Method for regulating and controlling production of human milk oligosaccharide through membrane protein

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for regulating and controlling production of human milk oligosaccharide through membrane protein. The method is realized by inactivating a membrane protein YgfX coding gene on a production strain. According to the human milk oligosaccharide production strain obtained in the mode that the membrane protein YgfX is inactivated after the coding gene of the membrane protein YgfX is knocked out or deleted from a 192nd guanine base through a gene editing technology, the human milk oligosaccharide production capacity of the human milk oligosaccharide production strain is obviously improved compared with that before gene editing, the yields of LNTII, LNnT, LNT, 3 '-SL and 6'-SL are improved by about 40%, 125%, 46%, 32% and 47% respectively, the effect is remarkable, and the human milk oligosaccharide production strain has a good application prospect. And a new method and thought are provided for widening the production way of the human milk oligosaccharide.

Description

Technical Field

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

[0002] Human Milk Oligosaccharides (HMOs), as a key type of oligosaccharides in breast milk, possess various important biological functions. They can act as prebiotics and play a role in regulating the ecological balance of the gut microbial community. At the same time, HMOs also have an immune defense regulatory function and play a positive promoting role in the brain development and cognitive development of infants and young children. Given the significant importance of HMOs to the growth and development of infants and young children, they can be added as functional ingredients to infant formula milk powder, showing broad market application prospects. According to different structures, there are more than 200 types of HMOs, and 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), etc. Currently, the production methods of HMOs mainly include chemical synthesis method, enzyme catalysis method, and microbial fermentation method, etc.

[0003] Escherichia coli has become a typical model strain in the field of microbial fermentation due to its clear genetic background, simple cultivation method, short growth cycle, relatively clear metabolic pathway, and mature gene manipulation technology, and is widely used in the biosynthesis of various HMOs. For example: in the process of synthesizing LNTII, LNnT, or LNT using Escherichia coli, usually glucose or glycerol is used as the carbon source, and lactose is used as the substrate. Glucose-6 -phosphate (Glc-6-P) undergoes an isomerization reaction under the catalysis of glucose-6-phosphate isomerase encoded by pgi the gene and is transformed into fructose-6-phosphate (F6P). F6P is gradually transformed under the catalysis of a series of enzymes. First, it is acted on by glmS glutamine-fructose-6-phosphate aminotransferase encoded by the gene, and then under the action of glmM phosphoglucosamine mutase encoded by the gene and glmU the gene encodes NUnder the catalysis of UDP-N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is finally generated. In another synthetic pathway of the precursor uridine diphosphate-galactose (UDP-Gal), Glc-6-P is pgm converted to UDP-Gal under the catalysis of phosphoglucomutase encoded by the galE gene and UDP-glucose-4-epimerase encoded by the lgtA gene. Subsequently, under the catalysis of β-1,3- N -N-acetylglucosamine aminotransferase encoded by the lgtB gene, lactose and UDP-GlcNAc generate the intermediate LNT II. LNT II combines with UDP-Gal under the catalysis of β-1,4-galactosyltransferase encoded by the wbgO gene to form LNnT; LNT II can also generate LNT under the catalysis of β-1,3-galactosyltransferase encoded by the

[0004] gene. When using Escherichia coli to synthesize sialylated human milk oligosaccharides, based on its own UDP-GlcNAc synthetic pathway, Escherichia coli synthesizes cytidine monophosphate-N-acetylneuraminic acid CMP-Neu5Ac under the synergistic catalysis of UDP-N-acetylglucosamine-2-epimerase NeuC, N-acetylneuraminic acid synthase NeuB, and N-acetylneuraminic acid cytidylyltransferase NeuA. CMP-Neu5Ac undergoes glycosylation reactions 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 that is mainly involved in the process of cytoskeleton formation in the strain. Currently, the function of YgfX is still not clearly understood. Some studies have shown that overexpression of YgfX causes the cell shape to change to lemon-shaped, while other studies have shown that overexpression of YgfX has no effect on cell growth (Masuda H, et al. Anovel membrane-bound toxin for cell division, CptA (YgfX), inhibitspolymerization 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] When producing human milk oligosaccharides at a high industrial level, various metabolic intermediates and end products accumulate in the fermentation environment. The strain has poor tolerance to this fermentation environment, and the cell morphology changes to some extent in the later stage of fermentation, and the growth and production capacity of the strain are limited. In this study, YgfX was regulated in the human milk oligosaccharide-producing strain, hoping to further improve the production performance of the human milk oligosaccharide-producing strain. Summary of the Invention

[0006] To solve the above technical problems, the present invention uses gene editing technology to edit the genes of Escherichia coli ygfx Through gene editing, LNTII, LNnT, LNT, 3'-SL, and 6'-SL production strains with increased yields were obtained.

[0007] One of the technical solutions provided by the present invention is a method for improving the production of human milk oligosaccharides, which is achieved by inactivating the membrane protein YgfX on the production strain; The membrane protein YgfX has an amino acid sequence as shown in SEQ ID NO.1; The coding gene of the membrane protein YgfX ygfx has a nucleotide sequence as shown in SEQ ID NO.2; The human milk oligosaccharides include, but are not limited to, LNTII, LNnT, LNT, 3'-SL, 6'-SL; The methods for inactivating the membrane protein YgfX include, but are not limited to: gene knockout, insertion or deletion of bases, etc.; Furthermore, by deleting the gene encoding the membrane protein YgfX ygfx the 192nd guanine base is inactivated.

[0008] The second technical solution provided by the present invention is an engineered bacterium for producing human milk oligosaccharides, which is obtained by using a human milk oligosaccharide-producing strain as the starting strain and inactivating the membrane protein YgfX on the production strain; The methods for inactivating the membrane protein YgfX include, but are not limited to: gene knockout, insertion or deletion of bases, etc.; Furthermore, the engineered bacterium is obtained by using a human milk oligosaccharide-producing strain as the starting strain and knocking out the gene encoding the membrane protein YgfX on the production strain ygfx ; Furthermore, the engineered bacterium is obtained by using a human milk oligosaccharide-producing strain as the starting strain and deleting the 192nd guanine base on the gene encoding the membrane protein YgfX ygfx ; Furthermore, the starting bacterium uses Escherichia coli K12 MG1655 as the host, knocks out the lacZ in the lactose operon sequence of the host, and overexpresses 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: lactyl- N -trisaccharide (Lacto-N-Triose, LNT II), lactyl- N -neotetraose (Lacto-N-Neotetraose, LNnT), lactose- N -tetrasaccharide (Lacto-N-tetraose, LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL), and 6'-sialyllactose (6'-Sialyllactose, 6'-SL); Even further, 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-PJ23119 - neuB - neuC -P trc - neuA - ist and pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 。

[0009] The third technical solution provided by the present invention is the application of the engineering bacteria described in the second technical solution in the production of human milk oligosaccharides; Further, the human milk oligosaccharides include, but are not limited to, LNTII, LNnT, LNT, 3'-SL, and 6'-SL.

[0010] The fourth technical solution provided by the present invention is a membrane protein YgfX mutant, and the coding gene of the mutant is obtained by deleting the 192nd guanine base on the basis of the coding gene of the wild-type membrane protein YgfX; ygfx of the wild-type membrane protein YgfX coding gene. The fifth technical solution provided by the present invention is the application of the membrane protein YgfX mutant described in the fourth technical solution, especially in the production of human milk oligosaccharides.

[0011] Beneficial effects: The present invention provides a method for improving the production capacity of human milk oligosaccharides, and the method is achieved by inactivating the coding gene of the membrane protein YgfX on the production strain. The human milk oligosaccharide production strain obtained by the present invention through gene editing technology to knock out or delete the 192nd guanine base of the coding gene of the membrane protein YgfX and then inactivate the membrane protein YgfX has significantly improved its human milk oligosaccharide production capacity compared with that before gene editing. Among them, the yields of LNTII, LNnT, LNT, 3'-SL, and 6'-SL are increased by about 40%, 125%, 46%, 32%, and 47% respectively, and the effect is remarkable, providing a new method and idea for broadening the production pathway of human milk oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 For ygfX PCR verification of the first-step homologous recombination colony of the mutant Figure 2 For ygfX PCR verification of the second-step homologous recombination colony of the mutant.

[0013] Figure 3 For knocking out ygfX PCR verification of the first-step homologous recombination colony.

[0014] Figure 4 For knocking out ygfX PCR verification of the second-step homologous recombination colony. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be further described below through specific implementation embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following implementation embodiments can be well-known to those skilled in the art, and suitable ones can be selected from the known means and materials that can solve the corresponding technical problems. In addition, the implementation embodiments should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation embodiments also fall within the protection scope of the present invention without departing from the essence and scope of the present invention.

[0016] The mutant of the membrane protein YgfX involved in the present invention is obtained by deleting the 192nd guanine base of the wild-type membrane protein coding gene ( ygfX , GeneID: 947379). Among them: The wild-type membrane protein YgfX has the amino acid sequence shown in SEQ ID NO.1: MVLWQSDLRVSWRAQWLSLLIHGLVAAVILLMPWPLSYTPLWMVLLSLVVFDCVRSQRRINARQGEIRLLMDGRLRWQGQEWSIVKAPWMIKSGMMLRLRSDGGKRQHLWLAADSMDEAEWRDLRRILLQQETQR* The wild-type membrane protein YgfX coding gene ygfX , has the nucleotide sequence shown in SEQ ID NO.2: GTGGTCCTGTGGCAATCTGATTTGCGCGTCTCCTGGCGCGCACAGTGGCTTTCCTTGCTGATTCATGGGCTGGTTGCCGCTGTTATTTTACTCATGCCCTGGCCACTCAGTTACACCCCGTTATGGATGGTGTTACTTTCGCTGGTGGTGTTTGATTGCGTTCGCAGCCAGCGGCGTATTAATGCTCGCCAGGGGGAAATTCGCTTGTTGATGGACGGGCGTTTGCGTTGGCAAGGGCAGGAGTGGAGCATCGTCAAAGCACCGTGGATGATTAAGAGCGGCATGATGCTGCGTTTACGTTCTGATGGCGGTAAACGGCAACATTTATGGCTGGCAGCCGACAGCATGGACGAAGCTGAATGGCGGGATTTACGGCGGATTTTGTTGCAACAAGAGACGCAAAGATAA The membrane protein mutant has a deletion of the guanine base at position 192 of the coding gene, resulting in subsequent codon rearrangement and ultimately three stop codons (marked with "*") in the amino acid sequence. The amino acid sequence of the membrane protein mutant is as follows: MVLWQSDLRVSWRAQWLSLLIHGLVAAVILLMPWPLSYTPLWMVLLSLVVFDCVRSQRRINARQGKFAC*WTGVCVGKGRSGASSKHRG*LRAA*CCVYVLMAVNGNIYGWQPTAWTKLNGGIYGGFCCNKRRKD (SEQ ID NO.3) The coding gene of the membrane protein mutant has the following nucleotide sequence: GTGGTCCTGTGGCAATCTGATTTGCGCGTCTCCTGGCGCGCACAGTGGCTTTCCTTGCTGATTCATGGGCTGGTTGCCGCTGTTATTTTACTCATGCCCTGGCCACTCAGTTACACCCCGTTATGGATGGTGTTACTTTCGCTGGTGGTGTTTGATTGCGTTCGCAGCCAGCGGCGTATTAATGCTCGCCAGGGGAAATTCGCTTGTTGATGGACGGGCGTTTGCGTTGGCAAGGGCAGGAGTGGAGCATCGTCAAAGCACCGTGGATGATTAAGAGCGGCATGATGCTGCGTTTACGTTCTGATGGCGGTAAACGGCAACATTTATGGCTGGCAGCCGACAGCATGGACGAAGCTGAATGGCGGGATTTACGGCGGATTTTGTTGCAACAAGAGACGCAAAGATAA (SEQ ID NO.4) The present invention will be further explained and illustrated by specific embodiments below.

[0017] Example 1 Construction of Strain Z1 It was constructed using Escherichia coli K12 MG1655( Escherichia coli K12 MG1655) as the starting strain. Using the 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), the lactose operon sequence in the starting strain was knocked out lacZ , and after the original lacZ site, trc was overexpressed under the P lacY promoter, named strain Z0. The specific construction process of this strain can be found in Example 1 of Patent CN 119464168A.

[0018] The strain carrying the YgfX mutant (with the 192nd guanine base deleted based on the wild-type coding gene) is Z1, and its specific construction method is as follows: 1. Construction of homologous recombination fragment Using the MG1655 wild-type strain preserved in the laboratory as a template, homologous recombination fragments were constructed using the primers in Table 1 respectively. Using ygfX-up-f / r and ygfX-down-f / r as primers, the upstream and downstream homologous arms of homologous recombination were amplified by PCR. Using the synthetic vector containing the chloramphenicol resistance gene cat , cat promoter and N20 sequence as a template, PCR amplification was carried out using the primer pair ygfX-cat-f / r to obtain a fragment with cat - N20 sequence (SEQ ID NO.5). Using the upstream and downstream homologous arms and the fragment with cat - N20 sequence as the templates, overlapping PCR was carried out using the primers ygfX-up-f and ygfX-down-r to obtain a homologous recombination fragment, which contained ygfX the mutation site of the gene (deletion of the 192nd guanine base).

[0019] 2. First-step homologous recombination The pCAGO plasmid was transformed into strain Z0 using the conventional plasmid transformation method to obtain strain Z0(pCAGO). The competent cells of Z0 (pCAGO) were prepared using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG, and the homologous recombination fragment obtained in step 1 was introduced by electroporation. The transformed bacterial solution was spread on an LB plate containing 100 mg / L ampicillin, 25 mg / L chloramphenicol, and 1% glucose, and cultured at 30 °C. The transformants were picked for colony PCR verification (the verification primers were: ygfX-yz-f / r). If the homologous recombination was successful, the band size was about 1342 bp. The verification result was as Figure 1 shown. The band was correct, indicating that the first-step homologous recombination was successful. The correct transformants were picked for the second-step homologous recombination.

[0020] 3. Second-step homologous recombination The strain with successful first-step recombination verification was inoculated into an 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 λ-red protein to complete the second recombination. The single colonies were separated by streaking in three zones on an LB plate containing ampicillin. The isolated single colonies were respectively spotted on a chloramphenicol-resistant LB plate and an ampicillin-resistant LB plate, and the single colonies that did not grow on the chloramphenicol medium and grew on the ampicillin medium were selected, and verified by colony PCR (the verification primers were: ygfX-yz-f / r). If the recombination was correct, the band size was about 407 bp. The verification result was as Figure 2As shown, the strip was correct, and the PCR product of the strip 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 therein, thereby obtaining a strain with ygfX a mutation, named Z1.

[0021] Table 1 Primers used to construct strain Z1

[0022] The cat - N20 sequence is shown in SEQ ID NO.5: ATTAATTAATCTCGAGTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGTCCATCGAACCGAAGTAAGG Example 2: Construction of Strain Z2 Based on strain Z0, the gene encoding YgfX was knocked out to obtain strain Z2. The specific construction method is as follows: 1. Construction of homologous recombination fragment Using the MG1655 wild-type strain preserved in the laboratory as a template, homologous recombination fragments were constructed using the primers in Table 2 respectively. Using ygfX-up-1-f / r and ygfX-down-1-f / r as primers, the upstream and downstream homologous arms of homologous recombination were amplified by PCR. Using the artificially synthesized vector containing the chloramphenicol resistance gene cat 、 cat promoter and N20 sequence as a template, PCR amplification was carried out using the primer pair ygfX-cat-1-f / r as primers to obtain a fragment with cat - N20 sequence. Using the upstream and downstream homologous arms and the fragment with cat - N20 sequence (SEQ ID NO.5), these three fragments were used as templates, and overlapping PCR was carried out using primers ygfX-up-1-f and ygfX-down-1-r to obtain the homologous recombination fragment.

[0023] 2. First-step homologous recombination The pCAGO plasmid was transformed into strain Z0 using the conventional plasmid transformation method to obtain strain Z0(pCAGO). The competent cells of Z0 (pCAGO) were prepared using LB medium containing 1% (m / v) glucose and IPTG at a concentration of 0.1 mM, and the homologous recombination fragment obtained in step 1 was introduced by the electrotransformation method. The transformed bacterial solution was spread on an LB plate containing 100 mg / L ampicillin, 25 mg / L chloramphenicol, and 1% glucose, and cultured at 30 °C. The transformants were picked for colony PCR verification (the verification primers were: ygfX-yz-1-f / r). If the homologous recombination was successful, the band size was about 2630 bp. The verification result was as Figure 3 shown. The band was correct, that is, the first-step homologous recombination was successful. The correct transformants were picked for the second-step homologous recombination.

[0024] 3. Second-step homologous recombination The strain with successful first-step recombination verification was inoculated into an 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 λ-red protein to complete the second recombination. The single colonies were separated by streaking in three zones on an LB plate containing ampicillin. The isolated single colonies were spotted on a chloramphenicol-resistant LB plate and an ampicillin-resistant LB plate respectively, and the single colonies that did not grow on the chloramphenicol medium and grew on the ampicillin medium were selected, and verified by colony PCR (the verification primers were: ygfX-yz-1-f / r). If the recombination was correct, the band size was about 1695 bp. The verification result was as Figure 4As shown, the strip was correct, and the PCR product of this strip 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 therein, thereby obtaining the knockout ygfX strain, named Z2.

[0025] Table 2 Primers used to construct strain Z2

[0026] Example 3 Construction of plasmid pTrc99a-P trc -lgtA lgtA The gene encodes β-1,3- N -N-acetylglucosaminyltransferase. The plasmid pTrc99a-P trc - lgtA was constructed using plasmid pTrc99a as a template. The specific construction process of the plasmid refers to Example 4 of Patent CN119464168A.

[0027] Example 4 Construction of plasmid pTrc99a-P trc -lgtB-lgtA lgtB The gene encodes lipooligosaccharide biosynthesis protein. On the basis of plasmid pTrc99a-P trc - lgtA the plasmid pTrc99a-P trc - lgtB - lgtA was constructed. The specific construction process of this plasmid refers to Example 5 of Patent CN119464168A.

[0028] Example 5 Construction of plasmid pTrc99a-P trc - wbgO-lgtA wbgO The gene encodes β-1,3-galactosyltransferase. On the basis of plasmid pTrc99a-P trc - lgtA the plasmid pTrc99a-P trc - wbgO - lgtA was constructed. The specific construction process of this plasmid refers to Example 6 of Patent CN119464168A.

[0029] Example 6 Construction of plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist Construct plasmid pTrc99a-P using plasmid pTrc99a as a template J23119 - neuB - neuC -P trc - neuA - ist , and the specific construction process of this plasmid refers to Example 2 of Patent CN117736280A.

[0030] Example 7 Construct plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 Using plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist as a template to construct plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 . The specific construction process of this plasmid refers to Example 3 of Patent CN117736280A.

[0031] Example 8 Construction and Fermentation Testing of Production Strains for LNT II, LNnT, LNT, 3'-SL, and 6'-SL Using the method of electroporation, 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 respectively introduced into strains Z0, Z1, and Z2 to construct: (1) Production strain for LNT II: B1[Z0 (pTrc99a-P trc - lgtA )]; B2[Z1 (pTrc99a-P trc - lgtA )]; B3[Z2 (pTrc99a-P trc - lgtA )]; (2) LNnT-producing strain: B4[Z0(pTrc99a-P trc - lgtB - lgtA )]; B5[Z1(pTrc99a-P trc - lgtB - lgtA )]; B6[Z2(pTrc99a-P trc - lgtB - lgtA )]; (3) LNT-producing strain: B7[Z0(pTrc99a-P trc - wbgO - lgtA )]; B8[Z1(pTrc99a-P trc - wbgO - lgtA )]; B9[Z2(pTrc99a-P trc - wbgO - lgtA )]; (4) 3'-SL-producing strain: B10[Z0(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist )]; B11[Z1(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist )]; B12[Z2(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist )]; (5) 6'-SL-producing strain: B13 [Z0(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 )]; B14[Z1(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 )]; B15[Z2(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 )]; Table 3 Strains and plasmids used

[0032] The production levels of the above strains were fermentatively tested separately. The media used were as follows: LB medium (1 L): 10 g of NaCl, 5 g of yeast extract, 10 g of peptone.

[0033] Fermentation medium (1 L): 3 g of KH2PO4, 8 g of yeast extract, 4 g of (NH4)2SO4, 1.7 g of citric acid, 2 g of MgSO4·7H2O, 10 mg of thiamine, 60 g of MOPS, 10 g of glycerol, 5 g of lactose, 1 mL of trace elements, adjusted to pH 7.0 with ammonia water.

[0034] Trace elements (1 L): 25 g of FeCl3·6H2O, 9.8 g of MnCl2·4H2O, 1.6 g of CoCl2·6H2O, 1 g of CuCl2·H2O, 1.9 g of H3BO3, 2.6 g of ZnCl2, 1.1 g of Na2M O O4·2H2O, 1.5 g of Na2SeO3, 1.5 g of NiSO4·6H2O.

[0035] The fermentation test process was as follows: Single colonies of LNT II, LNnT, LNT, 3'-SL, and 6'-SL producing strains were separately picked and cultured overnight at 37°C and 220 rpm / min in LB liquid medium containing 50 mg / L ampicillin. The overnight cultured bacterial solution was used as the seed solution, and the bacterial solution was transferred to a 24-well plate containing 1 mL of fermentation medium at an inoculation amount of 2% (v / v). The fermentation medium contained 50 mg / L ampicillin and 0.1 mmol / L IPTG, and fermentation was carried out at 37°C and 800 rpm. Each strain was cultured in parallel for 3 replicates. During the fermentation process, the growth of the bacteria (OD 600), Yield of LNT II, LNnT, LNT, 3'-SL and 6'-SL: The concentrations of LNT II, LNnT, LNT, 3'-SL and 6'-SL in the samples were detected by high performance liquid chromatography (HPLC), and the sample concentrations were quantified using the standard curves of LNT II, LNnT, LNT, 3'-SL and 6'-SL. The HPLC detection conditions for LNT II, LNnT, LNT, 3'-SL and 6'-SL refer to Example 4 of Patent CN117736280A. The results are shown in Tables 4 to 8 as follows: Table 4 Test Results of LNT II Production by Different Strains

[0036] Table 5 Test Results of LNnT Production by Different Strains

[0037] Table 6 Test Results of LNT Production by Different Strains

[0038] Table 7 Test Results of 3'-SL Production by Different Strains

[0039] Table 8 Test Results of 6'-SL Production by Different Strains

[0040] As can be seen from the above results, ygfx Both gene mutation and knockout have significantly improved the ability of the strains to ferment human milk oligosaccharides, ultimately resulting in a substantial increase in the yield of human milk oligosaccharides. It is speculated that the YgfX mutant and YgfX knockout may affect the cell morphology, promote the changes in the internal metabolic space and the distribution of substances in the cells, and thus increase the yield of human milk oligosaccharides. This work provides a new idea for the construction of strains for the efficient synthesis of human milk oligosaccharides. The present invention only describes the guanine base deletion mutant at position 192 of YgfX and YgfX knockout as examples, and other ways of inactivating YgfX caused by base insertion, deletion, etc. are also within the scope of protection.

[0041] Although the present invention has been disclosed above with 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 the form and details of these embodiments without departing from the spirit and principle of the present invention. The scope of the present invention 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 production strain; the membrane protein YgfX has an amino acid sequence as shown in SEQ ID NO.

1.

2. The method for improving the production capacity of human milk oligosaccharides according to claim 1, characterized in that, The human milk oligosaccharides include: LNTII, LNnT, LNT, 3'-SL, 6'-SL.

3. A method for improving the production capacity of human milk oligosaccharides according to claim 1, characterized in that, The methods for inactivating the membrane protein YgfX include, but are not limited to: the coding gene of the membrane protein YgfX ygfx is subjected to gene knockout, and the coding gene ygfx is subjected to insertion or deletion of bases, or point mutation.

4. The method for improving the production capacity of human milk oligosaccharides according to claim 3, characterized in that, Inactivating by deleting the membrane protein YgfX encoding gene ygfx inactivating the 192nd guanine base on it; the encoding gene of the membrane protein YgfX ygfx , and the nucleotide sequence is as shown in SEQ ID NO.

2.

5. An engineered bacterium for producing human milk oligosaccharides, characterized in that, The engineered bacterium is obtained by using a human milk oligosaccharide production strain as the starting strain and inactivating the membrane protein YgfX on the production strain.

6. The engineered bacterium for producing human milk oligosaccharide according to claim 5, characterized in that, The starting bacterium uses Escherichia coli K12 MG1655 as the host, and knocks out the lacZ in the lactose operon sequence of the host, and overexpresses lacY ; on this basis, the strain also contains a human milk oligosaccharide production pathway.

7. The engineered bacterium for producing human milk oligosaccharides according to claim 6, wherein, 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 和pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 。 8. Use of the engineered bacterium according to any one of claims 5 - 7 in the production of human milk oligosaccharides.

9. A mutant of membrane protein YgfX, characterized in that, The coding gene of the mutant is obtained by deleting the 192nd guanine base on the basis of the coding gene of the wild-type membrane protein YgfX shown in SEQ ID NO.2 ygfx ​ 10. Use of the membrane protein YgfX mutant according to claim 9, characterized in that, It is the use in the production of human milk oligosaccharides.

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