UDP-N-acetylmannosamine epimerase mutant as well as recombinant bacteria and application thereof

Abstract

The invention provides a UDP-N-acetylmannosamine epimerase mutant as well as a recombinant strain and application of the UDP-N-acetylmannosamine epimerase mutant. Compared with a wild type, the UDP-N-acetylmannosamine epimerase mutant disclosed by the invention has higher enzyme activity, can be used for efficiently preparing N-acetylneuraminic acid, and has the potential of industrial application.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to a UDP-N-acetylmnosamine epimerase mutant, its recombinant strain, and its applications. Background Technology

[0002] Sialic acids (SA) are a large class of acids with a 9-carbon skeleton. α - Ketoacids. To date, approximately 90 natural forms of sialic acid have been identified from various biological sources. N -acetylneuraminic acid ( N -acetylneuraminicacid, Neu5Ac), N -Hydroxyacetylneuraminic acid ( N Sialic acid consists of three major molecules: α-glycolylneuraminic acid (NeuGc) and ketodeoxynononic acid (Kdn).

[0003] N α-Acetylneuraminic acid is the most ubiquitous monosaccharide unit in sialic acid, often serving as a precursor in the biosynthesis of other sialic acids. Neu5Ac is naturally found in many organisms, including viruses, bacteria, fungi, protozoa, and higher animals. In human cells, Neu5Ac primarily binds to proteins, lipids, and oligosaccharides to form complex sialylated structures. Neu5Ac is also a key monosaccharide unit in sialylated human milk oligosaccharides (HMOs). For example, 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL) are two of the simplest sialylated HMOs, which are composed of... N -Acetylneuraminic acid residues via α 2,3- and α It is obtained by binding 2,6-glycosidic bonds to the galactose residues of lactose. N Acetylneuraminic acid has anti-wrinkle, antioxidant, moisturizing, anti-inflammatory, and wound-healing effects, and has great commercial application potential in the fields of medicine, cosmetics, food additives, and medical care.

[0004] From 2017 to 2024, N- Acetylneuraminic acid, as a functional monosaccharide developed in recent years, has been approved by domestic and foreign regulatory agencies for use in cosmetics, food and other fields. In 2016, the U.S. Food and Drug Administration (FDA) approved Neu5Ac as a "Generally recognized as safe" (GRAS) food additive for infant formula and general food according to the announcement No. 602. In 2017, the European Food Safety Authority (EFSA) approved Neu5Ac as a Novel Food (NF) ingredient according to the announcement No. 258 / 97. In June of the same year, China also approved Neu5Ac as a new food raw material. In addition, in 2022, NMPA approved the expansion N - Application of acetylneuraminic acid in the field of cosmetics. With the continuous introduction of favorable policies at home and abroad and the continuous expansion of its application-based research, N - The application field and scope of acetylneuraminic acid are continuously expanding, accompanied by a surge in downstream demand, N - The market demand for acetylneuraminic acid continues to grow, and the scale is increasing. It is expected that in the future N - Acetylneuraminic acid will have a good market prospect and market development space.

[0005] N - Acetylneuraminic acid can be extracted from natural resources or synthesized by chemical method, but these methods have the disadvantages of complex production process, low production efficiency, poor stereoselectivity, serious environmental pollution and high cost. Whole-cell catalytic production depends on the efficient expression and purification of the required enzymes, and the use of relatively expensive N - Acetylglucosamine (GlcNAc) and pyruvate as substrates, so it is not suitable for large-scale production.

[0006] N-acetylglucosamine-2-epimerase is the key starting enzyme and rate-limiting enzyme for the synthesis of N-acetylneuraminic acid in organisms. It catalyzes the first and decisive isomerization step in the synthesis pathway, converting N-acetylglucosamine to N-acetylmannosamine, thus providing the necessary precursor for the subsequent condensation reaction. Therefore, the catalytic efficiency of N-acetylglucosamine-2-epimerase is the key to improving the yield of NeuAc synthesis.

[0007] The cell factory method based on metabolic engineering strategy synthesizes NAcetylneuraminic acid, which is easy to scale up, is a more promising high-efficiency production strategy. The genetic background of Escherichia coli is clear, and it can efficiently express foreign genes. The related technical operation is simple, and the culture cycle is short. It is one of the most commonly used model chassis microorganisms for engineering production of chemicals. Therefore, it is of great significance to construct an engineered Escherichia coli strain for high-yield production of acetylneuraminic acid. N The engineered Escherichia coli strain for acetylneuraminic acid has important significance for N The engineered Escherichia coli strain for acetylneuraminic acid has important significance for SUMMARY

[0008] In order to solve the problem of low catalytic efficiency of N-acetylglucosamine-2-epimerase known in the prior art, the present application provides a N-acetylglucosamine-2-epimerase mutant, a recombinant strain thereof and an application thereof.

[0009] Specifically, the present application relates to the following aspects: 1. A UDP-N-acetylmannosamine epimerase mutant, wherein the amino acid sequence of the mutant comprises a sequence obtained by mutating the amino acid residues in the sequence shown in SEQ ID NO. 1, and the mutated sites are selected from any one or more of the following sites: 008, 109, 135, 209, 326.

[0010] 2. The UDP-N-acetylmannosamine epimerase mutant according to item 1, wherein the mutation is selected from one or more of the following mutations: T at position 008 is mutated to N, A at position 109 is mutated to E, R at position 135 is mutated to C, T at position 209 is mutated to I, and A at position 326 is mutated to V. Preferably, the mutation is selected from one or two of the following mutations: A at position 109 is mutated to E, and T at position 209 is mutated to I.

[0011] 3. A biological material, wherein the biological material comprises any one of the following materials: A1), a nucleic acid molecule encoding the UDP-N-acetylmannosamine epimerase mutant according to item 1 or 2; A2), an expression cassette containing the nucleic acid molecule according to A1); A3), a recombinant vector containing the nucleic acid molecule according to A1) and / or containing the expression cassette according to A2); A4), a recombinant microorganism containing the nucleic acid molecule according to A1), containing the expression cassette according to A2), and / or containing the recombinant vector according to A3), preferably, the recombinant microorganism comprises at least one of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae; A5), a recombinant cell containing the nucleic acid molecule according to A1), containing the expression cassette according to A2), and / or containing the recombinant vector according to A3); A6) A whole-cell catalyst containing the nucleic acid molecule described in A1), the expression cassette described in A2), the recombinant vector described in A3), the recombinant microorganism described in A4), and / or the recombinant cell described in A5).

[0012] 4. The biological material according to claim 3, wherein the nucleic acid molecule comprises any one or more nucleotide sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0013] 5. An Escherichia coli that produces N-acetylneuraminic acid, wherein the Escherichia coli comprises at least the following modifications: expressing and / or enhancing the expression of the UDP-N-acetylmannosamine epimerase mutant described in item 1 or 2, or introducing a nucleic acid molecule encoding the UDP-N-acetylmannosamine epimerase mutant described in item 1 or 2.

[0014] 6. The *Escherichia coli* according to claim 5, wherein the *Escherichia coli* further comprises at least one of the following modifications: (1) Silencing and / or inhibiting the expression of one or more of the following: β-galactosidase (lacZ), UDP-glucose lipotransferase (wcaJ), N-acetylneuraminic acid aldolase gene nanA and / or N-acetylmannosamine kinase gene nanK; (2) Enhance the expression of β-galactosidase permease (lacY); (3) Express or enhance the expression of the N-acetylneuraminic acid synthase gene neuB.

[0015] 7. The *Escherichia coli* according to claim 5 or 6, wherein the *Escherichia coli* further comprises at least one and more of the following modifications: (1) Silence and / or inhibit one or more of the following genes: nanT (sialic acid transporter), nanE (N-acetylmnosamine-6-P epimerase), and nagB (glucosamine-6-phosphate deaminase); (2) Integrate one or more copies of the UDP-GlcNAc synthesis pathway genes glmM, glmU and glmS.

[0016] 8. The *Escherichia coli* of claim 6, wherein the N-acetylneuraminic acid synthase gene neuB comprises the nucleotide sequence shown in SEQ ID NO. 8.

[0017] 9. Use of the mutant described in item 1 or 2, the biological material described in item 3 or 4, and / or the *Escherichia coli* described in any one of items 5-8, wherein said use is selected from any one of the following: A) Production of N-acetylneuraminic acid; B) Increase the content of N-acetylneuraminic acid in microorganisms.

[0018] 10. A method for producing N-acetylneuraminic acid, comprising at least the step of culturing Escherichia coli as described in any one of items 5-8.

[0019] Beneficial effects of this application This application's UDP- N The -acetylmannosamine epimerase mutant can be efficiently expressed in engineered bacteria, exhibiting a higher N-acetylneuraminic acid yield compared to the wild type.

[0020] The engineered strain in this application integrates exogenous UDP- N - Acetylmannosamine epimerase gene neuC or its mutant genes and N - Acetylneuraminidase synthase gene neuB , constructed N - Acetylneuraminic acid synthesis pathway. Furthermore, the competing pathway genes on the *E. coli* genome were knocked out. N -acetylmannosamine-6-P epimerase gene nanE sialic acid transporter gene nanT and glucosamine-6-phosphate deaminase gene nagB and RNaseE adaptor protein gene rapZ It also expressed multiple copies of the endogenous UDP-GlcNAc synthesis pathway gene. glmM and glmUS To enhance the UDP-GlcNAc supply.

[0021] Finally, the engineered strains of this application were used to prepare... N The optimal yields of acetylneuraminic acid were achieved in shake-flask fermentation and 10L fermenter fermentation, reaching 13.20 g / L and 117.64 g / L, respectively. Therefore, the engineered strain of this application has the potential for industrial application. Attached Figure Description

[0022] Figure 1 For recombinant Escherichia coli N Comparison chart of 10 L fermenter yields of acetylneuraminic acid. Detailed Implementation

[0023] Definitions As used herein, the term "wildtype" has the meaning commonly understood by those skilled in the art as referring to the typical form of an organism, strain, gene, or trait that distinguishes it from mutants or variants when it exists in nature. It can be isolated from resources in nature and is not deliberately modified.

[0024] As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to human involvement. The term "engineered strain" or "engineered strain cell" refers to a bacterial cell that has been genetically modified from its natural state. For example, an engineered bacterial cell can have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into its DNA. These genetic modifications can be present in the chromosome of the bacterium or bacterial cell, or on a plasmid in the bacterium or bacterial cell. The engineered bacterial cells of the present disclosure can comprise exogenous nucleotide sequences on a plasmid. Alternatively, the recombinant bacterial cells can comprise exogenous nucleotide sequences stably integrated into their chromosome.

[0025] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Thus, a gene includes, but is not limited to, coding sequences and / or regulatory sequences required for their expression. A gene can also include unexpressed segments of DNA, which for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloned from a source of interest or synthesized from known or predicted sequence information, and can include sequences designed with desired parameters.

[0026] As used herein, the terms "exogenous" or "heterologous" are used interchangeably and refer to a substance from a source different from its native source. For example, the term "exogenous protein" or "exogenous gene" refers to a protein or gene from a non-native source or location and has been provided to the biological system by artificial means.

[0027] As used herein, the term "knockout" refers to reducing, inhibiting, or eliminating expression of a corresponding gene sequence and / or reducing, inhibiting, or eliminating activity of a protein encoded by the gene. In addition to interrupting the expression of a certain gene, gene knockout also includes introducing a new gene and introducing a site-directed mutation. It can be either knocking out the corresponding normal gene with a mutant gene or other gene, or knocking out the corresponding mutant gene with a normal gene. At present, gene knockout technology has derived from the traditional homologous recombination strategy to more and more new methods, such as Red homologous recombination system, Cre / LoxP site-specific recombination, insertion mutation, RNA interference, zinc finger nuclease (ZFN) technology and transcription activator-like effector nuclease technology, CRISPR / Cas9 gene editing system, etc.

[0028] As used herein, the terms "integration" or "knockin" refer to the replacement of an endogenous gene with an exogenous or heterologous gene or portion thereof. For example, in some embodiments, the terms "integration" or "knockin" refer to the introduction of a nucleic acid sequence encoding a desired protein into a target locus by homologous recombination, thereby resulting in the expression of the desired protein.

[0029] As used herein, the term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using routine techniques for detecting proteins (e.g., detection of enzymatic activity, SDS-Page, ELISA, Western blot, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0030] As used herein, the terms "polynucleotide," "nucleotide sequence," and "nucleic acid molecule" are used interchangeably. They refer to polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs, that can be DNA, such as a cDNA, genomic DNA, or recombinant DNA; or that can be RNA, such as a gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0031] As used herein, the term "encoding" refers to i) the inclusion of genetic information in a DNA sequence that can be transcribed into an RNA molecule, and / or ii) the inclusion of genetic information in an RNA molecule that can be translated into an amino acid sequence. Thus, as used herein, an "encoding sequence" can be used to refer to a ribonucleotide (RNA) sequence or a fragment thereof in an mRNA precursor or mature mRNA that can be translated into a protein, or to the complement or a fragment thereof of a deoxyribonucleotide (DNA) sequence that serves as a template to transcribe the mRNA precursor or mature mRNA. In addition, an "encoding sequence" of the present application can further include a polynucleotide sequence encoding a protein, a functional nucleic acid, or a fragment thereof, such as a miRNA, shRNA, dsRNA, guide RNA, Poly(A) tail, 5' UTR, 3' UTR, etc. Among them, the DNA molecule containing genetic information that can be transcribed into an RNA molecule is referred to as the "encoding nucleic acid" of the RNA molecule; the RNA molecule containing genetic information that can be translated into an amino acid sequence is referred to as the "encoding nucleic acid" of the amino acid sequence.

[0032] As used herein, the term "vector" is used to describe a nucleic acid molecule that can be engineered (e.g., to contain a polynucleotide or polynucleotides that can be expanded in a host cell) to introduce foreign nucleic acid into a host cell. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded or partially double-stranded; that comprise one or more free ends, or that are closed (e.g., circular); that comprise DNA, RNA, or both; and other varieties known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0033] As used herein, the term "recombinant microorganism" includes a microorganism (e.g., bacteria, yeast, algae, fungi, etc.) or strain of microorganism that has been genetically altered, modified, or engineered (e.g., genetically engineered) so that it displays an altered, modified, or different genotype and / or phenotype as compared to the naturally occurring microorganism or "parent" microorganism from which it was derived (e.g., when the genetic modification affects a coding nucleic acid sequence of the microorganism).

[0034] The term "recombinant" when referring to, for example, a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or alteration of a native nucleic acid or protein, or that the cell is derived from such a modified cell. For example, a recombinant protein is a protein produced from a recombinant nucleic acid molecule. The nucleic acid molecule can include genetic material from multiple sources, thus including non-naturally occurring sequences. Recombinant DNA can be produced by methods known in the art of molecular biology, or by synthetic methods. Thus, for example, a recombinant cell expresses a gene not found in the native (non-recombinant) form of the cell, or expresses a native gene that is abnormally expressed, under-expressed, or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0035] As used herein, the term "expression cassette" refers to a nucleotide sequence or corresponding polynucleotide or nucleic acid molecule comprising a desired coding sequence and operably linked regulatory or control sequences, such that a host transformed or transfected with these sequences is able to produce the encoded protein or host cell metabolite. Exemplary expression control sequences can include any of a promoter, ribosome binding site, transcriptional or translational initiation sequence, and termination sequence, or enhancer or activator sequence.

[0036] In view of the problems existing in the prior art, in one aspect, the present application provides a UDP- N- acetylmannosamine epimerase mutants, wherein the amino acid sequence of the mutant comprises a sequence obtained by mutating the amino acid residue at a position in the sequence set forth in SEQ ID NO. 1, the mutated position being selected from any one or more of the following positions: position 008, position 109, position 135, position 209, position 326.

[0037] In some embodiments, the mutation is selected from one or more of the following mutations: T at position 008 to N, A at position 109 to E, R at position 135 to C, T at position 209 to I, A at position 326 to V.

[0038] In some embodiments, the mutation is selected from one or more of the following mutations: A at position 109 to E, T at position 209 to I.

[0039] In another aspect, the present application also provides a biological material, which comprises any one of the following materials: A1), a nucleic acid molecule encoding the above-mentioned UDP-N-acetylmannosamine epimerase mutant; A2), an expression cassette comprising the nucleic acid molecule of A1); A3), a recombinant vector comprising the nucleic acid molecule of A1) and / or comprising the expression cassette of A2); A4), a recombinant microorganism comprising the nucleic acid molecule of A1), comprising the expression cassette of A2), and / or comprising the recombinant vector of A3); A5), a recombinant cell comprising the nucleic acid molecule of A1), comprising the expression cassette of A2), and / or comprising the recombinant vector of A3); A6), a whole-cell catalyst comprising the nucleic acid molecule of A1), comprising the expression cassette of A2), comprising the recombinant vector of A3), comprising the recombinant microorganism of A4), and / or comprising the recombinant cell of A5).

[0040] In some embodiments, the recombinant microorganism comprises at least one of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae.

[0041] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of any one or more of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7.

[0042] In another aspect, the present application also provides an engineered strain, in particular an engineered Escherichia coli strain for producing N-acetylneuraminic acid, which comprises the following modifications: expressing and / or enhancing expression of the above-mentioned UDP-N-acetylmannosamine epimerase mutant or introducing a nucleic acid molecule encoding the above-mentioned UDP-N-acetylmannosamine epimerase mutant.

[0043] In some embodiments, the nucleotide sequence of the gene encoding the above-mentioned UDP-N-acetylmannosamine epimerase mutant is as shown in any one of SEQ ID NO. 3-7.

[0044] In some embodiments, the nucleotide sequence of the N-acetylneuraminic acid synthase gene neuB is as shown in SEQ ID NO. 8.

[0045] Preferably, the engineering of the engineered bacteria further comprises at least one of the following modifications: (1) silencing and / or inhibiting expression of one or more of the following: β-galactoside permease (lacZ), UDP-glucose lipid carrier transferase (wcaJ), N-acetylneuraminic acid aldolase gene nanA, N-acetylmannosamine kinase gene nanK; (2) enhancing expression of β-galactoside permease (lacY); (3) expressing or enhancing expression of N-acetylneuraminic acid synthase gene neuB.

[0046] Preferably, silencing and / or inhibiting expression of β-galactoside permease (lacZ) is achieved by knocking out the β-galactoside permease (lacZ) gene.

[0047] Preferably, silencing and / or inhibiting expression of UDP-glucose lipid carrier transferase (wcaJ) is achieved by knocking out the UDP-glucose lipid carrier transferase (wcaJ) gene.

[0048] Preferably, silencing and / or inhibiting expression of N-acetylneuraminic acid aldolase gene nanA is achieved by knocking out.

[0049] Preferably, the engineered bacteria silences and / or inhibits expression of N-acetylmannosamine kinase gene nanK by knocking out.

[0050] Other methods for gene editing can also be used by those skilled in the art as long as the purpose of silencing and / or inhibiting expression of the target protein is achieved.

[0051] Preferably, the method of overexpressing β-galactoside permease (lacY) comprises replacing the promoter controlling the expression of the β-galactoside permease gene with a strong promoter.

[0052] More preferably, the strong promoter can be selected from one or more of PJ23119, Ptac, PJ23100.

[0053] More preferably, the strong promoter can be PJ23119.

[0054] In some embodiments, the E. coli further comprises at least one and more of the following modifications: (1) silencing and / or inhibiting one or more of the sialic acid transporter gene nanT, the N-acetylmannosamine-6-P epimerase gene nanE, and the glucosamine-6-phosphate deaminase gene nagB; (2) integrating one or more copies of the UDP-GlcNAc synthesis pathway genes glmM, glmU, and the glucosamine-6-phosphate synthase gene glmS.

[0055] Preferably, the silencing and / or inhibiting of the above genes is achieved by knockout.

[0056] In some embodiments, the N-acetylneuraminate synthase gene neuB comprises the nucleotide sequence as set forth in SEQ ID NO. 8.

[0057] In another aspect, the present application also provides uses of the above-mentioned mutants, the above-mentioned biological materials, and / or the above-mentioned engineered strains, wherein the uses are selected from any one of the following: A) producing N-acetylneuraminate; B) increasing the content of N-acetylneuraminate in a microorganism.

[0058] In another aspect, the present application also provides a method for producing N-acetylneuraminate, comprising culturing the above-mentioned engineered strain.

[0059] In some embodiments, the culture medium used for culturing the engineered strain comprises glucose.

[0060] In some embodiments, the culture medium used for culturing the engineered strain is: 20 g / L glucose, 5 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L di-ammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, and 10 ml / L trace metal elements; the trace metal elements comprise: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate.

[0061] In some embodiments, the culture medium used for culturing the engineered strain is: 25 g / L glucose, 5.0 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L di-ammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, and 10 ml / L trace metal elements; the trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L copper sulfate anhydrous, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate.

[0062] It can be understood by those skilled in the art that the specific components of the culture medium and the specific culture conditions can be adjusted according to actual needs.

[0063] The engineered strain of the present application is constructed by integrating an exogenous UDP- N - acetylmannosamine epimerase gene neuC or a mutant gene thereof and N - acetylneuraminic acid synthase gene neuB , to construct N - acetylneuraminic acid synthesis pathway. Further, by knocking out the N - acetylmannosamine-6-P epimerase gene nanE , sialic acid transporter gene nanT and glucosamine-6-phosphate deaminase gene nagB on the genome of E. coli, and integrating the UDP-GlcNAc synthesis pathway genes glmM and glmUS to enhance the supply of UDP-GlcNAc.

[0064] Using the engineered strain of the present application, especially the engineered strain constructed by the UDP- N - acetylmannosamine epimerase mutant gene of the present application, the production of N - acetylneuraminic acid can have very high yield. For example, the yield can reach 117.64 g / L in a 10 L fermenter, which has the potential for industrial application.

[0065] Examples The present application will be further illustrated below in conjunction with examples, which should be understood that the examples are only used to further illustrate and explain the present application, and are not used to limit the present application.

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although essentially similar or equivalent methods and materials known to those in the art can be used in the practice or testing of the present application, methods and materials that are particularly suited to the purposes of the present application are described below. In case of conflict between the definitions and uses of terms in this disclosure and those contained in the prior art, the definitions and uses contained in this disclosure will control. The present application will be further described with reference to the following examples, but it is not intended that the present application be limited to the specific examples.

[0067] The plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercially available products, and the specific operations are carried out according to the kit instructions. The methods of colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells and extraction of bacterial genomes are carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition). The sequencing of plasmids and DNA products is completed by Genewiz (Suzhou). The target genes neuC The relevant mutant plasmids are obtained by site-directed mutagenesis technology using plasmid pTargetT-yghWX-neuBC as a template.

[0068] (I) Culture medium (1) LB liquid medium: yeast extract 5 g / L, protein peptone 10 g / L, sodium chloride 10 g / L.

[0069] (2) LB solid medium: 10 g / L protein peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0070] (3) Fermentation medium: 20 g / L glucose, 5 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L dihydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate.

[0071] (4) Fermentation tank medium: 25 g / L glucose, 5.0 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L di-ammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; the trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L copper sulfate anhydrous, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate.

[0072] (5) Fed-batch fermentation feed solution: 800 g / L glucose, 20 g / L magnesium sulfate heptahydrate, 0.2 g / L thiamine. pH control: 14% ammonia water (w / v).

[0073] (II) N - Fermentation production of acetylneuraminic acid (1) N - Acetylneuraminic acid flask fermentation process: the constructed strain was inoculated into 4 mL LB liquid medium, 37°C, 200 rpm, and cultured overnight for 12 h to obtain a seed solution, 500 μL of the seed solution was inoculated into 25 mL fermentation medium, 37°C, 200 rpm, and cultured until OD 600 was 0.6-0.8, and then 30°C, 200 rpm was continued for induction culture for 72 h.

[0074] (2) N - Acetylneuraminic acid fed-batch fermentation process: a single colony of recombinant E. coli was picked from a plate and inoculated into 4 mL LB medium for overnight culture as a primary seed solution; 4 mL of the primary seed solution was inoculated into 400 mL of fermentation medium and expanded at 37°C, 200 rpm until OD 600 was 2, and then transferred to a 10 L fermentation tank for culture. Fed-batch fermentation was carried out in a 10 L fermentation tank containing 4 L of fermentation medium. The initial temperature was maintained at 37°C. When OD 600 reached about 40, the temperature was lowered to 30°C. Subsequently, the glucose was maintained at a final concentration of 1-5 g / L by flow feeding of the fed-batch fermentation feed solution, the pH was maintained at 6.8±0.2 throughout, and the foam was controlled by adding an antifoam agent. The dissolved oxygen was controlled by adjusting the stirring speed (100-750 rpm) and the aeration amount (0.5-2 vvm).

[0075] (III) N - Detection of acetylneuraminic acid: 1 mL of the fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken for HPLC determination.

[0076] HPLC detection conditions: by high performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II); column: Zorbax NH2; detector: Agilent 1260 Infinity II VWD detector; mobile phase: 50% acetonitrile, 2mM MgCl2, 10 mM H3PO4; flow rate: 0.8 mL / min; column temperature: 35°C; injection volume: 10 μL.

[0077] (Four) Plasmids and strains CRISPR / Cas9 double-plasmid gene editing system has been disclosed in the literature: Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S. Multigene Editing in the Escherichia Coli Genome via the CRISPR / Cas9 System[J]. Applied and Environmental Microbiology, 2015, 81(7), 2506-2514. Example 1: Construction of recombinant expression vector In order to use the CRISPR-Cas9 double-plasmid gene integration system in Escherichia coli to yghWX Site-integrated genes neuBC ( neuB and neuC The recombinant expression vector pTargetT-yghWX-neuBC was constructed as follows (the primer sequences involved are shown in Table 1): (1) Using the original pTargetF plasmid as the template, yghWX-N 20 -F / R as primers, the N 20 sequences on the original pTargetF plasmid were replaced with N 20 sequences complementary to the yghWX sequence by PCR amplification, obtaining the pTargetF plasmid with targeted genes. yghWX After removing the template DNA with DpnI, the PCR product was transformed into Escherichia coli JM109 competent cells, spread on LB plates (containing spectinomycin), and cultured at 37°C to extract plasmids and perform sequencing.

[0078] (2) Using the Escherichia coli MG1655 genome as the template, the upper and lower fragments of neuB were amplified by PCR using primers yghWX- UP-F / R and yghWX-DH-F / R; the codon-optimized yghWX neuB gene was synthesized. N. meningitidisSource of genes neuBC Using the nucleotide sequence shown in SEQ ID NO.9 as a template, the resulting protein was amplified by PCR using primers neuB-F and neuC-R. neuBC Fragment; using pTargetF plasmid as a template, the pTargetF vector fragment was amplified by PCR using primers yghWX-VF / R.

[0079] (3) The pTargetF vector fragment was assembled using Gibson. yghWX Upstream and downstream homologous fragments and neuBC The fragments were assembled to obtain the plasmid pTargetT-yghWX-neuBC.

[0080] To utilize the CRISPR-Cas9 dual-plasmid gene integration system in E. coli yghWX Site integration genes neuBC The NeuC mutant plasmids were obtained by PCR using pTargetT-yghWX-neuBC plasmid as the vector template and primers designed for each desired mutated amino acid site (primer sequences are shown in Table 1). After removing template DNA with DpnI, the PCR products were transformed into E. coli JM109 competent cells, plated on LB agar plates (containing spectinomycin), and cultured at 37°C to extract plasmids and sequence them.

[0081] In E. coli manX Site integration genes neuBCT209I The specific steps for constructing the recombinant expression vector pTargetT-manX-neuBCT209I are the same as described above.

[0082] Based on the above steps, the integration on the genome was obtained. neuBC The plasmids required for their mutants are pTargetT-yghWX-neuBC, pTargetT-yghWX-neuBCA109E, pTargetT-yghWX-neuBCA326V, pTargetT-yghWX-neuBCT008N, pTargetT-yghWX-neuBCR135C, pTargetT-yghWX-neuBCT109I, and pTargetT-manX-neuBCT109I.

[0083] Table 1

[0084]

[0085]

[0086]

[0087] Example 2: Integration of genes in recombinant strains N - acetylneuraminic acid synthesis competition pathway genes Using CRISPR-Cas9 double-plasmid gene integration system in E. coli yghWX 、 poxB 、 hylE 、 yahO 、 ldhA Integrate genes neuBC ( and mutants thereof), glmM 、 glmUS to integrate yghWX site neuBC For example, the specific steps are as follows (the primer sequences involved are shown in Table 1): (1) Take pCas plasmid into the transformation competent cells of E. coli MGC02 (constructed according to the method in CN117305211A), and spread the transformed bacterial solution on LB plates containing kanamycin, and incubate in a 37°C incubator overnight to obtain MGC02-pCas.

[0088] (2) Pick a single colony of MGC02-pCas in step (1) in LB medium, and incubate at 30°C for 1.0 h, and then add L-arabinose to a final concentration of 10 mM to induce the expression of pCas-λ-red system. When the OD 600 reaches 0.6, prepare MGC08-pCas competent cells.

[0089] (3) Electroporate 300 ng of pTargetT-yghWX-neuBC plasmid of Example 1 into the MGC08-pCas competent cells of step (2), and spread on LB plates (containing kanamycin and spectinomycin), and incubate at 30°C for 24 h. Verify the integration effect of the gene neuBC on yghWX , neuBC which is integrated on yghWX is a positive clone.

[0090] (4) Pick the positive clone colony obtained in step (3) into 4 mL LB liquid test tube, and add IPTG to a final concentration of 1 mM and 50 mg / L kanamycin, and incubate at 30°C for 8-16 h to remove the pTargetT-yghWX-neuBC plasmid, and then incubate at 42°C for 12 h to remove the pCas plasmid. Obtain the MGN01 strain neuBC integrated on yghWX .

[0091] (5) Use the same method to integrate yghWX,poxB , hylE , yahO , ldhA respectively integrated genes neuBC mutants (neuBCA109E, neuBCA326V, neuBCT008N, neuBCR135C, neuBCT109I), glmM and glmUS (Concatenated genes) glmU and glmS , to obtain the corresponding recombinant strains.

[0092] Example 3: Knockout of acetylneuraminic acid in recombinant strains N - Example 4: Knockout of genes in MGN06 Knocking out genes in E. coli using CRISPR-Cas9 double-plasmid gene knockout system nanT , nanE , nagB , to knockout genes in MGN06 nanT , for example, the specific steps are as follows (the primer sequences involved are shown in Table 1): (1) Using the original pTargetF plasmid as the template, and using nanT-N 20 -F / R as the primers, the N 20 sequence on the original pTargetF plasmid is replaced with the N 20 sequence complementary to the N nanT sequence by PCR amplification, to obtain the pTargetF plasmid with the targeted gene nanT . After removing the template DNA from the PCR product, the E. coli JM109 competent cells are transformed, and the LB plates (containing spectinomycin) are coated, and the plasmid is extracted and sequenced after being cultured at 37°C. DpnI

[0093] (2) Using the E. coli MG1655 genome as the template, and using the primers nanT-UP-F / R and nanT-DH-F / R, the upper and lower homologous fragments of nanT are amplified by PCR; using the pTargetF plasmid as the template, and using the primer nanT-V-F / R, the pTargetF vector fragment is amplified by PCR.

[0094] (3) The pTargetF vector fragment is assembled with the upper and lower homologous fragments of nanT by Gibson assembly, to obtain the plasmid pTargetT-nanT.

[0095] (4) The pCas plasmid is transformed into the competent cells of E. coli MGN06, and the transformed bacterial liquid is coated on the LB plate containing kanamycin, and cultured overnight in a 37°C incubator to become MGN06-pCas.​

[0096] (5) Pick a single colony of MGN06-pCas from step (4) to LB medium, incubate at 30℃ for 1.0 h, add L-arabinose to a final concentration of 10 mM to induce the expression of pCas-λ-red system. When the OD 600 of the culture reaches 0.6, prepare MGN06-pCas competent cells.

[0097] (6) Electroporate 300 ng of pTargetT-nanT plasmid from step (3) into MGN06-pCas competent cells from step (5), and plate on LB plates (kanamycin and spectinomycin) at 30℃ for 24 h. Verify the knockout effect of the gene nanT by PCR, nanT and the knockout is the positive clone.

[0098] (7) Pick the positive clone from step (6) to 4 mL LB liquid tube, add IPTG to a final concentration of 1 mM and 50 mg / L kanamycin, incubate at 30℃ for 8-16 h to remove the pTargetT-nanT plasmid, and then incubate at 42℃ for 12 h to remove the pCas plasmid. Obtain the MGN07 strain with the gene nanT knocked out.

[0099] (8) Use the same method to knock out the genes nanE , nagB in the strain in turn, and obtain the corresponding knockout strains.

[0100] The obtained strains and their corresponding information are shown in Table 2.

[0101] Table 2 Recombinant strains and corresponding N - colitose production information

[0102] Example 4: Shake flask fermentation of recombinant strains to produce N colitose The recombinant strains constructed in Examples 2 and 3 (see Table 2 for details) were inoculated into LB liquid medium containing the corresponding antibiotics, incubated at 37℃, 200 rpm, overnight for 12 h to obtain seed liquid, and 500 μL of seed liquid was inoculated into 25 mL of fermentation medium, incubated at 37℃, 200 rpm, until the OD 600 reached 0.8, and then incubated at 30℃, 200 rpm for 72 h for induction. 1 mL of fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was used for HPLC determination. The results are shown in Table 2, wherein N the highest yield of colitose was 11.9 g / L.

[0103] Example 5: Fermentation tank batch feeding culture production N - Acetylneuraminic acid Strain MGN12 was selected for fermentation in a 10 L fermenter. N -Federated fermentation experiment of acetylneuraminic acid. Single colonies of recombinant Escherichia coli MGN12 were picked from plates and inoculated into 4 mL of LB medium and cultured overnight as primary seed culture. 4 mL of the primary seed culture was then inoculated into 400 mL of fermentation medium and cultured at 37°C and 200 rpm until OD reached... 600 After 2 hours, the culture medium was transferred to a 10 L fermenter for further cultivation. Fed-batch fermentation was carried out in a 10 L fermenter containing 4 L of culture medium. The initial temperature was maintained at 37°C. When OD... 600 When the temperature reached approximately 40°C, it was cooled to 30°C. Subsequent fed-batch fermentation was used to maintain the glucose concentration at a final level of 1-5 g / L, and the pH was maintained at 6.8±0.2 throughout the process. Foaming was controlled by adding an antifoaming agent. Dissolved oxygen was controlled by adjusting the stirring speed (100-750 rpm) and aeration rate (0.5-2 vvm). After 84 hours of fermentation, strain MGN12... N - Acetylneuraminic acid production was the highest, reaching 117.92 g / L, OD 600 The highest reached 268 ( Figure 1 ).

[0104] Related sequence information SEQ ID NO.1 NeuC MKRILCITGTRADFGKLKPLLAYIENHPDLELHLIVTGMHMMKTYGRTYKEVTRENYQHTYLFSNQIQGEPMGAVLGNTITFISRLSDEIEPDMVMIHGDRLEALAGAAVGALSSRLVCHIEGGELSGTVDDSIRHSISKLSHIHLVANEQAVTRLVQMGEKRKHIHIIGSPDLDVMASSTLPSLEEV KEYYGLPYENYGISMFHPVTTEAHLMPQYAAQYFKALELSGQNIISIYPNNDTGTESILQELLKYQSDKFIAFPSIRFEYFLVLLKHAKFMVGNSSAGIREAPLYGVPSIDVGTRQSNRHMGKSIIHTDYETKNIFDAIQQACSLGKFEADDTFNGGDTRTSTERFAEVINNPETWNVSAQKRFIDLNL SEQ ID NO. 2 neuC SEQ ID NO. 3 neuCT008N SEQ ID NO. 4 neuCA109E SEQ ID NO. 5 neuCR135C SEQ ID NO. 6 neuCT209I SEQ ID NO. 7 neuCA326V SEQ ID NO. 8 neuB SEQ ID NO. 9 neuBC SEQ ID NO. 10 P J23119 TCTGTGCGGTATTTCACACCGCATATGCTGGATCCTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC SEQ ID NO. 11 glmM MSNRKYFGTDGIRGRVGDAPITPDFVLKLGWAAGKVLARHGSRKIIIGKDTRISGYMLESALEAGLAAAGLSALFTGPMPTPAVAYLTRTFRAEAGIVISASHNPFYDNGIKFFSIDGTKLPDAVEEAIEAEMEKEISCVDSAELGKASRIVDAAGRYIEFCKATFPNELSLSELKIVVDCANGATYHIAPNVLRELGANVIAIGCEPNGVNINAEVGATDVRALQARVLAEKADLGIAFDGDGDRVIMVDHEGNKVDGDQIMYIIAREGLRQGQLRGGAVGTLMSNMGLELALKQLGIPFARAKVGDRYVLEKMQEKGWRIGAENSGHVILLDKTTTGDGIVAGLQVLAAMARNHMSLHDLCSGMKMFPQILVNVRYTAGSGDPLEHESVKAVTAEVEAALGNRGRVLLRKSGTEPLIRVMVEGEDEAQVTEFAHRIADAVKAV* SEQ ID NO. 12 glmU MLNNAMSVVILAAGKGTRMYSDLPKVLHTLAGKAMVQHVIDAANELGAAHVHLVYGHGGDLLKQALKDDNLNWVLQAEQLGTGHAMQQAAPFFADDEDILMLYGDVPLISVETLQRLRDAKPQGGIGLLTVKLDDPTGYGRITRENGKVTGIVEHKDATDEQRQIQEINTGILIANGADMKRWLAKLTNNNAQGEYYITDIIALAYQEGREIVAVHPQRLSEVEGVNNRLQLSRLERVYQSEQAEKLLLAGVMLRDPARFDLRGTLTHGRDVEIDTNVIIEGNVTLGHRVKIGTGCVIKNSVIGDDCEISPYTVVEDANLAAACTIGPFARLRPGAELLEGAHVGNFVEMKKARLGKGSKAGHLTYLGDAEIGDNVNIGAGTITCNYDGANKFKTIIGDDVFVGSDTQLVAPVTVGKGATIAAGTTVTRNVGENALAISRVPQTQKEGWRRPVKKK* SEQ ID NO. 13 glmS MCGIVGAIAQRDVAEILLEGLRRLEYRGYDSAGLAVVDAEGHMTRLRRLGKVQMLAQAAEEHPLHGGTGIAHTRWATHGEPSEVNAHPHVSEHIVVVHNGIIENHEPLREELKARGYTFVSETDTEVIAHLVNWELKQGGTLREAVLRAIPQLRGAYGTVIMDSRHPDTLLAARSGSPLVIGLGMGENFIASDQLALLPVTRRFIFLEEGDIAEITRRSVNIFDKTGAEVKRQDIESNLQYDAGDKGIYRHYMQKEIYEQPNAIKNTLTGRISHGQVDLSELGPNADELLSKVEHIQILACGTSYNSGMVSRYWFESLAGIPCDVEIASEFRYRKSAVRRNSLMITLSQSGETADTLAGLRLSKELGYLGSLAICNVPGSSLVRESDLALMTNAGTEIGVASTKAFTTQLTVLLMLVAKLSRLKGLDASIEHDIVHGLQALPSRIEQMLSQDKRIEALAEDFSDKHHALFLGRGDQYPIALEGALKLKEISYIHAEAYAAGELKHGPLALIDADMPVIVVAPNNELLEKLKSNIEEVRARGGQLYVFADQDAGFVSSDNMHIIEMPHVEEVIAPIFYTVPLQLLAYHVALIKGTDVDQPRNLAKSVTVE SEQ ID NO. 14 nanT MSTTTQNIPWYRHLNRAQWRAFSAAWLGYLLDGFDFVLIALVLTEVQGEFGLTTVQAASLISAAFISRWFGGLMLGAMGDRYGRRLAMVTSIVLFSAGTLACGFAPGYITMFIARLVIGMGMAGEYGSSATYVIESWPKHLRNKASGFLISGFSVGAVVAAQVYSLVVPVWGWRALFFIGILPIIFALWLRKNIPEAEDWKEKHAGKAPVRTMVDILYRGEHRIANIVMTLAAATALWFCFAGNLQNAAIVAVLGLLCAAIFISFMVQSAGKRWPTGVMLMVVVLFAFLYSWPIQALLPTYLKTDLAYNPHTVANVLFFSGFGAAVGCCVGGFLGDWLGTRKAYVCSLLASQLLIIPVFAIGGANVWVLGLLLFFQQMLGQGIAGILPKLIGGYFDTDQRAAGLGFTYNVGALGGALAPIIGALIAQRLDLGTALASLSFSLTFVVILLIGLDMPSRVQRWLRPEALRTHDAIDGKPFSGAVPFGSAKNDLVKTKS* SEQ ID NO. 15 nanE MSLLAQLDQKIAANGGLIVSCQPVPDSPLDKPEIVAAMALAAEQAGAVAIRIEGVANLQATRAVVSVPIIGIVKRDLEDSPVRITAYIEDVDALAQAGADIIAIDGTDRPRPVPVETLLARIHHHGLLAMTDCSTPEDGLACQKLGAEIIGTTLSGYTTPETPEEPDLALVKTLSDAGCRVIAEGRYNTPAQAADAMRHGAWAVTVGSAITRLEHICQWYNTAMKKAVL SEQ ID NO. 16 nanB MRLIPLTTAEQVGKWAARHIVNRINAFKPTADRPFVLGLPTGGTPMTTYKALVEMHKAGQVSFKHVVTFNMDEYVGLPKEHPESYYSFMHRNFFDHVDIPAENINLLNGNAPDIDAECRQYEEKIRSYGKIHLFMGGVGNDGHIAFNEPASSLASRTRIKTLTHDTRVANSRFFDNDVNQVPKYALTVGVGTLLDAEEVMILVLGSQKALALQAAVEGCVNHMWTISCLQLHPKAIMVCDEPSTMELKVKTLRYFNELEAENIKGL SEQ ID NO. 17 neuB MQNNNEFKIGNRSVGYNHEPLIICEIGINHEGSLKTAFEMVDAAYNAGAEVVKHQTHIVEDEMSDEAKQVIPGNADVSIYEIMERCALNEEDEIKLKEYVESKGMIFISTPFSRAAALRLQRMDIPAYKIGSGECNNYPLIKLVASFGKPIILSTGMNSIESIKKSVEIIREAGVPYALLHCTNIYPTPYEDVRLGGMNDLSEAFPDAIIGLSDHTLDNYACLGAVALGGSILERHFTDRMDRPGPDIVCSMNPDTFKELKQGAHALKLARGGKKDTIIAGEKPTKDFAFASVVADKDIKKGELLSGDNLWVKRPGNGDFSVNEYETLFGKVAACNIRKGAQIKKTDIE*

Claims

1. A UDP- N - acetylmannosamine epimerase mutant, wherein the amino acid sequence of said mutant comprises a sequence obtained by mutating an amino acid residue in the sequence shown in SEQ ID NO. 1 at a position selected from any one or more of the following positions: position 008, position 109, position 135, position 209, position 326.​ 2. The UDP- according to claim 1 N - acetylmannosamine epimerase mutants, wherein the mutation is selected from one or more of the following mutations: T008 to N, A109 to E, R135 to C, T209 to I, A326 to V; Preferably, the mutation is selected from one or both of the following mutations: mutation of A at position 109 to E, mutation of T at position 209 to I.

3. The biological material, wherein the biological material comprises any one of the following: A1) a nucleic acid molecule encoding the mutant UDP-N-acetylmannosamine epimerase of claim 1 or 2; A2) an expression cassette comprising the nucleic acid molecule of A1); A3) a recombinant vector comprising the nucleic acid molecule of A1) and / or comprising the expression cassette of A2); A4) a recombinant microorganism comprising the nucleic acid molecule of A1), comprising the expression cassette of A2), and / or comprising the recombinant vector of A3), preferably the recombinant microorganism comprises at least one of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae; A5) a recombinant cell comprising the nucleic acid molecule of A1), comprising the expression cassette of A2), and / or comprising the recombinant vector of A3); A6) a whole cell catalyst comprising the nucleic acid molecule of A1), comprising the expression cassette of A2), comprising the recombinant vector of A3), comprising the recombinant microorganism of A4), and / or comprising the recombinant cell of A5).

4. The biological material of claim 3, wherein the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and any combination thereof.

5. An Escherichia coli producing N UDP-N-acetylmannosamine, wherein said Escherichia coli comprises at least the following modification: expression and / or enhanced expression of a UDP-N-acetylmannosamine epimerase mutant according to claim 1 or 2 or introduction of a nucleic acid molecule encoding a UDP-N-acetylmannosamine epimerase mutant according to claim 1 or 2.

6. The Escherichia coli of claim 5, wherein the Escherichia coli further comprises at least one of the following modifications: (1) silencing and / or inhibiting expression of one or more of the following genes: beta-galactoside permease (lacZ), UDP-glucose lipid carrier transferase (wcaJ), N-acetylneuraminate aldolase gene nanA, N-acetylmannosamine kinase gene nanK; (2) enhancing expression of beta-galactoside permease (lacY); (3) expressing or enhancing expression of N-acetylneuraminate synthase gene neuB.

7. The Escherichia coli of claim 5 or 6, wherein the Escherichia coli further comprises at least one or more of the following modifications: (1) silencing and / or inhibiting one or more of the following genes: sialic acid transporter gene nanT, N-acetylmannosamine-6-P epimerase gene nanE, glucosamine-6-phosphate deaminase gene nagB; (2) integrating one or more copies of the following genes: UDP-GlcNAc synthesis pathway genes glmM, glmU, and glucosamine-6-phosphate synthase gene glmS.

8. The Escherichia coli of claim 6, wherein the N-acetylneuraminate synthase gene neuB comprises the nucleotide sequence as set forth in SEQ ID NO.

8.

9. Use of the mutant of claim 1 or 2, the biological material of claim 3 or 4, and / or the Escherichia coli of any one of claims 5-8, wherein the use is selected from any one of the following: A) producing N-acetylneuraminate; B) increasing N-acetylneuraminate content in a microorganism. ​ 10. A method of producing N-acetylneuraminic acid, comprising at least the step of culturing the E. coli of any one of claims 5-8.

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