Mutant of uridine 5 '-diphosphate-N-acetylglucosamine 2 epimerase and application thereof

Abstract

The invention relates to the technical field of fermentation engineering, in particular to a mutant of uridine 5 '-diphosphate-N-acetylglucosamine 2 epimerase and application of the mutant. The mutant comprises any one of the following components on the basis of an amino acid sequence of uridine 5 '-diphosphate-N-acetylglucosamine 2 epimerase: i) mutation S263Q or mutation S263L; and ii) further comprising one of mutation F266W, F266Y, F266I or F266L on the basis of the i). A plurality of mutation modes of uridine 5 '-diphosphate-N-acetylglucosamine 2 epimerase are found through research, the yield of sialyllactose can be effectively increased by applying the uridine 5'-diphosphate-N-acetylglucosamine 2 epimerase to construction of escherichia coli for producing sialyllactose, and the uridine 5 '-diphosphate-N-acetylglucosamine 2 epimerase has important application value in the field of sialyllactose production.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a mutant of uridine 5'-diphosphate-N-acetylglucosamine 2 epimerase and application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk and are considered to be the first prebiotic beneficial to infant health. Among HMOs, sialyllactose (SL) accounts for approximately 13% of the total HMOs. 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are the two most abundant and representative sialyllactoses in breast milk. Many efficacy studies have shown that SLs have potential prebiotic effects, especially in promoting the proliferation of bifidobacteria and shaping the intestinal microbiota. In addition, SLs also have anti-adhesion and antibacterial capabilities, antiviral activity, prevention of necrotizing enterocolitis, immunomodulatory activity, regulation of intestinal epithelial cell responses, promotion of brain development, and improvement of cognition. Both 3'-SL and 6'-SL are commercially added to infant formula.

[0003] The synthesis of SLs by enzymatic or microbial methods has been widely studied. Enzymatic synthesis of SLs can be achieved by two types of enzymes: sialic acid synthases with trans-sialyl esterase activity and sialyltransferases. Microbial synthesis can be achieved by multiple recombinant bacteria expressing enzymes involved in the SL synthesis pathway individually or in combination in a one-pot reaction, or by de novo synthesis during fermentation by metabolically engineered strains. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a mutant of uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase and its application.

[0005] The microbial synthesis of sialyllactose mainly depends on engineered strains, which generally use glycerol, glucose and lactose as substrates and express related enzymes in the synthesis pathways of sialyllactose (SL) and sialic acid cytidine monophosphate (CMP-Neu5Ac).

[0006] The synthesis of sialyllactose is achieved by transferring the sialic acid group in CMP-NeuAc to lactose (st). CMP-NeuAc can be synthesized based on NeuAc, which is transported to the engineering host by the sialic acid permease NanT and further converted into CMP-NeuAc by sialic acid cytidine monophosphate synthase (neuA). There are two synthetic pathways for NeuAc, which can be produced by acetylmannosamine (ManNAc) through sialic acid synthase (neuB) using phosphoenolpyruvate (PEP) as a co-substrate, or by ManNAc through sialic acid lyase (NanA) using pyruvate as a substrate. There are three synthetic pathways for ManNAc: the first is that uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is irreversibly converted to ManNAc under the catalysis of hydrolytic UDP-GlcNAc 2-epimerase (neuC); the second is that acetylglucosamine (GlcNAc) is reversibly catalyzed by N-acetylglucosamine 2-epimerase (age) to synthesize N-acetylglucosamine; the third is that 6-phosphoacetylglucosamine is reversibly converted into 6-phosphoacetylmanosamine by N-acetylmannosamine-6-phosphate 2-epimerase (nanE), and then further enzymatically converted into ManNAc. All three pathways use glycerol and glucose as the initial substrates and metabolize their precursors.

[0007] like Figure 1 The figure shows a typical synthesis pathway of sialyllactose, in which the uridine 5′-diphospho-N-acetylglucosamine 2 epimerase (NeuC) gene encodes UDP-acetylglucosamine epimerase, which converts UDP-acetylglucosamine (UDP-GlcNAc) into acetylmannosamine (ManNAc). This step is crucial in the biosynthesis of sialic acid (SA, NeuAc) and sialyllactose (SL) and is a necessary step for the biosynthesis of sialic acid and sialyllactose.

[0008] The present invention explores the key transformation sites of neuC and obtains corresponding mutants. After replacing the original neuC, the mutants significantly optimize the catalytic efficiency thereof, and the sialyllactose content of the obtained engineered strain is significantly increased.

[0009] In a first aspect, the present invention provides a mutant of uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase, wherein the mutant is based on the amino acid sequence of uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase and includes any one of the following: i) mutation S263Q or mutation S263L; ii) the method according to i) further comprising one of the mutations F266W, F266Y, F266I or F266L.

[0010] Mutations F266W, F266Y, F266I and F266L can all increase the production of sialyllactose, among which mutation F266W has the best effect.

[0011] The above expression of mutations in the present invention is a commonly used expression in the art. Taking S263Q as an example, the position of the mutation at position 263 on uridine 5′-diphospho-N-acetylglucosamine 2-epimerase, S is the amino acid type before mutation (serine), and Q is the amino acid type after mutation (glutamine), that is, the serine at position 263 is replaced by glutamine.

[0012] Correspondingly, S263L means that the serine at position 263 is replaced by leucine; F266W means that the phenylalanine at position 266 is replaced by tryptophan; F266Y means that the phenylalanine at position 266 is replaced by tyrosine; F266I means that the phenylalanine at position 266 is replaced by isoleucine; and F266L means that the phenylalanine at position 266 is replaced by leucine.

[0013] Furthermore, the uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase is derived from Campylobacter jejuni.

[0014] Furthermore, the uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids, and having the same functional protein.

[0015] Furthermore, the gene encoding uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase comprises any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

[0016] In a second aspect, the present invention provides the use of the aforementioned mutant in producing sialyllactose in Escherichia coli.

[0017] Furthermore, the application is to increase the level of sialyllactose production by Escherichia coli.

[0018] Furthermore, the sialyllactose comprises: 3'-sialyllactose and / or 6'-sialyllactose.

[0019] Furthermore, the Escherichia coli is an Escherichia coli capable of producing sialyllactose, and the mutant is introduced or integrated into the genome of the Escherichia coli.

[0020] Preferably, the Escherichia coli also has neuB, neuA and sialyltransferase genes (ST), which are integrated independently or in any combination into the genome of the genetically engineered bacteria, or exist in a recombinant plasmid carried by the genetically engineered bacteria.

[0021] Further preferably, the Escherichia coli reduces the expression level of one or more genes among the lacZ, nanA, nanK, nanE and nanT genes.

[0022] in, l The acZ gene encodes β-galactosidase, which, when inactivated or missing, prevents the degradation of lactose into glucose and galactose; nA The gene encodes N-acetylmannosamine lyase, and its inactivation or deletion can prevent the degradation of N-acetylneuraminic acid and N-acetylmannosamine; K The gene encodes N-acetylmannosamine kinase, and its inactivation or deletion prevents the degradation of N-acetylmannosamine-6-phosphate; E The gene encodes N-acetylmannosamine-6-phosphate-2-epimerase, and its inactivation or deletion prevents the degradation of glucosamine-6-phosphate; T The gene encodes sialic acid transporter, and inactivation or deletion can prevent the intracellular and extracellular transport of sialic acid; neuA is the CMP-N-acetylneuraminic acid synthetase gene, and neuB is the sialic acid synthase gene.

[0023] The reduction of the expression level of lacZ, nanA, nanK, nanE and nanT genes described in the present invention can be achieved by adopting the technical means commonly used in the art to reduce the gene expression level, such as gene knockout to cause the deletion of gene fragments or complete knockout of genes, or by adopting means such as RNA interference. At the same time, reducing the gene expression level includes different degrees of gene level reduction, including complete loss of function. In a third aspect, the present invention provides a method for improving the ability of Escherichia coli to produce sialyllactose, comprising: using the aforementioned mutant to replace the uridine 5'-diphosphate-N-acetylglucosamine 2 epimerase in Escherichia coli; or, introducing or integrating the aforementioned mutant into the genome of the Escherichia coli.

[0024] Furthermore, the use of the aforementioned mutant to replace the uridine 5′-diphospho-N-acetylglucosamine 2 epimerase in Escherichia coli allows the neuC mutant to be directly used for strain construction when constructing a sialic acid production strain.

[0025] Furthermore, the mutant is introduced into the Escherichia coli by one or more of plasmid transfection, CaCl2 transformation, electroporation or phage transformation.

[0026] Furthermore, the Escherichia coli is an Escherichia coli capable of producing sialyllactose.

[0027] In a fourth aspect, the present invention provides Escherichia coli prepared by the aforementioned method.

[0028] The present invention has the following beneficial effects: The present invention overcomes the shortcomings of the prior art, and improves the efficiency of sialyllactose production by modifying the amino acid sequence of neuC, a key synthase of sialic acid and sialyllactose (providing a variety of neuC mutants). In a specific experiment, based on the above modification, the 3'-SL synthetic engineering strain constructed by the present invention has a maximum 3'-SL yield of 46.85g / L in a 5L tank, and no sialic acid residues are detected; the constructed 6'-SL synthetic engineering strain has a maximum 6'-SL yield of 45.26g / L in a 5L tank, and no sialic acid residues are detected. The mutants provided by the present invention are very effective in improving the ability of Escherichia coli to synthesize sialyllactose, and have a high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The schematic diagram of the synthesis route of sialyllactose provided by the present invention is shown in FIG. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0033] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0034] Example 1 1. Obtaining mutants The uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase mutant provided by the present invention uses neuC (GenBank: AAK91727.1) from Campylobacter jejuni as a template (the amino acid sequence of neuC is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2), and includes mutation modes: S263Q, S263L, S263Q+F266W, S263Q+F266Y, S263Q+F266I, and S263Q+F266L.

[0035] Table 1 Nucleotide changes corresponding to each mutation pattern

[0036] 2. Construction of sialyllactose-producing strain (1) Chassis strains A strain capable of producing sialyllactose can be constructed using existing technology. In this embodiment, the strain prepared by the following method is preferred: The present invention uses Escherichia coli BL21 as a starting strain, and knocks out SL synthesis precursor substance related metabolic genes sialic acid lyase gene (N-acetylneuraminate lyase, nanA), N-acetylmannosamine kinase gene (N-acetylmannosamine kinase, nanK), N-acetylmannosamine-6-phosphate 2-isomerase (N-acetylmannosamine-6-P epimerase, nanE), sialic acid transporter (sialic acid transporter, nanT), and beta-D-galactosidase (beta-D-galactosidase, lacZ) in the genome to obtain a chassis strain.

[0037] (2) Construction of plasmid The gene sequences for neuB, neuC, neuA from Campylobacter jejuni (AF400048), 6'-sialyltransferase 6ST from Photobacterium damselae (BAA25316.1), and 3'-sialyltransferase 3ST from Neisseriagonorrheae (U60664) were targeted. The neuB, neuC, and neuA genes were combined and inserted into the NcoⅠ restriction site of pRSFDuet-1 to obtain the plasmid pRSF-neuB-neuC-neuA; based on pRSF-neuB-neuC-neuA, 3ST / 6ST was inserted into the NdeⅠ restriction site of the plasmid to obtain the plasmid pRSF-neuB-neuC-neuA-3ST / 6ST, and the plasmid pRSF-neuB-neuC-neuA-3ST / 6ST was transformed into the obtained chassis strain to obtain strains named FS301 / FS601, which were used to study the production of 3'-SL / 6'-SL.

[0038] 3. Shake flask fermentation Inoculate 50 mL of fresh LB medium with 0.5 mL of overnight culture of recombinant E. coli (FS301 / FS601 described above). 600 ) reached 0.6-0.8, 1 mL of the obtained culture was inoculated into 100 mL of the specified culture medium in a 500 mL shake flask. The indicated medium contained (per liter) 30 g of glycerol, 17.9 g of Na2HPO4·12H2O, 3.1 g of KH2PO4, 2.0 g of NH4C, 1.0 g of (NH4)2HPO4, 1.7 g of citric acid, 15 mg of CaCl2, 2 g of yeast extract, 15 g of Oxoid tryptone, 2.2 g of C6H5Na3O7·2H2O, 1 g of MgSO4·7H2O, 0.3 mL of 100× Triton-X, 10 mg of vitamin B1, 10 mL of trace element solution (25 g of FeCl3·6H2O, 2 g of CaCl2·2H2O, 2.0 g of ZnCl2, 1.9 g of CuSO4·5H2O, 0.42 g of MnCl·H2O, 2 g of Na2B4O7·10H2O, and 2 g of The culture was incubated at 37°C with a flow cytometry of 1% Na2MoO4·2H2O until the OD600 was 0.6-0.8, and isopropyl β-D-1-thiogalactoside and lactose were added to a final concentration of 0.2 mM and 6 g / L. The culture was then incubated at 28°C for 48 h. All experiments were repeated three times.

[0039] The results showed that the shake flask yields of 3'-SL / 6'-SL were 1.61 / 1.89 g / L, corresponding to sialic acid yields of 1.17 / 1.13 g / L.

[0040] 4. Application of mutants (1) The present invention uses the aforementioned mutants based on strain FS301 / FS601, specifically targeting plasmid pRSF-neuB-neuC-neuA-3ST / 6ST, in which neuC is replaced with the aforementioned mutants after the two mutations S263Q and S263L.

[0041] The strains after S263L mutation were 3FS02 and 6FS02, and the production of sialyllactose reached 1.88g / L and 2.08g / L, which were 16.7% and 10.6% higher than those of strains 3FS01 and 6FS01, respectively.

[0042] The strains with S263Q mutation were 3FS03 and 6FS03, and the production of sialyllactose reached 2.26 g / L and 2.45 g / L, which were 40.37% and 29.62% higher than those of strains 3FS01 and 6FS01, respectively.

[0043] (2) As can be seen from the above results, the S263Q mutation has a better effect. Therefore, based on the S263Q mutation, we further performed F266W, F266Y, F266I and F266L mutations. Similarly, neuC in pRSF-neuB-neuC-neuA-3ST / 6ST was replaced with a mutant (S263Q+F266W, S263Q+F266Y, S263Q+F266I, S263Q+F266L).

[0044] After mutation, strains 3FS03W / 6FS03W, 3FS03Y / 6FS03Y, 3FS03I / 6FS03I and 3FS03L / 6FS03L were obtained in turn. The results are shown in Table 1. Among them, 3FS03W / 6FS03W had the best effect, with 3'-SL and 6'-SL production of 2.53 g / L and 2.84 g / L, which were 57.1% and 50.3% higher than those of strains 3FS01 and 6FS01, respectively, and the residual sialic acid was significantly reduced.

[0045] Table 2 3'-SL production of each strain

[0046] Table 3 6'-SL production of each strain

[0047] 5. 5L tank fed-batch fermentation experiment The invention performs fed-batch fermentation in a 5L bioreactor, pre-loads 2.5L of culture medium, wherein the culture medium contains 10g / L initial glycerol, 4.0g / L (NH4)2SO4, 9.2g / L K2HPO4, 8.2g / L KH2PO4, 0.3g / L citric acid, 6.0g / L tryptone, 2.0g / L yeast extract, 10mg / L thiamine, 2.0g / L MgSO4·7H2O, 0.02g / L CaCl2 and 10mL / L trace element solution. After culturing 0.15mL of culture solution (containing 3FS03W / 6FS03W) in a 1L shaking flask containing 150mL LB culture medium for 6h, the culture solution is transferred to the bioreactor to prepare a seed culture. During the whole culturing process, the pH of the fermentation solution is adjusted to 6.8 by automatically adding 28% (v / v) NH4OH, and the dissolved oxygen is maintained between 30% and 50% by automatically controlling the stirring speed.

[0048] During the cultivation phase, the culture temperature was 37 °C, the aeration rate was 2 VVM, and the rotation speed was 900 rpm. When the initial glycerol was completely consumed, a feed solution containing 800 g / L glycerol and 5 g / L MgSO4·7H2O was added to the bioreactor at a constant rate of 3.8 g / L / h. At the same time, the temperature was shifted to 29.5 °C. After another 4 h of incubation, isopropyl β-d-1-thiogalactoside (final concentration 0.2 mM) and 70 g lactose were added. After another 12 h of cultivation, 70 g lactose was added. The medium was sampled every 12 h after replenishment to detect OD600 and lactose and corresponding SL levels.

[0049] After 86 h of fermentation, the yields of 3FS03W and 6FS03W were 46.85 g / L 3'-SL and 45.26 g / L 6'-SL, respectively, and no residual sialic acid was detected.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant of uridine 5′-diphospho-N-acetylglucosamine 2 epimerase, characterized in that: The mutant is based on the amino acid sequence of uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase, including any one of the following: i) mutation S263Q or mutation S263L; ii) the method according to i) further comprising one of the mutations F266W, F266Y, F266I or F266L.

2. The mutant according to claim 1, characterized in that The uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase is derived from Campylobacter jejuni.

3. The mutant according to claim 2, characterized in that The uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids, and having the same functional protein.

4. The mutant according to claim 3, characterized in that The gene encoding uridine 5′-diphosphate-N-acetylglucosamine 2 epimerase includes any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

5. Use of the mutant according to any one of claims 1 to 4 in producing sialyllactose in Escherichia coli.

6. The use according to claim 5, characterized in that: The sialyllactose comprises: 3'-sialyllactose and / or 6'-sialyllactose.

7. The use according to claim 5 or 6, characterized in that: The Escherichia coli is an Escherichia coli capable of producing sialyllactose, and the mutant is introduced or integrated into the genome of the Escherichia coli; Preferably, the Escherichia coli further comprises: neuB, neuA and sialyltransferase genes. Further preferably, the Escherichia coli reduces the expression level of one or more genes among the lacZ, nanA, nanK, nanE and nanT genes.

8. A method for improving the ability of Escherichia coli to produce sialyllactose, characterized in that: include: Using the mutant according to any one of claims 1 to 4 to replace the uridine 5′-diphospho-N-acetylglucosamine 2 epimerase in Escherichia coli; or, The mutant according to any one of claims 1 to 4 is introduced or integrated into the genome of the Escherichia coli.

9. The method according to claim 8, characterized in that The mutants are introduced into the E. coli by one or more of plasmid transfection, CaCl2 transformation, electroporation or phage transformation.

10. An Escherichia coli, characterized in that The Escherichia coli is prepared by the method according to claim 8 or 9.

Images