A method for regulating human milk oligosaccharide production by membrane proteins

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a method for regulating human milk oligosaccharide production through membrane proteins. The method is achieved by inactivating the membrane protein YgfX coding gene on a production strain. The human milk oligosaccharide production strain obtained by the method is obtained by knocking out or deleting the 192th guanine base of the membrane protein YgfX coding gene through gene editing technology, and then inactivating the membrane protein YgfX. The human milk oligosaccharide production capacity of the strain is obviously improved compared with that before gene editing. The production of LNTII, LNnT, LNT, 3'-SL and 6'-SL is increased by about 40%, 125%, 46%, 32% and 47% respectively, and the effect is significant. The application provides a new method and idea for widening the human milk oligosaccharide production pathway.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a method for regulating production of human milk oligosaccharides by membrane proteins. BACKGROUND

[0002] Human milk oligosaccharides (HMOs for short) are a class of key oligosaccharides in breast milk, and have a variety of important biological functions. As prebiotics, HMOs play a regulatory role in the ecological balance of intestinal microbial communities; at the same time, HMOs also have immune defense regulation functions, and play a positive promoting role in the brain development and cognitive development of infants. In view of the important significance of HMOs for the growth and development of infants, HMOs can be added as functional ingredients into infant formula milk powder, and have shown a broad market application prospect. According to the different structures, the types of HMOs are more than 200, and the common ones include lacto-N-triose (LNT II), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) and the like. At present, the production methods of HMOs mainly include chemical synthesis method, enzyme catalysis method and microbial fermentation method and the like.

[0003] Escherichia coli has the advantages of clear genetic background, simple culture method, short growth cycle, relatively clear metabolic pathway and mature gene operation technology, and has become a typical model strain in the field of microbial fermentation, and is widely used in the biosynthesis of a variety of HMOs. For example: in the process of synthesizing LNTII, LNnT or LNT by using E. coli, glucose or glycerol is usually used as a carbon source, and lactose is used as a substrate. Glucose-6-phosphate (Glc-6-P) is isomerized to fructose-6-phosphate (F6P) under the catalysis of glucose-6-phosphate isomerase encoded by pgi glmS glutamine-fructose-6-phosphate aminotransferase encoded by glmM phosphoglucomutase encoded by glmU N ​​Catalyzed by acetylglucosamine-1-phosphate uridine transferase / glucosamine-1-phosphate acetyltransferase, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is ultimately produced. In the synthetic pathway of another precursor, uridine diphosphate-galactose (UDP-Gal), Glc-6-P... pgm Gene-encoded glucose phosphomutase and galE The gene-encoded UDP-glucose-4-isomerase catalyzes the conversion to UDP-Gal. Subsequently, lgtA Gene-encoded β-1,3- N Under the catalysis of α-acetylglucosamine aminotransferase, lactose reacts with UDP-GlcNAc to form the intermediate LNT II. LNT II in... lgtB Under the catalytic action of the gene-encoded β-1,4-galactosyltransferase, it binds with UDP-Gal to generate LNnT; LNT II can also... wbgO LNTs are generated under the catalysis of gene-encoded β-1,3-galactosyltransferase. In the synthesis of sialylated human lactose oligosaccharides using *E. coli*, *E. coli* utilizes its own UDP-GlcNAc synthetic pathway, with the synergistic catalysis of UDP-N-acetylglucosamine-2-epimerase NeuC, N-acetylneuraminic acid synthase NeuB, and N-acetylneuraminic acid cytyltransferase NeuA, to synthesize cytidine monophosphate-N-acetylneuraminic acid CMP-Neu5Ac. CMP-Neu5Ac then undergoes glycosylation with lactose under the catalysis of α2,3-sialyltransferase α2,3-SiaT and α2,6-sialyltransferase α2,6-SiaT, respectively, to synthesize 3'-SL and 6'-SL.

[0004] YgfX (also known as CptA, cytoskeleton polymerization inhibitory toxin) is a membrane-associated protein mainly involved in the process of cytoskeleton formation of the strain. The current research on the function of YgfX is still unclear. Some studies have shown that overexpression of YgfX leads to cell shape change to lemon shape, and some studies have shown that overexpression of YgfX has no effect on cell growth (Masuda H, et al. A novel membrane-bound toxin for cell division, CptA (YgfX), inhibits polymerization of cytoskeleton proteins, FtsZ and MreB, in Escherichia coli [J]. FEMS microbiology letters, 2012, 328(2): 174-181; McNeil, et al. YgfX (CptA) is a multimeric membrane protein that interacts with the succinatedehydrogenase assembly factor SdhE (YgfY) 2013, 159, 1352-1365).

[0005] In the industrial high-level production of human milk oligosaccharides, various metabolic intermediates and end products accumulate in the fermentation environment. The strain has poor tolerance to the fermentation environment, and the morphology of the strain changes to a certain extent in the later stage of fermentation, and the growth and production capacity of the strain are limited to a certain extent. In this study, YgfX in the human milk oligosaccharide producing strain was regulated, and it was expected to further improve the production performance of the human milk oligosaccharide producing strain. SUMMARY

[0006] To solve the above technical problems, the gene editing technology is used to edit the gene of Escherichia coli ygfx The gene editing technology is used to edit the gene of Escherichia coli

[0007] One of the technical solutions provided by the present application is a method for improving the production of human milk oligosaccharides, which is realized by inactivating the membrane protein YgfX on the production strain.

[0008] The membrane protein YgfX has an amino acid sequence as shown in SEQ ID NO. 1.

[0009] The coding gene of the membrane protein YgfX ygfx has a nucleotide sequence as shown in SEQ ID NO. 2.

[0010] The human milk oligosaccharide includes but is not limited to LNTII, LNnT, LNT, 3'-SL, 6'-SL;

[0011] The method for inactivating the membrane protein YgfX includes but is not limited to gene knockout, insertion or deletion of bases and the like;

[0012] Further, the engineering bacteria is obtained by deleting the gene encoding the membrane protein YgfX of the human milk oligosaccharide production strain; ygfx The 192th guanine base is inactivated.

[0013] The second technical solution of the present application provides an engineering bacteria for producing human milk oligosaccharide, wherein the engineering bacteria is obtained by inactivating the membrane protein YgfX of a human milk oligosaccharide production strain;

[0014] The method for inactivating the membrane protein YgfX includes but is not limited to gene knockout, insertion or deletion of bases and the like;

[0015] Further, the engineering bacteria is obtained by knocking out the gene encoding the membrane protein YgfX of the human milk oligosaccharide production strain; ygfx Further, the engineering bacteria is obtained by deleting the gene encoding the membrane protein YgfX of the human milk oligosaccharide production strain;

[0016] Further, the engineering bacteria is obtained by deleting the gene encoding the membrane protein YgfX of the human milk oligosaccharide production strain; ygfx The 192th guanine base is inactivated.

[0017] Further, the engineering bacteria is obtained by knocking out the gene encoding the membrane protein YgfX of the human milk oligosaccharide production strain; lacZ Further, the engineering bacteria is obtained by knocking out the gene encoding the membrane protein YgfX of the human milk oligosaccharide production strain; lacY ; on this basis, the strain further comprises a human milk oligosaccharide production pathway;

[0018] Further, the human milk oligosaccharide includes but is not limited to lacto-N-triose (Lacto-N-Triose, LNT II), lacto-N-neotetraose (Lacto-N-Neotetraose, LNnT), lacto-N-tetraose (Lacto-N-tetraose, LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL) and 6'-sialyllactose (6'-Sialyllactose, 6'-SL); N N N

[0019] Further, the human milk oligosaccharide production pathway is any one of the following plasmids:

[0020] 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 .

[0021] The third technical solution provided by the present application is the application of the engineering bacteria in the production of human milk oligosaccharides.

[0022] Further, the human milk oligosaccharides include but are not limited to LNTII, LNnT, LNT, 3'-SL and 6'-SL.

[0023] The fourth technical solution provided by the present application is a membrane protein YgfX mutant, and the coding gene of the mutant is obtained by deleting the 192th guanine base on the basis of the wild-type membrane protein YgfX coding gene. ygfx

[0024] The fifth technical solution provided by the present application is the application of the membrane protein YgfX mutant in the production of human milk oligosaccharides.

[0025] Beneficial effects:

[0026] The present application provides a method for improving the production capacity of human milk oligosaccharides, which is achieved by inactivating the membrane protein YgfX coding gene on the production strain. The human milk oligosaccharide production strain obtained by inactivating the membrane protein YgfX through gene editing technology to knockout or delete the 192th guanine base of the membrane protein YgfX coding gene has a significant improvement in the production capacity of human milk oligosaccharides, and the production of LNTII, LNnT, LNT, 3'-SL and 6'-SL is increased by about 40%, 125%, 46%, 32% and 47% respectively, which has a significant effect and provides a new method and idea for widening the production route of human milk oligosaccharides. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 For ygfX Mutant first step homologous recombination colony PCR verification

[0028] Figure 2 For ygfX ​The mutant was validated by homologous recombination colony PCR in the second step.

[0029] Figure 3 To knock out ygfX The first step was to verify the homologous recombination colony PCR.

[0030] Figure 4 To knock out ygfX The second step is homologous recombination colony PCR verification. Detailed Implementation

[0031] 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.

[0032] The mutant of the membrane protein YgfX involved in this invention is a variant of the wild-type membrane protein encoding gene ( ygfX It was obtained by deleting the guanine base at position 192 of GeneID: 947379. Among them:

[0033] The wild-type membrane protein YgfX has the amino acid sequence shown in SEQ ID NO.1:

[0034] MVLWQSDLRVSWRAQWLSLLIHGLVAAVILLMPWPLSYTPLWMVLLSLVVFDCVRSQRRINARQGEIRLLMDGRLRWQGQEWSIVKAPWMIKSGMMLRLRSDGGKRQHLWLAADSMDEAEWRDLRRILLQQETQR*

[0035] Wild-type membrane protein YgfX encoding gene ygfX The nucleotide sequence is shown in SEQ ID NO.2:

[0036] GTGGTCCTGTGGCAATCTGATTTGCGCGTCTCCTGGCGCGCACAGTGGCTTTCCTTGCTGATTCATGGGCTGGTTGCCGCTGTTATTTTACTCATGCCCTGGCCACTCAGTTACACCCCGTTATGGATGGTGTTACTTTCGCTGGTGGTGTTTGATTGCGTTCGCAGCCAGCGGCGTATTAATGCTCGCCAGGGGGAAATTCGCTTGTTGATGGACGGGCGTTTGCGTTGGCAAGGGCAGGAGTGGAGCATCGTCAAAGCACCGTGGATGATTAAGAGCGGCATGATGCTGCGTTTACGTTCTGATGGCGGTAAACGGCAACATTTATGGCTGGCAGCCGACAGCATGGACGAAGCTGAATGGCGGGATTTACGGCGGATTTTGTTGCAACAAGAGACGCAAAGATAA

[0037] The membrane protein mutant, due to the deletion of the 192th guanine base of the encoding gene, causes the subsequent codon rearrangement, and finally results in three stop codons (“*” marked) in the amino acid sequence, and the amino acid sequence of the membrane protein mutant is as follows:

[0038] MVLWQSDLRVSWRAQWLSLLIHGLVAAVILLMPWPLSYTPLWMVLLSLVVFDCVRSQRRINARQGKFAC*WTGVCVGKGRSGASSKHRG*LRAA*CCVYVLMAVNGNIYGWQPTAWTKLNGGIYGGFCCNKRRKD (SEQ ID NO. 3)

[0039] The nucleotide sequence of the encoding gene of the membrane protein mutant is as follows:

[0040] GTGGTCCTGTGGCAATCTGATTTGCGCGTCTCCTGGCGCGCACAGTGGCTTTCCTTGCTGATTCATGGGCTGGTTGCCGCTGTTATTTTACTCATGCCCTGGCCACTCAGTTACACCCCGTTATGGATGGTGTTACTTTCGCTGGTGGTGTTTGATTGCGTTCGCAGCCAGCGGCGTATTAATGCTCGCCAGGGGAAATTCGCTT GTTGATGGACGGGCGTTTGCGTTGGCAAGGGCAGGAGTGGAGCATCGTCAAAGCACCGTGGATGATTAAGAGCGGCATGATGCTGCGTTTACGTTCTGATGGCGGTAAACGGCAACATTTATGGCTGGCAGCCGACAGCATGGACGAAGCTGAATGGCGGGATTTACGGCGGATTTTGTTGCAACAAGAGACGCAAAGATAA (SEQ ID NO.4)

[0041] The present invention will be further explained and illustrated below through specific embodiments.

[0042] Example 1 Construction of strain Z1

[0043] With Escherichia coli K12 MG1655 ( Escherichia coli Using K12MG1655 as the starting strain, the lactose operon sequence of the starting strain was knocked out using CRISPR / Cas9 technology (Zhao D, et al. CRISPR / Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. SciRep 7, 16624). lacZ , in the original lacZ Following the site with P trc Promoter overexpression lacY The strain was named Z0. For details of the construction process of this strain, please refer to Example 1 of patent CN 119464168A.

[0044] The strain carrying the YgfX mutant (with the guanine base deleted at position 192 in the wild-type coding gene) is designated Z1, and its specific construction method is as follows:

[0045] 1. Construction of homologous recombination fragments

[0046] The laboratory preserved MG1655 wild type strain was used as a template to construct the homologous recombination fragment using the primers in Table 1. The upper and lower homologous arms of the homologous recombination were obtained by PCR amplification using primers ygfX-up-f / r and ygfX-down-f / r. The fragment containing the chloramphenicol resistance gene cat 、 cat The promoter and N20 sequence vector were used as templates to obtain the fragment with the N20 sequence (SEQ ID NO. 5) by PCR amplification using the primer pair ygfX-cat-f / r. The three fragments, the upper and lower homologous arms, and the fragment with the N20 sequence, were used as templates to obtain the homologous recombination fragment by overlap PCR using the primers ygfX-up-f and ygfX-down-r. The homologous recombination fragment contains the mutation site (deletion of the 192th guanine base) of the ygfX gene. cat- cat- The three fragments, the upper and lower homologous arms, and the fragment with the N20 sequence, were used as templates to obtain the homologous recombination fragment by overlap PCR using the primers ygfX-up-f and ygfX-down-r. The homologous recombination fragment contains the mutation site (deletion of the 192th guanine base) of the ygfX gene. ygfX

[0047] 2. First step homologous recombination

[0048] The pCAGO plasmid was transformed into the strain Z0 by conventional plasmid transformation to obtain the strain Z0 (pCAGO). The LB medium containing 1% (m / v) glucose and 0.1 mM IPTG was used to prepare the Z0 (pCAGO) competent cells, and the homologous recombination fragment obtained in step 1 was introduced by electroporation. The transformed bacterial solution was spread on the LB plate containing 100 mg / L ampicillin and 25 mg / L chloramphenicol, and 1% glucose, and cultured at 30°C. The transformants were picked for colony PCR verification (verification primers: ygfX-yz-f / r). If the recombination is successful, the band size is about 1342 bp, and the verification result is shown in Figure 1 . The correct band indicates that the first homologous recombination is successful, and the correct transformants were picked for the second step homologous recombination.

[0049] 3. Second step homologous recombination

[0050] The strain with successful verification of the first recombination was inoculated into the LB test tube containing 100 μg / mL AMP 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 the λ-red protein to complete the second recombination. The single colonies were separated by drawing three zones on the LB plate containing ampicillin, and the separated single colonies were spotted on the chloramphenicol-resistant LB plate and the ampicillin-resistant LB plate, respectively. The single colonies that did not grow on the chloramphenicol medium and grew on the ampicillin medium were selected, and the colony PCR verification (verification primers: ygfX-yz-f / r) was performed. If the recombination is correct, the band size is about 407 bp, and the verification result is shown in​​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, and the pCAGO plasmid in the strain was lost, thereby obtaining a mutant strain, named Z1. ygfX

[0051] Table 1 Primers used for constructing strain Z1

[0052]

[0053] The cat- N20 sequence, as shown in SEQ ID NO. 5:

[0054] ​ATTAATTAATCTCGAGTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGTCCATCGAACCGAAGTAAGG

[0055] Example 2 Construction of strain Z2

[0056] The YgfX-encoding gene was knocked out based on strain Z0, and the obtained strain was Z2. The specific construction method is as follows:

[0057] 1. Construction of homologous recombination fragment

[0058] The laboratory preserved MG1655 wild type strain was used as a template to construct the homologous recombination fragment using the primers in Table 2. The upper and lower homologous arms of the homologous recombination were obtained by PCR amplification using primers ygfX-up-1-f / r and ygfX-down-1-f / r. The fragment with N20 sequence was obtained by PCR amplification using the primer pair ygfX-cat-1-f / r with the template of the artificially synthesized vector containing the chloramphenicol resistance gene cat , cat promoter and N20 sequence. The above and lower homologous arms, the fragment with N20 sequence (SEQ ID NO. 5) were used as templates to obtain the homologous recombination fragment by overlap PCR using primers ygfX-up-1-f and ygfX-down-1-r. cat- cat- N20 sequence.

[0059] 2. First step homologous recombination

[0060] The pCAGO plasmid was transformed into strain Z0 by conventional plasmid transformation method to obtain strain Z0 (pCAGO). The LB medium containing 1% (m / v) glucose and 0.1 mM IPTG was used to prepare the Z0 (pCAGO) competent cells, and the homologous recombination fragment obtained in step 1 was introduced by electroporation method. The transformed bacterial solution was spread on the LB plate containing 100 mg / L ampicillin and 25 mg / L chloramphenicol, and 1% glucose, and incubated at 30°C. The transformants were picked for colony PCR verification (verification primers: ygfX-yz-1-f / r). If the recombination is successful, the band size is about 2630 bp, and the verification result is shown in Figure 3 , the band is correct, i.e. the first homologous recombination is successful, and the correct transformants were picked for the second step homologous recombination.

[0061] 3. Second step homologous recombination

[0062] ​The once recombination verified strain is inoculated into LB test tube containing 100 μg / mL AMP and 0.1 mM IPTG, and cultured at 30°C for more than 6 h on a shaker to induce the expression of CRISPR / Cas9 system and λ-red protein to complete the second recombination. Three regions are separated respectively on LB plate containing ampicillin, and the separated single colonies are respectively spotted on chloramphenicol resistant LB plate and ampicillin resistant LB plate. The single colony which does not grow on chloramphenicol medium and grows on ampicillin medium is selected, and colony PCR verification (verification primer: ygfX-yz-1-f / r) is carried out. If the recombination is correct, the band size is about 1695 bp, the verification result is shown in Figure 4 , the band is correct, and the PCR product of the band is sequenced, the sequencing result is correct, and the second step homologous recombination strain is obtained. The second step homologous recombination strain is further cultured at 37°C, and the pCAGO plasmid in it is lost, so that the strain of the knock-out ygfX is obtained, which is named as Z2.

[0063] Table 2 Primers used for constructing strain Z2

[0064]

[0065] Example 3 Construction of plasmid pTrc99a-P trc -lgtA

[0066] lgtA Gene encoding β-1,3- N acetylglucosamine transferase, plasmid pTrc99a-P trc - lgtA is constructed by using plasmid pTrc99a as a template. The specific construction process of the plasmid is referred to patent CN119464168A embodiment 4.

[0067] Example 4 Construction of plasmid pTrc99a-P trc -lgtB-lgtA

[0068] lgtB Gene encoding lipid oligosaccharide biosynthesis protein, plasmid pTrc99a-P trc - lgtA is constructed on the basis of plasmid pTrc99a-P trc - lgtB - lgtA . The specific construction process of the plasmid is referred to patent CN119464168A embodiment 5.

[0069] Example 5 Construction of plasmid pTrc99a-P trc - wbgO-lgtA

[0070] wbgO The gene encodes β-1, 3-galactosyltransferase, and the plasmid pTrc99a-P trc - lgtA The plasmid pTrc99a-P trc - wbgO - lgtA The specific construction process of the plasmid is referred to Example 6 of the patent CN119464168A.

[0071] Example 6 Construction of plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist

[0072] The plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist is constructed by using the plasmid pTrc99a as a template. The specific construction process of the plasmid is referred to Example 2 of the patent CN117736280A.

[0073] Example 7 Construction of plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6

[0074] The plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist is constructed by using the plasmid pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6 as a template. The specific construction process of the plasmid is referred to Example 3 of the patent CN117736280A.

[0075] Example 8 Construction and fermentation test of LNT II, LNnT, LNT, 3'-SL and 6'-SL production strains

[0076] The 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 were introduced into Z0, Z1 and Z2 strains respectively, and the following strains were constructed respectively:

[0077] (1) LNT II producing strain:

[0078] B1 [Z0 (pTrc99a-P trc - lgtA )];

[0079] B2 [Z1 (pTrc99a-P trc - lgtA )];

[0080] B3 [Z2 (pTrc99a-P trc - lgtA )];

[0081] (2) LNnT producing strain:

[0082] B4 [Z0 (pTrc99a-P trc - lgtB - lgtA )];

[0083] B5 [Z1 (pTrc99a-P trc - lgtB - lgtA )];

[0084] B6 [Z2 (pTrc99a-P trc - lgtB - lgtA )];

[0085] (3) LNT producing strain:

[0086] B7 [Z0 (pTrc99a-P trc wbgO - lgtA )];

[0087] B8 [Z1 (pTrc99a-P trc wbgO - lgtA )];

[0088] B9 [Z2 (pTrc99a-P trc wbgO - lgtA )];

[0089] (4) 3'-SL producing strain:

[0090] B10 [Z0 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist )];

[0091] B11 [Z1 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist )];

[0092] B12 [Z2 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist )];

[0093] (5) 6'-SL production strain:

[0094] B13 [Z0 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA - ST6 )];

[0095] B14 [Z1 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA - ST6 )];

[0096] B15 [Z2 (pTrc99a-P J23119 - neuB-neuC -P trc - neuA - ST6 )];

[0097] Table 3 Strains and plasmids used

[0098]

[0099] The production levels of the above strains were tested by fermentation, respectively. The medium used was:

[0100] LB medium (1 L): NaCl 10 g, yeast powder 5 g, peptone 10 g.

[0101] 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, adjust pH to 7.0 with ammonia water.

[0102] 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, Na2MoO4·2H2O 1.1 g, Na2SeO3 1.5 g, NiSO4·6H2O 1.5 g. O

[0103] The fermentation test process is as follows:

[0104] Single colonies of LNT II, LNnT, LNT, 3'-SL and 6'-SL production strains were picked respectively, and cultured in LB liquid medium containing 50 mg / L ampicillin at 37°C, 220 rpm / min, overnight. The bacterial liquid cultured overnight was used as seed liquid, and the bacterial liquid was transferred to a 24-well plate containing 1 mL fermentation medium at a inoculation amount of 2% (v / v), and the fermentation medium contained 50 mg / L ampicillin and 0.1 mmol / L IPTG. The fermentation was carried out at 37°C, 800 rpm. Each strain was cultured in parallel for 3 times. During the fermentation process, the growth (OD 600 ) of the bacterial cells, the yield of LNT II, LNnT, LNT, 3'-SL and 6'-SL were determined, and the concentration of LNT II, LNnT, LNT, 3'-SL and 6'-SL in the sample was detected by high performance liquid chromatography. The sample concentration was quantified by using the standard curve of LNT II, LNnT, LNT, 3'-SL and 6'-SL. The high performance liquid chromatography detection conditions of LNT II, LNnT, LNT, 3'-SL and 6'-SL are referred to the example 4 of the patent CN117736280A. The results are shown in Tables 4 to 8:

[0105] Table 4 Test results of different strains producing LNT II

[0106]

[0107] Table 5 Test results of different strains producing LNnT

[0108]

[0109] Table 6 Test results of different strains producing LNT ​

[0110]

[0111] Table 7 Test results of different strains producing 3'-SL

[0112]

[0113] Table 8 Test results of different strains producing 6'-SL

[0114]

[0115] From the above results, it can be seen that, ygfx The gene mutation and knockout both greatly improve the ability of the strain to ferment human milk oligosaccharides, and finally greatly improve the yield of human milk oligosaccharides. It is speculated that the YgfX mutant and the YgfX knockout can affect the cell morphology, promote the change of the distribution of the internal metabolic space and substances of the cell, and thus improve the yield of human milk oligosaccharides. This work provides a new idea for the construction of strains for efficient synthesis of human milk oligosaccharides. The present application only takes the YgfX 192th guanine base deletion mutant and the YgfX knockout as examples for illustration, and other ways of causing YgfX inactivation such as base insertion, deletion, etc. are also within the scope of protection.

[0116] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various forms and details of changes, modifications, replacements and variations to these embodiments without departing from the spirit and principles of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A method for improving the production capacity of human milk oligosaccharides, characterized in that, The method is achieved by inactivating the membrane protein YgfX on the producing strain; the amino acid sequence of the membrane protein YgfX is shown in SEQ ID NO.1; The human milk oligosaccharides are: LNTII, LNnT, LNT, 3'-SL, or 6'-SL.

2. The method for improving human milk oligosaccharide production capacity as described in claim 1, characterized in that, The method for inactivating the membrane protein YgfX is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] ygfx Gene knockout, or by deleting the gene encoding the membrane protein YgfX. ygfx Inactivation occurs at the 192nd guanine base; The gene encoding the membrane protein YgfX ygfx The nucleotide sequence is shown in SEQ ID NO.

2.

3. An engineered bacterium for producing human milk oligosaccharides, characterized in that, The engineered bacteria were obtained by inactivating the membrane protein YgfX on the human milk oligosaccharide producing strain as the starting strain. The starting strain used *Escherichia coli* K12 MG1655 as the host, with the lactose operon sequence knocked out. lacZ and overexpression lacY In addition, the strain also contains 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 和pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6 ; The membrane protein YgfX has the amino acid sequence shown in SEQ ID NO.1; the human milk oligosaccharides are: LNTII, LNnT, LNT, 3'-SL, 6'-SL.

4. The application of the engineered bacteria described in claim 3 in the production of human milk oligosaccharides.

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