Method for producing lactose-N-tetraose and lactose-N-neotetraose by using corynebacterium glutamicum
Through the genetic modification and specific culture conditions of recombinant Corynebacterium glutamicum, the safety and cost problems of Lactose-N-tetrasaccharide and Lactose-N-newtetrasaccharide are solved in E. coli, and efficient and safe lactose-N-N-newtetrasaccharides are achieved. -Tetrasugar and lactose-N-new tetrasugar production.
Patent Information
- Application Number
- CN202380039271.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-05-10
- Filing Date
- 2023-05-11
- Publication Date
- 2025-01-07
AI Technical Summary
The existing methods of using E. coli to produce lactose-N-tetrasaccharides and lactose-N-newtetrasaccharides have safety problems and high costs, and E. coli cells die due to the action of lactose osmosis enzymes, which limits its application.
Using recombinant Corynebacterium glutamicum, by introducing and overexpressing specific genes such as lactose permease, β-1,3-N-acetylglucosamine transferase, and β-1,4-galactosyl Transferases, etc., improve the productivity of lactose-N-tetrasaccharides and lactose-N-neotetrasaccharides, and cultivate them in a medium containing glucose to promote their synthesis.
It has achieved high concentration, high yield and high productivity production of lactose-N-tetrasaccharides and lactose-N-neotetrasaccharides, and is safer than E. coli, and is suitable as a production host for food and drug materials.
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Abstract
Description
Technical Field
[0001] The present invention relates to a method for producing lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) by using Corynebacterium glutamicum, and more particularly to a recombinant Corynebacterium glutamicum and a method for producing LNT and LNnT by using the same. The recombinant Corynebacterium glutamicum is transformed so that a gene introduced from the outside is expressed in Corynebacterium glutamicum and a gene possessed by Corynebacterium glutamicum itself is overexpressed to improve the productivity of LNT and LNnT. Background Art
[0002] Human milk oligosaccharides (HMOs) are oligosaccharides contained in breast milk and are the third most abundant component in breast milk after lactose and fat. There are more than 200 different types of human milk oligosaccharides, and representative examples of human milk oligosaccharides include 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lactose-N-triose (Lacto-N-triose) II, lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT), lactose-N -Lacto-N-fucopentaose (LNFP), Lacto-N-neofucopentaose, Lacto-N-hexaose (LNH), Lacto-N-neohexaose (LNnH), 6'-galactosyl lactose (6'-galactosylactose) and 3'-galactosyl lactose (3'-galactosylactose), etc.
[0003] Since human milk oligosaccharides have various advantages such as enhancing immune function or having a beneficial effect on children's development and behavior, it is necessary to continuously study the technology for producing various human milk oligosaccharides. In existing research, the technology of using microorganisms to produce human milk oligosaccharides has been studied, among which recombinant Escherichia coli is used. However, although Escherichia coli is not actually a pathogen, it is generally considered to be a harmful bacterium by consumers, and because the cell membrane components of Escherichia coli may act as endotoxins, the cost of separating and purifying the produced human milk oligosaccharides is very high, and because Escherichia coli cells die due to the action of lactose permease (Lactose permease) ("lactose killing"), its use is subject to certain restrictions. Therefore, there is a continuous need for the technology of using new microorganisms to produce human milk oligosaccharides. Summary of the invention
[0004] Technical issues
[0005] The present invention aims to develop and provide a method for producing lactose-N-tetraose (Lacto-N-tetraose, LNT) and lactose-N-neotetraose (LNnT) as food and pharmaceutical materials with high concentration, high yield and high productivity using Corynebacterium glutamicum, which is safer than Escherichia coli, as a host cell.
[0006] Technical Solution
[0007] The present invention provides a recombinant Corynebacterium glutamicum, characterized in that the recombinant Corynebacterium glutamicum is transformed so that a gene encoding lactose permease, a gene encoding β-1,3-N-acetylglucosaminyltransferase, and a gene encoding β-1,3-galactosyltransferase introduced from the outside are expressed in the Corynebacterium glutamicum, and the recombinant Corynebacterium glutamicum is transformed so that a gene encoding glutamine-fructose-6-phosphate aminotransferase, a gene encoding phosphoglucosamine mutase, a gene encoding glucosamine-1-phosphate N-acetyltransferase, which are selected from genes possessed by the genus Corynebacterium itself, are expressed. One or more genes among a gene encoding UDP-N-acetyltransferase, a gene encoding UDP-N-acetylglucosamine pyrophosphorylase, a gene encoding phosphoglucomutase, a gene encoding UTP-glucose-1-phosphate uridylyltransferase, and a gene encoding UDP-glucose-4-epimerase are overexpressed.
[0008] Furthermore, the present invention provides a method for producing lactose-N-tetraose, characterized in that the recombinant Corynebacterium glutamicum is cultured in a culture medium supplemented with lactose.
[0009] On the other hand, in the method for producing lactose-N-tetraose of the present invention, it is preferred that the culture medium further contains glucose.
[0010] Furthermore, the present invention provides a recombinant Corynebacterium glutamicum, characterized in that the recombinant Corynebacterium glutamicum is transformed so that a gene encoding lactose permease, a gene encoding β-1,3-N-acetylglucosaminyltransferase, and a gene encoding β-1,4-galactosyltransferase introduced from the outside are expressed in Corynebacterium glutamicum, and the recombinant Corynebacterium glutamicum is transformed so that a gene encoding glutamine-fructose-6-phosphate aminotransferase, a gene encoding phosphoglucosamine mutase, a gene encoding glucosamine-1-phosphate N-acetyltransferase, which are selected from genes possessed by the genus Corynebacterium itself, are expressed. One or more genes among a gene encoding UDP-N-acetyltransferase, a gene encoding UDP-N-acetylglucosamine pyrophosphorylase, a gene encoding phosphoglucomutase, a gene encoding UTP-glucose-1-phosphate uridylyltransferase, and a gene encoding UDP-glucose-4-epimerase are overexpressed.
[0011] Furthermore, the present invention provides a method for producing lactose-N-neotetraose, characterized in that the recombinant Corynebacterium glutamicum is cultured in a culture medium supplemented with lactose.
[0012] On the other hand, in the method for producing lactose-N-neotetraose of the present invention, it is preferred that the culture medium further contains glucose.
[0013] Effects of the Invention
[0014] The present invention can utilize Corynebacterium glutamicum to produce lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) at high concentration, high yield and high productivity and more safely than the existing Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The figure shows the pathway for biosynthesis of lactose-N-tetraose (LNT) in the recombinant Corynebacterium glutamicum strain of the present invention.
[0016] Figure 2 The figure shows the biosynthesis pathway of lacto-N-neotetraose (LNnT) in the recombinant Corynebacterium glutamicum strain of the present invention.
[0017] Figure 3 The present invention is a graph showing the results of comparing the production of lactose-N-triose II (Lacto-N-triose II, LNTII) by recombinant Corynebacterium glutamicum strains prepared by overexpressing glmS, glmM, and glmU in the production pathway of UDP-N-acetylglucosamine (UDP-N-acetylglucosamine) as a precursor substance.
[0018] Figure 4 The present invention is a graph showing the results of comparing the LNT / LNnT production (final production) of recombinant Corynebacterium glutamicum strains prepared by overexpressing pgm, galU, and galE in the production pathway of UDP-galactose as a precursor substance.
[0019] Figure 5 The present invention is a graph showing the results of confirming the temporal changes in the LNT / LNnT production of the recombinant Corynebacterium glutamicum strain prepared by overexpressing pgm, galU, and galE in the production pathway of UDP-galactose as a precursor substance. DETAILED DESCRIPTION
[0020] Since human milk oligosaccharides have various advantages such as enhancing immune function or having beneficial effects on children's development and behavior, technologies for producing various human milk oligosaccharides are being continuously studied. In existing research, technologies for producing human milk oligosaccharides using microorganisms have been studied, and there is a great need to produce various human milk oligosaccharides using new microorganisms.
[0021] In the present invention, Corynebacterium glutamicum is used as host cell to produce lactose-N-neotetraose (Lacto-N-neotetraose, LNnT) and lactose-N-tetraose (Lacto-N-tetraose, LNT). Different from the Escherichia coli mainly used in existing research, this is not only recognized as a strain generally recognized as safe (generally recognized as safe, GRAS), but also a strain widely used in the industrial production of amino acids and nucleic acids as food additives. In addition, Escherichia coli is generally considered to be harmful bacteria by consumers, and the cell membrane components of Escherichia coli may play the role of endotoxins, so there are limitations in the high cost of separating and purifying the produced human milk oligosaccharides. In addition, due to the phenomenon of Escherichia coli cell death (" lactose kills ") due to the effect of lactose permease (Lactose permease), its use is subject to certain restrictions. Therefore, the Corynebacterium glutamicum used in the present invention can be said to be safe and suitable for producing food and pharmaceutical materials.
[0022] Therefore, the present invention provides a recombinant Corynebacterium glutamicum, characterized in that the recombinant Corynebacterium glutamicum is transformed so that a gene encoding lactose permease, a gene encoding β-1,3-N-acetylglucosaminyltransferase, and a gene encoding β-1,3-galactosyltransferase introduced from the outside are expressed in Corynebacterium glutamicum, and the recombinant Corynebacterium glutamicum is transformed so that a gene encoding glutamine-fructose-6-phosphate aminotransferase, a gene encoding phosphoglucosamine mutase, a gene encoding glucosamine-1-phosphate N-acetyltransferase, which are genes possessed by the genus Corynebacterium itself, are expressed. The present invention provides a method for producing lactose-N-tetraose, characterized in that the recombinant Corynebacterium glutamicum is cultured in a culture medium supplemented with lactose.
[0023] Furthermore, the present invention provides a recombinant Corynebacterium glutamicum, characterized in that the recombinant Corynebacterium glutamicum is transformed so that a gene encoding lactose permease, a gene encoding β-1,3-N-acetylglucosaminyltransferase, and a gene encoding β-1,4-galactosyltransferase introduced from the outside are expressed in Corynebacterium glutamicum, and the recombinant Corynebacterium glutamicum is transformed so that a gene encoding glutamine-fructose-6-phosphate aminotransferase, a gene encoding phosphoglucosamine mutase, a gene encoding glucosamine-1-phosphate N-acetyltransferase, which are selected from genes possessed by the genus Corynebacterium itself, are expressed. The present invention provides a method for producing lactose-N-neotetraose, characterized in that the recombinant Corynebacterium glutamicum is cultured in a culture medium supplemented with lactose.
[0024] The process of producing LNT and LNnT using the recombinant Corynebacterium glutamicum of the present invention is as follows: Figure 1 and Figure 2As shown. When lactose meets UDP-N-acetylglucosamine (UDP-N-GlcNAc), one of the precursor substances, lactose-N-triose II (Lacto-N-trioseII, LNTII) is produced by β-1,3-N-acetylglucosaminyltransferase (β-1,3-N-acetylglucosaminyltransferase, encoded by lgtA). The produced LNTII meets UDP-galactose (UDP-galactose), which is another precursor substance. In this case, LNT is produced by β-1,3-galactosyltransferase (β-1,3-galactosyltransferase, encoded by WbgO). Figure 1 ), mediated by β-1,4-galactosyltransferase (encoded by lgtB) to produce LNnT ( Figure 2 ).
[0025] On the other hand, the recombinant Corynebacterium glutamicum of the present invention is transformed to express a gene encoding lactose permease, which is an enzyme involved in delivering lactose present outside the strain to the inside of the strain, preferably, a gene derived from Escherichia coli is used. For example, LacY can be used.
[0026] On the other hand, the recombinant Corynebacterium glutamicum of the present invention is transformed to express the gene encoding β-1,3-N-acetylglucosaminyltransferase (lgtA), for example, can be derived from Neisseria meningitidis or Neisseria cinerea. More preferably, the gene derived from Neisseria meningitidis M98 or Neisseria cinerea ATCC 14685 can be used.
[0027] On the other hand, the recombinant Corynebacterium glutamicum of the present invention is transformed to express a gene encoding β-1,3-N-acetylglucosaminyltransferase to produce LNT, for example, lgtA, preferably derived from Neisseria cinerea. In addition, it is transformed to express a gene encoding β-1,3-galactosyltransferase, for example, WbgO, preferably, derived from Lutiella nitroferrum. More preferably, a gene derived from Lutiella nitroferrum ATCC BAA-1479 can be used.
[0028] Furthermore, the recombinant Corynebacterium glutamicum of the present invention is transformed to express a gene encoding β-1,3-N-acetylglucosaminyltransferase to produce LNnT, for example, lgtA, preferably, derived from Neisseria meningitidis. Furthermore, it is transformed to express a gene encoding β-1,4-galactosyltransferase, for example, lgtB, preferably, derived from Neisseria cinerea.
[0029] On the other hand, the recombinant Corynebacterium glutamicum of the present invention is transformed so that a gene encoding glutamine-fructose-6-phosphate aminotransferase, a gene encoding phosphoglucosamine mutase, a gene encoding glucosamine-1-phosphate N-acetyltransferase, a gene encoding UDP-N-acetylglucosamine pyrophosphorylase, a gene encoding phosphoglucomutase, a gene encoding UTP-glucose-1-phosphate uridyltransferase, a gene encoding UDP-N-acetylglucosamine pyrophosphorylase, a gene encoding phosphoglucomutase, a gene encoding UTP-glucose-1-phosphate uridyltransferase, a gene encoding UDP-N-acetylglucosamine pyrophosphorylase, a gene encoding phosphoglucomutase, a gene encoding UDP ... It is preferred that at least one of the genes encoding UDP-uridylyltransferase and UDP-glucose-4-epimerase be overexpressed.
[0030] In this case, the gene encoding glutamine-fructose-6-phosphate aminotransferase is preferably glmS, and the gene encoding phosphoglucosamine mutase is preferably glmM. Furthermore, the gene encoding glucosamine-1-phosphate N-acetyltransferase and the gene encoding UDP-N-acetylglucosamine pyrophosphorylase are preferably glmU. In this case, glmU is a gene encoding a bifunctional enzyme having both UDP-N-acetylglucosamine pyrophosphorylase and glucosamine-1-phosphate N-acetyltransferase activities (see Figure 1 and Figure 2). In addition, the gene encoding phosphoglucomutase is preferably pgm, the gene encoding UTP-glucose-1-phosphate uridylyltransferase is preferably galU, and the gene encoding UDP-glucose-4-epimerase is preferably galE. As described above, by overexpressing the genes possessed by Corynebacterium glutamicum itself to mass-produce UDP-N-acetylglucosamine (UDP-N-acetylglucosamine, UDP-N-GlcNAc) and lactose-N-triose II (Lacto-N-trioseII, LNTII), which are precursors of LNT and LNnT, the productivity of LNT and LNnT can be improved.
[0031] On the other hand, the term "expression" used in the present invention refers to the artificial expression of an enzyme that the Corynebacterium glutamicum strain of the present invention cannot express itself by introducing a gene from an external source into the strain and expressing it, and the term "overexpression" means that the Corynebacterium glutamicum strain of the present invention itself has a gene encoding the corresponding enzyme and can express the enzyme by itself, but overexpression is achieved by artificially increasing the expression level of the enzyme to increase its expression level to achieve the purpose of mass production.
[0032] On the other hand, in the method for producing lactose-N-tetraose or lactose-N-neotetraose of the present invention, it is preferable that the above-mentioned culture medium further contains glucose. As mentioned above, by adding additional culture medium components to activate the growth of the strain, lactose-N-tetraose or lactose-N-neotetraose can be produced with higher productivity.
[0033] On the other hand, according to the following experiment, the recombinant Corynebacterium glutamicum of the present invention is prepared by overexpressing glmS, glmM, and glmU in the production pathway of UDP-N-acetylglucosamine (UDP-N-GlcNAc) as a precursor, thereby significantly increasing the production of LNTII as a precursor of LNT / LNnT, and by overexpressing pgm, galU, and galE in the production pathway of UDP-galactose (UDP-galactose) as another precursor, thereby significantly increasing the production of LNT / LNnT. As described above, the recombinant Corynebacterium glutamicum of the present invention can be used to produce lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) at high concentration, high yield, and high productivity, and more safely than the existing Escherichia coli.
[0034] Hereinafter, the content of the present invention will be described in more detail by the following embodiments. However, the scope of the present invention is not limited to the following embodiments, and also includes the deformation of the technical concept equivalent thereto.
[0035] Example 1: Preparation of recombinant Corynebacterium glutamicum and plasmid
[0036] 1. Construction of LNT and LNnT production strains
[0037] Escherichia coli TOP10 and Corynebacterium glutamicum ATCC 13032 were used to construct plasmids and produce lactose-N-triose II (Lacto-N-triose, LNTII), lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT), respectively.
[0038] (1) Construction of pAY as LNTII production plasmid (construction of plasmid for expressing lgtA-lacY)
[0039] The gene (lgtA) encoding β-1,3-N-acetylglucosaminyltransferase was amplified from Neisseria meningitidis M98 by PCR reaction using two DNA primers 21RBS-lgtA F, lgtA R. In addition, the lacY gene was amplified from the genomic DNA of Escherichia coli K-12MG1655 by PCR reaction using two DNA primers RBS-lacYF, LacY R, and then the lgtA-lacY DNA fragment was synthesized by overlapping PCR reaction using two DNA primers 21RBS-lgtA F and LacY R, and then inserted into the plasmid pCN013 treated with restriction enzyme EcoRI to construct the pAY plasmid.
[0040] (2) Construction of pABY as a LNnT production plasmid (construction of a plasmid for expressing lgtA-lgtB-lacY)
[0041] The gene (lgtA) encoding β-1,3-N-acetylglucosaminyltransferase was amplified from Neisseria meningitidis M98 by PCR reaction using two DNA primers lgtA_t F and lgtA_20BR, and the gene (lgtB) encoding β-1,4-galactosyltransferase was amplified from Neisseria cinerea ATCC 14685 by PCR reaction using two DNA primers 20_B1 F and 15_B1 R, and then the lgtA-lgtB DNA fragments were synthesized by overlapping PCR reaction using two DNA primers lgtA_t F and 15_B1 R. Then, the lacY gene was amplified from the genomic DNA of Escherichia coli K-12MG1655 by a PCR reaction using two DNA primers lacY_B F and 20ABY R3, and then the lgtA-lgtB-lacY DNA fragment was synthesized by overlapping PCR reaction using two DNA primers lgtA_t F and 20ABY R3, and then inserted into the plasmid pCN013 treated with restriction enzyme EcoRI to construct the pABY plasmid.
[0042] (3) Construction of pAWY as an LNT production plasmid (Construction of a plasmid for expressing lgtA-WbgO-lacY)
[0043] The pgk promoter was amplified from Corynebacterium glutamicum ATCC 13032 by PCR reaction using two DNA primers pgkF and pgkR. The gene encoding β-1,3-N-acetylglucosaminyl transferase (lgtA, i.e., NclgtA; Nc indicates that lgtA is derived from Neisseria cinerea) was amplified from Neisseria cinerea ATCC 14685 by a PCR reaction using two DNA primers 21NcA F and NcA R, and the gene encoding β-1,3-galactosyltransferase (WbgO, i.e., LnWbgO; Ln indicates that WbgO is derived from Lutiella nitroferrum) was amplified from Lutiella nitroferrum ATCC BAA-1479 by a PCR reaction using two DNA primers LnW F and LnW R. The lacY gene was amplified from the genomic DNA of Escherichia coli K-12MG1655 by a PCR reaction using two DNA primers 20ABY F3 and 20ABY R3, and then the pgk-lgtA-WbgO-lacY (i.e., pgk-NclgtA-LnWbgO-lacY; wherein Nc indicates that lgtA is derived from Neisseria cinerea, and Ln indicates that WbgO is derived from Lutiella nitroferrum) DNA fragment was synthesized by overlapping PCR reaction using two DNA primers pgk F and 20ABY R3, and then inserted into the pCN013 plasmid treated with restriction enzymes EcoRⅠ and EcoRV to construct the pAWY plasmid.
[0044] 2. Construction of UDP-N-acetylglucosamine (UDP-N-GlcNAc) as a precursor of LNT and LNnT and its production strain
[0045] In order to construct LNT and LNnT production strains, strains that overproduce "UDP-N-acetylglucosamine (UDP-N-GlcNAc)" as a precursor substance were constructed. Figure 1 and Figure 2As shown, a total of three integration plasmids, pK19mobsacB-tuf-glmS, pK19mobsacB-tuf-glmM, and pK19mobsacB-tuf-glmU, were prepared to overexpress glmS, glmM, and glmU in the biosynthetic pathway.
[0046] (1) Construction of pK19mobsacB-tuf-glmS plasmid (construction of a plasmid for overexpressing glmS)
[0047] Three genes were amplified from the genomic DNA of Corynebacterium glutamicum by PCR reaction using three pairs of primers (glmS F1, glmS R1)(glmS F2, glmS R2)(glmS F3, glmS R3), and then DNA fragments were synthesized using two DNA primers glmS F1, glmS R3 and by overlapping PCR reaction, and then inserted into the plasmid pK19mobsacB treated with XbaI to construct the pK19mobsacB-tuf-glmS plasmid.
[0048] (2) Construction of pK19mobsacB-tuf-glmM plasmid (construction of a plasmid for overexpressing glmM)
[0049] Three genes were amplified from the genomic DNA of Corynebacterium glutamicum using three pairs of primers (glmM F1, glmM R1)(glmM F2, glmM R2)(glmM F3, glmM R3), and then DNA fragments were synthesized using two DNA primers glmM F1 and glmM R3 by overlapping PCR reaction, and then inserted into plasmid pK19mobsacB treated with HindIII and EcoRI to construct pK19mobsacB-tuf-glmM plasmid.
[0050] (3) Construction of pK19mobsacB-tuf-glmU plasmid (construction of a plasmid for overexpressing glmU)
[0051] Three genes were amplified from the genomic DNA of Corynebacterium glutamicum using three pairs of primers (glmU F1, glmU R1)(glmU F2, glmU R2)(glmU F3, glmU R3), and then DNA fragments were synthesized using two DNA primers glmU F1, glmU R3 and by overlapping PCR reaction, which were then inserted into the plasmid pK19mobsacB treated with XbaI to construct the pK19mobsacB-tuf-glmU plasmid.
[0052] 3. Construction of UDP-galactose as a precursor of LNT and LNnT and its production strain
[0053] A strain that overproduces UDP-galactose as another precursor was constructed for the biosynthesis of LNT and LNnT. Figure 1 and Figure 2 As shown, a total of three integration plasmids, pK19mobsacB-tuf-pgm, pK19mobsacB-tuf-galU1, and pK19mobsacB-tuf-galE, were prepared to overexpress pgm, galU1, and galE in the biosynthetic pathway.
[0054] (1) Construction of pK19mobsacB-tuf-pgm plasmid (construction of a plasmid for overexpressing pgm)
[0055] Three genes were amplified from the genomic DNA of Corynebacterium glutamicum by PCR reaction using six DNA primers (pgm F1, pgm R1), (pgm F2, pgm R2), (pgm F3, pgm R4), and then DNA fragments were synthesized by overlapping PCR reaction using two DNA primers pgm F1, pgmR4, and then inserted into plasmid pK19mobsacB treated with Xba I to construct pK19mobsacB-tuf-pgm plasmid.
[0056] (2) Construction of pK19mobsacB-tuf-galU1 plasmid (construction of a plasmid for overexpressing galU)
[0057] Three genes were amplified from the genomic DNA of Corynebacterium glutamicum by PCR reaction using six DNA primers (galU1 F1, galU1 R1), (galU1 F2, galU1 R2), (galU1F3, galU1 R3), and then DNA fragments were synthesized using two DNA primers galU1 F1, galU1 R3 and by overlapping PCR reaction, and then inserted into the plasmid pK19mobsacB treated with XbaI to construct the pK19mobsacB-tuf-galU1 plasmid.
[0058] (3) Construction of pK19mobsacB-tuf-galE plasmid (construction of a plasmid for overexpressing galE)
[0059] Three genes were amplified from the genomic DNA of Corynebacterium glutamicum by PCR reaction using six DNA primers (galE F1, galE R1), (galE F2, galE R2), and (galE F3, galER3), and then the DNA fragments were synthesized using two DNA primers galEF1 and galE R3 and by overlapping PCR reaction, and then inserted into the plasmid pK19mobsacB treated with XbaI to construct the pK19mobsacB-tuf-galE plasmid.
[0060] On the other hand, the primers, strains, plasmids, and gene sequences used in this example are shown in Tables 1 to 5 below.
[0061] Table 1
[0062] Primers
[0063]
[0064] Table 2
[0065] Primers
[0066]
[0067]
[0068] Table 3 Strains
[0069]
[0070]
[0071] Table 4 Plasmid
[0072]
[0073]
[0074] Table 5 Gene sequence
[0075]
[0076] Example 2: Cultivation conditions and methods of recombinant Corynebacterium glutamicum
[0077] For the culture of seed bacteria, a glass test tube filled with 4 mL of Brain Heart Infusion (BHI) medium containing appropriate antibiotics (25 μg / mL of kanamycin) was used and cultured at 30°C with a stirring speed of 250 rpm for 12 hours.
[0078] The main culture was cultured in flasks using 40 mL of CGXII (5 g / L urea, 0.25 g / L MgSO4, 42 g / L MOPS, 1 g / L potassium phosphate monobasic, 1 g / L potassium phosphate dibasic, 10 mg / L CaCl2, 0.2 mg / L biotin, 30 mg / L protocatechuic acid) containing appropriate antibiotics (25 μg / mL kanamycin). acid), 10 mg / L FeSO47H2O, 10 mg / L MnSO4H2O, 1 mg / L ZnSO47H2O, 0.2 mg / L CuSO4, 0.02 mg / L NiCl26H2O, 20 g / L glucose (Glucose), 5 g / L lactose (Lactose), pH 7.0) culture medium, at a temperature of 25°C and a stirring speed of 200 rpm for 72 hours.
[0079] Experimental Example 1: Determine the concentration of cells and metabolites and compare productivity
[0080] 1) Experimental methods to determine the concentration of cells and metabolites and compare productivity
[0081] In order to conduct a comparative experiment on the productivity of LNT, LNnT, and LNTII, a glass test tube containing 4 mL of brain heart infusion (BHI) medium containing antibiotics (25 μg / mL of kanamycin) was used, and cultured for 12 hours at a temperature of 30°C and a stirring speed of 250 rpm, and then inoculated with an initial optical density (OD) of 0.3 in a shake flask containing 40 mL of CGXII medium containing 25 μg / mL of kanamycin. Cultured for 72 hours at a culture temperature of 25°C and a stirring speed of 200 rpm. After 72 hours of culture, 1 ml of the culture solution was inoculated in a 1.7 ml tube and then boiled at 95°C. The boiled culture solution was centrifuged at 15,000 rpm for 1 minute, and the supernatant was aliquoted 100 times, and then the concentration was analyzed by HPLC. The concentrations of LNT, LNnT, LNTII, lactose, lactate, glucose and acetic acid were determined by high performance liquid chromatography (HPLC) (Agilent 1260, USA) equipped with a "Carbohydrate Analysis column" (Aminex HPX87H column, Bio-rad) and a "refractive index, RI" detector. 20 μl of culture medium was analyzed by applying a column heated to 60° C. A 5 mM H2SO4 solution with a flow rate of 0.6 mL / min was used as the mobile phase.
[0082] 2) Comparison of the productivity of LNTII
[0083] In order to produce LNT / LNnT, the strain of the above-mentioned Example 1 was prepared by overexpressing glmS, glmM, and glmU in the production pathway of UDP-N-acetylglucosamine (UDP-N-acetylglucosamine) as a precursor substance, and the production of LNTII, which is a precursor of LNT / LNnT, was compared using the above-mentioned productivity comparison experimental method.
[0084] The results are as follows Figure 3 As shown, it was confirmed that the production amount of LNTII was significantly increased in glmSMU O / E in which glmS, glmM, and glmU in the production pathway of UDP-N-acetylglucosamine were simultaneously overexpressed.
[0085] 3) Comparison of the productivity of LNT and LNnT
[0086] In order to produce LNT / LNnT, the strain of the above-mentioned Example 1 prepared by overexpressing pgm, galU, and galE in the production pathway of UDP-galactose (UDP-galactose) as a precursor substance was used, and the production amounts of LNT / LNnT were compared using the above-mentioned productivity comparison experimental method (PU O / E: pgm GalU O / E; PE O / E: pgm GalE O / E; UE O / E: GalU GalE O / E; PUE O / E: pgm GalU GalE O / E).
[0087] The results are as follows Figure 4 As shown, it can be confirmed that for the production of LNT, the production amount (final production amount) is maximized when pgm, galU, and galE are overexpressed simultaneously (PUEO / E), and it can be confirmed that for the production of LNnT, the production amount (final production amount) is maximized when pgm and galU are overexpressed (PU O / E) compared with when pgm, galU, and galE are overexpressed simultaneously (PUE O / E).
[0088] On the other hand, when confirming the change of LNT and LNnT production over time, such as Figure 5 As shown, it can be confirmed that the production of LNT is most improved when pgm, galU, and galE are overexpressed simultaneously (PUE O / E), and it can be confirmed that the production of LNnT is most improved when pgm and galU are overexpressed compared to when pgm, galU, and galE are overexpressed simultaneously (PUE O / E).
Claims
1. A recombinant Corynebacterium glutamicum, characterized in that The recombinant Corynebacterium glutamicum is transformed so that the gene encoding lactose permease, the gene encoding β-1,3-N-acetylglucosaminyltransferase, and the gene encoding β-1,3-galactosyltransferase introduced from the outside are expressed in Corynebacterium glutamicum, The above-mentioned recombinant Corynebacterium glutamicum is transformed so as to overexpress one or more genes selected from the group consisting of a gene encoding glutamine-fructose-6-phosphate aminotransferase, a gene encoding phosphoglucosamine mutase, a gene encoding glucosamine-1-phosphate N-acetyltransferase, a gene encoding UDP-N-acetylglucosamine pyrophosphorylase, a gene encoding phosphoglucosomutase, a gene encoding UTP-glucose-1-phosphate uridyltransferase, and a gene encoding UDP-glucose-4-epimerase, which are genes possessed by the genus Corynebacterium itself.
2. A recombinant Corynebacterium glutamicum, characterized in that The recombinant Corynebacterium glutamicum is transformed so that the gene encoding lactose permease, the gene encoding β-1,3-N-acetylglucosaminyltransferase, and the gene encoding β-1,4-galactosyltransferase introduced from the outside are expressed in Corynebacterium glutamicum, The above-mentioned recombinant Corynebacterium glutamicum is transformed so as to overexpress one or more genes selected from the group consisting of a gene encoding glutamine-fructose-6-phosphate aminotransferase, a gene encoding phosphoglucosamine mutase, a gene encoding glucosamine-1-phosphate N-acetyltransferase, a gene encoding UDP-N-acetylglucosamine pyrophosphorylase, a gene encoding phosphoglucosomutase, a gene encoding UTP-glucose-1-phosphate uridyltransferase, and a gene encoding UDP-glucose-4-epimerase, which are genes possessed by the genus Corynebacterium itself.
3. A method for producing lactose-N-tetraose, characterized in that: The recombinant Corynebacterium glutamicum according to claim 1 is cultured in a culture medium supplemented with lactose.
4. The method for producing lactose-N-tetraose according to claim 3, characterized in that The above culture medium also contains glucose.
5. A method for producing lactose-N-neotetraose, characterized in that: The recombinant Corynebacterium glutamicum according to claim 2 is cultured in a culture medium supplemented with lactose.
6. The method for producing lactose-N-neotetraose according to claim 5, characterized in that: The above culture medium also contains glucose.