WO2024109169A1 - 合成HMOs的成熟多肽序列及应用 - Google Patents

合成HMOs的成熟多肽序列及应用 Download PDF

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WO2024109169A1
WO2024109169A1 PCT/CN2023/112040 CN2023112040W WO2024109169A1 WO 2024109169 A1 WO2024109169 A1 WO 2024109169A1 CN 2023112040 W CN2023112040 W CN 2023112040W WO 2024109169 A1 WO2024109169 A1 WO 2024109169A1
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WO2024109169A9 (zh
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方诩
牛康乐
唐琪
广濑芳彦
李宾
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山东恒鲁生物科技有限公司
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Definitions

  • the present application relates to a mature polypeptide sequence of a synthetic oligosaccharide and its expression in a microorganism, and belongs to the technical field of enzyme engineering/genetic engineering.
  • HMOs human milk oligosaccharides
  • lactose-N-tetraose lactose-N-tetraose
  • lactose-N-neotetraose LNT
  • lactose-N-neotetraose LNnT
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • LNT and LNnT have similar structures, and their molecular weights are both 707.63.
  • the chemical structure of LNT is as follows:
  • LNnT The chemical structure of LNnT is as follows:
  • HMOs Since the supply of HMOs that meet the requirements is limited, effective industrial production is very much needed. Direct extraction of HMOs from natural breast milk is certainly not an ideal approach.
  • researchers have developed some chemical methods and bioenzymatic methods for HMOs.
  • the preparation methods disclosed in the existing literature have the problems of high production cost, low yield and high price.
  • the chemical synthesis method can obtain oligosaccharides with defined structures through lengthy multiple protection-deprotection operations, it has the problems of cumbersome work, large workload and low product yield.
  • the use of heavy metals increases food safety risks. Therefore, there is an urgent need for HMOs synthesis technology with stable process, green safety, low cost, high production efficiency and large-scale industrial production to meet the public's demand for HMOs.
  • Chinese invention patent 201810206004.3 discloses a recombinant Bacillus subtilis and a method for preparing lacto-N-neotetraose.
  • the lactose permease gene and the corresponding genes of ⁇ -1,3-N-glucosaminyltransferase and ⁇ -1,4-galactosyltransferase were recombinantly integrated into the genome of Bacillus subtilis 168.
  • the obtained recombinant Bacillus subtilis contained 1071 mg/L lactose-N-neotetraose in the supernatant of the fermentation broth.
  • Chinese patent 201180071512.1 reports a method for producing lactose-N-tetraose and lactose-N-neotetraose from lactose-N-triose using active cell extracts.
  • the existing technology can only synthesize lactose-N-tetraose (LNT) or lactose-N-neotetraose (LNnT).
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • the present application provides a mature polypeptide sequence that can simultaneously synthesize lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) and a preparation method thereof, as well as a method for using these mature polypeptides to produce specific oligosaccharides in human breast milk.
  • the present application also provides related nucleic acid products, enzyme products and preparation methods and applications of the polypeptide.
  • This application uses computer-aided design technology to screen out for the first time the mature polypeptide sequence shown in SEQ ID NO:1.
  • the mature polypeptide sequence shown in SEQ ID NO: 1 is a derivative of 1,3- ⁇ -galactosyl-N-acetylhexosamine phosphorylase.
  • the species source may be: Aspergillus sp., Kluyveromyces sp., Bacillus sp., Bifidobacterium sp., etc.
  • a further preferred species source is Bifidobacterium longum, or microorganisms classified as Bifidobacterium longum, wild strains isolated from nature, or mutants after physical and chemical mutagenesis, or engineered bacteria after genetic engineering.
  • the inventors of the present application have carried out a series of modifications to the polypeptide shown in SEQ ID NO:1, and obtained mutants of the mature polypeptide shown in SEQ ID NO:1.
  • the ability of the series of derivative polypeptides thus obtained to catalyze the synthesis of LNT and LNnT is greatly improved, and they have more excellent performance in the ability to simultaneously catalyze the synthesis of LNT and LNnT.
  • the present application modifies at least one of positions 233, 271, 312, 336, 342, 345, 456, 457, 458, 459, and 460 of the mature polypeptide sequence shown in SEQ ID NO: 1, thereby obtaining a mature polypeptide sequence having the ability to catalyze the synthesis of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • the modification refers to any chemical modification of the polypeptide or its homologous sequence of the polypeptide composition, and the genetic manipulation of the DNA encoding the polypeptide.
  • the modification can be the replacement, deletion and/or insertion of one or more (several) amino acids, and the replacement of one or more (several) amino acid side chains.
  • the mature polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid residue at at least one of positions 233, 271, 312, 336, 342, 345, 456, 457, 458, 459 and 460 is replaced by at least one selected from a phenylalanine residue, an alanine residue, a leucine residue and a tyrosine residue.
  • the mature polypeptide has an amino acid sequence selected from the following:
  • amino acid sequence shown in SEQ ID NO: 1 is substituted with a phenylalanine residue at position 233 to obtain the amino acid sequence shown in SEQ ID NO: 2; or
  • amino acid sequence shown in SEQ ID NO: 1 is substituted with an alanine residue at position 271 to obtain an amino acid sequence shown in SEQ ID NO: 3; or
  • the 312th position of the amino acid sequence shown in SEQ ID NO:1 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6; or
  • the 336th position of the amino acid sequence shown in SEQ ID NO: 1 is replaced by a phenylalanine residue or an alanine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8; or
  • the threonine residue at position 342 is replaced by a phenylalanine residue or a leucine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10; or
  • the methionine residue at position 345 is replaced by a phenylalanine residue or a leucine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12; or
  • the phenylalanine residue at position 456 is replaced by a tyrosine residue, a leucine residue, or an alanine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; or
  • the methionine residue at position 457 in the polypeptide sequence shown in SEQ ID NO:1 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:16, or SEQ ID NO:17, or SEQ ID NO:18; or
  • the glycine residue at position 458 is replaced by a phenylalanine residue, or a leucine residue, or an alanine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:19, or SEQ ID NO:20, or SEQ ID NO:21; or
  • the glycine residue at position 459 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:22, or SEQ ID NO:23, or SEQ ID NO:24; or
  • the asparagine residue at position 460 is replaced by a phenylalanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:25; or
  • the tryptophan residue at position 233 is replaced by a phenylalanine residue and the phenylalanine residue at position 456 is replaced by a tyrosine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:26; or
  • the tryptophan residue at position 233 is replaced by a phenylalanine residue and the methionine residue at position 457 is replaced by an alanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:27; or
  • the tryptophan residue at position 233 is replaced by a phenylalanine residue and the asparagine residue at position 460 is replaced by a phenylalanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:28; or
  • position 233 is replaced from a tryptophan residue to a phenylalanine residue
  • position 342 is replaced from a threonine residue to a leucine residue
  • position 460 is replaced from an asparagine residue to a phenylalanine residue, thereby obtaining an amino acid sequence shown in SEQ ID NO:29.
  • the mature polypeptide sequence preferably, comprises an amino acid sequence identical to SEQ ID NO:2 and/or SEQ ID NO:3 and/or SEQ ID NO:4 and/or SEQ ID NO:5 and/or SEQ ID NO:6 and/or SEQ ID NO:7 and/or SEQ ID NO:8 and/or SEQ ID NO:9 and/or SEQ ID NO:10 and/or SEQ ID NO:11 and/or SEQ ID NO:12 and/or SEQ ID NO:13 and/or SEQ ID NO:14 and/or SEQ ID NO:15 and/or SEQ ID NO:16 and/or SEQ ID NO:17 and/or SEQ ID NO:18
  • the amino acid sequence of the mature polypeptide is at least selected from the sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.
  • the present application provides a polynucleotide encoding the polypeptide as described in the first aspect above.
  • the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and one or more regulatory sequences operably linked thereto, wherein the regulatory sequences can guide the production of the polypeptide in a suitable expression host.
  • the present application provides an expression vector comprising the polynucleotide as described in the second aspect above, or comprising the nucleic acid construct as described in the third aspect above.
  • the present application provides a transformed host cell, in which the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above is transformed.
  • the host cell includes any one of bacteria, mold and yeast.
  • the bacteria include: Escherichia or Bacillus.
  • Escherichia sp. cells include: Escherichia coli, Escherichia hermanii, and Escherichia fergusonii cells.
  • Bacillus sp. cell includes: Bacillus mycoides, Bacillus cereus, Bacillus polymyxa, Bacillus licheniformis, Bacillus brevis, Bacillus circulans or Bacillus subtilis; further preferably, the host cell is Bacillus circulans or Bacillus subtilis.
  • the mold includes: Aspergillus oryzae, Aspergillus fumigatu, Aspergillus niger, Aspergillus flavus, Trichoderma reesei, Trichoderma viride, or Trichoderma koningii;
  • the yeast cell includes: a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell;
  • yeast cells include: Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxinus, Yarrowia lipolytica, Rhodotorula graminis, Saccharomyces pastorianus and the like;
  • yeast cells include: Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxinus cells.
  • the method for expressing the mature polypeptide sequence includes a wild strain isolated from nature, or a mutant strain after physical and chemical mutagenesis, or an engineered bacterium after genetic engineering.
  • the transformed host cell is a genetically engineered bacterium transformed with the polynucleotide described in the second aspect, the nucleic acid construct described in the third aspect, or the expression vector described in the fourth aspect (so as to express the polypeptide described in the first aspect), preferably a Kluyveromyces lactis, a Buddleija spp. Bacillus subtilis or Aspergillus oryzae.
  • the present application provides a composition comprising the polypeptide as described in the first aspect above.
  • composition preferably contains one or more mutants of the mature polypeptide sequence.
  • the composition is preferably an enzyme or an enzyme composition.
  • the present application provides the use of mutants of the mature polypeptide sequence or a composition thereof in the production of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT);
  • the use comprises the step of using a mutant of the mature polypeptide sequence described in the present application or a combination thereof as a catalyst to catalyze the production of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT);
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • the reaction substrate is any one or any combination of galactose-1-phosphate and/or lactose and/or galactose and acetylglucosamine and/or a carbohydrate with an acetylglucosamine group;
  • the reaction substrate is selected from galactose-1-phosphate, lactose-N-triose (LNTII), lactose, galactose, acetylglucosamine and carbohydrates with acetylglucosamine groups;
  • reaction substrates are galactose-1-phosphate and lactose-N-triose (LNTII);
  • the reaction solvent is an aqueous solvent or a solution containing solids
  • the application uses a culture fluid of the mutant containing a mature polypeptide sequence
  • the mutant of the mature polypeptide sequence is purified before use.
  • the present application provides a method for expressing the mature polypeptide sequence described in the first aspect, comprising the following steps:
  • the method further comprises: (b) a step of recovering the mature polypeptide sequence.
  • step (a) a recombinant plasmid encoding a mutant gene of the mature polypeptide sequence is first constructed and then introduced into a host cell;
  • the step (a) further comprises the step of culturing the obtained recombinant engineering strain.
  • the step (b) of recovering the mutant of the mature polypeptide sequence comprises the step of separating and purifying the mutant of the mature polypeptide sequence as described above.
  • the host cell is selected from yeast cells, filamentous fungal cells, bacterial cells, etc.
  • the type of the host cell is as defined above.
  • the present application provides a method for producing the polypeptide as described in the first aspect above, comprising:
  • the transformed host cell is as defined above.
  • the step (1) comprises: firstly introducing a nucleic acid construct or a recombinant expression vector encoding the polypeptide as described in the first aspect above into a host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the polypeptide.
  • the step (2) includes the step of separating and purifying the polypeptide from the culture.
  • Host cells are cultured in a nutrient medium suitable for producing the mature polypeptide sequence using methods known in the art.
  • cells can be cultured by shaking flasks, or in a suitable medium and under conditions that allow the mature polypeptide sequence to be expressed and/or separated, small-scale or large-scale fermentations (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) are carried out in a laboratory or industrial fermentor tank.
  • Cultivation occurs in a suitable nutrient medium using procedures known in the art, and the medium comprises carbon and nitrogen sources and inorganic salts.
  • Suitable medium can be purchased through commercial channels, or prepared according to disclosed compositions.
  • the mature polypeptide sequence can be recovered using methods known in the art.
  • the mature polypeptide sequence can be recovered from the nutrient medium by a variety of conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.
  • the mature polypeptide sequence can be purified by various procedures known in the art to obtain pure mature polypeptide sequences, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility methods (e.g., ammonium sulfate precipitation), SDS-PAGE, salting out, etc. or a combination thereof for separation or purification. Further preferably, purification can also be performed by Ni column affinity chromatography.
  • chromatography e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography
  • electrophoresis procedures e.g., preparative isoelectric focusing
  • differential solubility methods e.g., ammonium sulfate precipitation
  • SDS-PAGE salting out, etc. or a combination thereof for separation
  • the present application provides a method for simultaneously producing lactose-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), comprising: using the polypeptide described in the first aspect above, or the transformed host cell (especially the transformed engineered bacteria) described in the fifth aspect above, or the enzyme or enzyme composition described in the sixth aspect above as a catalyst, and lactose and its derivatives as reaction substrates, through catalytic or fermentation reactions, to generate LNT and LNnT.
  • LNT lactose-N-tetraose
  • LNnT lacto-N-neotetraose
  • the transformed host cell is Kluyveromyces lactis, Bacillus subtilis or Aspergillus oryzae into which the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect is transformed;
  • the reaction substrate is any one or any combination of galactose-1-phosphate and/or lactose and/or galactose and acetylglucosamine and/or a carbohydrate with an acetylglucosamine group;
  • the reaction substrate is selected from galactose-1-phosphate, lactose-N-triose (LNTII), lactose, galactose, acetylglucosamine and carbohydrates with acetylglucosamine groups;
  • reaction substrates are galactose-1-phosphate and lactose-N-triose;
  • the catalytic or fermentation reaction is carried out in a reaction solvent, and the reaction solvent is an aqueous solvent or a solution containing solids;
  • the mature polypeptide can use a culture medium containing a mature polypeptide sequence
  • the mature polypeptide sequence is used after purification.
  • the present application provides a polypeptide as described in the first aspect above, or a transformed host cell (particularly a transformed).
  • the invention relates to an enzyme or enzyme composition as described in the sixth aspect as a catalyst in the production of lactose-N-tetraose (LNT) and/or lacto-N-neotetraose (LNnT);
  • the transformed host cell is Kluyveromyces lactis, Bacillus subtilis or Aspergillus oryzae transformed with the polynucleotide as described in the second aspect, or the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect;
  • the method for producing lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) is as described in the eighth aspect above.
  • sequence identity The relatedness between two amino acid sequences is described by the parameter “sequence identity”.
  • expression includes any step involved in the production of a mature polypeptide sequence, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, etc. Expression can be measured by techniques known in the art, such as measuring the concentration or activity of mRNA and/or translated mature polypeptide sequences.
  • host cell refers to any cell type susceptible to transformation, transfection, transduction, etc. with an expression vector.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to substitutions that occur during replication.
  • mature polypeptide sequence means a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, etc.
  • a mature polypeptide sequence includes a protein whose amino acid sequence is shown as SEQ ID NO: 1, SEQ ID NO: 2-29.
  • the present application provides a series of mature polypeptide sequences, which have improved catalytic activity for the synthesis of LNT and LNnT.
  • the prior art has studied the use of some enzymes to synthesize and prepare HMOs, there are many problems such as the catalytic efficiency needs to be further improved, the substrate is difficult to obtain or expensive, or the reaction specificity is poor, resulting in complex downstream separation and purification processes; currently, there is no technology disclosed for the simultaneous catalytic synthesis of LNT and LNnT.
  • the mature polypeptide sequence described in the present application can be used to simultaneously catalyze the synthesis of LNT and LNnT, eliminating the need to compound LNT and LNnT in dairy products, and the catalytic efficiency reaches an industrial level.
  • the present application provides a mature polypeptide sequence capable of generating LNT and synthesizing LNnT and an expression method thereof; and also provides a method for catalyzing the synthesis of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) using the mature polypeptide sequence.
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • FIG. 1 HPLC graph of the reaction liquid after purification of LNT and LNnT catalyzed by LNBP-M3-W271A.
  • the products obtained from the catalytic reaction of the mature polypeptide sequence were analyzed or identified using HPLC analysis methods and ion chromatography.
  • HPLC analysis conditions are as follows: High performance liquid chromatography (HPLC) analysis conditions are as follows: chromatographic column model: Cosmosil Sugar-D amino column (nacalaitesque, INC.), mobile phase is 70% acetonitrile in water, column temperature is 30°C, injection volume is 10 ⁇ L, UV detector (Hitachi Chromaster), wavelength is 210nm, flow rate is 1.0mL/min.
  • chromatographic column MetroSep Carb2 (4.0mm ⁇ 250mm), eluent: 140mM NaOH/20mM NaAc, isocratic elution, flow rate: 0.500mL/min, amperometric detector, column temperature 40°C, injection volume 20 ⁇ L, running time 50min.
  • Lactose-N-triose (LNTII), lactose-N-tetraose (LNT), and lactose-N-neotetraose (LNnT) standards were produced by ELICITYL, France.
  • Example 1 Expression of mature polypeptide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2-29 in Escherichia coli.
  • a gene synthesis company was commissioned to obtain the genes corresponding to the mature polypeptide sequences SEQ ID NO: 1 and SEQ ID NO: 2-29 through codon optimization and DNA synthesis. Specifically, based on the nucleotides shown in SEQ ID NO: 80 (which encodes the mature polypeptide sequence shown in SEQ ID NO: 1), PCR amplification was performed using the PCR amplification primers shown in Table 1 to obtain the coding genes for the polypeptides shown in SEQ ID NO: 2-29.
  • the pET32a-lnbpX recombinant plasmid series (pET32a-lnbp1, pET32a-lnbp2-29) were constructed.
  • the primers are shown in Table 1.
  • the above recombinant plasmids were transformed into Escherichia coli BL21 (DE3) as follows: Take the prepared Escherichia coli BL21 (DE3) competent cells, place them on ice for 30 minutes to thaw, take 100 ⁇ L of competent cells and 10 ⁇ L of pET32a-lnbpX recombinant plasmid (concentration 50 ng/ ⁇ L) and mix them.
  • the cells were placed in a 42°C water bath for 45 seconds for heat shock, and then immediately cooled in an ice bath for 2 minutes.
  • 1 mL of fresh LB medium (LB medium: 1.0% peptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar powder on the plate) was added, and the cells were revived and cultured at 37°C and 100 rpm for 1 hour.
  • 100 ⁇ L of bacterial solution was spread on an LB plate containing ampicillin (100 ⁇ g/mL), and cultured in a 37°C constant temperature incubator for 12 hours.
  • Single colonies were picked for colony PCR (PCR amplification primers are shown in Table 1) to screen positive transformants. Further, after culturing the correct transformants, their plasmids were extracted, and double restriction digestion and gene sequencing were used to verify whether the pET32a-lnbpX recombinant plasmid was successfully introduced into E. coli.
  • the correct transformant was inoculated into LB liquid medium, placed on a shaker at 37°C and 200rpm for 12h to obtain seed liquid; then the seed liquid was inoculated into fresh LB medium at an inoculum of 1% (v/v), shaken and cultured at 37°C until OD 600 was 0.8, and induced by isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), the final concentration of IPTG was 0.1mmol/L, induced at 16°C for 12h, and the speed was 200rpm. After induction of expression, the fermentation broth was centrifuged at 5000r/min for 30min at 4°C to collect the bacteria.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the bacteria were resuspended in 20mM pH7.4 PBS buffer, they were ultrasonically broken at a frequency of plus on 5s/off 5s for 30min; the broken liquid was centrifuged at 13000 ⁇ g and 4°C for 30min to remove cell debris and collect the supernatant.
  • the soluble mature polypeptide sequence was purified by nickel column affinity chromatography. The process is as follows: deionized water was added to the nickel column to the top of the column. After natural elution, it was eluted with 5 volumes of Binding buffer. The crude enzyme solution filtered by 0.45 ⁇ m filter membrane was loaded onto the column. The sample was fully combined with the nickel column at a flow rate of 1.5 mL/min. After the sample was dried, it was continuously eluted with 5 column volumes of Washing buffer to remove impurities. Finally, the target protein was eluted with 5 times Elution buffer and the eluate was collected. Then SDS-PAGE was used to analyze the expression of the target protein.
  • the obtained proteins were the mature polypeptide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2-29, respectively, as shown in Table 2.
  • the reaction solution of the mature polypeptide (LNBP-M3-G312F) shown in SEQ ID NO:4 showed a strong absorption peak near 25.7 min, which was consistent with the peak time of the lactose-N-neotetraose (LNnT) standard, indicating that lactose-N-neotetraose (LNnT) was synthesized in the reaction.
  • reaction solutions of mature polypeptides shown in SEQ ID NO:2-3 and SEQ ID NO:5-29 showed strong absorption peaks at around 29.8 min and 25.7 min, which were consistent with the peak times of lactose-N-tetraose (LNT) standard and lactose-N-neotetraose (LNnT) standard, indicating that lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) were synthesized in the reaction.
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • the mature polypeptide sequence shown in SEQ ID NO:1 can catalyze the synthesis of lactose-N-tetraose (LNT) from LNTII and galactose-1-phosphate, but cannot synthesize lactose-N-neotetraose (LNnT).
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose
  • LNBP-M3-G312F shown in SEQ ID NO:4 lost the ability to synthesize lactose-N-tetraose (LNT), but was able to synthesize lactose-N-neotetraose (LNnT).
  • Example 3 Expression of the mature polypeptide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 26 in Kluyveromyces lactis (CICC1773).
  • the construction method of the recombinant plasmid pKLAC1-LNBP-M3-W233F or pKLAC1-LNBP-M3-W233F/F456Y is as follows:
  • KLAC1-LNBP-M3-W233F or KLAC1-LNBP-M3-W233F/F456Y expression plasmid was transformed into Kluyveromyces lactis.
  • the yeast transformation method is as follows:
  • (1) First prepare competent Kluyveromyces lactis cells Take a small amount of frozen yeast strains and streak them on a flat solid culture medium, and invert and culture at 30°C for 2 days. Pick a single yeast colony in 50mL liquid culture medium, and culture at 30°C, 220rpm until OD600 is between 0.8-1.5. Collect the bacteria and wash them with 25mL sterile water, centrifuge at 1500 ⁇ g for 10min at room temperature, and discard the supernatant. Add 1mL of 100mM lithium chloride buffer, resuspend the precipitate, centrifuge at 12000rpm for 30s, and discard the supernatant. Add 400 ⁇ L of 100mM lithium chloride buffer again, resuspend the precipitate, and obtain the competent yeast cells, and divide them into 50 ⁇ L/tube for transformation.
  • the plasmid DNA is KLAC1-LNBP-M3-W233F or KLAC1-LNBP-M3-W233F/F456Y expression plasmid.
  • Shake flask fermentation is as follows: 1) Preparation of primary species: The above-mentioned target Kluyveromyces lactis genetic engineering strain single colony is cultured overnight in 10mL YGD liquid culture medium at 30°C and 200rpm, and the obtained strain is the primary species. 2) Preparation of secondary species: The primary species is inoculated in 1L YGD liquid culture medium and cultured at 30°C and 200rpm until OD600 is about 7.
  • the protein is purified according to the method in Example 1, and the obtained protein is the mature polypeptide sequence of KL-LNBP-M3-W233F (SEQ ID NO: 2) or KL-LNBP-M3-W233F/F456Y (SEQ ID NO: 26).
  • KL-LNBP-M3-W233F and KL-LNBP-M3-W233F/F456Y obtained in this example were respectively placed in a water bath at 37°C for 24 hours according to the catalytic synthesis method described in Example 2.
  • the yields of LNnT and LNT in the reaction solution were measured by ion chromatography, and the results are recorded in Table 4.
  • Example 4 Expression of mature polypeptides SEQ ID NO: 2 and SEQ ID NO: 26 in Bacillus subtilis 168
  • the coding genes of the polypeptides SEQ ID NO:2 and SEQ ID NO:26 obtained in Example 1 were cloned into the pEB03 plasmid by restriction endonucleases and T4 ligase, respectively, to construct a recombinant expression plasmid; specifically, the gene fragment encoding the polypeptide and the plasmid pEB03 were double-digested with restriction endonucleases EcoR I and NotI, respectively, and then the two were connected with T4 ligase, and the connected plasmids were transformed into Escherichia coli DH5 ⁇ . After screening and identification, the recombinant expression plasmids pEB03 ⁇ LNBP-M3-W233F and pEB03 ⁇ LNBP-M3-W233F/F456Y were obtained.
  • the transformation method of pEB03 ⁇ LNBP-M3-W233F and pEB03 ⁇ LNBP-M3-W233F/F456Y plasmids in Bacillus subtilis is as follows: take a full loop of Bacillus subtilis glycerol bacteria and streak it on an LB plate (LB medium: 1.0% peptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar powder on the plate), and culture it in a 37°C incubator overnight.
  • LB medium 1.0% peptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar powder on the plate
  • the treated bacterial solution was divided into 80 ⁇ L per tube, 1 ⁇ L (about 50 ng) of plasmid was added, and the solution was transferred to a pre-cooled electroporation cup and placed on ice for 1-1.5 min. Then, the electroporation instrument was used for electrotransformation.
  • the electroporation parameters were set as follows: voltage 2000 V, resistance 200 ⁇ , followed by an ice bath for 2 min, and then 1 mL of recovery medium (recovery medium: LB medium with 0.5 M sorbitol and 0.38 M mannitol) was added and cultured at 37 ° C and 200 rpm for 90 min.
  • recovery medium recovery medium: LB medium with 0.5 M sorbitol and 0.38 M mannitol
  • the bacterial solution was coated on a chloramphenicol (5 ⁇ g/mL) LB plate, and the transformant was picked to obtain the target Bacillus subtilis genetically engineered strain.
  • the protein was purified according to the method in Example 1.
  • the molecular weight of the band was basically consistent with the expected molecular weight of 103 kDa.
  • the obtained protein was the mature polypeptide shown in SEQ ID NO: 2 and SEQ ID NO: 26, and was named BS-LNBP-M3-W233F and BS-LNBP-M3-W233F/F456Y.
  • the obtained BS-LNBP-M3-W233F and BS-LNBP-M3-W233F/F456Y were respectively placed in a water bath at 37°C for 24 hours according to the catalytic synthesis method described in Example 2.
  • the yields of LNnT and LNT in the reaction solution were measured by ion chromatography, and the results are recorded in Table 5.
  • Example 5 Expression of mature polypeptides SEQ ID NO: 2 and SEQ ID NO: 26 in Aspergillus oryzae (CCTCC AF 93036).
  • RNA was extracted from Aspergillus oryzae, and cDNA was produced by reverse transcription.
  • the Aspergillus oryzae amylase promoter PamyB and glycosidase terminator TagdA were amplified using Aspergillus oryzae cDNA as a template.
  • pCAMBIA001-AO-LNBP-M3-W233F and pCAMBIA001-AO-LNBP-M3-W233F/F456Y plasmids were constructed by restriction digestion and ligation, and sequencing verification results showed that the target gene was correct.
  • Preparation of competent Agrobacterium tumefaciens EHA105 1) Take Agrobacterium tumefaciens EHA105 activated on solid YEB plate and inoculate it into 20mL liquid YEB medium, to which rifampicin antibiotics have been added. Shake and culture at 180rpm for 24h at 28°C. 2) Inoculate Agrobacterium tumefaciens into 50mL liquid YEB medium with rifampicin added at 1% inoculum, shake and culture at 28°C, 180rpm until OD600 is 0.8, then it can be used.
  • the plasmid was transformed into competent EHA105 using the freeze-thaw method: 1) Thaw the competent EHA105 of Agrobacterium tumefaciens on ice, add 10 ⁇ L of the recombinant plasmid, mix well, and place on ice for 30 minutes. 2) Quickly put it in liquid nitrogen for 5 minutes, then immediately put it in a 28°C constant temperature water bath and incubate it for 5 minutes. 3) Place it on ice for 5 minutes. 4) Add 400 ⁇ L of YEB liquid medium without rifampicin to this mixture, and shake and culture it at 28°C at 180 rpm for 2 hours.
  • Transformation of Aspergillus oryzae by Agrobacterium tumefaciens-mediated expression vector 1) Inoculate the EHA105 bacterial suspension containing different expression vectors into liquid LB medium supplemented with rifampicin and kanamycin, and culture at 28°C and 180rpm for 24h. 2) Take 2mL of bacterial suspension and add it to a mixed medium consisting of 10mL of liquid LB medium and 10mL of basal medium, and culture at 28°C and 180rpm for 48h. 3) Take 2mL of bacterial suspension and add it to 20mL of induction medium, and culture at 28°C and 180rpm until OD600 is 0.8, then co-culture with Aspergillus oryzae.
  • Aspergillus oryzae engineered bacteria were activated, they were cultured at 30°C for 3 days, the spores were washed with sterile water, and a bacterial suspension was prepared for inoculation. Different strains were inoculated into liquid fermentation medium at 2%, cultured at 30°C, 200 rpm for 6 days, centrifuged at 12000 rpm, the supernatant was collected, and concentrated using a 10 kDa ultrafiltration membrane.
  • the protein was purified according to the method in Example 1.
  • the molecular weight of the band was basically consistent with the expected molecular weight of 103 kDa.
  • the obtained proteins were mature polypeptides shown as SEQ ID NO: 2 and SEQ ID NO: 26, and were called AO-LNBP-M3-W233F and AO-LNBP-M3-W233F/F456Y.
  • AO-LNBP-M3-W233F and AO-LNBP-M3-W233F/F456Y were respectively placed in a water bath at 37°C for 24 hours according to the catalytic synthesis method described in Example 2.
  • the yields of LNnT and LNT in the reaction solution were measured by ion chromatography, and the results are recorded in Table 6.
  • Example 6 Using lactose as a substrate, the mature polypeptide LNBP-M3-W233F was used to catalyze the synthesis of lactose-N-tetraose (LNT) and lactose-N-tetraose (LNnT).
  • LNT lactose-N-tetraose
  • LNnT lactose-N-tetraose
  • Lactase was purchased from Weilan Biotechnology. Referring to the method provided in the paper (Immobilization of ⁇ -galactosidase and its application in the preparation of low-lactose milk, Food Industry, 2018, 39(9): 105-110), 20 mmol/L lactose was added to 100 mmol/L Tris-HCl buffer (pH 6.5), and 120 U of lactase was added per gram of lactose. The mixture was mixed and reacted at 37°C for 5 hours to obtain a mixed solution A.
  • Example 7 Using galactose as substrate, the mature polypeptide LNBP-M3-W233F catalyzes the synthesis of lactose-N-tetraose (LNT) and lactose-N-tetraose (LNnT)
  • lactose-N-triose (Lacto-N-trioseII), 1mmol/L MgCl 2 , and 2.2mg/mL of LNBP-M3-W233F obtained in Example 1 were added, mixed, and heated to 45°C for 0.5 hours. After the reaction was terminated, the reaction solution was detected by the above ion chromatography method, wherein the yield of lactose-N-tetraose (LNT) was 1.67 mg/mL, and the yield of lactose-N-neotetraose (LNnT) was 0.49 mg/mL.
  • LNT lactose-N-tetraose
  • LNnT lactose-N-neotetraose

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Abstract

本申请描述了一系列成熟多肽序列，其氨基酸序列为在如SEQ ID NO:1所示成熟多肽序列中的第233、271、312、336、342、345、456、457、458、459、460位中的至少一个位点发生修饰，所得修饰后的成熟多肽序列具有催化合成乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)的能力。本申请还提供了SEQ ID NO:1、SEQ ID NO:2-29所示成熟多肽序列的表达方法；还提供了使用所述成熟多肽序列催化合成乳糖-N-四糖(LNT)和合成LNnT的方法。

Description

合成HMOs的成熟多肽序列及应用
交叉引用
本申请要求于2022年11月21日提交的、申请号为202211473306.X、发明名称为“合成HMOs的成熟多肽序列及应用”的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本申请涉及合成寡糖的成熟多肽序列及其在微生物中的表达，属于酶工程/基因工程技术领域。
背景技术
母乳对婴幼儿的成长起着至关重要的作用。与母乳喂养的婴儿相比，奶瓶喂养的婴儿存活率要低，感染几率更高，这一现象引起了科学家对母乳成分的研究兴趣。21世纪以来，研究者发现人类初乳中总HMOs(酸性和中性低聚糖)浓度会随泌乳期延长而下降；还发现HMOs对婴儿的生长发育以及后期的健康非常重要，其可促进婴儿肠道微生态平衡，促进肠道内有益菌的增殖、抑制有害菌的生长、抵御病原菌感染、调节免疫系统和促进婴儿的认知发育等，母乳的许多有益作用都归功于人乳寡糖(human milk oligosaccharides，HMOs)。
HMOs中的2-岩藻糖基乳糖(2’-FL)、乳糖-N-三糖(Lacto-N-triose II，LNTII)、乳糖-N-四糖(Lacto-N-tetraose，LNT)和乳糖-N-新四糖(Lacto-N-neotetraose，LNnT)被发现是HMOs中的主要成分，发挥双歧因子的作用。研究证明，以牛乳或羊乳作为主要原料的婴幼儿配方奶粉中添加HMOs，可以在婴幼儿的营养、免疫等方面接近母乳哺育的效果。因此，向婴幼儿奶粉、一些人造奶、乳制品、婴儿食品以及功能保健品或特殊人群食品中添加具有生理功能的HMOs，将大大提高相应人群的身体素质，提高其生活质量，具有明显的社会和经济价值。
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。
LNT和LNnT结构相似，分子量均为707.63。LNT化学结构式如式下：
LNnT化学结构式如式下：
由于符合要求的HMOs的供给量有限，因此非常需要有效的工业化生产。从自然母乳中直接提取HMOs当然不是一个理想的途径，研究人员已经开发了一些HMOs的化学方法和生物酶促方法。现有文献公开的制备方法中，存在生产成本高、产量低、价格昂贵的问题。化学合成法虽然可以通过冗长的多重保护-脱保护操作获得结构确定的寡糖，但存在工作繁琐、工作量大、产品收率低的问题，同时重金属的使用增加了食品安全风险。因此，亟需具有工艺稳定、绿色安全、成本低、生产效率高并可大规模工业化生产的HMOs合成技术，满足社会公众对HMOs的需求。
中国发明专利201810206004.3公开了重组枯草芽孢杆菌及其应用于制备乳-N-新四糖的方法。在枯草芽孢杆菌168基因组上重组整合乳糖透性酶基因，及对应β-1,3-N-葡糖氨基转移酶和β-1,4-半乳糖转移酶的基因，获得的重组枯草芽孢杆菌在发酵24小时后，发酵液上清中含有1071mg/L乳糖-N-新四糖。
此外，酶促反应由于其操作简单，产物专一，环境友好等特点，被广泛应用合成寡糖。中国专利201180071512.1，报道了利用活性细胞提取物从乳糖-N-丙糖生产乳糖-N-四糖和乳糖-N-新四糖的方法。
综上，为了实现酶促合成LNT的规模化生产，以期更快地推进HMOs应用于乳制品、烘焙食品、饮料等各种食品中。虽然现有技术提供了各种HMOs的生产方法，但处于惠及社会公众及产品迭代的需求，酶促反应的效率需要进一步提高，人们仍在研究具有更多优势的HMOs生产方法。
发明内容
发明目的：针对现有技术的不足，提供一种能同时合成乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)的成熟多肽序列以及制备方法，以及利用它们生产人类母乳中特定存在的低聚糖的方法。
现有技术只能合成乳糖-N-四糖(LNT)或乳糖-N-新四糖(LNnT)，而针对现有市场对这两种糖的需求，本申请提供了一种能同时合成乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)的成熟多肽序列及其制备方法，以及应用这些成熟多肽生产人类母乳中特定低聚糖的方法。
此外，本申请还提供了所述多肽的相关核酸产品、酶产品及其制备方法和应用。
技术方案：
本申请通过计算机辅助设计技术，首次筛选出了氨基酸序列如SEQ ID NO:1所示成熟多肽序列。
所述SEQ ID NO:1所示成熟多肽序列为1,3-β-半乳糖苷-N-乙酰己糖胺磷酸化酶(1,3-beta-galactosyl-N-acetylhexosamine phosphorylase)的衍生物。所述1,3-β-半乳糖苷-N-乙酰己糖胺磷酸化酶， 其物种来源可以是：曲霉属(Aspergillus sp.)、克鲁维酵母属(Kluyveromyces sp.)、芽孢杆菌属(Bacillus sp.)、双歧杆菌属(Bifidobacterium sp.)等，进一步优选的物种来源为长双歧杆菌(Bifidobacterium longum)，或分类为长双歧杆菌微生物，来自自然界分离的野生株，或者物理化学诱变后的变异株，或者基因工程改造后的工程菌。
本申请的发明人对如SEQ ID NO:1所示的多肽进行了一系列改造，获得了SEQ ID NO:1所示成熟多肽的突变体，由此获得的一系列衍生多肽催化合成LNT和LNnT的能力大大提高，其在同时催化合成LNT和LNnT的能力方面有着更优异的性能。
具体地，第一方面，本申请对SEQ ID NO:1所示成熟多肽序列的第233、271、312、336、342、345、456、457、458、459、460位中至少一个位点进行修饰，获得了具有催化合成乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)的能力的成熟多肽序列。
所述修饰是指多肽组成的多肽或其同源序列的任何化学修饰，以及对编码所述多肽的DNA的遗传操作。所述修饰可以是一个或多个(几个)氨基酸的置换、缺失和/或插入，以及一个或多个(几个)氨基酸侧链的置换。
具体地，所述成熟多肽，其氨基酸序列为如SEQ ID NO:1所示的氨基酸序列中的第233位、第271位、第312位、第336位、第342位、第345位、第456位、第457位、第458位、第459位和第460位中的至少一个位点上的氨基酸残基置换为选自苯丙氨酸残基、丙氨酸残基、亮氨酸残基和酪氨酸残基中的至少一个而得到的序列。
优选的，所述成熟多肽，其具有选自以下的氨基酸序列：
SEQ ID NO:1所示氨基酸序列的第233位从色氨酸酸残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:2所示的氨基酸序列；或
SEQ ID NO:1所示氨基酸序列的第271位从色氨酸残基置换为丙氨酸残基，由此获得如SEQ ID NO:3所示的氨基酸序列；或
SEQ ID NO:1所示的氨基酸序列的第312位从甘氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:4、或SEQ ID NO:5、或SEQ ID NO:6所示的氨基酸序列；或
SEQ ID NO:1所示的氨基酸序列的第336位从丝氨酸残基置换为苯丙氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:7、或SEQ ID NO:8所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第342位从苏氨酸残基置换为苯丙氨酸残基、或亮氨酸残基，由此获得如SEQ ID NO:9、或SEQ ID NO:10所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第345位从甲硫氨酸残基置换为苯丙氨酸残基、或亮氨酸残基，由此获得如SEQ ID NO:11、或SEQ ID NO:12所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第456位从苯丙氨酸残基置换为酪氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:13、或SEQ ID NO:14、或SEQ ID NO:15所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中第457位从甲硫氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:16、或SEQ ID NO:17、或SEQ ID NO:18所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第458位从甘氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:19、或SEQ ID NO:20、或SEQ ID NO:21所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第459位从甘氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:22、或SEQ ID NO:23、或SEQ ID NO:24所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第460位从天冬酰胺残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:25所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第456位从苯丙氨酸残基置换为酪氨酸残基，由此获得如SEQ ID NO:26所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第457位从甲硫氨酸残基置换为丙氨酸残基，由此获得如SEQ ID NO:27所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第460位从天冬酰胺残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:28所示的氨基酸序列；或
SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第342位从苏氨酸残基置换为亮氨酸残基和第460位从天冬酰胺残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:29所示的氨基酸序列。
所述成熟多肽序列，优选的，包括氨基酸序列与SEQ ID NO:2和/或SEQ ID NO:3和/或SEQ ID NO:4和/或SEQ ID NO:5和/或SEQ ID NO:6和/或SEQ ID NO:7和/或SEQ ID NO:8和/或SEQ ID NO:9和/或SEQ ID NO:10和/或SEQ ID NO:11和/或SEQ ID NO:12和/或SEQ ID NO:13和/或SEQ ID NO:14和/或SEQ ID NO:15和/或SEQ ID NO:16和/或SEQ ID NO:17和/或SEQ ID NO:18和/或SEQ ID NO:19和/或SEQ ID NO:20和/或SEQ ID NO:21和/或SEQ ID NO:22和/或SEQ ID NO:23和/或SEQ ID NO:24和/或SEQ ID NO:25和/或SEQ ID NO:26和/或SEQ ID NO27和/或SEQ ID NO:28和/或SEQ ID NO:29所示序列具有至少60％，至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少98％、至少99％、或100％序列一致性的多肽。
优选的，所述成熟多肽的氨基酸序列至少选自如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28或SEQ ID NO:29所示的序列。
第二方面，本申请提供了一种多核苷酸，其编码如上述第一方面所述的多肽。
第三方面，本申请提供了一种核酸构建体，其包含如上述第二方面所述的多核苷酸，以及与之可操作地连接的一个或多个调控序列，所述调控序列可指导多肽在适当表达宿主中生产。
第四方面，本申请提供了一种表达载体，其包含如上述第二方面所述的多核苷酸，或者包含如上述第三方面所述的核酸构建体。
第五方面，本申请提供了一种转化的宿主细胞，其在宿主细胞中转化了如上述第二方面所述的多核苷酸、或第三方面所述的核酸构建体、或如上述第四方面所述的表达载体。
作为优选，所述宿主细胞包括：细菌、霉菌和酵母菌中的任意一种。
优选地，所述细菌包括：埃希氏杆菌属细菌芽或孢杆菌属。
进一步地，所述埃希氏杆菌属(Escherichia sp.)细胞，包括：大肠埃希菌(Escherichia coli)、赫尔曼埃希菌(Escherichia hermanii)、弗格森埃希菌(Escherichia fergusonii)细胞。
进一步地，所述芽孢杆菌属(Bacillus sp.)细胞，包括：蕈状芽孢杆菌(Bacillus mycoides)、蜡样芽孢杆菌(Bacillus cereus)、多黏芽孢杆菌(Bacillus polymyxa)、地衣芽孢杆菌(Bacillus licheniformis)、短芽孢杆菌(Bacillus brevis)、环状芽孢杆菌(Bacillus circulans)或枯草芽孢杆菌(Bacillus subtilis)；进一步优选的，所述宿主细胞是环状芽孢杆菌(Bacillus circulans)或枯草芽孢杆菌(Bacillus subtilis)。
优选的，所述霉菌，包括：米曲霉(Aspergillus oryzae)、烟曲霉(Aspergillus fumigatu)、黑曲霉菌(Aspergillus niger)、黄曲霉(Aspergillus flavus)、里氏木霉(Trichoderma reesei)、绿色木霉(Trichoderma viride)、或康宁木霉(Trichoderma koningii)；
优选的，所述酵母细胞，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，所述酵母细胞，包括：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(Saccharomyces pastorianus)等细胞；
进一步优选，所述酵母细胞，包括：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)细胞。
所述成熟多肽序列的表达方法，所述宿主细胞包括自然界分离的野生株，或者物理化学诱变后的变异株，或者基因工程改造后的工程菌。
在进一步优选的具体实施方案中，所述转化的宿主细胞为其中转化了如上述第二方面所述的多核苷酸、或如上述第三方面所述的核酸构建体、或如上述第四方面所述的表达载体(从而表达如上述第一方面所述的多肽)的基因工程菌，优选为含有如第一方面所述多肽的编码基因的乳酸克鲁维酵母菌(Kluyveromyces lactis)、枯草芽 孢杆菌(Bacillus subtilis)或米曲霉菌(Aspergillus oryzae)。
第六方面，本申请提供了一种组合物，其包含如上述第一方面所述的多肽。
所述组合物，优选的，含有所述成熟多肽序列的突变体中的一种或多种。
所述组合物，优选的，为酶剂或酶组合物。
第七方面，本申请提供了所述成熟多肽序列的突变体或其组合物在生产乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)中的应用；
优选的，所述应用，包括以本申请所述成熟多肽序列的突变体或其组合物作为催化剂催化生成乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)的步骤；
优选的，所述应用，反应底物为半乳糖-1-磷酸和/或乳糖和/或半乳糖中的任意一种或任意组合与乙酰氨基葡萄糖和/或带有乙酰氨基葡萄糖基团的碳水化合物；
优选的，所述反应底物选自半乳糖-1-磷酸、乳糖-N-三糖(LNTII)、乳糖、半乳糖、乙酰氨基葡萄糖和带有乙酰氨基葡萄糖基团的碳水化合物；
进一步优选的，所述应用，反应底物为半乳糖-1-磷酸与乳糖-N-三糖(LNTII)；
优选的，所述应用，反应溶剂为含水溶剂或含有固形物的溶液；
优选的，所述应用，使用含有成熟多肽序列的突变体的培养液；
优选的，所述应用，成熟多肽序列的突变体纯化后使用。
第八方面，本申请提供了上述第一方面中所述成熟多肽序列的表达方法，包括如下步骤：
(a)在适合于表达成熟多肽序列的条件下培养宿主细胞；
优选的，还包括：(b)回收所述成熟多肽序列的步骤。
优选的，所述步骤(a)，首先构建编码所述成熟多肽序列的突变体基因的重组质粒，然后导入到宿主细胞中；
所述步骤(a)，还包括培养所得重组工程菌株的步骤。
优选的，所述步骤(b)回收成熟多肽序列的突变体的步骤，包括以上所述成熟多肽序列的突变体的分离纯化步骤。
优选的，以上所述成熟多肽序列的突变体的表达方法，所述宿主细胞选自酵母细胞、丝状真菌细胞、细菌细胞等。所述宿主细胞的种类如上文中所定义。
第九方面，本申请提供了如上述第一方面所述的多肽的生产方法，其包括：
(1)在适合表达所述多肽的条件下，培养转化的宿主细胞以产生多肽；所述转化的宿主细胞如上述第五方面中所述；和
(2)回收所述多肽。所述转化的宿主细胞如上文中所定义。
在具体实施方案中，所述步骤(1)包括：首先将包含编码如上述第一方面所述的多肽的核酸构建体、或重组表达载体导入宿主细胞中，构建表达所述多肽的工程化宿主细胞；然后，培养所述工程化宿主细胞，并诱导其表达所述多肽。
在具体实施方案中，所述步骤(2)包括将所述多肽从培养物中分离、纯化的步骤。使用本领域已知的方法在适合于产生所述成熟多肽序列的营养培养基中培养宿主细胞。例如，可以通过摇瓶培养，或者在适合的培养基中并在允许成熟多肽序列表达和/或分离的条件下在实验室或工业发酵罐中进行小规模或大规模发酵(包括连续发酵、分批发酵、分批给料发酵或固态发酵)来培养细胞。培养是使用本领域已知的程序，在适合的营养培养基中发生，所述培养基包含碳和氮来源及无机盐。适合的培养基可以通过商业渠道购买，或根据公开的组成制备。
所述成熟多肽序列，可以使用本领域已知的方法回收。例如，可以通过多种常规程序从营养培养基中回收所述成熟多肽序列，所述常规程序包括但不限于收集、离心、过滤、提取、喷雾干燥、蒸发或沉淀。
可以通过本领域已知的多种程序来纯化成熟多肽序列以获得纯的成熟多肽序列，所述程序包括但不限于色谱法(例如，离子交换色谱、亲和色谱、疏水作用色谱、凝胶过滤色谱法)、电泳程序法(例如，制备型等电点聚焦)、差别溶解度法(例如，硫酸铵沉淀)、SDS-PAGE法、盐析等或其组合进行分离或纯化。进一步优选的，还可通过Ni柱亲和层析法进行纯化。
第十方面，本申请提供了一种同时生产乳糖-N-四糖(LNT)和乳-N-新四糖(LNnT)的方法，其包括：以如上第一方面所述的多肽、或如上第五方面所述的转化的宿主细胞(特别是转化的工程菌)、或如上第六方面所述的酶剂或酶组合物作为催化剂，以乳糖及其衍生物作为反应底物，经过催化或发酵反应，生成LNT和LNnT。
上述方法中，优选地，所述转化的宿主细胞为其中转化了如上述第二方面所述的多核苷酸、或如上述第三方面所述的核酸构建体、或如上述第四方面所述的表达载体的乳酸克鲁维酵母菌(Kluyveromyces lactis)、枯草芽孢杆菌(Bacillus subtilis)或米曲霉菌(Aspergillus oryzae)；
优选的，所述方法中，反应底物为半乳糖-1-磷酸和/或乳糖和/或半乳糖中的任意一种或任意组合与乙酰氨基葡萄糖和/或带有乙酰氨基葡萄糖基团的碳水化合物；
优选的，所述反应底物选自半乳糖-1-磷酸、乳糖-N-三糖(LNTII)、乳糖、半乳糖、乙酰氨基葡萄糖和带有乙酰氨基葡萄糖基团的碳水化合物；
进一步优选的，所述方法中，反应底物为半乳糖-1-磷酸与乳糖-N-三糖；
优选的，所述方法中，催化或发酵反应在反应溶剂中进行，所述反应溶剂为含水溶剂或含有固形物的溶液；
优选的，所述方法中，所述成熟多肽可以使用含有成熟多肽序列的培养液；
优选的，所述方法中，成熟多肽序列在纯化后使用。
第九方面，本申请提供了如上述第一方面所述的多肽、或如上第五方面所述的转化的宿主细胞(特别是转 化的工程菌)、或如上第六方面所述的酶剂或酶组合物作为催化剂在生产乳糖-N-四糖(LNT)和/或乳-N-新四糖(LNnT)中的应用；
优选地，所述转化的宿主细胞为转化了如上述第二方面所述的多核苷酸、或如上述第三方面所述的核酸构建体、或如上述第四方面所述的表达载体的乳酸克鲁维酵母菌(Kluyveromyces lactis)、枯草芽孢杆菌(Bacillus subtilis)或米曲霉菌(Aspergillus oryzae)；
优选地，所述生产乳糖-N-四糖(LNT)和乳-N-新四糖(LNnT)的方法如上述第八方面所述。
定义：
术语“序列一致性”：两个氨基酸序列之间的相关性，由参数“序列一致性”描述。
术语“表达”包括在成熟多肽序列的产生中涉及的任何步骤，包括但不局限于，转录、转录后修饰、翻译、翻译后修饰等。可以通过本领域已知的技术对表达进行测量，例如测量mRNA和/或翻译的成熟多肽序列的浓度或活性。
术语“宿主细胞”是指对于用表达载体进行的转化、转染、转导等时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置换而与亲本细胞不同的亲本细胞的任何后代。
术语“成熟多肽序列”意指在翻译和任何翻译后的修饰如N-末端加工、C-末端截短等之后处于其最终形式的多肽。在一个方面中，成熟多肽序列包括氨基酸序列如SEQ ID NO:1、SEQ ID NO:2-29所示的蛋白。
有益效果
本申请提供了一系列成熟多肽序列，其具有提高的催化合成LNT和LNnT的活性。虽然现有技术有研究利用一些酶催化合成制备了HMOs，但是其存在催化效率有待进一步提高、底物获得困难或价格昂贵、或者反应专一性不佳导致下游分离纯化处理工艺复杂等很多问题；当前，没有同时催化合成LNT和LNnT的技术公开。
利用本申请中所述成熟多肽序列能够同时催化合成LNT和合成LNnT，省去了在乳品中复配LNT和LNnT的过程，且催化的效率达到了工业化的水平。
本申请提供了具有生成LNT和合成LNnT的成熟多肽序列及其表达方法；还提供了使用所述成熟多肽序列催化合成乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)的方法。本申请技术方案对于人乳寡糖的工业化生产有积极的意义，该方法绿色、高效、可持续，有利于工业化大规模生产的应用。
附图说明：
图1.LNBP-M3-W271A催化合成LNT和LNnT纯化后反应液HPLC图。
图2.LNBP-M3-W233F/F456Y催化合成LNT和LNnT纯化后反应液HPLC图。
图3.LNBP-M3-W271A催化合成LNT和LNnT纯化后反应液离子色谱图。
图4.LNBP-M3-W233F/F456Y催化合成LNT纯化后反应液离子色谱图。
图5.LNBP-M3-W271A催化合成反应液rt＝11.34峰物质的质谱图。
具体实施方式
下述实施例中所使用的实验方法，如无特殊说明，均为常规方法；所有的材料、试剂等，如无特殊说明，均可从商业途径获得。
下面通过实施例对本申请进行进一步详细说明，应当理解，此处所描述的具体实施方式仅用以解释本申请，并不用于限定本申请，在不偏离本申请的结构思路、使用范围下对本技术方案的细节和形式进行修改或替换均落入本申请的保护范围内。
除非有其他定义，本说明书中使用的所有技术术语以及科学术语与本领域技术人员通常理解的术语具有相同的意义。一般情况下，本说明书中使用的命名法以及以下记述的实验方法，是在本领域中众所周知且通常被使用的。
如下实施例中，成熟多肽序列催化反应所得产物用HPLC分析方法和离子色谱法进行分析或鉴定。
HPLC分析条件如下：利用高效液相色谱(HPLC)分析，条件如下：色谱柱型号：Cosmosil Sugar-D氨基柱(nacalaitesque,INC.)，流动相为70％乙腈的水溶液，柱温30℃，进样体积10μL，紫外检测器(日立Chromaster)，波长210nm，流速1.0mL/min。
离子色谱法分析条件如下：色谱柱：MetroSep Carb2(4.0mm×250mm)，淋洗液：140mM NaOH/20mM NaAc，等度洗脱，流速：0.500mL/min，安培检测器，柱温40℃，进样体积20μL，运行时间50min。
乳糖-N-三糖(LNTII)、乳糖-N-四糖(LNT)、乳糖-N-新四糖(LNnT)标准品产自法国ELICITYL公司。
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申请的示例性实施方式。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，本申请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，本申请实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本申请的保护范围。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。
实施例1.SEQ ID NO:1、SEQ ID NO:2-29所示成熟多肽序列在大肠杆菌中的表达。
委托基因合成公司，通过密码子优化和DNA合成，获得成熟多肽序列SEQ ID NO:1、SEQ ID NO:2-29对应的基因。具体地，基于如SEQ ID NO:80所示的核苷酸(其编码SEQ ID NO:1所示成熟多肽序列)，通过表1所示的PCR扩增引物进行PCR扩增，获得SEQ ID NO:2-29所示多肽的编码基因。
参照《分子克隆实验指南》方法，构建pET32a-lnbpX重组质粒系列(pET32a-lnbp1、pET32a-lnbp2-29)，引物详见表1。按如下步骤将上述重组质粒转化到大肠杆菌BL21(DE3)中：取制备好的大肠杆菌BL21(DE3)感受态细胞，置于冰上30min融化，取100μL感受态细胞与10μL的pET32a-lnbpX重组质粒(浓度50ng/μL)混 合，置于42℃水浴中热激45s，随后立即冰浴2min冷却，加入1mL的新鲜LB培养基(LB培养基：蛋白胨1.0％，酵母膏0.5％，NaCl 1.0％，平板加1.5％琼脂粉)，于37℃、100rpm条件下复苏培养1h，之后取100μL菌液涂布于含有氨苄青霉素(100μg/mL)的LB平板，37℃恒温培养箱培养12h后挑取单菌落进行菌落PCR(PCR扩增引物见表1)，筛选出阳性转化子。进一步，培养正确的转化子后提取其质粒，利用双酶切以及基因测序验证pET32a-lnbpX重组质粒是否成功导入大肠杆菌。
然后，将正确的转化子接种到LB液体培养基中，置于37℃、200rpm摇床上振荡培养12h，得种子液；再以1％(v/v)的接种量将种子液接种到新鲜的LB培养基中，于37℃振荡培养至OD 600为0.8，经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导，IPTG终浓度0.1mmol/L，于16℃诱导12h，转速200rpm。诱导表达后，在4℃下将发酵液以5000r/min离心30min收集菌体。将菌体用20mM pH7.4PBS缓冲液重悬后，以plus on 5s/off 5s的频率下超声30min中以破碎菌体；将破碎后液体于13000×g、4℃离心30min去除细胞碎片后收集上清液。
利用镍柱亲和层析法纯化得到可溶性成熟多肽序列，过程如下：向镍柱加入去离子水至柱顶端，自然洗脱后，用5倍体积的Binding buffer洗脱；再将经0.45μm滤膜过滤的粗酶液上柱，以1.5mL/min流速使样品与镍柱充分结合，待样品流干后分别用5倍柱体积的Washing buffer连续梯度洗脱去除杂蛋白，最后用5倍Elution buffer洗脱目的蛋白，收集洗脱液。然后利用SDS-PAGE分析目的蛋白表达情况。基因工程菌经诱导后有明显的特异表达条带，条带分子量与预期分子量103kDa基本一致，所得蛋白分别为SEQ ID NO:1、SEQ ID NO:2-29所示成熟多肽序列，详见表2。
表1.重组质粒所用的引物


表2.质粒、成熟多肽序列及氨基酸序列编号对应关系

实施例2.SEQ ID NO:1、SEQ ID NO:2-29所示成熟多肽序列催化合成LNT和/或LNnT
分别将20mM半乳糖-1-磷酸(Gal-1-P)，20mM乳糖-N-三糖(LNT II)，1mmol/L MgCl2，0.1-2.2mg/mL成熟多肽序列(SEQ ID NO:1、SEQ ID NO:2-29所示成熟多肽序列的具体用量详见表3)，100mM Tri-HCl缓冲液(pH为7)混匀，37℃反应24小时，终止反应，用凝胶柱法对其进行纯化。通过HPLC、离子色谱和LC-MS分析和鉴定成熟多肽序列催化反应所产生的物质。
HPLC分析条件如前所示，分析结果显示：
(1)LNTII标准品出峰时间rt为8.79min，LNT和LNnT标准品出峰时间rt均为11.34min。
(2)反应液均在11.34min附近出现了强吸收峰，与LNT和LNnT标准品的出峰时间一致。
LC-MS分析条件如下所示，分析结果显示：
色谱柱型号：Cosmosil Sugar-D氨基柱(nacalaitesque,INC.)，检测器：紫外检测器(日立Chromaster))，检测波长：210nm，进样量：10μL，流速：1.0mL/min，柱温：30℃，流动相为乙腈:水＝70:30；H-ESI模式，分子量扫描范围400～900。
利用LC-MS对反应液HPLC色谱峰在rt＝11.34附近的产物进行分析，其图谱如附图5所示，LNT+/H的分子量为708.2579；LNT+/Na的分子量为730.2393。质谱结果检索可以得到结果如下：Compound CID:440993，Chemcial Formula:C26H45NO21，Extract Mass:707.63，与LNT和LNnT的分子量相同。
离子色谱如前所示，分析结果显示：
(1)乳糖-N-三糖(LNTII)标准品出峰时间为16.9min；乳糖-N-新四糖(LNnT)标准品出峰时间为25.7min；乳糖-N-四糖(LNT)标准品出峰时间为29.8min。
(2)SEQ ID NO:1所示成熟多肽(LNBP-M3)反应液纯化后，在29.8min有强吸收峰，与乳糖-N-四糖(LNT)标准品的出峰时间一致，说明反应合成了乳糖-N-四糖(LNT)。
SEQ ID NO:4所示成熟多肽(LNBP-M3-G312F)反应液纯化后，在25.7min附近均有强吸收峰，与乳糖-N-新四糖(LNnT)标准品的出峰时间一致，说明反应合成了乳糖-N-新四糖(LNnT)。
SEQ ID NO:2-3、SEQ ID NO:5-29所示成熟多肽反应液纯化后，在29.8min附近和25.7min附近均有强吸收峰，与乳糖-N-四糖(LNT)标准品和乳糖-N-新四糖(LNnT)标准品的出峰时间一致，说明反应合成了乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)。
综合以上HPLC、离子色谱和LC-MS分析结果，反应液中LNT和/或LNnT浓度检测结果记录于表3。
表3.SEQ ID NO:1、SEQ ID NO:2-29所示成熟多肽序列催化合成LNT和/或LNnT研究

说明：(LNT或LNnT)产率(mg/mg)＝(LNT或LNnT)产量(mg/ml)/酶添加量(mg/ml)
表3数据说明：在以半乳糖-1-磷酸(Gal-1-P)和乳糖-N-三糖(Lacto-N-trioseⅡ)为底物情况下：
(1)SEQ ID NO:1所示成熟多肽序列能够催化LNTII与半乳糖-1-磷酸合成乳糖-N-四糖(LNT)，但不能合成乳糖-N-新四糖(LNnT)。
(2)SEQ ID NO:4所示LNBP-M3-G312F失去了合成乳糖-N-四糖(LNT)的能力，但能够合成乳糖-N-新四糖(LNnT)。
(3)SEQ ID NO:2-3、SEQ ID NO:5-29所示成熟多肽序列能够同时催化合成乳糖-N-新四糖(LNnT)和乳糖-N-四糖(LNT)。
实施例3.SEQ ID NO:2和SEQ ID NO:26所示成熟多肽序列在乳酸克鲁维酵母(CICC1773)中的表达。
重组质粒pKLAC1-LNBP-M3-W233F或pKLAC1-LNBP-M3-W233F/F456Y的构建方法如下：
根据成熟多肽SEQ ID NO:2、SEQ ID NO:26的氨基酸序列，进行DNA合成，通过酶切连接，构建KLAC1-LNBP-M3-W233F、或KLAC1-LNBP-M3-W233F/F456Y表达质粒。
随后，将KLAC1-LNBP-M3-W233F、或KLAC1-LNBP-M3-W233F/F456Y表达质粒转化到乳酸克鲁维酵母中，酵母转化方法具体如下：
(1)先制备乳酸克鲁维酵母感受态细胞：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1mL 100mM的氯化锂缓冲液，重悬沉淀，12000rpm离心30s，弃上清。再次加入100mM的氯化锂缓冲液400μL，重悬沉淀，得酵母细胞感受态，按50μL/管分装，待转化用。
同时，煮沸1mL鲑鱼精DNA 5min，迅速冰浴以制备单链担体DNA。
(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500μLYPD液体培养基，30℃摇床孵育；1～4h后，取25～100μL菌液涂布于G418抗性的选择性培养基平板，于30℃倒置培养，平板静置培养2-3天，长出的转化子即为目标乳酸克鲁维基因工程菌株。
所述质粒DNA，为KLAC1-LNBP-M3-W233F、或KLAC1-LNBP-M3-W233F/F456Y表达质粒。摇瓶发酵如下：1)一级种的制备：将上述目标乳酸克鲁维基因工程菌株单菌落在10mL YGD液体培养基30℃、200rpm培养过夜，所得的菌种为一级种。2)二级种的制备：一级种接种于1LYGD液体培养基，于30℃、200rpm培养至OD600到7左右。3)将YGD培养基离心，获得目的蛋白粗酶液后，按照实施例1中的方法纯化蛋白，所得蛋白为KL-LNBP-M3-W233F(SEQ ID NO:2)、或KL-LNBP-M3-W233F/F456Y(SEQ ID NO:26)成熟多肽序列。
分别取本实施例所得KL-LNBP-M3-W233F、KL-LNBP-M3-W233F/F456Y，按照实施例2所述催化合成方法，分别将反应体系置于水浴锅中37℃反应24h。离子色谱法测得反应液中LNnT和LNT的产量，结果记录于表4。
表4.乳酸克鲁维酵母表达的KL-LNBP-M3-W233F和KL-LNBP-M3-W233F/F456Y催化合成LNT

实施例4.成熟多肽SEQ ID NO:2、SEQ ID NO:26在枯草芽孢杆菌(Bacillus subtilis 168)中的表达
参照《分子克隆实验指南》的方法，通过限制性内切酶和T4连接酶，分别将实施例1中所获得的多肽SEQ ID NO:2和SEQ ID NO:26的编码基因克隆到pEB03质粒中，以构建重组表达质粒；具体地，分别用限制性内切酶EcoR I、NotI对多肽编码的基因片段和质粒pEB03进行双酶切，然后用T4连接酶将二者进行连接，将连接后的质粒转化到大肠杆菌DH5α中，经筛选鉴定，获得重组表达质粒pEB03□LNBP-M3-W233F、pEB03□LNBP-M3-W233F/F456Y。
pEB03□LNBP-M3-W233F、pEB03□LNBP-M3-W233F/F456Y质粒在枯草芽孢杆菌中的转化方法如下：取一满环枯草芽孢杆菌甘油菌划LB平板(LB培养基：蛋白胨1.0％，酵母膏0.5％，NaCl 1.0％，平板加1.5％琼脂粉)，37℃培养箱培养过夜。挑单菌落至1mL LB培养基中，37℃，200rpm培养过夜后，取200μL培养液转接至200mL生长培养基中(生长培养基：LB培养基加入0.5M山梨醇)，37℃，200rpm培养至OD600达到0.85-0.95，冰浴10min，4℃，5000rpm离心5min，接着利用预冷的电转培养基洗4次(电转培养基：LB培养基加入0.5M山梨醇，10％甘油)，最后利用2mL预冷的电转培养基重悬。将上述处理后的菌液分装成80μL每管，加入1μL(约50ng)质粒，转移至预冷的电转杯，冰上放置1-1.5min，接着利用电转仪进行电转化，电转化参数设置如下：电压2000V，电阻200Ω，接着冰浴2min，然后加入1mL恢复培养基(恢复培养基：LB培养基加入0.5M山梨醇，0.38M甘露醇)于37℃，200rpm培养90min，取菌液涂布氯霉素(5μg/mL)LB平板，挑取转化子即得目标枯草芽孢杆菌基因工程菌株。
从含5μg/mL氯霉素的LB平板挑取阳性转化单菌落接种于20mL种子培养基(酵母浸粉0.5％，胰蛋白胨0.5％，葡萄糖1％，K2HPO4 1.8％，氯霉素5μg/mL)中，37℃、220rpm振荡培养8h。取2.5mL接种到50mL发酵培养基(胰蛋白胨1.0％，酵母粉0.5％，NaCl 1.0％)中，34℃、250rpm振荡培养60h，取上清液进行SDS-PAGE电泳检测。利用SDS-PAGE分析目的蛋白表达情况。获得目的蛋白粗酶液后，按照实施例1中的方法纯化蛋白，条带分子量与预期分子量103kDa基本一致，所得蛋白为SEQ ID NO:2、SEQ ID NO:26所示成熟多肽，命名为为BS-LNBP-M3-W233F、BS-LNBP-M3-W233F/F456Y。
分别取所得BS-LNBP-M3-W233F、BS-LNBP-M3-W233F/F456Y，分别按照实施例2所述催化合成方法，将反应体系置于水浴锅中37℃反应24h。离子色谱法测得反应液中LNnT和LNT的产量，结果记录于表5。
表5.BS-LNBP-M3-W233F、BS-LNBP-M3-W233F/F456Y催化合成LNT
实施例5.成熟多肽SEQ ID NO:2、SEQ ID NO:26在米曲霉菌(CCTCC AF 93036)中的表达。
根据成熟多肽SEQ ID NO:2、SEQ ID NO:26的氨基酸序列，进行密码子优化并DNA合成，获得AO-LNBP-M3-W233F和AO-LNBP-M3-W233F/F456Y的基因。从米曲霉中提取RNA，通过反转录生产cDNA，以米曲霉cDNA为模板扩增米曲霉淀粉酶启动子PamyB及糖苷酶终止子TagdA。通过酶切连接，构建pCAMBIA001-AO-LNBP-M3-W233F、pCAMBIA001-AO-LNBP-M3-W233F/F456Y质粒，测序验证结果显示目的基因正确。
感受态根瘤农杆菌EHA105的制备：1)取经固体YEB平板上活化的农杆菌EHA105，接种于20mL液体YEB培养基中，培养基中已添加利福平抗生素。28℃下，180rpm振荡培养24h。2)按1％接种量将根瘤农杆菌接于50m L添加利福平的液体YEB培养基中，28℃下，180rpm，震荡培养至OD600为0.8，即可使用。3)取50mL菌液冰上放置30min，4℃下，5000rpm离心10min，弃去上清液。4)加入10mL预冷的0.1M浓度的CaCl2溶液，重悬菌体，冰上放置30min；4℃下，5000rpm离心10min，弃去上清。5)加入2mL预冷0.1M浓度的CaCl2和2mL甘油，重悬EHA105细胞，每100μL分装使用，保存在超低温冰箱-70℃备用。
采用冻融法使质粒转化感受态EHA105：1)冰上融化根瘤农杆菌感受态EHA105，加入10μL重组质粒，混匀，冰上放置30min。2)迅速放入液氮中冷刺5min后，立即放入28℃恒温水浴锅中，孵育5min。3)冰上放置5min。4)向此混合液中加入400μL未添加利福平的YEB液体培养基，28℃下，180rpm振荡培养2h。5)取200mL菌液涂布含有利福平和卡那霉素的固体YEB平板，28℃下，倒置培养48h，观察菌落的形成。6)挑取单个菌落至20mL含有利福平和卡那霉素的YEB液体培养基中，28℃下，180rpm振荡摇培48h。筛选阳性克隆。从阳性克隆中提取质粒DNA，进行质粒PCR检测。
根瘤农杆菌介导表达载体转化米曲霉：1)分别将含有不同表达载体的农杆菌EHA105菌液，接入到添加了利福平和卡那霉素的液体LB培养基，28℃下，180rpm培养24h。2)取2mL菌液加入到由10mL液体LB培养基和10mL基础培养基组成的混合培养基中，28℃下，180rpm振荡培养48h。3)取2mL菌液加入到20mL诱导培养基中，28℃下，180rpm振荡培养至OD600为0.8时可与米曲霉进行共培养。4)制备米曲霉分生孢子悬液。5)取100μL孢子悬液和100μL EHA105菌液混和均匀后，涂布在铺有微孔滤膜的固体诱导培养基上，诱导培养基中已添加乙酰丁香酮，28℃下，共培养3天。6)揭下微孔滤膜，反铺到含有潮霉素B和头孢霉素的查氏培养基上，进行抗生素筛选培养，30℃下培养3天。7)移除微孔滤膜，继续培养直至有抗性菌落生长，将筛选出具有抗性的菌落转移至另一选择培养基上，继续进行筛选培养。将筛选出的抗性转化子进行基因组发酵验证。
构建的米曲霉工程菌活化后于30℃恒温培养3天，用无菌水洗下孢子，制成菌悬液接种。将不同菌株按2％分别接种液体发酵培养基，在30℃，200rpm条件下，培养6天，12000rpm离心，收集上清，利用10kDa的超滤膜进行浓缩。
获得目的蛋白粗酶液后，按照实施例1中的方法纯化蛋白，条带分子量与预期分子量103kDa基本一致， 所得蛋白为所示成熟多肽SEQ ID NO:2、SEQ ID NO:26，称为AO-LNBP-M3-W233F和AO-LNBP-M3-W233F/F456Y。
分别取所得AO-LNBP-M3-W233F、AO-LNBP-M3-W233F/F456Y，分别按照实施例2所述催化合成方法，分别将反应体系置于水浴锅中37℃反应24h。离子色谱法测得反应液中LNnT和LNT的产量，结果记录于表6。
表6.AO-LNBP-M3-W233F、AO-LNBP-M3-W233F/F456Y催化合成LNT和LNnT
实施例6.以乳糖为底物，利用成熟多肽LNBP-M3-W233F催化合成乳糖-N-四糖(LNT)和乳糖-N-四糖(LNnT)。
乳糖酶采购自蔚蓝生物，参照论文(β-半乳糖苷酶的固定化及其在制备低乳糖牛奶中的应用，食品工业，2018，39(9)：105-110)提供的方法，将20mmol/L乳糖加入100mmol/L Tris-HCl缓冲液(pH 6.5)中，按照每克乳糖添加120U的乳糖酶加入酶后混均，在37℃下反应5小时，获得混合液A。
取实施例1所得LNBP-M3-W233F和混合液A混合后，参照中国发明专利201911055516.5以及论文(Lacto-N-biose Synthesis via a Modular Enzymatic Cascade with ATP Regeneration.iScience 24(3):102236)提供的方法，将pH值调至7.0后，加入20mmol/L ATP，20U/mL半乳糖激酶(Galk；EC 2.7.1.6)，于30℃反应60分钟。然后，将10mmol/L乳糖-N-三糖(Lacto-N-trioseⅡ)，1mmol/L MgCl2加入混匀，在37℃反应24小时。终止反应后，用离子色谱法检测所得反应液，其中乳糖-N-四糖(LNT)产量2.51mg/mL，乳糖-N-新四糖(LNnT)产量0.59mg/mL。
实施例7.以半乳糖为底物，成熟多肽LNBP-M3-W233F催化合成乳糖-N-四糖(LNT)和乳糖-N-四糖(LNnT)
参照中国发明专利201911055516.5以及论文(Lacto-N-biose Synthesis via a Modular Enzymatic Cascade with ATP Regeneration.iScience 24(3):102236)提供的方法，将20mmol/L半乳糖和20mmol/L ATP加入100mmol/L Tris-HCl缓冲液(pH 7.0)混合后，加入20U/mL半乳糖激酶(Galk；EC 2.7.1.6)，于30℃反应60分钟。然后，将10mmol/L乳糖-N-三糖(Lacto-N-trioseⅡ)，1mmol/L MgCl2，取实施例1所得LNBP-M3-W233F 2.2mg/mL，加入混匀，升温至45℃反应0.5小时。终止反应后，用以上离子色谱法法检测所得反应液，其中乳糖-N-四糖(LNT)产量1.67mg/mL，乳糖-N-新四糖(LNnT)产量0.49mg/mL。
应注意的是，以上实例仅用于说明本申请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申请进行了详细说明，但是本领域的普通技术人员可根据需要对本申请的技术方案进行修改或者等同替换，而不脱离本申请技术方案的范围。

Claims (10)

  1. 一种成熟多肽，其氨基酸序列中包括在如SEQ ID NO:1所示多肽序列中的第233、271、312、336、342、345、456、457、458、459、460位中的至少一个位点发生修饰，其中，所述修饰包括：
    SEQ ID NO:1所示的氨基酸序列的第233位从色氨酸酸残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:2所示的氨基酸序列；或
    SEQ ID NO:1所示的氨基酸序列的第271位从色氨酸残基置换为丙氨酸残基，由此获得如SEQ ID NO:3所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，SEQ ID NO:1所示的氨基酸序列的第312位从甘氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:4、或SEQ ID NO:5、或SEQ ID NO:6所示的氨基酸序列；或
    SEQ ID NO:1所示的氨基酸序列的第336位从丝氨酸残基置换为苯丙氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:7、或SEQ ID NO:8所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第342位从苏氨酸残基置换为苯丙氨酸残基、或亮氨酸残基，由此获得如SEQ ID NO:9、或SEQ ID NO:10所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第345位从甲硫氨酸残基置换为苯丙氨酸残基、或亮氨酸残基，由此获得如SEQ ID NO:11、或SEQ ID NO:12所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第456位从苯丙氨酸残基置换为酪氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:13、或SEQ ID NO:14、或SEQ ID NO:15所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中第457位从甲硫氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:16、或SEQ ID NO:17、或SEQ ID NO:18所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第458位从甘氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:19、或SEQ ID NO:20、或SEQ ID NO:21所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第459位从甘氨酸残基置换为苯丙氨酸残基、或亮氨酸残基、或丙氨酸残基，由此获得如SEQ ID NO:22、或SEQ ID NO:23、或SEQ ID NO:24所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第460位从天冬酰胺残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:25所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第456位从苯丙氨酸残基置换为酪氨酸残基，由此获得如SEQ ID NO:26所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第457位从甲硫氨酸残基置换为丙氨酸残基，由此获得如SEQ ID NO:27所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第460位从天冬酰胺残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:28所示的氨基酸序列；或
    SEQ ID NO:1所示多肽序列中，第233位从色氨酸酸残基置换为苯丙氨酸残基和第342位从苏氨酸残基置换为亮氨酸残基和第460位从天冬酰胺残基置换为苯丙氨酸残基，由此获得如SEQ ID NO:29所示的氨基酸序列。
  2. 一种多核苷酸，其编码如权利要求1所述的成熟多肽。
  3. 一种核酸构建体，其包含如权利要求2所述的多核苷酸，以及与之可操作地连接的一个或多个调控序列，所述调控序列可指导多肽在适当表达宿主中生产。
  4. 一种表达载体，其包含如权利要求2所述的多核苷酸，或者包含如权利要求3所述的核酸构建体。
  5. 一种转化的宿主细胞，其在宿主细胞中转化了如权利要求2所述的多核苷酸、或如权利要求3所述的核酸构建体、或如权利要求4所述的表达载体。
  6. 根据权利要求5所述的转化的宿主细胞，其中，所述宿主细胞为细菌、霉菌或酵母菌；
    优选地，所述细菌选自：芽孢杆菌属和埃希氏杆菌属细菌；
    进一步优选地，所述芽孢杆菌属细菌选自：蕈状芽孢杆菌(Bacillus mycoides)、蜡样芽孢杆菌(Bacillus cereus)、多黏芽孢杆菌(Bacillus polymyxa)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、地衣芽孢杆菌(Bacillus licheniformis)、短芽孢杆菌(Bacillus brevis)、环状芽孢杆菌(Bacillus circulans)和枯草芽孢杆菌(Bacillus subtilis)；
    所述埃希氏杆菌属细菌选自大肠埃希菌(Escherichia coli)、赫尔曼埃希菌(Escherichia hermanii)和弗格森埃希菌(Escherichia fergusonii)，
    所述细菌更优选为环状芽孢杆菌(Bacillus circulans)、枯草芽孢杆菌(Bacillus subtilis)或地衣芽孢杆菌(Bacillus licheniformis)；
    优选地，所述霉菌选自：曲霉属和木霉属霉菌；
    进一步优选地，所述霉菌选自：绿色木霉(Trichoderma viride)、里氏木霉(Trichoderma reesei)、哈茨木霉(Trichoderma harzianum)、米曲霉(Aspergillus oryzae)、烟曲霉(Aspergillus fumigatu)、黑曲霉(Aspergillus niger)和黄曲霉(Aspergillus flavus)，更优选为米曲霉(Aspergillus oryzae)；
    优选地，所述酵母选自：假丝酵母属、汉逊酵母属、克鲁维酵母属、毕赤酵母属、酵母属、裂殖酵母属和耶氏酵母属酵母；
    进一步优选地，所述酵母选自：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(Saccharomyces pastorianus)，更优选为酿酒酵母(Saccharomyces cerevisiae)、乳酸克鲁维酵母(Kluyveromyces lactis)或马克斯克鲁维酵母(Kluyveromyces marxinus)；
    更进一步优选地，所述转化的宿主细胞为其中转化有如权利要求2所述的多核苷酸、或如权利要求3所述的核酸构建体、或如权利要求4所述的表达载体。
  7. 一种组合物，其包含如权利要求1所述的成熟多肽；
    优选的，所述组合物为酶剂或酶组合物。
  8. 如权利要求1所述的多肽的生产方法，其包括：
    (1)在适合表达所述多肽的条件下，培养如权利要求5或权利要求6所述的转化的宿主细胞以产生多肽；和
    (2)回收所述多肽。
  9. 一种生产乳糖-N-四糖和乳-N-新四糖的方法，其包括：以如权利要求1所述的成熟多肽或如权利要求5或6所述的转化的宿主细胞、或如权利要求7所述的酶剂或酶组合物作为催化剂，以乳糖及其衍生物作为反应底物，经过催化或发酵反应，生成糖-N-四糖和乳-N-新四糖；
    所述方法，优选的，反应底物为半乳糖-1-磷酸和/或乳糖和/或半乳糖中的任意一种或任意组合与乙酰氨基葡萄糖和/或带有乙酰氨基葡萄糖基团的碳水化合物；
    所述方法，优选的，反应底物为半乳糖-1-磷酸与乳糖-N-三糖(LNTII)；
    所述方法，优选的，催化或发酵反应在反应溶剂中进行，所述反应溶剂为含水溶剂或含有固形物的溶液；
    所述方法，优选的，所述成熟多肽可以为含有成熟多肽的培养液；
    所述方法，优选的，所述成熟多肽在纯化后使用。
  10. 如权利要求1所述的成熟多肽、或如权利要求5或6所述的转化的宿主细胞、或如权利要求7所述的组合物作为催化剂在生产乳糖-N-四糖和乳-N-新四糖中的应用；
    优选地，所述转化的宿主细胞为在宿主细胞中转化了如权利要求2所述的多核苷酸、或如权利要求3所述的核酸构建体、或如权利要求4所述的表达载体的乳酸克鲁维酵母菌(Kluyveromyces lactis)、枯草芽孢杆菌(Bacillus subtilis)或米曲霉菌(Aspergillus oryzae)；
    优选地，所述生产乳糖-N-四糖和乳-N-新四糖的方法如权利要求9中所述。
PCT/CN2023/112040 2022-11-21 2023-08-09 合成HMOs的成熟多肽序列及应用 WO2024109169A1 (zh)

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