Engineering bacterium for efficiently producing lactose-N-tetrasaccharide, biological material and application

Abstract

The invention provides an engineering bacterium for efficiently producing lactose-N-tetrasaccharide, a biological material and application, and relates to the technical field of biological engineering. The invention provides galactosyl transferase expressed by engineering bacteria. The galactosyl transferase comprises an amino acid sequence as shown in SEQ ID NO. 1 or an amino acid sequence with at least 98% sequence identity with the amino acid sequence as shown in SEQ ID NO. 1. Related galactosyl transferase is mined through a public database and transferred into engineering bacteria, the yield of lactose-N-tetrasaccharide (LNT) and lactose-N-trisaccharide (LNT II) is preliminarily verified through shake flask fermentation, the galactosyl transferase and the engineering bacteria which are high in LNT yield and capable of efficiently converting the LNT II to synthesize the LNT are successfully screened, the galactosyl transferase and the engineering bacteria are applied to LNT production, and the engineering bacteria have the advantages of being high in LNT yield and capable of efficiently converting the LNT II to synthesize the LNT. The proportion of a byproduct (LNT II) can be greatly reduced, and the problems that the proportion of the byproduct is too high and the byproduct is difficult to separate in the prior art are solved.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to an engineering bacterium, a biomaterial and an application for efficiently producing lactose-N-tetraose. 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] 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, enzymatic synthesis and microbial fermentation. The biofermentation method based on engineered bacteria such as Escherichia coli and Corynebacterium glutamicum has the advantages of low production cost and low environmental pollution, and is a synthetic method actively used in commercial products today. Lactose-N-tetraose (LNT) is a neutral non-fucosylated sugar and one of the important core structures of HMOs. Its microbial fermentation method uses relatively cheap lactose as a substrate. Through the action of β-1,3-N-acetylglucosamine transferase and β-1,3-galactosyltransferase, LNT is generated through a two-step reaction. The intermediate metabolite lactose-N-triose (LNT II) is the precursor structure of LNT. Because LNT 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 LNT 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, Zhang Tao's team at Jiangnan University used exogenous expression of lgtA and wbgO in Escherichia coli to combine and regulate the expression of galE, galT and galK in the LNT synthesis pathway and knock out the lacZ gene of the Escherichia coli host. The production of LNT in the fermenter reached 31.56g / L, but the content of the by-product LNT II was 5.68g / L, accounting for more than 15%. In the same year, Mu Wanmeng's team at Jiangnan University achieved 57.5g / L of LNT production in Escherichia coli, but the content of the by-product LNT II reached 21.58g / L, accounting for 27%. In terms of regulations, LNT produced by Glycom from Denmark and Jennewein from Germany have been approved as GRAS products by the US FDA in 2019 and 2021, respectively. In the LNT quality standard proposed by Glycom, the content of LNT shall not be less than 70%, and LNT II shall not be higher than 10%; in the LNT quality standard proposed by Jennewein, the content of LNT shall not be less than 75%, and LNT II shall not be higher than 5%. It can be seen from the above that when LNT is produced by microbial fermentation, the byproduct LNT II still accounts for a relatively high proportion. The high proportion of LNT II will bring at least three problems: first, it makes it difficult for the final product to achieve a high purity; second, it causes the conversion rate of the substrate lactose to be low, that is, lactose is not completely converted into the target product LNT, which will increase the cost of raw materials; third, it brings challenges to the subsequent separation and purification, and increases the cost of separation and purification. In fact, the molecular weights of LNT and LNT II are 707.63 and 545.49, respectively, and the difference between the two is small. The effect of separation using ordinary membrane separation devices is not good. If the two are to achieve a better separation effect, multiple process procedures need to be added, which will not only increase the cost, but also reduce the final product yield.

[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 LNT, 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 β-1,3-galactosyltransferases reported to be more common at present are not strong enough to catalyze LNT II to produce LNT. The proportion of LNT II in the fermentation process is basically maintained at 15%-40%. Therefore, it is urgent to screen suitable β-1,3-galactosyltransferases to reduce the proportion of byproducts (LNT II) in the LNT production process. Summary of the invention

[0006] At present, in the synthesis process of human milk oligosaccharide LNT, the substrate lactose can synthesize a large amount of LNT II under the catalysis of β-1,3-N-acetylglucosamine transferase, but the conversion rate of LNT II to LNT under the action of β-1,3-galactosyltransferase is still at a low level, which limits the synthesis of LNT and the proportion of byproduct LNT II is too high. LNT and LNT II are difficult to separate, resulting in the need to separate and purify the product separately in the future to reduce the proportion of LNT II in the product to make it meet the relevant industry regulations, which adds a lot of costs to industrial production. At present, there are very few galactosyltransferases reported in the literature for the synthesis of human milk oligosaccharides.

[0007] In response to the above problems, the present invention mined relevant galactosyltransferases through public databases and transferred them into engineered bacteria, preliminarily verified the production of LNT and LNT II through shake flask fermentation, and compared them with WbgO from Escherichia coli, which is currently more commonly used, to obtain engineered bacteria with a low proportion of by-product LNT II.

[0008] On the one hand, the present application provides an engineered bacterium for efficiently producing lactose-N-tetraose, 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.

[0009] 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.

[0010] More preferably, the amino acid sequence of the galactosyltransferase is as shown in SEQ ID NO.1.

[0011] Furthermore, the nucleic acid molecule encoding the galactosyltransferase 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.

[0012] Preferably, the nucleic acid molecule encoding 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 with SEQ ID NO.2.

[0013] More preferably, the nucleotide sequence of the nucleic acid molecule encoding the galactosyltransferase is shown as SEQ ID NO.2.

[0014] Furthermore, the engineered bacteria expresses β-1,3-N-acetylglucosamine transferase; preferably, the engineered bacteria also has a β-galactosidase gene knocked out;

[0015] Preferably, the engineered bacteria also express UDP-galactose-4-epimerase.

[0016] Preferably, the β-1,3-N-acetylglucosamine transferase comprises the amino acid sequence shown in SEQ ID NO.13; more preferably, the nucleic acid molecule encoding the β-1,3-N-acetylglucosamine transferase comprises the nucleotide sequence shown in SEQ ID NO.14.

[0017] More preferably, the β-galactosidase comprises the amino acid sequence shown in SEQ ID NO.15.

[0018] More preferably, the UDP-galactose-4-epimerase comprises the amino acid sequence shown in SEQ ID NO.11; more preferably, the nucleic acid molecule encoding the UDP-galactose-4-epimerase comprises the nucleotide sequence shown in SEQ ID NO.12.

[0019] 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;

[0020] The β-galactosidase gene is lacZ;

[0021] 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.

[0022] In a preferred embodiment, the preparation method of the engineered bacteria EB-LNT2 comprises the following steps:

[0023] 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.

[0024] 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.

[0025] Furthermore, the engineered bacteria is one of Corynebacterium glutamicum, Bacillus subtilis, and Escherichia coli; preferably, Escherichia coli.

[0026] In a preferred embodiment, wild-type Escherichia coli BL21 (DE3) is used as the starting strain.

[0027] On the other hand, the present application also provides a biomaterial, wherein the biomaterial is any one of the following A1)-A5):

[0028] A1) a nucleic acid molecule encoding a galactosyltransferase as described above;

[0029] A2) an expression cassette, the expression cassette comprising the nucleic acid molecule described in A1);

[0030] A3) a recombinant vector, the recombinant vector comprising the nucleic acid molecule described in A1) and / or the expression cassette described in A2);

[0031] 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);

[0032] A5) A whole cell catalyst, wherein the whole cell catalyst comprises the recombinant cell described in A4) or the engineered bacteria described above.

[0033] Preferably, the expression vector is a pET expression vector; more preferably, the expression vector is a pETDuet-1 vector.

[0034] Furthermore, the nucleic acid molecule comprises 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.

[0035] 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 production of lactose-N-tetraose.

[0036] Furthermore, the process of increasing the yield of lactose-N-tetraose includes the process of converting lactose-N-triose into lactose-N-tetraose.

[0037] In the present application, a galactosyltransferase with high LNT yield and the ability to efficiently convert LNT II to synthesize LNT was screened and an engineered strain with high LNT yield was constructed, which effectively increased the LNT yield, greatly reduced the content of by-products (LNT II) in the LNT product, and improved the product purity.

[0038] On the other hand, the present application also provides a method for producing lactose-N-tetraose, which comprises fermenting and producing lactose-N-tetraose using the engineered bacteria as described above or the biomaterial as described above.

[0039] In a preferred embodiment, a method for producing lactose-N-tetraose 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.

[0040] 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-tetraose.

[0041] The present invention has the following beneficial effects:

[0042] 1. The present invention successfully screened a galactosyltransferase that can produce high LNT yield and efficiently convert LNT II to synthesize LNT. The amino acid sequence of the galactosyltransferase is shown in SEQ ID NO.1. The galactosyltransferase effectively improves the efficiency of converting LNT II to LNT and greatly reduces the content of the by-product (LNT II);

[0043] 2. The present invention also constructs an engineered bacterium capable of expressing the above-mentioned galactosyltransferase, which has good adaptability, high yield and low by-product ratio, and provides a new synthetic pathway for the synthesis of lactose-N-tetraose. The final yield of LNT produced by shake flask fermentation using the engineered bacterium is 2.50 g / L, and the proportion of the by-product LNT II is only 9%, which greatly reduces the proportion of by-products in the product, saves subsequent purification costs, and solves the problem of too high a by-product ratio and difficulty in separating by-products in the prior art, and has high economic value and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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:

[0045] Figure 1 This is a diagram of the shake flask fermentation results of EB0-EB5. DETAILED DESCRIPTION

[0046] Technical terms

[0047] 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.

[0048] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Engineered strains (recombinant microorganisms): Fungal cell strains that use genetic engineering methods to efficiently express exogenous genes.

[0053] 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.

[0054] 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.

[0055] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0056] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0057] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed in accordance with the kit instructions.

[0058] 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).

[0059] Example 1 Construction of genetically engineered bacteria

[0060] Construction of gene expression vectors from different sources:

[0061] The inventors of the present application screened genes in public databases and selected 4 gene sequences encoding galactosyltransferases from different sources and wbgO from Escherichia coli for synthesis, and used the Escherichia coli high-efficiency expression system BL21 (DE3) / pETDuet-1 to construct genetically engineered bacteria.

[0062] Specifically, the four gene sequences encoding galactosyltransferases from different sources include the amino acid sequence of the galactosyltransferase from Helicobacter pylori as shown in SEQ ID NO.1, and the gene sequence as shown in SEQ ID NO.2; the amino acid sequence of the galactosyltransferase from Pseudogulbenkiania ferrooxidans as shown in SEQ ID NO.3, and the gene sequence as shown in SEQ ID NO.4; the amino acid sequence of the galactosyltransferase from Chromobacterium violaceum as shown in SEQ ID NO.5, and the gene sequence as shown in SEQ ID NO.6; the amino acid sequence of the galactosyltransferase from Streptococcus agalactiae as shown in SEQ ID NO.7, and the gene sequence as shown in SEQ ID NO.8.

[0063] The construction process of the recombinant expression vector in the E. coli high-efficiency expression system BL21 (DE3) / pETDuet-1 includes: taking the wbgO gene as an example, inserting a BamH I site at the N-terminus of the gene and a Hind III site at the C-terminus through gene synthesis. Then, the sequence is connected to the vector pETDuet-1 by enzyme digestion and ligation to obtain the recombinant plasmid pETDuet-1-wbgO. The amino acid sequence of WbgO is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.

[0064] The above method was used to construct galactosyltransferase expression vectors from Helicobacter pylori, Pseudogulbenkiania ferrooxidans, Bacillus purpurogenus and Streptococcus agalactiae, respectively. The only difference was that four gene sequences encoding galactosyltransferases from different sources were used to replace wbgO from Escherichia coli.

[0065] Construction of the LNT II producing strain (EB-LNT2):

[0066] Using wild-type Escherichia coli BL21 (DE3) as the starting strain, the β-galactosidase gene lacZ (amino acid sequence as shown in SEQ ID NO.15) was knocked out by homologous recombination method assisted by CRISPR-Cas system to prevent the decomposition of 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 corresponding to 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 capable of producing LNT II, ​​the amino acid sequence corresponding to the lgtA gene is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.14. The above strain capable of producing LNT II was named EB-LNT2.

[0067] Transformation of plasmid expression vector of LNT genetic engineering bacteria:

[0068] Taking the transformation of pETDuet-1-wbgO 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, precool 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% precooled 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-wbgO, named EB1. The galactosyltransferase expression vectors from other sources mentioned above were transformed in the same way and named EB2-EB5 respectively, and the strain transformed with the pETDuet-1 empty plasmid was named EB0, as shown in Table 1.

[0069] Table 1

[0070] Engineering bacteria number Galactosyltransferase gene source Amino acid sequence Nucleotide sequence EB0 pETDuet-1 empty plasmid - - EB1 Escherichia coli SEQ ID NO.9 SEQ ID NO.10 EB2 Pseudogulbenkiania ferrooxidans SEQ ID NO.3 SEQ ID NO.4 EB3 Chromobacterium violaceum SEQ ID NO.5 SEQ ID NO.6 EB4 Streptococcus agalactiae SEQ ID NO.7 SEQ ID NO.8 EB5 Helicobacter pylori SEQ ID NO.1 SEQ ID NO.2

[0071] Example 2 Genetically engineered bacteria shake flask fermentation test

[0072] In this example, the engineered bacteria constructed in Example 1 were used to carry out fermentation experiments.

[0073] The specific method is as follows: single colonies EB0-EB5 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)6Mo7O2 4·4H2O 0.1 g / L and Na2B4O7·10H2O 0. 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, sterile thiamine (filter sterilized) was added to the culture medium at a final concentration of 4.5mg / L), cultured at 37℃, 200rpm, OD 600 When the concentration 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 hours. After the fermentation was completed, the cell concentration was measured again (OD 600Represents bacterial concentration), take 2mL fermentation broth, 12,000g, centrifuge for 10min, take the supernatant, which is the sample extracellular detection sample, and detect the LNT and LNTII content in the sample. All samples need to pass through a 0.22μm PES water filter.

[0074] LNT and LNT II detection conditions: 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.

[0075] The pETDuet-1 empty plasmid was transferred into the engineered bacteria as a blank control, and the yields of LNT and LNT II produced by the shake flask fermentation of the four genes and wbgO were compared. The bacterial concentration and the yields of LNT and LNT II are shown in Table 2.

[0076] Table 2

[0077]

[0078]

[0079] Plot the data in Table 2 to obtain Figure 1 , according to Table 2 and Figure 1 The results show that the yield of LNT II in the blank control is 1.59 g / L, and the yield of LNT produced by shake flask fermentation of EB1 in the WbgO control is 2.01 g / L, and the proportion of LNT II is about 17%. The yield of the engineered bacteria EB3 expressing the galactosyltransferase gene from Bacillus purpurogenus is 1.71 g / L, and the proportion of LNT II is about 20%. The engineered bacteria EB2 and EB4 engineered strains expressing the galactosyltransferase gene from Pseudogulbenkiania ferrooxidans and Streptococcus agalactiae have a low LNT yield of only 0.2-0.4 g / L, which cannot meet the needs of industrialization.

[0080] Among the above-mentioned engineered bacteria, the engineered bacteria EB5 expressing the galactosyltransferase gene from Helicobacter pylori has the highest yield of LNT, about 2.50g / L, and the yield of LNT II is about 0.25g / L, accounting for 9%. It is proved that during the process of EB5 fermentation to produce LNT, LNT II is basically completely converted into LNT, that is, EB5 has the advantages of high LNT yield and high conversion rate, which can effectively simplify the subsequent separation and purification process and reduce costs.

[0081] 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-tetraose, characterized in that: The engineered bacteria express 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.

2. The engineered bacteria according to claim 1, characterized in that The nucleic acid molecule encoding the galactosyltransferase comprises 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.

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 have 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, the engineered bacteria is 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 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.

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-tetraose.

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

9. A method for producing lactose-N-tetraose, characterized in that: The method comprises fermenting and producing lactose-N-tetraose 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-tetraose.

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