A genetically engineered strain for high-yield sialyllactose and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a genetically engineered strain for producing sialyllactose in high yield and application thereof. The genetically engineered strain is obtained by any one or more of the following gene editing methods on the basis of an Escherichia coli strain for 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) increasing the expression level of tolA and / or tolB while decreasing the expression level of tolR. The present application screens a plurality of gene editing methods for increasing the expression level of sialyllactose on the basis of an Escherichia coli strain for expressing sialyllactose, and the gene editing methods of tolA, tolB and tolR are the most optimal, which can significantly improve the efficiency of the Escherichia coli strain for producing sialyllactose.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a genetically engineered strain that produces high levels of 3'-sialic acid lactose and its applications. Background Technology

[0002] Currently, the main methods for synthesizing sialyl lactose (SL) are chemical synthesis and biosynthesis. Chemical synthesis, due to its complex process, is primarily limited to laboratory-scale research. In contrast, biosynthesis, with its shorter production cycle and diverse substrate pathways, opens up new avenues for industrial production. Biosynthesis mainly includes enzymatic catalysis and fermentation. In fermentation, it is necessary to construct the biometabolic pathways of SL in the bacterial strain. These pathways are mainly divided into three types: the neuC pathway, the AGE pathway, and the nanE pathway. Currently, SL production mainly relies on the optimization and modification of the neuC pathway. However, during fermentation, the concentration difference between intracellular and extracellular 3'-SL becomes significant with prolonged fermentation time. This invention hypothesizes that this may be due to a higher intracellular residue, leading to cell rupture in the later stages of fermentation, thus limiting the synthesis efficiency of SL and affecting the overall fermentation effect. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a genetically engineered strain that produces high levels of 3'-sialic acid lactose and its applications. This application claims priority to Chinese Patent Application No. 202411696307X, filed on November 25, 2024.

[0004] To increase the production of sialyl lactose (SL), this invention screened for genes related to the outer membrane structure of *E. coli* (OmpC, OmpA, OmpF, MdtM, MdtL, MdtG, tolR, tolA, and tolB). These genes were overexpressed using the PT7 promoter and then integrated into the *E. coli* genome. intQ and ydeU Sites. Ultimately, increased SL expression levels were discovered, particularly with combined knockout. tolR At the same time, overexpression tolA , tolB The optimal results were achieved. This strategy not only increased the yield of SL, but also provided a new technological 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 sialic acid lactose production by Escherichia coli, comprising: performing one or more improvements on Escherichia coli according to the following gene editing methods:

[0006] i) Increase the expression levels of OmpC, OmpA and / or OmpF;

[0007] ii) Increase the expression levels of MdfA, MdtM and / or MdtL;

[0008] iii) In reducing tolR While improving the level of expression, tolA and / or tolB The level of expression.

[0009] Furthermore, the gene editing method is as follows: in reducing tolR While improving the level of expression, tolA and tolB The level of expression.

[0010] Furthermore, the sialyl lactose is 3'-sialyl lactose or 6'-sialyl lactose.

[0011] Furthermore, the improvement of gene expression level described in this invention can be carried out using methods commonly used in the art, such as exogenous expression of corresponding genes from other sources, or endogenous expression of corresponding genes from the strain itself.

[0012] Furthermore, the reduction of gene expression levels described in this invention can be achieved using methods commonly used in the art, such as gene knockout to inactivate or delete the gene. Inactivation can be the complete or partial loss of gene function, while deletion can be the deletion of part or all of the nucleotide sequence of the gene.

[0013] Secondly, the present invention provides a genetically engineered strain that produces high levels of sialyl lactose, wherein the genetically engineered strain is obtained by any one or more of the following modifications based on Escherichia coli expressing sialyl lactose:

[0014] i) Increase the expression levels of OmpC, OmpA and / or OmpF;

[0015] ii) Increase the expression levels of MdfA, MdtM and / or MdtL;

[0016] iii) In reducing tolR While improving the level of expression, tolA and / or tolB The level of expression.

[0017] Furthermore, the gene editing method is as follows: in reducing tolR While improving the level of expression, tolA and tolB The level of expression.

[0018] Furthermore, the *E. coli* expressing sialyl lactose is obtained from *E. coli* that does not have the ability to produce sialyl lactose through the following method:

[0019] i) Improve neuB, neuC and neuA Improve the level of expression nST The level of expression;

[0020] ii) Reduce lacZ The level of expression;

[0021] neuA The gene encodes CMP-N-acetylneuraminic acid synthase. neuB The gene encodes acetylneuraminic acid synthase. neuC The gene encodes N-acetylglucosamine isomerase. CMP-N-acetylneuraminic acid synthase is responsible for activating N-acetylneuraminic acid (Neu5Ac) and converting it into CMP-Neu5Ac; acetylneuraminic acid synthase can catalyze the conversion of N-acetylmnosamine (GlcNAc) to N-acetylneuraminic acid (Neu5Ac), participating in the sialylation process of glycoproteins and glycolipids on the cell surface; N-acetylglucosamine isomerase can convert N-acetylglucosamine (GlcNAc) to N-acetylmannosamine (ManNAc); all three enzymes play important roles in the biosynthetic pathway of sialylated human lactose oligosaccharides. 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. Preferably, as described above. neuB , neuC and neuA You can choose between endogenous overexpression or exogenous overexpression, as shown above. nST This is due to exogenous overexpression.

[0022] The sialyl lactose is 3'-sialyl lactose or 6'-sialyl lactose.

[0023] In one specific embodiment of the present invention, by constructing the method described herein, 3'-sialyl transferase was introduced to construct Escherichia coli that produces 3'-sialyl lactose (3'-SL). Those skilled in the art will understand that by changing the type of sialyl transferase, Escherichia coli that produces 6'-sialyl lactose (6'-SL) can be constructed.

[0024] Furthermore, it also includes the following methods:

[0025] reduce nanA , nanK , nanE , nanT , wcaJ , fucI or fucK One or more levels of expression.

[0026] wcaJThe gene encodes UDP-glucose lipotransferase. Inactivation or deletion of this enzyme can prevent the metabolic synthesis pathway of capsular heteropolysaccharide acid, thereby increasing the accumulation of the precursor UDP-galactose. lacZ The gene encodes β-galactosidase, and its inactivation or deletion can prevent lactose from being degraded into glucose and galactose. fucI Genes and fucK The genes encode L-fucoisomerase and L-fucokinase, respectively. Inactivation or deletion of these genes can prevent L-fucose from entering the glycolysis pathway. nanA The gene encodes N-acetylmnosamine lyase, and its inactivation or deletion can prevent the degradation of N-acetylneuraminic acid and N-acetylmnosamine. nanK The gene encodes N-acetylmannosamine kinase, and its inactivation or deletion can prevent the degradation of N-acetylmannosamine-6-phosphate. nanE The gene encodes N-acetylmnosamine-6-phosphate-2-epimerase, which can prevent the degradation of glucosamine-6-phosphate after inactivation or deletion. nanT The gene encodes sialic acid transporter, and its inactivation or deletion can prevent the intracellular and extracellular transport of sialic acid.

[0027] Further, the *E. coli* is *E. coli* BL21, *E. coli* BL21(DE3), or *E. coli* BL21star(DE3). Existing technologies have constructed *E. coli* capable of expressing sialic acid lactose; in a specific embodiment of the present invention, this is achieved through… lacZ Campylobacter jejuni introduced into gene-deleted Escherichia coli BL21star(DE3) Campylobacter jejuni of neuB , neuC , neuA Genes (e.g., AF400048), and Neisseria meningitidis. Neisseria gonorhoeae (U60664) nST Genes, all of which are in P T7 Expressed under the regulation of the promoter, and integrated into the E. coli genome as a single copy. caiB site and hlyE The neuC pathway was constructed by identifying the site; simultaneously, the pathway involved in Neu5Ac catabolism was further knocked out. nanA , nanK , nanE and nanT Genes were used to obtain the basic E. coli strain that expresses sialic acid lactose.

[0028] Thirdly, the present invention provides a microbial inoculant, comprising the aforementioned genetically engineered strains.

[0029] Fourthly, the present invention provides the application of the aforementioned genetically engineered strains or the aforementioned microbial agents in the production of sialic acid lactose.

[0030] The present invention has the following beneficial effects:

[0031] This invention screened various gene editing methods to enhance sialyl lactose expression levels in *E. coli* expressing sialyl lactose, among which... tolA , tolB and tolR The gene editing method is the most effective and can significantly improve the efficiency of E. coli in producing sialic acid lactose, especially 3'-sialic acid lactose.

[0032] The gene editing method provided by this invention has important value in the field of microbial synthesis of sialic acid lactose. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0035] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0036] Example 1

[0037] I. Construction of Escherichia coli expressing 3'-sialic acid lactose

[0038] 1. The construction of Escherichia coli expressing 3'-sialic acid lactose can be achieved using existing technologies, mainly by improving upon existing Escherichia coli. neuB , neuC and neuA Improve the level of expression nST The level of expression is sufficient, and the method used in this embodiment is as follows:

[0039] (1) Knockout of the lacZ gene in Escherichia coli BL21star(DE3) using CRISPR / Cas9 technology, including the following steps:

[0040] Based on the data indexed in GenBank:CP001509.3 on NCBI lacZThe target gene sequence was obtained by searching online at http: / / chopchop.cbu.uib.no / and designing the gRNA. The sgRNA was ligated to the plasmid pEcgRNA, and the plasmid was transformed into DH5α and cultured overnight. Positive clones were selected and cultured, and the plasmid was extracted using the Tiangen Biotech Mini-Prep Kit to obtain the plasmid pEcgRNA::lacZ-N20.

[0041] Homologous fragment preparation: Designed based on genome information from GenBank (CP001509.3) published in the NCBI database. lacZ Primers required for gene knockout should be selected. Escherichia coli Using BL21star(DE3) genomic DNA as a template, homologous arm sequences lacZ-1 and lacZ-2 were amplified using upstream homologous arm primers lacZ-up-F / lacZ-up-R and downstream homologous arm primers lacZ-down-F / lacZ-down-R, respectively. After purification and recovery of the products, fragments lacZ-1 and lacZ-2 were amplified and ligated using overlap PCR with primers lacZ-up-F / lacZ-down-R to obtain the gene homologous repair arm. The pEcCas plasmid was then transformed... Escherichia coli BL21star(DE3) was used to select positive clones for electroporation competence preparation. The constructed plasmid pEcgRNA and the fusion fragment were co-electroporated. Escherichia coli BL21star(DE3) / pEcCas, coated on 50 μg·mL -1 Kanamycin and 50 μg·mL -1 Spectinomycin-containing double antibody plates were incubated overnight at 37 °C. Positive recombinants were identified by PCR using lacZ-up-F and lacZ-down-R. Successfully knocked-out strains were then cultured in 50 μg / mL solutions. -1 Kanamycin, 10 mmol·L -1 In rhamnose LB broth, cultured for 6 h to induce elimination of plasmid pEcgRNA, subcultured at 37 ℃ for 2 h, and streaked onto 10 g·L⁻¹ medium. -1 Sucrose solid plates were used to eliminate plasmid pEcCas, resulting in strain BS0. (This is followed by a seemingly unrelated sentence about stacked knockouts.) nanAKET The gene manipulation steps are the same as described above. lacZ Knockout yields strain BS1.

[0042] (2) Design primer pairs neuBCA-F / neuBCA-R and nST-F / nST-R clones from Campylobacter jejuni of neuB , neuC , neuAGenes and from Neisseria gonorhoeae (U60664) nST The gene was integrated into the pRSFDuet-1 plasmid, and the constructed plasmids were pRSFDuet-neuBCA and pRSFDuet-nST.

[0043] (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 transport protein genes from Escherichia coli, and use homologous recombination technology to link the cloned fragments to the first T7 promoter of plasmid pRSFDuet-1 to construct plasmids pRSFDuet-ompC, pRSFDuet-ompA, pRSFDuet-ompF, pRSFDuet-MdfA, pRSFDuet-MdtM, pRSFDuet-MdtL, pRSFDuet-tolA, and pRSFDuet-tolB.

[0044] As mentioned above, the plasmids involved... ompC , ompA , ompF , mdfA , mdtM , mdtL , mdtG , tolA , tolB Derived from Escherichia coli BL21star(DE3), the genome sequence of this strain has been fully sequenced and annotated.

[0045] (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 above plasmids with T7 promoters. Gene fragments that regulate the expression of genes were cloned and ligated into the pSPIN plasmid using 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.

[0046] (5) The steps for inserting the target gene into the genome of the recipient strain using CRISPR / Cas9 technology are as follows:

[0047] The recipient strain was prepared into competent cells using the CaCl2 method. The pSPIN-caiB-neuBCA plasmid was transformed into 100 μL of BS1 competent cells. After transformation, the bacterial culture was evenly spread onto LB agar plates containing 25 μg / mL kanamycin and incubated at 30°C for 30 h. Single colonies were picked and cultured in LB liquid medium containing 50 μg / mL kanamycin at 37°C in a shaker until the logarithmic growth phase. PCR amplification was performed using transposable DF and transposable DR as primers, and the PCR products were detected by gel electrophoresis. The positive bacterial culture identified in the previous step was inoculated at a 1% inoculum into antibiotic-free LB liquid medium and incubated at 37°C for 12 h. The culture was then inoculated at a 1% inoculum into fresh antibiotic-free LB liquid medium and incubated at 37°C for 12 h. The resulting culture was streaked onto antibiotic-free LB agar plates and incubated at 37°C for 10–16 h. Half of the single colonies were inoculated onto kanamycin plates, and the other half were inoculated into antibiotic-free LB broth. Incubation was performed at 37°C for 10–16 h. Cells with pSPIN plasmid eliminated were sent to Universal Biotech for sequencing (caiB-DF, caiB-DR) to obtain BS2, which was then superimposed on… hlyE Site transposition nST The gene manipulation steps are the same as described above. neuBCA strain BS3 was obtained.

[0048] 2. Gene editing was performed on strain BS3 using the same method as described above, based on strain BS3. intQ Site transposition ompC , ompA , ompF , mdfA , mdtM , mdtL BS4-BS9 strains were obtained respectively.

[0049] 3. Gene editing was performed on strain BS3 using the same method as described above. intQ Site transposition tolA , ydeU Site transposition tolB BS10 and BS11 strains were obtained, and strain BS10 was used as a base in... ydeU Site transposition tolB BS12 strain was obtained.

[0050] 4. Gene editing was performed on strain BS3 using the same method as described above, based on strain BS3, to knock out... tolR BS13 strain was obtained, and based on strain BS12, it was knocked out. tolR BS14 strain was obtained.

[0051] II. Fermentation verification of strain BS3-14

[0052] 1. The composition of the culture medium in a 250mL shake flask is shown in the table below.

[0053] Table 1. Composition of shake flask culture medium

[0054]

[0055] 2. The following raw materials need to be added to every 100 mL of culture medium before inoculation.

[0056] Table 2 Culture medium components

[0057]

[0058] The trace elements include 25 g / L FeCl3·6H2O, 2 g / L CaCl2·2H2O, 2.0 g / L ZnCl2, 1.9 g / L CuSO4·5H2O, 0.42 g / L MnCl2·H2O, 2 g / L Na2B4O7·10H2O, and 2 g / L Na2MoO4·2H2O.

[0059] The bacterial culture was inoculated at a rate of 1% and cultured at 37°C and 230 rpm for 5 hours. IPTG was then added to a final concentration of 0.2 mM to begin induction, and the culture was induced at 28°C and 230 rpm for 48 hours.

[0060] III. Technical Effects

[0061] Table 3 Comparison of strain yield

[0062]

[0063] Based on the results above, compared with the control strain BS3, overexpression... ompC , ompA , ompF The yield of 3'-SL was increased to varying degrees after gene sequencing.

[0064] Table 4 Comparison of strain yield

[0065]

[0066] The results show that, compared with the control strain BS3, overexpression mdfA , mdtM , mdtL , mdtG After genes, except mdfA Production decreased, while production of the other 3'-SL all increased to varying degrees.

[0067] Table 5 Comparison of strain yield

[0068]

[0069] The results show that, compared with the control strain BS3, the knockout strain... tolR Simultaneous overexpression of genes tolA , tolB Subsequently, the yield of 3'-SL was significantly increased, and was significantly higher than that of other strains and the yield of engineered strains obtained by other gene editing methods mentioned above.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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 sialic acid lactose, characterized in that, include: The following methods were used to improve E. coli: While knocking out the expression level of tolR, the expression levels of tolA and tolB were increased; The sialyl lactose is 3'-sialyl lactose; The Escherichia coli mentioned is Escherichia coli BL21 star(DE3).

2. A genetically engineered strain that produces high levels of sialic acid lactose, characterized in that, The genetically engineered strain was obtained from Escherichia coli expressing sialyl lactose through the following method: While knocking out the expression level of tolR, the expression levels of tolA and tolB were increased; The *E. coli* expressing sialic acid lactose was obtained from *E. coli* through the following method: i) Increase the expression levels of neuB, neuC, and neuA, and increase the expression level of nST; ii) Reduce the expression level of lacZ; The sialyl lactose is 3'-sialyl lactose.

3. The genetically engineered strain according to claim 2, characterized in that, It also includes the following methods: Reduce the expression levels of one or more of nanA, nanK, nanE, nanT, wcaJ, fucI, or fucK.

4. The genetically engineered strain according to claim 2, characterized in that, The Escherichia coli is Escherichia coli BL21, Escherichia coli BL21(DE3) or Escherichia coli BL21 star(DE3).

5. A microbial inoculant, characterized in that, Includes the genetically engineered strains as described in any one of claims 2-4.

6. The use of the genetically engineered strain according to any one of claims 2-4, or the microbial agent according to claim 5, in the production of sialic acid lactose.

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