Engineering bacterium for efficiently producing lactose-N-neotetraose and application of engineering bacterium

Abstract

The invention provides an engineering bacterium for efficiently producing lactose-N-neotetraose and application of the engineering bacterium, and relates to the technical field of biological engineering. Galactosyl transferase is expressed by the engineering bacterium; the galactosyl transferase comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence which has at least 98% sequence identity with the SEQ ID NO.1; and / or an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence with at least 98% sequence identity with the SEQ ID NO.3. The galactosyl transferase which is high in yield of lactose-N-neotetraose and can efficiently convert lactose-N-triose to synthesize the lactose-N-neotetraose is successfully screened in the invention; and a new engineering enzyme is provided for the process of converting the lactose-N-trisaccharide into the lactose-N-neotetraose.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to an engineering bacterium for efficiently producing lactose-N-neotetraose and its application. Background Art

[0002] Human milk has a significant effect on the growth and development of infants and young children, and contains many active biological molecules that are not found in other milk sources (cow's milk, goat's milk), such as bioactive peptides, innate glycoproteins, free fatty acids and cytokines, etc. It has the function of promoting the healthy growth of infants and young children, protecting infants from infection and inflammation, and promoting the development of immune function and organ maturation. Human milk oligosaccharides (HMOs) are the third largest substance in human milk after lactose and lipids, with a content of 5-15g / L, and are the main biologically active substances in human milk. To date, more than 200 different types of HMOs have been identified in human milk. In recent years, with the approval of foreign countries to add HMOs to infant formula, improving the nutritional value of infant food and dairy products has become a topic of increasing concern.

[0003] The types of HMOs are mainly divided into three categories: neutral fucosylation, neutral non-fucosylation and acidic sialylated sugars. There are three methods for their synthesis: chemical synthesis, enzyme synthesis and microbial fermentation. Microbial fermentation is the mainstream method for producing HMOs. Lactose-N-neotetraose (LNnT) is a neutral non-fucosylated sugar and one of the important core structures of HMOs. The microbial fermentation method synthesizes LNnT using relatively cheap lactose as a substrate. Through the action of β-1,3-N-acetylglucosamine transferase and β-1,4-galactosyltransferase, LNnT is generated through a two-step reaction. The intermediate metabolite lactose-N-triose (LNT II) is the precursor structure of LNnT. Because the LNnT listing regulations require that the LNT II content in the product must be within a certain range, if the concentration of LNT II in the fermentation broth is high, it will have a great impact on the subsequent LNnT separation and purification and product purity, greatly increasing the complexity of the purification process and the high requirements for purification equipment, thereby increasing production costs. Therefore, the intermediate metabolite LNT II can be regarded as a by-product.

[0004] In 2022, Mu Wanmeng's team at Jiangnan University successfully screened out two gene sequences that can synthesize LNnT through gene screening, which were derived from Histophilus somni and Aggregatibacter actinomycetemcomitans. The E. coli strain expressing the second gene sequence produced 12.10 g / L of LNnT in a fermenter, but the byproduct LNT II was 9.36 g / L, accounting for 43.6%. In 2023, Zhang Tao's team at Jiangnan University also used exogenous expression of lgtA and lgtB in E. coli, combined regulation of the expression of glmU, glmS and glmM in the LNnT synthesis pathway and knocked out the lacZ and wecB genes of the E. coli host. The yield of LNnT in the fermenter reached 13.25 g / L, but the yield of the byproduct LNT II was still above 6 g / L, accounting for about 31%. In terms of regulations, as early as 2016, the US FDA approved LNnT produced by microbial fermentation as a GRAS product. In the existing LNnT quality standards, the content of LNnT is generally not less than 92%, and the content of LNT II is generally not more than 3%. Therefore, if the proportion of LNT II in the fermentation broth is too high, it will cost a lot of separation and purification costs to meet the quality standards. In addition to causing low purity of the final product and increasing separation and purification costs, a high proportion of LNT II will also result in a low conversion rate of the substrate lactose, that is, lactose is not completely converted into the target product LNnT, which will also increase the cost of raw materials.

[0005] It is reported that the separation and purification cost of biomanufacturing products generally accounts for at least 60% of the total cost, and can be as high as 80%. For the production of LNnT, reducing the accumulation of the byproduct LNT II is of great significance to reducing the separation and purification cost. It can also improve the conversion rate of the substrate lactose and improve the purity of the final product. The ability of the β-1,4-galactosyltransferase reported to catalyze LNT II to produce LNnT is not strong enough, and the proportion of LNT II in the fermentation process is basically maintained at more than 30%. Therefore, it is urgent to screen suitable β-1,4-galactosyltransferases to reduce the proportion of byproducts (LNT II) in the LNnT production process. Summary of the invention

[0006] To solve the above problems, the present invention mined relevant galactosyltransferases in public databases and transferred them into engineering bacteria. The production of LNnT and LNT II was preliminarily verified by shake flask fermentation. The engineering bacteria that reduced the proportion of the byproduct LNT II were screened by comparing with the galactosyltransferase LgtB, and an engineering bacterium that can efficiently convert LNT II into LNnT was obtained.

[0007] On the one hand, the present application provides an engineered bacterium for efficiently producing lactose-N-neotetraose, wherein the engineered bacterium expresses galactosyltransferase; the galactosyltransferase comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 98% sequence identity with SEQ ID NO.1, and / or an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 98% sequence identity with SEQ ID NO.3.

[0008] Preferably, the galactosyltransferase comprises an amino acid sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO.1, and / or an amino acid sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO.3.

[0009] More preferably, the galactosyltransferase comprises the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.3.

[0010] Furthermore, the nucleic acid molecule encoding the galactosyltransferase includes a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.2, and / or a nucleotide sequence as shown in SEQ ID NO.4 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.4.

[0011] Preferably, the nucleic acid molecule of the galactosyltransferase comprises a nucleotide sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO.2, and / or a nucleotide sequence having at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO.4.

[0012] More preferably, the nucleic acid molecule of the galactosyltransferase comprises the nucleotide sequence shown in SEQ ID NO.2 and / or SEQ ID NO.4.

[0013] Among them, the nucleic acid molecule encoding the galactosyltransferase described in SEQ ID NO.1 is shown in SEQ ID NO.2; the nucleic acid molecule encoding the galactosyltransferase described in SEQ ID NO.3 is shown in SEQ ID NO.4.

[0014] Further, the engineered bacteria expresses β-1,3-N-acetylglucosamine transferase; preferably, the β-1,3-N-acetylglucosamine transferase includes the amino acid sequence shown in SEQ ID NO.13; more preferably, the nucleic acid molecule encoding the β-1,3-N-acetylglucosamine transferase includes the nucleotide sequence shown in SEQ ID NO.14;

[0015] Preferably, the engineered bacteria also has a β-galactosidase gene knocked out; more preferably, the β-galactosidase comprises an amino acid sequence as shown in SEQ ID NO.15;

[0016] Preferably, the engineered bacteria also expresses UDP-galactose-4-isomerase; more preferably, the UDP-galactose-4-isomerase comprises the amino acid sequence shown in SEQ ID NO.11; more preferably, the nucleic acid molecule encoding the UDP-galactose-4-isomerase comprises the nucleotide sequence shown in SEQ ID NO.12.

[0017] Preferably, the β-1,3-N-acetylglucosamine transferase is a β-1,3-N-acetylglucosamine transferase from Neisseria meningitidis, and the gene of the β-1,3-N-acetylglucosamine transferase is lgtA;

[0018] The β-galactosidase gene is lacZ;

[0019] The UDP-galactose-4-epimerase is a UDP-galactose-4-epimerase derived from Escherichia coli MG1655, and the gene of the UDP-galactose-4-epimerase is galE.

[0020] In a preferred embodiment, the preparation method of the engineered bacteria EB-LNT2 comprises the following steps:

[0021] Using wild-type Escherichia coli BL21 (DE3) as the starting strain, the CRISPR-Cas-assisted homologous recombination method was used to knock out the β-galactosidase gene lacZ to prevent the decomposition of the substrate lactose; then the galE gene from Escherichia coli MG1655 was integrated at the recA site to promote the production of UDP-galactose; finally, two copies of the β-1,3-N-acetylglucosamine transferase gene lgtA from Neisseria meningitidis were integrated at the IS186 site to obtain a strain that can produce LNT II.

[0022] Those skilled in the art will appreciate that the above-mentioned transformation can be carried out using known gene editing methods, such as the CRISPR-Cpf1 gene editing system, the CRISPR-Cas9 gene editing system, RNA interference (RNAi)-mediated gene knockout, etc.

[0023] Furthermore, the engineered bacteria is one of Corynebacterium glutamicum, Bacillus subtilis, and Escherichia coli; preferably, Escherichia coli.

[0024] In a preferred embodiment, wild-type Escherichia coli BL21 (DE3) is used as the starting strain.

[0025] The present application also provides a biomaterial, which is any one of the following A1)-A5):

[0026] A1) a nucleic acid molecule encoding a galactosyltransferase as described above;

[0027] A2) an expression cassette, the expression cassette comprising the nucleic acid molecule described in A1);

[0028] A3) a recombinant vector, the recombinant vector comprising the nucleic acid molecule described in A1) and / or the expression cassette described in A2);

[0029] A4) a recombinant cell, the recombinant cell comprising the nucleic acid molecule described in A1), the expression cassette described in A2) and / or the recombinant vector described in A3);

[0030] A5) A whole cell catalyst, wherein the whole cell catalyst comprises the recombinant cell described in A4) or the engineered bacteria described above.

[0031] Preferably, the expression vector is a pET expression vector; more preferably, the expression vector is a pETDuet-1 vector.

[0032] Further, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.2, and / or the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.4.

[0033] On the other hand, the present application also provides the use of the above-mentioned engineered bacteria or the above-mentioned biological material in increasing the yield of lactose-N-neotetraose.

[0034] Furthermore, the process of increasing the yield of lactose-N-neotetraose includes the process of converting lactose-N-triose into lactose-N-neotetraose.

[0035] In the present application, a galactosyltransferase with high LNnT yield and high efficiency in converting LNT II to synthesize LNnT was screened, which effectively increased the LNnT yield, greatly reduced the content of byproducts (LNT II) in the LNnT product, and improved the product purity.

[0036] On the other hand, the present application also provides a method for producing lactose-N-neotetraose, which comprises fermenting and producing lactose-N-neotetraose using the above-mentioned engineered bacteria or the above-mentioned biological material.

[0037] In a preferred embodiment, a method for producing lactose-N-neotetraose comprises the following steps: inoculating the engineered bacteria into LB medium (containing 100 μg / mL Amp) at 37°C, 200 rpm overnight culture, inoculating the same inoculum into DM fermentation medium at 37°C, 200 rpm, OD 600 When the pH value reached 0.6-0.8, the temperature was lowered to 25°C, 0.2 mM isopropylthiogalactoside (IPTG) was added for induction, and 5 g / L lactose was added at the same time. After sampling, the culture was continued for 72 h.

[0038] On the other hand, the present application also provides the use of the above-mentioned engineered bacteria or the above-mentioned biological material or the above-mentioned method in the preparation of medicines, foods or cosmetics containing lactose-N-neotetraose.

[0039] The present invention has the following beneficial effects:

[0040] 1. The present invention successfully screened a galactosyltransferase that can produce high LNnT and efficiently convert LNT II to synthesize LNnT. The amino acid sequence of the galactosyltransferase is shown in SEQ ID NO.1 or SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.2 or SEQ ID NO.4. A new engineered enzyme is provided for the process of converting lactose-N-triose into lactose-N-neotetraose. The enzyme effectively improves the efficiency of converting LNT II into LNnT and greatly reduces the content of by-products (LNT II);

[0041] 2. The present invention also constructs an engineered bacterium capable of expressing the above-mentioned galactosyltransferase, which has good adaptability, high yield and no by-product generation, and provides a new synthetic pathway for the synthesis of lactose-N-neotetraose. The final yield of LNnT produced by shake flask fermentation using the engineered bacterium is 1.09 g / L, and the yield of LNT II is 0.07 g / L. The proportion of by-products is only 6%, which greatly reduces the proportion of by-products in the product, saves subsequent purification costs, and solves the problem of excessively high by-product proportions in the prior art. It has high economic value and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0043] Figure 1 This is a diagram of the shake flask fermentation results of E0-E5. DETAILED DESCRIPTION

[0044] Technical terms

[0045] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in molecular evolution research.

[0046] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0047] Expression cassette: An expression cassette is a group of DNA sequences consisting of a promoter, a target gene, and a reporter gene, which can be expressed in specific tissues and easily detected.

[0048] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0049] Whole-cell catalyst: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The corresponding complete biological organism involved in the catalytic process is the whole-cell catalyst.

[0050] Engineered strains (recombinant microorganisms): Fungal cell strains that use genetic engineering methods to efficiently express exogenous genes.

[0051] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0052] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0053] If not otherwise specified, in the following embodiments, reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially. If specific conditions are not indicated in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and specific operations are carried out according to the kit instructions.

[0054] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0055] Example 1 Construction of LNnT production strain

[0056] In this embodiment, the inventors screened genes in public databases and selected 4 gene sequences from different sources and lgtB from Neisseria meningitidis for synthesis, and used the Escherichia coli high-efficiency expression system BL21 (DE3) / pETDuet-1 to construct genetically engineered bacteria.

[0057] Construction of gene expression vectors from different sources:

[0058] Taking the lgtB gene as an example, the restriction sites (BamHI, HindIII) in the gene base sequence were removed by gene synthesis, and a BamHI site was inserted at the N-terminus of the gene and a HindIII site was inserted at the C-terminus. The sequence was then connected to the vector pETDuet-1 by enzyme cutting and ligation to obtain the recombinant plasmid pETDuet-1-lgtB. The amino acid sequence of LgtB is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.6.

[0059] The above method was used to construct galactosyltransferase expression vectors derived from Aggregatibacter actinomycetemcomitans, Neisseria meningitidis and two galactosyltransferases from Helicobacter pylori, respectively. The only difference was that four gene sequences encoding galactosyltransferases from different sources were used to replace lgtB from Neisseria meningitidis.

[0060] Among them, the amino acid sequences of two galactosyltransferases from Helicobacter pylori are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively, and the nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively. The amino acid sequence of the galactosyltransferase from Aggregatibacter actinomycetemcomitans is shown in SEQ ID NO.7, and the gene sequence is shown in SEQ ID NO.8. The amino acid sequence of the galactosyltransferase from Neisseria meningitidis is shown in SEQ ID NO.9, and the gene sequence is shown in SEQ ID NO.10.

[0061] Construction of LNT II producing strain:

[0062] Using wild-type Escherichia coli BL21 (DE3) as the starting strain, the β-galactosidase gene lacZ (amino acid sequence as SEQ ID NO.15) was knocked out by CRISPR-Cas-assisted homologous recombination to prevent the decomposition of the substrate lactose; then the galE gene from Escherichia coli MG1655 was integrated at the recA site to promote the production of UDP-galactose, the amino acid sequence of the galE gene is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12; finally, two copies of the β-1,3-N-acetylglucosamine transferase gene lgtA from Neisseria meningitidis were integrated at the IS186 site to obtain a strain that can produce LNTII, the amino acid sequence of the lgtA gene is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.14. The strain that can produce LNT II obtained in this step is named EB-LNT2.

[0063] Transformation of plasmid expression vector of genetically engineered bacteria for producing LNnT:

[0064] Taking the transformation of pETDuet-1-lgtB as an example, the EB-LNT2 glycerol bacteria stored in the laboratory were inoculated into 5 mL of LB medium and cultured at 37°C, 200 rpm overnight. The seed liquid was inoculated into 100 mL of LB medium at a 1% inoculum and cultured at 37°C, 200 rpm until OD 600 =0.6-0.8, pre-cool the bacterial solution to stop bacterial growth, collect the bacterial cells by centrifugation at 5000rpm / min for 5min, wash the bacterial cells three times with 10% pre-cooled glycerol, and finally resuspend with a small amount of 10% glycerol and aliquot for use. Add an appropriate amount of plasmid to the prepared competent state for electrotransformation (2.5kV), quickly add 1mL of LB culture medium to the electrotransformation cup after the electric shock, transfer to the Ep tube at 37°C, 200rpm for 1h after simple aspiration, apply LB plate containing ampicillin (Amp) resistance, and then place in a 37°C incubator for overnight culture. The grown monoclonal clone is EB-LNT2 / pETDuet-1-lgtB, named E1. EB-LNT2 was transformed with galactosyltransferase expression vectors from other sources and named E2-E5 respectively, and the strain transformed with the pETDuet-1 empty plasmid was named E0, see Table 1 for details.

[0065] Table 1 Gene sequences, gene sources and strains involved in Example 1

[0066] Engineering bacteria number Galactosyltransferase gene source Galactosyltransferase amino acid sequence Galactosyltransferase nucleotide sequence E0 pETDuet-1 empty plasmid - - E1 Neisseria meningitidis (lgtB) SEQ ID NO.5 SEQ ID NO.6 E2 Aggregatibacter actinomycetemcomitans SEQ ID NO.7 SEQ ID NO.8 E3 Neisseria meningitidis SEQ ID NO.9 SEQ ID NO.10 E4 Helicobacter pylori SEQ ID NO.3 SEQ ID NO.4 E5 Helicobacter pylori SEQ ID NO.1 SEQ ID NO.2

[0067] Example 2 Genetically engineered bacteria shake flask fermentation test

[0068] In this example, the engineered bacteria constructed in Example 1 were used to carry out fermentation experiments.

[0069] The specific method is as follows: single colonies E0-E5 were picked from the plate and inoculated into 5 mL of LB medium (containing 100 μg / mL Amp) at 37°C and 200 rpm overnight, the bacterial concentration was determined by a spectrophotometer, and the same inoculum volume was inoculated into 25 mL of DM fermentation medium (glycerol 20 g / L, KH2PO4 13.5 g / L, MgSO4·7H2O 1.4 g / L, citric acid 1.7 g / L, trace element solution (FeSO4·7H2O 10.0 g / L, CaCl2 2.0 g / L, ZnSO4·7H2O 2.2 g / L, MnSO4·H2O 0.38 g / L, CuSO4·5H2O 1.0 g / L, (NH4)6Mo7O 2 4·4H2O 0.1g / L and Na2B4O7·10H2O 0.02g / L, dissolved in 5M HCl) 10.0mL / L, pH adjusted to 6.8 before sterilization. All culture media need to be sterilized at 115℃ for 30min. After sterilization, add sterile thiamine (filter sterilized) at a final concentration of 4.5mg / L) and culture at 37℃, 200rpm, OD 600 When the concentration reaches 0.6-0.8, cool down to 25℃, add 0.2mM isopropylthiogalactoside (IPTG) for induction, and add 5g / L lactose at the same time, take samples and continue to culture for 72h. After the fermentation is completed, measure the bacterial concentration again, take 2mL of fermentation liquid, 12,000g, centrifuge for 10min, and take the supernatant, which is the sample for extracellular detection. All samples need to pass through a 0.22μm PES water filter.

[0070] Detection conditions of LNnT and LNTII: The samples were analyzed by high performance liquid chromatography (HPLC) system and Carbohydrate Analysis (AMINEX HPX-87H Organic Acid Analysis Column) column, differential detector. Mobile phase: 5mmol / L H2SO4; flow rate: 0.6mL / min; column temperature: 60℃; injection volume: 10μL.

[0071] The engineered bacteria were transformed with the pETDuet-1 empty plasmid as a blank control, and the yield of LNnT produced by the four genes and lgtB was compared by shake flask fermentation. 600 represents bacterial concentration) and the yields of LNnT and LNTII are shown in Table 2.

[0072] LNT II ratio (%) = LNT II yield (mol / L) / LNT II yield (mol / L) + LNnT yield (mol / L)

[0073] Table 2

[0074]

[0075]

[0076] The blank control (E0) was the pETDuet-1 empty plasmid transferred into the engineered bacteria, and the yield of LNnT produced by the four genes and lgtB was compared by shake flask fermentation. Figure 1 The results are shown in Table 2 and Figure 1 As shown, the yield of LNT II in the blank control was 1.59 g / L, the yield of LNnT produced by shake flask fermentation in E1 was 0.21 g / L, the yield of LNT II was 0.62 g / L, and the proportion of LNT II was about 75%. The proportion of LNT II in the two engineered bacteria E2 and E3, which were derived from the galactosyltransferase genes of Aggregatibacteractinomycetemcomitans and Neisseria meningitidis, was 56% for E2 and 77% for E3. Therefore, the E1-E3 engineered bacteria all have the problem of too low LNnT yield or too high proportion of LNTII, which cannot meet the needs of industrialization. The engineered bacteria E4 and E5, which express two genes from Helicobacter pylori, show good conversion efficiency, among which E5 has the highest yield, about 1.09 g / L, and the proportion of LNT II is about 6%. In industrial production, the subsequent separation and purification process can be greatly simplified and the cost can be reduced.

[0077] The above results prove that the present invention has finally successfully screened out a galactosyltransferase and an engineering strain that can produce high LNnT and efficiently convert LNT II into LNnT.

[0078] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. An engineered bacterium for efficiently producing lactose-N-neotetraose, characterized in that: The engineered bacteria express galactosyltransferase; the galactosyltransferase includes an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 98% sequence identity with SEQ ID NO.1, and / or an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 98% sequence identity with SEQ ID NO.

3.

2. The engineered bacteria according to claim 1, characterized in that The nucleic acid molecule encoding the galactosyltransferase includes the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.2, and / or the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.

4.

3. The engineered bacteria according to claim 1, characterized in that The engineered bacteria express β-1,3-N-acetylglucosamine transferase; Preferably, the engineered bacteria also has the β-galactosidase gene knocked out; Preferably, the engineered bacteria also express UDP-galactose-4-epimerase.

4. The engineered bacteria according to claim 1, characterized in that The engineered bacteria is one of Corynebacterium glutamicum, Bacillus subtilis and Escherichia coli; preferably, Escherichia coli.

5. Biomaterial, characterized in that The biological material is any one of the following A1)-A5): A1) a nucleic acid molecule encoding a galactosyltransferase according to any one of claims 1 to 4; A2) an expression cassette, the expression cassette comprising the nucleic acid molecule described in A1); A3) a recombinant vector, the recombinant vector comprising the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) a recombinant cell, the recombinant cell comprising the nucleic acid molecule described in A1), the expression cassette described in A2) and / or the recombinant vector described in A3); A5) A whole-cell catalyst, which contains the recombinant cell described in A4) or the engineered bacteria described in any one of claims 1 to 4.

6. The biomaterial according to claim 5, characterized in that The nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.2, and / or the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.

4.

7. Use of the engineered bacteria according to any one of claims 1 to 4 or the biomaterial according to claim 5 or 6 in increasing the yield of lactose-N-neotetraose.

8. The use according to claim 7, characterized in that: The process of increasing the yield of lactose-N-neotetraose includes the process of converting lactose-N-triose into lactose-N-neotetraose.

9. A method for producing lactose-N-neotetraose, characterized in that: The method comprises fermenting and producing lactose-N-neotetraose by using the engineered bacteria according to any one of claims 1 to 4 or the biological material according to claim 5 or 6.

10. Use of the engineered bacteria according to any one of claims 1 to 4, or the biomaterial according to claim 5 or 6, or the method according to claim 9 in the preparation of medicines, foods or cosmetics containing lactose-N-neotetraose.

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