Genetic engineering strain for high yield of sialyllactose and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a genetic engineering strain for high-yield production of sialyllactose and application of the genetic engineering strain. On the basis of escherichia coli expressing sialyllactose, the genetic engineering strain is obtained through any one or more of the following gene editing modes: i) improving the expression level of OmpC, OmpA and / or OmpF; ii) increasing the expression level of MdfA, MdtM and / or MdtL; and iii) the expression level of tolA and / or tolB is improved while the expression level of tolR is reduced. On the basis of escherichia coli expressing sialic acid lactose, multiple gene editing modes for improving the expression level of sialic acid lactose are obtained through screening, particularly, the effect of the gene editing modes of tolA, tolB and tolR is optimal, and the sialic acid lactose production efficiency of escherichia coli can be remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a genetic engineering strain with high yield of 3'-sialyllactose and application thereof. Background Art

[0002] At present, the methods for synthesizing sialyllactose (SL) are mainly chemical synthesis and biosynthesis. The chemical synthesis method is mainly limited to laboratory-scale research due to its cumbersome process. In contrast, the biosynthesis method has opened up a new path for industrial production with its short production cycle and diversity of substrate pathways. The biosynthesis method mainly includes enzyme catalysis and fermentation. In the fermentation method, it is necessary to construct the biological metabolic pathway of SL in the strain. These pathways are mainly divided into three, namely, the neuC pathway, the AGE pathway and the nanE pathway. At present, the production of SL mainly depends on the optimization and transformation of the neuC pathway. However, during the fermentation process, as the fermentation time increases, the concentration of 3'-SL inside and outside the cell is significantly different. The present invention speculates that this may be due to the large amount of intracellular residues, which causes the cells to rupture in the late fermentation period, thereby limiting the synthesis efficiency of SL and affecting the overall fermentation effect. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the present invention provides a genetically engineered strain with high yield of 3'-sialyllactose and its application. This application claims priority to Chinese patent application No. 202411696307X filed on November 25, 2024.

[0004] In order to increase the production of sialyllactose (SL), the present invention screened genes related to the outer membrane structure of Escherichia coli (OmpC, OmpA, OmpF, MdtM, MdtL, MdtG, tolR, tolA, tolB). The overexpression of each gene was regulated by the PT7 promoter, and they were integrated into the Escherichia coli genome. <h2 style=";text-align:left;direction:ltr">intQ and <h2 style=";text-align:left;direction:ltr"> ydeU Finally, it was found that the expression level of SL was increased, especially by knocking out the combination <h2 style=";text-align:left;direction:ltr"> tolR , while overexpressing <h2 style=";text-align:left;direction:ltr"> tolA , <h2 style=";text-align:left;direction:ltr"> tolB This strategy not only increases the yield of SL, but also provides a new technical path for the application of microbial fermentation in industrial production.

[0005] In a first aspect, the present invention provides a method for improving the efficiency of Escherichia coli in producing sialyllactose, comprising: performing one or more of the following gene editing improvements on Escherichia coli: i) increasing the expression level of OmpC, OmpA and / or OmpF; ii) increasing the expression level of MdfA, MdtM and / or MdtL; iii) In reducing <h2 style=";text-align:left;direction:ltr"> tolR While increasing the expression level of <h2 style=";text-align:left;direction:ltr"> tolA and / or <h2 style=";text-align:left;direction:ltr"> tolB expression level.

[0006] Furthermore, the gene editing method is: <h2 style=";text-align:left;direction:ltr"> tolR While increasing the expression level of <h2 style=";text-align:left;direction:ltr"> tolA and <h2 style=";text-align:left;direction:ltr"> tolB expression level.

[0007] Furthermore, the sialyllactose is 3'-sialyllactose or 6'-sialyllactose.

[0008] Furthermore, the improvement of gene expression level described in the present invention can be carried out by commonly used methods in the art, such as exogenously expressing corresponding genes from other sources, or endogenously expressing corresponding genes of the strain itself.

[0009] Furthermore, the reduction of gene expression level in the present invention can be carried out by a common method in the art, such as inactivating or deleting a gene by gene knockout. Inactivation can be the total or partial loss of gene function, and deletion can be the deletion of part or all of the nucleotide sequence of a gene.

[0010] In a second aspect, the present invention provides a genetically engineered strain with high sialyllactose production, wherein the genetically engineered strain is obtained by any one or more of the following improvements based on Escherichia coli expressing sialyllactose: i) increasing the expression level of OmpC, OmpA and / or OmpF; ii) increasing the expression level of MdfA, MdtM and / or MdtL; iii) In reducing <h2 style=";text-align:left;direction:ltr"> tolR While increasing the expression level of <h2 style=";text-align:left;direction:ltr"> tolA and / or <h2 style=";text-align:left;direction:ltr"> tolB expression level.

[0011] Furthermore, the gene editing method is: <h2 style=";text-align:left;direction:ltr"> tolR While increasing the expression level of <h2 style=";text-align:left;direction:ltr"> tolA and <h2 style=";text-align:left;direction:ltr"> tolB expression level.

[0012] Furthermore, the Escherichia coli expressing sialyllactose is obtained on the basis of Escherichia coli without the ability to produce sialyllactose by the following method: i) Improve <h2 style=";text-align:left;direction:ltr"> neuB , <h2 style=";text-align:left;direction:ltr"> neuC and <h2 style=";text-align:left;direction:ltr"> neuA The expression level of <h2 style=";text-align:left;direction:ltr"> nST The expression level of ii) Reduce <h2 style=";text-align:left;direction:ltr"> lacZ The expression level of <h2 style=";text-align:left;direction:ltr"> neuA The gene encodes CMP-N-acetylneuraminic acid synthetase, <h2 style=";text-align:left;direction:ltr"> neuB The gene encodes acetylneuraminic acid synthase, <h2 style=";text-align:left;direction:ltr"> neuC The gene encodes N-acetylglucosamine isomerase. CMP-N-acetylneuraminic acid synthetase is responsible for activating N-acetylneuraminic acid (Neu5Ac) and converting it into CMP-Neu5Ac; acetylneuraminic acid synthetase can catalyze the conversion of N-acetylmannosamine (GlcNAc) into N-acetylneuraminic acid (Neu5Ac), and participates in the sialylation process of glycoproteins and glycolipids on the cell surface; N-acetylglucosamine isomerase can convert N-acetylglucosamine (GlcNAc) into N-acetylmannosamine (ManNAc); the above three enzymes all play an important role in the biosynthesis pathway of sialylated human milk oligosaccharides. <h2 style=";text-align:left;direction:ltr"> nST Sialyltransferases are distinguished by the glycosidic bonds they form, such as sialyltransferases that form α-2,3-, α-2,6- and α-2,8-bonds. <h2 style=";text-align:left;direction:ltr"> neuB , <h2 style=";text-align:left;direction:ltr"> neuC and <h2 style=";text-align:left;direction:ltr"> neuA Endogenous overexpression or exogenous overexpression can be selected, as above <h2 style=";text-align:left;direction:ltr"> nST Exogenous overexpression.

[0013] The sialyllactose is 3'-sialyllactose or 6'-sialyllactose.

[0014] In a specific embodiment of the present invention, the method of the present invention is constructed and 3'-sialyltransferase is introduced to construct Escherichia coli that produces 3'-sialyllactose (3'-SL). Those skilled in the art will understand that by changing the type of sialyltransferase, Escherichia coli that produces 6'-sialyllactose (6'-SL) can be constructed.

[0015] Furthermore, the following methods are also included: reduce <h2 style=";text-align:left;direction:ltr"> nanA , <h2 style=";text-align:left;direction:ltr"> nanK , <h2 style=";text-align:left;direction:ltr"> nanE , <h2 style=";text-align:left;direction:ltr"> nanT , <h2 style=";text-align:left;direction:ltr"> wcaJ , <h2 style=";text-align:left;direction:ltr"> fucI or <h2 style=";text-align:left;direction:ltr"> fucK The expression level of one or more of the

[0016] <h2 style=";text-align:left;direction:ltr"> wcaJ The gene encodes UDP-glucose lipid carrier transferase, and inactivation or deletion can block the metabolic synthesis branch of colanic acid, thereby increasing the accumulation of the precursor substance UDP-galactose; <h2 style=";text-align:left;direction:ltr"> lacZ The gene encodes β-galactosidase, which, when inactivated or missing, prevents the degradation of lactose into glucose and galactose; <h2 style=";text-align:left;direction:ltr"> fucI Genes and <h2 style=";text-align:left;direction:ltr"> fucKThe genes encode L-fucose isomerase and L-fucokinase, respectively, and their inactivation or deletion can prevent L-fucose from entering the glycolytic pathway; <h2 style=";text-align:left;direction:ltr"> nanA The gene encodes N-acetylmannosamine lyase, and its inactivation or deletion can prevent the degradation of N-acetylneuraminic acid and N-acetylmannosamine; <h2 style=";text-align:left;direction:ltr"> nanK The gene encodes N-acetylmannosamine kinase, and its inactivation or deletion prevents the degradation of N-acetylmannosamine-6-phosphate; <h2 style=";text-align:left;direction:ltr"> nanE The gene encodes N-acetylmannosamine-6-phosphate-2-epimerase, and its inactivation or deletion prevents the degradation of glucosamine-6-phosphate; <h2 style=";text-align:left;direction:ltr"> nanT The gene encodes sialic acid transporter, and inactivation or deletion of the gene prevents the intracellular and extracellular transport of sialic acid.

[0017] Furthermore, the Escherichia coli is Escherichia coli BL21, Escherichia coli BL21 (DE3) or Escherichia coli BL21star (DE3). <h2 style=";text-align:left;direction:ltr"> lacZ Introduction of Campylobacter jejuni into gene-deficient Escherichia coli BL21star(DE3) <h2 style=";text-align:left;direction:ltr"> Campylobacter jejuni of <h2 style=";text-align:left;direction:ltr"> neuB , <h2 style=";text-align:left;direction:ltr"> neuC , <h2 style=";text-align:left;direction:ltr"> neuA genes (e.g., AF400048), and Neisseria meningitidis <h2 style=";text-align:left;direction:ltr"> Neisseria gonorrhoeae (U60664) <h2 style=";text-align:left;direction:ltr"> nST genes, all of which are in P T7 The gene is expressed under the control of the promoter and integrated into the Escherichia coli genome in a single copy. <h2 style=";text-align:left;direction:ltr"> caiB Site and <h2 style=";text-align:left;direction:ltr"> hlyE The neuC pathway was constructed by knocking out the mitochondria involved in Neu5Ac catabolism. <h2 style=";text-align:left;direction:ltr"> nanA , <h2 style=";text-align:left;direction:ltr"> nanK , <h2 style=";text-align:left;direction:ltr"> nanE and <h2 style=";text-align:left;direction:ltr"> nanT Gene, thereby obtaining a basic Escherichia coli strain expressing sialyllactose.

[0018] In a third aspect, the present invention provides a microbial agent comprising the aforementioned genetically engineered strain.

[0019] In a fourth aspect, the present invention provides the use of the aforementioned genetically engineered strain or the aforementioned microbial agent in the production of sialyllactose.

[0020] The present invention has the following beneficial effects: The present invention screened and obtained a variety of gene editing methods for improving the expression level of sialyllactose based on Escherichia coli expressing sialyllactose, among which the method is particularly effective. <h2 style=";text-align:left;direction:ltr"> tolA , <h2 style=";text-align:left;direction:ltr"> tolB and <h2 style=";text-align:left;direction:ltr"> tolRThe gene editing method is the most effective and can significantly improve the efficiency of Escherichia coli in producing sialyllactose, especially 3'-sialyllactose.

[0021] The gene editing method provided by the present invention has important value in the field of microbial synthesis of sialyllactose. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0024] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0025] Example 1 1. Construction of Escherichia coli expressing 3'-sialyllactose 1. The method of constructing Escherichia coli expressing 3'-sialyllactose can be achieved by using existing technology, which mainly improves the <h2 style=";text-align:left;direction:ltr"> neuB , <h2 style=";text-align:left;direction:ltr"> neuC and <h2 style=";text-align:left;direction:ltr"> neuA The expression level of <h2 style=";text-align:left;direction:ltr"> nST The expression level of the expression level is as follows, and the method used in this embodiment is as follows: (1) The lacZ gene of Escherichia coli BL21star (DE3) was knocked out using the CRISPR / Cas9 technology, including the following steps: The NCBI GenBank: CP001509.3 <h2 style=";text-align:left;direction:ltr"> lacZ The gene sequence is the target gene sequence, which is retrieved online at http: / / chopchop.cbu.uib.no / . The gRNA of the gene is designed, and the sgRNA is connected to the plasmid pEcgRNA. The plasmid is transformed into DH5α and cultured overnight. The positive clones are selected for culture, and the plasmid is extracted using the Tiangen Biochemical Extraction Kit to obtain the plasmid pEcgRNA::lacZ-N20.

[0026] Homologous fragment preparation: Designed according to the GenBank: CP001509.3 genome information published by NCBI database <h2 style=";text-align:left;direction:ltr"> lacZ Primers required for gene knockout, selected<h2 style=";text-align:left;direction:ltr"> Escherichia coli BL21star (DE3) genomic DNA was used as a template, and the upstream homology arm primers lacZ-up-F / lacZ-up-R and the downstream homology arm primers lacZ-down-F / lacZ-down-R were used to amplify the homology arm sequences lacZ-1 and lacZ-2, respectively. After the product was purified and recovered, the overlapping PCR technology was used to amplify and connect the fragments lacZ-1 and lacZ-2 with primers lacZ-up-F / lacZ-down-R to obtain the gene homology repair arm. <h2 style=";text-align:left;direction:ltr"> Escherichia coli BL21star(DE3), select positive clones for electroporation preparation. Co-electroporate the constructed plasmid pEcgRNA and fusion fragment <h2 style=";text-align:left;direction:ltr"> Escherichia coli BL21star(DE3) / pEcCas, coated at 50 μg·mL -1 Kanamycin and 50 μg mL -1 The culture was carried out on a spectinomycin double-resistance plate at 37 °C overnight, and the positive recombinants were identified by PCR using lacZ-up-F and lacZ-down-R. The knockout strains were selected and plated at 50 μg mL -1 Kanamycin, 10mmol·L -1 The cells were cultured in rhamnose LB liquid medium for 6 h to induce elimination of plasmid pEcgRNA, subcultured at 37 °C for 2 h, and streaked at 10 g·L -1 Sucrose solid plate, eliminate plasmid pEcCas, and obtain strain BS0. <h2 style=";text-align:left;direction:ltr"> nanAKET The gene manipulation steps are the same as above <h2 style=";text-align:left;direction:ltr"> lacZ Knockout, resulting in strain BS1.

[0027] (2) Design primer pairs neuBCA-F / neuBCA-R and nST-F / nST-R to clone <h2 style=";text-align:left;direction:ltr"> Campylobacter <h2 style=";text-align:left;direction:ltr"> Jejuni of <h2 style=";text-align:left;direction:ltr"> neuB , <h2 style=";text-align:left;direction:ltr"> neuC , <h2 style=";text-align:left;direction:ltr"> neuA Genes and from <h2 style=";text-align:left;direction:ltr"> Neisseria gonorrhoeae (U60664) <h2 style=";text-align:left;direction:ltr"> nST The gene was integrated into the pRSFDuet-1 plasmid, and the constructed plasmids were pRSFDuet-neuBCA and pRSFDuet-nST.

[0028] (3) Design primer pairs ompC-F / ompC-R, ompA-F / ompA-R, ompF-F / ompF-R, mdfA-F / mdfA--R, mdtM-F / mdtM-R, mdtL-F / mdtL-R, tolA-F / tolA-R, tolB-F / tolB-R to clone the transporter gene from Escherichia coli, and ligate the cloned fragments to the first T7 promoter of plasmid pRSFDuet-1 through homologous recombination technology to construct plasmids pRSFDuet-ompC, pRSFDuet-ompA, pRSFDuet-ompF, pRSFDuet-MdfA, pRSFDuet-MdtM, pRSFDuet-MdtL, pRSFDuet-tolA, and pRSFDuet-tolB.

[0029] As mentioned above, the plasmid <h2 style=";text-align:left;direction:ltr"> ompC , <h2 style=";text-align:left;direction:ltr"> ompA , <h2 style=";text-align:left;direction:ltr"> ompF , <h2 style=";text-align:left;direction:ltr"> mdfA , <h2 style=";text-align:left;direction:ltr"> mdtM , <h2 style=";text-align:left;direction:ltr"> mdtL , <h2 style=";text-align:left;direction:ltr"> mdtG , <h2 style=";text-align:left;direction:ltr"> tolA , <h2 style=";text-align:left;direction:ltr"> tolB It is derived from Escherichia coli BL21star (DE3), the genome sequence of which has been completely sequenced and annotated.

[0030] (4) Design primer pairs neuBCA-T7F / neuBCA-T7R, nST-T7F / nST-T7R, ompC-T7F / ompC-T7R, ompA-T7F / ompA-T7R, ompF-T7F / ompT7F-R, mdfA-T7F / mdfA-T7R, mdtM-T7F / mdtM-T7R, mdtL-T7F / mdtL-T7R, tolA-T7F / tolA-T7R, tolB-T7F / tolB-T7R to clone the T7 promoter of the above plasmids. The gene fragments regulated by the expression of the promoter were cloned, and the cloned fragments were connected to the plasmid pSPIN by homologous recombination technology to obtain pSPIN-caiB-neuBCA, pSPIN-hlyE-nST, pSPIN-intQ-ompC, pSPIN-intQ-ompA, pSPIN-intQ-ompF, pSPIN-intQ-mdfA, pSPIN-intQ-mdtM, ​​pSPIN-intQ-mdtL, pSPIN-intQ-tolA, and pSPIN-ydeU-tolB.

[0031] (5) The steps for inserting the target gene into the genome of the recipient strain using CRISPR / Cas9 technology are as follows: The recipient strain was prepared into competent cells by the CaCl2 method, and the pSPIN-caiB-neuBCA plasmid was transferred into 100 μL BS1 competent cells; after transformation, the bacterial solution was evenly spread on LB plates containing 25 μg / mL kanamycin and cultured at 30°C for 30 h; a single colony was picked in LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C on a shaking table until the logarithmic phase, and PCR amplification was performed using post-transposition DF and post-transposition DR as primers, and the PCR product was detected by gel electrophoresis; the bacterial solution identified as positive in the previous step was inoculated in an anti-resistant LB liquid medium at a 1% inoculation amount, and after culturing at 37°C for 12 h, the culture was inoculated in a fresh anti-resistant LB liquid medium at a 1% inoculation amount, and cultured at 37°C for 12 h. The obtained culture was streaked on an anti-resistant LB plate and cultured at 37°C for 10-16 h. Pick a single colony and inoculate half of it on a kana plate, and the other half inoculate it in an antibiotic-free LB liquid medium and culture it at 37°C for 10-16 h; pick the bacteria that have eliminated the pSPIN plasmid and send them to Universal Biology for sequencing (caiB-DF, caiB-DR), and get BS2, which is superimposed on <h2 style=";text-align:left;direction:ltr"> hlyE Site transposition <h2 style=";text-align:left;direction:ltr"> nST The gene manipulation steps are the same as above <h2 style=";text-align:left;direction:ltr"> neuBCA , and obtained strain BS3.

[0032] 2. Gene editing was performed on the basis of strain BS3. The same method as above was used to edit the gene on the basis of strain BS3. <h2 style=";text-align:left;direction:ltr"> intQ Site transposition <h2 style=";text-align:left;direction:ltr"> ompC , <h2 style=";text-align:left;direction:ltr"> ompA , <h2 style=";text-align:left;direction:ltr"> ompF , <h2 style=";text-align:left;direction:ltr"> mdfA , <h2 style=";text-align:left;direction:ltr"> mdtM , <h2 style=";text-align:left;direction:ltr"> mdtL The BS4-BS9 strains were obtained respectively.

[0033] 3. Gene editing was performed on the basis of strain BS3. The same method as above was used to edit the gene on the basis of strain BS3. <h2 style=";text-align:left;direction:ltr"> intQ Site transposition <h2 style=";text-align:left;direction:ltr"> tolA , <h2 style=";text-align:left;direction:ltr"> ydeU Site transposition <h2 style=";text-align:left;direction:ltr"> tolB BS10 and BS11 strains were obtained, and strain BS10 was used as the basis for <h2 style=";text-align:left;direction:ltr"> ydeU Site transposition <h2 style=";text-align:left;direction:ltr"> tolB The BS12 strain was obtained.

[0034] 4. Gene editing was performed on the basis of strain BS3. The same method as above was used to knock out the <h2 style=";text-align:left;direction:ltr"> tolR The BS13 strain was obtained, based on the BS12 strain, with the <h2 style=";text-align:left;direction:ltr"> tolR The BS14 strain was obtained.

[0035] 2. Fermentation verification of strain BS3-14 1. The composition of 250mL shake flask culture medium is as follows.

[0036] Table 1 Shake flask medium composition

[0037] 2. Before inoculation, add the following raw materials to every 100 mL of culture medium.

[0038] Table 2 Culture medium components

[0039] The trace elements include 25g / L FeCl3·6H2O, 2g / L CaCl2·2H2O, 2.0g / L ZnCl2, 1.9g / LCuSO4·5H2O, 0.42 g / L MnCl2·H2O, 2g / L Na2B4O7·10H2O and 2g / L Na2MoO4·2H2O.

[0040] The bacterial solution was inoculated at a 1% inoculation rate and cultured at 37°C and 230 rpm for 5 h. IPTG was added to a final concentration of 0.2 mM to initiate induction. The induction culture was carried out at 28°C and 230 rpm for 48 h.

[0041] 3. Technical Effect Table 3 Comparison of strain yields

[0042] From the above results, compared with the control strain BS3, the overexpression <h2 style=";text-align:left;direction:ltr"> ompC , <h2 style=";text-align:left;direction:ltr"> ompA , <h2 style=";text-align:left;direction:ltr"> ompF The 3'-SL production increased to varying degrees after gene expression.

[0043] Table 4 Comparison of strain yields

[0044] The results showed that compared with the control strain BS3, the overexpression <h2 style=";text-align:left;direction:ltr"> mdfA , <h2 style=";text-align:left;direction:ltr"> mdtM , <h2 style=";text-align:left;direction:ltr"> mdtL , <h2 style=";text-align:left;direction:ltr"> mdtG After gene <h2 style=";text-align:left;direction:ltr"> mdfA The yield of the other 3'-SLs decreased, while the yields of the other 3'-SLs increased to varying degrees.

[0045] Table 5 Comparison of strain yields

[0046] The results showed that compared with the control strain BS3, the knockout <h2 style=";text-align:left;direction:ltr"> tolRGene overexpression <h2 style=";text-align:left;direction:ltr"> tolA , <h2 style=";text-align:left;direction:ltr"> tolB After that, the 3'-SL production was significantly improved, which was significantly higher than the production of other strains and the production of engineered strains obtained by the aforementioned other gene editing methods.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the efficiency of Escherichia coli in producing sialyllactose, characterized in that: include: The Escherichia coli is improved in one or more of the following ways: i) increasing the expression level of OmpC, OmpA and / or OmpF; ii) increasing the expression level of MdfA, MdtM and / or MdtL; iii) while reducing the expression level of tolR, increasing the expression level of tolA and / or tolB.

2. The method according to claim 1, characterized in that The method is: while reducing the expression level of tolR, increasing the expression levels of tolA and tolB.

3. The method according to claim 1, characterized in that The sialyllactose is 3'-sialyllactose or 6'-sialyllactose.

4. A genetically engineered strain with high sialyllactose production, characterized in that: The genetically engineered strain is obtained by any one or more of the following methods based on Escherichia coli expressing sialyllactose: i) increasing the expression level of OmpC, OmpA and / or OmpF; ii) increasing the expression level of MdfA, MdtM and / or MdtL; iii) while reducing the expression level of tolR, increasing the expression level of tolA and / or tolB.

5. The genetically engineered strain according to claim 4, characterized in that The method is: while reducing the expression level of tolR, increasing the expression levels of tolA and tolB.

6. The genetically engineered strain according to claim 4, characterized in that The Escherichia coli expressing sialyllactose is obtained on the basis of Escherichia coli by the following method: i) increasing the expression levels of neuB, neuC and neuA, and increasing the expression level of nST; ii) reduce the expression level of lacZ; The sialyllactose is 3'-sialyllactose or 6'-sialyllactose.

7. The genetically engineered strain according to claim 6, characterized in that It also includes the following methods: The expression level of one or more of nanA, nanK, nanE, nanT, wcaJ, fucI or fucK is reduced.

8. The genetically engineered strain according to claim 6, characterized in that: The Escherichia coli is Escherichia coli BL21, Escherichia coli BL21 (DE3) or Escherichia coli BL21 star (DE3).

9. A microbial agent, characterized in that: The invention comprises the genetically engineered strain according to any one of claims 4 to 8.

10. Use of the genetically engineered strain according to any one of claims 4 to 8 or the microbial agent according to claim 9 in the production of sialyllactose.

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