A mutant of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase and its application

Abstract

This invention relates to the field of fermentation engineering technology, and particularly to a mutant of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase and its application. The mutant is based on the amino acid sequence of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase, including any one of the following: i) a mutation of S263Q or S263L; ii) in addition to i), a mutation of one of F266W, F266Y, F266I, or F266L. This invention has discovered multiple mutation modes of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase, and its application in the construction of sialyl lactose-producing *Escherichia coli* can effectively increase the yield of sialyl lactose, which has significant application value in the field of sialyl lactose production.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a mutant of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase and its application. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk and are considered the primary prebiotics beneficial to infant health. Sialactose (SL) accounts for approximately 13% of all HMOs. 3'-Sialactose (3'-SL) and 6'-Sialactose (6'-SL) are the two most abundant and representative sialyl lactoses in breast milk. Numerous efficacy studies have shown that SLs have potential prebiotic effects, particularly in promoting the proliferation of Bifidobacteria and shaping the gut microbiota. In addition, SLs also possess anti-adhesion and antibacterial properties, 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 lactate synthases (SLs) using enzymatic or microbial methods has been extensively studied. Enzymatic synthesis of SLs can be achieved using two types of enzymes: sialic acid synthases with trans-sialic acid esterase activity and sialic acid transferases. Microbial synthesis can be achieved by expressing enzymes involved in the SL synthesis pathway individually or in combination in a one-pot reaction using multiple recombinant strains, or by achieving de novo synthesis during fermentation using metabolically engineered strains. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a mutant of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase and its applications.

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

[0006] The synthesis of sialyl lactose is achieved through the transfer of sialic acid groups from CMP-NeuAc to lactose (st). CMP-NeuAc, on the other hand, can be synthesized based on NeuAc. NeuAc is transported to the engineered host via the sialic acid permease NanT, and further converted to CMP-NeuAc by sialyl cytidine monophosphate synthase (neuA). There are two synthetic pathways for NeuAc: it can be produced from acetylmannosamine (ManNAc) using phosphoenolpyruvate (PEP) as a co-substrate via sialic acid synthase (neuB), or it can be produced from ManNAc using sialic acid lyase (NanA) with pyruvate as a substrate. There are three synthetic pathways for ManNAc: First, uridine 5'-bisphosphate-N-acetylglucosamine (UDP-GlcNAc) is irreversibly converted to ManNAc under the catalysis of hydrolytic UDP-GlcNAc 2-epimerase (neuC); second, acetylglucosamine (GlcNAc) is reversibly catalyzed by N-acetylglucosamine 2-epimerase (age) to synthesize N-acetylglucosamine N-acetylglucosamine; and third, acetylglucosamine 6-phosphate is reversibly converted to acetylglucosamine 6-phosphate via N-acetylglucosamine-6-phosphate 2-epimerase (nanE), which is then further enzymatically converted to ManNAc. All three pathways use glycerol and glucose as initial substrates, metabolizing them to obtain their precursors.

[0007] like Figure 1 The diagram illustrates a typical biosynthetic pathway of sialyl lactose. The uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase (NeuC) gene encodes UDP-acetylglucosamine epimerase, which converts UDP-acetylglucosamine (UDP-GlcNAc) into acetylmmannosamine (ManNAc). This step is crucial in the biosynthesis of sialic acid (SA, NeuAc) and sialyl lactose (SL) and is a necessary step for the biosynthesis of both.

[0008] This invention identifies key modification sites in neuC to obtain corresponding mutants. These mutants significantly optimize the catalytic efficiency after replacing the original neuC, and the resulting engineered strains show a significant increase in sialic acid lactose content.

[0009] In a first aspect, the present invention provides a mutant of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase, said mutant comprising, based on the amino acid sequence of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase, any one of the following:

[0010] i) Mutate S263Q or mutate S263L;

[0011] ii) In addition to i), it also includes one of the mutations F266W, F266Y, F266I or F266L.

[0012] Mutants F266W, F266Y, F266I, and F266L can all increase the yield of sialic acid lactose, with the F266W mutation showing the best effect.

[0013] The above-described mutation is a commonly used expression in the art. Taking S263Q as an example, the mutation at position 263 is located on the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase. S represents the amino acid type before the mutation (serine), and Q represents the amino acid type after the mutation (glutamine). That is, the serine at position 263 is replaced by glutamine.

[0014] Correspondingly, in S263L, serine at position 263 is replaced by leucine; in F266W, phenylalanine at position 266 is replaced by tryptophan; in F266Y, phenylalanine at position 266 is replaced by tyrosine; in F266I, phenylalanine at position 266 is replaced by isoleucine; and in F266L, phenylalanine at position 266 is replaced by leucine.

[0015] Furthermore, the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase is derived from Campylobacter jejuni.

[0016] Furthermore, the uridine 5′-bisphosphate-N-acetylglucosamine 2-epomerase comprises any one of the following amino acid sequences:

[0017] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0018] (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids as shown in SEQ ID NO.1.

[0019] Furthermore, the gene encoding the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase includes any one of the following nucleotide sequences:

[0020] (1) The nucleotide sequence shown in SEQ ID NO.2;

[0021] (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2;

[0022] (3) A nucleotide sequence that can hybridize with a nucleotide sequence as shown in SEQ ID NO.2 under strict conditions.

[0023] Secondly, the present invention provides the application of the aforementioned mutant in the production of sialic acid lactose by Escherichia coli.

[0024] Furthermore, the application is to increase the level of sialic acid lactose production by Escherichia coli.

[0025] Further, the sialyl lactose comprises:

[0026] 3′-sialyl lactose and / or 6′-sialyl lactose.

[0027] Furthermore, the *E. coli* is an *E. coli* species capable of producing sialic acid lactose, and the mutant is introduced into or integrated into the genome of the *E. coli*.

[0028] Preferably, the *Escherichia coli* also possesses neuB, neuA, and sialyl transferase (ST) genes, which are independently integrated into each other or in any combination integrated into the genome of the genetically engineered bacterium, or exist in a recombinant plasmid carried by the genetically engineered bacterium.

[0029] More preferably, the *E. coli* reduces the expression level of one or more of the lacZ, nanA, nanK, nanE, and nanT genes.

[0030] in, l The acZ gene encodes β-galactosidase, and its inactivation or deletion can prevent lactose from being degraded into glucose and galactose. nanA The gene encodes N-acetylmnosamine lyase, and its inactivation or deletion can prevent the degradation of N-acetylneuraminic acid and N-acetylmnosamine. nanK The gene encodes N-acetylmannosamine kinase, and its inactivation or deletion can prevent the degradation of N-acetylmannosamine-6-phosphate. nanE The gene encodes N-acetylmnosamine-6-phosphate-2-epimerase, which can prevent the degradation of glucosamine-6-phosphate after inactivation or deletion. nanT The gene encodes sialic acid transporter, which can prevent the intracellular and extracellular transport of sialic acid when inactivated or deleted; neuA is the CMP-N-acetylneuraminic acid synthase gene, and neuB is the sialic acid synthase gene.

[0031] The reduction of expression levels of lacZ, nanA, nanK, nanE, and nanT genes described in this invention can be achieved using techniques commonly used in the field to reduce gene expression levels, such as gene knockout causing the deletion of gene fragments or complete gene knockout, or RNA interference. Furthermore, the reduction of gene expression levels includes varying degrees of gene level reduction, including complete loss of function. Thirdly, this invention provides a method for improving the ability of *E. coli* to produce sialyl lactose, comprising: replacing the aforementioned mutant with the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase in *E. coli*; or, introducing or integrating the aforementioned mutant into the genome of *E. coli*.

[0032] Furthermore, the aforementioned mutant can be used to replace the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase in Escherichia coli, allowing the neuC mutant to be used directly for strain construction when constructing sialic acid-producing strains.

[0033] Furthermore, the mutant is introduced into the *E. coli* through one or more of the following methods: plasmid transfection, CaCl2 transformation, electroporation, or phage transformation.

[0034] Furthermore, the *Escherichia coli* is an *Escherichia coli* capable of producing sialic acid lactose.

[0035] Fourthly, the present invention provides Escherichia coli prepared by the aforementioned method.

[0036] The present invention has the following beneficial effects:

[0037] This invention overcomes the shortcomings of existing technologies by modifying the amino acid sequence of neuC, a key synthase for sialic acid and sialyl lactose (providing multiple neuC mutants), thereby improving the production efficiency of sialyl lactose. In specific experiments, based on the above modifications, the 3'-SL engineered strain constructed in this invention achieved a maximum 3'-SL yield of 46.85 g / L in a 5L tank, with no detected sialic acid residue; the constructed 6'-SL engineered strain achieved a maximum 6'-SL yield of 45.26 g / L in a 5L tank, with no detected sialic acid residue. The mutants provided by this invention have a significant effect on improving the ability of *E. coli* to synthesize sialyl lactose and have high application value. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 This is a schematic diagram of the synthesis pathway of sialic acid lactose provided by the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0042] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0043] Example 1

[0044] 1. Obtaining mutants

[0045] The uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase mutant provided by this 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), including the following mutation modes: S263Q, S263L, S263Q+F266W, S263Q+F266Y, S263Q+F266I, and S263Q+F266L.

[0046] Table 1. Nucleotide changes corresponding to each mutation mode

[0047]

[0048] 2. Construction of sialic acid lactose-producing strains

[0049] (1) The chassis strain can be constructed using existing technology to produce sialic acid lactose. In this embodiment, the strain prepared by the following method is preferred:

[0050] This invention uses Escherichia coli BL21 as the starting strain and knocks out the following metabolic genes related to SL synthesis precursors in the genome: N-acetylneuraminate lyase (nanA), N-acetylmannosamine kinase (nanK), N-acetylmannosamine-6-phosphate epimerase (nanE), sialic acid transporter (nanT), and beta-D-galactosidase (lacZ) to obtain a chassis strain.

[0051] (2) Construction of plasmids

[0052] Gene sequences of neuB, neuC, and neuA (AF400048) from Campylobacter jejuni, 6'-sialyltransferase 6ST (BAA25316.1) from Photobacterium damselae, and 3'-sialyltransferase 3ST (U60664) from Neisseriagonorrheae were extracted. 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. The obtained chassis strain was transformed with the plasmid pRSF-neuB-neuC-neuA-3ST / 6ST, and the resulting strain was named FS301 / FS601 for the study of 3'-SL / 6'-SL production.

[0053] 3. Shake-flask fermentation

[0054] Inoculate 50 mL of fresh LB medium with 0.5 mL of overnight culture of recombinant Escherichia coli (FS301 / FS601). The absorbance (OD) at 600 nm was measured. 600When the culture reaches 0.6-0.8, 1 mL of the resulting culture is inoculated into 100 mL of the specified culture medium in a 500 mL shake flask. The specified culture medium contains (per liter) 30 g glycerol, 17.9 g Na2HPO4·12H2O, 3.1 g KH2PO4, 2.0 g NH4C, 1.0 g (NH4)2HPO4, 1.7 g citric acid, 15 mg calcium chloride, 2 g yeast extract, 15 g Oxoid tryptone, 2.2 g C6H5Na3O7·2H2O, 1 g MgSO4·7H2O, 0.3 mL 100×Triton-X, 10 mg vitamin B1, 10 mL of trace element solution (25 g FeCl3·6H2O, 2 g CaCl2·2H2O, 2.0 g ZnCl2, 1.9 g CuSO4·5H2O, 0.42 g MnCl·H2O, 2 g Na2B4O7·10H2O, and 2 g... When the culture medium (Na₂MoO₄·2H₂O) reached an OD₆₀ of 0.6–0.8 at 37 °C, isopropyl β-D-1-thiogalactoside and lactose were added to a final concentration of 0.2 mM and 6 g / L, respectively. The culture was then incubated at 28 °C for 48 h. All experiments were repeated three times.

[0055] The results showed that the shake flask yields of 3'-SL and 6'-SL were 1.61 and 1.89 g / L, respectively, corresponding to sialic acid yields of 1.17 and 1.13 g / L.

[0056] 4. Application of mutants

[0057] (1) Based on strain FS301 / FS601, this invention applies the aforementioned mutants, specifically targeting plasmid pRSF-neuB-neuC-neuA-3ST / 6ST, replacing neuC with the aforementioned mutants obtained through the two mutation methods S263Q and S263L.

[0058] The S263L mutant strains, 3FS02 and 6FS02, produced sialyl lactose yields of 1.88 g / L and 2.08 g / L, respectively, which were 16.7% and 10.6% higher than those of strains 3FS01 and 6FS01.

[0059] The S263Q mutant strains, 3FS03 and 6FS03, produced sialyl lactose yields of 2.26 g / L and 2.45 g / L, respectively, which were 40.37% and 29.62% higher than those of strains 3FS01 and 6FS01.

[0060] (2) As can be seen from the above results, the S263Q mutation is more effective. Therefore, based on the S263Q mutation, further F266W, F266Y, F266I and F266L mutations were performed. Similarly, the neuC in pRSF-neuB-neuC-neuA-3ST / 6ST was replaced with mutants (S263Q+F266W, S263Q+F266Y, S263Q+F266I, S263Q+F266L).

[0061] After mutation, strains 3FS03W / 6FS03W, 3FS03Y / 6FS03Y, 3FS03I / 6FS03I, and 3FS03L / 6FS03L were obtained in sequence. The results are shown in Table 1. Among them, 3FS03W / 6FS03W showed the best performance, with 3'-SL and 6'-SL yields of 2.53 g / L and 2.84 g / L, respectively, which were 57.1% and 50.3% higher than those of strains 3FS01 and 6FS01, and the sialic acid residue was significantly reduced.

[0062] Table 2. 3'-SL yield of each strain

[0063]

[0064] Table 3. 6'-SL yield of each strain

[0065]

[0066] 5.5L tank batch feeding fermentation experiment

[0067] This invention utilizes fed-batch fermentation in a 5L bioreactor, pre-loaded with 2.5L of culture medium containing 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. 0.15mL of the culture medium (containing 3FS03W / 6FS03W) is cultured for 6 hours in a 1L shake flask containing 150mL LB medium, then transferred to the bioreactor to prepare a seed culture. Throughout the culture process, the pH of the fermentation broth 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.

[0068] During the cultivation phase, the cultivation temperature was 37℃, the aeration rate was 2 VVM, and the rotation speed was 900 rpm. After the initial glycerol was completely depleted, 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. Simultaneously, the temperature was adjusted to 29.5℃. After another 4 hours of incubation, isopropyl β-d-1-thiogalactoside (final concentration 0.2 mM) and 70 g lactose were added. After another 12 hours of cultivation, 70 g lactose was added again. Samples were taken every 12 hours after culture medium replenishment to detect OD600, lactose, and the corresponding SL levels.

[0069] After 86 hours 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 sialic acid residue was detected in either.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant of uridine 5′-bisphosphate-N-acetylglucosamine 2-episomerase, characterized in that, The mutants are based on the amino acid sequence of uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase, with mutations S263Q and F266W; the amino acid sequence of the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase is shown in SEQ ID NO.

1.

2. The mutant according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase is shown in SEQ ID NO.

2.

3. The use of the mutant according to claim 1 or 2 in the production of sialic acid lactose by Escherichia coli; wherein the sialic acid lactose is 3′-sialic acid lactose and / or 6′-sialic acid lactose.

4. The application according to claim 3, characterized in that, The *E. coli* is an *E. coli* species capable of producing sialic acid lactose, and the mutant is introduced into the cells of the *E. coli* or integrated into the genome of the *E. coli*; the *E. coli* also includes: neuB, neuA, and sialic acid transferase genes; The *E. coli* strain reduced the expression levels of the lacZ, nanA, nanK, nanE, and nanT genes.

5. A method for improving the ability of Escherichia coli to produce sialic acid lactose, characterized in that, include: The mutant described in claim 1 or 2 is used to replace the uridine 5′-bisphosphate-N-acetylglucosamine 2 epimerase in Escherichia coli; or, The mutant of claim 1 or 2 is introduced into the cells of the Escherichia coli or integrated into the genome of the Escherichia coli.

6. The method according to claim 5, characterized in that, The mutant was introduced into the cells of the *E. coli* via one or more of the following methods: plasmid transfection, CaCl2 transformation, electroporation, or phage transformation.

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