Method for producing human milk oligosaccharide

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to mutants of murA and ygeA genes and application of the mutants in production of human milk oligosaccharides. According to the invention, murA and ygeA genes of escherichia coli are edited by using a gene editing technology, so that a genetic engineering chassis strain carrying murA and ygeA mutants is obtained. Further, the chassis strain is applied to the construction process of production strains of LNT II, LNnT, LNT, 3 '-SL and 6'-SL, and the yield of the LNT II, the LNnT, the LNT, the 3 '-SL and the 6'-SL is further increased.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and specifically relates to a murA and ygeA Mutants of the gene and their application in the production of human milk oligosaccharides. Background Art

[0002] Human milk oligosaccharides (HMOs) are an important class of oligosaccharides present in breast milk. HMOs can be used as prebiotics to maintain the balance of the intestinal microbial environment, have immune defense regulation, and stimulate the brain development and cognition of infants and young children. They can be added to infant formula as functional ingredients, and play an important role in the growth and development of infants and young children, and have broad market prospects. 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. More than 200 HMOs have been discovered so far, including lactoyl- N -trisaccharide (lacto- N -triose, LNT II), lactoyl- N -New tetrasaccharide (Lacto- N -neotetraose, LNnT), lactose- N -tetraose (Lacto-N-tetraose, LNT), 3'-sialyllactose (3'-sialyllactose, 3'-SL) and 6'-sialyllactose (6'-sialyllactose, 6'-SL), etc. At present, the production methods of HMOs include chemical synthesis, enzyme catalysis and microbial fermentation, among which microbial fermentation has the advantages of simple operation, environmentally friendly production method and low cost during the fermentation process, and is more suitable for large-scale industrial production.

[0003] Escherichia coli has become a typical model strain in microbial fermentation due to its advantages such as clear background, simple culture method and short growth cycle, relatively clear metabolic pathway and mature gene manipulation technology, and is used to synthesize a variety of HMOs. In Escherichia coli, glucose or glycerol is used as the carbon source and lactose is used as the substrate when synthesizing LNnT. Glucose-6-phosphate (Glc-6-P) is a kind of pgi The encoded glucose-6-phosphate isomerase is converted into fructose-6-phosphate (F6P), which is then glmS Encoded glutamine-fructose-6-phosphate aminotransferase, glmM Encoded by phosphoglucosamine mutase, glmUThe enzyme N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase encodes the catalytic conversion of Glc-6-P into uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). In the synthesis pathway of another precursor, uridine diphosphate-galactose (UDP-Gal), Glc-6-P is converted into pgm Encoded glucose 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. wbgO In the synthesis of sialylated human milk oligosaccharides, Escherichia coli uses the UDP-GlcNAc synthesis pathway as the basis, and in the presence of UDP- N -acetylglucosamine-2-epimerase NeuC, N -Cytidine monophosphate is synthesized by acetylneuraminic acid synthase NeuB and N-acetylneuraminic acid cytidylyltransferase NeuA 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'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-sialyllactose, 6'-SL), respectively.

[0004] At present, a lot of work has been done on the construction of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL synthesis pathways, and relevant production strains have been obtained. How to further improve the production level of strains has become an urgent problem to be solved. Summary of the invention

[0005] In order to solve the above technical problems, our research group found that UDP- N -acetylglucosamine 1-carboxyvinyltransferase (MurA, a gene essential for cell growth murA Encoded by , catalyzes the first step in the synthesis of the cell wall component peptidoglycan) and moderately weakened the amino acid racemase (YgeA, encoded by ygeAGene encoding, catalyzing the conversion of L-type and D-type amino acids, with broad substrate specificity) can be mutated to increase the production level of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL. The present invention uses gene editing technology to modify Escherichia coli murA and ygeA Gene editing has been carried out to obtain murA and ygeA The mutant gene engineering chassis strain is then applied to the construction of production strains of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL, further improving the yields of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL.

[0006] One of the technical solutions provided by the present invention is a MurA mutant, wherein the mutant is obtained by mutation of the first amino acid from Met to Val based on the wild-type MurA (MurA, Protein Id: AAC76221.1) shown in SEQ ID NO.3, and the amino acid sequence of the MurA mutant is shown in SEQ ID NO.1; Furthermore, the nucleotide sequence of the gene encoding the MurA mutant is shown in SEQ ID NO.2.

[0007] The second technical solution provided by the present invention is a YgeA mutant, which is obtained by mutation of the 198th amino acid from Thr to Ala on the basis of the wild-type YgeA (YgeA, Protein Id: AAC75879.1) shown in SEQ ID NO.7, and the amino acid sequence of the YgeA mutant is shown in SEQ ID NO.5; Furthermore, the nucleotide sequence of the gene encoding the YgeA mutant is shown in SEQ ID NO.6.

[0008] The third technical solution provided by the present invention is the application of the above-mentioned MurA mutant and YgeA mutant, especially in the production of human milk oligosaccharides, more particularly in the production of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL; Furthermore, using Escherichia coli as the starting strain, the lactose operon sequence of the starting strain was deleted. lacZ Afterwards, overexpression lacY , and further weakening the expression of the coding gene of MurA, and / or mutating the coding gene of YgeA, to obtain a base plate bacteria that produces human milk oligosaccharides, and further, the human milk oligosaccharides are LNT II, ​​LNnT, LNT, 3'-SL or 6'-SL; Furthermore, the method for weakening the expression of the MurA coding gene is to mutate it into the MurA mutant coding gene shown in SEQ ID NO.2; Furthermore, the method of mutating the gene encoding YgeA is to mutate it into the gene encoding the YgeA mutant shown in SEQ ID NO.6; Furthermore, the starting strain is Escherichia coli K12 MG1655; Furthermore, the β-1,3- N -Acetylglucosamine transferase encoding gene lgtA , a strain producing LNT II was obtained; Furthermore, the β-1,3- N -Acetylglucosamine transferase encoding gene lgtA and the gene encoding the lipooligosaccharide biosynthesis protein lex-1 lgtB , a strain producing LNnT was obtained; Furthermore, the β-1,3- N -Acetylglucosamine transferase encoding gene lgtA and β-1,3-galactosyltransferase encoding genes wbgO , a strain producing LNT was obtained; Furthermore, based on the above-mentioned chassis bacteria, further overexpression N -Acetylneuraminic acid synthase encoding neuB 、UDP- N -Acetylglucosamine 2-differentiase encoding gene neuC , N -Acylneuraminic acid cytidylyltransferase encoding gene neuA and sialyltransferase encoding genes ist , obtain a strain producing 3'-SL; preferably, express pTrc99a-P on the basis of the above-mentioned base bacteria J23119 - neuB - neuC -P trc - neuA - ist plasmid, and a strain producing 3'-SL was obtained; Furthermore, based on the above-mentioned chassis bacteria, further overexpression N -Acetylneuraminic acid synthase encoding neuB 、UDP- N -Acetylglucosamine 2-differentiase encoding gene neuC , N -Acylneuraminic acid cytidylyltransferase encoding gene neuA and sialyltransferase encoding genesST6 , obtain a strain producing 6'-SL; preferably, express pTrc99a-P on the basis of the above-mentioned base bacteria J23119 - neuB - neuC -P trc - neuA - ST6 Plasmid, a strain producing 6'-SL was obtained; Furthermore, the lgtA The nucleotide sequence of the gene is shown in SEQ ID NO.10; Furthermore, the lgtB The nucleotide sequence of the gene is shown in SEQ ID NO.11; Furthermore, the wbgO The nucleotide sequence of the gene is shown in SEQ ID NO.12; pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist Plasmids and pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 The specific construction process of the plasmid refers to patent CN117736280A.

[0009] Beneficial effects: The invention obtains MurA and YgeA mutants by gene editing technology, and applies the mutants to Escherichia coli for producing LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL, so that the yields of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Knockout lacZ Overexpression lacY The first step is PCR verification of recombinant colonies.

[0011] Figure 2 Knockout lacZ Overexpression lacY The second step is PCR verification of recombinant colonies.

[0012] Figure 3 for murA The first step is to verify homologous recombination by colony PCR.

[0013] Figure 4 for murA The second step is colony PCR verification of homologous recombination.

[0014] Figure 5 forygeA The first step is to verify homologous recombination by colony PCR.

[0015] Figure 6 for ygeA The second step is colony PCR verification of homologous recombination. DETAILED DESCRIPTION

[0016] The present invention is 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 suitable ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications of the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0017] 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, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0018] 2. Identification of Mutants The mutated amino acid in the mutant is represented by "original amino acid + position + replaced amino acid". For example, M1V in the MurA mutant indicates that the amino acid at position 1 is replaced by Val from Met in the wild-type MurA, and the position number corresponds to the amino acid sequence number of the wild-type MurA in SEQ ID NO.3. For example, T198A in the YgeA mutant indicates that the amino acid at position 198 is replaced by Ala from Thr in the wild-type YgeA, and the position number corresponds to the amino acid sequence number of the wild-type YgeA in SEQ ID NO.7.

[0019] The specific information is shown in the following table.

[0020]

[0021] The amino acid sequence of the MurA mutant involved in the present invention is shown in SEQ ID NO.1: VDKFRVQGPTKLQGEVTISGAKNAALPILFAALLAEEPVEIQNVPKLKDVDTSMKLLSQLGAKVERNGSVHIDARDVNVFCAPYDLVKTMRASIWALGPLVARFG QGQVSLPGGCTIGARPVDLHISGLEQLGATIKLEEGYVKASVDGRLKGAHIVMDKVSVGATVTIMCAATLAEGTTIIENAAREPEIVDTANFLITLGAKISGQGT DRIVIEGVERLGGGVYRVLPDRIETGTFLVAAAISRGKIICRNAQPDTLDAVLAKLRDAGADIEVGEDWISLDMHGKRPKAVNVRTAPHPAFPTDMQAQFTLLNL VAEGTGFITETVFENRFMHVPELSRMGAHAEIESNTVICHGVEKLSGAQVMATDLRASASLVLAGCIAEGTTVVDRIYHIDRGYERIEDKLRALGANIERVKGE.

[0022] The amino acid sequence of the YgeA mutant involved in the present invention is shown in SEQ ID NO.5: MKTIGLLGGMSWESTIPYYRLINEGIKQRLGGLHSAQVLLHSVDFHEIEECQRRGEWDKTGDILAEAALGLQRAGAEGIVLCTNTMHKVADAIESRCTLPFLHIADATGRAITGA GMTRVALLGTRYTMEQDFYRGRLTEQFSINCLIPEADERAKINQIIFEELCLGQFTEASRAYCAQVIARLAEQGAQGVIFGCAEIGLLVPEERSVLPVFDTAAIHAEDAVAFMLS.

[0023] The present invention will be further explained below through specific implementation modes.

[0024] Example 1 Construction of strain H0 Escherichia coli K12 MG1655 ( Escherichia coliK12 MG1655) was constructed as the starting strain, and 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) was used to knock out the lactose operon sequence of the starting strain. lacZ , in the original lacZ P trc Promoter overexpression lacY , strain H0 was obtained.

[0025] The specific construction method of strain H0 is as follows: The homologous recombination fragments were constructed by using the wild-type strain MG1655 preserved in the laboratory as a template and primers in Table 1. The upstream and downstream homology arms of homologous recombination were obtained by PCR amplification using primers lac-up-f / r and lac-down-f / r. The promoter P was obtained by using the pTrc99a plasmid (GeneBank: U13872) as a template and primers lac-trc-f / r as a template. trc Sequence fragment. Artificially synthesized chloramphenicol resistance gene cat , cat The promoter and N20 sequence vector were used as templates, and PCR amplification was performed using the primer pair lac-cat-f / r to obtain a cat - N20 sequence (SEQ ID NO.9). trc Promoter fragment with cat - The four fragments of N20 sequence were used as templates, and overlapping PCR was performed using primers lac-up-f and lac-down-r to obtain homologous recombinant fragments.

[0026] In the first step, homologous recombination was carried out by transforming the pCAGO plasmid into Escherichia coli K12 MG1655 using conventional plasmid transformation methods to obtain the strain Escherichia coli K12 MG1655 (pCAGO). The competent Escherichia coli K12 MG1655 (pCAGO) was prepared using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG. The homologous recombination fragment obtained in step 1 was introduced using the electroporation method. The transformed bacterial solution was spread on an LB plate containing 100 mg / L ampicillin and 25 mg / L chloramphenicol, as well as 1% glucose, and cultured at 30°C. The transformants were picked for colony PCR verification (verification primers: lac-yz-f / r). If the recombination was successful, the band size was about 2732 bp, and the verification results were 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.

[0027] In the second step, homologous recombination, the strain that has been successfully verified for the first recombination is 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 hours to induce the expression of the CRISPR / Cas9 system and λ-red protein to complete the second recombination. Three zones are drawn on the LB plate containing ampicillin to separate single colonies, and the separated single colonies are picked and spotted on the chloramphenicol-resistant LB plate and the ampicillin-resistant LB plate respectively. The single colonies that do not grow on the chloramphenicol medium but grow on the ampicillin medium are selected and verified by colony PCR (the verification primers are: lac-yz-f / r). If the recombination is correct, the band size is about 1797 bp, and the verification result is as follows Figure 2 As shown, 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 is lost, thereby obtaining a knockout strain. lacZ And in the original lacZ P trc Promoter overexpression lacY The strain was named H0.

[0028] Table 1 Knockout constructs lacZ Use P trc Overexpression lacY Primers used for strains

[0029] Example 2 Construction of strain H1 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 murA The first nucleotide of the gene, adenine, mutated to guanine (A mutated to G), and strain H1 was obtained. The specific construction method of strain H1 is as follows: The homologous recombination fragments were constructed using the wild-type strain MG1655 preserved in the laboratory as a template, and the primers in Table 2 were used to construct the homologous recombination fragments. The upstream and downstream homology arms of homologous recombination were obtained by PCR amplification using murA-up-f / r and murA-down-f / r as primers. cat , cat The promoter and N20 sequence vector were used as templates, and PCR amplification was performed using primer pair murA-cat-f / r to obtain a fragment with cat - A fragment of the N20 sequence (SEQ ID NO.9). The upper and downstream homology arms, with cat - The three fragments were used as templates to perform overlapping PCR using primers murA-up-f and murA-down-r to obtain homologous recombinant fragments containing murA The mutation site of the gene, i.e. the wild type murA The first nucleotide adenine in the nucleotide sequence is mutated to guanine.

[0030] In the first step of homologous recombination, the pCAGO plasmid was transformed into strain H0 using conventional plasmid transformation methods to obtain strain H0 (pCAGO). H0 (pCAGO) competent cells were prepared using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG, and homologous recombination fragments were introduced using electroporation. The transformed bacterial solution was spread on LB plates containing 100 mg / L ampicillin and 25 mg / L chloramphenicol, as well as 1% glucose, and cultured at 30°C. Transformants were picked for colony PCR verification (verification primers: murA-yz-f / r). If the recombination was successful, the band size was about 1800 bp, and the verification results were as follows: Figure 3 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.

[0031] In the second step, homologous recombination, the strain that has been successfully verified for the first recombination is 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 hours to induce the expression of the CRISPR / Cas9 system and λ-red protein to complete the second recombination. Three zones are drawn on the LB plate containing ampicillin to separate single colonies, and the separated single colonies are picked and spotted on the chloramphenicol-resistant LB plate and the ampicillin-resistant LB plate respectively. The single colonies that do not grow on the chloramphenicol medium but grow on the ampicillin medium are selected and verified by colony PCR (verification primers are: murA-yz-f / r). If the recombination is correct, the band size is about 900 bp, and the verification result is as follows: Figure 4 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 murA The strain with the mutation (SEQ ID NO.2) was named H1.

[0032] Table 2 Construction murA Primers used for gene mutation strains

[0033] Example 3 Construction of strains H2 and H3 Based on strains H0 and H1, 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 modify the ygeA The nucleotide adenine at position 592 of the gene was mutated to guanine (A to G), i.e. the threonine at position 198 was mutated to alanine, and strains H2 and H3 were obtained.

[0034] The specific construction methods of strains H2 and H3 are as follows: The wild-type strain MG1655 preserved in the laboratory was used as a template, and the primers in Table 3 were used to construct homologous recombination fragments. The upstream and downstream homology arms of homologous recombination were obtained by PCR amplification using ygeA-up-f / r and ygeA-down-f / r as primers. cat , cat The promoter and N20 sequence vector were used as templates, and PCR amplification was performed using the primer pair ygeA-catf / r to obtain acat - A fragment of the N20 sequence (SEQ ID NO.9). The upper and downstream homology arms, with cat - The three fragments were used as templates to perform overlapping PCR using primers ygeA-up-f and ygeA-down-r to obtain homologous recombinant fragments containing ygeA The mutation site of the gene, i.e. the wild type ygeA The 592nd nucleotide adenine in the nucleotide sequence was mutated to guanine.

[0035] The obtained recombinant fragments were transformed into H0 and H1 strains respectively by electroporation. The specific operation process of gene editing is referred to Example 1 of this application. Figure 5 and Figure 6 As shown, the PCR band size of the colony with successful primary recombination is about 2600 bp (verification primers: ygeA-yz-f / r), and the PCR band size of the colony with successful secondary recombination is about 1600 bp (verification primers: ygeA-yz-f / r). The homologous recombinant strain obtained in the second step was further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining strains H2 and H3.

[0036] Table 3 Construction ygeA Primers used for gene mutation strains

[0037] Example 4 Construction of plasmid pTrc99a-P trc - lgtA lgtA Genes encoding β-1,3- N -acetyl glucosamine transferase, the nucleotide sequence of which is shown in SEQ ID NO.10. Plasmid pTrc99a was used as a template to construct plasmid pTrc99a-P trc - lgtA The plasmid construction involved P trcThe promoter sequence is: gcgcaacgcaattaatgtgagttagcgcgaattgatctggtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgt cgctcaaggcgcactcccgttctggataatgtttttgcgccgacatcataacggttctggcaaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatctcacacaggaaacagacc.

[0038] Using plasmid pTrc99a as template and 99a-F / R in Table 4 as primers, PCR amplification was performed to obtain the linear vector pTrc99a-P trc ; artificially synthesized and codon-optimized lgtA The sequence was used as a template and lgtA-F / R in Table 4 was used as primers for PCR amplification to obtain a linear gene fragment. lgtA (shown in SEQ ID NO.10). The linear vector and linear gene fragment obtained by the above PCR were purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA .

[0039] Table 4 Construction of plasmid pTrc99a-P trc - lgtA Primers used

[0040] Example 5 Construction of plasmid pTrc99a-P trc - lgtB - lgtA lgtB The gene encodes the lipooligosaccharide biosynthesis protein lex-1, and its nucleotide sequence is shown in SEQ ID NO.11. trc - lgtA The plasmid pTrc99a-P was constructed based on trc -lgtB - lgtA . With plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using lgtA-F and 99a-R in Table 5 as primers to obtain the linear vector pTrc99a-P trc -lgtA; artificially synthesized and codon-optimized lgtB The sequence was used as a template and lgtB-F / R in Table 5 was used as primers for PCR amplification to obtain a linear gene fragment. lgtB The linear vector and linear gene fragment obtained by the above PCR were purified and recovered, connected using the ClonExpress Ⅱ recombination ligation kit, transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtB - lgtA .

[0041] Table 5 Construction of plasmid pTrc99a-P trc - lgtB - lgtA Primers used

[0042] Example 6 Construction of plasmid pTrc99a-P trc - wbgO - lgtA wbgO The gene encodes β-1,3-galactosyltransferase, and its nucleotide sequence is shown in SEQ ID NO.12. trc - lgtA The plasmid pTrc99a-P was constructed based on trc - wbgO - lgtA . With plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using lgtA-F and 99a-R in Table 6 as primers to obtain the linear vector pTrc99a- trc -lgtA; wbgO The sequence in Table 6 was used as a template. wbgO -F / R as primers for PCR amplification to obtain linear gene fragments wbgOThe linear vector and linear gene fragment obtained by the above PCR were purified and recovered, connected using the ClonExpress Ⅱ recombination ligation kit, transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - wbgO - lgtA .

[0043] Table 6 Construction of plasmid pTrc99a-P trc - wbgO - lgtA Primers used

[0044] Example 7 Construction and fermentation test of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production strains The plasmid 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 (Plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist and pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 The specific construction process refers to patent CN117736280A Example 2 and Example 3) are respectively introduced into H0, H1, H2 and H3 to construct: (1) LNT II production strain: G1[H0 (pTrc99a-P trc - lgtA )]; G2[H1 (pTrc99a-P trc -lgtA )]; G3[H2 (pTrc99a-P trc - lgtA )]; G4[H3 (pTrc99a-P trc - lgtA )]; (2) LNnT production strain: G5[H0(pTrc99a-P trc - lgtB - lgtA )]; G6[H1(pTrc99a-P trc - lgtB - lgtA )]; G7[H2(pTrc99a-P trc - lgtB - lgtA )]; G8[H3(pTrc99a-P trc - lgtB - lgtA )]; (3) LNT production strains: G9[H0(pTrc99a-P trc - wbgO - lgtA )]; G10[H1(pTrc99a-P trc - wbgO - lgtA )]; G11[H2(pTrc99a-P trc - wbgO - lgtA )]; G12[H3(pTrc99a-P trc - wbgO - lgtA )]; (4) 3'-SL producing strains: G13[H0(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist )]; G14[H1(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist )]; G15[H2(pTrc99a-PJ23119 - neuB - neuC -P trc - neuA - ist )]; G16[H3(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist )]; (5) 6'-SL producing strains: G17[H0(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 )]; G18[H1(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 )]; G19[H2(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 )]; G20[H3(pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 )].

[0045] The strains and plasmids used in this patent are shown in Table 7.

[0046] Table 7 Strains and plasmids used in this application

[0047] 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.

[0048] Fermentation medium (1 L): KH2PO4 3 g, yeast powder 8 g, (NH4)2SO4 4 g, citric acid 1.7 g, MgSO4·7H2O2 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.

[0049] Trace elements (1 L): FeCl3·6H2O 25 g, MnCl2·4H2O 9.8 g, CoCl2·6H2O 1.6 g, CuCl2·H2O 1 g, H3BO31.9 g, ZnCl22.6 g, Na2M O O4·2H2O 1.1 g, Na2SeO31.5 g, NiSO4·6H2O 1.5 g.

[0050] The fermentation test process is: Single colonies of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production strains were picked and cultured overnight in LB liquid medium containing 50 mg / L ampicillin at 37°C and 220 rpm. The overnight culture was used as seed liquid and inoculated into a 24-well plate containing 1 mL of fermentation medium at a 2% (v / v) inoculation volume. The fermentation medium contained 50 mg / L ampicillin and 0.1 mM IPTG at 37°C and 800 rpm. Three parallel cultures were cultured for each strain.

[0051] During the fermentation process, the growth of the bacteria (OD 600 ), LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production, the concentrations of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL in the samples were detected by high performance liquid chromatography, and the sample concentrations were quantified using the standard curves 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 refer to Example 4 of patent CN117736280A. The results are shown in Tables 8 to 12: It can be seen from Tables 8 to 12 that the expression murA and ygeA Gene mutants and combinatorial expression murA and ygeA After the mutants of the gene were modified, the yields of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL were all increased to a certain extent.

[0052] Table 8 Test results of LNT II produced by different strains

[0053] Table 9 Test results of LNnT produced by different strains

[0054] Table 10 Test results of LNT production by different strains

[0055] Table 11 Test results of 3'-SL production by different strains

[0056] Table 12 Test results of 6'-SL production by different strains

[0057] murA It is a key gene for cell wall synthesis. The weakening of its expression level is more likely to affect the cell wall synthesis level of the strain, thereby affecting growth and production. In fact, in the process of weakening MurA, we also used a variety of methods such as modifying the promoter and ribosome binding site, but none of them achieved the desired effect. It can be seen that by murA The method of mutating the start codon ATG of the gene to GTG can increase the production of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL while having little effect on the strain. Therefore, this method is currently a better way to weaken expression. ygeA The gene mutant (threonine at position 198 mutated to alanine) also increased the production of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL, and was similar to murA The weakening of gene expression has a synergistic effect. This work provides a new idea for the construction of strains that efficiently synthesize human milk oligosaccharides. In addition to E. coli, other commonly used industrial chassis, such as Corynebacterium glutamicum and yeast, also have enzymes with the same function. It can be predicted that after corresponding modification, it is also possible to increase the yield of the corresponding strains.

[0058] Although the present invention has been disclosed as 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 chassis strain for producing human milk oligosaccharides, characterized in that: The strain is based on Escherichia coli as the starting strain, and the lactose operon sequence of the starting strain is deleted. lacZ Afterwards, overexpression lacY , further weakening the expression of the MurA coding gene, and / or mutating the YgeA coding gene to obtain it.

2. The chassis strain for producing human milk oligosaccharides according to claim 1, characterized in that: The method for weakening the expression of the MurA coding gene is to mutate it into the MurA mutant coding gene shown in SEQ ID NO.2; The method for mutating the YgeA coding gene is to mutate it into the coding gene of the YgeA mutant shown in SEQ ID NO.

6.

3. The chassis strain for producing human milk oligosaccharides according to claim 1, characterized in that The strain is based on Escherichia coli K12 MG1655.

4. Use of the chassis strain described in claim 1 in the production of human milk oligosaccharides.

5. The use according to claim 4, characterized in that: The human milk oligosaccharide is lactoyl- N- trisaccharide, lactoyl- N - Neotetraose, lactose- N -tetrasaccharide, 3'-sialyllactose or 6'-sialyllactose.

6. The use according to claim 5, characterized in that: Based on the bottom plate bacteria of claim 1, β-1,3- N -Acetylglucosamine transferase encoding gene oeLh , obtain a strain producing LNT II; or, Based on the bottom plate bacteria of claim 1, β-1,3- N -Acetylglucosamine transferase encoding gene oeLh and the gene encoding the lipooligosaccharide biosynthesis protein lex-1 lgB , a strain producing LNnT was obtained; or, Based on the bottom plate bacteria of claim 1, β-1,3- N -Acetylglucosamine transferase encoding gene oeLh and β-1,3-galactosyltransferase encoding genes wxya , obtain a strain producing LNT; or, Overexpression based on the bottom plate bacteria of claim 1 N -Acetylneuraminic acid synthase encoding neB 、UDP- N -Acetylglucosamine 2-differentiase encoding gene neC , N -Acylneuraminic acid cytidylyltransferase encoding gene neA and sialyltransferase encoding genes ist , obtain a strain producing 3'-SL; or, Overexpression based on the bottom plate bacteria of claim 1 N -Acetylneuraminic acid synthase encoding neB 、UDP- N -Acetylglucosamine 2-differentiase encoding gene neC , N -Acylneuraminic acid cytidylyltransferase encoding gene neA and sialyltransferase encoding genes ST6 , and obtained a strain producing 6'--SL.

7. The use according to claim 6, characterized in that: Said oeLh The nucleotide sequence of the gene is shown in SEQ ID NO.10; Said lgB The nucleotide sequence of the gene is shown in SEQ ID NO.11; Said wxya The nucleotide sequence of the gene is shown in SEQ ID NO.

12.

8. A MurA mutant, characterized in that The amino acid sequence of the MurA mutant is shown in SEQ ID NO.

1.

9. A YgeA mutant, characterized in that The amino acid sequence of the YgeA mutant is shown in SEQ ID NO.

5.

10. Use of the MurA mutant according to claim 8 and / or the YgeA mutant according to claim 9 in producing human milk oligosaccharides.

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