backbone structures in human milk oligosaccharides:trans-glycosylation by metagenomicβ-N-acetylhexosaminidases.Applied Microbiology and Biotechnology,2015；99:7997-8009)。。
生物合成技术因特异性高、可规模化量产等优点近年来受到广泛关注。目前关于人乳寡糖的合成主要有细胞工厂和酶催化两种策略。酶催化法虽然具有反应副产物少、可控性强的优势，但是其催化效率较低，如专利CN202010386045.2公开了Haloferula sp.来源的β-N-乙酰氨基己糖苷酶在合成LNTII中的应用，其利用底物-几丁质作为氨糖供体，其转化率仅有14％以下。而全细胞催化直接利用体内高能的活化底物形式，如UDP-GlcNAc，是一种更容易实现高转化效率的方法，如中国专利CN201680052611.8公开了一种经遗传修饰的微生物宿主细胞产生所需寡糖的方法，CN202010834657.3公开了一种提高LNTII的基因工程菌及生产方法。目前，已报道生产LNTII的宿主的主要以大肠杆菌居多，但大肠杆菌内毒素的去除是大规模工业生产中的一个重大挑战。因此，寻找产量稳定、生产效率高，且安全性好的宿主合成LNTII对于人乳寡糖的产业化应用和推广具有十分重要的价值。
发明内容
发明目的：针对现有的技术难点及存在的问题，提供一种安全性好、特异性高并且绿色可持续的生物合成法。
本文描述的是包含与乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase基因相关的修饰的微生物菌株。
在一个方面，本发明提供了用于催化生成乳糖-N-三糖(LNTII)的酶。所述用于催化生成乳糖-N-三糖(LNTII)的酶，根据Carbohydrate-Active-Enzymes即CAZy数据库分类，属于糖苷水解酶28家族。所述糖苷水解酶28家族的酶，为乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase。
所述催化生成乳糖-N-三糖(LNTII)的酶，优选的，来自于脑膜炎奈瑟氏菌(Neisseria meningitidis)，EC No:2.4.1.56；Genebank编号AAC44084.1，其氨基酸序列如SEQ ID NO:1所示；或包含SEQ ID NO:1所示氨基酸序列，或与SEQ ID NO:1具有至少70％的氨基酸序列一致性，或包含与SEQ ID NO:1具有至少70％的氨基酸序列一致性。
所述催化生成乳糖-N-三糖(LNTII)的酶，优选的，来源于幽门螺旋杆菌Helicobacter pylori，EC No:2.4.1.129；Genebank编号WP000199766.1；其氨基酸序列如SEQ ID NO:2所示；或与SEQ ID NO:2具有至少70％的氨基酸序列一致性，或包含SEQ ID NO:2所示氨基酸序列，或包含与SEQ ID NO:2具有至少70％的氨基酸序列一致性。
所述催化生成乳糖-N-三糖(LNTII)的酶，优选的，来源于肠道沙门氏菌(Salmonella  enterica),EC No:2.4.1.155；Genebank编号WP_140239824.1，其具有SEQ ID NO:3所示氨基酸序列；或与SEQ ID NO:3具有至少70％的氨基酸序列一致性，或包含SEQ ID NO:3所示氨基酸序列，或包含与SEQ ID NO:3具有至少70％的氨基酸序列一致性。
所述生成乳糖-N-三糖的酶，包括氨基酸序列与SEQ ID NO:1、或SEQ ID NO:2、或SEQ ID NO:3具有至少70％、至少71％、至少72％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、至少100％的序列一致性的酶，且具有产生乳糖-N-三糖的活性。
如附图1所示，SEQ ID NO:1与SEQ ID NO:2与SEQ ID NO:3的差异较大。SEQ ID NO:1与SEQ ID NO:2的氨基酸序列一致性为12.5％。SEQ ID NO:1与SEQ ID NO:3的氨基酸序列一致性为44.44％，SEQ ID NO:2与SEQ ID NO:3的氨基酸序列一致性为5.28％。
在一些实施例中，本发明描述了一种重组细胞，其包含一种或多种(例如两种、若干种)异源多核苷酸。优选的，所述异源多核苷酸选自编码乙酰氨基葡萄糖基转移酶的异源多核苷酸，其具有生成LNTII的能力。
所述重组细胞，优选的，包含上述SEQ ID NO:1和/或SEQ ID NO:2和/或SEQ ID NO:3所示用于生成乳糖-N-三糖的酶的氨基酸序列及其编码核酸序列
在一些实施例中，本发明描述了以酵母细胞为宿主，借助其自身的代谢途径并导入异源的糖苷水解酶28家族的乙酰氨基葡萄糖基转移酶，高效合成了乳糖-N-三糖(Lac-N-Triose II，LNTII)。
在一些实施例中，所述重组细胞是酵母细胞，例如假丝酵母属、汉逊酵母属、克鲁维酵母属、毕赤酵母属、酵母属、裂殖酵母属、或耶氏酵母属细胞；进一步优选的，为酿酒酵母(Saccharomyces cerevisiae)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxinus)、解脂耶氏酵母(Yarrowia lipolytica)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccha romyces pastorianus)等细胞；酿酒酵母(Saccharomyces cerevisiae)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxinus)和解脂耶氏酵母(Yarrowia lipolytica)均为通过美国食品药物管理局GRAS食品安全认证的酵母细胞，在食品或食品添加剂安全管理方面，更容易被接受。
本发明提供了乳糖-N-三糖(LNTII)的新型生产工艺，可以通过发酵简便合成乳糖-N-三糖(LNTII)，既简化了合成步骤，同时还提高了目的产物的安全性。
在一个方面，所述重组酵母，包含(1)能够产生乳糖-N-三糖的乙酰氨基葡萄糖基转移酶和 (2)对标志物基因的破坏。
术语“破坏”意指参考基因的编码区和/或控制序列被部分或完全修饰(例如通过缺失、***和/或取代一个或多个核苷酸)，从而使得编码的多肽的表达不存在(失活)或降低，和/或编码的多肽的酶活性不存在或降低。可以使用本领域已知的技术测量破坏效果，如使用来自在此参考的无细胞提取物测量值检测乳糖消耗量的不存在或降低。
所述标志物基因，包含β-半乳糖苷酶或者β-半乳糖苷酶的部分氨基酸序列，与SEQ ID NO:4和/或SEQ ID NO:5至少50％、至少51％、至少52％、至少53％、至少54％、至少55％、至少60％、至少61％、至少62％、至少63％、至少64％、至少65％、至少66％、至少67％、至少68％、至少69％、至少70％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、至少100％的氨基酸序列一致性。
优选的，所述重组酵母，为乳酸克鲁维重组酵母细胞；进一步优选的，其包含至少一个标志物基因被破坏，并且含有所述标志物的氨基酸序列，以及编码所述标志物的核酸序列。所述标志物基因的核酸序列Genebank编号M84410.1；其氨基酸序列Genebank编号AAA35265.1。
优选的，所述重组酵母，为马克斯克鲁维重组酵母细胞；进一步优选的，其包含至少一个标志物基因被破坏，并且包括所述标志物的氨基酸序列，以及编码所述标志物的核酸序列。所述标志物基因的核酸序列Genebank编号XM_022818497.1；其氨基酸序列Genebank编号XP_022675157.1。
在一个实施例中，所述标志物基因为β-半乳糖苷酶基因lac4。所述标志物基因的破坏，优选的，是指lac4基因的破坏。所述lac4基因的破坏，优选的，是指部分标志物基因的破坏。
所述重组乳酸克鲁维酵母菌株，优选的，为乳酸克鲁维重组酵母菌株KL-NMLgtA和/或KL-SELgtA和/或KL-HPLgtA。
所述重组马克斯克鲁维重组酵母菌株，优选的，为马克斯克鲁维重组酵母菌株KM-NMLgtA和/或KM-SELgtA和/或KM-HPLgtA。
优选的，本发明所述重组乳酸克鲁维酵母菌株StrainKL-2-02，其分类命名为：Kluyveromyces lactis，保藏编号为:CCTCC NO:M 2022118。
优选的，本发明所述重组酵母，可通过紫外诱变方法对其进行遗传学改造，进一步提高LNTII的产量。
本发明还提供了所述的重组酵母细胞的表达方法，包括：(a)培养亲本菌株；(b)破坏在(a)的亲本菌株中的标志物基因；(c)导入能够产生乳糖-N-三糖的酶；优选的，还包括(d)分离所述重组菌株的步骤。
所述“亲本”或“亲本菌株”，是指对其进行破坏以产生在此描述的重组菌株的菌株。亲本可以是天然存在的(野生型)或预先修饰的菌株。
优选的，所述酵母菌株为克鲁维酵母(Kluyreromyces sp.)菌株；更优选的，为乳酸克鲁维酵母菌株K.lactic和/或马克斯克鲁维酵母K.marxianus；进一步优选的，为乳酸克鲁维酵母菌株K.lactic DSM70799和/或马克斯克鲁维酵母K.marxianus DMKU3-1042。
本发明所述乳酸克鲁维重组酵母菌株以及马克斯克鲁维重组酵母菌株在制备乳糖-N-三糖LNTII中的应用，其自身代谢合成的UDP-乙酰葡萄糖胺(UDP-GlcNAc)和乳糖为反应供体，在异源导入的28家族糖苷水解酶的催化下生成乳糖-N-三糖(LNTII)。
本发明还提供了所述乳酸克鲁维重组酵母和/或马克斯克鲁维重组酵母在生成乳糖-N-三糖方面的应用。
优选的，所述应用的培养基是含水溶液；更优选的，所述应用的培养基是含有乳糖和/或葡萄糖和/或果糖和/或蔗糖和/或半乳糖的水溶液。优选的，所述含水溶液需添加所述乳酸克鲁维重组酵母菌株和/或马克斯克鲁维重组酵母菌株。
优选的，所述应用的培养温度为15-60℃；进一步优选的，所述应用的培养温度为28-32℃。优选的，所述重组酵母在生成乳糖-N-三糖方面的应用，培养基中还含有无机盐和/或微量元素；优选的，所述无机盐和/或微量元素选自磷酸氢二钾、硫酸镁、硫酸铵、硫酸锰、硫酸钾或它们的任意组合；优选的，所述无机盐或微量元素的终浓度为3-7mM。
本发明还提供了所述乳糖-N-三糖(LNTII)的生产方法，该方法包括：(a)在适合的条件下，在可发酵的培养基中培养所述重组酵母细胞，以生产乳糖-N-三糖(LNTII)；并且(b)回收该乳糖-N-三糖(LNTII)。
所述乳糖-N-三糖(LNTII)的生产方法，优选的，通过添加活性炭等方法进行分离纯化后，得到的LNTII。
所述乳糖-N-三糖(LNTII)的生产方法，优选的，利用所述重组酵母进行混合发酵，获得LNTII。
术语解释：术语“序列一致性”是指两个氨基酸序列之间或两个核苷酸序列之间的相关性，通过参数“序列一致性”来描述，又称“序列一致性”。
术语“宿主细胞”意指对用核酸构建体或表达载体进行的转化、转染、转导等是易感的任何细胞类型。术语“宿主细胞”涵盖由于复制期间发生的突变而与亲本细胞不同的亲本细胞的 任何后代。
术语“重组细胞”定义为包括一种或多种(例如，两种、若干种)异源多核苷酸的非天然存在的宿主细胞。宿主细胞可以是在此描述的突变菌株，或被进一步破坏以提供在此描述的突变菌株。
有益技术效果：
本发明提供了一种新的乳糖-N-三糖(LNTII)的制造方法，利用重组克鲁维酵母(乳酸克鲁维重组酵母和***克鲁维重组酵母)为细胞工厂，合成人乳寡糖中的LNTII。与以往的方法，整个制造过程简单可控，生物安全性高，易扩大规模，原料来源广泛，产品制备成本低，实用价值高，以乳糖为底物合成乳糖-N-三糖(LNTII)的转化率更高。
本发明证明了重组酵母细胞在实现人乳寡糖的高效生产方面具有巨大潜力，为乳糖-N-三糖(LNTII)及其类似物的大规模工业生产奠定了基础，具有重要的经济价值和社会效益。除了安全性外，本发明所用原料易得，方法简便易行，适合产业化生产，具有优异的应用前景。
附图说明
图1.SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示氨基酸序列的比对结果图。
图2.为实施例3中乳酸克鲁维酵母原始菌株、对照菌株StrainKL-1合成产物的HPLC色谱图。
图3.为实施例4中乳酸克鲁维酵母菌株StrainKL-2-01和实施例6中重组菌株StrainKL-2-02发酵液的合成产物及LNTII标准品的HPLC色谱图。
图4.为实施例4中乳酸克鲁维重组酵母菌株StrainKL-2-01发酵液纯化后合成产物及LNTII标准品的HPLC色谱图。
图5.为实施例6中乳酸克鲁维酵母重组菌株StrainKL-2-02发酵液纯化后合成产物及LNTII标准品的HPLC色谱图。
图6.LNTII标准品LC-MS分析色谱图。
图7.为图5中乳酸克鲁维重组酵母菌株StrainKL-2-02发酵液出峰时间为12.10min产物的LC-MS分析图谱。
图8.为图5中乳酸克鲁维重组酵母菌株StrainKL-2-02发酵液出峰时间为12.10min产物的1H-NMR分析图谱。
图9.为图5中乳酸克鲁维重组酵母菌株StrainKL-2-02发酵液出峰时间为12.10min产物的13C-NMR分析图谱。
具体实施方式
应该指出，以下说明和实施例都是示例性的，旨在对本申请提供进一步的说明。除非另 有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的含义相同。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列具体实施方式中如果未注明具体条件的实验方法，通常按照本领域技术内的分子生物学的常规方法和条件，这种技术和条件在文献中有完整解释。参见例如Sambrook等人，《分子克隆：实验手册》中所述的技术和条件，或按照制造厂商所建议的条件。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件进行。
本发明的具体实施方式中，以利用生物安全性好且工业应用广泛的乳酸克鲁维酵母和/或马克斯克鲁维酵母为宿主。敲除乳酸克鲁维酵母和/或马克斯克鲁维酵母的β-半乳糖苷酶基因lac4，避免乳糖在酵母菌株内被消耗；并将来自于Neisseria meningitidis的编码乙酰氨糖转移酶的基因NMLgtA和/或来自于Salmonella enterica的编码乙酰氨糖转移酶的基因(SELgtA)和/或来自于Helicobacter pylori的编码乙酰氨糖转移酶的基因(HPLgtA)基因分别整合进乳酸克鲁维酵母或马克斯克鲁维酵母基因组，获得合成乳糖-N-三糖(LNTII)的菌株。
对酵母细胞进行两阶段培养。第一阶段为菌体生长期：以葡萄糖为碳源培养细胞，进行生物量和酶的积累，至细胞生长进入对数后期或稳定期。第二阶段为产物合成期：首先是30℃，200rpm下震荡培养至40h时，再向培养基中补加2g/L的乳糖、20g/L的葡萄糖和终浓度为3-7mM的金属离子,最终生成乳糖-N-三糖(LNTII)。
宿主细胞产生的蛋白质可以通过任何已知的用于分离和纯化蛋白质的工艺获得。这些工艺包含但不限于盐析和溶剂沉淀、超滤、凝胶电泳、离子交换色谱、亲和色谱、反相高效液相色谱、疏水相互作用色谱、混合模式色谱、羟基磷灰石色谱和等电聚焦。
本发明的具体实施方式中还提供了一种合成乳糖-N-三糖(LNTII)的方法。
具体的，所述生产方法包括：分别将来源于脑膜炎奈瑟氏菌(Neisseria meningitidis)、肠道沙门氏菌(Salmonella enterica)和幽门螺旋杆菌(Helicobacter pylori)的编码乙酰氨基葡萄糖基转移酶的基因增幅并构建适用于乳酸克鲁维酵母和/或马克斯克鲁维酵母的表达盒；将表达盒导入乳酸克鲁维酵母DSM70799和/或马克斯克鲁维酵母DMKU3-1042中，整合到菌 株的基因组中；筛选出阳性酵母转化子；基因工程菌发酵培养，获得乳糖-N-三糖(LNTII)；利用高效液相色谱、质谱和核磁共振色谱对发酵液及其纯化后产物进行鉴定。
以下实施例中，大肠杆菌BL21和表达载体pET-28a购自于生工生物工程(上海)股份有限公司。下列实施例中未注明具体条件的实验方法，按照常规条件进行，例如《分子克隆：实验室手册》中所述的条件，或按照相应生物学试剂的制造厂商所建议的条件。PCR扩增反应的反应程序可以为常规的PCR扩增反应程序。酿酒酵母(Saccharomyces cerevisiae)在ATCC菌种保藏中心保藏号为CICC1964。
出于清楚理解的目的，已经通过说明以及实例的方式相当详细描述了本发明，本领域普通技术人员将清楚的是，可以实施任何等效方面或修饰。因此，该说明和实例不应当解释为限制本发明的范围。
高效液相(HPLC)色谱检测：
仪器：126-四级杆飞行时间高分辨质谱仪(液质联用)。
高效液相(HPLC)色谱检测条件：色谱柱：Asahipak NH2P-504E 4.6mmx250mm，5μm，流动相：乙腈:水＝65:35，流速：1mL/min，检测器：UV-VIS检测器，波长：210nm，柱温：30℃，进样量：10μl，运行时间30min。
LC-MS检测：H-ESI模式，分子量扫描范围400～900。
NMR分析：
仪器：安捷伦DD2-600光谱仪。
检测方法：室温下，1H-NMR谱(600MHz)；13C-NMR谱(150MHz)。化学位移以百万分之一(ppm)表示，以D2O内部四甲基硅烷为基准(0ppm)往后开始计算。化学位移和耦合常数从光谱的一级分析计算。
LNTII标准品购买于法国ELICITYL公司，如上HPLC色谱条件，出峰时间rt＝12.10min。
实施例1.ΔLAC4标志物KL和/或KM敲除盒以及糖苷水解酶28家族的酶基因的KL和/或KM表达盒的构建。
1.标志物KL-ΔLAC4和/或KM-ΔLAC4敲除盒的构建：
1.1引物设计见表1：
表1

1.2 PCR扩增
以乳酸克鲁维酵母基因组或马克斯克鲁维酵母基因组为模板，以LAC4Pro-Nde1和LAC4Pro-Not1为引物PCR扩增LAC4启动子区，以LAC4Tef-Kpn1和LAC4Tef-Bamh1为引物PCR扩增LAC4终止序列区。以带有遗传霉素(Geneticin)抗性基因KanMX4(核苷酸序列如SEQ ID NO:9所示)的重组质粒pUG6的DNA为模板，经PCR扩增抗性基因KanMX4的DNA片段。琼脂糖凝胶电泳验证条带大小正确后切取条带，用OMEGA凝胶提取试剂盒回收基因片段。
PCR扩增组分：Phanta Super-Fidelity DNA Polymerase 1μL，2×Phanta Max Buffer 25μL，dNTPs(10mM)1μL，引物F 2μL，引物R 2μL，模板DNA 1μL，ddH2O 18μL，总体积50μL。
PCR扩增步骤：(1)预变性95℃、3min；(2)变性95℃、0.25min；(3)退火54℃、0.5min；(4)延伸72℃、0.5min；步骤(2)-(4)循环30次；(5)终延伸72℃、5min；(6)保存4℃。
以PUG6质粒为模板，PUG6-Kpn1、PUG6-Bamh1为引物(PUG6上无Kpn1、Bamh1的酶切位点，需PCR扩增引入所需酶切位点)进行PCR扩增。
PCR扩增组分：Phanta Super-Fidelity DNA Polymerase 1μL，2×Phanta Max Buffer 25μL，dNTPs(10mM)1μL，引物F 2μL，引物R 2μL，模板DNA 1μL，ddH2O 18μL，总体积50μL。
PCR扩增步骤：(1)预变性95℃、3min；(2)变性95℃、0.25min；(3)退火54℃、0.5min；(4)延伸72℃、3min；步骤(2)-(4)循环30次；(5)终延伸72℃、5min；(6)保存4℃。
PCR反应程序结束后，进行琼脂糖凝胶电泳鉴定，将大小符合预期的条带利用凝胶回收试剂盒进行纯化回收。
1.3构建标志物KL-ΔLAC4和KM-ΔLAC4敲除盒
通过两次酶切连接先后将LAC4启动子区和终止序列区连入PUG6载体，构建KL-ΔLAC4和KM-ΔLAC4敲除盒。
第一次酶切连接：将1.2步骤中获得的终止序列与PUG6载体分别用Kpn1、Bamh1进行 双酶切，酶切温度37℃、时间约2h。酶切体系：DNA片段XμL(DNA总量约2-3ng)，Kpn1 2μL，Bamh1 2μL，Buffer 5μL，ddH2O 41-XμL，总体积50μL。
酶切结束后进行琼脂糖凝胶电泳鉴定，将大小符合预期的条带利用凝胶回收试剂盒进行纯化回收。将纯化回收的片段用T4DNA连接酶进行连接：温度22℃、时间约30min。连接体系：DNA片段XμL(终止序列摩尔数：PUG6载体摩尔数＝3:1)，Buffer 1μL，ddH2O 9-XμL，总体积10μL。
连接完成后及时进行大肠杆菌转化。挑取正确的转化子进行质粒提取，以此质粒为新的载体进行第二次酶切连接。
第二次酶切连接：将已连入终止序列的PUG6载体(载体中本身携带Nde1、Not1酶切位点)与启动子区分别用Nde1、Not1进行双酶切，酶切温度37℃、时间约2h。酶切结束后进行琼脂糖凝胶电泳鉴定，将大小符合预期的条带利用凝胶回收试剂盒进行纯化回收。将纯化回收的片段用T4DNA连接酶进行连接。连接完成后及时进行大肠杆菌转化。挑取正确的转化子进行质粒提取，获得携带ΔLAC4敲除盒的敲除载体。
2.含有乙酰氨糖转移酶编码基因的表达盒lgtA KL和lgtA KM的建立:
所述lgtA KL表达盒，包括其KL-ΔLAC4::NMLgtA、KL-ΔLAC4::HPLgtA、KL-ΔLAC4::SELgtA、KL-18S::NMLgtA、KL-18S::HPLgt A、KL-18S::SELgt A表达盒。
所述lgtA KM表达盒，包括其KM-ΔLAC4::NMLgtA、KM-ΔLAC4::HPLgtA、KM-ΔLAC4::SELgtA、KM-18S::NMLgtA、KM-18S::HPLgt A、KM-18S::SELgt A表达盒。
2.1NMLgtA/SELgtA/HPLgtA编码区、ADH1终止区、半乳糖苷酶LAC4启动子区融合序列的获取：
全基因合成NMLgtA、HPLgtA、SELgtA，其氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示。
启动子区序列：如SEQ ID NO:6所示，来源于乳酸克鲁维酵母LAC4启动子区序列；如SEQ ID NO:7所示，来源于马克斯克鲁维酵母LAC4启动子区序列。
ADH1终止区序列，如SEQ ID NO:8所示，来源于酿酒酵母
分别利用NMLgtA/HPLgtA/SELgtA基因、乳酸克鲁维酵母基因组、马克斯克鲁维基因组、酿酒酵母基因组为模板进行PCR扩增。
PCR扩增组分：Phanta Super-Fidelity DNA Polymerase 1μL，2×Phanta Max Buffer 25μL，dNTPs(10mM)1μL，引物F 2μL，引物R 2μL，模板DNA 1μL，ddH2O 18μL，总体积50μL。
PCR扩增步骤：(1)预变性95℃、3min；(2)变性95℃、0.25min；(3)退火54℃、 0.5min；(4)延伸72℃、1.5min；步骤(2)-(4)循环30次；(5)终延伸72℃、5min；(6)保存4℃。
PCR反应程序结束后，进行琼脂糖凝胶电泳鉴定，将大小符合预期的条带利用凝胶回收试剂盒进行纯化回收。将回收后产物利用微量测定仪进行DNA浓度测量，利用ddH2O稀释其浓度至1ng/μL。以稀释后的DNA片段为模板，ADH1-Not1和LAC4Pro-Nde1/LAC4Pro-S为引物进行融合PCR扩增。
PCR扩增组分：Phanta Super-Fidelity DNA Polymerase 1μL，2×Phanta Max Buffer 25μL，dNTPs(10mM)1μL，引物F 2μL，引物R 2μL，模板DNA 1μL，ddH2O 18μL，总体积50μL。
PCR扩增
步骤：(1)预变性95℃、3min；(2)变性95℃、0.25min；(3)退火54℃、0.5min；(4)延伸72℃、2.5min；步骤(2)-(4)循环30次；(5)终延伸72℃、5min；(6)保存4℃。
PCR反应程序结束后，进行琼脂糖凝胶电泳鉴定，将大小符合预期的条带利用凝胶回收试剂盒进行纯化回收，得到NMLgtA/SELgtA/HPLgtA编码区、ADH1终止区、半乳糖苷酶LAC4启动子区融合序列。
2.2.通过PCR扩增获得启动子区、终止序列区、PUG6载体序列、PUG6载体抗性序列
以乳酸克鲁维酵母基因组、马克斯克鲁维基因组和PUG6载体为模板进行PCR扩增。
PCR扩增组分：Phanta Super-Fidelity DNA Polymerase 1μL，2×Phanta Max Buffer 25μL，dNTPs(10mM)1μL，引物F 2μL，引物R 2μL，模板DNA 1μL，ddH2O 18μL，总体积50μL。
PCR扩增：
步骤：(1)预变性95℃、3min；(2)变性95℃、0.25min；(3)退火54℃、0.5min；(4)延伸72℃、1.5min；步骤(2)-(4)循环30次；(5)终延伸72℃、5min；(6)保存4℃。
PCR反应程序结束后，进行琼脂糖凝胶电泳鉴定，将大小符合预期的条带利用凝胶回收试剂盒进行纯化回收。
2.3表达盒的获取
将2.1步骤获得的融合序列与ΔLAC4敲除载体分别用Nde1、Not1进行双酶切，酶切温度37℃、时间约2h。酶切结束后进行琼脂糖凝胶电泳鉴定，将大小符合预期的条带利用凝胶回收试剂盒进行纯化回收。将纯化回收的片段用T4DNA连接酶进行连接。连接完成后及时 进行大肠杆菌转化。挑取正确的转化子进行质粒提取，获得携带NMLgtA和/或SELgtA和/或HPLgtA表达盒的表达载体KL-ΔLAC4::NMLgtA和/或KL-ΔLAC4::SELgtA和/或KL-ΔLAC4::HPLgtA，以及KM-ΔLAC4::NMLgtA和/或KM-ΔLAC4::SELgtA和/或KM-ΔLAC4::HPLgtA。
将2.1步骤获得的融合序列与2.2步骤的上下游同源臂序列、骨架序列、抗性序列按照总体积5μL、摩尔比1:1:1:1:1的比例混合后加入5μL无缝克隆MIX进行连接。连接温度50℃，时间30-60min。连接完成后及时进行大肠杆菌转化，挑取正确的转化子进行质粒提取，获得携带NMLgtA和/或SELgtA和/或HPLgtA表达盒的表达载体KL-18S::NMLgtA和/或KL-18S::SELgtA和/或KL-18S::HPLgtA，以及KM-18S::NMLgtA和/或KM-18S::SELgtA和/或KM-18S::HPLgtA(酶切、连接、大肠杆菌转化方法均同实施例1中的1.3)。
本实施例所用试剂及试剂盒来源如表2所示
表2
本实施例所用溶剂及缓冲液配方如下所示
50×TAE溶液：使用ddH2O配制，其中含有2M Tris，100mM Na2EDTA·H2O，2％SDS，调pH至8.5。
基因组抽提buffer：使用ddH2O配制，其中含有200mM Tris-HCl，250mM NaCl，2％SDS，25mM EDTA，调pH至8.0。
大肠杆菌培养基：10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠和水配制而成，固体培养基需额外添加20g/L琼脂粉。采用经121℃高温蒸汽灭菌20min后备用。
酵母培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖、加入水配制而成，固体培养基需额外添加20g/L琼脂粉。采用经115℃高温蒸汽灭菌30min后备用。
本实施例所用培养条件：
大肠杆菌在温度设置为37℃的恒温培养箱中进行固体平板培养，在温度设置为37℃，转速设定为200rpm的摇床中进行摇瓶培养。
酵母在温度设置为30℃的恒温培养箱中进行固体平板培养，在温度设置为30℃，转速设定为200rpm的摇床中进行摇瓶培养。
本实施例酵母基因组提取方法：
(1)将乳酸克鲁维酵母单菌落挑取至1mL YPD(10mL离心管)培养基中，30℃，200rpm过夜培养；
(2)取600μL菌液于1.5毫升EP管中，10000rpm离心1min，去上清；
(3)加入600-800μL的基因组抽提buffer，100μL石英砂，充分震荡5min；
(4)65℃水浴30min，每10min颠倒一次；
(5)取上清于新的1.5毫升EP管中，加入等体积的DNA提取液，吹打混匀；
(6)13000rpm离心10min。取400μL上清于新的1.5毫升EP管中；
(7)加入0.6倍体积的异丙醇和40μL 3M醋酸钠，混匀，-20℃静置30min；
(8)13000rpm离心10min，弃上清，得到酵母基因组；
(9)用70％的乙醇洗涤两次，待乙醇挥发后溶于ddH2O中，-20℃保存。
实施例2：乳酸克鲁维酵母和马克斯克鲁维酵母重组菌株的构建：
1.ΔLAC4敲除盒构建成功后进行酵母转化，分别得到对照菌株StrainKL-1和StrainKM-1，酵母转化方法见本实施例。
2.KL-ΔLAC4::NMLgtA和/或KL-ΔLAC4::SELgt A和/或KL-ΔLAC4::HPLgt A，以及KL-18S::NMLgtA和/或KL-18S::SELgt A和/或KL-18S::HPLgt A表达盒构建成功后分别进行酵母转化，得到重组菌株StrainKL-2-01/StrainKL-2-05/StrainKL-2-02和重组菌株StrainKL-2-03/StrainKL-2-05/StrainKL-2-04。
KM-ΔLAC4::NMLgtA和/或KM-ΔLAC4::SELgt A和/或KM-ΔLAC4::HPLgt A，以及KM-18S::NMLgtA和/或KM-18S::SELgt A和/或KM-18S::HPLgt A表达盒构建成功后分别进行酵母转化，得到重组菌株StrainKM-2-01/StrainKM-2-05/StrainKM-2-02和重组菌株StrainKM-2-03/StrainKM-2-06/StrainKM-2-04。
酵母转化方法具体如下：
(1)先制备酵母感受态细胞：取少量酵母菌株冻存物在平板固体培养基上划线，30℃倒置培养2天。挑取酵母单菌落于50mL液体培养基中，30℃，220rpm培养至OD600在0.8-1.5之间。收集菌体用25mL无菌水洗涤，室温1500×g离心10min，弃上清。加入1mL100mM的氯化锂缓冲液，重悬沉淀，12000rpm离心30s，弃上清。再次加入400μL 100mM 的氯化锂缓冲液，重悬沉淀，得酵母细胞感受态，按50μL/管分装，待转化用。
(2)转化：将上述制备的感受态酵母菌离心，以Tips去除残余的氯化锂溶液。对于每一个转化，按以下顺序加入：50％PEG3350(240μL)；1M LiCl(36μL)；2mg/mL单链Salmon sperm DNA(25μL)；5～10μg/50μL H2O质粒DNA(50μL)，剧烈旋涡混匀，直至沉淀菌体完全分布均匀；30℃水浴孵育30min；42℃水浴热休克20～25min；8000rpm离心10min后，收集酵母菌体；然后，重悬酵母于500μL液体培养基，30℃摇床孵育；1～4h后，取25～100μL菌液涂布于选择性培养基平板，于30℃倒置培养。
所述酵母感受态细胞，KL-ΔLAC4::NMLgtA和/或KL-ΔLAC4::SELgtA和/或KL-ΔLAC4::HPLgtA表达盒导入的是乳酸克鲁维原始出发菌株StrainKL-0；KM-18S::NMLgtA和/或KM-18S::SELgt A和/或KM-18S::HPLgtA表达盒导入的是马克斯克鲁维原始出发菌株StrainKM-0。
所述质粒DNA，为实施例1构建的KL-ΔLAC4敲除盒，和/或KL-ΔLAC4::NMLgtA和/或KL-ΔLAC4::SELgtA和/或KL-ΔLAC4::HPLgt A，以及KL-18S::NMLgtA和/或KL-18S::SELgtA和/或KL-18S::HPLgt A表达盒。
所述质粒DNA，为实施例1构建的KM-ΔLAC4敲除盒，和/或KM-ΔLAC4::NMLgtA和/或KM-ΔLAC4::SELgtA和/或KM-ΔLAC4::HPLgt A，以及KM-18S::NMLgtA和/或KM-18S::SELgtA和/或KM-18S::HPLgt A表达盒。
3.乳酸克鲁维酵母和马克斯克鲁维酵母重组菌株的验证：
平板静置培养2-3天，对长出的转化子单菌落进行验证，验证方法如下：
挑取转化子单菌落于1.5mL液体培养基1，30℃、200rpm震荡培养过夜后分别取50μL菌液转接于1.5mL液体培养基1和液体培养基2，30℃、200rpm震荡培养过夜。观察其生长情况，选取在固体培养基1中正常生长而在固体培养基2几乎无法生长的转化子保种，认为其为乳酸克鲁维重组菌株StrainKL-2-01、StrainKL-2-02、StrainKL-2-03、StrainKL-2-04、StrainKL-2-05、StrainKL-2-06以及马克斯克鲁维重组菌株StrainKM-2-01、StrainKM-2-02、StrainKM-2-03、StrainKM-2-04、StrainKM-2-05、StrainKM-2-06，其与表达盒的对应关系详见表3-4。
表3.乳酸克鲁维重组菌株与表达盒的对应关系

表4.马克斯克鲁维重组菌株与表达盒的对应关系
本实施例中培养基具体情况如下：
固体培养基1：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖、20g/L琼脂粉加入水配制而成，经115℃高温蒸汽灭菌30min后备用。
液体培养基1：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖加入水配制而成，经115℃高温蒸汽灭菌30min后备用。
固体培养基2：20g/L胰蛋白胨、10g/L酵母提取物、20g/L乳糖、20g/L琼脂粉加入水配制而成，经115℃高温蒸汽灭菌30min后备用。
液体培养基2：20g/L胰蛋白胨、10g/L酵母提取物、20g/L乳糖加入水配制而成，经115℃高温蒸汽灭菌30min后备用。
实施例3：乳酸克鲁维酵母细胞原始菌株及其对照株、马克斯克鲁维酵母细胞原始菌株及其对照株发酵实验。
分别对乳酸克鲁维酵母细胞原始菌株及其对照株、马克斯克鲁维酵母细胞原始菌株及其对照株采用两段式培养进行LNTII的合成验证实验。
第一阶段为菌体生长期：以葡萄糖为碳源培养细胞，分别进行菌体量和酶量的积累，至细胞生长进入对数末期或稳定期。将菌株划线培养于固体培养基中，30℃培养2-3天后挑取单菌落接种于1.5mL液体培养基，30℃、200rpm震荡培养过夜。随后按2％的接种量接种于50mL液体培养基摇瓶中，30℃、200rpm培养至OD600＝1，按2％的接种量接种于5L液体培养基中(10L体积发酵罐)，30℃、200rpm震荡培养进行菌体量积累。
第二阶段为产物合成期：30℃，200rpm下震荡培养至40h时，开始流加补料(记录流 加补料的量)，总发酵时长72h。发酵72h结束后，离心发酵液，使用高压匀浆破碎仪进行菌体破碎，离心除去蛋白，上清过0.22μm滤膜除杂后，用HPLC方法测定其中LNTII含量，并用生物传感器测量乳酸克鲁维、马克斯克鲁维原始出发菌株StrainKL-0发酵液中乙醇含量，结果记录于表5。
表5.乳酸克鲁维、马克斯克鲁维原始出发菌株发酵液分析
以上说明：乳酸克鲁维原始出发菌株及对照菌株、马克斯克鲁维原始出发菌株及对照菌株没有生成LNTII的能力。
本实施例中培养基具体情况如下：
固体培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖、20g/L琼脂粉，经115℃高温蒸汽灭菌30min后备用。
液体培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖，8g/L乳糖、终浓度分别为5mM的磷酸氢二钾、硫酸镁、硫酸铵和硫酸锰，经115℃高温蒸汽灭菌30min后备用。
补料：2g/L的乳糖、20g/L的葡萄糖、终浓度分别为5mM的磷酸氢二钾、硫酸镁、硫酸铵和硫酸锰。
实施例4：乳酸克鲁维重组酵母菌株StrainKL-2-01、马克斯克鲁维重组酵母菌株StrainKM-2-01催化合成LNTII。
对重组菌株StrainKL-2-01和/或StrainKM-2-01也采用两段式培养进行LNTII的制备。
第一阶段为菌体生长期：以葡萄糖为碳源培养细胞，分别进行菌体量和酶量的积累，至细胞生长进入对数末期或稳定期。将菌株划线培养于固体培养基中，30℃培养2-3天后挑取单菌落接种于1.5mL液体培养基，30℃、200rpm培养至OD600＝1，按2％(v/v)的接种量接种于5L液体培养基中(10L体积发酵罐)，30℃、200rpm震荡培养过夜。随后按2％(v/v)的接种量接种于50mL液体培养基摇瓶中，30℃、200rpm震荡培养进行菌体量积累。
第二阶段为产物合成期：除重组菌株外，其他同实施例3第二阶段。
HPLC法测得StrainKL-2-01、或StrainKM-2-01发酵液中LNTII含量分别为8.53g/L和8.75g/L，乳糖合成LNTII转化率分别为51.53％和53.53％。重组菌株StrainKL-2-01原始发酵 液的HPLC图谱见附图2曲线B，纯化后发酵液的的HPLC图谱见附图3。
重组菌株StrainKM-2-01合成产物的HPLC图谱与StrainKL-2-01合成产物的HPLC图谱相同。
另外，使用生物传感器测得StrainKL-2-01和/或StrainKM-2-01发酵液中乙醇含量分别为约为25.1g/L和25.7g/L。
本实施例中固体培养基、液体培养基、补料同实施例3。
实施例5：乳酸克鲁维重组酵母菌株StrainKL-2-03、马克斯克鲁维重组酵母菌株StrainKM-2-03催化合成LNTII。
对重组菌株StrainKL-2-03和/或StrainKM-2-03也采用两段式培养进行LNTII的制备。
第一阶段为菌体生长期：除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：除重组菌株外，其他同实施例4第二阶段。
HPLC法测得StrainKL-2-03和/或StrainKM-2-03发酵液中LNTII含量分别为7.21g/L和7.15g/L，乳糖合成LNTII转化率分别为45.24％和44.21％。另外，使用生物传感器测得乙醇含量分别约为25g/L和24.3g/L。
本实施例中固体培养基、液体培养基、补料同实施例3
实施例6：乳酸克鲁维重组酵母菌株StrainKL-2-02、马克斯克鲁维重组酵母菌株StrainKM-2-02催化合成LNTII。
对重组菌株StrainKL-2-02和/或StrainKM-2-02也采用两段式培养进行LNTII的制备。
第一阶段为菌体生长期：除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：除重组菌株外，其他同实施例4第二阶段。
HPLC法测得StrainKL-2-02和/或StrainKM-2-02发酵液中LNTII含量分别为15.23g/l和15.27g/l，乳糖合成LNTII转化率分别为95.57％和95.71％。重组菌株StrainKL-2-02原始发酵液的HPLC图谱见附图2曲线C，纯化后发酵液的HPLC图谱见附图4。
重组菌株StrainKM-2-02合成产物的HPLC图谱与StrainKL-2-02合成产物的HPLC图谱相同。
另外，使用生物传感器测得StrainKL-2-02和/或StrainKM-2-02发酵液中乙醇含量分别为约为25.2g/l和25.6g/l。
本实施例中固体培养基、液体培养基、补料同实施例3。
实施例7：乳酸克鲁维重组酵母菌株StrainKL-2-04、马克斯克鲁维重组酵母菌株StrainKM-2-04催化合成LNTII。
对重组菌株StrainKL-2-04和/或StrainKM-2-04也采用两段式培养进行LNTII的制备。
第一阶段为菌体生长期：除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：除重组菌株外，其他同实施例4第二阶段。
HPLC法测得StrainKL-2-04和/或StrainKM-2-04发酵液中LNTII含量分别为14.57g/L和14.71g/L，乳糖合成LNTII转化率分别为91.42％和92.02％。另外，使用生物传感器测得乙醇含量分别约为24.6g/L和24.9g/L。
本实施例中固体培养基、液体培养基、补料同实施例3。
实施例8：乳酸克鲁维重组酵母菌株StrainKL-2-05、马克斯克鲁维重组酵母菌株StrainKM-2-05催化合成LNTII。
对重组菌株StrainKL-2-05和/或StrainKM-2-05也采用两段式培养进行LNTII的制备。
第一阶段为菌体生长期：除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：除重组菌株外，其他同实施例4第二阶段。
HPLC法测得StrainKL-2-05和/或StrainKM-2-05发酵液中LNTII含量分别为3.23g/L和3.27g/L，乳糖合成LNTII转化率分别为20.27％和20.52％。
重组菌株StrainKM-2-05合成产物的HPLC图谱与StrainKL-2-05合成产物的HPLC图谱相同。
另外，使用生物传感器测得StrainKL-2-05和/或StrainKM-2-05发酵液中乙醇含量分别为约为26.1g/L和26.3g/L。
本实施例中固体培养基、液体培养基、补料同实施例3。
实施例9：乳酸克鲁维重组酵母菌株StrainKL-2-06、马克斯克鲁维重组酵母菌株StrainKM-2-06催化合成LNTII。
对重组菌株StrainKL-2-06和/或StrainKM-2-06也采用两段式培养进行LNTII的制备。
第一阶段为菌体生长期：除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：除重组菌株外，其他同实施例4第二阶段。
HPLC法测得StrainKL-2-06和/或StrainKM-2-06发酵液中LNTII含量分别为2.45g/L和2.17g/L，乳糖合成LNTII转化率分别为15.37％和13.61％。另外，使用生物传感器测得乙醇含量分别约为26.3g/L和27.1g/L。
本实施例中固体培养基、液体培养基、补料同实施例3。
实施例10：乳酸克鲁维重组酵母菌株StrainKL-2-01、马克斯克鲁维重组酵母菌株StrainKM-2-01催化合成LNTII。
第一阶段除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：在15℃，200rpm下震荡培养至40h时，开始流加补料，总发 酵时长72h。发酵72h结束后，离心发酵液，使用高压匀浆破碎仪，离心除去蛋白，上清过0.22μm滤膜除杂后用HPLC方法测定其中LNTII含量。
HPLC法测得StrainKL-2-01和/或StrainKM-2-01发酵液中LNTII含量分别为4.73g/L和4.67g/L，乳糖合成LNTII转化率分别为29.81％和29.68％。另外，使用生物传感器测得乙醇含量分别约为21.4g/L和21.5g/L。
本实施例中培养基具体情况如下：
固体培养基：同实施例3。
液体培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖，8g/L乳糖、终浓度分别为3mM的硫酸铵、硫酸锰，经115℃高温蒸汽灭菌30min后备用。
补料：2g/L的乳糖、20g/L的葡萄糖、终浓度分别为3mM的硫酸铵、硫酸锰。
实施例11：乳酸克鲁维重组酵母菌株StrainKL-2-01、马克斯克鲁维重组酵母菌株StrainKM-2-01催化合成LNTII。
第一阶段除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：在60℃，200rpm下震荡培养至40h时，开始流加补料进行LNTII发酵，总发酵时长72h。发酵72h结束后，离心发酵液，使用高压匀浆破碎仪，离心除去蛋白，上清过0.22μm滤膜除杂后用HPLC方法测定其中LNTII含量。
HPLC法测得StrainKL-2-01和/或StrainKM-2-01发酵液中LNTII含量分别为1.12g/L和1.15g/L，乳糖合成LNTII转化率分别为7.02％和7.07％。另外，使用生物传感器测得乙醇含量分别约为18.0g/L和18.2g/L。
本实施例中培养基具体情况如下：
固体培养基：同实施例3。
液体培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖，8g/L乳糖、终浓度分别为7mM的磷酸氢二钾、硫酸镁，经115℃高温蒸汽灭菌30min后备用。
补料：2g/L的乳糖、20g/L的葡萄糖、终浓度分别为7mM的磷酸氢二钾、硫酸镁。
实施例12：乳酸克鲁重组酵母菌株StrainKL-2-01、马克斯克鲁维维重组酵母菌株StrainKM-2-01催化合成LNTII。
第一阶段除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：在28℃，200rpm下震荡培养至40h时，开始流加补料进行LNTII发酵，总发酵时长72h。发酵72h结束后，离心发酵液，使用高压匀浆破碎仪，离心除去蛋白，上清过0.22μm滤膜除杂后用HPLC方法测定其中LNTII含量。
HPLC法测得StrainKL-2-01和/或StrainKM-2-01发酵液中LNTII含量分别为7.89g/L 和7.87g/L，乳糖合成LNTII转化率分别为49.51％和49.49％。另外，使用生物传感器测得乙醇含量分别约为24.8g/L和24.7g/L。
本实施例中培养基具体情况如下：
固体培养基：同实施例3。
液体培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖，8g/L乳糖、终浓度为6mM的硫酸镁，经115℃高温蒸汽灭菌30min后备用。
补料：2g/L的乳糖、20g/L的葡萄糖、终浓度为6mM的硫酸镁。
实施例13：乳酸克鲁维重组酵母菌株StrainKL-2-01、马克斯克鲁维重组酵母菌株StrainKM-2-01催化合成LNTII。
第一阶段除菌体外，其他同实施例4第一阶段。
第二阶段为产物合成期：除液体培养基和补料外，其他同实施例10第二阶段。
HPLC法测得StrainKL-2-01和/或StrainKM-2-01发酵液中LNTII含量分别为7.17g/L和7.15g/L，乳糖合成LNTII转化率分别为44.99％和44.97％。另外，使用生物传感器测得乙醇含量分别约为24.3mg/L和24.5mg/L。
本实施例中培养基具体情况如下：
固体培养基：同实施例3。
液体培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖，8g/L乳糖、终浓度为6mM的硫酸钾，经115℃高温蒸汽灭菌30min后备用。
补料：2g/L的乳糖、20g/L的葡萄糖、终浓度为6mM的硫酸钾。
实施例14.纯化实施例6中的合成产物。
使用活性炭吸附的方法来纯化实施例6中的合成产物，具体步骤如下：
(1)取100ml实施例6发酵液，按溶液终体积：质量＝1-2％加入活性炭，摇匀；
(2)放入摇床中，在30-40℃下150-250rpm振摇1-3h；
(3)低温冻干得到粉末，HPLC测定重组菌株StrainKL-2-02和/或StrainKM-2-02催化合成所得产物LNTII的纯度分别为97.2％和97.3％。
实施例15.紫外诱变导致的突变随机性很强，突变产生的效果也是随机的，为了获得有效的正突变，通常需要进行多轮紫外诱变进行筛选，其工作量较大，且存在无法获得有效正突变的可能性。但因为紫外诱变所需设备简单、费用少，且在短时间内能够获得大量突变体，因此，是一种常用的诱变选育方法。
申请人分别以重组菌株StrainKL-2-01、StrainKL-2-02以及StrainKM-2-01、StrainKM-2-02为出发菌株，通过紫外诱变方法对其进行遗传学改造，进一步提高其LNTII的产量。
分别将重组菌株StrainKL-2-01、StrainKL-2-02以及StrainKM-2-01、StrainKM-2-02以葡萄糖为碳源培养细胞，进行菌体量和酶量的积累，至细胞生长进入对数末期或稳定期。将菌株划线培养于固体培养基中，30℃培养2-3天后挑取单菌落接种于1.5mL液体培养基，30℃、200rpm震荡培养过夜。使用无菌水洗菌体，制成悬浮液，稀释至1×106个/mL，紫外灯(40W)照射2-10min，距离约22cm，致死率达到90％以上。涂布平板，30℃培养48h。
第一轮紫外诱变，StrainKL-2-01/StrainKM-2-01均获得了约200个突变菌单菌落，StrainKL-2-02/StrainKM-2-02均获得了约215个突变菌单菌落，分别将各个单菌落分别接种于装有200μl液体培养基1的96孔板，30℃、200rpm振荡培养24h后，离心去掉上层培养基，再加入200μl液体养基2，30℃200rpm振荡培养72h。以出发菌StrainKL-2-01/StrainKL-2-02和StrainKM-2-01/StrainKM-2-02为对照，筛选合成LNTII产量显著提高的突变菌株。
结果显示，第一轮紫外诱变筛选获得的突变菌中，没有一株突变菌发酵上清液中LNTII产量高于出发菌。申请人又按照上述方法继续进行了8轮诱变筛选，最终各获得1株LNTII产量显著高于出发菌的突变菌株，分别命名为StrainKL-2-07/StrainKL-2-08和StrainKM-2-07/StrainKM-2-08。.
将上述筛选获得的突变菌StrainKL-2-07/StrainKL-2-08和为StrainKM-2-07/StrainKM-2-08转接于液体培养基1中，30℃，200rpm振荡培养24h后，离心去掉上层培养基，再转入液体培养基2中，30℃，200rpm下震荡培养至40h时，开始流加补料，总发酵时长72h。发酵72h结束后，离心发酵液，使用高压匀浆破碎仪进行菌体破碎，离心除去蛋白，上清过0.22μm滤膜除杂后用HPLC方法测定其中LNTII含量。结果如下：StrainKL-2-07/StrainKL-2-08发酵液中LNTII的浓度分别为10.32g/L和20.44g/L，乳糖合成LNTII转化率分别为64.76％和98.76％；StrainKM-2-07/StrainKM-2-08发酵液中LNTII的产量分别为10.81g/L和20.67g/L，乳糖合成LNTII转化率分别为67.84％和99.06％，均明显高于出发菌株。
固体培养基：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖、20g/L琼脂粉，经115℃高温蒸汽灭菌30min后备用。
液体培养基1：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖加入水配制而成，经115℃高温蒸汽灭菌30min后备用。
液体培养基2：20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖，8g/L乳糖、终浓度分别为5mM的磷酸氢二钾、硫酸镁、硫酸铵和硫酸锰，经115℃高温蒸汽灭菌30min后备用。
补料：5g/L的乳糖、20g/L的葡萄糖、终浓度分别为5mM的磷酸氢二钾、硫酸镁、硫 酸铵和硫酸锰
实施例16：重组菌株StrainKL-2-01和/或StrainKL-2-02与StrainKM-2-01和/或StrainKM-2-02催化合成LNTII。
对重组菌株StrainKL-2-01和/或StrainKL-2-02与StrainKM-2-01和/或StrainKM-2-02采用两段式培养进行LNTII的制备。
第一阶段为菌体生长期：以葡萄糖为碳源培养细胞，进行菌体量和酶量的积累，至细胞生长进入对数末期或稳定期。将菌株划线培养于固体培养基中，30℃培养2-3天后挑取单菌落接种于1.5mL液体培养基，30℃、200rpm培养至OD600＝1，按2％的接种量接种于5L液体培养基中(10L体积发酵罐)，30℃、200rpm震荡培养过夜。随后按2％的接种量接种于50mL液体培养基摇瓶中，30℃、200rpm震荡培养进行菌体量积累。
第二阶段为产物合成期：同实施例3。。
重组菌株StrainKL-2-01和/或StrainKM-2-01/发酵液中LNTII含量分别为4.21mg/L和4.25mg/L，乳糖合成LNTII转化率分别为26.41％和26.42％，使用生物传感器测得原始出发菌株StrainKM-0发酵液中乙醇含量分别约为22.0g/L和22.2g/L。重组菌株StrainKL-2-02和/或StrainKM-2-02/发酵液中LNTII含量分别为7.18g/L和7.28g/L，乳糖合成LNTII转化率分别为45.68％和45.88％，使用生物传感器测得原始出发菌株StrainKM-0发酵液中乙醇含量分别约为24.3g/L和24.1g/L。
本实施例中培养基具体情况如下：
固体培养基：同实施例3。
液体培养基：同实施例3。
补料：2g/L的乳糖、20g/L的葡萄糖。
补料：2g/L的乳糖、20g/L的葡萄糖、终浓度分别为5mM的磷酸氢二钾、硫酸镁、硫酸铵和硫酸锰。
实施例17.合成产物的鉴定
(1)高效液相色谱分析实施例4-9中的合成产物的成分。
分别对实施例4-9中乳酸克鲁维重组酵母细胞、马克斯克鲁维重组酵母细胞的发酵产物进行高效液相分析(HPLC)，检测条件如前所述。
LNTII标准品的出峰时间是12.10min。
附图2HPLC图谱显示，实施例4、6中重组菌株StrainKL-2-01和/或StrainKM-2-01、StrainKL-2-02和/或StrainKM-2-02的发酵产物在12.10min有较强峰出现，与LNTII标准品的出峰时间一致。
附图3-4HPLC图谱清晰的显示，实施例4、6中重组菌株StrainKL-2-01和/或StrainKM-2-01、StrainKL-2-02和/或StrainKM-2-02的纯化后发酵液在12.10min处有强吸收峰出现，与LNTII标准品的出峰时间一致，初步判断实施例4、6中在12.10min吸收峰可能为乳糖-N-三糖(LNTII)，发酵过程产生了LNTII。
实施例5重组菌株StrainKL-2-03、StrainKM-2-03，实施例7重组菌株StrainKL-2-04、StrainKM-2-04纯化后发酵液的HPLC图谱与实施例4、实施例6重组菌株发酵液的HPLC图谱一致。
通过比较重组菌株和LNTII标准品的HPLC检测图谱，初步判断实施例4-7中乳酸克鲁维重组酵母菌株和马克斯克鲁维重组酵母菌株催化合成了LNTII。
(2)对LNTⅡ标准品和实施例6合成产物中HPLC图谱吸收峰在12.10min的成分进行质谱分析，质谱图见附图6。
LNTⅡ分子式为C20H35NO16，分子量为545.495，LNTⅡ+H分子量为546.2029，LNTⅡ+Na分子量为568.485，LNTⅡ+K分子量为584.593。
附图5显示，LNTⅡ标准品LNTⅡ+H分子量为546.2029，LNTⅡ+Na分子量为568.1851，LNTⅡ+K分子量为584.1552。
附图6显示，实施例6StrainKL-2-02和/或StrainKM-2-02发酵所得合成产物中，在12.10min有吸收峰的物质成分，其质谱分析图谱中分子量为546.2039的峰与标准品的LNTⅡ+H相近，分子量为568.1859的峰与标准品的LNTⅡ+Na相近，分子量为584.1559的峰与标准品的LNTⅡ+K相近。
综上，经分析认为实施例6所得合成产物中，在12.10min有吸收峰的物质成分质谱分析结果与LNTⅡ标准品一致，与理论分子质量基本一致，说明实施例6所得合成产物中，在12.10min有吸收峰的物质为LNTII。
综上，通过高效液相色谱和质谱的联用分析，判定重组菌株StrainKL-2-02和StrainKM-2-02确实合成了LNTII。
由此可以推断，在12.10min同样有吸收峰的重组菌株StrainKL-2-01、StrainKL-2-03、StrainKL-2-04、StrainKL-2-05、StrainKL-2-06、StrainKM-2-01、StrainKM-2-03、StrainKM-2-04、StrainKM-2-05、StrainKM-2-06也合成了与重组菌株StrainKL-2-02、StrainKM-2-02相同的物质，即重组菌株StrainKL-2-01、StrainKL-2-02、StrainKL-2-03、StrainKL-2-04、StrainKL-2-05、StrainKL-2-06、StrainKM-2-01、StrainKM-2-02、StrainKM-2-03、StrainKM-2-04、StrainKM-2-05、StrainKM-2-06催化合成了LNTII。
(3)对实施例6合成产物中吸收峰在12.10min的物质成分进行NMR分析，结果见附 图7-8。
检测结果如附图7和8所示。13C-NMR谱(102.8ppm)与1H-NMR谱(3.56ppm)并存代表了GlcNAc残基与Gal残基之间的β1-3键；13C-NMR谱(102.8ppm)与1H-NMR谱(3.48ppm)并存代表了Gal和Glc残基的β1-4键。由此进一步明确了重组菌株StrainKL-2-02和StrainKM-2-02合成产物为GlcNAc-β1-3Gal-β1-4Glc(也即LNTII)。
实施例18.对照菌株StrainKL-1与原始菌株StrainKL-0的生长曲线对比。
按照实施例3描述的发酵过程第一阶段(即菌体生长阶段)的试验方法，分别以20g/L乳糖或20g/L葡萄糖与20g/L乳糖的混合物为碳源，并于第0、12、24、36、48、60、72取样检测对照菌株StrainKL-1与原始菌株StrainKL-0的生长情况，结果分别记录于表6-7。
表6.以20g/L乳糖为碳源时原始菌株StrainKL-0、对照菌株StrainKL-1生长情况
表6数据说明：以20g/L乳糖为碳源时，对照菌株StrainKL-1的生长与原始菌株StrainKL-0相比，明显要慢很多。
表7.以20g/L葡萄糖与20g/L乳糖混合物为碳源时，对照菌株StrainKL-1与原始菌株StrainKL-0的生长情况
表7数据说明：以20g/L葡萄糖与20g/L乳糖混合物为碳源时，对照菌株StrainKL-1与原始菌株StrainKL-0的生长速度基本一致。
综上，表6-7数据说明，菌株StrainKL-1对乳糖的利用率极低，达到了本发明减少乳糖消耗的目的。
综上，本发明首次使用乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase的重组酵母合成LNTII。所述含有乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase的重组酵母具有非常优异的合成LNTII的能力。其中，含有来源于幽门螺旋杆菌Helicobacter pylori的乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase的重组酵母，其乳糖合成 LNTII的转化率远高于含有来源于脑膜炎奈瑟氏菌Neisseria meningitidis的乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase的重组酵母和来源于肠道沙门氏菌(Salmonella enterica)的乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase的重组酵母，且高于在大肠杆菌中乳糖合成LNTII的转化率。
应注意的是，以上实例仅用于说明本发明的技术方案而非对其进行限制。尽管参照所给出的实例对本发明进行了详细说明，但是本领域的普通技术人员可根据需要对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (10)

  1. 用于生成乳糖-N-三糖的酶，根据Carbohydrate-Active-Enzymes即CAZy数据库分类，属于糖苷水解酶28家族的酶，其特征在于，为乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase；
  2. 如权利要求1所述生成乳糖-N-三糖的酶，包括氨基酸序列与SEQ ID NO:1、或SEQ ID NO:2、或SEQ ID NO:3具有至少70％、至少71％、至少72％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、至少100％的序列一致性的酶，且具有产生乳糖-N-三糖的活性。
  3. 如权利要求1-2所述用于生成乳糖-N-三糖的酶，其特征在于，为来源于脑膜炎奈瑟氏菌Neisseria meningitidis的乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase；
    所述用于生成乳糖-N-三糖的乙酰氨基葡萄糖基转移酶，优选的，其氨基酸序列如SEQ ID NO:1所示；或包含SEQ ID NO:1所示氨基酸序列，或与SEQ ID NO:1具有至少70％的氨基酸序列相同性，或包含与SEQ ID NO:1具有至少70％的氨基酸序列相同性。
  4. 如权利要求1-2所述用于生成乳糖-N-三糖的酶，其特征在于，为来源于幽门螺旋杆菌Helicobacter pylori的乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase；
    所述用于生成乳糖-N-三糖的乙酰氨基葡萄糖基转移酶，优选的，其氨基酸序列如SEQ ID NO:2所示；或包含SEQ ID NO:2所示氨基酸序列，或与SEQ ID NO:2具有至少70％的氨基酸序列相同性，或包含SEQ ID NO:2所示氨基酸序列，或包含与SEQ ID NO:2具有至少70％的氨基酸序列相同性。
  5. 如权利要求1-2所述用于生成乳糖-N-三糖的酶，其特征在于，为来源于肠道沙门氏菌(Salmonella enterical)的乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase；所述用于生成乳糖-N-三糖的乙酰氨基葡萄糖基转移酶，其具有SEQ ID NO:3所示氨基酸序列；或与SEQ ID NO:3具有至少70％的氨基酸序列一致性，或包含SEQ ID NO:3所示氨基酸序列，或包含与SEQ ID NO:3具有至少70％的氨基酸序列一致性。
  6. 一种生成乳糖-N-三糖(lacto-N-triose II)的重组酵母，其特征在于，含有权利要求1-5所述的乙酰氨基葡萄糖基转移酶β-N-acetylglucosaminyl transferase的氨基酸序列及其编码核酸序列；
    所述重组细胞是酵母细胞，优选的，包括假丝酵母属、汉逊酵母属、克鲁维酵母属、毕赤酵母属、酵母属、裂殖酵母属、或耶氏酵母属细胞；进一步优选的，为酿酒酵母 (Saccharomyces cerevisiae)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxinus)、解脂耶氏酵母(Yarrowia lipolytica)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccha romyces pastorianus)等细胞；
    所述重组酵母为重组克鲁维酵母；优选的，为乳酸克鲁维重组酵母菌株或重组马克斯克鲁维重组酵母菌株。
  7. 如权利要求6所述重组酵母，其特征在于，还包含标志物基因的破坏；
    所述破坏，优选的，是指部分标志物基因的破坏；
    所述标志物基因，优选的，包含β-半乳糖苷酶或者β-半乳糖苷酶的部分氨基酸序列，与SEQ ID NO:4和/或SEQ ID NO:5至少50％、至少51％、至少52％、至少53％、至少54％、至少55％、至少60％、至少61％、至少62％、至少63％、至少64％、至少65％、至少66％、至少67％、至少68％、至少69％、至少70％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、至少100％的氨基酸序列一致性；
    所述重组酵母，为乳酸克鲁维重组酵母细胞；优选的，其包含至少一个标志物基因被破坏，并且含有所述标志物的氨基酸序列，以及编码所述标志物的核酸序列；
    所述重组酵母，为马克斯克鲁维重组酵母细胞；优选的，其包含至少一个标志物基因被破坏，并且包括所述标志物的氨基酸序列，以及编码所述标志物的核酸序列。
  8. 如权利要求6-7所述重组酵母，其特征在于，在发酵产生乙醇的同时，产生乳糖-N-三糖(Lacto-N-triose II)。
    优选的，权利要求6-7所述重组酵母细胞的表达方法，包括：(a)培养亲本菌株；(b)破坏在(a)的亲本菌株中的标志物基因；(c)导入能够产生乳-N-三糖的酶；优选的，还包括(c)分离所述重组菌株的步骤。
  9. 如权利要求6-7任一项所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，包括：(a)在适合的条件下，在可发酵的培养基中培养所述重组酵母细胞，以生产乳-N-三糖(LNTII)；并且(b)回收该乳-N-三糖(LNTII)；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，培养基是含水溶液；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，的培养基是含有乳糖和/或葡萄糖和/或果糖和/或蔗糖和/或半乳糖的水溶液；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，培养温度为15-60℃；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，培养温度为28-32℃；
    所述重组酵母在生成乳糖-N-三糖方面的应用，优选的，培养基中还含有无机盐和/或微量元素；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，所述无机盐和/或微量元素选自磷酸氢二钾、硫酸镁、硫酸铵、硫酸锰、硫酸钾或它们的任意组合；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，所述无机盐或微量元素的终浓度为3-7mM；
    所述重组酵母在生成乳糖-N-三糖(Lacto-N-triose II)方面的应用，优选的，通过添加活性炭等方法进行分离纯化后，得到的LNTII。
  10. 权利要求6-7所述重组酵母的紫外诱变突变体，其特征在于，具有进一步提高的合成LNTII的能力。
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