CgGlmS mutant and application thereof in synthesis of GlcNAc by corynebacterium glutamicum

Abstract

The invention discloses a CgGlmS mutant and application of the CgGlmS mutant in synthesis of N-acetylglucosamine (GlcNAc) from corynebacterium glutamicum, and belongs to the technical field of biology. According to the invention, a bottleneck enzyme CgGlmS in a synthetic route of N-acetylglucosamine is subjected to deep rational analysis and semi-rational transformation. Through systematic research on the structure and function of an original CgGlmS enzyme from corynebacterium glutamicum S9114, the efficient CgGlmS mutant A253M is successfully screened out, and the enzyme activity, stability and catalytic efficiency of the mutant are obviously improved compared with those of a wild type. The mutant CgGlmS is overexpressed in corynebacterium glutamicum S9114 by using plasmids, so that the yield of N-acetylglucosamine in corynebacterium glutamicum is increased. The CgGlmS enzyme is directionally modified, so that the catalytic efficiency of the CgGlmS enzyme is improved, accumulation of N-acetylglucosamine in corynebacterium glutamicum is further promoted, and powerful support is provided for efficient production of the corynebacterium glutamicum.

Description

Technical Field

[0001] The present invention relates to a CgGlmS mutant and application thereof in GlcNAc synthesis by Corynebacterium glutamicum, belonging to the field of biotechnology. Background Art

[0002] N-acetylglucosamine (GlcNAc) is a monosaccharide widely found in organisms, with significant distribution in bacteria, fungi, plants, and animals. It is a precursor for the disaccharide unit of glycosaminoglycans (GAGs) and plays a key role in the repair and maintenance of cartilage and joint tissue function. In recent years, N-acetylglucosamine has seen increasing application in medicine, cosmetics, and food. It is often a key ingredient in medications for diseases such as arthritis, effectively alleviating joint pain and inflammation. In cosmetics, N-acetylglucosamine is used in skincare products to enhance skin moisturizing and elasticity. Currently, progress has been made in the production of N-acetylglucosamine using Corynebacterium glutamicum. Existing research focuses on regulating metabolic pathways through metabolic engineering to enhance product synthesis. However, relying solely on metabolic pathway remodeling can make it difficult to achieve coordinated intracellular metabolic fluxes. This is particularly true when it is unclear whether key enzymes constitute a bottleneck. This can lead to increased metabolic load and accumulation of byproducts, which can affect the synthesis efficiency of the target product and the growth of host cells. While some studies have attempted to improve product levels by engineering key enzymes in the synthesis pathway, the identification and in-depth mechanistic understanding of the rate-limiting enzyme remains relatively limited. The presence of rate-limiting enzymes is often a key factor limiting product synthesis. Targeted engineering of these enzymes, focusing on their catalytic efficiency and substrate affinity, is expected to significantly enhance N-acetylglucosamine synthesis.

[0003] Furthermore, current research on modifying key enzymes in the N-acetylglucosamine biosynthesis pathway is relatively limited. Existing studies have primarily focused on improving product synthesis efficiency by modifying the activity and substrate specificity of enzymes involved in glucosamine synthesis. For example, Zheng-Bing Guan et al. modified the binding pocket of the BsGlmS enzyme from Bacillus subtilis, enabling it to directly synthesize glucosamine using fructose as a substrate. This study provides a new pathway for the synthesis of N-acetylglucosamine. Another study, by Piwu Li et al., performed saturation mutagenesis targeting the fructose-6-phosphate binding pocket and, through rational analysis, identified a superior mutant of the Bacillus subtilis-derived GlmS enzyme. The results showed that the mutant L593S exhibited significantly increased enzyme activity, from 5 U / mL to 48 U / mL, demonstrating its potential for industrial production. However, in research on GlcNAc synthesis in Corynebacterium glutamicum, literature has reported on screening the key enzyme GlmS from different sources to enhance biosynthesis capacity, and found that endogenous GlmS from Corynebacterium glutamicum exhibited superior compatibility and catalytic efficiency. Although heterologous enzymes may possess high activity, expression in non-native hosts is often limited by multiple factors, including protein stability, folding efficiency, cofactor compatibility, and differences in regulatory mechanisms. Therefore, targeted engineering of the endogenous GlmS in Corynebacterium glutamicum can avoid the compatibility issues associated with heterologous expression while also facilitating further enhancement of enzyme activity within the host context, offering greater feasibility and potential for application (DENG C, LV X, LIUY, et al. Metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis).

[0004] Therefore, the present invention is proposed based on the above two points. Summary of the Invention

[0005] For solving the above problems, the present invention expects to transform the key enzyme that fails to fully optimize in the prior art to improve the output of N-acetylglucosamine in Corynebacterium glutamicum. The present invention, through a semi-rational transformation strategy, explores potential transformation sites in conjunction with virtual saturation mutation, and in conjunction with a high-throughput enzyme activity screening method, successfully screens out the CgGlmS mutant with significant advantages. Further, by technologies such as kinetic simulations, the mutant and the original enzyme are systematically compared, verifying the lifting of its catalytic efficiency and the enhancing of its stability. Finally, the present invention successfully applies the optimized CgGlmS mutant to Corynebacterium glutamicum S9114, significantly improving the output of N-acetylglucosamine.

[0006] The first object of the present invention is to provide a 6-phosphoglucosamine synthetase (CgGlmS) mutant derived from Corynebacterium glutamicum, the amino acid sequence of which is shown in SEQ ID NO.3 (relative to the wild type, the mutation site is alanine at position 253 mutated to methionine).

[0007] The second object of the present invention is to provide a nucleic acid molecule encoding the 6-phosphoglucosamine synthetase mutant.

[0008] Furthermore, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0009] The third object of the present invention is to provide a gene expression cassette or recombinant plasmid carrying the nucleic acid molecule.

[0010] Furthermore, the backbone vector of the recombinant plasmid includes pJYW4 plasmid.

[0011] Furthermore, the gene expression cassette or recombinant plasmid contains a promoter, and the promoter includes the constitutive promoter tac (the promoter used in the embodiment of the present invention).

[0012] The fourth object of the present invention is to provide a recombinant cell expressing the 6-phosphoglucosamine synthetase mutant.

[0013] Furthermore, the host of the recombinant cell includes bacteria, fungi or animal cells.

[0014] Furthermore, the host of the recombinant cell is preferably Escherichia coli or Corynebacterium glutamicum.

[0015] The fifth object of the present invention is to provide the use of the 6-phosphoglucosamine synthetase mutant, nucleic acid molecule, gene expression cassette or recombinant plasmid or recombinant cell in the preparation of N-acetylglucosamine.

[0016] The sixth object of the present invention is to provide a recombinant Corynebacterium glutamicum, wherein the recombinant Corynebacterium glutamicum overexpresses the gene glmS encoding the 6-phosphoglucosamine synthetase mutant.

[0017] Furthermore, the host bacteria of the recombinant Corynebacterium glutamicum includes Corynebacterium glutamicum S9114.

[0018] Furthermore, the recombinant Corynebacterium glutamicum also overexpresses the N-acetylglucosamine transferase encoding gene GNA1.

[0019] Furthermore, the N-acetylglucosamine transferase encoding gene GNA1 is numbered as Gene ID: 850529.

[0020] The seventh object of the present invention is to provide a method for producing N-acetylglucosamine, comprising the step of fermentation production using the recombinant Corynebacterium glutamicum.

[0021] Beneficial effects of the present invention:

[0022] The present invention successfully obtained a mutant A253M with excellent performance by carrying out semi-rational transformation to the CgGlmS enzyme in Corynebacterium glutamicum S9114. Compared with the control group, the enzyme activity increased by 9.3% compared to the wild type. Further, this mutant was applied to the metabolic process of Corynebacterium glutamicum S9114, successfully improving the output of N-acetylglucosamine, with the output increase reaching 12%, from 5.9g / L to 6.6g / L. Showing that this mutant has a remarkable effect in increasing output. In addition, the present invention also improves the stability and catalytic efficiency of the CgGlmS enzyme, improves the production capacity of the target product, and has good industrial application prospects. The successful implementation of the present invention provides new ideas for further optimizing the production of N-acetylglucosamine, especially achieving breakthrough progress in the transformation of key enzymes, and provides important reference value for the transformation and promotion of other N-acetylglucosamine production strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Detection of GlcNAc precursor concentration and Ramachandran plot analysis of CgGlmS: (a) detection of the content of the precursor in the fermentation broth; (b) Ramachandran plot analysis of CgGlmS.

[0024] Figure 2 The mechanism of action and molecular docking of CgGlmS: (a) three-dimensional structure of CgGlmS;

[0025] (b) Schematic diagram of the catalytic mechanism of CgGlmS; (c) Molecular docking results of CgGlmS.

[0026] Figure 3For the transformation process, virtual saturation mutagenesis and enzyme activity detection results: (a) mutant screening process; (b) results of virtual saturation mutagenesis of the F6P active pocket amino acid of CgGlmS; (c) results of virtual saturation mutagenesis of the Gln active pocket amino acid of CgGlmS; (d) crude enzyme activity detection results of the mutant library.

[0027] Figure 4 Results of virtual saturation mutation of the loop region and crude enzyme activity detection: (a) Results of virtual saturation mutation of amino acids in the non-conserved loop region of CgGlmS; (b) Results of crude enzyme activity detection of the mutant library.

[0028] Figure 5 The enzyme activity of the dominant mutant was detected and the yield of GlcNAc produced by fermentation was determined.

[0029] Figure 6 These are the results of mutant molecular docking, dynamics simulation and relative binding free energy analysis.

[0030] (a) Docking results of the mutant and product GlcN6P; (b) Docking results of the mutant and product Glu; (c) RMSD analysis of the mutant and control group; (d) RMSF analysis of the mutant and control group; (e) Relative binding free energy of the mutant and control group with small molecules during 100ns of molecular dynamics. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0032] The methods and materials involved in the following examples are as follows:

[0033] (1) Culture medium

[0034] E. coli culture medium LB (g / L): yeast powder 5.0, peptone 10.0, NaCl 10.0

[0035] Seed activation liquid medium BHIS (g / L): Brain Heart Infusion 37.0, Sorbitol 91.0.

[0036] Seed activation solid medium BHIS (g / L): brain heart infusion 37.0, sorbitol 91.0, nutrient agar 20.0.

[0037] Competent medium (g / L): peptone 10.0, yeast extract 5.0, NaCl 10.0, glycine 30.0, isoniazid 4.0, and 10 mL of Tween 80.

[0038] Recovery medium BHIS (g / L) after electroporation transformation: 5.0% peptone, 2.5% yeast extract, 5.0% NaCl, 18.5% brain heart infusion, and 91.0% sorbitol.

[0039] Solid BHIS medium for transformant coating (g / L): 5.0% peptone, 2.5% yeast extract, 5.0% NaCl, 18.5% brain heart infusion, 91.0% sorbitol, and 20.0% nutrient agar.

[0040] Sterilization conditions: 115°C, 20 min. Add 25 mg / L kanamycin sulfate to all culture media used for transformant detection or recombinant bacterial culture.

[0041] (2) PCR method

[0042] The PCR reaction system consisted of 25 μL of PrimeSTAR Max (purchased from Takara), 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 1 μL of template DNA, and 22 μL of double-distilled water. PCR amplification conditions were: initial denaturation at 98°C for 5 minutes, followed by 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 3 minutes, followed by a final incubation at 72°C for 5 minutes.

[0043] (III) Method for preparing competent E. coli cells

[0044] E. coli competent cells were prepared using the Competent Cell Preparation Kit (purchased from Takara). The preparation method is described in the kit instructions.

[0045] (IV) Plasmid chemical transformation into E. coli competent cells

[0046] Thaw competent E. coli cells stored in a freezer on ice; add 6 μL of PCR product, mix gently, and incubate on ice for 45 minutes. Heat shock the competent cell tube in a 42°C water bath for 90 seconds. Transfer the tube to an ice bath and allow the cells to cool for 2 minutes. Add 800 μL of LB culture medium and incubate at 37°C for 1 hour. Centrifuge to remove a small amount of supernatant, resuspend the cells, and spread the culture medium on an LB plate containing the corresponding antibiotic. Incubate inverted at 37°C for 10-12 hours. Observe the colonies and select a single colony to verify the positive clone using colony PCR. Culture the positive single colony in LB liquid medium overnight, extract the plasmid, and verify by sequencing.

[0047] (V) Preparation of electroporated Corynebacterium glutamicum competent cells

[0048] (1) Corynebacterium glutamicum was inoculated into BHIS medium (selection was required on a freshly cultured slant, otherwise the growth of the bacteria would be affected), placed on a circulating shaker (200 rpm), and cultured at 30°C for 16 h until the OD600 reached 3.0.

[0049] (2) Transfer the bacterial solution from (1) to the competent culture medium at a 10% inoculum volume until the OD600 reaches 0.3. Place the culture on a roving shaker (200 rpm) and culture at 30°C until the OD600 reaches 0.9 (culture for about 3-5 hours, and it is in the logarithmic growth phase. Generally, if the bacterial concentration remains low at around 0.6, the subsequent steps can be continued). The general concentration multiple is 100 times (50 mL of competent culture medium is concentrated to 0.5 mL to prepare 5 tubes of competent cells).

[0050] (3) Place the bacterial solution in an ice-water bath for 15 min, centrifuge at 4000 rpm and 4°C for 10 min, and discard the supernatant.

[0051] (4) Fully suspend the cells in 30 mL of pre-cooled 10% glycerol, centrifuge at 4000 rpm, 4°C for 10 min, discard the supernatant, and repeat the washing and resuspending process four times.

[0052] (5) Resuspend the cells in 500 μL of pre-chilled 10% glycerol (concentrated 100 times) and dispense into 1.5 mL sterile centrifuge tubes, with 90-100 μL per tube.

[0053] (6) Store the aliquoted competent cells at -80°C until use.

[0054] (VI) Electroporation transformation of Corynebacterium glutamicum

[0055] (1) Thaw the competent bacteria of Corynebacterium glutamicum stored at -80°C in an ice bath.

[0056] (2) Add 1-5.0 μL of plasmid, mix well, and incubate on ice for 5-10 minutes.

[0057] (3) Add the sample to a pre-cooled 0.1 cm electric shock cup and shock twice with 1.8 kV and 5 ms.

[0058] (4) Quickly add 1.0 mL of culture medium (BHIS) preheated at 46°C, mix well, and transfer to a new 1.5 mL sterile centrifuge tube. Incubate in a 46°C water bath for 6 min.

[0059] (5) Place the bacteria on a rotating shaker (220 rpm) and incubate at 30°C for 2 h.

[0060] (6) Centrifuge at 4000 rpm for 2 min at room temperature, discard part of the supernatant, resuspend the remaining bacterial solution and spread it on a solid plate coated with the transformant with the corresponding resistance, and culture in a constant temperature incubator at 30°C for 2-3 days.

[0061] (VII) LCMS detection of intermediate metabolites

[0062] Sample preparation for LCMS analysis of intracellular products is as follows: First, take 1.1 mL of fermentation broth and centrifuge at 12,000 rpm for 10 minutes. After discarding the supernatant, freeze the pellet at -80°C and resuspend it in an equal volume of water before use. Next, prepare a 1:1 mixture of acetonitrile and methanol and pre-cool it. Next, take 800 μL of the extract and mix it with 200 μL of fermentation broth to a final ratio of 4:4:2. Label the sample and freeze-extract it overnight at -40°C to ensure thorough resuspension and dispersion of the sample. After overnight extraction, centrifuge at maximum speed for 10 minutes. The supernatant is then lyophilized, typically for 2 hours. The remaining approximately 100 μL of water can be retained and does not require complete lyophilization. Next, add ultrapure water to 1 mL and centrifuge at maximum speed for 10 minutes. After filtering through a water membrane, the sample is transferred to a liquid chromatography vial containing a glass-lined tube with a pre-cut cap for LCMS analysis.

[0063] The liquid chromatography conditions are as follows: an amino column (ACQUITY UPLC BEH Amide Column, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation) is used for separation, which is suitable for separating small molecule metabolites with strong polarity. Mobile phase A is 95% acetonitrile and 5% 10 mM ammonium bicarbonate aqueous solution, and mobile phase B is an aqueous solution containing 10 mM ammonium carbonate and 0.2% ammonium hydroxide (final pH is 10.4). The mobile phase is prepared by first preparing the mother liquor, filtering it, and then diluting it. The ammonium hydroxide aqueous solution is prepared by adding 0.8 mL of liquid-grade ammonia water to every 100 mL of mobile phase. After the AB phase solution is prepared, it needs to be ultrasonically treated for 5 minutes to ensure uniform mixing. Note that too long of an ultrasonic time may cause the ammonia water to volatilize and change the pH. The gradient elution conditions were: 10% B at 0 min, 10% B at 2 min, 45% B at 3 min, 48% B at 8 min, 60% B at 8.1 min, 60% B at 11 min, 10% B at 11.5 min, and 10% B at 15 min. The flow rate for the entire process was 0.2 mL / min.

[0064] (8) CgGlmS crude enzyme activity detection

[0065] LB liquid culture medium containing 50 μg·mL-1 ampicillin was injected into a 96-well deep-well plate at 200 μL / well as a master plate, and a sterile toothpick was used to randomly pick a single colony for inoculation. The first well was inoculated with the wild-type CgGlmS strain, and the last well was reserved as a blank control. After culturing at 37°C and 200 rpm for 8-10 h, it was stored at 4°C for use. When preparing the daughter plate, 800 μL LBamp medium was injected into each well, and 50 μL of the master plate bacterial solution was transferred for inoculation. After culturing at 37°C and 200 rpm until OD600 reached 0.6-0.8, a final concentration of 0.2 mmol·L-1 IPTG was added, and the expression was induced at 22°C and 200 rpm for 24 h, and the bacteria were collected by centrifugation. The bacteria were washed twice with 500 μL phosphate buffer (pH 7.5, 100 mM) and resuspended in 250 μL of the same system buffer. 20 mM D-fructose, 25 mM KCl, 30 mM A mixed reaction solution of L-glutamine and 0.3% v / v TritonX-100 was catalyzed at 30°C and 100 rpm for 6 hours, and then the final hydrochloric acid concentration was adjusted to 0.2 M to terminate the reaction; the modified Elson-Morgan method was used for detection: 50 μL of the reaction solution was mixed with 450 μL of deionized water, 100 μL of acetylacetone solution was added, and the mixture was acetylated at 90°C for 25 minutes and then cooled. 300 μL of anhydrous ethanol and 100 μL of DMAB color developer were added in sequence. After color development at 60°C for 1 hour, 200 μL of the reaction solution was transferred to a 96-well plate and the absorbance at 530 nm was measured; mutant strains with significantly increased absorbance were screened, and the strains were preserved in glycerol tubes and stored at -80°C.

[0066] (IX) CgGlmS pure enzyme activity detection

[0067] Single colonies of the wild-type and mutant strains were inoculated into 50 mL of LBamp medium, shaken and cultured at 37°C and 200 rpm for 16 hours, and 10 mL of culture medium was transferred to 800 mL of LBamp medium and continued to be cultured until the OD600 reached 0.4-0.6; after cooling in an ice bath for 30 minutes, IPTG was added to a final concentration of 0.2 mM and induced at 22°C and 200 rpm for 16 hours. The cells were collected by centrifugation at 4°C and 8000 rpm for 10 minutes; the cells were resuspended in 10 mL of 0.1 mol / L phosphate buffer (pH 7.5) and ultrasonically disrupted in an ice bath (500 W, 2 seconds on / 3 seconds off, total time 15 minutes), followed by centrifugation at 4°C and 8000 rpm for 5 minutes. The supernatant was filtered through a 0.22 μm filter to obtain a crude enzyme solution; the crude enzyme solution was passed through a HisTrap HP nickel column affinity chromatography purification was performed, and the AKTA avant system was used to sequentially perform low-concentration imidazole buffer equilibration, constant flow rate loading, low-concentration imidazole elution of impurities, and gradient increasing imidazole concentration specific elution of the target protein. The eluate was collected and the purity was verified by 12% SDS-PAGE, and the protein concentration was determined by Bradford method; the enzyme activity detection system contained 100 mM phosphate buffer (pH 7.5), 20 mM D-fructose, 25 mM KCl, 30 mM L-glutamine, and 1.5 U / L purified enzyme solution. After reacting at 30°C for 2 hours, an equal volume of acetonitrile was added to terminate the reaction and the final concentration of hydrochloric acid was adjusted to 0.2 M; 500 μL of the reaction solution was taken and the volume was adjusted to 5 mL, and 1 mL of acetylacetone solution was added in sequence to react in a 90°C water bath for 25 minutes. After cooling in an ice-water bath, 10 mL of anhydrous ethanol and 1 mL DMAB colorimetric reagent was used. After color development at 60°C for 1 hour, the absorbance at 530 nm was measured. The system in which the enzyme solution was replaced by deionized water was used as a blank control. The experiment was repeated three times and the GlcN concentration was calculated based on the standard curve.

[0068] (10) Construction of mutant library

[0069] Using the pET-28a(+) plasmid as a template, upstream primers (HP-PETDuet-F) and downstream primers (HP-PETDuet-R) were designed to amplify the linearized plasmid fragment via PCR. Using Corynebacterium glutamicum 9114 as a template, upstream primers (CgGlmS-F) and downstream primers (CgGlmS-R) were designed to amplify the CgGlmS gene via PCR. The expression vector and gene fragment were ligated using the ClonExpress II One-Step Cloning Kit (Novozymes Biotechnology). The linearized vector and the target gene fragment with vector-homologous ends were recovered from the gel and mixed in a 3:1 molar ratio. 4 μL of 5×CE II Buffer and 2 μL of Exnase II were added, and then ddH2O was added to bring the total volume of the ligation system to 20 μL. The reaction was incubated at 37°C for 30 min, then the temperature was reduced to 4°C for incubation. Then take 10 μL of the connection system to transform E. coli JM109 competent cells (for details on the competent preparation method, please refer to the instructions of the Takara E. coli competent kit). Select the correct transformants by colony PCR and send them to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification to obtain the recombinant plasmid pET-28a (+) -CgGlmS. After that, the upstream primers (15-F) and the downstream primers (15-R) were designed respectively to obtain the pET-28a (+) -CgGlmS linear plasmid fragment by PCR amplification. Use the same method to connect the transformation plasmids and finally obtain the plasmid pET-28a (+) -CgGlmS-WK containing the corresponding mutant library. The mutant library plasmid was transformed into Escherichia coli DE3, and the colonies on the plate were randomly picked. The enzyme activity was detected according to the crude enzyme activity detection method. The construction of other mutants is similar.

[0070] The dominant mutant was then tested for pure enzyme activity. Because the key enzyme CgGlmS contains active pockets at both its N- and C-termini, a HIS fragment was ligated to the C-terminus and then a restriction enzyme site was added. After protein purification, the HIS tag was removed using enzyme digestion to prevent degradation of enzyme activity. Plasmid reconstruction for the dominant mutant was performed as follows: Using the pET-28a(+) plasmid as a template, an upstream primer (CgGlmS-tag-F) and a downstream primer (CgGlmS-tag-R) were designed. A linearized plasmid fragment was amplified by PCR. The expression vector and gene fragment were ligated using the ClonExpress II One-Step Cloning Kit from Novozymes Biotechnology. The linearized vector and the target gene fragment with homologous ends to the vector were recovered from the gel and mixed in a 3:1 molar ratio. 4 μL of 5× CE II Buffer and 2 μL of Exnase II were added, and the total volume of the ligation system was brought to 20 μL by adding ddH2O. The reaction was allowed to proceed at 37°C for 30 min, then the temperature was reduced to 4°C for incubation. Then take 10 μL of the ligation system to transform E. coli JM109 competent cells (for details on the competent preparation method, please refer to the instructions of the Takara E. coli competent kit). Select the correct transformants by colony PCR and send them to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification to obtain the recombinant plasmid pET-28a(+)-CgGlmS-tag with supplemented HIS tag and restriction site. Then use the pure enzyme detection method to detect the enzyme activity. The results of the pure enzyme detection are as follows Figure 3 shown.

[0071] (11) Sequence

[0072] The wild-type CgGlmS gene sequence is shown in SEQ ID NO.1:

[0073]

[0074] The gene sequence of the mutant CgGlmSA 253M is shown in SEQ ID NO.2:

[0075]

[0076] The amino acid sequence of the mutant CgGlmSA253M is shown in SEQ ID NO.3:

[0077] MRMCGIVGYIGQAGDSRDYFALDVVLEGLRRLEYRGYDSAGVAVHANGEISYRKKAGKVAALDAEIARAPLADSILAIGHTRWATHGGPTDANAHPHVVDGGKLAVVHNGIIENFAELRAELSAKGYNFVSVTDTEVAATLLAEIYNTQANGDLTK AMQLTGQRLEGAFTLLAIHADHDDRIVAARRNSPLVIGLGEGENFLGSDVSGFIDYTRKAVEMGNDQIVTITANDYQITNFDGSEATGKPFDVEWDMAAAEKGGFDSFMDKEIHDQPAAVRDTLLGRLDEDGKLVLDELRIDEATLRSVNKIIVVA CGTAAYAGQVARYAIEHWCRIPTEVELAHEFRYRDPIVNEKTLVVALSQSGETMDTLMAVRHAREQGAKVIAICNTVGSTLPREAADASLYTYAGPEIAVASTKAFLAQITASYLLGLYLAQLRGNKFADEVSSILDSLREMPEKIQQVIDAEEQIK KLGQDMSDAKSVLFLGRHVGFPVALEGALKLKEIAYLHAEGFAAGELKHGPIALVEEGQPVFVIVPSPRGRDSLHSKVVSNIQEIRARGAVTIVIAEEGDEAVNDYANFIIRIPQAPTLMQPLLSTVPLQIFACAVATAKGYNVDQPRNLAKSVTVE

[0078] Primer sequences:

[0079]

[0080]

[0081] Example 1: Determination of rate-limiting enzymes in metabolic pathways

[0082] In order to identify the metabolic bottleneck, liquid chromatography-mass spectrometry (LC-MS) was used to analyze the intracellular intermediate metabolites in the fermentation broth. Figure 1In a study (shown in Figure 1), the content of GlcN6P, a product generated by the CgGlmS-catalyzed reaction, was found to be almost zero. This indicates that once this product is generated intracellularly, it is rapidly converted to GlcNAc6P, suggesting that there may be a metabolic bottleneck in this reaction step. Therefore, the key enzyme CgGlmS plays a limiting role in the GlcNAc synthesis pathway. Modification of CgGlmS is needed to eliminate this bottleneck.

[0083] Since the crystal structure of GlmS (CgGlmS) from C. glutamicum S9114 has not yet been determined experimentally, we used Alphfold2 to predict the protein model. The model was then verified using Ramachandranplot analysis, which showed that 94.9% of the residues were located within the optimal region, indicating that the model was highly reliable and could be used for subsequent simulations (such as Figure 1 b). We then use The software performed induced fit docking on the CgGlmS-F6P / Gln complex. CgGlmS was defined as the receptor protein and docked with the small molecule Gln. The docked CgGlmS-Gln complex was used as the receptor and then induced fit docking was performed with the small molecule F6P (fructose-6-phosphate). The docking results are shown in Figure 2. Figure 2 As shown, the substrate binding pocket is composed of the hydrophobic cavity of the glutaminase domain and the polar residues of the sugar isomerase domain, which together form a stable catalytic environment.

[0084] Example 2: Construction and screening of CgGlmS mutants

[0085] In order to improve the catalytic efficiency of CgGlmS, we Figure 3 The experimental process shown in a was used to construct and screen mutants. We first identified the substrate binding pockets of F6P and Gln. The amino acids in the range were subjected to virtual saturation mutation using the Residuescanning module, and the results were as follows: Figure 3 As shown in Figure b. Mutations at positions 360, 361, and 517 significantly affected F6P binding, resulting in a significant decrease in the affinity of the small molecule F6P, suggesting that these sites may play a key catalytic role in the catalytic pocket. This result is consistent with the active site identified by molecular docking. We experimentally mutated positions 360, 361, and 517 to alanine and subsequently assayed crude enzyme activity, finding a near-complete loss of enzymatic activity. This further demonstrates the importance of these sites in enzyme catalysis and validates the accuracy of our molecular docking. Similarly, we performed the same analysis for key residues in the Gln binding pocket.

[0086] We then selected sites whose substrate affinity decreased after pseudo-saturation mutagenesis, indicating a positive effect on small molecule binding, to construct a saturation mutant library. Specifically, we performed saturation mutagenesis experiments at sites 312, 316, 362, 367, and 412 of the F6P binding pocket, and 109, 110, 135, and 623 of the Gln binding pocket. First, the recombinant plasmid pET28a-CgGlmS was constructed as a template for mutant library construction. Subsequently, degenerate primers NNK were used to construct saturation mutant libraries: CgGlmS312, CgGlmS316, CgGlmS362, CgGlmS367, CgGlmS412, CgGlmS109, CgGlmS110, CgGlmS135, and CgGlmS623. The crude enzyme activities of the mutants were then assayed, with mutant #1 representing the wild-type CgGlmS. The experimental results show that ( Figure 3 c) The crude enzyme activity test did not find any mutants with significantly improved enzyme activity.

[0087] We speculate that this may be due to the high conservation of the active pocket region of CgGlmS. Mutations in the amino acids within the pocket significantly perturb the catalytic process of small molecules, and the mutants are unable to maintain catalytic activity. Since semi-rational modification of the small molecule pocket failed to yield advantageous mutants, we shifted our research focus to the non-conserved loop region of CgGlmS. As the most flexible and least conserved region, the loop structure plays an important role in various dynamic processes by regulating substrate binding, active site pocket orientation, and conformational changes. In recent years, rational and semi-rational design of loop regions has played an increasingly important role in enzyme modification. Therefore, by aligning the sequences of CgGlmS from E. coli and B. subtilis, we screened for loop regions unique to CgGlmS and again used the Residue Scanning module to perform virtual saturation mutagenesis on these sites to evaluate residues that have a significant impact on protein stability. Specifically, we performed virtual saturation mutagenesis on the amino acids in the six loop regions 13-16, 51-56, 73-77, 535-541, 565-576, and 253-256 ( Figure 4 a) Based on the functional plasticity hypothesis of the loop region, we selected sites that are beneficial to enzyme stability for experimental verification and tested crude enzyme activity according to the above method. That is, saturation mutagenesis was performed on sites 15, 51, 73, 540, 541, 573, 576, 253, and 254. The results of the mutant enzyme activity test are as follows: Figure 4 As shown in b, there are 6 mutants showing improved enzyme activity compared to the wild type: D15R, D15M, D73Y, A253M, A253R, and A254E.

[0088] Example 3: Characterization and application of CgGlmS mutants

[0089] The yield of the dominant mutant was characterized. Since the synthesis of GlcNAc requires the catalysis of the GNA1 enzyme, and the GNA1 enzyme does not exist in Corynebacterium glutamicum, the GNA1 enzyme from Saccharomyces cerevisiae (Gene ID: 850529) was expressed together with the mutant of CgGlmS to verify the effect of the mutant on the yield of GlcNAc. Upstream primers (CgGlmS-F) and downstream primers (CgGlmS-R) were designed, and the gene fragment of the CgGlmS mutant was obtained by PCR amplification. The GNA1 gene was synthesized using Jinweizhi's gene synthesis service. Upstream primers (GNA1-F) and downstream primers (GNA1-R) were designed, and the gene fragment of GNA1 was obtained by PCR amplification. The expression vector and gene fragment were connected using the ClonExpress II One Step Cloning Kit rapid cloning kit from Novozymes Biotech Co., Ltd. The linearized vector obtained by PCR and the target gene fragment with homologous ends of the vector were recovered by gel and mixed in a molar ratio of 3 to 1. At the same time, 4 μL of 5×CE II Buffer and 2 μL of Exnase II were added, and then ddH2O was added to make the total volume of the connection system reach 20 μL. The reaction was carried out at 37°C for 30 minutes and then reduced to 4°C for insulation. Then 10 μL of the connection system was taken to transform E. coli JM109 competent cells (for details on the competent preparation method, please refer to the instructions of the Takara Escherichia coli competent kit). The correct transformants in the colony PCR were selected and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification to obtain the recombinant plasmid pJYW4-GNA1-CgGlmSmut. They were transformed into Corynebacterium glutamicum S9114 for fermentation verification, and the yield results were as follows. Figure 5 As shown, the yield of the mutant strain increased by 12% compared with the wild-type strain.

[0090] Example 4: Analysis of CgGlmS mutants

[0091] In order to further analyze the effect of the A253M mutation on the enzyme, induced fit docking was first performed on the mutant A253M and small molecules to analyze the binding between the enzyme and the small molecule. Specifically, the wild-type CgGlmS and mutant A253M were used to perform induced fit docking with the substrates F6P, Gln and the products GlcN6P, Glu, respectively. The results showed that there was no obvious change in the binding pocket between the small molecule and the mutant. Therefore, we speculated that the change in the binding stability of the small molecule and the enzyme led to a change in its catalytic efficiency. We then used the MM-GBSA module to calculate the binding energy of the mutant with the substrates Gln, F6P and the products Glu, GlcN6P. The calculation results are shown in Table 1. The binding energy of the mutant with the substrates Gln and F6P is slightly different from that of the wild type, that is, the mutant has no obvious effect on the binding of the substrate. However, the binding energy of the mutant with the product Glu increased significantly, from 3.32 kcal·mol -1 Significantly increased to 23.51 kcal·mol -1 The product Glu is less conducive to the binding of the enzyme mutant, which accelerates the separation of the product and thus improves the catalytic efficiency of the enzyme.

[0092] Table 1 Comparison of binding energy between enzyme, substrate and product (unit: kcal·mol -1 )

[0093]

[0094] like Figure 6 To further verify our hypothesis, we performed 100 ns molecular dynamics simulations on the docked complexes of the wild-type and mutant A253M. The dynamics simulation results showed that the RMSD of the mutant A253M protein backbone stabilized in the late stage of the dynamics simulation (>50 ns) and was more stable than the wild-type. It is speculated that this may be due to the introduction of a hydrophobic side chain after the replacement of alanine (Ala) at position 253 with methionine (Met). The hydrophobic residue can form stronger van der Waals forces with the adjacent α-helix, thereby improving the stability of the enzyme. This is also confirmed by the RMSF results. The mutant showed a reduced amplitude of change in the 600-800 residue region of the dimer, indicating that the mutant is more stably bound in the F6P pocket.

[0095] Finally, we analyzed the relative binding free energy during the 100 ns kinetic simulation to determine the change in the binding free energy between the product and the enzyme. We calculated the relative binding free energy every 2 ns and found that in the later stages of the kinetic simulation (60-100 ns), the relative binding free energy between the mutant A253M and the product as a whole was approximately -25 kcal·mol -1 , while the wild type is about -50 kcal·mol -1The overall binding free energy of the mutant with the product is significantly higher than that of the wild type, indicating that it is more difficult for it to bind to the product, thereby accelerating the release of the product so that the enzyme can quickly carry out the next catalytic reaction, thereby improving the catalytic efficiency of the enzyme.

[0096] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A 6-phosphoglucosamine synthetase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding the 6-phosphoglucosamine synthetase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A gene expression cassette or recombinant plasmid carrying the nucleic acid molecule according to claim 2 or 3.

5. A recombinant cell expressing the 6-phosphoglucosamine synthetase mutant according to claim 1.

6. Use of the 6-phosphoglucosamine synthetase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the gene expression cassette or recombinant plasmid according to claim 4, or the recombinant cell according to claim 5 in the preparation of N-acetylglucosamine.

7. A recombinant Corynebacterium glutamicum, characterized in that The recombinant Corynebacterium glutamicum overexpresses the gene glmS encoding the 6-phosphoglucosamine synthetase mutant according to claim 1.

8. The recombinant Corynebacterium glutamicum according to claim 7, characterized in that The host bacteria of the recombinant Corynebacterium glutamicum include Corynebacterium glutamicum S9114.

9. The recombinant Corynebacterium glutamicum according to claim 7, characterized in that The recombinant Corynebacterium glutamicum also overexpresses the N-acetylglucosamine transferase encoding gene GNA1.

10. A method for producing N-acetylglucosamine, characterized in that: The method comprises the steps of fermenting and producing by using the recombinant Corynebacterium glutamicum according to any one of claims 7 to 9.

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