Application of OpgG and mutant thereof in production of human milk oligosaccharide

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to applications of OpgG and a mutant thereof in production of human milk oligosaccharide. The invention provides a dextran biosynthetic protein OpgG mutant, and the OpgG mutant is obtained by mutating the 443rd amino acid from threonine to proline on the basis of a wild type dextran biosynthetic protein OpgG as shown in SEQ ID NO.1. The invention also provides a preparation method of the dextran biosynthetic protein OpgG mutant. An opgG gene and / or a T443P mutant coding gene are / is introduced into a human milk oligosaccharide production strain and applied to production of human milk oligosaccharide, and the yield of 3 '-SL, 6'-SL, LNT II, LNnT and LNT is greatly increased.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to the application of OpgG and its mutants in the production of human milk oligosaccharides. Background Art

[0002] Human milk oligosaccharides (HMOs) are an important class of bioactive components in breast milk. HMOs have multiple physiological functions, including establishing a balanced infant intestinal microbiome, strengthening the gastrointestinal barrier, preventing infection, and potentially supporting the immune system. According to their different structures, they are divided into fucosylated 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), lactoyl- N -trisaccharide II (lacto- N- triose, LNT II), lactoyl- N -New tetrasaccharide (Lacto- N -neotetraose, LNnT) and lactoyl- N -tetrasaccharide (Lacto- N Currently, HMOs are produced by chemical synthesis, enzymatic catalysis, and microbial fermentation. 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 HMOs microbial cell factory strategy has achieved certain results, the production volume still needs to be improved. N -acetylglucosamine isomerase gene ( neuC) , acetylneuraminic acid synthetase gene ( neuB ), CMP-acetylneuraminic acid synthetase gene ( neuA ), synthetic cytidine monophosphate- N -Acetylneuraminic acid (CMP-Neu5Ac). CMP-Neu5Ac combines with 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. When synthesizing LNnT, glucose is used as the carbon source, and glucose-6-phosphate (Glc-6-P) is pgi Glucose-6-phosphate isomerase is catalyzed by the gene encoding glucose-6-phosphate isomerase, which is then converted into fructose-6-phosphate (F6P). glmS Encoded glutamine-fructose-6-phosphate aminotransferase, glmM Encoded phosphoglucosamine mutase, glmU Encoded N -acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase catalyzes the conversion of uridine diphosphate to N -acetylglucosamine (UDP-GlcNAc); in the synthesis pathway of another precursor uridine diphosphate-galactose (UDP-Gal), Glc-6-P pgm Encoded phosphoglucomutase and galE UDP-glucose-4-isomerase is converted into UDP-Gal. lgtA Encoded β-1,3- N Under the catalysis of acetyl glucosamine aminotransferase, lactose reacts with UDP-GlcNAc to generate the intermediate product LNT II. lgtB Under the catalysis of the encoded β-1,4-galactosyltransferase, it combines with UDP-Gal to generate LNnT. w.b.g.O. LNT is produced by the encoded β-1,3-galactosyltransferase.

[0004] Glucan biosynthesis protein OpgG is a periplasmic protein that is required 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 is still unclear (XavierHanoulle, 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 human milk oligosaccharide production strains opgG Gene mutation and / or overexpression opgG , hoping to further increase the production of human milk oligosaccharides. Summary of the Invention

[0006] The present invention utilizes gene editing technology to obtain opgGGenetically engineered chassis strains or overexpression of mutants opgG strains, and then applied the above chassis strains to the construction of production strains of 3'-SL, 6'-SL, LNT II, LNnT and LNT, further improving the yields of 3'-SL, 6'-SL, LNT II, LNnT and LNT.

[0007] One of the technical solutions provided by the present invention is a mutant of the glucan biosynthesis protein OpgG, wherein the OpgG mutant is obtained by mutating the amino acid at position 443 from threonine to proline based on the wild-type glucan biosynthesis protein OpgG shown in SEQ ID NO.1; the OpgG mutant is named T443P mutant, and the amino acid sequence is shown in SEQ ID NO.3; The present invention also provides a gene encoding the T443P mutant; Furthermore, the coding gene is opgG T443P , the nucleotide sequence is shown in SEQ ID NO.4. The second technical solution provided by the present invention is to include opgG T443P Recombinant vectors or recombinant strains of genes; Furthermore, the expression plasmid used in the recombinant vector is psb4k5 plasmid; Furthermore, the expression host used by the recombinant strain is Escherichia coli, preferably MG1655.

[0008] The third technical solution provided by the present invention is the application of the T443P mutant described in the first technical solution, especially in the production of human milk oligosaccharides; Furthermore, the application is to mutate the glucan biosynthesis protein OpgG encoding gene on the genome of the human milk oligosaccharide production strain to opgG T443P , and / or in human milk oligosaccharide producing strains opgG T443P Gene expression.

[0009] The fourth technical solution provided by the present invention is an engineered bacterium for producing human milk oligosaccharides, wherein the engineered bacterium is obtained by overexpressing the gene encoding the glucan biosynthesis protein OpgG on the genome based on a human milk oligosaccharide-producing strain; or The engineering bacteria is based on the human milk oligosaccharide production strain as the starting strain, and the glucan biosynthesis protein OpgG encoding gene on the genome is mutated to opgG T443P obtain; or, The engineering bacteria is based on the human milk oligosaccharide production strain as the starting strain, and the strain contains the glucan biosynthesis protein OpgG encoding gene and opgG T443P Gene; or The engineering bacteria is based on the human milk oligosaccharide production strain as the starting strain, and the glucan biosynthesis protein OpgG encoding gene on the genome is mutated to opgG T443P At the same time opgG T443P Obtained by overexpression; Furthermore, the human milk oligosaccharide-producing strain is a strain capable of producing LNTII, LNnT, LNT, 3'-SL, or 6'-SL; Furthermore, the human milk oligosaccharide production strain uses Escherichia coli K12 MG1655 as a host and knocks out the host lactose operon sequence. lacZ , and overexpression lacY ; On this basis, the strain also contains a human milk oligosaccharide production pathway; Furthermore, the human milk oligosaccharides include but are not limited to: lactoyl- N -trisaccharide (Lacto-N-Triose, LNT II), lactoyl- N -Lacto-N-Neotetraose (LNnT), lactose- N -Lacto-N-tetraose (LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL) and 6'-sialyllactose (6'-Sialyllactose, 6'-SL); Furthermore, 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 - w.b.g.O. - lgtA、 pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist and pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ST6 .

[0010] The fifth technical solution provided by the present invention is the use of the engineered bacteria described in the fourth technical solution in the production of human milk oligosaccharides; Furthermore, the human milk oligosaccharide includes: LNTII, LNnT, LNT, 3'-SL, and 6'-SL.

[0011] Beneficial effects: The present invention provides a mutant of a glucan biosynthesis protein OpgG, wherein the OpgG mutant is obtained by mutating the amino acid at position 443 from threonine to proline based on the wild-type glucan biosynthesis protein OpgG shown in SEQ ID NO.1. opgG Gene, and / or T443P mutant encoding gene, and applied them to the production of human milk oligosaccharides, the yield of 3'-SL, 6'-SL, LNT II, LNnT and LNT was greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the first step of homologous recombination colony PCR verification.

[0013] Figure 2 For the second step homologous recombination colony PCR verification. DETAILED DESCRIPTION

[0014] The present invention will be further described below by specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments may be well known to those skilled in the art, and appropriate ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, various changes or modifications made to the material composition and dosage in these embodiments also fall within the scope of protection of the present invention.

[0015] The following definitions are used in the present invention: 1. Nomenclature of amino acid and DNA sequences The generally accepted IUPAC nomenclature for amino acid residues is used, using either the single-letter or three-letter code. DNA nucleic acid sequences use the generally accepted IUPAC nomenclature.

[0016] 2. Identification of mutants of the glucan biosynthesis protein OpgG 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 substituted from the wild-type threonine to proline. The position numbering corresponds to the amino acid sequence numbering of the wild-type OpgG in SEQ ID NO. 1.

[0017] In this invention, lowercase italics opgG Indicates the gene encoding the wild-type glucan biosynthesis protein OpgG, in lowercase italics opgG T443P It represents the gene encoding the mutant T443P. The specific information is shown in the following table.

[0018]

[0019] The amino acid sequence of the glucan biosynthesis protein OpgG involved in the present invention is shown in SEQ ID NO.1: MMKMRWLSAAVMLTLYTSSSWAFSIDDVAKQAQSLAGKGYETPKSNLPSVFRDMKYADYQQQIQFNHDKAYWNNLKTPFKLEFYHQGMYFDTPVKINEVTATAVKRIKYSPDYFTFGDVQHDKDTVKDL GFAGFKVLYPINSKDKNDEIVSMLGASYFRVIGAGQVYGLSARGLAIDTALPSGEEFPRFKEFWIERPKPTDKRLTIYALLDSPRATGAYKFVVMPGRDTVVDVQSKIYLRDKVGKLGVAPLTSMFLF GPNQPSPANNYRPELHDSNGLSIHAGNGEWIWRPLNNPKHLAVSSFSMENPQGFGLLQRGRDFSRFEDLDDRYDLRPSAWVTPKGEWGKGSVELVEIPTNDETNDNIVAYWTPDQLPEPGKEMNFKYT ITFSRDEDKLHAPDNAWVQQTRRSTGDVKQSNLIRQPDGTIAFVVDFTGAEMKKLPEDTPVTAQTSIGDNGEIVESTVRYNPVTKGWRLVMRVKVKDAKKTTEMRAALVNADQTLSETWSYQLPANE* The glucan biosynthesis protein OpgG mutant involved in the present invention is obtained by mutating the threonine at position 443 to proline based on the wild-type glucan biosynthesis protein OpgG. The amino acid sequence is shown in SEQ ID NO.3: MMKMRWLSAAVMLTLYTSSSWAFSIDDVAKQAQSLAGKGYETPKSNLPSVFRDMKYADYQQQIQFNHDKAYWNNLKTPFKLEFYHQGMYFDTPVKINEVTATAVKRIKYSPDYFTFGDVQHDKDTVKDL GFAGFKVLYPINSKDKNDEIVSMLGASYFRVIGAGQVYGLSARGLAIDTALPSGEEFPRFKEFWIERPKPTDKRLTIYALLDSPRATGAYKFVVMPGRDTVVDVQSKIYLRDKVGKLGVAPLTSMFLF GPNQPSPANNYRPELHDSNGLSIHAGNGEWIWRPLNNPKHLAVSSFSMENPQGFGLLQRGRDFSRFEDLDDRYDLRPSAWVTPKGEWGKGSVELVEIPTNDETNDNIVAYWTPDQLPEPGKEMNFKYT ITFSRDEDKLHAPDNAWVQQTRRSTGDVKQSNLIRQPDGTIAFVVDFTGAEMKKLPEDPPVTAQTSIGDNGEIVESTVRYNPVTKGWRLVMRVKVKDAKKTTEMRAALVNADQTLSETWSYQLPANE* The present invention will be further explained below through specific embodiments.

[0020] Example 1 Construction of strain G1 Based on Escherichia coli H0, CRISPR / Cas9 gene editing technology was used to modify the glucan biosynthesis protein gene on the genome. opgG (Gene ID: 945005) was mutated, and the nucleotide adenine at position 1327 was mutated to cytosine (A was mutated to C), and the corresponding amino acid threonine at position 443 was mutated to proline, thereby obtaining strain G1.

[0021] Among them, E. coli H0 is based on E. coli K12 MG1655 ( Escherichia coli K12 MG1655) was constructed as the starting strain, and the lactose of the starting strain was knocked out. lac P in the operator sequence lac Promoter sequences and regulatory genes lacZ , in the original lacZ P trc Promoter overexpression lacY The specific construction process of this strain is detailed in Example 1 of CN119464168A.

[0022] The specific construction method of strain G1 is as follows: Based on the H0 strain, 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 nucleotide adenine at position 1327 of the gene was mutated to cytosine (A to C), resulting in strain G1. The specific construction method of strain G1 is as follows: 1. Construction of homologous recombination fragments Using the wild-type strain MG1655 preserved in the laboratory as a template, the primers listed in Table 1 were used to construct homologous recombination fragments. opgG -up-f / r and opgG -down-f / r as primers, PCR amplification obtained the upstream and downstream homology arms of homologous recombination. cat 、 cat Promoter and N20 nucleotide vector as template, using primer pair opgG -cat-f / r were PCR amplified to obtain cat- A fragment of the N20 sequence (SEQ ID NO.5). The upper and downstream homology arms, with cat- The fragments of N20 sequence, these three fragments are used as templates, using primers opgG -up-f and opgG -down-r overlap PCR was performed to obtain a homologous recombinant fragment containing opgG The mutation site of the gene, i.e. the wild type opgG The nucleotide adenine at position 1327 of the nucleotide sequence was mutated to cytosine.

[0023] 2. First step: homologous recombination The pCAGO plasmid was transformed into strain H0 using conventional plasmid transformation methods to obtain strain H0 (pCAGO). Competent strain H0 (pCAGO) was prepared using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG. The homologous recombination fragment was introduced by electroporation. The transformed bacterial suspension was plated on LB plates containing 100 mg / L ampicillin, 25 mg / L chloramphenicol, and 1% glucose and incubated at 30°C. Transformants were selected for colony PCR verification (verification primers: opgG -yz-f / r), if the recombination is successful, the band size is about 2660 bp, and the verification result is as follows Figure 1 As shown, the bands are correct, that is, the first homologous recombination is successful, and the correct transformants are picked for the second step of homologous recombination.

[0024] 3. Second step homologous recombination The strain that successfully underwent the first recombination test was inoculated into a LB tube containing 100 μg / mL ampicillin and 0.1 mM IPTG. The strain was cultured on a shaker at 30°C for more than 6 hours to induce the expression of the CRISPR / Cas9 system and the λ-red protein to complete the second recombination. Three separate colonies were streaked on an LB plate containing ampicillin. The isolated colonies were then spotted on a chloramphenicol-resistant LB plate and an ampicillin-resistant LB plate. Single colonies that did not grow on the chloramphenicol medium but grew on the ampicillin medium were selected for verification by colony PCR (verification primers: opgG -yz-f / r). If the recombination is correct, the band size is about 1725 bp, and the verification result is as follows Figure 2 The band was correct, and the PCR product of the 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 ° C to lose the pCAGO plasmid, thereby obtaining a strain with opgG The mutant strain was named G1.

[0025] Table 1 Construction opgG Primers used for gene mutation strains

[0026] Example 2 Construction of strains H1, H2, G2 and G3 (1) Construction of plasmid psb4k5- opgG Plasmid psb4k5 was used as template and psb4k5-fp-F / R in Table 2 was used as primers for PCR amplification to obtain linear vector psb4k5. opgG The sequence in Table 2 was used as a template. opgG -F / R as primers for PCR amplification to obtain linear gene fragments opgG The linear vector and linear gene fragment obtained by PCR were purified and recovered, ligated using the ClonExpress II Recombination Ligation Kit (Novozymes Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, and cultured on LB plates containing 50 mg / L kanamycin. Transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid psb4k5- opgG .

[0027] (2) Construction of plasmid psb4k5- opgG T443P Using plasmid psb4k5- opgG As a template, PCR amplification was performed using fp443-f / r in Table 2 as primers to obtain opgG Linear vector psb4k5- encoding gene with T443P mutation (SEQ ID NO.4) opgG T443P The linear vector obtained by PCR was purified and recovered, ligated using the ClonExpress II Recombination Ligation Kit (Novozymes Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, and cultured on LB plates containing 50 mg / L kanamycin. Transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid psb4k5- opgG T443P .

[0028] Table 2 Construction of plasmid psb4k5- opgG All primers for mutants

[0029] The above plasmid psb4k5- opgG 、psb4k5- opgG T443P were introduced into H0 to obtain strains H1 and H2; plasmid psb4k5- opgG 、psb4k5- opgG T443P They were introduced into G1 respectively to obtain strains G2 and G3.

[0030] Example 3 Construction of plasmid pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist Plasmid pTrc99a was used as a template to construct plasmid pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist The specific construction process of this plasmid refers to Example 2 of patent CN117736280A.

[0031] Example 4 Construction of plasmid pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ST6 Plasmid pTrc99a-P J23119 - neuB–neuC -Ptrc - neuA-ist The plasmid pTrc99a-P was constructed as a template J23119 - neuB–neuC -P trc - neuA-ST6 The specific construction process of this plasmid refers to Example 3 of patent CN117736280A.

[0032] Example 5 Construction of plasmid pTrc99a-P trc - lgtA lgtA Gene encoding β-1,3- N -acetyl glucosamine transferase, plasmid pTrc99a was used as a template to construct plasmid pTrc99a-P trc - lgtA The specific construction process of the plasmid is referred to Example 4 of patent CN 119464168A.

[0033] Example 6 Construction of plasmid pTrc99a-P trc -lgtB-lgtA lgtB The gene encoding lipooligosaccharide biosynthesis protein is located in the plasmid pTrc99a-P trc - lgtA The plasmid pTrc99a-P was constructed based on trc - lgtB - lgtA The specific construction process of this plasmid is referred to Example 5 of patent CN 119464168A.

[0034] Example 7 Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA w.b.g.O. The gene encoding β-1,3-galactosyltransferase is located in the plasmid pTrc99a-P trc - lgtA Plasmid pTrc99a-P was constructed based on trc - w.b.g.O. - lgtA The specific construction process of this plasmid is referred to Example 6 of patent CN 119464168A.

[0035] Example 8 Construction and fermentation testing of 3'-SL, 6'-SL, LNT II, LNnT and LNT production strains 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 - w.b.g.O. - lgtA were introduced into H0, H1, H2, G1, G2 and G3 respectively to construct the following production strains: (1) 3'-SL production strain: Y1 [H0 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )]; Y2 [H1 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )]; Y3 [H2 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )]; Y4 [G1 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist ) ]; Y5 [G2 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )]; Y6 [G3 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )]; (2) 6'-SL production strain: Y7 [H0 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )]; Y8 [H1 (pTrc99a-P J23119 - neuB–neuC -Ptrc - neuA - ST6 )]; Y9 [H2(pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )]; Y10 [G1 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )]; Y11 [G2 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )]; Y12 [G3 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )]; (3) LNT II production strain: Y13 [H0 (pTrc99a-P trc - lgtA )]; Y14 [H1 (pTrc99a-P trc - lgtA )]; Y15 [H2 (pTrc99a-P trc - lgtA )]; Y16 [G1 (pTrc99a-P trc - lgtA )]; Y17 [G2 (pTrc99a-P trc - lgtA )]; Y18 [G3 (pTrc99a-P trc - lgtA )] (4) LNnT production strains: Y19 [H0 (pTrc99a-P trc - lgtB - lgtA )]; Y20 [H1 (pTrc99a-P trc - lgtB - lgtA )]; Y21[ H2 (pTrc99a-P trc - lgtB - lgtA )]; Y22 [G1 (pTrc99a-P trc - lgtB - lgtA )]; Y23 [G2 (pTrc99a-P trc - lgtB - lgtA )]; Y24 [G3 (pTrc99a-P trc - lgtB - lgtA )]; (5) LNT production strains: Y25 [H0 (pTrc99a-P trc - w.b.g.O. - lgtA )]; Y26 [H1 (pTrc99a-P trc - w.b.g.O. - lgtA )]; Y27 [H2 (pTrc99a-P trc - w.b.g.O. - lgtA )]; Y28 [G1 (pTrc99a-P trc - w.b.g.O. - lgtA )]; Y29 [G2 (pTrc99a-P trc - w.b.g.O. - lgtA )]; Y30 [G3 (pTrc99a-P trc - w.b.g.O. - lgtA )] The strains and plasmids used in this patent are shown in Table 3.

[0036] Table 3 Strains and plasmids used in this application

[0037] The production levels of the above strains were tested by fermentation. The culture medium used was: LB medium (1 L): NaCl 10 g, yeast powder 5 g, peptone 10 g.

[0038] 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, ammonia water to adjust pH to 7.0.

[0039] 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.

[0040] The fermentation test process is: Single colonies of 3'-SL, 6'-SL, LNT II, LNnT and LNT production strains were picked and cultured overnight in LB liquid medium containing 50 mg / L ampicillin and kanamycin at 37°C and 220 rpm / min. The overnight culture liquid was used as seed liquid and transferred to a 24-well plate containing 2 mL fermentation medium at a 1% inoculum volume. The fermentation medium contained 50 mg / L ampicillin and kanamycin and 0.1 mmol / L IPTG. Fermentation was carried out at 37°C and 800 rpm. Three samples were cultured in parallel for each strain. During the fermentation process, the growth of the bacteria (OD 600 ) and 3'-SL, 6'-SL, LNTII, LNnT, and LNT content in the samples were determined by HPLC. The HPLC detection conditions for LNTII, LNnT, LNT, 3'-SL, and 6'-SL were similar to those in Example 4 of CN117736280A. The results are shown in Tables 4 to 8: Table 4 Test results of 3'-SL production by different strains

[0041] Table 5 Test results of 6'-SL production by different strains

[0042] Table 6 Test results of LNT II production by different strains

[0043] Table 7 Test results of LNnT production by different strains

[0044] Table 8 Test results of LNT production by different strains

[0045] The results showed that after the amino acid at position 443 of the OpgG protein was mutated from threonine to proline, the strain carrying only the OpgG genome mutation showed a certain advantage in synthesizing human milk oligosaccharides compared with the wild-type strain. opgG The wild type and / or mutant type were verified, and the results showed that compared with the wild type OpgG, the production of 3'-SL, 6'-SL, LNT II, LNnT and LNT human milk oligosaccharides were increased to varying degrees, and finally carried opgG Strains with genomic mutations and plasmids overexpressing wild-type and / or mutant opgG The strain with the gene significantly increased the total production of human milk oligosaccharides. This research result has opened up a new idea for constructing strains that can efficiently synthesize human milk oligosaccharides.

[0046] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details 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 glucan biosynthesis protein OpgG, characterized in that: The OpgG mutant is obtained by mutating the amino acid at position 443 from threonine to proline based on the wild-type glucan biosynthesis protein OpgG shown in SEQ ID NO.1; the OpgG mutant is named T443P mutant, and the amino acid sequence is shown in SEQ ID NO.

3.

2. The gene encoding the OpgG mutant according to claim 1.

3. The coding gene according to claim 2, wherein The coding gene is OPgG T443P , the nucleotide sequence is shown in SEQ ID NO.

4.

4. Inclusion OPgG T443P Recombinant vector or recombinant strain of the gene.

5. Use of the OpgG mutant according to claim 1 in producing human milk oligosaccharides, characterized in that: The application is to mutate the glucan biosynthesis protein OpgG encoding gene on the genome of the human milk oligosaccharide production strain to OPgG T443P , and / or in human milk oligosaccharide producing strains OPgG T443P Gene expression.

6. An engineered bacterium for producing human milk oligosaccharides, characterized in that: The engineered bacteria is obtained by overexpressing the glucan biosynthesis protein OpgG encoding gene on the genome using a human milk oligosaccharide production strain as a starting strain; or The engineering bacteria is based on the human milk oligosaccharide production strain as the starting strain, and the glucan biosynthesis protein OpgG encoding gene on the genome is mutated to OPgG T443P obtain; or, The engineering bacteria is based on the human milk oligosaccharide production strain as the starting strain, and the strain contains the glucan biosynthesis protein OpgG encoding gene and OPgG T443P Gene; or The engineering bacteria is based on the human milk oligosaccharide production strain as the starting strain, and the glucan biosynthesis protein OpgG encoding gene on the genome is mutated to OPgG T443P At the same time OPgG T443P Obtained by overexpression.

7. The engineered bacteria for producing human milk oligosaccharides according to claim 6, wherein: The human milk oligosaccharide production strain uses Escherichia coli K12 MG1655 as a host and knocks out the host lactose operon sequence. lacZ , and overexpression lacY ; On this basis, the strain also contains a human milk oligosaccharide production pathway.

8. The engineered bacteria according to claim 7, characterized in that The human milk oligosaccharides include: lactoyl- N -trisaccharide (Lacto-N-Triose, LNT II), lactoyl- N -Lacto-N-Neotetraose (LNnT), lactose- N -tetraose (Lacto-N-tetraose, LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL) and 6'-sialyllactose (6'-Sialyllactose, 6'-SL).

9. The engineered bacteria according to 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 - wxya - lgtA, pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist and pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6 .

10. Use of the engineered bacteria according to claim 9 in producing human milk oligosaccharides.

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