Recombinant microorganism expressing fucosyltransferase and method for producing 2'-fucosyllactose using the same

Abstract

The present invention relates to a microorganism into which a 2'-fucosyltransferase has been introduced and a method for producing 2'-fucosyllactose using the same. The microbial fucosyltransferase of the present invention is safer and more active than the conventional enzyme derived from Helicobacter pylori.

Description

[Technical field]

[0001] The present invention relates to a recombinant microorganism transformed to express fucosyltransferase, and a method for producing 2'-fucosyllactose using the same. [Background technology]

[0002] 137 types of oligosaccharides, including the three with the highest content in breast milk, are fucosylated, accounting for about 77%, and the remaining oligosaccharides are mostly sialylated (39), accounting for about 28%. Among them, 2'-fucosyllactose and 3'-fucosyllactose have prebiotic effects, inhibitory effects on the adhesion of pathogenic bacteria to the intestines, and immune system regulation effects. The prebiotic effect has been the most studied to date, and breast milk oligosaccharides selectively promote the growth of Bifidobacterium bifidum, which grows as a dominant species in the intestines of infants in the early stages, but are not used by harmful bacteria. In addition, breast milk oligosaccharides are known to inhibit the adhesion of pathogenic bacteria to the intestines, because the cell wall polysaccharide structure of pathogenic bacteria that bind to intestinal lectins is sometimes similar to some structures of breast milk oligosaccharides. Therefore, it is believed that breast milk oligosaccharides provide water-soluble ligand analogues that allow breast-fed infants to resist infection by pathogenic bacteria. However, it is known that about 20% of women cannot synthesize 2'-fucosyllactose in their bodies due to mutations in the fucosyltransferase that synthesizes it. This has led to the need for industrial production of 2'-fucosyllactose.

[0003] Some of the human milk oligosaccharides are also found in small amounts in the milk of most mammals (primates, cows, pigs, goats, sheep, and elephants). Of these, goat milk has the most similar composition to human milk oligosaccharides, and a large-scale membrane-based separation technique has been proposed from whey by-products in the goat milk cheese production process. In addition, the recently developed solid-phase chemical synthesis method allows the production of Lewis X-Lewis Y sugars within a day, and crystalline intermediate technology allows the rapid synthesis of 2'-fucosyllactose. However, despite the development of the above separation and chemical synthesis methods, there remain several obstacles to the industrial mass production of human milk oligosaccharides, because the low stereoselectivity, low total yield, and use of toxic reagents of this technology make it unsuitable for the food or pharmaceutical industry. Therefore, a more environmentally friendly and high-yield production method is needed, and recently, a trend has been reported in which a method for producing high-concentration 2'-FL using microorganisms has been reported. However, the 2'-fucosyllactose biosynthesis gene is not suitable for domestic production and sales approval because it may affect consumer perception negatively when applied mainly to breastfeeding substitute powdered milk and infant foods using 2'-fucosyllactose obtained from Bio Safety Level 2 strain Helicobacter pylori. Therefore, it is necessary to apply a more stable gene as a food additive and produce 2'-fucosyllactose by gene transfer of Bio Safety Level 1 strain. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides a recombinant microorganism having a transferase that produces fucosyllactose, which is a food, cosmetic, and pharmaceutical material, and which has a 2'-fucosyltransferase derived from a Biosafety level 1 strain, such as Bacillus megaterium or Akkermansia muciniphila, inserted therein, and a method for effectively producing 2'-fucosyllactose using the same. [Means for solving the problem]

[0005] One example of the present invention relates to a recombinant microorganism transformed to express α-1,2-fucosyltransferase. The α-1,2-fucosyltransferase may include one or more selected from the group consisting of SEQ ID NOs: 1 to 5. Effect of the Invention

[0006] The present invention provides a recombinant microorganism into which a fucosyltransferase derived from a biosafety level 1 strain has been introduced, which does not pose a concern to consumers in terms of stability, and a method for producing 2'-fucosyllactose using the same. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows the results of SDS-PAGE protein expression and predicted protein sizes of the enzymes selected according to the embodiments of the present invention. [Diagram 2] FIG. 2 is a graph showing the 2FL production tendency in a flask reaction (upper row) and a test tube reaction (lower row) according to an embodiment of the present invention. [Diagram 3] FIG. 1 shows the results of comparing the 2FL production activity of an α-1,2-fucosyltransferase according to an example of the present invention using E. coli host. [Figure 4]FIG. 1 shows the results of comparing the productivity of various 2FL strains using B. megaterium 14581 as a host. [Diagram 5] FIG. 1 shows a GDP-fucose cassette according to an embodiment of the present invention. [Figure 6] FIG. 1 shows the productivity of 2FL confirmed using a strain into which a GDP-fucose cassette according to an embodiment of the present invention has been introduced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In the present invention, the polypeptide having 2-fucosyltransferase activity is not limited to the amino acid sequences of SEQ ID NOs: 1 to 5, but includes amino acid sequences formed by substitution, insertion or deletion of some amino acid residues in the amino acid sequences of SEQ ID NOs: 1 to 5. If such amino acid-modified proteins or polypeptides have 2-fucosyltransferase activity, they can convert lactose and GDP-fucose as substrates into fucosyllactose.

[0009] The nucleic acid sequence encoding the α-1,2-fucosyltransferase may comprise, for example, one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 14-18.

[0010] The recombinant microorganism may be a GRAS (Generally Recognized As Safe) microorganism. The gene used to produce 2'-fucosyllactose to date is not stable because it uses 2'-fucosyllactose obtained from Helicobacter pylori, and in order to solve this problem, the recombinant microorganism according to the present invention may be a Bio Safety Level 1 microorganism. Fucosyltransferase derived from Helicobacter pylori is not suitable as an enzyme for producing food materials because it is Biosafety Level 2, but the fucosyltransferase according to the present invention is Biosafety Level 1, so it has the advantage of not causing any consumer concerns about stability.

[0011] The recombinant microorganism according to the present invention has a significantly increased 2'-fucosyllactose productivity compared to a control group containing α-1,2-fucosyltransferase derived from Helicobactor pyori, and thus allows for improved stability and productivity.

[0012] Specifically, the recombinant microorganism according to one embodiment of the present invention may have 2 times or more, 2.1 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, 6 times or more, 6.5 times or more, 7 times or more, or 7.5 times or more 2'-fucosyllactose productivity compared to a control group containing α-1,2-fucosyltransferase derived from Helicobactor pyori. Therefore, the recombinant microorganism can be useful for producing α-1,2-fucose and can improve the production yield of α-1,2-fucose.

[0013] A recombinant microorganism according to one embodiment of the present invention may not have the ability to produce 2'-fucosyllactose before recombination, and may have the ability to produce 2'-fucosyllactose through recombination.

[0014] The method for transforming the recombinant microorganism may be any transformation method known in the art, without particular limitation, and may be selected from bacterial protoplast fusion, electroporation, projectile bombardment, and infection using a viral vector.

[0015] A recombinant microorganism according to the present invention may be a Bacillus sp. microorganism, a Corynebacterium sp. microorganism, an Escherichia sp. microorganism, or a yeast.

[0016] Specifically, the Bacillus sp. microorganism may be Bacillus megaterium, Bacillus subtilis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, or Bacillus stearothermophilus.

[0017] Specifically, the Corynebacterium sp. microorganism may be Corynebacterium glutamicum.

[0018] Specifically, the Escherichia sp. microorganism may be Escherichia coli.

[0019] Specifically, the yeast may be Saccharomyces cerevisiae or Candida utilis.

[0020] The recombinant microorganism according to one embodiment of the present invention may have fucose synthesis ability or may have improved fucose synthesis ability. In order to produce 2'-fucosyllactose, all microorganisms basically require the introduction of an exogenous α-1,2-fucosyltransferase, but due to differences in the genetic characteristics of the strains of each microorganism, Escherichia coli and Bacillus megaterium can produce 2'-fucosyllactose even if only α-1,2-fucosyltransferase is inserted, while Bacillus subtilis, Corynebacterium, yeast microorganisms, etc. can additionally introduce fucose synthase.

[0021] Specifically, the recombinant microorganism according to one embodiment of the present invention may be characterized by possessing or overexpressing one or more selected from the group consisting of GDP-D-mannose-4,6-dehydratase, GDP-L-fucose synthase (WcaG), phosphomannomutase, and GTP-mannose-1-phosphate guanylyltransferase. Thus, the recombinant microorganism according to one embodiment of the present invention has improved fucose synthesis ability together with α-1,2-fucosyltransferase expression characteristics, and thus can further improve the α-1,2-fucose production yield.

[0022] For example, the fucose synthase may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 19-22.

[0023] For example, the GTP mannose-1-phosphate guanylyltransferase may comprise the amino acid sequence of SEQ ID NO:19 or be encoded by the nucleic acid sequence of SEQ ID NO:23.

[0024] For example, the phosphomannomutase may comprise the amino acid sequence of SEQ ID NO:20 or be encoded by the nucleic acid sequence of SEQ ID NO:24. For example, the GDP-L-fucose synthase (WcaG) may comprise the amino acid sequence of SEQ ID NO:21 or be encoded by the nucleic acid sequence of SEQ ID NO:25.

[0025] For example, the GDP-D-mannose-4,6-dehydratase may comprise the amino acid sequence of SEQ ID NO:22 or be encoded by the nucleic acid sequence of SEQ ID NO:26.

[0026] The recombinant microorganism according to one embodiment of the present invention may express or overexpress a lactose membrane transport protein. The lactose membrane transport protein may be capable of introducing lactose, which is a substrate for 2'-fucosyllactose, into cells to improve 2'-fucosyllactose production ability.

[0027] Specifically, the lactose membrane transport protein may be Lac12 and / or LacY.

[0028] For example, the Lac12 may have the amino acid sequence of SEQ ID NO: 27. For example, the nucleic acid encoding the Lac12 may comprise the nucleic acid sequence of SEQ ID NO: 29. As an example, the Lac12 may be encoded by the nucleic acid sequence of SEQ ID NO: 29.

[0029] For example, the LacY may have the amino acid sequence of SEQ ID NO: 28. For example, the nucleic acid sequence encoding the LacY may comprise the nucleic acid sequence of SEQ ID NO: 30. As an example, the LacY may be encoded by the nucleic acid sequence of SEQ ID NO: 30.

[0030] Another example of the present invention relates to a composition for producing 2'-fucosyllactose, comprising at least one selected from the group consisting of α-1,2-fucosyltransferase obtained by using the recombinant microorganism of the present invention, the cells of the microorganism, the culture of the microorganism, the disruption of the microorganism, and the extract of the disruption or culture. The recombinant microorganism, the α-1,2-fucosyltransferase, the 2'-fucosyllactose, etc. are as described above.

[0031] The culture contains the enzyme produced by the recombinant microorganism, and may be in a cell-free form that does not contain the recombinant microorganism or the recombinant microorganism. The lysate refers to a lysate obtained by lysing the recombinant microorganism or a supernatant obtained by centrifuging the lysate, and contains the enzyme produced by the recombinant microorganism. In this specification, unless otherwise specified, the recombinant microorganism used for producing 2'-fucosyllactose is used to mean one or more selected from the group consisting of the bacterial cell of the microorganism, the culture of the microorganism, and the lysate of the microorganism.

[0032] Yet another example of the present invention relates to a method for producing 2'-fucosyllactose, comprising the step of culturing a recombinant microorganism according to the present invention in a medium containing lactose.

[0033] The production method may involve reacting one or more selected from the group consisting of α-1,2-fucosyltransferase obtained from a culture, cells of the microorganism, a culture of the microorganism, a disrupted product of the microorganism, and an extract of the disrupted product or culture with a lactose-containing raw material to produce 2'-fucosyllactose. EXAMPLES

[0034] The present invention will now be described in more detail with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0035] Example 1. Screening for a-1,2-fucosyltransferase using bioinformatics tools Using bioinformatics tools, we selected candidates for a-1,2-fucosyltransferase enzymes (hereafter referred to as 2FT) derived from Biosafety level 1. Specifically, we removed sequences with >90% homology to Bacteria and GRAS bacteria from enzymes predicted to have a-1,2-fucosyltransferase function according to NCBI (https: / / www.ncbi.nlm.nih.gov / protein / ), and used a protein morphology visualization program (http: / / wlab.ethz.ch / protter) to determine whether or not they contained membrane proteins. A group of 32 candidates was selected using computer bioinformatics tools (Table 1). In addition, when the presence or absence of membrane proteins that may be problematic in protein expression was predicted using a program, no significant membrane proteins were predicted for the remaining 31 enzymes among the 32 candidates, excluding those derived from Lactococcus lactis.

[0036] [Table 1]

[0037] Among the candidates, Biosafety level 1 Bm2FT was subsequently confirmed to be reactive, and an additional candidate group was searched for enzymes derived from Bacillus megaterium based on Bm2FT protein sequence homology. Based on the sequence information of Bm2FT, the enzymes were searched for using the BLAST program (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The list of selected enzyme candidates is shown in Table 2.

[0038] [Table 2-1]

[0039] [Table 2-2]

[0040] Comparative Example 1. Preparation of conventional α-1,2-fucosyltransferase 2FT (FutC) derived from Helicobactor pyori was selected as a control because it has the highest activity among 2FT enzymes and has been used for 2FL production in various publications. In the paper "Search for bacterial α1,2-fucosyltransferases for whole-cell biosynthesis of 2'-fucosyllactose in recombinant Escherichia coli," Microbiol Res. 2019 May;222:35-42, 2FT reactivity derived from Thermosynechococcus elongates was confirmed using E. coli, and this was selected as the control group. Although the reactivity of 2FT derived from Akkermansia muciniphila could not be confirmed in WO2015 / 175801, it was selected as a control group to reexamine productivity. The list and information of the selected control enzymes are shown in Table 3.

[0041] [Table 3]

[0042] Example 2. Cloning of expression plasmids for E. coli In order to confirm the activity of the enzyme selected in Example 1 and the control enzyme in Comparative Example 1 in E. coli, they were cloned into an enzyme expression plasmid. Specifically, in order to optimize the codons of the enzyme sequence selected in Example 1 for E. coli, the sequence was input into a codon optimization program (http: / / genomes.urv.es / OPTIMIZER / ), and then Escherichia_coli was selected to perform a guided random codon optimization search. The confirmed enzyme sequence was synthesized, and PCR was performed using the primers listed in Table 5 according to the conditions to amplify the synthetic gene. In the case of amplifying the pET24ma plasmid, TAG_PET24ma_F or TAA_PET24ma_F PCR primer was used depending on whether the stop codon sequence of the synthetic gene was TAG or TAA. Primer sequence information and name for each enzyme are shown in Table 4.

[0043] [Table 4]

[0044] For Te2FT, Bt2FT, Bm2FT, Lg2FT, Ls2FT, and Ll2FT synthetic genes, after gene purification, each was treated with 1 μL NEB restriction enzyme (Nde1 / Xho1) and stored at 37°C for 3 hours. After that, to remove the restriction enzyme and buffer, the gene was purified again, and ligation was performed for 4 hours with pET24ma treated with the same restriction enzyme at 16°C. All the remaining synthetic genes except for the Te2FT, Bt2FT, Bm2FT, Lg2FT, Ls2FT, and Ll2FT synthetic genes were gel prepped for cloning by the Gibson assembly method. After prep, the concentration of each gene was confirmed using a nano dropper, and 2X NEBuilder® HiFi DNA Assembly Master Mix was added so that the vector to insert ratio was 1:2 and the total reaction volume was 8μL. The reaction was then carried out at 50℃ for 1 hour. The cloning gene was transformed into DH10b E.coli competent cells, which were then smeared on a plate containing Km antibiotic and grown overnight. Five colonies were selected from each plate and colony PCR was performed using the primers in Table 5 that correspond to the genes. Colonies that showed bands of the corresponding size on the DNA gel were inoculated into 3 ml of LB medium containing Km antibiotic and grown overnight. The next day, the cells were collected, and DNA was extracted and sequenced.

[0045] Example 3. Confirmation of enzyme protein expression We confirmed whether each gene cloned in E. coli expressed protein. E. coli BL21(DE3)△fucl△fucK::AprR lacZ△m15::PlacUV5 competent cells were transformed with each 2FT vector and pCDFm::PT7manC-manB and PT7-gmd-wcaG, which have the enzyme producing GDP-fuc. Selection was performed by spreading on plates containing Km and Amp antibiotics. The colony was inoculated into 3 mL of medium containing Km and Amp antibiotics, and the seeds were grown overnight. 200 μL seeds and 0.1 mM IPTG were added to 4 mL of LB, and the medium was grown at 30°C for 16 hours. The cells grown in 4 mL were collected by centrifugation, and the supernatant was discarded. After adding 500 μL of buffer, the cells were disrupted with a bead beater, and centrifuged at 13,000 rpm for 20 minutes to collect the soluble proteins. SDS-PAGE gel was loaded with a size marker. The results of SDS-PAGE of total and soluble protein expression of the selection enzymes (left side of Figure 1) and the predicted protein sizes (right side of Figure 1) are shown in Figure 1. In Figure 1, ManB, ManC, Gmd, and WcaG are proteins expressed by the additional pCDFm::PT7manC-manB+PT7-gmd-wcaG plasmid. As shown in Figure 1 , overexpression was confirmed for eight enzymes among the proteins compared through SDS-PAGE: Bm2FT, Te2FT, Hp2FT, Am2FT_2, Fp2FT, Ri2FT, Bm2FT_2, and Bm2FT_3.

[0046] Example 4. Optimization of reaction analysis using test tubes 2FL productivity was confirmed through a total of 50mL culture in a 250mL flask. When there are many enzyme candidates, it is necessary to reproduce the experiment three or more times for a reliable comparative analysis, but the existing 50mL culture conditions have the disadvantage of requiring more effort than the amount required for analysis (1mL). In order to grasp the fast reaction tendency of various candidates, we will establish a method to confirm the enzyme reaction by culturing 4mL in a 10mL test tube. The plasmids in which Hp2FT, Bm2FT, and Bt2FT were cloned were transformed into E. coli strains together with the pCDFm::PT7manC-manB+PT7-gmd-wcaG plasmid. Three colonies were inoculated onto each plate and overnight seed culture was performed. 500μL of the seeds were induced into a 50mL flask culture. OD 600 When the pH reached about 0.8, 0.1 mM IPTG and 10 mM lactose were added, and the mixture was analyzed by HPLC 20 hours later. For the test tube reaction, 200 μL of seed was added to 4 mL of medium supplemented with 0.1 mM IPTG and 10 mM lactose. The step of checking the OD of the flask can be omitted for the test tube reaction. After 20 hours, the reaction was analyzed by HPLC, and the 2FL production trends for the flask reaction (top row of Figure 2) and the test tube reaction (bottom row of Figure 2) are shown in Figure 2. As shown in Figure 2, the established test tube reaction conditions produced 1 / 3 the productivity of 2FL compared to the flask condition, but the reaction trends were the same, demonstrating that the reaction is suitable for the comparative analysis of various enzymes at the same time.

[0047] Example 5. Comparison of 2FL productivity through HPLC analysis The reactivity of the enzyme selected in Example 1 and three control enzymes was compared. The plasmids to be compared were transformed into E. coli and grown at 37°C. Three colonies were inoculated onto each plate and overnight seed culture was performed. 200 μL of the seeds were added to 4 mL of medium supplemented with 0.1 mM IPTG and 10 mM lactose. After 20 hours, the results were analyzed by HPLC. The results of the comparison of 2FL production activity of various 2FTs using E. coli host are shown in Figure 3. As shown in Figure 3, a total of five 2FL-producing enzymes were obtained, including three enzymes with equal or higher activity than FutC: Am2FT_2, Bm2FT, Bm2FT_2, Bm2FT_3, and Bs2FT were confirmed to produce 122%, 207%, 84%, 100%, and 21% 2FL, respectively, compared to FutC, through HPLC analysis. The sequences of the five 2FL-producing enzymes are shown in Table 5.

[0048] [Table 5]

[0049] Example 6. Comparison of 2'-FL productivity using fucosyltransferase in Corynebacterium strains Corynebacterium glutamicum ATCC13032 was used to construct the plasmid and produce 2'-fucosyllactose (2'-FL). To construct the pMBC plasmid, the manB gene was amplified from the genomic DNA of Corynebacterium glutamicum ATCC13032 through PCR using two DNA primers, and then inserted into the pCES208 plasmid. The manC gene was amplified from the constructed plasmid again through PCR using two DNA primers from the genomic DNA of Corynebacterium glutamicum ATCC13032, and then inserted into the plasmid to construct pMBC. To construct the pEGW plasmid, the gmd-wcaG gene cluster was amplified from the genomic DNA of Escherichia coli K-12 through PCR using two DNA primers, and then inserted into the plasmid to construct pEGW. The fucosyltransferase of SEQ ID NO: 2 was also introduced. When the C. glutamicum host was used, the strain carrying Bm2FT and the GDP-fucose cassette was added to 4 mL of medium containing 10 mM lactose to produce 2FL, and after 60 hours of culture, approximately 120 mg / L of 2FL was produced. The strains carrying pMBC and pEGW empty vectors did not produce 2FL.

[0050] Example 8. Comparison of 2'-FL productivity through fucosyltransferase in Bacillus megaterium strains The Am2FT_2 and Bm2FT enzymes, which showed the greatest increase in activity compared to FutC when using an E. coli host, were each transformed into a B. megaterium 14581 (Korea Center for Microorganisms (KCCM) accession number 40441) host, and the 2FL productivity of each was compared. To accurately confirm the reactivity, b. megaterium 14581 into which the empty vector p1525 without the 2FT gene was inserted was used as a control. Figure 4 shows the results of comparing various 2FL productivity using the B. megaterium 14581 host. As shown in Figure 4, when using B. megaterium host, the strain containing the p1525 empty vector did not produce 2FL, while Bm2FT and FutC produced an average of 40.3 mg / L and 18.8 mg / L of 2FL, respectively, after 64 hours of reaction, confirming that the Bm2FT enzyme showed a 2.1-fold increase in productivity compared to FutC. To date, no research has been reported on the production of human milk oligosaccharides using a B. megaterium strain. Since the B. megaterium 14581 host is a biosafety level 1 strain and is harmless to the human body, the Bm2FT established in this invention was introduced into a GRAS strain, achieving more than twice the productivity compared to the existing enzyme (FucT).

[0051] Example 9. Comparison of 2'-FL productivity through fucosyltransferase in Bacillus subtilis strains The Bm2FT enzyme, which showed the highest increase in activity compared to FutC when using the E. coli host, was transformed into a Bacillus subtilis host, and the 2'-FL productivity of each enzyme was compared. To accurately confirm the reactivity, B. subtilis 168 (Accession No.: (ATCC (registered trademark) 23857) transformed with the empty vector pBE-S in which the 2'-FT gene was not cloned was used as a control. TM) was used. The vector used was pBE-S for B. subtilis secretion, which contains the aprE promoter that allows constitutive expression. Since the secretion function is not necessary during cloning, the aprE signal peptide was removed when performing 2'-FT cloning. Since B. subtilis 168 does not have the four enzymes that produce GDP-fucose, genes encoding the four enzymes were cloned into one vector at once. The four genes (manB, manC, gmd, and wcaG) were derived from E. coli and were obtained by gene amplification through PCR. The vector used to clone the four genes at once was p3STOP1623-2RBShp, an E. coli-Bacillus shuttle vector. This vector can be used not only in B. megaterium but also in B. subtilis. This vector has two RBSs, making it very easy to clone multiple genes. The promoter of this vector is the xylose inducible promoter PxylA. The GDP-fucose cassette was constructed in the gene order shown in Figure 5. The Gibson assembly method was applied to the vector and the four genes, as in the Bm2FT cloning described above. However, since the size of the vector is about 6.6 Kb, PCR may cause sequence errors if performed all at once, so a primer was created in the middle of the vector and PCR was performed by dividing it in half. To purify a total of six PCR fragments, the entire PCR mixture was electrophoresed and gel extracted. The purified PCR fragments were measured for DNA concentration, and the content was adjusted according to concentration and size, and Gibson assembly was performed. Then, E. coli was transformed in the same way to obtain single colonies, and the insertion of the plasmid was confirmed through colony PCR. The colonies confirmed by PCR were cultured in LB, and the plasmid was extracted, and the four genes were confirmed to be sufficiently cloned through sequence analysis. The plasmids confirmed by sequencing were transformed into B. subtilis 168 to confirm productivity. The insertion of the plasmid was confirmed by colony PCR in the same way as in the method of transformation into E. coli, and then cultured for comparison of 2'-FL productivity. As shown in Figure 6, when using B. subtilis host, the strain containing pBE-S and p3STOP1623-2RBShp empty vector did not produce 2FL, but the strain containing Bm2FT and GDP-fucose cassette produced about 150mg / L of 2FL after 63 hours of reaction. This is a result of an increase in productivity of more than 7 times compared to the average 2FL production of 18.8mg / L by conventional FutC.

[0052] Example 10. Comparison of 2'-FL productivity through fucosyltransferase in Saccaromyces Cerevisiae strains Saccharomyces cerevisiae CEN.PK2-1c was used to construct the plasmid and produce 2'-fucosyllactose (2'-FL). The GDP-L-fucose gene, which is not present in Saccharomyces cerevisiae, was cloned from E. coli K-12 and used. To construct the pRSK426 plasmid, the gmd-wcaG gene cluster was amplified from E. coli K-12 genomic DNA using two DNA primers in a PCR reaction, and then inserted into the plasmid to construct pRSK426. Am2FT_2 and Bm2FT enzymes, which showed the greatest increase in activity compared to FutC when using E. coli host, were transformed into S. cerevisiae host, respectively, and the 2FL productivity of each was compared. The three recombinant plasmids were transformed into Saccharomyces cerevisiae CEN.PK2-1c strain and selected on YPD solid medium containing G418 antibiotic. The selected strains were cultured in 50mL liquid production medium containing G418 antibiotic for 2 days, and 2FL productivity was measured by HPLC analysis. When using S.cerevisiae CEN.PK2-1c host, the strain containing pRSK426 empty vector did not produce 2FL, while the strains containing Bm2FT, Am2FT_2 and GDP-fucose cassette produced 80mg / L and 40mg / L of 2FL, respectively, after 48 hours of culture.

Claims

1. A recombinant microorganism which has been transformed so as to express α-1,2-fucosyltransferase comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 and 2.

2. The recombinant microorganism of claim 1 , wherein the recombinant microorganism is a GRAS (Generally Recognized As Safe) microorganism.

3. 2. The recombinant microorganism of claim 1, wherein the recombinant microorganism is a Bacillus sp. microorganism, a Corynebacterium sp. microorganism, an Escherichia sp. microorganism, or a yeast.

4. The Bacillus sp. microorganism is Bacillus megaterium, Bacillus subtilis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, or Bacillus stearothermophilus; The Corynebacterium sp. microorganism is Corynebacterium glutamicum; The Escherichia sp. microorganism is Escherichia coli; 4. The recombinant microorganism of claim 3, wherein the yeast is Saccharomyces cerevisiae or Candida utilis.

5. The recombinant microorganism according to claim 1, wherein the microorganism does not have the ability to produce 2'-fucosyllactose before recombination and has the ability to produce 2'-fucosyllactose by recombination.

6. The recombinant microorganism of claim 1 , wherein the recombinant microorganism expresses a fucose synthesis gene.

7. The recombinant microorganism according to claim 6, wherein the fucose synthesis gene expresses one or more selected from the group consisting of GDP-D-mannose-4,6-dehydratase, GDP-L-fucose synthase, phosphomannomutase, and GTP-mannose-1-phosphate guanylyltransferase.

8. The recombinant microorganism of claim 1 , wherein the recombinant microorganism expresses a lactose membrane transport protein.

9. The recombinant microorganism according to claim 8, wherein the lactose membrane transport protein is one or more selected from the group consisting of Lac12 and LacY.

10. The recombinant microorganism according to claim 1, wherein the recombinant microorganism has a 2'-fucosyllactose productivity that is at least twice as high as that of a control group containing α-1,2-fucosyltransferase derived from Helicobacter pylori.

11. A method for producing 2'-fucosyllactose, comprising culturing a recombinant microorganism according to any one of claims 1 to 10 in a medium containing lactose.

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