A cgglm s mutant and its application in synthesis of glcnac by corynebacterium glutamicum

Abstract

The application discloses a CgGlmS mutant and application thereof in synthesis of N-acetylglucosamine (GlcNAc) in corynebacterium glutamicum, and belongs to the technical field of biotechnology. In-depth rational analysis and semi-rational modification are carried out on the bottleneck enzyme CgGlmS in the N-acetylglucosamine synthesis pathway. Through systematic research on the structure and function of the original CgGlmS enzyme from corynebacterium glutamicum S9114, a high-efficiency CgGlmS mutant A253M is successfully screened, and the enzyme activity, stability and catalytic efficiency of the mutant are obviously improved compared with those of the wild type. The mutant CgGlmS is overexpressed in corynebacterium glutamicum S9114 by using a plasmid, and the yield of N-acetylglucosamine in corynebacterium glutamicum is improved. Through the directional modification of the CgGlmS enzyme, the catalytic efficiency is improved, and the accumulation of N-acetylglucosamine in corynebacterium glutamicum is promoted, which provides strong support for the efficient production of N-acetylglucosamine.

Description

Technical Field

[0001] This invention relates to a CgGlmS mutant and its application in the synthesis of GlcNAc by Corynebacterium glutamicum, belonging to the field of biotechnology. Background Technology

[0002] N-acetylglucosamine (GlcNAc), a monosaccharide widely distributed in organisms, is found in bacteria, fungi, plants, and animals. It is a precursor for the synthesis of disaccharide units of glycosaminoglycans (GAGs) and plays a crucial role in the functional repair and maintenance of cartilage and joint tissues. In recent years, N-acetylglucosamine has seen increasingly widespread applications in medicine, cosmetics, and food. In drugs treating diseases such as arthritis, N-acetylglucosamine is often used as a main ingredient, effectively relieving joint pain and inflammation. In cosmetics, it is used in skincare products to enhance skin hydration and elasticity. Currently, some progress has been made in the production of N-acetylglucosamine using Corynebacterium glutamicum, with existing research mainly focusing on regulating metabolic pathways through metabolic engineering to improve product synthesis levels. However, relying solely on metabolic pathway reconstruction may not be sufficient to coordinate intracellular metabolic flux, especially when it is unclear whether key enzymes constitute a bottleneck. This can easily lead to increased metabolic load and byproduct accumulation, thus affecting the synthesis efficiency of the target product and the growth status of the host cell. While existing research has attempted to improve product yields by engineering key enzymes in synthetic pathways, the identification and in-depth mechanistic study of rate-limiting enzymes remain relatively weak. The presence of rate-limiting enzymes is often one of the key factors restricting product synthesis. Precise modification of their catalytic efficiency and substrate affinity holds promise for significantly enhancing the synthesis of N-acetylglucosamine.

[0003] Furthermore, current research on the modification of key enzymes in the N-acetylglucosamine synthesis pathway is relatively limited. Existing studies mainly focus on improving product synthesis efficiency by modifying the enzyme activity and substrate specificity related to 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 research provides a new pathway for the synthesis of N-acetylglucosamine. Another study by Piwu Li et al. saturated the binding pocket of fructose hexaphosphate and screened a superior Bacillus subtilis-derived GlmS enzyme mutant through rational analysis. The results showed that the enzyme activity of mutant L593S was significantly increased, from the original 5 U / mL to 48 U / mL, demonstrating its potential in industrial production. However, in the study of GlcNAc synthesis by Corynebacterium glutamicum, existing literature reports on improving the synthetic capacity by screening key enzymes GlmS from different sources, and found that endogenous GlmS from Corynebacterium glutamicum itself exhibits better adaptability and catalytic effect. Although heterologous enzymes may possess high activity, their expression in non-natural hosts is often limited by multiple factors, including protein stability, folding efficiency, cofactor compatibility, and differences in regulatory mechanisms. Therefore, targeted modification of Corynebacterium glutamicum S9114 based on its endogenous GlmS in Corynebacterium glutamicum can avoid the adaptation problems caused by heterologous expression and is more conducive to further enhancing enzyme activity in the host context, thus possessing greater feasibility and application potential (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, this invention is proposed based on the above two points. Summary of the Invention

[0005] To address the aforementioned issues, this invention aims to improve the yield of N-acetylglucosamine in *Corynebacterium glutamicum* by modifying key enzymes that have not been adequately optimized in existing technologies. This invention employs a semi-rational modification strategy, combining virtual saturation mutagenesis to explore potential modification sites with high-throughput enzyme activity screening methods, successfully identifying the CgGlmS mutant with significant advantages. Furthermore, a systematic comparison between the mutant and the original enzyme was conducted using kinetic simulations and other techniques, verifying the improved catalytic efficiency and enhanced stability. Finally, the optimized CgGlmS mutant was successfully applied to *Corynebacterium glutamicum* S9114, significantly increasing the yield of N-acetylglucosamine.

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

[0007] A second objective of this invention is to provide a nucleic acid molecule encoding the 6-phosphate glucose synthase mutant.

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

[0009] A third objective 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 the pJYW4 plasmid.

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

[0012] A fourth objective of this invention is to provide recombinant cells expressing the 6-phosphate glucose synthase mutant.

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

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

[0015] A fifth objective of this invention is to provide the use of the aforementioned 6-phosphoglucosamine synthase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the preparation of N-acetylglucosamine.

[0016] The sixth objective of this invention is to provide a recombinant Corynebacterium glutamicum that overexpresses the glmS gene encoding the 6-phosphate glucose synthase mutant.

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

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

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

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

[0021] The beneficial effects of this invention are:

[0022] This invention successfully obtained a mutant, A253M, with superior performance by semi-rationally modifying the CgGlmS enzyme in Corynebacterium glutamicum S9114. Compared with the control group, the enzyme activity was increased by 9.3% compared with the wild type. Further application of this mutant to the metabolic process of Corynebacterium glutamicum S9114 successfully increased the yield of N-acetylglucosamine by 12%, from 5.9 g / L to 6.6 g / L. This indicates that the mutant has a significant effect on increasing yield. In addition, this invention also improved the stability and catalytic efficiency of the CgGlmS enzyme, increasing the production capacity of the target product and showing good prospects for industrial application. The successful implementation of this invention provides new ideas for further optimizing the production of N-acetylglucosamine, especially achieving a breakthrough in the modification of key enzymes, and providing important reference value for the modification and improvement of other N-acetylglucosamine producing strains. Attached Figure Description

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

[0024] Figure 2 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 3To modify the process, the results of virtual saturation mutation and enzyme activity detection are as follows: (a) the mutant screening process; (b) the results of virtual saturation mutation of amino acids in the F6P active pocket of CgGlmS; (c) the results of virtual saturation mutation of amino acids in the Gln active pocket of CgGlmS; and (d) the results of crude enzyme activity detection of the mutant library.

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

[0028] Figure 5 To detect the enzyme activity of the dominant mutant and to measure the yield of GlcNAc produced by fermentation.

[0029] Figure 6 Results of mutant molecular docking, kinetic simulation, and relative binding free energy analysis.

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

[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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

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

[0033] (I) Culture medium

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

[0035] Seed activation liquid culture medium BHIS (g / L): brain heart extract 37.0, sorbitol 91.0.

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

[0037] Competent culture 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 added.

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

[0039] Transformant plating medium solid BHIS (g / L): peptone 5.0, yeast extract 2.5, NaCl 5.0, brain heart extract 18.5, sorbitol 91.0, nutrient agar 20.0.

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

[0041] (II) PCR Method

[0042] The PCR reaction mixture consisted of: 25 μL PrimeSTAR Max (Takara), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL template DNA, and 22 μL double-distilled water. The PCR amplification conditions were: 98℃ pre-denaturation for 5 min; followed by 30 cycles of 98℃ for 10 s, 55℃ for 15 s, and 72℃ for 3 min; and finally, incubation at 72℃ for 5 min.

[0043] (III) Methods for preparing competent Escherichia coli cells

[0044] Escherichia coli competent cells were prepared using the Competent Cell Preparation Kit (purchased from Takara). The preparation method is described in the kit's instruction manual.

[0045] (iv) Plasmid chemical transformation of E. coli competent cells

[0046] E. coli competent cells stored in an ultra-low temperature freezer were thawed on ice; 6 μL of PCR product was added and gently mixed, and the mixture was incubated on ice for 45 min. Centrifuge tubes containing competent cells were heat-shocked in a 42°C water bath for 90 s, and then transferred to an ice bath to cool the cells for 2 min; 800 μL of LB culture medium was added and cultured at 37°C for 1 h; after centrifugation to remove a small amount of supernatant, the bacterial suspension was resuspended, and the culture medium was spread on LB agar plates containing the appropriate antibiotics. The plates were incubated upside down at 37°C for 10-12 h, and colonies were observed. Single colonies were picked and colony PCR was used to verify positive clones. Positive single colonies were picked and cultured overnight in LB liquid medium, and then plasmids were extracted and sequenced for verification.

[0047] (V) Preparation of Corynebacterium glutamicum electrocompetent cells

[0048] (1) Corynebacterium glutamicum was inoculated on BHIS medium (selection should be made on fresh culture slant, otherwise it will affect the growth of the bacteria), placed on a circulating shaker (200 rpm), and cultured at 30℃ for 16 h until OD600 reached 3.0.

[0049] (2) Transfer the bacterial culture from (1) to competent culture medium at an inoculation rate of 10% until the OD600 reaches 0.3. Place the medium on a circulating shaker (200 rpm) and incubate at 30°C until the OD600 reaches 0.9 (incubate for about 3-5 hours, until the culture is in the logarithmic growth phase; if the bacterial concentration remains low at around 0.6, subsequent operations can continue). The general concentration factor is 100 times (50 mL of competent culture medium is concentrated to 0.5 mL to prepare 5 tubes of competent cells).

[0050] (3) Incubate the bacterial culture in an ice water bath for 15 min, centrifuge at 4000 rpm and 4℃ for 10 min, and discard the supernatant.

[0051] (4) The bacterial cells were fully suspended in 30 mL of pre-cooled 10% glycerol, centrifuged at 4000 rpm and 4℃ for 10 min, the supernatant was discarded, and the cells were washed and resuspended four times.

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

[0053] (6) The competent cells were aliquoted and stored at -80℃ for later use.

[0054] (vi) Electroporation transformation of Corynebacterium glutamicum

[0055] (1) Competent cells of Corynebacterium glutamicum stored at -80℃ were thawed in an ice bath.

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

[0057] (3) Add to a pre-cooled 0.1cm shock cup, and shock twice with a voltage of 1.8KV for 5ms.

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

[0059] (5) Place the bacterial cells on a circulating shaker (220 rpm) and incubate at 30°C for 2 hours.

[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 onto a solid plate containing the corresponding resistant transformant, and incubate at 30℃ for 2-3 days.

[0061] (vii) LCMS detection of intermediate metabolites

[0062] The sample preparation method for LCMS determination of intracellular products is as follows: First, take 1.1 mL of fermentation broth, centrifuge at 12000 rpm for 10 minutes, remove the supernatant, and freeze the precipitate at -80℃. Resuspend in an equal volume of water before use. Next, prepare a 1:1 mixture of acetonitrile and methanol and pre-cool it. Then, mix 800 μL of the extract with 200 μL of fermentation broth to form a 4:4:2 ratio, label it, and freeze-extract at -40℃ overnight, ensuring thorough resuspending and sample dispersion. After overnight extraction, centrifuge at maximum speed for 10 minutes, collect the supernatant, and freeze-dry it. Freeze-drying is usually sufficient for 2 hours; approximately 100 μL of water can be retained, and complete freeze-drying is not necessary. Next, add ultrapure water to 1 mL, centrifuge at maximum speed for 10 minutes, filter through a water membrane, and add the sample to a LC-HPLC vial with a glass liner. The vial cap should be pre-cut for LCMS analysis.

[0063] The liquid chromatography conditions were as follows: separation was performed using an amino column (ACQUITY UPLC BEH Amide Column, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation), suitable for separating highly polar small molecule metabolites. Mobile phase A consisted of 95% acetonitrile and 5% 10 mM ammonium bicarbonate aqueous solution, and mobile phase B consisted of an aqueous solution containing 10 mM ammonium carbonate and 0.2% ammonium hydroxide (final pH 10.4). The mobile phases were prepared by first preparing a stock solution, filtering, and then diluting. The ammonium hydroxide aqueous solution was prepared by adding 0.8 mL of liquid-grade ammonia to every 100 mL of mobile phase. After preparing the A and B phase solutions, they were sonicated for 5 minutes to ensure homogeneous mixing. Note that excessive sonication may cause ammonia evaporation and alter the pH. The gradient elution conditions were: 0 min 10% B, 2 min 10% B, 3 min 45% B, 8 min 48% B, 8.1 min 60% B, 11 min 60% B, 11.5 min 10% B, and 15 min 10% B. The flow rate for the entire process was 0.2 mL / min.

[0064] (viii) Detection of crude enzyme activity of CgGlmS

[0065] LB liquid medium containing 50 μg·mL⁻¹ ampicillin was injected at 200 μL / well into a 96-well deep-well plate as a mother plate. Single colonies were randomly picked and inoculated using sterile toothpicks. The first well was inoculated with wild-type CgGlmS strain, and the last well was reserved as a blank control. After incubation at 37℃ and 200 rpm for 8-10 h, the culture was stored at 4℃ for later use. When preparing daughter plates, 800 μL of LB medium was injected into each well, and 50 μL of the mother plate bacterial culture was transferred for inoculation. After incubation at 37℃ and 200 rpm until the OD600 reached 0.6-0.8, 0.2 mmol·L⁻¹ IPTG was added. After induction of expression at 22℃ and 200 rpm for 24 h, the bacterial cells were collected by centrifugation. The bacterial cells were washed twice with 500 μL of phosphate buffer (pH 7.5, 100 mM) and resuspended in 250 μL of the same buffer solution. Each well was then inoculated with 20 mM D-fructose, 25 mM KCl, and 30 mM... A mixture of L-glutamine and 0.3% v / v Triton X-100 was catalyzed at 30℃ and 100 rpm for 6 h, and the final concentration of hydrochloric acid was adjusted to 0.2 M to terminate the reaction. A modified Elson-Morgan method was used for detection: 50 μL of the reaction solution was mixed with 450 μL of deionized water, and 100 μL of acetylacetone solution was added. After acetylation at 90℃ for 25 min and cooling, 300 μL of anhydrous ethanol and 100 μL of DMAB colorimetric reagent were added sequentially. After color development at 60℃ for 1 h, 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, preserved in glycerol tubes, and stored at -80℃.

[0066] (ix) Detection of enzyme activity of CgGlmS purified enzyme

[0067] Wild-type and mutant strains were inoculated into 50 mL LBamp medium and cultured at 37°C with shaking at 200 rpm for 16 hours. 10 mL of the culture was then transferred to 800 mL LBamp medium and cultured until the OD600 reached 0.4-0.6. After cooling on ice for 30 minutes, 0.2 mM IPTG was added, and the cells were induced at 22°C with shaking at 200 rpm for 16 hours. The cells were then collected by centrifugation at 4°C with 8000 rpm for 10 minutes. The cells were resuspended in 10 mL of 0.1 mol / L phosphate buffer (pH 7.5) and sonicated on ice (500 W, 2 seconds on, 3 seconds off, total 15 minutes). The cells were then centrifuged at 4°C with 8000 rpm for 5 minutes. The supernatant was filtered through a 0.22 μm filter to obtain the crude enzyme solution. The crude enzyme solution was then passed through a HisTrap filter. HP nickel column affinity chromatography purification was performed using an AKTAavant system. The process involved equilibration with low-concentration imidazole buffer, constant flow rate loading, elution of impurities with low-concentration imidazole, and specific elution of the target protein with a gradient increasing imidazole concentration. The eluent was collected, and its purity was verified by 12% SDS-PAGE. Protein concentration was determined using the Bradford method. The enzyme activity assay consisted of 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 hydrochloric acid concentration was adjusted to 0.2 M. 500 μL of the reaction solution was brought to a final volume of 5 mL, and 1 mL of acetylacetone solution was added sequentially. The mixture was then incubated in a 90°C water bath for 25 minutes, cooled in an ice-water bath, and 10 mL of anhydrous ethanol and 1 mL of... The absorbance at 530 nm was measured after DMAB colorimetric reagent was developed at 60℃ for 1 hour. A system with deionized water instead of enzyme solution was used as a blank control. The experiment was repeated three times and the GlcN concentration was calculated according to the standard curve.

[0068] (x) Construction of mutant libraries

[0069] Using pET-28a(+) plasmid as a template, upstream primer (HP-PETDuet-F) and downstream primer (HP-PETDuet-R) were designed to amplify the linear plasmid fragment via PCR. Using Corynebacterium glutamicum 9114 as a template, upstream primer (CgGlmS-F) and downstream primer (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 from Novizan Biotechnology Co., Ltd. The linearized vector obtained by PCR and the target gene fragment with homologous ends were gel-recovered and mixed at a 3:1 molar ratio. 4 μL of 5×CE II Buffer and 2 μL of Exnase II were added, followed by ddH2O to bring the total ligation volume to 20 μL. The mixture was incubated at 37°C for 30 min and then cooled to 4°C for further incubation. Then, 10 μL of the ligation system was transformed into E. coli JM109 competent cells (for the preparation method of competent cells, please refer to the Takara E. coli competent cell kit instructions). Transformants with correct colony PCR results were selected and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing verification, yielding the recombinant plasmid pET-28a(+)-CgGlmS. Subsequently, upstream primers (15-F) and downstream primers (15-R) were designed, and the pET-28a(+)-CgGlmS linear plasmid fragment was amplified by PCR. The transformed plasmid was ligated using the same method, finally obtaining the plasmid pET-28a(+)-CgGlmS-WK containing the corresponding mutant library. The mutant library plasmid was transformed into E. coli DE3, and colonies were randomly picked from the plates. Enzyme activity was detected according to the crude enzyme activity assay method. The construction of other mutants was carried out in the same manner.

[0070] The dominant mutant was then subjected to enzyme activity testing. Since the key enzyme CgGlmS contains activity pockets at both its N-terminus and C-terminus, the HIS fragment was ligated to the C-terminus, followed by ligation of the restriction enzyme site. After protein purification, the HIS tag was removed using enzyme digestion to prevent interference with enzyme activity. The method for reconstructing the plasmid from the dominant mutant is 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. The linear plasmid fragment was obtained by PCR amplification. The expression vector and gene fragment were ligated using the ClonExpress II One Step Cloning Kit from Novizan Biotechnology Co., Ltd. The linearized vector obtained by PCR and the target gene fragment with homologous ends were gel-recovered and mixed at a 3:1 molar ratio. 4 μL of 5×CE II Buffer and 2 μL of Exnase II were added, followed by ddH2O to bring the total ligation volume to 20 μL. The reaction was carried out at 37°C for 30 min and then incubated at 4°C. Then, 10 μL of the ligation system was transformed into E. coli JM109 competent cells (for the preparation method of competent cells, please refer to the Takara E. coli competent cell kit instructions). Transformants with correct colony PCR results were selected and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing verification, yielding the recombinant plasmid pET-28a(+)-CgGlmS-tag with supplemented HIS tags and restriction enzyme sites. Enzyme activity was then detected using pure enzyme assays. The results of the pure enzyme assay are shown below. Figure 3 As shown.

[0071] (XI) 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] To identify metabolic bottlenecks, intracellular intermediate metabolites in the fermentation broth were analyzed using liquid chromatography-mass spectrometry (LC-MS). Based on the detection results of intermediate metabolites in the fermentation broth (e.g....), Figure 1As shown in Figure a), the content of GlcN6P, the product catalyzed by CgGlmS, was found to be almost zero. This indicates that once generated intracellularly, this product is rapidly converted to GlcNAc6P, suggesting a possible 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 remove this bottleneck.

[0083] Since the crystal structure of GlmS (CgGlmS) derived from C. glutamicum S9114 has not been experimentally determined, we used Alphfold2 to predict the protein model. Model validation was then performed using a Ramachandran plot, which showed that 94.9% of the residues were located within the optimal region, indicating high model reliability for subsequent simulations (e.g., ...). Figure 1 (as shown in b). We then utilized... The software was used to induce fitting docking of the CgGlmS-F6P / Gln complex. CgGlmS was defined as the receptor protein and docked with the small molecule Gln. Using the docked CgGlmS-Gln complex as the receptor, further induced fitting docking was performed with the small molecule F6P (fructose-6-phosphate). The docking results are as follows: Figure 2 As shown, the substrate binding pocket is composed of the hydrophobic cavity of the glutaminase domain and the polar residues of the glycoisomerase domain, which together form a stable catalytic environment.

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

[0085] To improve the catalytic efficiency of CgGlmS, we followed... Figure 3 The experimental procedure shown in a was used to construct and screen mutants. We first tested the substrate binding pockets of F6P and Gln. The amino acids within the range were subjected to virtual saturation mutagenesis using the Residuescanning module, and the results are as follows: Figure 3 As shown in b, mutations at sites 360, 361, and 517 significantly affected F6P binding, resulting in a significant decrease in the affinity of small-molecule F6P. This suggests that these sites may play a crucial catalytic role in the catalytic pocket, a result consistent with the active sites identified in the molecular docking results. We experimentally mutated sites 360, 361, and 517 to alanine and then measured the crude enzyme activity, finding that the enzyme activity was almost completely lost. This further demonstrates the importance of these sites in enzyme catalysis and verifies the accuracy of our molecular docking. Similarly, we performed the same analysis on key residues in the Gln binding pocket.

[0086] Subsequently, we selected sites where substrate affinity decreased after virtual saturation mutation, i.e., sites with a positive effect on small molecule binding, to further construct saturation mutant libraries. Specifically, we performed saturation mutation experiments at sites 312, 316, 362, 367, and 412 of the F6P binding pocket, and sites 109, 110, 135, and 623 of the Gln binding pocket. First, we constructed the recombinant plasmid pET28a-CgGlmS as a template for mutant library construction. Then, using degenerate primers NNK, we constructed saturation mutant libraries CgGlmS312, CgGlmS316, CgGlmS362, CgGlmS367, CgGlmS412, CgGlmS109, CgGlmS110, CgGlmS135, and CgGlmS623, respectively. We then tested the crude enzyme activity of the mutants, with mutant number 1 being the wild-type CgGlmS. Experimental results show that ( Figure 3 c) No mutants with significantly increased enzyme activity were found in the detection of crude enzyme activity.

[0087] We hypothesize that this may be due to the high conservation of the active pocket region in CgGlmS. Mutations in the amino acids within the pocket significantly perturb the catalytic process of small molecules, and mutants cannot maintain catalytic activity. Since semi-rational modification of the small molecule pocket failed to yield dominant mutants, we shifted our focus to the non-conserved loop region of CgGlmS. As the most flexible and least conserved region, the loop structure plays a crucial role in various dynamic processes by regulating substrate binding, active site pocket orientation, and conformational changes. In recent years, rational and semi-rational design targeting the loop region has played an increasingly important role in enzyme modification. Therefore, we sequence-aligned CgGlmS from E. coli and B. subtilis to identify CgGlmS-specific loop regions, and then used the Residue Scanning module to perform virtual saturation mutagenesis on these sites to evaluate residues that significantly affect protein stability. Specifically, we performed virtual saturation mutations on the amino acids in 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 favorable to enzyme stability for experimental verification and conducted crude enzyme activity detection according to the aforementioned method. Specifically, we performed saturation mutation experiments at sites 15, 51, 73, 540, 541, 573, 576, 253, and 254. The mutant enzyme activity detection results are as follows: Figure 4 As shown in b, there are 6 mutants that show improved enzyme activity compared to wild type: D15R, D15M, D73Y, A253M, A253R, and A254E.

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

[0089] The yield of the dominant mutant was characterized. Since GlcNAc synthesis requires the catalysis of the GNA1 enzyme, and GNA1 is absent in *Corynebacterium glutamicum*, a GNA1 enzyme from *Saccharomyces cerevisiae* (Gene ID: 850529) was co-expressed with the CgGlmS mutant to verify the effect of the mutant on GlcNAc yield. Upstream primers (CgGlmS-F) and downstream primers (CgGlmS-R) were designed, and the gene fragment of the CgGlmS mutant was amplified by PCR. The GNA1 gene was synthesized using Genewiz's gene synthesis service. Upstream primers (GNA1-F) and downstream primers (GNA1-R) were designed, and the GNA1 gene fragment was amplified by PCR. The expression vector and gene fragment were ligated using the ClonExpress II One Step Cloning Kit from Novizan Biotechnology Co., Ltd. The linearized vector obtained from PCR and the target gene fragment with homologous ends were gel-recovered and mixed at a 3:1 molar ratio. 4 μL of 5×CE II Buffer and 2 μL of Exnase II were added, followed by ddH2O to bring the total ligation volume to 20 μL. The mixture was incubated at 37°C for 30 min and then cooled to 4°C. 10 μL of the ligation mixture was then transformed into E. coli JM109 competent cells (competent cell preparation method is detailed in the Takara E. coli competent cell kit instructions). Transformants with correct colony PCR results were selected and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing verification, yielding the recombinant plasmid pJYW4-GNA1-CgGlmSmut. This plasmid was then transformed into Corynebacterium glutamicum S9114 for fermentation verification, and the yield results are shown below. Figure 5 As shown, the mutant strain increased yield by 12% compared to the wild strain.

[0090] Example 4: Analysis of CgGlmS mutant

[0091] 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 of the enzyme to the small molecules. Specifically, wild-type CgGlmS and mutant A253M were used to induce fit docking with substrates F6P and Gln, and products GlcN6P and Glu, respectively. The results showed that the binding pocket of the small molecules and the mutant was not significantly changed. Therefore, we speculated that the change in the binding stability of the small molecules and the enzyme led to the change in its catalytic efficiency. Subsequently, we used the MM-GBSA module to calculate the binding energies of the mutant with substrates Gln and F6P, and products Glu and GlcN6P. The calculation results are shown in Table 1. The binding energies of the mutant with substrates Gln and F6P were not significantly different from those of the wild type, that is, the mutant had no significant effect on the binding of the substrates. However, the binding energy of the mutant with product Glu increased significantly, from 3.32 kcal·mol⁻¹. -1 Significantly increased to 23.51 kcal·mol -1 The product Glu is less conducive to the binding of enzyme mutants, which accelerates the product's release and thus improves the enzyme's catalytic efficiency.

[0092] Table 1 Comparison of binding energies of enzymes to substrates and products (unit: kcal·mol) -1 )

[0093]

[0094] like Figure 6 As shown, to further verify our hypothesis, we performed 100 ns molecular dynamics simulations on the docking complexes of wild-type and mutant A253M. The simulation results showed that the RMSD of the mutant A253M protein backbone tended to stabilize in the later stages of the simulation (>50 ns), and was more stable than that of the wild-type. This is speculated to be due to the introduction of a hydrophobic side chain after the alanine (Ala) at position 253 was replaced by methionine (Met). The hydrophobic residues can form stronger van der Waals forces with adjacent α-helices, thereby improving enzyme stability. The RMSF results also confirmed this, showing a reduced variation in the 600-800 residue region of the dimer in the mutant, indicating that the mutant binds more stably 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. The results showed that in the later stages of the kinetic simulation (60-100 ns), the relative binding free energy between the mutant A253M and the overall product was approximately -25 kcal·mol⁻¹. -1 The wild type is approximately -50 kcal·mol⁻¹ -1The overall binding free energy of the mutant to the product is significantly higher than that of the wild type, indicating that it is more difficult for the mutant to bind to the product, thereby accelerating the release of the product so that the enzyme can quickly carry out the next catalytic reaction, thus improving the enzyme's catalytic efficiency.

[0096] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A mutant of glucosamine 6-phosphate synthase, characterized in that, The amino acid sequence is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding the 6-phosphate glucosamine synthase mutant of 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 of claim 2 or 3.

5. Recombinant cells expressing the 6-phosphate glucose synthase mutant of claim 1.

6. The use of the 6-phosphoglucosamine synthase mutant of claim 1, the nucleic acid molecule of claim 2 or 3, the gene expression cassette or recombinant plasmid of claim 4, or the recombinant cell of claim 5 in the preparation of N-acetylglucosamine, characterized in that, The application involves the preparation of N-acetylglucosamine in Corynebacterium glutamicum.

7. A recombinant Corynebacterium glutamicum, characterized in that, The recombinant Corynebacterium glutamicum overexpressed the encoding gene of the 6-phosphate glucose synthase mutant of claim 1. glmS .

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

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

10. A method for producing N-acetylglucosamine, characterized in that, The step includes fermentation production using the recombinant Corynebacterium glutamicum as described in claim 9.

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