Recombinant escherichia coli for synthesizing fucosyllactose and construction method and application thereof

Abstract

The application relates to the technical field of engineering bacteria, and particularly discloses recombinant Escherichia coli for synthesizing fucosyllactose as well as a construction method and application thereof. In the genome of the recombinant Escherichia coli, phosphofructokinase genes or phosphofructokinase genes and pyruvate kinase genes are knocked out, so that glucose carbon flow is reduced to flow to a tricarboxylic acid cycle required for cell growth, and the intake of glucose carbon flow is forced to flow more to the synthesis of products (GDP-L-fucose in the synthesis of FL), and the synthesis efficiency of the products is improved. Meanwhile, the glucose carbon flow flowing to the cell growth is greatly reduced, and the glucose effect can be removed, so that the tolerance of the fermentation process to excessive glucose residues is improved, and the difficulty of downstream production amplification is reduced. The recombinant Escherichia coli uses glycerol and glucose compound carbon sources as raw materials, uses lactose as a substrate, and has a 3-fucosyllactose yield of 73.88 g / L in a 5L tank fermentation, which is the highest level reported at present.

Description

Technical Field

[0001] This application relates to the field of engineered bacteria technology, and in particular to a recombinant Escherichia coli that synthesizes fucose-based lactose, its construction method, and its application. Background Technology

[0002] Fucosyllactose (FL) is the most abundant human milk oligosaccharide in breast milk. There are two types of fucosyllactose in breast milk: 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL). Generally speaking, 2'-FL is the most abundant in breast milk, while 3-FL is the most widely distributed (present in all breast milk). 2'-FL and 3-FL have some functional similarities but also differences. Currently, food safety regulatory agencies in the United States, the European Union, and countries such as China, Singapore, and the Philippines have approved the addition of fucosyllactose as a nutritional supplement in infant formula. Fucosyllactose, as a high-value nutritional supplement, has broad prospects in the future markets for infant formula and dietary supplements.

[0003] Currently, the main methods for producing FL include chemical synthesis, enzymatic synthesis, and biosynthesis, with de novo biosynthesis becoming the mainstream method. This method typically uses *E. coli* as the primary producing bacterium, employing glycerol, glucose, or sucrose as a single carbon source for substrate and energy supply. Starting with fructose-6-phosphate, an important intermediate from glycolysis, it catalyzes the synthesis of GDP-L-fucose through five enzymatic cascade steps (ManA, ManB, ManC, Gmd, and WcaG / Fcl). GDP-L-fucose then reacts with lactose under the catalysis of α-1,2-fucosyltransferase or α-1,3-fucosyltransferase to synthesize fucosyllactose. GDP-L-fucose, as an essential precursor, is a crucial factor determining the yield of fucosyllactose. The biosynthesis of fucoidan requires fructose-6-phosphate from the glycolysis pathway as a precursor. Therefore, there is a competitive relationship between the product synthesis process and cell growth metabolism. Appropriate carbon flux allocation between the two becomes a key challenge for the efficient synthesis of fucoidan in host cells.

[0004] Recombinant Escherichia coli that synthesizes fucoidan lactose has been developed by knocking out the β-galactosidase gene lacZ and the UDP-glucose lipotransferase gene wcaJ from the chassis cell E. coli BL21(DE3) genome. It also exogenously expresses the phosphogannatase-mannose-1-phosguanine syltransferase gene cluster manC-manB, the GDP-mannose-6-dehydrogenase-GDP-fucose synthase gene cluster gmd-fcl, the lactose permease lacY, and codon-optimized α-1,2-fucosyltransferase WcfB or α-1,3-fucosyltransferase cafF.

[0005] However, existing recombinant E. coli technologies use glucose as a single carbon source to synthesize FL. Since glucose supports both growth and product synthesis, the carbon flux taken up needs to be allocated between growth and product synthesis, resulting in low product synthesis efficiency. Furthermore, residual glucose in the fed-batch fermentation system of existing technologies inhibits intracellular enzyme synthesis, thus suppressing product synthesis and reducing efficiency (commonly known as the glucose effect). Therefore, the process control of fed-batch fermentation requires stricter control (ensuring sufficient but not excessive glucose supplementation), resulting in lower operational flexibility and significantly reducing stability and reproducibility during scale-up fermentation and changes in personnel or location. In addition, existing strains are highly dependent on nutrient-rich organic nitrogen-based media (such as LB medium containing peptone and yeast extract), and yields are significantly reduced when using low-mineral-content media, leading to higher production costs. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a recombinant Escherichia coli for synthesizing fucose-based lactose, its construction method, and its application.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0008] This application provides a recombinant Escherichia coli that synthesizes fucoidan lactose, wherein the recombinant Escherichia coli is formed by knocking out (a) and (b) or knocking out (a) and (c) in the Escherichia coli genome and overexpressing an exogenous fucoidan transferase gene;

[0009] (a) β-galactosidase gene lacZ and UDP-glucose lipid transporter transferase gene WcaJ;

[0010] (b) Phosphofructokinase gene;

[0011] (c) Phosphofructokinase gene and pyruvate kinase gene.

[0012] This application significantly reduces the flow of glucose carbon to the tricarboxylic acid cycle (TCA cycle) required for cell growth by knocking out the phosphofructokinase or pyruvate kinase gene. This forces more of the taken-up glucose carbon to be used for product synthesis (GDP-L-fucose synthesis in FL synthesis), thereby improving product synthesis efficiency. Simultaneously, significantly reducing the flow of glucose carbon to the TCA cycle also eliminates the glucose effect, increasing the fermentation process's tolerance to excess glucose residue and reducing the difficulty of downstream production scale-up.

[0013] As a preferred embodiment of the recombinant Escherichia coli that synthesizes fucose-syl lactose described in this application, the recombinant Escherichia coli also overexpresses the lactose permease gene LacY and genes related to the GDP-L-fucose synthesis pathway;

[0014] The genes related to the GDP-L-fucose synthesis pathway include phosphogannatase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4,6-dehydratase Gmd, and GDP-fucose synthase WcaG.

[0015] Overexpression of lactose permease gene LacY, phosphogannatase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4,6-dehydrase Gmd, and GDP-fucose synthase WcaG can improve the synthesis of fucose-syllactose.

[0016] As a preferred embodiment of the recombinant Escherichia coli that synthesizes fucose-L-lactose as described in this application, the GDP-L-fucose synthesis pathway-related genes also include the 6-phosphate mannose isomerase gene ManA.

[0017] This application also overexpresses the 6-phosphate mannose isomerase gene ManA, which can better synthesize 2'-FL.

[0018] As a preferred embodiment of the recombinant Escherichia coli that synthesizes fucose-based lactose according to this application, the phosphofructokinase gene includes phosphofructokinase genes pfkA and / or pfkB; the pyruvate kinase gene includes pyruvate kinase gene pykF;

[0019] The exogenous fucosyltransferase gene includes the fucosyltransferase gene WcfB from Bacteroides fragilis or the fucosyltransferase gene cafF from Akkermansia myxophilus.

[0020] The optimized codon sequence of the WcfB gene, a fucosyltransferase gene derived from Bacteroides fragilis, is shown in SEQ ID NO: 1, and the optimized codon sequence of the cafF gene, a fucosyltransferase gene derived from Akkermansia myxotropicis, is shown in SEQ ID NO: 2.

[0021] The technical solution of this application knocks out the pfkA gene, which significantly inhibits the role of glucose as a growth energy source (measurements showed that cell growth rate was significantly reduced compared to wild type when glucose was added alone as a carbon source). Knocking out the pfkB gene further enhances this inhibitory effect, thus effectively reducing the glucose effect. Experiments have shown that excessive glucose during fed-batch fermentation does not inhibit product synthesis; on the contrary, it has a positive promoting effect because it facilitates glucose uptake. Therefore, it greatly reduces the operational difficulty of the fed-batch control process in actual fermentation and improves the stability and reproducibility of actual production.

[0022] In the technical solution of this application, when the pykF gene is knocked out, E. coli uses glycerol to grow, and carbon flow is blocked. It is necessary to couple the glucose transport process to successfully convert PEP into pyruvate and enter the tricarboxylic acid cycle. Therefore, it can promote the expression of glucose transport protein, thereby increasing the glucose uptake rate and accelerating the synthesis of the product.

[0023] Knocking out the pfkA and pfkB genes or the pykF gene in the *E. coli* genome makes the strain tolerant to the glucose effect, thus making the fed-batch fermentation process more flexible and fault-tolerant in large-scale production. During fed-batch fermentation, only the glycerol addition flow rate needs to be fixed, and glucose can be added in excess.

[0024] In the technical solution of this application, it is preferable to knock out the phosphofructokinase gene pfkA, or knock out the phosphofructokinase genes pfkA and pfkB, or knock out the phosphofructokinase gene pfkA and the pyruvate kinase gene pykF. This can better and more significantly reduce the flow of glucose carbon to the tricarboxylic acid cycle required for cell growth, forcing more of the taken-up glucose carbon to flow to product synthesis, improving the product synthesis efficiency, and also removing the glucose effect, increasing the tolerance of the fermentation process to excessive glucose residue, and reducing the difficulty of downstream production scale-up.

[0025] As a preferred embodiment of the recombinant Escherichia coli that synthesizes fucose-based lactose according to this application, the Escherichia coli includes Escherichia coli BL21(DE3).

[0026] In some specific embodiments, the Escherichia coli is selected from Escherichia coli BL21(DE3), including those with Plac-T7RNAP and PlacUV5-T7RNAP gene backgrounds.

[0027] The overexpression methods described in this application include replacing the endogenous promoter sequence before the start codon on the genome with a structurally active promoter and using an exogenous E. coli expression vector.

[0028] As a preferred embodiment of the recombinant Escherichia coli that synthesizes fucosylated lactose described in this application, the overexpression of the exogenous fucosylated transferase gene is achieved through a plasmid vector.

[0029] As a preferred embodiment of the recombinant Escherichia coli that synthesizes fucose-syl lactose described in this application, the overexpression of the lactose permease gene LacY, phosphogannatase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4,6-dehydratase Gmd, and GDP-fucose synthase WcaG is achieved through a plasmid vector.

[0030] Overexpression of the 6-phosphate mannose isomerase gene ManA is achieved by replacing the endogenous promoter in the genome with a structurally active promoter.

[0031] As a preferred embodiment of the recombinant Escherichia coli that synthesizes fucose-based lactose as described in this application, the plasmid vectors used for overexpression include pCDFDuet1 and pCOLADuet1, and the structurally active promoters used for overexpression include PJ23100 and PJ23119.

[0032] In some specific embodiments, overexpression of the lactose permease gene LacY and the exogenous fucosyltransferase gene is achieved using the pCDFDuet1 plasmid vector; overexpression of GDP-L-fucose synthesis pathway-related genes (including phosphogannase mutase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4,6-dehydrase Gmd, and GDP-fucose synthase WcaG) is achieved using the pCOLADuet1 plasmid vector; and overexpression of the 6-phosphogannase isomerase gene ManA is achieved by replacing the endogenous promoter on the genome with the structurally active promoter PJ23100 or PJ23119.

[0033] This application also provides a method for constructing the above-mentioned recombinant Escherichia coli containing synthetic fucoidosyl lactose, comprising the following steps:

[0034] S1. Knock out the β-galactosidase gene lacZ, the UDP-glucose lipotransferase gene WcaJ, and the phosphofructokinase gene of Escherichia coli to obtain recombinant bacteria;

[0035] Alternatively, knocking out the β-galactosidase gene lacZ, the UDP-glucose lipotransferase gene WcaJ, the phosphofructokinase gene, and the pyruvate kinase gene of Escherichia coli can yield recombinant bacteria.

[0036] S2. Recombinant Escherichia coli was obtained by overexpressing the lactose permease gene LacY, phosphogluconomutase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4,6-dehydratase Gmd, GDP-fucose synthase WcaG, and exogenous fucosyltransferase gene using a plasmid vector of Escherichia coli.

[0037] Preferably, in step S1, the 6-phosphate mannose isomerase gene ManA is also overexpressed.

[0038] In a preferred embodiment of the method for constructing recombinant Escherichia coli as described in this application, in step S1, the knockout of the β-galactosidase gene lacZ, the UDP-glucose lipotransferase gene WcaJ, and the phosphofructokinase gene or pyruvate kinase gene, as well as the overexpression of the 6-phosphomannose isomerase gene ManA, is accomplished using the CRISPR / Cas9 system.

[0039] In a preferred embodiment of the method for constructing recombinant Escherichia coli as described in this application, in step S1, the overexpression of the 6-phosphate mannose isomerase gene ManA is accomplished by replacing its endogenous promoter with a structurally active promoter.

[0040] Preferably, the structurally active promoters used for overexpressing the 6-phosphate mannose isomerase gene ManA include the PJ23100 and PJ23119 promoters.

[0041] In a preferred embodiment of the method for constructing recombinant Escherichia coli as described in this application, in step S2, the vectors used for overexpressing the lactose permease gene LacY, phosphogannatase ManB, GDP-mannose pyrophosphorylase ManC, GDP-mannose 4,6-dehydratase Gmd, GDP-fucose synthase WcaG, and exogenous fucosyltransferase gene include pCOLADuet1 and pCDFDuet1.

[0042] As a preferred embodiment of the method for constructing recombinant Escherichia coli according to this application, in step S1, the nucleotide sequence of the target sequence of the gRNA of the UDP-glucose lipotransferase gene WcaJ knocked out is shown in SEQ ID NO: 19, and the primer sequences used for Doner fragment amplification are shown in SEQ ID NO: 13-16.

[0043] The nucleotide sequence of the target sequence of the gRNA that knocks out the β-galactosidase gene lacZ is shown in SEQ ID NO: 26, and the primer sequences used for Doner fragment amplification are shown in SEQ ID NO: 20-23.

[0044] The nucleotide sequence of the target sequence of the gRNA that knocks out the phosphofructokinase gene pfkA is shown in SEQ ID NO: 42, and the primer sequences used for Doner fragment amplification are shown in SEQ ID NO: 36-39.

[0045] The nucleotide sequence of the target sequence of the gRNA that knocks out the phosphofructokinase gene pfkB is shown in SEQ ID NO: 49, and the primer sequences used for Doner fragment amplification are shown in SEQ ID NO: 43-46.

[0046] The nucleotide sequence of the target sequence of the gRNA that knocks out the pyruvate kinase gene pykF is shown in SEQ ID NO: 56, and the primer sequences used for Doner fragment amplification are shown in SEQ ID NO: 50-53.

[0047] The nucleotide sequences of the target sequences of the upstream promoter of the 6-phosphate mannose isomerase gene ManA are shown in SEQ ID NO: 35. The primer sequences used for amplification of the Doner fragment used for the PJ23100 promoter are shown in SEQ ID NO: 27-29 and SEQ ID NO: 32. The primer sequences used for amplification of the Doner fragment used for the PJ23119 promoter are shown in SEQ ID NO: 27 and SEQ ID NO: 30-32.

[0048] This application also provides the application of the above-mentioned recombinant Escherichia coli for the synthesis of fucoidosyl lactose in the fermentation synthesis of fucoidosyl lactose.

[0049] This application also provides a method for fermenting and synthesizing fucoidan lactose, using glucose and glycerol as dual carbon sources and lactose as a common substrate, and employing the aforementioned recombinant Escherichia coli for fucoidan synthesis. During the fermentation process, a feed carbon source and lactose are added to maintain the glucose residue in the fermentation broth at 1-15 g / L.

[0050] The recombinant Escherichia coli of this application can be fermented using inexpensive inorganic salt culture media, which can significantly reduce production costs and is suitable for large-scale industrial production of fucoidan.

[0051] The recombinant *E. coli* strain described in this application uses glucose and glycerol as dual carbon sources for fermentation, with lactose added as a substrate for FL synthesis. The fermentation temperature is 25–30°C. During fermentation, biomass growth is controlled by adjusting the glycerol flow rate. 600 After reaching 80-100, gradually reduce the glycerol flow rate to slow cell growth but maintain bacterial turnover, while simultaneously adding sufficient glucose and lactose (maintaining a residual glucose level of 1-15 g / L and a residual lactose level of 15-25 g / L).

[0052] Analysis revealed that the strain described in this application is a dual-carbon-source fermentation strain. The strain utilizes glycerol for energy growth and converts glucose into GDP-L-fucose, which participates in FL synthesis. Even when using an inorganic salt medium like M9, it can efficiently synthesize the product.

[0053] Compared with the prior art, this application has the following beneficial effects:

[0054] This application provides a recombinant *E. coli* strain for synthesizing fucoidan, its construction method, and its applications. By knocking out the phosphofructokinase gene or both the phosphofructokinase and pyruvate kinase genes, this application significantly reduces the flow of glucose carbon to the tricarboxylic acid cycle required for cell growth, forcing more of the taken-up glucose carbon to flow towards product synthesis (GDP-L-fucose synthesis in FL synthesis), thus improving product synthesis efficiency. Simultaneously, significantly reducing the flow of glucose carbon to the tricarboxylic acid cycle also eliminates the glucose effect, increasing the fermentation process's tolerance to excess glucose residue and reducing the difficulty of downstream production scale-up. Furthermore, this *E. coli* strain can efficiently synthesize the product even in low-salt inorganic media, which contributes to cost reduction in large-scale production. Attached Figure Description

[0055] Figure 1 This is a metabolic pathway diagram of recombinant Escherichia coli.

[0056] Figure 2 OD values ​​of 2'-FL synthesizing strains corresponding to different chassis during 24 hours of fermentation with pure glucose as a single carbon source. 600nm Value results graph;

[0057] Figure 3 The results of fructose-6-phosphate content in four recombinant Escherichia coli strains with different gene backgrounds are shown in the figure.

[0058] Figure 4 The image shows the fermentation results of the 3-FL recombinant strain corresponding to chassis 1 in a 5L tank with poor culture medium.

[0059] Figure 5 This is a magnified image of the fermentation results of the 50L tank for producing recombinant strains of 3-FL corresponding to chassis 1.

[0060] Figure 6 This is a magnified image of the fermentation results of the 3-FL recombinant strain produced in a 500L tank corresponding to chassis 1. Detailed Implementation

[0061] To better illustrate the purpose, technical solution, and advantages of this application, the following will provide further explanation of this application in conjunction with specific embodiments and accompanying drawings.

[0062] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

[0063] In the following embodiments, the sequences used are shown in Table 1.

[0064] Table 1

[0065]

[0066]

[0067]

[0068]

[0069] Example 1: Gene Acquisition

[0070] In this embodiment, the following genes were obtained: manB (Genome Accession Number GI: 946574), manC (Genome Accession Number GI: 946580), gmd (Genome Accession Number GI: 946562), fcl (Genome Accession Number GI: 946563), and lacY (Genome Accession Number GI: 949083) from Escherichia coli MG1655; wcfB (Genome Accession Number GI: 3287270) from Bacteroides fragilis NCTC9343; and cafF (Genome Accession Number GI: 6274963) from Akkermansia ATCC BAA-835.

[0071] In this embodiment, genomic DNA of *E. coli* MG1655 was extracted using the Sigma-Aldrich bacterial genomic DNA extraction kit according to the kit's instructions. The *manC-manB* gene cluster, *gmd-fcl* gene cluster, and *lacY* gene were successfully obtained. Genes with optimized codons were synthesized and cloned into the *E. coli* pCDFDuet1 vector plasmid. The *wcfB* gene (SEQ ID NO: 1) from *Bacteroides fragilis* NCTC9343 and the *cafF* gene (SEQ ID NO: 2) from *Ackermania ATCC BAA-835* were successfully obtained.

[0072] Example 2: Preparation of recombinant plasmids

[0073] The manC-manB gene cluster obtained from Escherichia coli MG1655 in Example 1 was amplified by PCR using the designed primers manC-manB-F / R (SEQ ID NO: 3-4). The amplified fragment was purified by gel excision and double-digested with NcoI and HindIII restriction endonucleases. The digested fragment was ligated with the plasmid pCOLADuet-1, which was also double-digested with NcoI and HindIII. The vector and target fragment were mixed at a molar ratio of 1:3, and T4 DNA ligase was added. The ligation was carried out at 22°C for 5 h. The ligation product was transformed into Escherichia coli DH5α competent cells and screened on kanamycin plates to obtain the recombinant plasmid pCOLA-CB.

[0074] The gmd-fcl gene cluster obtained from Escherichia coli MG1655 in Example 1 was amplified by PCR using the designed primers gmd-fcl-F / R (SEQ ID NO: 5-6). The amplified fragment was purified by gel excision and double-digested with NdeI and XhoI. The digested fragment was ligated with the plasmid pCOLA-CB, which was also double-digested with NdeI and XhoI. The vector and target fragment were mixed at a molar ratio of 1:3, and T4 DNA ligase was added. The ligation was carried out at 22°C for 5 h. The ligation product was transformed into Escherichia coli DH5α competent cells and screened on kanamycin plates to obtain the recombinant plasmid pCOLA-CBGF.

[0075] The gene lacY from Escherichia coli MG1655 obtained in Example 1 was amplified by PCR using the designed primers lacY-F / R (SEQ ID NO: 7-8). The amplified fragment was purified by gel excision and double-digested with NdeI and XhoI. The digested fragment was ligated with the plasmid pCDFDuet-1, which was also double-digested with NdeI and XhoI. The vector and the target fragment were mixed at a molar ratio of 1:3, and T4 DNA ligase was added. The ligation was carried out at 22°C for 5 h. The ligation product was transformed into Escherichia coli DH5α competent cells and screened on streptomycin plates to obtain the recombinant plasmid pCDF-lacY.

[0076] The wcfB gene obtained from Bacteroides fragilis in Example 1 was amplified by PCR using the designed primers WcfB-F / R (SEQ ID NO: 9-10). The amplified fragment was purified by gel excision and double-digested with NcoI and BamHI. The digested fragment was ligated with the plasmid pCDF-lacY, which was also double-digested with NcoI and BamHI. The vector and target fragment were mixed at a molar ratio of 1:3, and T4 DNA ligase was added. The ligation was carried out at 22°C for 5 h. The ligation product was transformed into Escherichia coli DH5α competent cells and screened on streptomycin plates to obtain the recombinant plasmid pCDF-WY.

[0077] The cafF gene obtained from Akkermansia myxophilus in Example 1 was amplified by PCR using the designed primers cafF-F / R (SEQ ID NO: 11-12). The amplified fragment was purified by gel excision and double-digested with NcoI and BamHI. The digested fragment was ligated with the plasmid pCDF-lacY, which was also double-digested with NcoI and BamHI. The vector and the target fragment were mixed at a molar ratio of 1:3, and T4 DNA ligase was added. The ligation was carried out at 22°C for 5 h. The ligation product was transformed into Escherichia coli DH5α competent cells and screened on streptomycin plates to obtain the recombinant plasmid pCDF-CY.

[0078] Example 3: Gene Editing

[0079] The gene editing (including gene knockout, promoter replacement, etc.) involved in this application was performed using the CRISPR gene editing system.

[0080] I. Gene knockout:

[0081] This embodiment uses the wcaJ gene as an example to illustrate the gene knockout steps in detail. The knockout of other genes is the same.

[0082] 1) Sequence information acquisition: The sequence information of the gene wcaJ to be knocked out and its upstream and downstream components was determined based on the genome sequence information of Escherichia coli BL21(DE3) included in NCBI (GenBank:CP001509.3).

[0083] 2) Design of sgRNA sequence: Using the http: / / chopchop.cbμ.μib.no / database, the sgRNA required for gene knockout wcaJ was designed and screened, namely the PAM site and its upstream 20bp nucleotide sequence, as shown in wcaJ-N20-PAM (SEQ ID NO: 19, as shown in Table 1).

[0084] 4) Preparation of homologous arms: Using primer pair wcaJ-UP-F / R (SEQ ID NO: 13-14) and Escherichia coli BL21(DE3) genome as templates, the upstream homologous arm sequence of the target gene wcaJ was amplified by PCR to obtain the wcaJ-UP fragment; using primer pair wcaJ-DO-F / R (SEQ ID NO: 15-16) and Escherichia coli BL21(DE3) genome as templates, the downstream homologous arm sequence of the target gene wcaJ was amplified by PCR to obtain the wcaJ-DO fragment.

[0085] 5) Preparation of the Donor fragment: Using primer pairs wcaJ-UP-F and wcaJ-DO-R, and with wcaJ-UP and wcaJ-DO fragments as a mixed template, the upstream and downstream homologous arms were fused by overlap PCR to obtain the wcaJ-Donor fragment.

[0086] 6) Preparation of guide plasmid pEcgRNA-wcaJ-N20:

[0087] ①Preparation of wcaJ-N20 oligo dsDNA: Oligo dsDNA double strands were formed by annealing complementary primer pairs wcaJ-N20-F / R (SEQ ID NO: 17-18). The reaction system consisted of 10 μL each of primers wcaJ-N20-F / R; 5 μL of T4 ligase bμffer; and 25 μL of deionized water, for a total volume of 50 μL. The annealing procedure was as follows: boil at 94℃ for 2 min, then allow to cool naturally to room temperature until it reached 16℃, and incubate for 10 min. Finally, the annealed product was diluted 200-fold and stored at -80℃ for later use.

[0088] ② Linearization of pEcgRNA: The guide plasmid pEcgRNA was digested with restriction endonuclease BsaI, and the digestion product was recovered by gel extraction and purified for later use.

[0089] ③ After enzyme digestion, the linearized pEcgRNA and wcaJ-N20 oligo dsDNA fragment were mixed at a molar ratio of 1:3. T4 DNA ligase was added and ligation was carried out at 22℃ for 2-5 hours. The ligation product was transformed into E. coli DH5α competent cells and screened on streptomycin plates to obtain the recombinant plasmid pEcgRNA-wcaJ-N20.

[0090] 7) Prepare electrocompetent cells of *E. coli* BL21(DE3) and transfer the plasmid pEcCas into them to obtain recombinant bacteria BL21(DE3) / pEcCas; then transfer the recombinant bacteria containing the pEcCas plasmid into LB liquid medium and incubate at 37°C and 220 rpm. When OD 600When the value reaches approximately 0.2, L-arabinose at a final concentration of 20 mM is added to induce recombinase expression, and the culture continues until OD200 reaches 0.2. 600 When the pH is 0.6–0.8, the cells are collected by low-temperature, low-speed centrifugation to prepare electrocompetent cells of recombinant Escherichia coli.

[0091] 8) Electroporation: 100 ng of guide plasmid pEcgRNA-wcaJ-N20 and 400 ng of Donor fragment were sequentially added to competent cells of recombinant bacteria containing pEcCas plasmid. After electroporation and recovery, the cells were plated with spectinomycin (50 μg / ml) and kanamycin (50 μg / ml). The mutant strain with the target gene deleted was confirmed by primer pair identification, PCR amplification and sequencing.

[0092] 9) Elimination of the edited plasmid: Successfully identified mutant bacteria were inoculated into a medium containing kanamycin (50 μg / ml) and 20 mM rhamnose to induce the loss of the guide plasmid pEcgRNA-wcaJ-N20. After incubation at 37℃ and 220 rpm for 16–24 h, single bacteria were isolated by streaking on solid plates containing 10 g / L sucrose to obtain mutant bacteria with the pEcCas plasmid loss. The wcaJ gene knockout strain was finally obtained.

[0093] Similarly, the β-galactosidase gene LacZ was knocked out using the above method. The difference is that the sgRNA and primers required for LacZ gene knockout are different. The sgRNA is lacZ-N20-PAM (SEQ ID NO: 26), and the primers are lacZ-UP-F (SEQ ID NO: 20), lacZ-UP-R (SEQ ID NO: 21), lacZ-DO-F (SEQ ID NO: 22), lacZ-DO-R (SEQ ID NO: 23), lacZ-N20-F (SEQ ID NO: 24), and lacZ-N20-R (SEQ ID NO: 25).

[0094] The phosphofructokinase gene pfkA was knocked out, with the difference being that the sgRNA and primers required for pfkA knockout were different. The sgRNA was pfkA-N20-PAM (SEQ ID NO: 42), and the primers were pfkA-UP-F (SEQ ID NO: 36), pfkA-UP-R (SEQ ID NO: 37), pfkA-DO-F (SEQ ID NO: 38), pfkA-DO-R (SEQ ID NO: 39), pfkA-N20-F (SEQ ID NO: 40), and pfkA-N20-R (SEQ ID NO: 41).

[0095] The phosphofructokinase gene pfkB was knocked out, with the difference being that the sgRNA and primers required for pfkB knockout were different. The sgRNA was pfkB-N20-PAM (SEQ ID NO: 49), and the primers were pfkB-UP-F (SEQ ID NO: 43), pfkB-UP-R (SEQ ID NO: 44), pfkB-DO-F (SEQ ID NO: 45), pfkB-DO-R (SEQ ID NO: 46), pfkB-N20-F (SEQ ID NO: 47), and pfkB-N20-R (SEQ ID NO: 48).

[0096] The pyruvate kinase gene pykF was knocked out, with the difference being that the sgRNA and primers required for pykF gene knockout were different. The sgRNA was pykF-N20-PAM (SEQ ID NO: 56), and the primers were pykF-UP-F (SEQ ID NO: 50), pykF-UP-R (SEQ ID NO: 51), pykF-DO-F (SEQ ID NO: 52), pykF-DO-R (SEQ ID NO: 53), pykF-N20-F (SEQ ID NO: 54), and pykF-N20-R (SEQ ID NO: 55).

[0097] II. Promoter replacement:

[0098] In this embodiment, the promoter replacement of the target gene is similar to the gene knockout operation in step one, except that the design and construction of the Donor fragment are slightly different, in which the promoter sequence to be replaced is fused in the middle.

[0099] When knocking in the promoter upstream of the manA gene site on the genome, the Donor fragment is designed and prepared as follows: The PJ23100 or PJ23119 promoter sequence is designed into the 3' end of the upstream homologous arm and the 5' end of the downstream homologous arm primers, as shown in primers manA-UP-PJ23100-R (SEQ ID NO: 28) and manA-DO-PJ23100-F (SEQ ID NO: 29). Using primer pairs manA-UP-F / manA-UP-PJ23100-R (SEQ ID NO: 27, SEQ ID NO: 28), manA-DO-PJ23100-F / manA-DO-R (SEQ ID NO: 29, SEQ ID NO: 32) and the genome of *E. coli* BL21(DE3) as templates, the upstream and downstream sequences of the gmd gene locus were amplified by PCR to obtain upstream and downstream homologous arm fragments manA-UP-PJ23100 and manA-DO-P23100, respectively. Using primer pairs manA-UP-F (SEQ ID NO: 27) and manA-DO-R (SEQ ID NO: 32), and with the manA-UP-PJ23100 and manA-DO-P23100 fragments as a mixed template, the upstream and downstream homologous arms were fused by overlap PCR to obtain the manA-Donor-PJ23100 fragment. Similarly, following the above method, the PJ23119 promoter sequence was designed into primers at the 3' end of the upstream homologous arm and the 5' end of the downstream homologous arm. The difference is that the primers involved are manA-UP-F (SEQ ID NO: 27), manA-UP-PJ23119-R (SEQ ID NO: 30), manA-DO-PJ23119-F (SEQ ID NO: 31), and manA-DO-R (SEQ ID NO: 32).

[0100] Example 4: Construction of chassis strains

[0101] In this application, the chassis strain was constructed through gene editing, which involved knocking out the β-galactosidase gene LacZ, the UDP-glucose lipotransferase gene WcaJ, the phosphofructokinase gene pfkA, the phosphofructokinase gene pfkB, and the pyruvate kinase gene pykF, and replacing the upstream regulatory sequence of the 6-phosphomannose isomerase gene ManA with the PJ23100 or PJ23119 promoter.

[0102] The obtained chassis strain numbers and their corresponding genotypes are shown in Table 2.

[0103] Example 5: Construction of recombinant Escherichia coli

[0104] The recombinant plasmids (pCOLA-CBGF and pCDF-WY or pCDF-CY) obtained in Example 2 were co-transformed into the *E. coli* chassis cells obtained in Example 4. Transformation required the preparation of competent chassis cells, using the same method as in Example 3 for preparing and electroporating competent cells. The transformed *E. coli* were then screened using a double-antibody plate (KAN resistance and Str resistance) to obtain recombinant *E. coli* that synthesize fucoidan-lactose. The metabolic pathway diagram of the recombinant *E. coli* is shown below. Figure 1 As shown.

[0105] Example 6: Shake-flask fermentation of recombinant Escherichia coli

[0106] The purpose of this embodiment is to verify the effect of knocking out the phosphofructokinase gene and / or pyruvate kinase gene on the efficiency of fucose-syl lactose synthesis in the strain.

[0107] In this embodiment, the recombinant Escherichia coli obtained in Example 5 was used as the fermentation strain, and a 250mL Erlenmeyer flask was used as the fermentation vessel. The fermentation medium used was the commonly used LB medium, which consists of 10g / L peptone, 5g / L yeast extract, and 5g / L sodium chloride.

[0108] The specific fermentation process is as follows: Recombinant *E. coli* was inoculated into LB liquid medium containing Kan and Str antibiotics and cultured overnight at 37°C. A 1% inoculum was then transferred to 50 ml of fresh LB medium and cultured at 30°C for 2 hours. 0.3 mM IPTG was added as an inducer, along with 0–1 g / L glycerol, 20–30 g / L glucose, and 12 g / L lactose. The mixture was then fermented in shake flasks at 25°C for 66–90 hours.

[0109] Fermentation yield determination: A certain volume of the final fermentation broth was taken, and acetonitrile was added at a 1:1 volume ratio. The mixture was centrifuged at 13,000g for 5 min, and the supernatant was collected. The supernatant was separated by HPLC and detected by an evaporative light detector. The final yield was calculated using a standard curve.

[0110] The shake-flask fermentation results of the recombinant strains involved in this application are shown in Table 2.

[0111] The data in the table show that knockout of the phosphofructokinase gene and / or pyruvate kinase gene significantly increases the yield of fucose-syl lactose in shake-flask fermentation.

[0112] Table 2. Types of bacterial strains in the chassis and their corresponding relative yields in shake-flask fermentation.

[0113]

[0114]

[0115] Example 7: Shake-flask fermentation of recombinant Escherichia coli in barren culture medium M9

[0116] The purpose of this embodiment is to test the advantages of the recombinant Escherichia coli strain in fermentation in a poor culture medium compared with the control strain in this application.

[0117] In this embodiment, the strain corresponding to chassis 1 obtained in Example 5 and the strain corresponding to control chassis 1 were fermented in shake flasks in LB medium and M9 medium, respectively. The formula of M9 medium was: 6.778 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 1 g / L ammonium chloride, 0.5 g / L sodium chloride, 0.24 g / L magnesium sulfate, 11 mg / L calcium chloride, and 10 ml / L trace element mixture. The specific implementation process of shake flask fermentation was the same as in Example 6. The results of shake flask fermentation are shown in Table 3.

[0118] As can be seen from Table 3, under M9 medium conditions, the fermentation yield of the control chassis was significantly lower than that under LB medium conditions (only about one-fifth of that under LB medium conditions). Although the fermentation yield of chassis 1 with pfkA knocked out was also not as high as that under LB medium conditions, it could reach 70% to 80% of that under LB medium conditions. This indicates that knocking out pfkA did indeed reduce the strain's dependence on organic nitrogen sources and showed stronger industrialization potential.

[0119] Table 3. Relative yields of shake-flask fermentation for control chassis and chassis strain 1 under LB and M9 medium conditions.

[0120]

[0121] Example 8: Growth of recombinant Escherichia coli under conditions where glucose is the sole carbon source.

[0122] The purpose of this embodiment is to test the growth of recombinant Escherichia coli in this application under the condition of using glucose as a single carbon source, so as to support our hypothesis of carbon flow allocation and the necessity of using dual carbon source fermentation in this application.

[0123] In this embodiment, 1 mL of 2'-FL synthetic recombinant E. coli corresponding to the control chassis, chassis 1, chassis 7, and chassis 8 during the plateau phase (density 5 OD) was taken from each chassis. 600nm The solution was diluted 50 times in M9 medium and incubated at 30°C for 3 hours. Then, 0.3 mM IPTG, 20 g / L glucose, and 12 g / L lactose were added, and the mixture was fermented at 25°C for 24 hours. The OD value of the culture was then measured. 600nm Value. Result as follows Figure 2 As shown. The results show that the growth rate (OD) 600nm The values ​​are sorted as follows: chassis > chassis1 ≥ chassis7 > chassis8.

[0124] This indicates that knocking out the pfkA gene significantly inhibits glucose's role as an energy source for growth, while knocking out the pfkB gene further enhances this inhibition, thereby forcing more fructose-6-phosphate to be diverted to GDP-L-fucose synthesis. On the other hand, since the knockout of the phosphofructokinase gene and / or pyruvate kinase leads to slower cell growth, it is necessary to add glycerol as an energy source to increase cell density during fermentation in tank fermentation.

[0125] Example 9: Determination of the content of fructose-6-phosphate, the GDP-L-fucose synthesis precursor, in recombinant Escherichia coli

[0126] The purpose of this embodiment is to detect the content of GDP-L-fucose precursor fructose-6-phosphate in recombinant Escherichia coli synthesizing 3-FL using control chassis, chassis 1, chassis 7 and chassis 8 as chassis strains in Example 5, so as to provide evidence for the beneficial effect on gene editing in this application.

[0127] The specific implementation steps are as follows:

[0128] Four recombinant *E. coli* strains were inoculated into LB liquid medium containing Kan and Str antibiotics and cultured overnight at 37°C. A 1% inoculum was then transferred to 50 ml of fresh LB medium and cultured at 30°C for 2 h. 0.3 mM IPTG was added as an inducer, along with 0–1 g / L glycerol and 20–30 g / L glucose (but no lactose). The culture was then fermented in shake flasks at 25°C for 24 h, and the bacterial culture was collected. The culture was diluted to 5 OD. 600nm For a concentration of / mL, take 1mL of bacterial culture and use the fructose-6-phosphate assay kit (Shanghai Qiyi Biotechnology Co., Ltd.) to detect the fructose-6-phosphate concentration according to the kit instructions.

[0129] The results are as follows Figure 3 As shown, knockout of the phosphofructokinase gene and / or pyruvate kinase gene significantly increased the intracellular content of fructose-6-phosphate, a precursor for GDP-L-fucose synthesis.

[0130] Example 10: Fed-feed fermentation of recombinant Escherichia coli

[0131] In this embodiment, the superior recombinant *Escherichia coli* obtained in Example 6 was used as the fermentation strain, and a 5L fermenter was used as the fermentation vessel. The fermentation medium was an organic nitrogen source medium based on LB medium. The formulation of the organic nitrogen source medium based on LB medium was as follows: 5 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, 5 g / L ammonium sulfate, 12.5 g / L potassium dihydrogen phosphate, 13.2 g / L dipotassium hydrogen phosphate trihydrate, 1 g / L citric acid, 2 g / L magnesium sulfate heptahydrate, 10 ml / L trace element mixture, and initial glucose and glycerol were both 20 g / L.

[0132] The specific fermentation process is as follows: Recombinant Escherichia coli was inoculated into LB liquid medium containing Kan and Str antibiotics and cultured overnight at 37°C. A 1% inoculum was then transferred to 200 ml of fresh LB medium as a secondary seed culture. When the OD... 600 When the concentration reaches approximately 4, the secondary seed culture is inoculated into a fermenter at a 2% inoculum for expansion culture at 37℃. During fermentation, the dissolved oxygen concentration is maintained at 20%-40%, the pH is controlled at 6.8-7.2, ammonia is used as a pH adjuster, and the aeration ratio is 2.5 vvm. When the cell OD... 600 When the temperature reaches 20-25°C, slowly cool it to 28°C, then add 0.3 mM IPTG as an inducer and 20 g / L lactose. During fermentation, lactose is fed in as a substrate, maintaining a lactose concentration of 10-25 g / L. Glucose and glycerol are simultaneously fed in during fermentation. The glucose concentration is maintained at 1-15 g / L in the early and middle stages, and feeding is stopped in the last 24 hours. The glycerol feeding rate is determined by the turbine rotation speed in the early and middle stages, maintaining a speed of 1000-1200 rpm, and then maintaining a speed of 800-1400 rpm after 42 hours.

[0133] Fermentation yield determination: A certain volume of the final fermentation broth was taken, and acetonitrile was added at a 1:1 volume ratio. The mixture was then diluted 2.5–20 times with distilled water, centrifuged at 13,000g for 5 min, and the supernatant was collected. The supernatant was separated by HPLC and detected using an evaporative light detector. The final yield was calculated using a standard curve.

[0134] The fermentation results of some recombinant strains in 5L tanks in nutrient-rich medium are shown in Table 4 (the control tank corresponding to the recombinant Escherichia coli fermented with glucose as the single carbon source).

[0135] As shown in Table 4, knockout of the phosphofructokinase gene significantly improved the yield and space-time yield of fucoidosyl lactose synthesized by the strain during fed-batch fermentation, and knockout of the pyruvate kinase pykF gene further improved the space-time yield of fucoidosyl lactose synthesized by the strain during fed-batch fermentation.

[0136] Table 4. Optimal strains for chassis fermentation and their corresponding 5L tank fermentation yields

[0137]

[0138]

[0139] Example 11: Determination of the effect of glucose residue on the yield of recombinant Escherichia coli fed-batch fermentation

[0140] This embodiment aims to detect the impact of glucose residue during fermentation on the fermentation of recombinant Escherichia coli in a fed tank.

[0141] In this embodiment, the recombinant Escherichia coli strains that synthesized 3-FL corresponding to the control chassis and chassis 1 were used as the strains. Two groups were set up, one with no glucose residue (residual concentration 0-1 g / L) and the other with glucose residue (residual concentration 1-15 g / L), for fermentation testing. The fermentation conditions and procedures were exactly the same as in Example 10, except for the glucose residue concentration.

[0142] The fermentation results are shown in Table 5. It can be seen from the table that glucose residue impairs the synthesis of 3-FL in the control strain, but promotes the synthesis of 3-FL in the pfkA knockout strain.

[0143] Table 5 shows the fermentation yield of the 3-FL synthetic strains corresponding to the control and control chassis 1 in a 5L tank under conditions with and without glucose residue.

[0144]

[0145] Example 12: Fed-feed fermentation of recombinant Escherichia coli in poor culture medium

[0146] This embodiment aims to test the fermentation performance of recombinant Escherichia coli in a fed-batch culture medium (inorganic nitrogen source medium) based on M9 medium.

[0147] In this embodiment, the recombinant Escherichia coli corresponding to the synthesis of 3-FL in chassis 1 was used as the strain, and fed-batch fermentation was carried out under almost the same fermentation conditions and steps as in Example 10. The only difference was that the culture medium was replaced by an organic nitrogen source medium based on LB medium in Example 10 with a barren medium (inorganic nitrogen source medium) based on M9 medium.

[0148] The depleted culture medium based on M9 medium was formulated as follows: 6.778 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 11 g / L ammonium chloride, 0.5 g / L sodium chloride, 0.24 g / L magnesium sulfate, 11 mg / L calcium chloride, and 10 ml / L trace element solution. The trace element solution contained 5 g / L EDTA, 0.83 g / L ferric chloride hexahydrate, 84 mg / L zinc chloride, 0.13 mg / L copper chloride dihydrate, 10 mg / L cobalt chloride dihydrate, 10 mg / L boric acid, and 1.6 mg / L manganese chloride tetrahydrate.

[0149] Fermentation results as follows Figure 4 As shown, the fermentation yield of recombinant Escherichia coli in the barren medium was slightly weaker than that in the organic nitrogen source medium, but it was still close to the highest level reported to date of 60.24 g / L, which proved the production capacity of this strain in the barren (inorganic nitrogen source) medium.

[0150] Example 13: Fermentation of recombinant Escherichia coli in 50L and 500L scale-up tanks

[0151] This embodiment aims to verify the industrial-scale production capacity of the recombinant Escherichia coli described in this application.

[0152] In this embodiment, the recombinant Escherichia coli synthesized with 3-FL corresponding to chassis 1 was used as the strain, and fed-batch fermentation was carried out under almost the same fermentation conditions and steps as in Example 10. The only difference was that the fermentation containers were changed to 50L and 500L fermenters.

[0153] The result of fermentation is as follows Figure 5 and Figure 6 As shown, although the fermentation yield decreased compared to the 5L tank, it still reached over 60g / L (50L) and over 50g / L (500L) respectively, proving its industrial-scale production capacity. The reasons why the yields of the 50L and 500L tanks were not higher than those of the 5L tank may be due to insufficient glucose control (there was no glucose remaining in the 50L tank, which may have been insufficient) and inadequate dissolved oxygen levels in the equipment (in the 500L tank).

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A recombinant Escherichia coli for synthesizing 3-fucosyllactose, characterized in that, The recombinant E. coli is knockout (a) and (b), knockout (a) and (c), or knockout (a) and (d) on the E. coli genome, and overexpresses a fucosyltransferase gene from Akkermansia muciniphila cafF ; (a) β - galactosidase genes lacZ and UDP-glucose lipid carrier transferase genes WcaJ ; (b) a phosphofructokinase gene pfkA ; (c) a phosphofructokinase gene pfkA and a phosphofructokinase gene pfkB ; (d) a phosphofructokinase gene pfkA and a pyruvate kinase gene pykF ; The recombinant E. coli also overexpresses a lactose permease gene LacY and genes associated with the GDP-L-fucose synthesis pathway. The GDP-L-fucose synthesis pathway-related genes include phosphomannomutase ManB GDP-mannose pyrophosphorylase ManC GDP-mannose 4,6-dehydratase Gmd and GDP-fucose synthase WcaG .

2. The recombinant E. coli synthesizing 3-fucosyllactose according to claim 1, characterized in that, The GDP-L-fucose synthesis pathway-related genes also include a 6-phosphomannose isomerase gene ManA .

3. The recombinant E. coli synthesizing 3-fucosyllactose according to claim 1, characterized in that, The overexpression of the gene of the mucinophilic akkermansia-derived fucosyltransferase cafF is achieved by means of a plasmid vector.

4. The recombinant E. coli synthesizing 3-fucosyllactose according to claim 2, characterized in that, The overexpression of lactose permease gene LacY phosphomannomutase ManB GDP-mannose pyrophosphorylase ManC GDP-mannose 4,6-dehydratase Gmd GDP-fucose synthetase WcaG is achieved by means of a plasmid vector; Overexpression of 6-phosphomannose isomerase gene ManA is achieved by replacing the endogenous promoter on the genome with a structurally active promoter.

5. The method for constructing a recombinant E. coli synthesizing 3-fucosyl lactose according to any one of claims 1 to 4, wherein comprising the steps of: S1, knocking out the galactoside permease gene of E. coli β - a galactoside permease gene lacZ , a UDP-glucose lipid carrier transferase gene WcaJ and a phosphofructokinase gene pfkA to obtain a recombinant bacterium; Alternatively, the galactoside hydrolase gene of E. coli is knocked out β - a phosphofructokinase gene lacZ , a UDP-glucose lipid carrier transferase gene WcaJ , a phosphofructokinase gene pfkA and a phosphofructokinase gene pfkB , to obtain a recombinant bacterium; Alternatively, the galactoside hydrolase gene of E. coli is knocked out β - a galactoside hydrolase gene lacZ , a UDP-glucose lipid carrier transferase gene WcaJ , a phosphofructokinase gene pfkA and a pyruvate kinase gene pykF , to obtain a recombinant bacterium; S2, overexpression of lactose permease gene using plasmid vector of Escherichia coli LacY , phosphomannomutase ManB , GDP-mannose pyrophosphorylase ManC , GDP-mannose 4,6-dehydratase Gmd , GDP-fucose synthetase WcaG and fucosyltransferase gene derived from Akkermansia muciniphila cafF , and obtaining recombinant Escherichia coli 6. The method for constructing a recombinant E. coli synthesizing 3-fucosyllactose according to claim 5, wherein, In the step S1, the 6-phosphomannose isomerase gene is also overexpressed ManA .

7. Use of the recombinant E. coli for synthesizing 3-fucosyllactose according to any one of claims 1 to 4 for fermentatively synthesizing 3-fucosyllactose.

8. A method for the fermentative synthesis of 3-fucosyl lactose, characterized in that, The recombinant E. coli for synthesizing 3-fucosyllactose according to any one of claims 1 to 4 is used for fermentation with glucose and glycerol as dual carbon sources and lactose as a common substrate, and the residual amount of glucose is maintained at 1 to 15 g / L during the fermentation process.

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