Engineering bacterium for preparing human milk oligosaccharide and application thereof

Abstract

The invention provides an engineering bacterium for preparing human milk oligosaccharide and application of the engineering bacterium, and relates to the technical field of genetic engineering. According to the invention, a beta-galactosidase (lacZ) gene is knocked out on a host genome, and key enzyme beta-1, 3-acetyl glucosamine transferase (lgtA) genes and beta-1, 3-galactosyl transferase (Pf) genes or beta-1, 4-galactosyl transferase (Aa) genes with different copy numbers are integrated. Experiments prove that when the ratio of lgtA to Pf or Aa is 3: 2, the yields of LNT and LNnT are the highest, meanwhile, the copy number of the key enzyme is (1-4): (4-1), and the yields of strains of which the copy numbers cannot be 1 at the same time can be increased by more than two times compared with those of single copy strains of the key enzyme. And a novel engineering bacterium is provided for LNT and LNnT. The engineering bacteria obtained in the invention are plasmid-free strains, the problem of poor genetic stability of the existing engineering bacteria is avoided, and the engineering bacteria have wide application scenarios.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to an engineering bacterium for preparing human milk oligosaccharides and its application. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third highest solid component in breast milk after lactose and lipids. They are considered natural prebiotics for infants. They can shape the intestinal microbiota, promote intestinal health, and help protect infant health, including promoting brain and immune system development, showing anti-adhesion and antibacterial activity, and reducing the incidence of enteritis. Therefore, the structure, physiological effects and synthesis methods of HMOs have attracted widespread attention in recent years. Adding HMOs to infant formula has become a future development trend. HMOs is a general term for soluble oligosaccharides in human milk, of which lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) have been approved by the US Food and Drug Administration and are commercially available. Some brands have added LNT or LNnT to commercial infant formula. For example, LNT is added to Nestlé BEBA Supreme Edition with concentrations of 30 mg / 100 mL, 10 mg / 100 mL and 9 mg / 100 mL for stages 1, 2 and 3; LNnT is added to Wyeth's Aptamil milk powder with contents of 0.5 g / L, 0.13 g / L, 0.11 g / L and 0.11 g / L for stages 1, 2, 3 and 4, respectively.

[0003] Both LNT and LNnT are composed of galactose, N-acetylglucosamine and glucose. The structure of LNT is Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc, and the structure of LNnT is Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc. At present, the main synthesis methods of LNT and LNnT are chemical synthesis, in vitro enzymatic method and microbial fermentation. The chemical synthesis method has a complex reaction process, expensive raw materials and high production cost, which is not suitable for industrial production. The in vitro enzymatic synthesis process requires nucleotide sugars and cofactors as substrates, which are expensive. In addition, the low enzyme conversion rate and other problems are not suitable for industrial large-scale production. Since the microbial fermentation method can use cheap carbon source substrates and meets the requirements of environmental protection and sustainable development, it has attracted much attention from researchers in recent years.

[0004] In 2014, et al. first developed an engineered strain for synthesizing LNT using Escherichia coli as a host, in which the β-galactosidase encoding gene lacZ was knocked out, and a bacterial β-1,3-acetylglucosamine transferase (LgtA, EC 2.4.1.149) was introduced from Neisseria meningitidis to synthesize LNTri II (GlcNAcβ1-3Galβ1-4Glc). LNTri II is an important precursor for the synthesis of LNT and LNnT. On this basis, the β-1,3-galactosyltransferase WbgO from Escherichia coli O55:H7 was introduced to catalyze the galactosylation of LNTri II to produce LNT; the β-1,4-galactosyltransferase LgtB was introduced to catalyze the galactosylation of LNTriII to produce LNnT. However, the production of LNT and LNnT still needs to be improved. Summary of the invention

[0005] In order to solve the problems of low yield of LNT and LNnT produced by existing microbial fermentation methods and strain instability caused by the use of antibiotics, the present invention integrates key enzyme genes with different copy numbers into the host genome, strengthens the LNTri II synthesis pathway and conversion pathway, thereby increasing the yield of LNT and LNnT.

[0006] On the one hand, the present application provides an engineered bacterium, which expresses β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase, wherein the galactosyltransferase includes β-1,3-galactosyltransferase (Pf) and / or β-1,4-galactosyltransferase (Aa), and the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and the galactosyltransferase is (1-4): (4-1), and their copy numbers cannot be 1 at the same time.

[0007] The engineered bacteria are engineered bacteria for preparing human milk oligosaccharides, and the human milk oligosaccharides include lactose-N-tetraose (LNT) and / or lactose-N-neotetraose (LNnT).

[0008] Among them, the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and galactosyl transferase can be any value of 1:4, 1:3, 1:2, 1:1, 2:4, 2:3, 2:2, 2:1, 3:4, 3:3, 3:2, 3:1, 4:4, 4:3, 4:2, and 4:1.

[0009] Preferably, the ratio of β-1,3-acetylglucosamine transferase (lgtA) to β-1,3-galactosyl transferase (Pf) is 1:1, 1:2, 2:1, 2:2, 2:3, 3:1, 3:2, 3:3.

[0010] More preferably, the ratio of β-1,3-acetylglucosamine transferase (lgtA) to β-1,3-galactosyl transferase (Pf) is 3:2.

[0011] Preferably, the ratio of β-1,3-acetylglucosamine transferase (lgtA) to β-1,4-galactosyl transferase (Aa) is 1:1, 1:2, 2:1, 2:2, 2:3, 3:1, 3:2, 3:3.

[0012] More preferably, the ratio of β-1,3-acetylglucosamine transferase (lgtA) to β-1,4-galactosyl transferase (Aa) is 3:2.

[0013] It is known to those skilled in the art that the genes encoding the β-1,3-acetylglucosamine transferase (lgtA) and galactosyl transferase can be selectively inserted into the genome of the starting strain, or can exist on a free plasmid, as long as gene expression or protease synthesis can be achieved.

[0014] Those skilled in the art may also make their own selections regarding the insertion position based on actual conditions, and this application does not impose any specific restrictions on this.

[0015] In a preferred embodiment,

[0016] The integration site of lgtA may be one or more of ldhA (genebank: WP_000762229.1), pta (genebank: WP_128449068.1), and lacA (genebank: WP_001320653.1).

[0017] The integration site of Pf can be one or more of nagB (genebank: WP_001237072.1), wecBC (wecB and wecC are two sites, genebank: WP_000866670.1, WP_130559483.1), and poxB (genebank: WP_000815335.1).

[0018] The integration site of Aa can be one or more of nagB (genebank: WP_001237072.1), wecBC (wecB and wecC are two sites, genebank: WP_000866670.1, WP_130559483.1), and poxB (genebank: WP_000815335.1).

[0019] Further, the β-1,3-acetylglucosamine transferase (lgtA) comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence that has at least 98% identity with SEQ ID NO.1; preferably, the nucleic acid molecule encoding the β-1,3-acetylglucosamine transferase (lgtA) comprises a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence that has at least 98% identity with SEQ ID NO.2.

[0020] Preferably, the β-1,3-acetylglucosamine transferase (lgtA) comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.1.

[0021] Preferably, the nucleic acid molecule encoding the β-1,3-acetylglucosamine transferase (lgtA) comprises a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.2.

[0022] Further, the β-1,3-galactosyltransferase (Pf) comprises an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence that has at least 98% identity with SEQ ID NO.3; preferably, the nucleic acid molecule encoding the β-1,3-galactosyltransferase (Pf) comprises a nucleotide sequence as shown in SEQ ID NO.4 or a nucleotide sequence that has at least 98% identity with SEQ ID NO.4.

[0023] Preferably, the β-1,3-galactosyltransferase (Pf) comprises an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.3.

[0024] Preferably, the nucleic acid molecule encoding the β-1,3-galactosyltransferase (Pf) comprises a nucleotide sequence as shown in SEQ ID NO.4 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.4.

[0025] Further, the β-1,4-galactosyltransferase (Aa) comprises an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence that has at least 98% identity with SEQ ID NO.5; preferably, the nucleic acid molecule encoding the β-1,4-galactosyltransferase (Aa) comprises a nucleotide sequence as shown in SEQ ID NO.6 or a nucleotide sequence that has at least 98% identity with SEQ ID NO.6.

[0026] Preferably, the β-1,4-galactosyltransferase (Aa) comprises an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.5.

[0027] Preferably, the nucleic acid molecule encoding the β-1,4-galactosyltransferase (Aa) comprises a nucleotide sequence as shown in SEQ ID NO.6 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.6.

[0028] Furthermore, the engineered bacteria does not express β-galactosidase (lacZ) and / or reduces the expression of β-galactosidase (lacZ).

[0029] Preferably, the lack of expression of β-galactosidase (lacZ) and / or reduced expression of β-galactosidase (lacZ) in the engineered bacteria is achieved by knocking out the β-galactosidase (lacZ) gene.

[0030] Those skilled in the art can also achieve non-expression or reduced expression of β-galactosidase (lacZ) by known methods, as long as the effect can be achieved, and this is not specifically limited in the present application.

[0031] In a preferred embodiment, the engineered bacteria for producing lactose-N-tetraose (LNT) express β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase, the galactosyltransferase includes β-1,3-galactosyltransferase (Pf), the copy number ratio of β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase is (1-4): (4-1), and the engineered bacteria does not express β-galactosidase (lacZ) and / or reduces the expression of β-galactosidase (lacZ).

[0032] In a preferred embodiment, the engineered bacteria producing lactose-N-neotetraose (LNnT) express β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase, the galactosyltransferase includes β-1,4-galactosyltransferase (Aa), the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and the galactosyltransferase is (1-4): (4-1), and the engineered bacteria does not express β-galactosidase (lacZ) and / or reduces the expression of β-galactosidase (lacZ).

[0033] Furthermore, the engineered bacteria is selected from one or more of Escherichia coli, Streptococcus, Bacillus, Saccharomyces cerevisiae, and Pichia pastoris.

[0034] Preferably, the engineered bacteria is Escherichia coli.

[0035] In a preferred embodiment, the engineered bacteria is Escherichia coli MG1655.

[0036] Those skilled in the art will appreciate that conventional fermentation strains or any known industrial strains may be used as starting strains, and no particular limitation is imposed on the specific strains.

[0037] It is understandable that those skilled in the art can select a suitable gene editing system and gene editing method according to actual conditions to complete the construction of the above-mentioned engineered bacteria.

[0038] Preferably, the recombinant microorganism does not contain a resistance selection marker gene.

[0039] On the other hand, the present application also provides a biomaterial, which comprises any one of the following A1)-A3):

[0040] A1) an expression cassette, wherein the expression cassette expresses β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase, wherein the galactosyltransferase includes β-1,3-galactosyltransferase (Pf) and / or β-1,4-galactosyltransferase (Aa), and the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and the galactosyltransferase is (1-4): (4-1);

[0041] A2) a recombinant vector, wherein the recombinant vector contains the expression cassette described in A1);

[0042] A3) a whole-cell catalyst, wherein the whole-cell catalyst comprises the expression cassette described in A1), the recombinant vector described in A2) and / or the engineered bacteria.

[0043] On the other hand, the present application also provides the use of the engineered bacteria or the biomaterial in producing lactose-N-tetraose (LNT) and / or lactose-N-neotetraose (LNnT), and increasing the yield of lactose-N-tetraose (LNT) and / or lactose-N-neotetraose (LNnT).

[0044] Those skilled in the art will appreciate that other commonly used expression elements can be adaptively added to the expression cassette described in the present application to assist in the expression of the target gene, such as adding tags, fluorescent protein markers, resistance screening markers, etc.

[0045] Optionally, the recombinant vector may include a nucleic acid molecule encoding the above protein, a promoter, and transcription and translation termination signals. The promoter or terminator for regulating expression in the present application may be a universal promoter or terminator, or a specific promoter or terminator in the host bacteria. Those skilled in the art may make adaptive selections according to actual conditions, and the present application does not impose mandatory restrictions on the sequence of the promoter or terminator.

[0046] The recombinant vector can be any vector (such as a plasmid or virus) that is convenient for recombinant DNA manipulation and expression of nucleic acid sequences. The selection of the vector usually depends on the compatibility of the vector with the host cell into which it will be introduced. The vector can be a linear or closed-loop plasmid. The vector can be an autonomous replicating vector (i.e., a complete structure present outside the chromosome that can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can include any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will be integrated into the genome and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid can be used, or two or more vectors or plasmids, or a transposon, that generally contain the entire DNA that will be introduced into the host cell genome.

[0047] On the other hand, the present application also provides a method for preparing lactose-N-tetraose (LNT) and / or lactose-N-neotetraose (LNnT), the method comprising: culturing the engineered bacteria.

[0048] Preferably, the engineered bacteria express β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase, the galactosyltransferase includes β-1,3-galactosyltransferase (Pf) and / or β-1,4-galactosyltransferase (Aa), and the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase is (1-4): (4-1).

[0049] Preferably, the engineered bacteria is Escherichia coli.

[0050] In a preferred embodiment, the engineered bacteria is Escherichia coli MG1655.

[0051] In a preferred embodiment, the preparation method of lactose-N-tetraose (LNT) and / or lactose-N-neotetraose (LNnT) comprises the following steps:

[0052] Step 1, constructing the engineered bacteria;

[0053] Step 2: inoculate the engineered bacteria into the culture medium at 25°C-37°C, 100-300rpm, and culture for 24-72h.

[0054] In a preferred embodiment, the preparation method of lactose-N-tetraose (LNT) comprises the following steps:

[0055] Step 1, constructing the engineered bacteria; the engineered bacteria expresses β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase, the galactosyltransferase includes β-1,3-galactosyltransferase (Pf), the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and the galactosyltransferase is (1-4): (4-1), and the engineered bacteria does not express β-galactosidase (lacZ) and / or reduces the expression of β-galactosidase (lacZ);

[0056] Step 2: inoculate the engineered bacteria into the culture medium at 25°C-37°C, 100-300rpm, and culture for 24-72h.

[0057] In a preferred embodiment, the preparation method of lactose-N-neotetraose (LNnT) comprises the following steps:

[0058] Step 1, constructing the engineered bacteria; the engineered bacteria expresses β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase, the galactosyltransferase includes β-1,4-galactosyltransferase (Aa), the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase is (1-4): (4-1), and the engineered bacteria does not express β-galactosidase (lacZ) and / or reduces the expression of β-galactosidase (lacZ);

[0059] Step 2: inoculate the engineered bacteria into the culture medium at 25°C-37°C, 100-300rpm, and culture for 24-72h.

[0060] The culture can be carried out using a general culture method for engineering bacteria.

[0061] It is understandable that the step 2 may also include a purification step, and the purification may be performed using a general method.

[0062] On the other hand, the present application also provides the use of β-1,3-acetylglucosamine transferase (lgtA) and galactosyltransferase for the synergistic preparation of lactose-N-tetraose (LNT) and / or lactose-N-neotetraose (LNnT), characterized in that the galactosyltransferase includes β-1,3-galactosyltransferase (Pf) and / or β-1,4-galactosyltransferase (Aa), and the copy number ratio of the β-1,3-acetylglucosamine transferase (lgtA) and the galactosyltransferase is (1-4): (4-1).

[0063] In this application, the key enzymes β-1,3-acetylglucosamine transferase (lgtA) and galactosyl transferase were co-expressed for the first time, and it was found that the co-expression of the two can synergistically increase the production of lactose-N-tetraose (LNT) or lactose-N-neotetraose (LNnT), and the production of strains with copy numbers of 2:3, 3:1, 3:2, and 3:3 is the most obvious, which can reach more than twice that of strains with a single copy of the key enzyme.

[0064] The present invention has the following beneficial effects:

[0065] The present invention knocks out the β-galactosidase (lacZ) gene on the host genome of Escherichia coli MG1655, and integrates different copy numbers of the key enzyme β-1,3-acetylglucosamine transferase (lgtA) gene and β-1,3-galactosyltransferase (Pf) gene or β-1,4-galactosyltransferase (Aa) gene. Experiments have shown that when the ratio of lgtA to Pf or Aa is 3:2, the yield of LNT and LNnT is the highest, and at the same time, the key enzyme copy number is (1-4): (4-1), and the yield of the strain whose copy number cannot be 1 at the same time can be increased by more than 2 times compared with the key enzyme single copy strain. A new engineering bacterium is provided for LNT and LNnT.

[0066] In addition, the engineered bacteria obtained in the present invention are all plasmid-free strains, which avoids the problem of poor genetic stability of existing engineered bacteria and has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] 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:

[0068] Figure 1 This is a statistical chart of the shake flask fermentation results of LNT series key enzyme multi-copy strains;

[0069] Figure 2 This is a statistical chart of the shake flask fermentation results of LNnT series key enzyme multi-copy strains. DETAILED DESCRIPTION

[0070] 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.

[0071] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0072] 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.

[0073] Engineered bacteria: Fungal cell strains that use genetic engineering methods to efficiently express exogenous genes.

[0074] (Gene) knockout: It is a foreign DNA introduction technology that uses a DNA fragment containing a certain known sequence to undergo homologous recombination with a gene with the same or similar sequence in the recipient cell genome, integrate into the recipient cell genome and express it. It changes the genetic genes of an organism for a sequence with a known sequence but unknown function, making a specific gene function ineffective, thereby shielding some functions.

[0075] Inoculation size: Inoculation size refers to the ratio of the volume of seed solution transferred in to the volume of culture solution after inoculation.

[0076] Free expression: Free expression is the expression of target genes using free plasmids. Free plasmids are independent DNA molecules that exist in cells and have the ability to replicate and transmit independently. They are widely used in genetic engineering and molecular biology research.

[0077] Integrative expression: Expression that occurs when a gene is integrated into the genome.

[0078] 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.

[0079] 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.

[0080] 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).

[0081] All plasmids used in the present invention are obtained by primer design, PCR and vector fragment recombination using conventional molecular biological methods, and all recombinant plasmids are sequenced and verified to be completely consistent with the target sequence.

[0082] Among them, 2×Hieff PCR Master mix was purchased from Yisheng Biotechnology, 2×High fidelity Mastermix was purchased from MCLAB, the plasmid mini-extraction kit was purchased from Omega, the universal DNA purification and recovery kit was purchased from Tiangen, and DpnⅠ was purchased from thermo.

[0083] The strains E. coli MG1655 and E. coil DH5α competent cells mentioned below were purchased from Beijing Quanshijin Company.

[0084] The plasmids pTargetF and pEcCas mentioned below were purchased from Addgene.

[0085] The amino acid sequence of β-1,3-acetylglucosamine transferase (lgtA) is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The amino acid sequence of β-1,3-galactosyl transferase (Pf) is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4. The amino acid sequence of β-1,4-galactosyl transferase (Aa) is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0086] The following culture medium components are as follows:

[0087] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride (add 15 g / L agar powder for solid medium).

[0088] DM medium: glycerol 20g / L, KH2PO4 13.5g / L, MgSO4·7H2O 1.4g / L, citric acid 1.7g / L, trace element solution (FeSO4·7H2O 10.0g / L, CaCl2 2.0g / L, ZnSO4·7H2O 2.2g / L, MnSO4·H2O 0.38g / L, CuSO4·5H2O 1.0g / L, (NH4)6Mo7O 24 ·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) to a final concentration of 4.5mg / L.

[0089] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as sterile water, ionized water, distilled water, ion exchanged water, double distilled water, high-purity water, and purified water.

[0090] In the following examples, unless otherwise specified, % means wt %, i.e. weight percentage.

[0091] Example 1 Preparation of competent cells for electroporation transformation

[0092] Preparation of competent E. coli MG1655, pEcCas strain:

[0093] Inoculate a single colony or glycerol stock of MG1655 or pEcCas strain into Kana-resistant LB medium and culture overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated into non-resistant LB medium at 1% inoculum at 37°C and 220 rpm and cultured until OD 600 =0.1-0.2, add 10 mM L-arabinose for induction, and continue to culture at 30°C and 220 rpm until OD 600 =0.6-0.8. After the bacterial solution was ice-bathed for 30 minutes, it was centrifuged at 4°C, 5000rpm for 5 minutes, and the supernatant was discarded. The bacterial precipitate was resuspended with 20mL of 10% glycerol and centrifuged again at 4°C, 5000rpm. This step was repeated three times. The final bacterial precipitate was resuspended with 500μL of 10% glycerol and divided into 10 tubes, and frozen at -80°C to obtain MG1655, pEcCas strain competent cells.

[0094] Example 2 Construction of pTargetF-N20 plasmid

[0095] Using pTargetF plasmid as template, PCR amplification was performed. The PCR system was as follows: 2.5 μL of pTargetF plasmid, 2.5 μL of corresponding forward primer and reverse primer, 25 μL of 2×High fidelity Master mix, 20 μL of sterile water, a total of 50 μL. If there are no impurities, the PCR product is purified, and then the template plasmid is digested with DpnI, and the PCR product is purified again; if there are impurities, only gel cutting and recovery can be performed or the PCR product can be re-amplified according to the situation. The linear plasmid was transformed into DH5α competent cells for circularization. The grown monoclonal clones were liquid activated, the plasmids were extracted, and sequencing verification N20 was performed to obtain the pTargetF plasmids of the corresponding sites, pTargetF-lacZ, pTargetF-ldhA, pTargetF-nagB, pTargetF-wecBC, pTargetF-pta, pTargetF-poxB, and pTargetF-lacA. The relevant primers are shown in Table 1 below.

[0096] Table 1 Primers for constructing pTargetF plasmid

[0097]

[0098]

[0099] Example 3 Gene knockout and integration

[0100] (1) Obtaining homology arms

[0101] Take lacZ (genebank: WP_268557137.1) as an example. Wild-type MG1655 was used as a template to amplify the upstream homology arm (lacZ up) (500bp) and the downstream homology arm (lacZ down) (500bp), respectively. The PCR system is as follows: MG1655 2.5μL, corresponding forward primer (lacZ up-F) and reverse primer (lacZ down-R) 2.5μL each, 2×High fidelity Master mix 25μL, sterile water 20μL, a total of 50μL. If there is no stray band in the PCR product, the PCR product is purified; if there is a stray band, the gel is cut and recovered or re-amplified according to the situation. The same method was used to obtain the homology arms of the ldhA, nagB, wecBC, pta, poxB, and lacA sites. The primers involved are shown in Table 2.

[0102] (2) Gene knockout

[0103] The following operation takes the knockout of lacZ as an example. The upstream and downstream homology arms are assembled by fusion PCR. The PCR system is as follows: 2.5μL of upstream homology arm (lacZ up), 2.5μL of downstream homology arm (lacZ down), 2.5μL of upstream fragment forward primer (lacZ up-F), 2.5μL of downstream reverse primer (lacZ down-R), 25μL of 2×High fidelity Master mix, 15μL of sterile water, a total of 50μL. If there is no stray band in the PCR product, purify the PCR product; if there is a stray band, cut the gel for recovery or re-amplify according to the situation to obtain the fused homology arm.

[0104] The homologous arms after the above fusion and the pTargetF-lacZ plasmid obtained in Example 2 were electroporated into the competent cells of MG1655 and pEcCas strains obtained in Example 1. 1 mL of anti-LB medium was added immediately after electroporation. After culturing at 37 ° C and 220 rpm for 1 hour, the bacterial solution was coated on LB+kana (100 mg / L)+spec (100 mg / L) plates and cultured at 37 ° C and 220 rpm for 12-16 hours. Pick a single colony and perform colony PCR verification to confirm the gene knockout. The system is as follows: lacZ up-F 1 μL, lacZ down-R 1 μL, 2×Hieff PCR Master mix 10 μL, sterile water 8 μL, a total of 20 μL. The above PCR system was run with DNA agarose gel electrophoresis to determine the gene knockout according to the band size.

[0105] (3) Gene integration

[0106] Based on the MG1655△lacZ strain, different copy numbers of lgtA and Pf were integrated respectively, and a total of 9 strains from KA01 to KA09 were obtained. The integration sites of lgtA were ldhA (genebank: WP_000762229.1), pta (genebank: WP_128449068.1), lacA (genebank: WP_001320653.1); the integration sites of Pf were nagB (genebank: WP_001237072.1), wecBC (wecB and wecC sites, genebank: WP_000866670.1, WP_130559483.1), and poxB (genebank: WP_000815335.1). Similarly, different copy numbers of lgtA and Aa were integrated into the MG1655△lacZ strain to obtain a total of 9 strains from KB01 to KB09. The integration sites of lgtA were ldhA, pta, lacA, and the integration sites of Aa were nagB, wecBC, and poxB. The gene integration method is as follows:

[0107] The following is an example of integrating lgtA into the ldhA site. First, pJET1.2-Ptac-lgtA (synthesized by Nanjing GenScript) was used as a template to amplify Ptac-lgtA, and Escherichia coli K12 MG1655 was used as a template to amplify 500bp upstream of ldhA (ldhA up) and 500bp downstream of ldhA (ldhA down). Ptac-lgtA, ldhA up and ldhA down were fused, and the fusion method was as follows: the first step was pre-fusion: Ptac-lgtA 2.5μL, ldhA up 2.5μL, ldhA down 2.5μL, 2×High fidelity Mastermix 25μL, sterile water 12.5μL, and 10 cycles were performed; the second step was fusion: on the basis of the above system, ldhA up-F 2.5μL and ldhA down-R 2.5μL were added, and 30 cycles were performed (see Table 3 below for detailed PCR process). The PCR product is excised and recovered to obtain the fused gene fragment.

[0108] The gene fragment after fusion in the previous step and the pTargetF-ldhA plasmid obtained in Example 2 were electrotransformed into MG1655, pEcCas strain competent cells. The obtained single colony was verified, and ldhA up-F and ldhA down-R were used as verification primers. The amplified product was verified by gel electrophoresis, and the integration was judged according to the band size.

[0109] The primers involved in the construction of the strain of the present invention are shown in Table 1, and the information of the engineered bacteria obtained by construction is shown in Table 4 and Table 5.

[0110] Table 2 Construction primers

[0111]

[0112]

[0113]

[0114] Table 3

[0115]

[0116] Table 4

[0117]

[0118]

[0119] Table 5

[0120]

[0121] The homologous fragment sequences involved in the gene editing process of the present invention are as follows: is the upstream homologous fragment, is the downstream homologous fragment, Key enzyme sequence

[0122] Knockout of lacZ:

[0123]

[0124]

[0125] lgtA integrates into the ldhA site:

[0126]

[0127]

[0128] IgtA integrates into the pta site:

[0129]

[0130]

[0131] IgtA integrates into the lacA site:

[0132]

[0133]

[0134] Pf integrates into the nagB site:

[0135]

[0136]

[0137] Pf integrated into the wecBC locus:

[0138]

[0139]

[0140] Pf integrates into the poxB site:

[0141]

[0142]

[0143] Aa integrates into nagB site:

[0144]

[0145]

[0146] Aa integrated into wecBC locus:

[0147]

[0148]

[0149] Aa integrated into the poxB locus:

[0150]

[0151]

[0152] Example 4 Shake flask fermentation verification

[0153] The strains KA01-KA09 and KB01-KB09 obtained in Example 3 were plate activated, and the plate activated strains were inoculated into a test tube containing 4 mL of LB medium without antibiotics and cultured overnight at 200 rpm and 37°C. The next day, the biomass of the overnight cultured seed solution was measured, and the seed solution was inoculated into 25 mL of DM medium to an initial OD of 600 =0.05, then cultured at 200 rpm and 37°C for about 72 h, and the fermentation was terminated.

[0154] Take 1 mL of fermentation broth, centrifuge at 8000 rpm for 2 min, and take the supernatant as the test sample. All samples were filtered through 0.22 μm PES water filter membrane, and the content of LNTri II and LNT in the samples was detected by HPLC.

[0155] HPLC detection conditions: using a high performance liquid chromatography (HPLC) system (Shimadzu LC20-ADXR) with a differential detector and chromatographic column HPX-87H, 300mm×7.8mm (Catalog#1250140) was used to analyze the samples. The mobile phase used was: 5mmol / L H2SO4; flow rate: 0.6mL / min; column temperature: 60℃; injection volume: 10μL. The LNT peak time was about 6.7min.

[0156] The test results are shown in Tables 6 and 7. Figure 1 , we can get from Table 7 Figure 2 .

[0157] Table 6

[0158]

[0159]

[0160] Table 7

[0161] strain LNnT(g / L) KB02 0.20 KB03 0.24 KB04 0.30 KB05 0.39 KB06 0.80 KB07 0.31 KB08 0.86 KB09 0.69

[0162] Depend on Figure 1 and Figure 2 As shown, when the ratio of lgtA to Pf or Aa is 3:2, the yield of LNT and LNnT reaches the highest value. Among them, the yield of LNT is 9.8 times higher than that of the key enzyme single copy (lgtA:Pf=1:1), which is 4.13g / L. The yield of LNnT is 4.3 times higher than that of the key enzyme single copy (lgtA:Aa=1:1), which is 0.86g / L. The yield of strains with key enzyme copy numbers of 2:3, 3:1, 3:2, and 3:3 can be increased by more than 2 times compared with the key enzyme single copy strain.

[0163] 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, characterized in that: The engineered bacteria express β-1,3-acetylglucosamine transferase and galactosyl transferase, wherein the galactosyl transferase includes β-1,3-galactosyl transferase and / or β-1,4-galactosyl transferase, and the copy number ratio of the β-1,3-acetylglucosamine transferase and the galactosyl transferase is (1-4): (4-1), and the copy numbers thereof cannot be 1 at the same time.

2. The engineered bacteria according to claim 1, characterized in that The β-1,3-acetylglucosamine transferase comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence that has at least 98% identity with SEQ ID NO.1; preferably, the nucleic acid molecule encoding the β-1,3-acetylglucosamine transferase comprises a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence that has at least 98% identity with SEQ ID NO.

2.

3. The engineered bacteria according to claim 1, characterized in that The β-1,3-galactosyltransferase comprises an amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 98% identity with SEQ ID NO.3; preferably, the nucleic acid molecule encoding the β-1,3-galactosyltransferase comprises a nucleotide sequence as shown in SEQ ID NO.4 or a nucleotide sequence having at least 98% identity with SEQ ID NO.

4.

4. The engineered bacteria according to claim 1, characterized in that The β-1,4-galactosyltransferase comprises an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence that has at least 98% identity with SEQ ID NO.5; preferably, the nucleic acid molecule encoding the β-1,4-galactosyltransferase comprises a nucleotide sequence as shown in SEQ ID NO.6 or a nucleotide sequence that has at least 98% identity with SEQ ID NO.

6.

5. The engineered bacteria according to any one of claims 1 to 4, characterized in that: The engineered bacteria do not express β-galactosidase and / or reduce the expression of β-galactosidase.

6. The engineered bacteria according to claim 1, characterized in that The engineering bacteria are selected from one or more of Escherichia coli, Streptococcus, Bacillus, Saccharomyces cerevisiae and Pichia pastoris.

7. Biomaterial, characterized in that The biological material includes any one of the following A1)-A3): A1) an expression cassette, wherein the expression cassette expresses β-1,3-acetylglucosamine transferase and galactosyl transferase, wherein the galactosyl transferase includes β-1,3-galactosyl transferase and / or β-1,4-galactosyl transferase, and the copy number ratio of the β-1,3-acetylglucosamine transferase and the galactosyl transferase is (1-4): (4-1); A2) a recombinant vector, wherein the recombinant vector contains the expression cassette described in A1); A3) A whole-cell catalyst, wherein the whole-cell catalyst comprises the expression cassette described in A1), the recombinant vector described in A2) and / or the engineered bacteria described in any one of claims 1-6.

8. Use of the engineered bacteria according to any one of claims 1 to 6 or the biomaterial according to claim 7 in producing lactose-N-tetraose and / or lactose-N-neotetraose, or in increasing the yield of lactose-N-tetraose and / or lactose-N-neotetraose.

9. A method for preparing lactose-N-tetraose and / or lactose-N-neotetraose, characterized in that: The method comprises: culturing the engineering bacteria as described in any one of claims 1-6.

10. Use of β-1,3-acetylglucosamine transferase and galactosyl transferase to synergistically prepare lactose-N-tetraose and / or lactose-N-neotetraose, characterized in that: The galactosyltransferase includes β-1,3-galactosyltransferase and / or β-1,4-galactosyltransferase, and the copy number ratio of the β-1,3-acetylglucosamine transferase and the galactosyltransferase is (1-4):(4-1).

Images