Mature polypeptide sequence for synthesizing HMOs and application

Abstract

The invention describes a series of mutants of a mature polypeptide sequence, the mutants are characterized in that at least one of 233, 271, 312, 336, 342, 345, 456, 457, 458, 459 and 460 sites in the mature polypeptide sequence as shown in SEQ ID NO: 1 is modified, and the mutants have the capability of catalytically synthesizing lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT). The invention also provides an expression method of the mutant of the mature polypeptide sequence as shown in SEQ ID NO: 1. Also provided are methods of catalytically synthesizing lactose-N-tetraose (LNT) and synthesizing LNnT using the mutants of the mature polypeptide sequence.

Description

This application is a divisional of the patent application with application number: 202211473306.X, and invention name: “Mature polypeptide sequences and applications of synthetic HMOs”. Technical Field

[0001] The invention relates to a mature polypeptide sequence of synthetic oligosaccharide and its expression in microorganisms, belonging to the technical field of enzyme engineering / genetic engineering. Background Art

[0002] Breast milk plays a vital role in the growth of infants and young children. Compared with breastfed infants, bottle-fed infants have a lower survival rate and a higher chance of infection, which has aroused scientists' interest in the study of breast milk components. Since the 21st century, researchers have found that the concentration of total HMOs (acidic and neutral oligosaccharides) in human colostrum is as high as 24g / L, but it decreases with the extension of lactation period; it is also found that HMOs are very important for the growth and development of infants and their health in the later period. It can promote the balance of infant intestinal microecology, promote the proliferation of beneficial bacteria in the intestine, inhibit the growth of harmful bacteria, resist pathogen infection, regulate the immune system and promote the cognitive development of infants. Many beneficial effects of breast milk are attributed to human milk oligosaccharides (HMOs).

[0003] With the continuous advancement of analytical tools and technologies for structural characterization, scientists are working to identify the components of breast milk, especially the rich and diverse HMOs, of which more than 200 have been identified. Among them, 2-fucosyllactose (2'-FL), lacto-N-triose II (LNTII), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) were found to be the main components of HMOs, playing the role of bifidobacterial factors. Studies have shown that adding HMOs to infant formula milk powder with cow's milk or goat's milk as the main raw material and feeding infants can approach the effect of breastfeeding in terms of nutrition and immunity of infants. Therefore, adding HMOs with physiological functions to infant formula milk powder, some artificial milk, dairy products, baby food, functional health products or special population foods will greatly improve the physical fitness of the corresponding population and improve their quality of life, which has obvious social and economic value.

[0004] Infant formula and dietary supplements added with HMOs are already sold in the markets of the United States, the European Union, Australia, New Zealand and other countries.

[0005] At present, both the EU EFSA and the US FDA have approved the marketing of lactose-N-tetraose (Lacto-N-tetraose, LNT) and lactose-N-neotetraose (LNnT). The approved lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) are produced by microbial fermentation.

[0006] LNT and LNnT have similar structures, and their molecular weights are both 707.63. The chemical structure of LNT is as follows: Formula 1. Chemical formula of lactose-N-tetraose (LNT)

[0007] The chemical structure of LNnT is as follows: Formula 2. Chemical formula of lactose-N-neotetraose (LNnT)

[0008] HMOs are approved for use in a variety of foods, including dairy products, baked goods, and beverages. The scope of application approved by the European Union has been expanded to dietary supplements, special medical formula foods, and milk-based beverages or similar products for infants only. 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 existing literature have the problems of high production cost, low yield, and high price. Although 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. At the same time, 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.

[0009] 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 β-1,3-N-glucosaminyltransferase and β-1,4-galactosyltransferase were recombinantly integrated into the genome of Bacillus subtilis. After 24 hours of fermentation, the obtained recombinant Bacillus subtilis contained 1071 mg / L lactose-N-neotetraose in the supernatant of the fermentation broth.

[0010] At the same time, enzymatic reactions are widely used to synthesize oligosaccharides due to their simple operation, product specificity, and environmental friendliness. Chinese patent 201180071512.1 reports a method for producing lactose-N-tetraose and lactose-N-neotetraose from lactose-N-triose using active cell extracts.

[0011] However, in order to achieve large-scale production of enzymatically synthesized LNT, and to promote the application of HMOs in various foods such as dairy products, baked goods, and beverages more quickly, although the existing technology provides various HMOs production methods, the efficiency of enzymatic reactions needs to be further improved in order to benefit the public and product iteration, and people are still studying HMOs production methods with more advantages. Summary of the invention

[0012] Purpose of the invention: In view of the shortcomings of the prior art, a mature polypeptide sequence and a preparation method for simultaneously synthesizing lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) are provided, as well as a method for using them to produce oligosaccharides specifically present in human breast milk.

[0013] The existing technology can only synthesize lactose-N-tetraose (LNT) or lactose-N-neotetraose (LNnT). In response to the current market demand for these two sugars, the present invention provides a mature polypeptide sequence and a preparation method that can simultaneously synthesize lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT), as well as a method for using them to produce oligosaccharides specifically present in human breast milk, which improves the existing technology and increases production efficiency.

[0014] Technical solution: As shown in SEQ ID NO: 1, the mature polypeptide sequence is 1,3-β-galactosyl-N-acetylhexosamine phosphorylase (1,3-beta-galactosyl-N-acetylhexosamine phosphorylase), also known as Lacto-N-biosephosphorylase, LNBP.

[0015] The mature polypeptide sequence shown in SEQ ID NO: 1 may be derived from species such as Aspergillus sp., Kluyveromyces sp., Bacillus sp., Bifidobacterium sp., etc. A further preferred species is Bifidobacterium longum, or a microorganism classified as Bifidobacterium longum, a wild strain isolated from nature, or a variant strain after physical and chemical mutagenesis, or an engineered bacterium after genetic engineering.

[0016] The present invention describes a mutant of the mature polypeptide sequence shown in SEQ ID NO: 1, including a mutant in which at least one of positions 233, 271, 312, 336, 342, 345, 456, 457, 458, 459, and 460 in the polypeptide sequence shown in SEQ ID NO: 1 is modified, and has the ability to catalyze the synthesis of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).

[0017] 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.

[0018] Preferably, the modification comprises: 1) the 233rd position of the amino acid sequence shown in SEQ ID NO: 1 is substituted from a tryptophan residue to a phenylalanine residue or one or two of an alanine residue; or 2) the 271st position of the amino acid sequence shown in SEQ ID NO: 1 is substituted from a tryptophan residue to an alanine residue or one or two of a leucine residues; or 3) the 312th position of the amino acid sequence shown in SEQ ID NO: 1 is substituted from a glycine residue to one or more of a phenylalanine residue, a leucine residue, or an alanine residue; or 4) the 336th position of the amino acid sequence shown in SEQ ID NO: 1 is replaced by a serine residue to a phenylalanine residue, or one or more of a leucine residue and an alanine residue; or 5) the 342nd position of the amino acid sequence shown in SEQ ID NO: 1 is substituted from the threonine residue to one or more of a phenylalanine residue, a leucine residue, or a tyrosine residue; or 6) the methionine residue at position 345 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by one or more of a phenylalanine residue, a leucine residue, or an alanine residue; or 7) the 456th position of the amino acid sequence shown in SEQ ID NO: 1 is substituted from a phenylalanine residue to one or more of a tyrosine residue, a leucine residue, or an alanine residue; or 8) the methionine residue at position 457 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by one or more of a phenylalanine residue, a leucine residue, or an alanine residue; or 9) the 458th position of the amino acid sequence shown in SEQ ID NO: 1 is substituted with one or more of a phenylalanine residue, a leucine residue, or an alanine residue; or 10) the 459th position of the amino acid sequence shown in SEQ ID NO: 1 is substituted from a glycine residue to one or more of a phenylalanine residue, a leucine residue, or an alanine residue; or 11) the 460th position of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a phenylalanine residue; or One or a combination of more than one of the substitution sites 1) to 11) above.

[0019] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence in which the tryptophan residue at position 233 of the amino acid sequence shown in SEQ ID NO:1 is replaced by a phenylalanine residue, such as the polypeptide sequence shown in SEQ ID NO:2.

[0020] The mutant of the mature polypeptide sequence is preferably a mutant in which the tryptophan residue at position 271 of the amino acid sequence shown in SEQ ID NO:1 is replaced by an alanine residue, and its amino acid sequence is shown in SEQ ID NO:3.

[0021] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which position 312 of the amino acid sequence shown in SEQ ID NO:1 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, and its amino acid sequence is shown in SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6.

[0022] The mutant of the mature polypeptide sequence is preferably a mutant in which the serine residue at position 336 of the amino acid sequence shown in SEQ ID NO:1 is replaced by a phenylalanine residue or an alanine residue, and its amino acid sequence is shown in SEQ ID NO:7 or SEQ ID NO:8.

[0023] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which the threonine residue at position 342 is replaced by a phenylalanine residue or a leucine residue, and its amino acid sequence is shown in SEQ ID NO:9 or SEQ ID NO:10.

[0024] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which the methionine residue at position 345 is replaced by a phenylalanine residue or a leucine residue, and its amino acid sequence is shown in SEQ ID NO:11 or SEQ ID NO:12.

[0025] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO: 1, in which the phenylalanine residue at position 456 is replaced by a tyrosine residue, a leucine residue, or an alanine residue, and its amino acid sequence is shown in SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0026] The mutant of the mature polypeptide sequence is preferably a mutant in which 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, and its amino acid sequence is shown in SEQ ID NO:16, or SEQ ID NO:17, or SEQ ID NO:18.

[0027] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which the glycine residue at position 458 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, and its amino acid sequence is shown in SEQ ID NO:19, or SEQ ID NO:20, or SEQ ID NO:21.

[0028] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which the glycine residue at position 459 is replaced by a phenylalanine residue, a leucine residue, or an alanine residue, and its amino acid sequence is shown in SEQ ID NO:22, or SEQ ID NO:23, or SEQ ID NO:24.

[0029] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which the asparagine residue at position 460 is replaced by a phenylalanine residue, and its amino acid sequence is shown in SEQ ID NO:25.

[0030] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which 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, and its amino acid sequence is shown in SEQ ID NO:26.

[0031] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which 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, and its amino acid sequence is shown in SEQ ID NO:27.

[0032] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which 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, and its amino acid sequence is shown in SEQ ID NO:28.

[0033] The mutant of the mature polypeptide sequence is preferably a polypeptide sequence shown in SEQ ID NO:1, in which the tryptophan residue at position 233 is replaced by a phenylalanine residue, the threonine residue at position 342 is replaced by a leucine residue, and the asparagine residue at position 460 is replaced by a phenylalanine residue, and its amino acid sequence is shown in SEQ ID NO:29.

[0034] The mutants of the mature polypeptide sequence preferably include amino acid sequences different from 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 and / or SEQ ID NO: 19 and / or SEQ ID NO: 20 and / or SEQ ID NO: 21 and / or SEQ ID NO: 22 and / or SEQ ID NO: 23 and / or SEQ ID NO: 24 and / or SEQ ID NO: 25 and / or SEQ ID NO: 26 and / or SEQ ID NO: 27 and / or SEQ ID NO: 28. NO:28 and / or a polypeptide having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity as shown in SEQ ID NO:29.

[0035] One embodiment of the present invention describes a method for expressing a mutant of the above-mentioned mature polypeptide sequence, comprising the following steps: (a) cultivating a host cell under conditions suitable for expressing a mutant of a mature polypeptide sequence; Preferably, the method further comprises: (b) a step of recovering a mutant of the mature polypeptide sequence.

[0036] Preferably, in 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.

[0037] Preferably, 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.

[0038] Preferably, in the method for expressing the mutant of the mature polypeptide sequence described above, the host cell is selected from yeast cells, filamentous fungal cells, bacterial cells and the like.

[0039] Preferably, the bacterial cells include: Escherichia and Bacillus, etc.

[0040] The Escherichia sp. cells are selected from the group consisting of Escherichia coli, Escherichia hermanii, and Escherichia fergusonii cells.

[0041] The 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.

[0042] Preferably, the filamentous fungi include Aspergillus and Trichoderma.

[0043] The Aspergillus sp. cell includes: Aspergillus oryzae, Aspergillus fumigatu, Aspergillus niger, or Aspergillus flavus cell; further preferably, the host cell is Aspergillus oryzae cell; The Trichoderma sp. cells include: Trichoderma reesei, Trichoderma viride, Trichoderma koningii cells; further preferably, the host cell is a Trichoderma reesei cell; Preferably, the yeast cell includes: Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells; more preferably, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxinus, Yarrowia lipolytica, Rhodotorula graminis, Saccharomyces pastorianus cells; more preferably, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxinus cells; The method for expressing the mutant of the mature polypeptide sequence, the host cell includes a wild strain isolated from nature, or a mutant strain after physical and chemical mutagenesis, or an engineered bacterium after genetic engineering.

[0044] Host cells can be cultivated in a nutrient medium suitable for producing the mutant of the mature polypeptide sequence using methods known in the art. For example, it is possible to culture by shaking bottles, or in a suitable medium and under the condition of allowing the mutant of the mature polypeptide sequence to express and / or separate, carry out small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) to cultivate cells in a laboratory or industrial fermentor. Cultivation is to use a program known in the art, which occurs in a suitable nutrient medium, and the medium comprises carbon and nitrogen sources and inorganic salts. Suitable medium can be purchased through commercial channels, or prepared according to disclosed composition.

[0045] The mutant of the mature polypeptide sequence can be recovered using methods known in the art. For example, the mutant of 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.

[0046] The mutants of the mature polypeptide sequence can be purified by various procedures known in the art to obtain pure mutants of the mature polypeptide sequence, 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 carried out by Ni column affinity chromatography.

[0047] The present invention also describes a composition comprising a mutant of the mature polypeptide sequence of the present invention.

[0048] The composition preferably contains one or more mutants of the mature polypeptide sequence.

[0049] Use of the mutant of the mature polypeptide sequence or a combination thereof in the production of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT); Preferably, the use comprises the steps of generating lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT); Preferably, in the application, 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; Further preferably, in the application, the reaction substrates are galactose-1-phosphate and lactose-N-triose (lacto-N-triose II, LNTII); Preferably, in the application, the reaction solvent is an aqueous solvent or a solution containing solids; Preferably, the application uses a culture fluid of the mutant containing a mature polypeptide sequence; Preferably, in the application, the mutant of the mature polypeptide sequence is purified before use.

[0050] definition: The term "sequence identity": The relatedness between two amino acid sequences is described by the parameter "sequence identity".

[0051] The term "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.

[0052] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with an expression vector. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to substitutions that occur during replication.

[0053] The term "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. In one aspect, mature polypeptides include proteins shown in SEQ ID NOs: 1-29. Beneficial Effects

[0054] The present invention provides a series of mutants of mature polypeptide sequences, which have the ability to catalyze the synthesis of LNT and LNnT and have good application prospects. Although the prior art has studied the preparation of HMOs using some enzymes for catalytic synthesis, 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.

[0055] The mutant of the mature polypeptide sequence of the present invention can simultaneously catalyze the synthesis of LNT and LNnT, and the catalytic efficiency reaches the industrial level.

[0056] The present invention provides a mutant having a mature polypeptide sequence for 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 mutant of the mature polypeptide sequence. The technical solution of the present invention has positive significance for the industrial production of human milk oligosaccharides. The method is green, efficient, sustainable, and conducive to the application of industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 .HPLC chart of the reaction liquid after purification of LNT and LNnT catalyzed by LNBP-M3-W271A.

[0058] Figure 2 .HPLC chart of the reaction liquid after purification of LNT and LNnT catalyzed by LNBP-M3-W233F / F456Y.

[0059] Figure 3 .Ion chromatogram of the reaction solution after purification of LNT and LNnT catalyzed by LNBP-M3-W271A.

[0060] Figure 4 .Ion chromatogram of the reaction liquid after purification of LNT catalyzed by LNBP-M3-W233F / F456Y.

[0061] Figure 5 .Mass spectrum of the peak substance at rt=11.34 in the LNBP-M3-W271A catalytic synthesis reaction solution. DETAILED DESCRIPTION

[0062] The experimental methods used in the following examples are conventional methods unless otherwise specified; all materials, reagents, etc., are commercially available unless otherwise specified.

[0063] The present invention is further described in detail below through examples. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention. Modifications or replacements to the details and forms of the technical solution without departing from the structural concept and scope of use of the present invention all fall within the protection scope of the present invention.

[0064] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art.

[0065] In the following examples, the products obtained by catalytic reaction of the mutants of the mature polypeptide sequence were analyzed or identified by HPLC analysis method and ion chromatography.

[0066] HPLC analysis conditions are as follows: High performance liquid chromatography (HPLC) analysis was performed under the following conditions: chromatographic column model: Cosmosil Sugar-D amino column (nacalaitesque, INC.), mobile phase was 70% acetonitrile in water, column temperature was 30°C, injection volume was 10 μL, UV detector (Hitachi Chromaster), wavelength was 210 nm, flow rate was 1.0 mL / min.

[0067] The ion chromatography analysis conditions were as follows: chromatographic column: MetroSep Carb2 (4.0 mm×250 mm), eluent: 140 mM NaOH / 20 mM NaAc, isocratic elution, flow rate: 0.500 mL / min, amperometric detector, column temperature 40°C, injection volume 20 μL, running time 50 min.

[0068] Lactose-N-triose (LNTII), lactose-N-tetraose (LNT), and lactose-N-neotetraose (LNnT) standards were produced by ELICITYL, France.

[0069] It should be noted that the terms used here are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are to describe specific embodiments, rather than to limit the scope of protection of the present invention. If the experimental methods of the specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology within the art are generally followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0070] Example 1. Expression of the mature polypeptide sequence shown in SEQ ID NO: 1 and the mature polypeptide sequence mutants shown in SEQ ID NO: 2-29 in Escherichia coli.

[0071] A gene synthesis company was commissioned to obtain the genes corresponding to the mature polypeptide sequences SEQ ID NO: 1-29 through codon optimization and DNA synthesis. The pET32a-lnbpX recombinant plasmid series (pET32a-lnbp1-29) were constructed with reference to the method of the Molecular Cloning Experiment Guide, as shown in Table 1 (consisting of Table 1-1, Table 1-2, Table 1-3, Table 1-4). The above recombinant plasmid was transformed into Escherichia coli BL21 (DE3) according to the following steps: the prepared Escherichia coli BL21 (DE3) competent cells were taken and placed on ice for 30 minutes to melt, 100 μL of competent cells were mixed with 10 μL of pET32a-lnbpX recombinant plasmid (concentration 50 ng / μL), placed in a 42°C water bath for heat shock for 45 seconds, and then immediately cooled in an ice bath for 2 minutes, 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, and 1.5% agar powder on the plate) was added, and the culture was resuscitated at 37°C and 100 rpm for 1 hour, and then 100 μL of the bacterial solution was spread on an LB plate containing ampicillin (100 μg / mL), and after culturing in a 37°C constant temperature incubator for 12 hours, a single colony was picked for colony PCR (PCR amplification primers are shown in Table 1) to screen positive transformants. Furthermore, the plasmid was extracted after culturing the correct transformant, and PCR amplification, double enzyme digestion and gene sequencing were used to verify whether the pET32a-lnbpX recombinant plasmid was successfully introduced into Escherichia coli.

[0072] Then, the correct transformant was inoculated into LB liquid medium, placed on a shaker at 37°C and 200rpm for 12h to obtain seed liquid; the seed liquid was then inoculated into fresh LB medium at a 1% (v / v) inoculation amount, shaken and cultured at 37°C until OD 600 was 0.8, and induced by isopropyl-β-D-thiogalactopyranoside (IPTG), with a final concentration of 0.1mmol / L of IPTG, induced at 16°C for 12h, and a speed of 200rpm. After induction of expression, the fermentation broth was centrifuged at 5000r / min for 30min at 4°C to collect the bacteria. After 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 to break the bacteria; the broken liquid was centrifuged at 13000×g and 4°C for 30min to remove cell debris and collect the supernatant.

[0073] The soluble mature polypeptide sequence was purified by nickel column affinity chromatography, and the process was as follows: deionized water was added to the nickel column to the top of the column, and after natural elution, it was eluted with 5 volumes of Binding buffer; the crude enzyme solution filtered by 0.45 μm filter membrane was then loaded onto the column, and the sample was fully combined with the nickel column at a flow rate of 1.5 mL / min. After the sample was drained, it was continuously gradient eluted with 5 column volumes of Washing buffer to remove impurities, and 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. After induction, the genetically engineered bacteria had obvious specific expression bands, and the molecular weight of the band was basically consistent with the expected molecular weight of 103 kDa. The obtained proteins were the mature polypeptide sequence shown in SEQ ID NO: 1, and the mutant of the mature polypeptide sequence shown in SEQ ID NO: 2-29, as shown in Table 2 (composed of Table 2-1 and Table 2-2).

[0074] Table 1-1. Primers used for recombinant plasmids

[0075] Table 1-2. Primers used for recombinant plasmids (continued from Table 1-1)

[0076] Table 1-3. Primers used for recombinant plasmids (continued from Table 1-2)

[0077] Table 1-4. Primers used for recombinant plasmids (continued from Table 1-3)

[0078] Table 2-1. Correspondence between plasmid, mature polypeptide sequence, mutant and amino acid sequence number Plasmid name Mature polypeptide sequence name Amino acid sequence number pET32a-lnb LNBP-M3 SEQ ID NO:1 pET32a-lnb LNBP-M3-W233F SEQ ID NO:2 pET32a-lnb LNBP-M3-W271A SEQ ID NO:3 pET32a-lnb LNBP-M3-G312F SEQ ID NO:4 pET32a-lnb LNBP-M3-G312L SEQ ID NO:5 pET32a-lnb LNBP-M3-G312A SEQ ID NO:8 pET32a-lnb LNBP-M3-S336F SEQ ID NO:7 pET32a-lnb LNBP-M3-S336A SEQ ID NO:8 pET32a-lnb LNBP-M3-T342F SEQ ID NO:9 pET32a-lnb LNBP-M3-T342L SEQ ID NO:10 pET32a-lnb LNBP-M3-M345F SEQ ID NO:11 pET32a-lnb LNBP-M3-M345L SEQ ID NO:12 pET32a-lnb LNBP-M3-F456Y SEQ ID NO:13 pET32a-lnb LNBP-M3-F456L SEQ ID NO:14 pET32a-lnb LNBP-M3-F456A SEQ ID NO:15

[0079] Table 2-2. Correspondence between plasmid, mature polypeptide sequence, mutant and amino acid sequence number (continued from Table 2-1) Plasmid name Mature polypeptide sequence name Amino acid sequence number pET32a-lnb LNBP-M3-M457F SEQ ID NO:16 pET32a-lnb LNBP-M3-M457L SEQ ID NO:17 pET32a-lnb LNBP-M3-M457A SEQ ID NO:18 pET32a-lnb LNBP-M3-G458F SEQ ID NO:19 pET32a-lnb LNBP-M3-G458L SEQ ID NO:20 pET32a-lnb LNBP-M3-G458A EQ ID NO:21 pET32a-lnb LNBP-M3-G459F SEQ ID NO:22 pET32a-lnb LNBP-M3-G459L SEQ ID NO:23 pET32a-lnb LNBP-M3-G459A SEQ ID NO:24 pET32a-lnb LNBP-M3-N460F SEQ ID NO:25 pET32a-lnb LNBP-M3-W233F / F456Y SEQ ID NO:26 pET32a-lnb LNBP-M3-W233F / M457A SEQ ID NO:27 pET32a-lnb LNBP-M3-W233F / N460F SEQ ID NO:28 pET32a-lnb LNBP-M3-W233F / T342L / N SEQ ID NO:29

[0080] Example 2. The mature polypeptide sequence shown in SEQ ID NO: 1 and the mutants of the mature polypeptide sequences shown in SEQ ID NO: 2-29 catalyze the synthesis of LNT and / or LNnT.

[0081] Specifically, 20 mM galactose-1-phosphate (Gal-1-P), 20 mM lactose-N-triose (lacto-N-trioseⅡ), 1 mmol / L MgCl2, 0.1-2.2 mg / mL mature polypeptide sequence (shown in SEQ ID NO: 1-29, see Table 3 for specific amounts), and 100 mM Tri-HCl buffer (pH 7) were mixed, reacted at 37°C for 24 hours, the reaction was terminated, and purified by gel column method. The substances produced by the catalytic reaction of the mature polypeptide sequence were analyzed and identified by HPLC, ion chromatography and LC-MS.

[0082] The HPLC analysis conditions were as shown above, and the analysis results showed: (1) The peak time rt of LNTII standard is 8.79 min, and the peak time rt of LNT and LNnT standard is 11.34 min.

[0083] (2) The reaction solutions all showed a strong absorption peak at around 11.34 min, which was consistent with the peak time of LNT and LNnT standards.

[0084] The LC-MS analysis conditions are as follows, and the analysis results show: Chromatographic column model: Cosmosil Sugar-D amino column (nacalaitesque, INC.), detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 1.0 mL / min, column temperature: 30°C, mobile phase: acetonitrile: water = 70:30; H-ESI mode, molecular weight scanning range 400-900.

[0085] The product at the HPLC chromatogram peak of the reaction solution near rt = 11.34 was analyzed by LC-MS, and its spectrum is attached. Figure 5As shown, the molecular weight of LNT+ / H is 708.2567; the molecular weight of LNT+ / Na is 730.2393. The mass spectrometry results can be retrieved to obtain the following results: Compound CID: 440993, Chemical Formula: C 26 H 45 NO 21 , Extract Mass:707.63, which is the same as the molecular weight of LNT and LNnT.

[0086] The ion chromatogram is shown above, and the analysis results show: (1) The peak elution time of lactose-N-triose (LNTII) standard was 16.9 min; the peak elution time of lactose-N-neotetraose (LNnT) standard was 25.7 min; and the peak elution time of lactose-N-tetraose (LNT) standard was 29.8 min.

[0087] (2) After the reaction solution of the mature polypeptide (LNBP-M3) shown in SEQ ID NO: 1 was purified, a strong absorption peak was found at 29.8 min, which was consistent with the peak time of the lactose-N-tetraose (LNT) standard, indicating that lactose-N-tetraose (LNT) was synthesized in the reaction.

[0088] After purification of the reaction solution of the mature polypeptide mutant (LNBP-M3-G312F) shown in SEQ ID NO: 4, a strong absorption peak was found around 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.

[0089] After purification, the reaction solutions of the mature polypeptide mutants shown in SEQ ID NOs: 2-3 and SEQ ID NOs: 5-29 showed strong absorption peaks near 29.8 min and 25.7 min, which were consistent with the peak times of the lactose-N-tetraose (LNT) standard and the lactose-N-neotetraose (LNnT) standard, indicating that lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) were synthesized in the reaction.

[0090] in conclusion The above HPLC, ion chromatography and LC-MS analysis results show that: Lactose-N-tetraose (LNT) was synthesized using galactose-1-phosphate (Gal-1-P) and lactose-N-triose (Lacto-N-trioseⅡ) as substrates using the mature polypeptide (LNBP-M3) shown in SEQ ID NO:1.

[0091] The mature polypeptide mutant (LNBP-M3-G312F) shown in SEQ ID NO:4 was used to catalyze the synthesis of lactose-N-neotetraose (LNnT) using galactose-1-phosphate (Gal-1-P) and lactose-N-triose (Lacto-N-trioseⅡ) as substrates, indicating that the mature polypeptide lost the ability to synthesize lactose-N-tetraose (LNT) after mutation.

[0092] The mature polypeptide mutants shown in SEQ ID NO: 2-3 and SEQ ID NO: 5-29 were used to catalyze the synthesis of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) using galactose-1-phosphate (Gal-1-P) and lactose-N-triose (Lacto-N-trioseⅡ) as substrates. The results of the detection of LNT and / or LNnT concentrations in the reaction solution are recorded in Table 3 (composed of Table 3-1 and Table 3-2).

[0093] Table 3-1. Study on the catalytic synthesis of LNT and / or LNnT by the mature polypeptide sequence shown in SEQ ID NO: 1 and the mutants of the mature polypeptide sequence shown in SEQ ID NO: 2-29

[0094] Table 3-2. Study on the catalytic synthesis of LNT and / or LNnT by the mature polypeptide sequence shown in SEQ ID NO: 1 and the mutants of the mature polypeptide sequence shown in SEQ ID NO: 2-29

[0095] Note: (LNT or LNnT) yield (mg / mg) = (LNT or LNnT) yield (mg / ml) / enzyme addition amount (mg / ml) Table 3 Data Description: (1) 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).

[0096] (2) 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).

[0097] (3) The mature polypeptide sequence mutants shown in SEQ ID NOs: 2-3 and SEQ ID NOs: 5-29 can simultaneously catalyze the synthesis of lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT).

[0098] Example 3. Expression of the mature polypeptide sequence mutants shown in SEQ ID NO: 2 and SEQ ID NO: 26 in Kluyveromyces lactis The construction method of the recombinant plasmid pKLAC1-LNBP-M3-W233F or pKLAC1-LNBP-M3-W233F / F456Y is as follows: According to the amino acid sequences of the mature polypeptides SEQ ID NO: 2 and SEQ ID NO: 26, codon optimization and DNA synthesis were performed, and the KLAC1-LNBP-M3-W233F or KLAC1-LNBP-M3-W233F / F456Y expression cassette was successfully constructed and verified by PCR amplification, enzyme digestion and gene sequencing.

[0099] Subsequently, the KLAC1-LNBP-M3-W233F or KLAC1-LNBP-M3-W233F / F456Y expression cassette was transformed into Kluyveromyces lactis. The yeast transformation method is as follows: (1) First, prepare competent cells of Kluyveromyces lactis: take a small amount of frozen yeast strains and streak them on a plate solid culture medium, invert and culture at 30°C for 2 days. Pick a single yeast colony in 50 mL liquid culture medium and culture at 30°C, 220 rpm until OD 600 Between 0.8 and 1.5. Collect the cells and wash with 25 mL of sterile water, centrifuge at room temperature at 1500 × g for 10 min, and discard the supernatant. Add 1 mL of 100 mM lithium chloride buffer, resuspend the precipitate, centrifuge at 12000 rpm for 30 s, and discard the supernatant. Add 400 μL of 100 mM lithium chloride buffer again, resuspend the precipitate, and obtain the competent yeast cells, and dispense them into 50 μL / tube for transformation.

[0100] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly place on ice to prepare single-stranded carrier DNA.

[0101] (2) Transformation: Centrifuge the competent yeast prepared above and remove the residual lithium chloride solution with tips. For each transformation, add in the following order: 50% PEG3350 (240 μL); 1M LiCl (36 μL); 2mg / mL single-stranded Salmonsperm DNA (25 μL); 5-10 μg / 50 μL H2O plasmid DNA (50 μL), vortex vigorously to mix until the precipitated bacteria are completely and evenly distributed; incubate in a 30°C water bath for 30 minutes; heat shock in a 42°C water bath for 20-25 minutes; after centrifugation at 8000rpm for 10 minutes, collect the yeast cells; then, resuspend the yeast in 500 μL YPD liquid culture medium and incubate in a shaker at 30°C; after 1-4 hours, take 25-100 μL of the bacterial solution and spread it on a G418-resistant selective culture medium plate, invert and culture at 30°C, and let the plate stand for 2-3 days. The transformed cells grown are the target lactic acid Kluyveromyces genetic engineering strains.

[0102] The plasmid DNA is a KLAC1-LNBP-M3-W233F or KLAC1-LNBP-M3-W233F / F456Y expression cassette. Shake flask fermentation is as follows: 1) Preparation of primary species: The above-mentioned target lactic acid Kluyveromyces genetic engineering strain single colony is cultured overnight in 10mL YGD liquid 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 1LYGD liquid medium and cultured at 30°C and 200rpm until OD600 is about 7. 3) The YGD medium is centrifuged to obtain the target protein crude enzyme solution, and the protein is purified according to the method in Example 1. 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).

[0103] KL-LNBP-M3-W233F and KL-LNBP-M3-W233F / F456Y obtained in this example were 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.

[0104] Table 4. LNT synthesis catalyzed by KL-LNBP-M3-W233F and KL-LNBP-M3-W233F / F456Y expressed by Kluyveromyces lactis Recombinant protein name Product LNnT concentration, mg / mL Product LNT concentration, mg / mL KL-LNBP-M3-W233F 0.92 7.82 KL-LNBP-M3-W233F / F456Y 1.03 9.34

[0105] Example 4. Expression of mature polypeptides (SEQ ID NO: 2 and SEQ ID NO: 26) in Bacillus subtilis The genome of the genetically engineered Escherichia coli in Example 1 was used as a template, and the primers in the table were used for PCR amplification to obtain the gene sequences corresponding to the mature polypeptides SEQ ID NO: 2 and SEQ ID NO: 26. PCR reaction system 50 μL: DNA template 1 μL (about 50 ng), 2×Phanta Max Buffer 25 μL, 10 pmol / μL dNTP Mix 1 μL, 10 pmol / μL forward and reverse primers 2 μL each, 1U / μL Phanta Max Super-Fidelity DNA Polymerase DNA polymerase 1 μL, add ultrapure water to 50 μL. PCR reaction program: 95°C, 3min; 95°C, 15s, 58°C, 15s, 72°C, 2.5min, 30 cycles; 72°C, 5min; 4°C storage. The PCR fragment and plasmid pEB03 were double-digested with restriction endonucleases EcoR I and Hind III, ligated with T4 ligase, and transformed into Escherichia coli DH5α. After screening and identification, the recombinant plasmid pEB03-LNBP-M3-W233F / pEB03-LNBP-M3-W233F / F456Y was obtained.

[0106] The transformation method of pEB03-LNBP-M3-W233F / pEB03-LNBP-M3-W233F / F456Y plasmid in Bacillus subtilis is as follows: take a full loop of Bacillus subtilis glycerol bacteria and streak it on LB plate (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, and 1.5% agar powder on the plate), and culture it in a 37°C incubator overnight. Pick a single colony into 1 mL LB medium, culture at 37°C, 200 rpm overnight, then transfer 200 μL of culture solution into 200 mL growth medium (growth medium: LB medium with 0.5 M sorbitol added), culture at 37°C, 200 rpm until OD600 reaches 0.85-0.95, ice bath for 10 min, centrifuge at 4°C, 5000 rpm for 5 min, then wash 4 times with pre-cooled electroporation medium (electroporation medium: LB medium with 0.5 M sorbitol, 10% glycerol), and finally resuspend with 2 mL pre-cooled electroporation medium. The treated bacterial solution was divided into 80 μL per tube, 1 μL (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 Ω, 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. The bacterial solution was coated on a chloramphenicol (5 μg / mL) LB plate, and the transformant was picked to obtain the target Bacillus subtilis genetically engineered strain.

[0107] Pick a positive transformation colony from the LB plate containing 5μg / mL chloramphenicol and inoculate it into 20mL seed culture medium (0.5% yeast extract, 0.5% tryptone, 1% glucose, 1.8% K2HPO4, 5μg / mL chloramphenicol), and culture it at 37℃, 220rpm for 8h. Take 2.5mL and inoculate it into 50mL fermentation medium (1.0% tryptone, 0.5% yeast powder, 1.0% NaCl), and culture it at 34℃, 250rpm for 60h. Take the supernatant for SDS-PAGE electrophoresis. Use SDS-PAGE to analyze the expression of the target protein. After obtaining the crude enzyme solution of the target protein, 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 or SEQ ID NO: 26, named BS-LNBP-M3-W233F or BS-LNBP-M3-W233F / F456Y.

[0108] The obtained BS-LNBP-M3-W233F and BS-LNBP-M3-W233F / F456Y were 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.

[0109] Table 5. Synthesis of LNT catalyzed by BS-LNBP-M3-W233F and BS-LNBP-M3-W233F / F456Y Recombinant protein Product LNnT concentration, mg / mL Product LNT concentration, mg / mL BS-LNBP-M3-W233F 1.01 7.67 BS-LNBP-M3-W233F / F456Y 0.99 9.03

[0110] Example 5. Expression of mature polypeptides SEQ ID NO: 2 and SEQ ID NO: 26 in Aspergillus oryzae According to the amino acid sequences of mature polypeptides SEQ ID NO:2 and SEQ ID NO:26, codon optimization and DNA synthesis were performed to obtain the genes of AO-LNBP-M3-W233F and AO-LNBP-M3-W233F / F456Y. RNA was extracted from Aspergillus oryzae, and cDNA was produced by reverse transcription. The Aspergillus oryzae cDNA was used as a template to amplify the Aspergillus oryzae amylase promoter PamyB and glycosidase terminator TagdA. pCAMBIA001-AO-LNBP-M3-W233F and The sequencing verification results of pCAMBIA001-AO-LNBP-M3-W233F / F456Y plasmid showed that the target gene was correct.

[0111] 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℃. 2) Inoculate Agrobacterium tumefaciens into 50mL liquid YEB medium with rifampicin added at 1% inoculum, shake and culture at 28℃, 180rpm until OD600 is 0.8, then it can be used. 3) Take 50mL bacterial solution and place it on ice for 30min, centrifuge it at 5000rpm for 10min at 4℃, and discard the supernatant. 4) Add 10mL pre-cooled 0.1M CaCl2 solution, resuspend the bacteria, place it on ice for 30min; centrifuge it at 5000rpm for 10min at 4℃, and discard the supernatant. 5) Add 2 mL of pre-cooled 0.1 M CaCl2 and 2 mL of glycerol, resuspend the EHA105 cells, and dispense into 100 μL portions for use. Store in an ultra-low temperature refrigerator at -70°C until use.

[0112] The plasmid was transformed into competent EHA105 by freeze-thaw method: 1) Thaw competent EHA105 of Agrobacterium tumefaciens on ice, add 10 μL of 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℃ constant temperature water bath and incubate it for 5 minutes. 3) Place it on ice for 5 minutes. 4) Add 400 μL of YEB liquid medium without rifampicin to this mixture, and shake and culture it at 28℃, 180rpm for 2 hours. 5) Take 200mL of bacterial liquid and spread it on a solid YEB plate containing rifampicin and kanamycin, invert and culture it at 28℃ for 48 hours, and observe the formation of colonies. 6) Pick a single colony and put it into 20mL of YEB liquid medium containing rifampicin and kanamycin, and shake and culture it at 28℃, 180rpm for 48 hours. Use colony PCR to identify and screen positive clones. Extract plasmid DNA from positive clones and perform plasmid PCR detection.

[0113] Transformation of Aspergillus oryzae with expression vector mediated by Agrobacterium tumefaciens: 1) Inoculate the EHA105 bacterial solution 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 solution 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 solution and add it to 20mL of induction medium, and culture at 28°C and 180rpm until OD600 is 0.8, and then co-culture with Aspergillus oryzae. 4) Prepare conidia suspension of Aspergillus oryzae. 5) Take 100μL of spore suspension and 100μL of EHA105 bacterial solution, mix them evenly, and spread them on a solid induction medium covered with a microporous filter membrane, to which acetosyringone has been added, and co-culture at 28°C for 3 days. 6) Remove the microporous filter membrane and invert it onto Cha's medium containing hygromycin B and cephalosporin for antibiotic screening culture at 30°C for 3 days. 7) Remove the microporous filter membrane and continue culturing until resistant colonies grow. Transfer the selected resistant colonies to another selection medium and continue screening culture. Perform genomic PCR verification on the selected resistant transformants.

[0114] After the constructed 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.

[0115] After obtaining the crude enzyme solution of the target protein, 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 SEQ ID NO: 2 and SEQ ID NO: 26, referred to as AO-LNBP-M3-W233F and AO-LNBP-M3-W233F / F456Y.

[0116] The obtained AO-LNBP-M3-W233F and AO-LNBP-M3-W233F / F456Y were 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.

[0117] Table 6. Synthesis of LNT and LNnT catalyzed by AO-LNBP-M3-W233F and AO-LNBP-M3-W233F / F456Y Recombinant protein Product LNnT concentration, mg / mL Product LNT concentration, mg / mL AO-LNBP-M3 0.94 7.62 AO-LNBP-M3-W233F / F456Y 1.04 9.19

[0118] Example 6. Synthesis of lactose-N-tetraose (LNT) and lactose-N-tetraose (LNnT) catalyzed by mature polypeptide LNBP-M3-W233F using lactose as substrate Lactase was purchased from Weilan Biotechnology. Referring to the method provided in the paper (Immobilization of β-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 enzyme was mixed and reacted at 37°C for 5 hours to obtain a mixed solution A.

[0119] After mixing LNBP-M3-W233F obtained in Example 1 and mixed solution A, refer to the method provided in Chinese invention patent 201911055516.5 and the paper (Lacto-N-biose Synthesis via a Modular Enzymatic Cascade with ATP Regeneration. iScience 24(3):102236), adjust the pH value to 7.0, add 20mmol / LATP, 20U / mL galactokinase (Galk; EC 2.7.1.6), and react at 30°C for 60 minutes. Then, 10mmol / L lactose-N-triose (Lacto-N-trioseⅡ) and 1mmol / L MgCl2 were added and mixed, and reacted at 37°C for 24 hours. After the reaction was terminated, the reaction solution was detected by ion chromatography, wherein the yield of lactose-N-tetraose (LNT) was 2.51 mg / mL and the yield of lactose-N-neotetraose (LNnT) was 0.59 mg / mL.

[0120] 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) Referring to the method provided in Chinese invention patent 201911055516.5 and the paper (Lacto-N-biose Synthesis via a Modular Enzymatic Cascade with ATP Regeneration. iScience 24(3):102236), 20mmol / L galactose and 20mmol / L ATP were added to 100mmol / L Tris-HCl buffer (pH 7.0) and mixed, and then 20U / mL galactokinase (Galk; EC 2.7.1.6) was added and reacted at 30°C for 60 minutes. Then, 10mmol / L lactose-N-triose (Lacto-N-trioseⅡ), 1mmol / L MgCl2, 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.

[0121] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A polypeptide whose amino acid sequence is shown in SEQ ID NO: 5 or 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.

2. A composition, characterized in that Contains the polypeptide according to claim 1.

3. The method for expressing the polypeptide according to claim 1, characterized in that: The method comprises the following steps: culturing host cells under conditions suitable for expressing the polypeptide.

4. The method for expressing a polypeptide according to claim 3, characterized in that: The host cell is selected from the group consisting of Escherichia coli, Kluyveromyces lactis, Bacillus subtilis and Aspergillus oryzae.

5. Use of the polypeptide according to claim 1 or the composition according to claim 2 in the production of lactose-N-tetraose and lactose-N-neotetraose.

6. A method for producing lacto-N-tetraose and lacto-N-neotetraose, characterized in that: Lactose-N-tetraose and lacto-N-neotetraose are synthesized by using the polypeptide according to claim 1 or the composition according to claim 2 as a catalyst and galactose-1-phosphate and lactose-N-triose as substrates.

7. A method for producing lactose-N-tetraose and lacto-N-neotetraose, comprising: Lactose and lactose-N-triose are used as substrates, and the polypeptide according to claim 1 or the composition according to claim 2 and galactokinase are used as catalysts to synthesize lactose-N-tetraose and lacto-N-neotetraose.

8. A method for producing lactose-N-tetraose and lacto-N-neotetraose, comprising: Galactose and lactose-N-triose are used as substrates, and the polypeptide according to claim 1 or the composition according to claim 2 and galactokinase are used as catalysts to synthesize lactose-N-tetraose and lacto-N-neotetraose.

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