Use of opgG and mutants thereof in the production of human milk oligosaccharides

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to the application of OpgG and its mutants in the production of human milk oligosaccharides. This invention provides a mutant of the dextran biosynthetic protein OpgG, which is obtained by mutating the 443rd amino acid from threonine to proline based on the wild-type dextran biosynthetic protein OpgG shown in SEQ ID NO.1. This mutant is introduced into human milk oligosaccharide-producing strains. opgG The gene, and / or the T443P mutant gene, was applied to the production of human milk oligosaccharides, resulting in a significant increase in the yield of 3'-SL, 6'-SL, LNT II, ​​LNnT, and LNT.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of OpgG and its mutants in the production of human milk oligosaccharides. Background Technology

[0002] Human milk oligosaccharides (HMOs) are an important class of bioactive components in breast milk. HMOs have various physiological functions, including establishing a balanced infant gut microbiota, strengthening the gastrointestinal barrier, preventing infection, and providing potential support to the immune system. Based on their structure, they are classified into fucoidylated neutral human milk oligosaccharides, sialylated human milk oligosaccharides, and non-fucosylated neutral human milk oligosaccharides. Currently, more than 200 HMOs have been discovered, including 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), lactyl- N - Trisaccharide II (lacto- N- triose, LNT II), lactoyl- N -New tetrasaccharide (Lacto- N -neotetraose, LNnT) and lactyl- N -Tetrasaccharide (Lacto- N HMOs are produced through various methods, including chemical synthesis, enzyme catalysis, and microbial fermentation. Among these, microbial fermentation offers advantages such as ease of operation, environmental friendliness, and low cost, making it more suitable for large-scale industrial production.

[0003] In recent years, although the development of HMO microbial cell factory strategies has achieved some success, there is still room for improvement in yield. When *E. coli* synthesizes sialylated human lactose oligosaccharides, the introduction of... N -acetylglucosamine isomerase gene ( neuC) Acetylneuraminic acid synthase gene ( neuB ), CMP-acetylneuraminic acid synthase gene ( neuA ), synthesizing cytidine monophosphate- N - Acetylneuraminic acid (CMP-Neu5Ac). CMP-Neu5Ac binds to lactose under the catalysis of α2,3-sialyltransferase α2,3-SiaT and α2,6-sialyltransferase α2,6-SiaT to synthesize 3'-SL and 6'-SL, respectively. In the synthesis of LNnT, glucose is used as the carbon source, and glucose-6-phosphate (Glc-6-P) is... pgi The encoded glucose-6-phosphate isomerase catalyzes the conversion to fructose-6-phosphate (F6P), which then sequentially... glmS Encoded glutamine-fructose-6-phosphate aminotransferase, glmM Encoded phosphoglucosamine mutase, glmU Encoded N Glucosamine-1-phosphate uridine transferase / glucosamine-1-phosphate acetyltransferase catalyzes its conversion to uridine diphosphate- N -Acetylglucosamine (UDP-GlcNAc); in the synthetic pathway of another precursor, uridine diphosphate-galactose (UDP-Gal), Glc-6-P... pgm Encoded glucose phosphomutase and galE The encoded UDP-glucose-4-isomerase catalyzes the conversion to UDP-Gal, in lgtA Encoded β-1,3- N Under the catalysis of α-acetylglucosamine aminotransferase, lactose reacts with UDP-GlcNAc to form the intermediate LNT II. Subsequently, LNT II... lgtB Catalyzed by the encoded β-1,4-galactosyltransferase, it binds to UDP-Gal to generate LNnT, which then... wbgO LNTs are generated under the catalysis of the encoded β-1,3-galactosyltransferase.

[0004] OpgG, a biosynthetic protein of dextran, is a periplasmic protein essential for the synthesis of periplasmic glucans (OPGs) (Bontemps-Gallo S, et al. Osmoregulated Periplasmic Glucans. EcoSal Plus. 2017 Jun;7(2):10.1128 / ecosalplus.ESP-0001). However, the exact function of OpgG remains unclear (Xavier Hanoulle, et al. Structural analysis of Escherichia coli OpgG, a protein required for the biosynthesis of osmoregulated periplasmic glucans. J MolBiol. 2004 Sep 3;342(1):195-205.).

[0005] This study introduced [a strain] into human milk oligosaccharide-producing strains. opgG Gene mutation and / or overexpression opgG It is hoped that the production of human milk oligosaccharides can be further increased. Summary of the Invention

[0006] This invention utilizes gene editing technology to obtain individuals carrying... opgGmutant genetically engineered chassis strains or overexpression opgG The strains were then used in the construction of production strains for 3'-SL, 6'-SL, LNT II, ​​LNnT and LNT, which further improved the yield of 3'-SL, 6'-SL, LNT II, ​​LNnT and LNT.

[0007] One of the technical solutions provided by this invention is a dextran biosynthetic protein OpgG mutant, which is obtained by mutating the 443rd amino acid from threonine to proline based on the wild-type dextran biosynthetic protein OpgG shown in SEQ ID NO.1; the OpgG mutant is named T443P mutant, and its amino acid sequence is shown in SEQ ID NO.3;

[0008] This invention also provides the encoding gene of the above-mentioned T443P mutant;

[0009] Furthermore, the coding gene is opgG T443P The nucleotide sequence is shown in SEQ ID NO.4.

[0010] The second technical solution provided by this invention includes... opgG T443P Recombinant vectors or recombinant strains of genes;

[0011] Furthermore, the expression plasmid used in the recombinant vector is the psb4k5 plasmid;

[0012] Furthermore, the recombinant strain uses Escherichia coli as its expression host; preferably MG1655.

[0013] The third technical solution provided by this invention is the application of the T443P mutant described in the first technical solution, particularly its application in the production of human milk oligosaccharides;

[0014] Furthermore, the application involves mutating the gene encoding the dextran biosynthesis protein OpgG on the genome of a human milk oligosaccharide-producing strain to... opgG T443P And / or in human milk oligosaccharide-producing strains opgG T443P Genes are expressed.

[0015] The fourth technical solution provided by this invention is an engineered bacterium for producing human milk oligosaccharides. This engineered bacterium is obtained by overexpressing the OpgG gene, which encodes the glucan biosynthesis protein, on the genome of a human milk oligosaccharide-producing strain; or...

[0016] The engineered bacteria were derived from human milk oligosaccharide-producing strains, with the gene encoding the dextran biosynthetic protein OpgG in the genome mutated to... opgG T443P Obtain; or,

[0017] The engineered bacteria are based on human milk oligosaccharide-producing strains, which simultaneously contain the gene encoding the glucan biosynthetic protein OpgG and... opgG T443P Genes; or,

[0018] The engineered bacteria were derived from human milk oligosaccharide-producing strains, with the gene encoding the dextran biosynthetic protein OpgG in the genome mutated to... opgG T443P At the same time opgG T443P Obtained through expression;

[0019] Furthermore, the human milk oligosaccharide producing strain is a strain capable of producing LNTII, LNnT, LNT, 3'-SL, or 6'-SL;

[0020] Furthermore, the human milk oligosaccharide-producing strain uses *Escherichia coli* K12 MG1655 as a host, and knocks out the lactose operon sequence in the host. lacZ and overexpression lacY Furthermore, the strain also contains a human milk oligosaccharide production pathway.

[0021] Furthermore, the human milk oligosaccharides include, but are not limited to: lactoyl- N - Trisaccharide (Lacto-N-Triose, LNT II), lactoyl- N - Neotetrasaccharide (Lacto-N-Neotetraose, LNnT), lactose- N -Tetrasaccharide (Lacto-N-tetraose, LNT), 3'-Sialyllactose (3'-SL) and 6'-Sialyllactose (6'-SL).

[0022] Furthermore, the human milk oligosaccharide production pathway is any one of the following plasmids:

[0023] pTrc99a-P trc - lgtA pTrc99a-P trc - lgtB - lgtA、 pTrc99a-P trc - wbgO - lgtA、pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist and pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6 .

[0024] The fifth technical solution provided by this invention is the application of the engineered bacteria described in the fourth technical solution in the production of human milk oligosaccharides;

[0025] Furthermore, the human milk oligosaccharides include: LNTII, LNnT, LNT, 3'-SL, and 6'-SL.

[0026] Beneficial effects:

[0027] This invention provides a mutant of the dextran biosynthetic protein OpgG, which is obtained by mutating the 443rd amino acid from threonine to proline based on the wild-type dextran biosynthetic protein OpgG shown in SEQ ID NO. 1. This mutant is introduced into human milk oligosaccharide-producing strains. opgG The gene, and / or the T443P mutant gene, was applied to the production of human milk oligosaccharides, resulting in a significant increase in the yield of 3'-SL, 6'-SL, LNT II, ​​LNnT, and LNT. Attached Figure Description

[0028] Figure 1 This is the first step in verifying homologous recombination colony PCR.

[0029] Figure 2 This is for the second step of homologous recombination colony PCR verification. Detailed Implementation

[0030] The present invention will be further described below through specific embodiments. Unless otherwise specified, the technical means and materials involved in the following embodiments are all known to those skilled in the art, and suitable means and materials that can solve the corresponding technical problems can be selected. In addition, the embodiments should be understood as illustrative, not limiting the scope of the present invention, and the essence and scope of the present invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the present invention also fall within the protection scope of the present invention.

[0031] In this invention, the following definitions are used:

[0032] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0033] The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing single-letter or three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0034] 2. Identification of the OpgG mutant of the dextran biosynthetic protein

[0035] The mutated amino acid in the OpgG mutant is represented by "original amino acid + position + substituted amino acid". For example, T443P indicates that the 443rd amino acid is replaced by proline instead of threonine in the wild type, and the position number corresponds to the amino acid sequence number of the wild type OpgG in SEQ ID NO.1.

[0036] In this invention, lowercase italics opgG Indicates the gene encoding the wild-type dextran biosynthetic protein OpgG, in lowercase italics. opgG T443P The following table shows the encoding gene for the mutant T443P.

[0037]

[0038] The amino acid sequence of the dextran biosynthetic protein OpgG involved in this invention is shown in SEQ ID NO.1:

[0039] MMKMRWLSAAVMLTLYTSSSWAFSIDDVAKQAQSLAGKGYETPKSNLPSVFRDMKYADYQQQIQFNHDKAYWNNLKTPFKLEFYHQGMYFDTPVKINEVTATAVKRIKYSPDYFTFGDVQHDKDTVKDL GFAGFKVLYPINSKDKNDEIVSMLGASYFRVIGAGQVYGLSARGLAIDTALPSGEEFPRFKEFWIERPKPTDKRLTIYALLDSPRATGAYKFVVMPGRDTVVDVQSKIYLRDKVGKLGVAPLTSMFLF GPNQPSPANNYRPELHDSNGLSIHAGNGEWIWRPLNNPKHLAVSSFSMENPQGFGLLQRGRDFSRFEDLDDRYDLRPSAWVTPKGEWGKGSVELVEIPTNDETNDNIVAYWTPDQLPEPGKEMNFKYT ITFSRDEDKLHAPDNAWVQQTRRSTGDVKQSNLIRQPDGTIAFVVDFTGAEMKKLPEDTPVTAQTSIGDNGEIVESTVRYNPVTKGWRLVMRVKVKDAKKTTEMRAALVNADQTLSETWSYQLPANE*

[0040] The OpgG mutant of the dextran biosynthetic protein involved in this invention is obtained by mutating threonine at position 443 to proline based on the wild-type OpgG dextran biosynthetic protein. The amino acid sequence is shown in SEQ ID NO.3:

[0041] MMKMRWLSAAVMLTLYTSSSWAFSIDDVAKQAQSLAGKGYETPKSNLPSVFRDMKYADYQQQIQFNHDKAYWNNLKTPFKLEFYHQGMYFDTPVKINEVTATAVKRIKYSPDYFTFGDVQHDKDTVKDL GFAGFKVLYPINSKDKNDEIVSMLGASYFRVIGAGQVYGLSARGLAIDTALPSGEEFPRFKEFWIERPKPTDKRLTIYALLDSPRATGAYKFVVMPGRDTVVDVQSKIYLRDKVGKLGVAPLTSMFLF GPNQPSPANNYRPELHDSNGLSIHAGNGEWIWRPLNNPKHLAVSSFSMENPQGFGLLQRGRDFSRFEDLDDRYDLRPSAWVTPKGEWGKGSVELVEIPTNDETNDNIVAYWTPDQLPEPGKEMNFKYT ITFSRDEDKLHAPDNAWVQQTRRSTGDVKQSNLIRQPDGTIAFVVDFTGAEMKKLPEDPPVTAQTSIGDNGEIVESTVRYNPVTKGWRLVMRVKVKDAKKTTEMRAALVNADQTLSETWSYQLPANE*

[0042] The present invention will be further explained and described below through specific embodiments.

[0043] Example 1 Construction of strain G1

[0044] Based on E. coli H0, CRISPR / Cas9 gene editing technology was used to edit the glucan biosynthesis protein gene in the genome. opgG (Gene ID: 945005) was mutated, with the nucleotide at position 1327 being adenine mutated to cytosine (A mutated to C), and the corresponding amino acid at position 443 being threonine mutated to proline, to obtain strain G1.

[0045] Among them, Escherichia coli H0 is Escherichia coli K12 MG1655 ( Escherichia coli K12 MG1655 was constructed using the starting strain, and lactose was knocked out of the starting strain. lac P in the manipulator sequence lac Promoter sequences and regulatory genes lacZ , in the original lacZ Following the site with P trc Promoter overexpression lacY For details on the construction process of this strain, please refer to Example 1 of CN119464168A.

[0046] The specific construction method of strain G1 is as follows:

[0047] Based on strain H0, CRISPR / Cas9 technology (Zhao D, et al. CRISPR / Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. Sci Rep 7,16624) was used to... opgG The adenine nucleotide at position 1327 of the gene was mutated to cytosine (A mutated to C), resulting in strain G1. The specific construction method of strain G1 is as follows:

[0048] 1. Construction of homologous recombination fragments

[0049] Using the wild-type MG1655 strain preserved in the laboratory as a template, homologous recombination fragments were constructed using the primers in Table 1. opgG -up-f / r and opgG Using -down-f / r primers, PCR amplification yielded the upstream and downstream homologous arms of homologous recombination. This was achieved using artificially synthesized chloramphenicol resistance genes. cat , cat Using the promoter and N20 nucleotide vector as templates, primer pairs were used... opgG PCR amplification was performed using -cat-f / r to obtain samples with... cat- A fragment of the N20 sequence (SEQ ID NO.5). Upstream and downstream homologous arms, carrying... cat- The N20 sequence fragments, these three fragments are used as templates, using primers... opgG -up-f and opgG -down-r performs overlap PCR to obtain homologous recombination fragments, which contain opgG The mutation site of the gene, i.e., the wild type opgG The nucleotide sequence at position 1327 is mutated from adenine to cytosine.

[0050] 2. First step: Homologous recombination

[0051] The pCAGO plasmid was transformed into strain H0 using a conventional plasmid transformation method to obtain strain H0 (pCAGO). Competent cells of H0 (pCAGO) were prepared using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG. Homologous recombination fragments were introduced via electroporation. The transformed bacterial culture was plated on LB agar plates containing 100 mg / L ampicillin, 25 mg / L chloramphenicol, and 1% glucose, and incubated at 30°C. Transformants were picked for colony PCR verification (verification primers were: opgG If recombination is successful (-yz-f / r), the band size will be approximately 2660 bp, as shown in the verification results. Figure 1 As shown, the bands are correct, indicating that the first homologous recombination was successful. The correct transformant is then selected for the second homologous recombination step.

[0052] 3. Second step: Homologous recombination

[0053] The strain that successfully underwent recombination verification was inoculated into LB tubes containing 100 μg / mL ampicillin and 0.1 mM IPTG, and cultured on a shaker at 30°C for more than 6 h to induce the expression of the CRISPR / Cas9 system and λ-red protein to complete the second recombination. Single colonies were isolated by streaking three regions on LB plates containing ampicillin. These isolated single colonies were then spotted onto chloramphenicol-resistant and ampicillin-resistant LB plates. Single colonies that did not grow on chloramphenicol medium but grew on ampicillin medium were selected for colony PCR verification (verification primers: [missing primers]). opgG -yz-f / r). If recombination is correct, the band size will be approximately 1725 bp, as shown in the verification results. Figure 2 As shown, the band is correct, and the PCR product of this band was sequenced. The sequencing result was correct, and the second-step homologous recombination strain was obtained. The second-step homologous recombination strain was further cultured at 37℃, resulting in the loss of the pCAGO plasmid, thus obtaining the strain carrying... opgG The mutated strain was named G1.

[0054] Table 1 Construction opgG Primers used for gene mutant strains

[0055]

[0056] Example 2: Construction of strains H1, H2, G2 and G3

[0057] (1) Constructing plasmid psb4k5- opgG

[0058] Using plasmid psb4k5 as a template and primers psb4k5-fp-F / R (Table 2) as primers, PCR amplification was performed to obtain the linear vector psb4k5; Escherichia coli MG1655... opgG Using the sequence as a template, and taking the sequence in Table 2 as an example. opgG -F / R primers were used for PCR amplification to obtain linear gene fragments. opgG The linear vector and linear gene fragment obtained by the above PCR were purified and recovered, and ligated using the ClonExpress II Recombinant Ligation Kit (Novizan Biotechnology Co., Ltd.). The ligation was then performed into *E. coli* DH5α competent cells, cultured on LB agar plates containing 50 mg / L kanamycin, and transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, named plasmid psb4k5- opgG .

[0059] (2) Construction of plasmid psb4k5- opgG T443P

[0060] With plasmid psb4k5- opgG Using fp443-f / r as a template and the primers in Table 2 as primers, PCR amplification was performed to obtain samples containing... opgG The linear vector psb4k5- encoding the gene (SEQ ID NO.4) that has the T443P mutation. opgG T443P The linear vector obtained from the above PCR was purified and recovered, and ligated using the ClonExpress II Recombinant Ligation Kit (Novizan Biotechnology Co., Ltd.). The ligation was then performed into *E. coli* DH5α competent cells, cultured on LB agar plates containing 50 mg / L kanamycin, and transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, named plasmid psb4k5- opgG T443P .

[0061] Table 2 Construction of plasmid psb4k5- opgG and all primers for the mutant

[0062]

[0063] Using electroconversion, the above plasmid psb4k5- opgG ,psb4k5- opgG T443P The plasmid psb4k5- was introduced into H0 to obtain strains H1 and H2 respectively; opgG ,psb4k5- opgG T443P The strains were introduced into G1 to obtain strains G2 and G3.

[0064] Example 3: Construction of plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist

[0065] plasmid pTrc99a-P was constructed using plasmid pTrc99a as a template. J23119 - neuB-neuC -P trc - neuA-ist The specific construction process of this plasmid is described in Example 2 of patent CN117736280A.

[0066] Example 4: Construction of plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6

[0067] With plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist constructing plasmid pTrc99a-P as template J23119 - neuB-neuC -P trc - neuA-ST6 The specific construction process of this plasmid is described in Example 3 of patent CN117736280A.

[0068] Example 5: Construction of plasmid pTrc99a-P trc - lgtA

[0069] lgtA Gene encodes β-1,3- N - Acetylglucosamine transferase, plasmid pTrc99a-P was constructed using plasmid pTrc99a as a template. trc - lgtA The specific construction process of the plasmid is described in Example 4 of patent CN 119464168A.

[0070] Example 6: Construction of plasmid pTrc99a-P trc -lgtB-lgtA

[0071] lgtB The gene encodes a lipid oligosaccharide biosynthetic protein in plasmid pTrc99a-P trc - lgtA Based on this, plasmid pTrc99a-P was constructed. trc - lgtB - lgtAThe specific construction process of this plasmid is described in Example 5 of patent CN 119464168A.

[0072] Example 7: Construction of plasmid pTrc99a-P trc - wbgO - lgtA

[0073] wbgO The gene encodes β-1,3-galactosyltransferase, which is expressed in plasmid pTrc99a-P. trc - lgtA Based on this, plasmid pTrc99a-P was constructed. trc - wbgO - lgtA The specific construction process of this plasmid is described in Example 6 of patent CN 119464168A.

[0074] Example 8: Construction and fermentation testing of 3'-SL, 6'-SL, LNT II, ​​LNnT and LNT producing strains

[0075] Using electroconversion, the following plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6、 pTrc99a-P trc - lgtA pTrc99a-P trc - lgtB - lgtA and pTrc99a-P trc - wbgO - lgtA The following production strains were constructed by introducing the strains into H0, H1, H2, G1, G2, and G3 respectively:

[0076] (1) 3'-SL production strain:

[0077] Y1 [H0 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist )];

[0078] Y2 [H1 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist )];

[0079] Y3 [H2 (pTrc99a - P J23119 - neuB-neuC -P trc - neuA-ist )];

[0080] Y4 [G1 (pTrc99a - P J23119 - neuB-neuC -P trc - neuA-ist ) ];

[0081] Y5 [G2 (pTrc99a - P J23119 - neuB-neuC -P trc - neuA-ist )];

[0082] Y6 [G3 (pTrc99a - P J23119 - neuB-neuC -P trc - neuA-ist )];

[0083] (2) 6'-SL production strain:

[0084] Y7 [H0 (pTrc99a - P J23119 - neuB-neuC -P trc - neuA - ST6 )];

[0085] Y8 [H1 (pTrc99a - P J23119 - neuB-neuC -P trc - neuA - ST6 )];

[0086] Y9 [H2(pTrc99a - P J23119 - neuB-neuC [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​)];

[0089] Y12 [G3 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA - ST6 )];

[0090] (3) LNT II producing strain:

[0091] Y13 [H0 (pTrc99a-P trc - lgtA )];

[0092] Y14 [H1 (pTrc99a-P trc - lgtA )];

[0093] Y15 [H2 (pTrc99a-P trc - lgtA )];

[0094] Y16 [G1 (pTrc99a-P trc - lgtA )];

[0095] Y17 [G2 (pTrc99a-P trc - lgtA )];

[0096] Y18 [G3 (pTrc99a-P trc - lgtA )]

[0097] (4) LNnT producing strain:

[0098] Y19 [H0 (pTrc99a-P trc - lgtB - lgtA )];

[0099] Y20 [H1 (pTrc99a-P trc - lgtB - lgtA )];

[0100] Y21[ H2 (pTrc99a-P trc - lgtB - lgtA )];

[0101] Y22 [G1 (pTrc99a-P trc - lgtB - lgtA )];

[0102] Y23 [G2 (pTrc99a-P trc - lgtB - lgtA )];

[0103] Y24 [G3 (pTrc99a-P trc - lgtB - lgtA )];

[0104] (5) LNT-producing strains:

[0105] Y25 [H0 (pTrc99a-P trc - wbgO - lgtA )];

[0106] Y26 [H1 (pTrc99a-P trc - wbgO - lgtA )];

[0107] Y27 [H2 (pTrc99a-P trc - wbgO - lgtA )];

[0108] Y28 [G1 (pTrc99a-P trc - wbgO - lgtA )];

[0109] Y29 [G2 (pTrc99a-P trc - wbgO - lgtA )];

[0110] Y30 [G3 (pTrc99a-P trc - wbgO - lgtA )]

[0111] The strains and plasmids used in this patent are shown in Table 3.

[0112] Table 3. Strains and plasmids used in this application

[0113]

[0114] The production levels of the above-mentioned strains were tested by fermentation. The culture medium used was:

[0115] LB medium (1 L): NaCl 10 g, yeast extract 5 g, peptone 10 g.

[0116] Fermentation medium (1 L): KH2PO4 3 g, yeast powder 8 g, (NH4)2SO4 4 g, citric acid 1.7 g, MgSO4·7H2O 2 g, thiamine 10 mg, MOPS 60 g, glycerol 10 g, lactose 5 g, 1 mL trace elements, and ammonia water to adjust pH to 7.0.

[0117] Trace elements (1 L): FeCl3·6H2O 25 g, MnCl2·4H2O 9.8 g, CoCl2·6H2O 1.6 g, CuCl2·H2O 1 g, H3BO3 1.9 g, ZnCl2 2.6 g, Na2M O O4·2H2O 1.1 g, Na2SeO31.5 g, NiSO4·6H2O 1.5 g.

[0118] The fermentation test process is as follows:

[0119] Single colonies of the 3'-SL, 6'-SL, LNT II, ​​LNnT, and LNT production strains were picked and incubated overnight at 37 ℃ and 220 rpm in LB broth containing 50 mg / L ampicillin and kanamycin. The overnight culture was used as a seed culture and transferred at a 1% inoculum to 24-well plates containing 50 mg / L ampicillin, kanamycin, and 0.1 mmol / L IPTG. Fermentation was carried out at 37 ℃ and 800 rpm. Three samples of each strain were cultured in parallel. During fermentation, cell growth (OD) was measured. 600 The concentrations of 3'-SL, 6'-SL, LNTII, LNnT, and LNT in the sample were determined by HPLC. The HPLC detection conditions for LNTII, LNnT, LNT, 3'-SL, and 6'-SL were as described in Example 4 of patent CN117736280A. The results are shown in Tables 4 to 8.

[0120] Table 4. Results of 3'-SL production tests by different strains

[0121]

[0122] Table 5. Results of 6'-SL production tests by different strains

[0123]

[0124] Table 6. Results of LNT II production tests by different strains

[0125]

[0126] Table 7. Results of LNnT production tests by different strains

[0127]

[0128] Table 8. Results of LNT production tests by different strains

[0129]

[0130] The results showed that, after the 443rd amino acid of the OpgG protein was mutated from threonine to proline, the strain carrying only the OpgG genome mutation exhibited a certain advantage in the synthesis of human milk oligosaccharides compared to the wild-type strain. Further investigation using plasmid overexpression of this mutant in the wild-type strain carrying the OpgG genome further demonstrated its superiority. opgG Validation was performed on wild-type and / or mutant types, and the results showed that, compared with wild-type OpgG, the production of human milk oligosaccharides 3'-SL, 6'-SL, LNT II, ​​LNnT, and LNT was increased to varying degrees, ultimately carrying... opgG Strains with genomic mutations and plasmid overexpression of wild-type and / or mutant strains. opgG The discovery of a strain that synthesizes human milk oligosaccharides significantly increased the total yield of these oligosaccharides. This research provides a novel approach for constructing strains that efficiently synthesize human milk oligosaccharides.

[0131] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A mutant of the dextran biosynthetic protein OpgG, characterized in that, The OpgG mutant is obtained by mutating the 443rd amino acid from threonine to proline based on the wild-type dextran biosynthetic protein OpgG shown in SEQ ID NO.1; the OpgG mutant is named T443P mutant, and its amino acid sequence is shown in SEQ ID NO.

3.

2. The encoding gene of the OpgG mutant as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, The encoding gene is opgG T443P The nucleotide sequence is shown in SEQ ID NO.

4.

4. Containing the contents of claim 3 opgG T443P Recombinant vectors or recombinant strains that encode genes.

5. The application of the OpgG mutant of claim 1 in the production of human milk oligosaccharides, characterized in that, The application involves mutating the OpgG gene encoding the dextran biosynthesis protein in the genome of human milk oligosaccharide-producing strains to... opgG T443P And / or in human milk oligosaccharide-producing strains opgG T443P Genes are expressed; The human milk oligosaccharide producing strain uses Escherichia coli K12 MG1655 as the host.

6. An engineered bacterium for producing human milk oligosaccharides, characterized in that, The engineered bacteria were obtained by overexpressing the OpgG gene, which encodes the glucan biosynthesis protein, in the genome of a human milk oligosaccharide-producing strain; or... The engineered bacteria are based on human milk oligosaccharide-producing strains, with the OpgG gene encoding the glucan biosynthesis protein in the genome mutated to the form described in claim 3. opgG T443P Obtain; or, The engineered bacteria are based on human milk oligosaccharide-producing strains, which simultaneously contain the OpgG gene encoding the dextran biosynthesis protein and the strain described in claim 3. opgG T443P Genes; or, The engineered bacteria are based on human milk oligosaccharide-producing strains, with the OpgG gene encoding the glucan biosynthesis protein in the genome mutated to the form described in claim 3. opgG T443P Simultaneously, regarding the claim 3 opgG T443P Obtained through expression; The human milk oligosaccharide producing strain uses Escherichia coli K12 MG1655 as its host.

7. The engineered bacteria for producing human milk oligosaccharides as described in claim 6, characterized in that, The human milk oligosaccharide-producing strain uses *Escherichia coli* K12 MG1655 as the host, and knocks out the lactose operon sequence in the host. lacZ and overexpression lacY Furthermore, the strain also contains a human milk oligosaccharide production pathway.

8. The engineered bacteria as described in claim 7, characterized in that, The human milk oligosaccharide includes: lactoyl- N - Trisaccharide (Lacto-N-Triose, LNT II), lactoyl- N - Neotetrasaccharide (Lacto-N-Neotetraose, LNnT), lactose- N -Tetrasaccharide (Lacto-N-tetraose, LNT), 3'-Sialyllactose (3'-SL) and 6'-Sialyllactose (6'-SL).

9. The engineered bacteria as described in claim 8, characterized in that, The human milk oligosaccharide production pathway is any one of the following plasmids: pTrc99a-P trc - lgtA 、pTrc99a-P trc - lgtB - lgtA、 pTrc99a-P trc - wbgO - lgtA、 pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist and pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6 .

10. The use of the engineered bacteria of claim 9 in the production of human milk oligosaccharides.

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