A recombinant bacterium for producing 2'-fucosyllactose by using kosakonia sp. strain, and a preparation method and application thereof

Abstract

The application discloses a kind of recombination bacteria for producing 2'-fucosyllactose by Kosakonia sp. Strain and its preparation method and application, to knock out wcaJ gene and heterologous expression futC and overexpression lacY of Kosakonia sp. CCTCC M2018092 as the bottom plate bacteria, knock out lacZ, ptsG, crr, edd, pgm, pfkA respectively to remodel glucose metabolic pathway, then based on the constructed genome scale metabolic network model, by flux balance analysis prediction and knock out key node gene gcd, the recombination bacteria can efficiently utilize glucose as sole carbon source fermentation production 2'-fucosyllactose, the yield of flask is increased by 101%, and the yield reaches 147.93±4.16 g / L in 5L fermenter for 108 h, which provides a new way of efficient and low-cost for industrial production of 2'-fucosyllactose.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant bacterium modified from Kosakonia sp. strain to produce 2′-fucosylated lactose, a preparation method thereof, and the application of the recombinant bacterium in the production of 2′-fucosylated lactose using glucose as a carbon source. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant naturally occurring solid component in breast milk, after lactose and fat. They possess important immune activity and significantly impact infant growth and development. As the most abundant functional component, fucoidan (2′-FL and 3-FL), composed of D-galactose, D-glucose, and L-fucose, has attracted considerable attention. Multiple studies have shown that fucoidan can significantly improve infant gut microbiota and influence cognitive development in young children. 2′-FL, which has the highest content, is currently approved for use as a nutritional supplement in infant formula. Furthermore, the prebiotic properties of 2′-fucoidan also make it a potentially effective active ingredient in cosmetics for skin care, demonstrating significant application potential.

[0003] Currently, the production of 2′-fucosyllactose mainly relies on the fermentation synthesis of *Escherichia coli*, a model microorganism using glycerol as a substrate. However, because the intracellular accumulation of the key precursor GDP-L-fucose requires the introduction of exogenous pathways, this chassis does not actually possess fermentation advantages for the large-scale production of 2′-fucosyllactose. High substrate costs, low carbon source utilization, long fermentation cycles, and high risk of strain degradation are a series of important problems currently facing the industrialization of 2′-fucosyllactose production.

[0004] Furthermore, metabolic modification with 2′-fucosylated lactose as the final product is still in the empirical accumulation stage, with a long and complex modification cycle. Genome-scale metabolic network models are mathematical models of metabolic networks systematically constructed based on genome annotation information. They are used to describe all known metabolic reactions and their interrelationships within a target organism, and can be used to analyze and predict the distribution of metabolic flux in target microorganisms, providing effective assistance in improving the production of metabolites from microbial fermentation. Currently, no reports have been found regarding genome-scale metabolic models that can guide the improvement of 2′-fucosylated lactose production. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a recombinant strain of Kosakonia sp. modified to produce 2′-fucosylated lactose; a second objective of the present invention is to provide a method for preparing the recombinant strain of Kosakonia sp. modified to produce 2′-fucosylated lactose; and a third objective of the present invention is to provide the application of the recombinant strain in the production of 2′-fucosylated lactose using glucose as a carbon source.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. A recombinant bacterium for producing 2′-fucosyllactose by modifying Kosakonia sp. strain, wherein the recombinant bacterium is obtained by modifying Kosakonia sp. CCTCC M2018092 strain, which has the wcaJ gene knocked out to block GDP-fucose flow to fucosyl, and simultaneously heterologously expresses the Helicobacter pylori fucosyltransferase futC and overexpresses the lactose permease lacY to facilitate metabolic flow to 2′-fucosyllactose, as follows: (1) Knock out the lacZ gene, the nucleotide sequence of which is shown in SEQ ID No.1; (2) Knock out the ptsG gene and the crr gene, wherein the nucleotide sequence of the ptsG gene is shown in SEQ ID No.2 and the nucleotide sequence of the crr gene is shown in SEQ ID No.3; (3) Knock out the edd gene, the nucleotide sequence of which is shown in SEQ ID No. 4; (4) Knock out the pgm gene, the nucleotide sequence of which is shown in SEQ ID No. 5; (5) Knock out the pfkA gene, the nucleotide sequence of which is shown in SEQ ID No. 6; (6) Knock out the gcd gene, the nucleotide sequence of which is shown in SEQ ID No.7.

[0008] In a preferred embodiment of the present invention, the recombinant bacteria produce 2′-fucosylated lactose using glucose as a carbon source, and the shake flask yield of 2′-fucosylated lactose reaches 24.78 ± 0.64 g / L.

[0009] In a preferred embodiment of the present invention, after the recombinant bacteria fermented in a 5L fermenter for 108 h, the final yield of 2′-fucosylated lactose reached 147.93 ± 4.16 g / L, and the glucose conversion rate reached 0.82 ± 0.04 g / g.

[0010] 2. A method for preparing a recombinant strain of Kosakonia sp. modified to produce 2′-fucosylated lactose, comprising the following steps: (1) In Kosakonia sp. CCTCC M2018092 strain, which had the wcaJ gene knocked out and heterologously expressed the fucosyltransferase futC of Helicobacter pylori and the lactose permease lacY, the lacZ, ptsG, crr, edd, pgm and pfkA genes were knocked out sequentially using the CRISPR / Cas9 gene editing system to obtain the recombinant strain K02; (2) In strain K02, the gcd gene was knocked out using the CRISPR / Cas9 gene editing system to shut down the glucose acidification reaction, thereby increasing the metabolic flux ratio of GDP-L-fucose, and obtaining the recombinant strain that was modified from Kosakonia sp. to produce 2′-fucosylated lactose.

[0011] In a preferred embodiment of the present invention, the plasmids used in the CRISPR / Cas9 gene editing system are pCas plasmid and pCB003 plasmid.

[0012] In a preferred embodiment of the present invention, the nucleotide sequence of the lacZ gene is shown in SEQ ID No. 1, the nucleotide sequence of the ptsG gene is shown in SEQ ID No. 2, the nucleotide sequence of the crr gene is shown in SEQ ID No. 3, the nucleotide sequence of the edd gene is shown in SEQ ID No. 4, the nucleotide sequence of the pgm gene is shown in SEQ ID No. 5, the nucleotide sequence of the pfkA gene is shown in SEQ ID No. 6, and the nucleotide sequence of the gcd gene is shown in SEQ ID No. 7.

[0013] 3. Application of the recombinant bacteria in the production of 2′-fucosylated lactose using glucose as a carbon source.

[0014] In a preferred embodiment of the present invention, the application includes the following steps: (1) Seed culture: The recombinant bacteria were inoculated into seed culture medium and cultured at 30 ℃ and 220 rpm for 24 hours; (2) Fermentation in a fermenter: The seed liquid obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 10%, and fermented for 108 hours at 30℃, pH 6.9 and 500 rpm. During the fermentation process, when the glucose concentration dropped to 20 g / L, glucose solution was added at a constant rate to maintain the glucose concentration at 5~20 g / L.

[0015] In a preferred embodiment of the present invention, the seed culture medium comprises: 30 g / L glucose, 3.6 g / L tryptone, and 7.2 g / L yeast extract.

[0016] In a preferred embodiment of the present invention, the fermentation medium comprises: 60 g / L glucose, 3.6 g / L tryptone, 7.2 g / L yeast extract, 0.7 g / L KH2PO4, 0.4 g / L (NH4)2HPO4, 1 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.8 g / L citric acid monohydrate, 3 mL / L Tween 80, 1 mL / L antifoaming agent, and 30 g / L lactose.

[0017] The beneficial effects of this invention are as follows: Guided by a genome-scale metabolic network model, this invention modifies a strain of Kosakonia sp. CCTCC M2018092 with a preference for GDP-L-fucose production. FBA analysis predicts the key glucose branch metabolic node gene gcd, resulting in a 101% increase in 2′-fucosyllactose yield in shake flasks. After 108 h of fermentation in a 5L fermenter, the final yield reaches 147.93 ± 4.16 g / L, with a glucose conversion rate of 0.82 ± 0.04 g / g, achieving efficient production of 2′-fucosyllactose using glucose as the carbon source. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Colony morphology of Kosakonia sp. CCTCC M2018092 on LB agar medium; Figure 2 The extracellular polysaccharide secretion characteristics of wild-type strain and recombinant strain K01 on Congo red agar medium; Figure 3 A map of the lacZ knockout plasmid; Figure 4 Electrophoresis verification image of gene knockout for pathway modification in the early stage; Figure 5 This is a metabolic modification plan for recombinant strain K01. Figure 6 A genome-scale metabolic model for Kosakonia sp. CCTCC M2018092 strain (K01); Figure 7 The FBA calculation results are for GDP-L-fucose as the final product; Figure 8 Electrophoresis verification image of GCD gene knockout; Figure 9 This is a verification result of the gcd gene knockout humidity experiment; Figure 10 The results of fed-batch fermentation of recombinant strain K03 in a 5L fermenter. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0020] The plasmids involved in this invention, pCas and pCB003, are both Escherichia coli CRISPR gene editing tools, derived from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Information on pCB003 plasmid can be found in the literature JIANG Y, CHEN B, DUAN CL, et al. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System [J]. Applied and Environmental Microbiology, 2015, 81(7):2506-2514:81. The wild-type strain involved in this invention refers to Kosakonia sp. CCTCC M2018092 (see Chinese Patent Application Publication No. CN 111234047 A, entitled "Extracellular Polysaccharides Rich in Fucosaccharide and Their Preparation Method and Application," whose whole genome sequence can be found in GenBank: CP034225.1). The recombinant strain K01 (Kosakonia (ΔwcaJ)) refers to the strain Kosakonia sp. CCTCC M2018092 in which the wcaJ gene is knocked out to block GDP-fucose flow to fucosaccharide, while simultaneously heterologously expressing the Helicobacter pylori fucosyltransferase futC and overexpressing the lactose permease lacY to direct metabolic flow towards 2'-fucosyllactose (construction method see Chinese Patent Application Publication No. CN 113025548 A, entitled "Recombinant Bacterium for Producing 2'-Fucosyllactose Based on Kosakonia sp. Strain and Its Method and Application").

[0021] The genes involved in this invention are as follows: lacZ gene (SEQ ID No. 1) encodes β-galactosidase, which catalyzes the breakdown of lactose into glucose and galactose. ptsG gene (SEQ ID No. 2) encodes EIICBGlc, and crr gene (SEQ ID No. 3) encodes EIIAGlc. This system plays a crucial role in bacterial metabolism, performing glucose transport and phosphorylation, and is a core component of the phosphoenolpyruvate-glucose phosphotransferase system (PTS). edd gene (SEQ ID No. 4) encodes 6-phosphoglucate dehydratase, catalyzing the dehydration of 6-phosphoglucate, an intermediate product in the glucose metabolism pathway, to 2-keto-3-deoxy-6-phosphogluconic acid. pgm gene (SEQ ID No. 5) encodes phosphoglucose mutase, whose main function is to catalyze the interconversion of glucose-1-phosphate and glucose-6-phosphate. pfkA gene (SEQ ID No. 6) encodes 6-phosphofructokinase A, a key rate-limiting enzyme in the glycolysis pathway, catalyzing the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. The gcd gene (SEQ ID No. 7) encodes glucose dehydrogenase, which catalyzes the oxidation of glucose to gluconic acid.

[0022] Example 1

[0023] Culture of wild strain and engineered recombinant strain K01

[0024] (1) Preparation of culture medium To prepare LB liquid culture medium, weigh out 5 g yeast powder, 10 g peptone, and 10 g sodium chloride, add 1 L of water and dissolve thoroughly. Dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 min, and cool before use.

[0025] To prepare LB agar medium, weigh out 5 g yeast powder, 10 g peptone, 10 g sodium chloride, and 20 g agar powder. Add 1 L of water to dissolve the mixture thoroughly, then dispense it into Erlenmeyer flasks. Sterilize at 121℃ for 20 min, then dispense it into petri dishes, cool, and store for later use.

[0026] To prepare TB agar medium, weigh out 12 g yeast powder, 24 g peptone, 60 g glucose, and 20 g agar powder, add 1 L of water to dissolve them completely, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 min, and cool before use.

[0027] (2) Morphological observation of wild strains After activating the wild-type strain with LB liquid medium, it was diluted and spread onto LB agar medium. The culture was then incubated upside down at 30°C for 2 days. The colony morphology was observed, and the results are shown in the attached figure. Figure 1 As shown: The strain grows well on LB agar medium, with round, yellow colonies that are easy to pick up.

[0028] (3) Comparison of phenotypic characteristics between wild-type strains and recombinant strains Wild-type strain and recombinant strain K01 were activated separately using LB liquid medium, then diluted and dropped onto TB agar medium, and incubated upside down at 30 ℃ for 2 days. After incubation, the samples were stained with Congo red for 15 minutes and eluted with sodium hydroxide solution. The results are shown in the attached figure. Figure 2 As shown: the wild strain secretes a large amount of extracellular polysaccharide to encapsulate the colonies in the culture medium, and the staining solution is not washed away, while the recombinant strain loses the ability to secrete polysaccharide and no longer secretes extracellular polysaccharide.

[0029] Example 2

[0030] Metabolic modification of recombinant strains with 2′-fucosylated lactose using glucose as carbon source

[0031] (1) Preparation of electrocompetent states Strain K01 was activated overnight in LB liquid medium, and the next day it was inoculated at 1% in 100 mL of LB liquid medium and cultured until OD500. 600 The concentration should be 0.6-0.8. Centrifuge at 4 ℃ and 6000 rpm for 8 minutes, discard the supernatant, wash twice with sterile pure water, and then wash once with sterile 10% glycerol solution. After thorough washing, suspend the bacterial cells in 1 mL of 10% glycerol solution, aliquot into sterile EP tubes, and store at -80 ℃ for later use.

[0032] (2) CRISPR / Cas9 toolbox-guided gene knockout Using the genome of recombinant strain K01 as a template, the lacZ, ptsG, crr, edd, pgm, and pfkA genes were knocked out using the CRISPR / Cas9 gene knockout system. Taking lacZ gene knockout as an example: The upstream and downstream homologous arms required for knockout are amplified using primers ΔlacZ-LA-F / R and ΔlacZ-RA-F / R, respectively, and then ligated using overlap PCR to obtain a fragment denoted as ΔlacZ-LRA; pCB003 is reverse-amplified using primers lacZ-N20-F and sgRNA-R to mutate the N20 sequence required for lacZ knockout, constructing a plasmid containing lacZ-N20, denoted as pCB003-lacZ-N20; the plasmid pCB003-lacZ-N20, double-digested with salⅠ and HindⅢ, is then cloned and recombinated with the ΔlacZ-LRA fragment in a one-step process to obtain the final plasmid containing the N20 sequence required for lacZ gene knockout and the upstream and downstream homologous arms, denoted as pCB003-lacZ-LRA. Figure 3 ).

[0033] Table 1 Target gene knockout primers

[0034] Plasmid pCas(kan) containing the Cas9 protein + ) and plasmid pCB003-lacZ-LRA(spc + The transformants were introduced into strain K01 via electroporation, and transformed individuals were screened using spectinomycin and kanamycin double antibody plates. Transformants were validated using primers V-lacZ-F / R; a shorter band compared to the control indicated successful knockout.

[0035] After successfully obtaining knockout positive transformants, they were first cultured in LB liquid medium containing kanamycin and IPTG at 30 °C and 220 rpm for 16 h to eliminate the pCB003-lacZ-LRA plasmid. Then, successful colonies were selected and transferred to new antibiotic-free LB liquid medium and cultured at 42 °C and 220 rpm for 16 h to eliminate the pCas plasmid.

[0036] Following the lacZ gene knockout method, the byproduct genes ptsG, crr, edd, pgm, and pfkA were sequentially knocked out using the CRISPR / Cas9 toolbox to complete the metabolic remodeling of strain K01, achieving efficient biosynthesis of 2′-fucosylated lactose using glucose as a carbon source. The modified strain was named K02: Kosakonia (ΔwcaJΔlacZΔptsGΔcrrΔeddΔpgmΔpfkA). The electrophoresis diagram verifying the gene knockout of the pathway modification is shown below. Figure 4 As shown in the attached diagram, the modification plan is as follows. Figure 5 As shown.

[0037] Example 3

[0038] Construction and application of a genome-scale metabolic model of Kosakonia sp. CCTCC M2018092

[0039] Model building The genome sequencing annotation results of Kosakonia sp. CCTCC M2018092 were obtained from the Uniport database, and a coarse model of the target strain (K01) was automatically constructed using CarveMe.

[0040] Manual simplification Based on literature review and analysis of the KEGG and BiGGModels databases, missing metabolic reactions in the coarse model were filled in, and stoichiometric non-conservation metabolic reactions were corrected. Then, the missing GDP-L-fucose synthesis reaction was added to construct a genome-scale metabolic network model incorporating the GDP-L-fucose synthesis pathway. The results are attached. Figure 6As shown, this model covers a total of 1,530 genes, 3,054 metabolic reactions, and 1,929 metabolites, and is named fuc101.

[0041] The fuc101 model has been uploaded to GitHub [https: / / github.com / y1zh2 / the-GEM-of-Kosakonia].

[0042] FBA Analysis The simplified metabolic model fuc101 of Kosakonia sp. CCTCC M2018092 was imported into the CAVE online platform (https: / / cave.biodesign.ac.cn / ). Using glucose as the carbon source and GDP-L-fucose as the end product, and with the on / off state of glucose acidification guided by the gcd gene identified in the model as the metabolic node, FBA metabolic analysis was performed. The results are attached. Figure 7 As shown, turning off the response guided by the gcd gene can save glucose consumption, increasing the proportion of its metabolic flux to GDP-L-fucose from 0.11% to 0.14%.

[0043] Wet test verification Following the lacZ gene knockout method, the gcd gene of strain K02 was knocked out using the CRISPR / Cas9 system described in Example 2. The electrophoresis image verifying the gcd gene knockout is shown below. Figure 8 As shown, the knockout strain K03: Kosakonia (ΔwcaJΔlacZΔptsGΔcrrΔeddΔpgmΔpfkAΔgcd) was obtained. Knockout strains K02 and K03, along with the control strain K01, were separately transferred to TB medium with glucose as the carbon source for shake-flask fermentation to verify the results, as shown in the attached figure. Figure 9 As shown, compared to strain K01, strain K02, obtained after the initial pathway modification, achieved highly efficient biosynthesis of 2′-fucosylated lactose using glucose as a carbon source, reaching 12.26 ± 0.09 g / L. Compared to strain K02, the 2′-fucosylated lactose yield of the gcd knockout strain increased by 101%, reaching 24.78 ± 0.64 g / L.

[0044] Example 4

[0045] Fermentation process of recombinant strain K03 Seed culture: The seed culture medium consisted of 30 g / L glucose, 3.6 g / L tryptone, and 7.2 g / L yeast extract. The seed culture process involved filling 500 mL shake flasks with 100 mL of liquid, incubating at 30 ℃, shaking at 220 rpm, and incubating for 24 hours.

[0046] The fermentation process in the upper tank was as follows: The fermentation formula consisted of glucose (60 g / L), tryptone (3.6 g / L), yeast powder (7.2 g / L), KH₂PO₄ (0.7 g / L), (NH₄)₂HPO₄ (0.4 g / L), MgSO₄·7H₂O (1 g / L), NaCl (0.5 g / L), citric acid monohydrate (0.8 g / L), Tween 80 (3 mL / L), defoamer (1 mL / L), and lactose (30 g / L). A 10% inoculum was added to the fermenter. Aeration was set at 5 L / min, pH was maintained at 6.9 with ammonia, the rotation speed was 500 rpm, the fermentation temperature was 30 ℃, and the fermentation time was 108 h. A 400 g / L glucose solution was prepared in the feeding tank. When the glucose concentration in the feeding tank reached 20 g / L, feeding was started at a uniform rate, maintaining a glucose concentration of 5–20 g / L until the end of fermentation. The fermentation results are shown in the attached figure. Figure 10 As shown, the final yield of 2′-fucosylated lactose reached 147.93 ± 4.16 g / L, the glucose conversion rate reached 0.82 ± 0.04 g / g, and the production rate was 1.37 ± 0.03 g / L·h.

[0047] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Furthermore, the present invention is applicable to the modification of 3-fucosylated lactose and other fucose derivatives. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A recombinant strain of Kosakonia sp. modified to produce 2′-fucosylated lactose, characterized in that, The recombinant strain was obtained by modifying Kosakonia sp. CCTCC M2018092, a strain that knocks out the wcaJ gene to block GDP-fucose flow to fucosaccharide, while heterologously expressing the Helicobacter pylori fucosyltransferase futC and overexpressing the lactose permease lacY to facilitate metabolic flow to 2′-fucosyllactose, as follows: (1) Knock out the lacZ gene, the nucleotide sequence of which is shown in SEQ ID No.1; (2) Knock out the ptsG gene and the crr gene, wherein the nucleotide sequence of the ptsG gene is shown in SEQ ID No.2 and the nucleotide sequence of the crr gene is shown in SEQ ID No.3; (3) Knock out the edd gene, the nucleotide sequence of which is shown in SEQ ID No. 4; (4) Knock out the pgm gene, the nucleotide sequence of which is shown in SEQ ID No. 5; (5) Knock out the pfkA gene, the nucleotide sequence of which is shown in SEQ ID No. 6; (6) Knock out the gcd gene, the nucleotide sequence of which is shown in SEQ ID No.

7.

2. The recombinant bacteria according to claim 1, characterized in that, The recombinant bacteria produced 2′-fucosylated lactose using glucose as a carbon source, and the shake flask yield of 2′-fucosylated lactose reached 24.78 ± 0.64 g / L.

3. The recombinant bacteria according to claim 1, characterized in that, After fermentation in a 5L fermenter for 108 h, the recombinant bacteria achieved a final yield of 147.93 ± 4.16 g / L of 2′-fucosylated lactose and a glucose conversion rate of 0.82 ± 0.04 g / g.

4. A method for preparing a recombinant strain for producing 2′-fucosylated lactose by modifying Kosakonia sp. strains as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) In Kosakonia sp. CCTCC M2018092 strain, which had the wcaJ gene knocked out and heterologously expressed the fucosyltransferase futC of Helicobacter pylori and the lactose permease lacY, the lacZ, ptsG, crr, edd, pgm and pfkA genes were knocked out sequentially using the CRISPR / Cas9 gene editing system to obtain the recombinant strain K02; (2) In strain K02, the gcd gene was knocked out using the CRISPR / Cas9 gene editing system to shut down the glucose acidification reaction, thereby increasing the metabolic flux ratio of GDP-L-fucose, and obtaining the recombinant strain that was modified from Kosakonia sp. to produce 2′-fucosylated lactose.

5. The preparation method according to claim 4, characterized in that, The CRISPR / Cas9 gene editing system uses pCas plasmid and pCB003 plasmid.

6. The preparation method according to claim 4, characterized in that, The nucleotide sequence of the lacZ gene is shown in SEQ ID No. 1, the nucleotide sequence of the ptsG gene is shown in SEQ ID No. 2, the nucleotide sequence of the crr gene is shown in SEQ ID No. 3, the nucleotide sequence of the edd gene is shown in SEQ ID No. 4, the nucleotide sequence of the pgm gene is shown in SEQ ID No. 5, the nucleotide sequence of the pfkA gene is shown in SEQ ID No. 6, and the nucleotide sequence of the gcd gene is shown in SEQ ID No.

7.

7. The use of the recombinant bacteria as described in any one of claims 1-3 in the production of 2′-fucosylated lactose using glucose as a carbon source.

8. The application according to claim 7, characterized in that, The application includes the following steps: (1) Seed culture: The recombinant bacteria were inoculated into seed culture medium and cultured at 30 ℃ and 220 rpm for 24 hours; (2) Fermentation in a fermenter: The seed liquid obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 10%, and fermented for 108 hours at 30 ℃, pH 6.9 and 500 rpm. During the fermentation process, when the glucose concentration dropped to 20 g / L, glucose solution was added at a constant rate to maintain the glucose concentration at 5~20 g / L.

9. The application according to claim 8, characterized in that, The seed culture medium contains: 30 g / L glucose, 3.6 g / L tryptone, and 7.2 g / L yeast extract.

10. The application according to claim 8, characterized in that, The fermentation medium contains: glucose 60 g / L, tryptone 3.6 g / L, yeast extract 7.2 g / L, KH2PO4 0.7 g / L, (NH4)2HPO4 0.4 g / L, MgSO4·7H2O 1 g / L, NaCl 0.5 g / L, citric acid monohydrate 0.8 g / L, Tween 80 3 mL / L, antifoaming agent 1 mL / L, and lactose 30 g / L.

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