Beta-1, 4 glucosyltransferase gme2697 and use thereof

Abstract

This invention relates to a β-1,4-glucosyltransferase, GME2697, and its applications, belonging to the field of fermentation technology. This invention provides a novel β-1,4-glucosyltransferase, GME2697, which possesses conserved domains catalyzing β-1,4-glycosidic bonds and β-1,6-glucan. Furthermore, this invention reveals that overexpression of GME2697 leads to a rearrangement of carbon metabolic flux, significantly enhancing the utilization efficiency of the carbon source glucose, directing more of it from cell growth to polysaccharide synthesis. GME2697 effectively upregulates extracellular polysaccharide synthesis, increasing extracellular polysaccharide yield. Simultaneously, overexpression promotes the synthesis of cell wall polysaccharides such as β-glucan and chitin. Chitin and other polysaccharides detach from the thickened cell wall and enter the fermentation broth, resulting in an increase in the proportion of water-insoluble polysaccharide components in the extracellular polysaccharide composition. Therefore, this invention provides a theoretical basis for elucidating the β-1,4-glucan synthesis mechanism in edible fungi.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a β-1,4-glucosyltransferase GME2697 and its applications. Background Technology

[0002] As a widely distributed polysaccharide in nature, β-glucan from different sources exhibits differences in molecular structure, including glycosidic bonds, molecular weight, degree of branching, and degree of polymerization. For example, in cereal β-glucan, glucose units are mainly linked by β-1,4 glycosidic bonds and spaced β-1,3 glycosidic bonds, forming an unbranched linear structure. Bacterial β-glucan, on the other hand, typically has a simple linear structure linked by β-1,3 bonds. β-glucan extracted from seaweed may simultaneously exist in two configurations: a pure β-1,3 backbone structure and a backbone with β-1,6-linked glucosyl side chains. In yeast and most fungal cell walls, β-glucan forms a backbone by linking sugar monomers with β-1,3 glycosidic bonds, while simultaneously exhibiting long branched structures linked by β-1,6 bonds.

[0003] Tiger Milk Mushroom (Pleurotus tuber-regium, PTR) is a basidiomycete with both edible and medicinal value, rich in polysaccharides, proteins, dietary fiber, and other bioactive substances. Hyperbranched β-glucan (HBG), a major component of the PTR cell wall, has been shown to possess multiple functions, including immunomodulation, antitumor activity, antihypertensive activity, and prebiotic activity, thus demonstrating broad application prospects in the development of pharmaceuticals and nutritional supplements. Notably, studies have found that the polysaccharide yield obtained from PTR sclerotia via alkaline extraction can reach as high as 41.18% of dry weight, confirming PTR as an excellent source of natural hyperbranched polysaccharides. Tiger Milk Mushroom hyperbranched β-glucan, due to its highly branched structure, exhibits high solubility, good dispersibility, and abundant terminal active groups, while also possessing good bioavailability, compatibility, and biodegradability, showing broad application prospects in the food, pharmaceutical, and nanomaterials fields. However, its synthesis mechanism remains unclear, and the key enzymes and their mechanisms of action are still unknown, which limits the further development and application of this polysaccharide. Therefore, elucidating the key enzymes in the hyperbranched β-glucan bioprocess of *Hylocereus undatus* can lay a theoretical foundation for the precise regulation of HBG synthesis and its structure. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of hyperbranched β-glucan synthase in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a β-1,4-glucosyltransferase and its applications. This invention screened a novel β-1,4-glucosyltransferase, GME2697, which is a hydrophilic and unstable protein with a molecular weight of 107.29 kDa. This protein possesses seven transmembrane domains and lacks a signal peptide, classifying it as a non-secretory membrane protein. Furthermore, GME2697 possesses conserved domains catalyzing β-1,4-glycosidic bonds (cellulose synthesis) and β-1,6-glucan (branching structure synthesis). Constructing a GME2697 homologous overexpression strain revealed that GME2697 gene overexpression leads to cell wall thickening and promotes extracellular polysaccharide synthesis, providing a theoretical basis for elucidating the β-1,4-glucan synthesis mechanism in edible fungi.

[0006] The first objective of this invention is to provide a β-1,4-glucosyltransferase GME2697, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, SEQ ID NO.1:

[0008] MDYDRWDAVLHYLFRQTQGDAWFRPDEQHISSGVAIRISPGGSTPDSPPEFRIFPYERQELEPFEQAVVGLNPEVAVKVRSAAVHAALAETSPDDRSIYVDANTRIQILDTMLHLPHADKEQSAAFIRDERVLVVWSHSLDAIIPTCHDFEERLIKLLWRSRPPVVTTPSVPASAAGSVSGHSLTHINSTNSGQRPGSRSGRRLINGASGMESDVEEKEKVGLGIETVSRSTDALGEDGGKEAKKQWRRNWYGKKVEVDGDIESLKAEKRPTMLYAPLYDGLSAGMAFLFVGNGLKVLLQEWTLDGAFLRFALIVALPLLYCVSLFFTIQLVQNLSMALGPVAHFHENSKYYSAIKPRPNKVVDNDLPHITIQMPVYKESLETVLTPSILSLKKAMQTYARQGGTSTIFINDDGLRAISEADRDERIAFYANHGIGWVARPKHDDSPDGFKRAGRFKKASNMNYGLNLSLKAEKHLETLVAAQKANPDKRSSTFSVHGSNSTSHSEPQYGMQYQHREGDDMQGMTAVGAEEDLEEKALNMAIEEVFQASGKKFRPWAANGKACRLGEIVLIVDSDTVVPEDCLRDAAREMRECPTVAIIQHESDVMQVAHHYFENGIAYFTRRVNRCISMACANGEVAPFMGHNAFLRWKAIQDAAFLDPVDGQEKIWSESNVSEDFDMALRLLQKGYIIRWATYSKGGFKEGVSLSVDDEINRWQKYAYGCNELLFNPLVQWLRKGPIARQVHRFVWSKAPLHYKFSTLAYMFSYYGISATVTIGIVNYVFLGFQFPTDGFYMHSFEIFLATTVVFYGSGNFCYTLLEYRLGHKQLLKGFLENIMWIPYFFFFFGGLAIPVSQALLAHLFSYNITWSATIKEVERSNFFKEVPKIAKRFWFPMLVSLVIIAGMIICSTPLVPYQWRVDGSSWAVIFPLAISTGCHILLPIVLNPWLMIFSY。

[0009] A second objective of this invention is to provide a gene encoding the aforementioned β-1,4-glucosyltransferase GME2697.

[0010] A third objective of this invention is to provide a recombinant plasmid carrying the aforementioned gene.

[0011] A fourth objective of this invention is to provide a recombinant cell expressing the aforementioned β-1,4-glucosyltransferase GME2697, wherein the host of the recombinant cell includes bacteria or fungi.

[0012] The fifth objective of this invention is to provide a method for preparing a transformant expressing the above-mentioned β-1,4-glucosyltransferase GME2697, comprising the following steps:

[0013] S1. Add lysozyme to Tiger Milk Mushroom to obtain Tiger Milk Mushroom protoplasts;

[0014] S2. Under the condition of transfection reagent, the recombinant plasmid is mixed with the protoplast of *Hylocereus undatus* and placed in an ice bath to obtain the transformant.

[0015] Furthermore, the transfection reagent includes polyethylene glycol.

[0016] The sixth objective of this invention is to provide a transformant prepared by the above-described preparation method.

[0017] A seventh object of the present invention is to provide the use of the above-mentioned β-1,4-glucosyltransferase GME2697, the above-mentioned gene, the above-mentioned recombinant plasmid, the above-mentioned recombinant cell or the above-mentioned transformant in the synthesis of β-glucan.

[0018] The eighth object of the present invention is to provide an application of the above-mentioned transformant in the production of high molecular weight polysaccharides.

[0019] Furthermore, the weight-average molecular weight of the high molecular weight polysaccharide is 4.621 × 10⁻⁶. 6 -2.242×10 8 g / mol.

[0020] The beneficial effects of this invention are:

[0021] The GME2697 overexpression described in this invention significantly affected the phenotype, growth, extracellular polysaccharide synthesis, and cell wall synthesis of PTR strains. This invention found that GME2697 overexpression led to a redistribution of carbon metabolic flux, significantly enhancing the utilization efficiency of the carbon source glucose (Glc), diverting more of it from cell growth to polysaccharide synthesis. This gene effectively upregulated extracellular polysaccharide synthesis, increasing extracellular polysaccharide (EPS) production. Furthermore, this gene overexpression promoted the synthesis of cell wall polysaccharides such as β-glucan and chitin, resulting in thickened and increased cell wall content. Chitin and other polysaccharides detached from the thickened cell wall and entered the fermentation broth, leading to an increase in the proportion of water-insoluble polysaccharide (WIP) components in EPS. In the later stages of fermentation, the degradation of extracellular polysaccharides served as a carbon source to maintain normal cell growth and metabolism. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0023] Figure 1 This is a secondary structure prediction for GME2697;

[0024] Figure 2 These are the protein structure prediction results from GME2697, where A represents the protein domains and B represents the protein's three-dimensional structure.

[0025] Figure 3 This is the result of a phylogenetic tree analysis of β-glycosyltransferases from different sources;

[0026] Figure 4 The construction of the GME2697 homologous overexpression plasmid;

[0027] Figure 5 These are the results of PCR verification of the genome of *Tiger Milk Mushroom* transformants;

[0028] Figure 6 The changes are in bacterial biomass, residual reducing sugar content and extracellular polysaccharide production; among them, WT is the wild-type Tiger Milk Mushroom strain, and 1-8 are 8 Tiger Milk Mushroom strains overexpressing GME2697 constructed in the previous experiment.

[0029] Figure 7 The quality of extracellular polysaccharides collected by different methods;

[0030] Figure 8 It describes the colony morphology and growth status;

[0031] Figure 9 The morphology and diameter distribution of Tiger Milk Mushroom mycelium balls on day 8 of liquid fermentation; AD were WT, MS, PTR-2697OE-2, and PTR-2697OE-4, respectively.

[0032] Figure 10 It is the average diameter of the mycelium pellets after 8 days of fermentation;

[0033] Figure 11 The differences are in biomass, fermentation residue sugar, and extracellular polysaccharides of *Lactarius deliciosus*, among which: A. WT; B. MS; C. PTR-2697OE-2; D. PTR-2697OE-4;

[0034] Figure 12 This is a graph showing the cell wall content of Tiger Milk Mushroom;

[0035] Figure 13 These are transmission electron micrographs and thickness diagrams of the cell walls of *Lactarius deliciosus* on day 8 of fermentation; among them, A. wild-type strain; B. empty vector transformant; C. PTR-2697OE-2; D. PTR-2697OE-4; E. comparison of cell wall thickness;

[0036] Figure 14 The determination of polysaccharide molecular weight is performed by size exclusion chromatography, where A) is a water-insoluble polysaccharide and B) is a polysaccharide obtained by 50% alcohol precipitation.

[0037] Figure 15 This is a diagram of the insoluble polysaccharide (WIP) of the GME2697 overexpression strain, where A. cell wall polysaccharide detachment; B. free flocculent polysaccharide;

[0038] Figure 16 This is a schematic diagram illustrating the effect of GME2697 overexpression on *Heliotropium indicum*. Detailed Implementation

[0039] 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.

[0040] The culture medium formula and preparation method used in this experiment are as follows:

[0041] (1) Liquid culture medium for Tiger Milk Mushroom (g / L): 20 g of anhydrous glucose, 4 g of Angel yeast extract, 1 g of potassium dihydrogen phosphate, and 0.6 g of magnesium sulfate heptahydrate. Sterilize at 115°C for 20 min.

[0042] (2) Solid PDA culture medium: 200g of potato, cut into small pieces, boiled in water, filtered out the liquid with gauze and added 20g of anhydrous glucose and 20g of agar, finally adjusted to 1L, sterilized at 115℃ for 20min.

[0043] (3) RCM medium (g / L): tryptone 2, yeast extract 2, glucose 20, magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 0.46, dipotassium hydrogen phosphate 1, add 20 g / L agar to make a solid medium. Add 0.6 M sucrose to make RCM regeneration medium, sterilize at 115℃ for 20 min.

[0044] (4) LB medium (g / L): yeast extract 5, peptone 10, sodium chloride 10, sterilized at 115℃ for 20 min.

[0045] Example 1: Structural Analysis of GME2697

[0046] In this embodiment, the glycosyltransferase GME2697 from *Lactarius deliciosus* was screened out. The following bioinformatics methods will be used to analyze the properties and structure of this enzyme.

[0047] The physicochemical properties of the *Lactarius delavayi* glycosyltransferase GME2697 were analyzed using the ProtParam tool on the ExPASy website. The results showed that GME2697 had an instability coefficient of 43.90, suggesting it is an unstable protein. Further prediction revealed that GME2697 had a lipid coefficient of 86.27 and an overall average hydrophilicity of -0.144, thus indicating it is a hydrophilic protein. In the secondary structure of this protein (… Figure 1 α-helices and random coils accounted for the largest proportions, at 48.95% and 42.75% respectively, while β-folds accounted for only 8.30%.

[0048] Table 1. Basic parameters and properties of GME2697

[0049]

[0050] This protein lacks a signal peptide and is a non-secretory protein. Subcellular localization analysis shows that it is a cell membrane protein, with only a small portion of its structure located in the cytoplasm, consistent with the well-studied distribution of the *Saccharomyces cerevisiae* FKS protein. However, compared to fungal β-1,3-glucan synthase, GME2697 has fewer transmembrane domains, only seven. Figure 2 The three-dimensional structure of the protein was predicted using AlphaFold 3.0 and visualized using Pymol. The seven transmembrane helices of the protein were clearly visible, with an electrostatic potential difference of -54.575 to +54.575.

[0051] Furthermore, by predicting protein domains using NCBI (such as...) Figure 2(As shown). GME2679 belongs to the glycosyltransferase family-2 (GT-2), possessing highly conserved Glyco_trans_2_3 and BcsA domains, as well as a less conserved CESA_CelA_like domain. Glyco_trans_2_3 is a prokaryotic glucosyltransferase involved in capsule synthesis. The BcsA superfamily of glycosyltransferases exhibits catalytic activities of cellulose synthase and poly-β-1,6-N-acetylglucosamine synthase, while the CESA_like superfamily of cellulose synthases shows activity in synthesizing β-1,4-glycosidic bonds. Analysis of the domains preliminarily demonstrates that glycosyltransferase GME2677 has catalytic activity in forming β-1,4-glycosidic bonds, and it is speculated that this enzyme may also possess β-1,6-glucanase activity.

[0052] BLAST was used to select sequences with high matching degrees from different sources for constructing a phylogenetic tree. Figure 3 The results showed that the glycosyltransferase GME2697 from *Lactarius delavayi* is highly similar to glycosyltransferases in basidiomycetes, indicating a close evolutionary relationship. Among them, it showed the highest homology (96.5%) with the GT-2 protein KAL4259244.1 from *Pleurotus pulmonarius*. Furthermore, compared to bacteria, fungal glycosyltransferases showed higher homology with enzymes from plants.

[0053] Example 2: Construction of recombinant bacteria overexpressing GME2697

[0054] 1. Construction of recombinant plasmids

[0055] like Figure 4 As shown, the laboratory previously constructed the *E. coli* overexpression plasmid p19T-msdhb and mutated the *E. coli* endogenous succinate dehydrogenase B subunit gene (sdhb) by changing the histidine codon (CAT) at position 241 to leucine (CTT), thus obtaining the carboxin resistance gene (msdhb). The plasmid was extracted from *E. coli* JM109 containing the p19T-msdhb-EGFP plasmid preserved in the laboratory, and its concentration was determined. PCR cloning was performed using p19T-msdhb-F and p19T-msdhb-R primers to obtain a linear plasmid with homologous sequences to the GME2697 gene. Using *E. coli* genomic DNA as a template, PCR cloning was performed using GME2697-F and GME2697-R primers to obtain a linear target gene with homologous sequences to the overexpression plasmid.

[0056] Table 2 Primers used in Example 2

[0057]

[0058] The plasmid and target gene were recovered via gel extraction and their concentrations determined. The p19T-msdhb-GME2697 plasmid was constructed using homologous recombination, in which GME2697 replaced the EGFP position on the original plasmid. The recombinant plasmid was introduced into E. coli JM109 competent cells, and selection was performed using ampicillin-resistant plates. Single colonies were collected for PCR verification to confirm successful plasmid introduction. Finally, positive colonies were selected for plasmid sequencing, thus confirming successful recombinant plasmid construction.

[0059] 2. Preparation of Tiger Milk Mushroom Protoplasts

[0060] (1) A 1×1cm specimen covered with tiger milk mushroom mycelium 2 The agar was inoculated into liquid culture medium and cultured at 30°C and 180 rpm for 4 days to obtain seed culture.

[0061] (2) Disperse the seed liquid with sterilized glass beads and a magnetic stirrer, take 10 mL and transfer it to 190 mL of Tiger Milk Mushroom liquid culture medium, incubate at 30℃, and shake twice a day.

[0062] (3) Take the fermented *Vibrio vulgaris* and filter it through a sterile nylon cloth. Wash the mycelium 2-3 times with 0.6M sucrose on the filter cloth. Then, take the mycelium into a 5mL centrifuge tube and add lysozyme (40mg of lysozyme dissolved in 0.6M sucrose solution, with a final concentration of 200mg / mL) for enzymatic hydrolysis. After enzymatic hydrolysis, separate the protoplasts using a 40μm cell filter. Centrifuge the filtrate at 4℃ and 4000×g for 10min to collect the precipitate, which is the *Vibrio vulgaris* protoplast. Wash the protoplasts once with STC buffer, and then resuspend them with an appropriate amount of STC buffer to obtain a protoplast suspension.

[0063] 3. PEG-mediated protoplast transformation

[0064] (1) Dilute the above protoplast suspension to a protoplast count of 10. 7 After reaching CFU / mL, add 20 μL of recombinant plasmid (approximately 10 mg), 20 μL of pre-chilled STC buffer, and 160 μL of bacterial suspension to a 1.5 mL centrifuge tube, mix well, and incubate on ice for 10 min. Then add 200 μL of PTC, gently tap the tube wall to mix, and incubate on ice for 10 min (repeat this step once). Finally, add 600 μL of PTC buffer, mix well, and incubate on ice for 30 min.

[0065] (2) Centrifuge the above solution slowly, add 1 mL of RCM regeneration medium to the precipitate and resuspend, and incubate at 30°C for 1 h.

[0066] (3) Centrifuge to remove the culture medium, then add 300 μL of RCM medium for resuspending. Finally, spread the resuspended solution onto antibiotic-free RCM regeneration plates and incubate at 28°C for 3-4 days. When the protoplasts grow into dot-like regenerated colonies, add a layer of 1 μg / mL carboxin-containing low-melting-point agarose regeneration medium and continue incubation at 28°C until transformants grow on the upper plate. Pick the transformants grown on the upper plate and passage them sequentially on plates containing 2 μg / mL carboxin resistance. Each concentration is passaged twice to obtain genetically stable transformants. Figure 5 ).

[0067] like Figure 6 As shown, the results indicated that, compared to the wild type, the transformant strain exhibited significantly increased extracellular polysaccharide production while decreasing biomass. This suggests that GME2697 overexpression can promote extracellular polysaccharide synthesis while inhibiting cell growth. Colony PCR and sequencing revealed that the genes in transformants 2 and 4 were correctly integrated into the genome. Therefore, transformant strains 2 and 4, with similar fermentation data and higher extracellular polysaccharide production, were ultimately selected for subsequent experiments.

[0068] Because the fermentation broth of the transformants on day 8 contained a large amount of translucent, gelatinous substance resembling polysaccharides, this experiment used a fractional alcohol precipitation method to collect the extracellular polysaccharides. The results are as follows: Figure 7 As shown in the figure, compared to the wild-type strain, the proportions of insoluble polysaccharides (obtained by direct centrifugation of fermentation broth) and polysaccharides obtained by 50% ethanol precipitation in the transformants were significantly increased. In the wild-type strain, the polysaccharide obtained by 75% ethanol precipitation accounted for the highest proportion of total extracellular polysaccharides, at 48.92%. Among the transformants, strain 3 had the highest proportion of polysaccharides obtained by 75% ethanol precipitation, but only reached 33.33%; transformant 7 had the lowest proportion (7.34%). The most abundant component of extracellular polysaccharides in the transformants was water-insoluble polysaccharide (WIP), with transformant 8 having the highest proportion at 82.44%, followed by transformants 7 and 5 at 78.65% and 62.09%, respectively. The previously selected transformants 2 and 4 also had WIP proportions of 49.53% and 47.66%, significantly higher than the 32.25% in the wild-type strain. Therefore, it is speculated that GME2697 overexpression can increase the molecular weight of extracellular polysaccharides.

[0069] Example 3: Effects of GME2697 overexpression on Tiger Milk Mushroom cells

[0070] 1. Screening of transformants

[0071] Eight strains of *Lactarius deliciosus* overexpressing the GME2697 gene were obtained through transformation. Two transformants with correct sequencing and good traits and extracellular polysaccharide production were then fermented. Five 1×1cm pieces were cut from the plates. 2Mycelial blocks were inoculated into 200 mL of Tiger Milk Mushroom liquid medium and cultured at 30℃ and 180 r / min for 4 days to obtain seed culture. 10 mL of the seed culture was added to 190 mL of Tiger Milk Mushroom liquid medium using a sterilized 5 mL pipette tip and cultured under the same conditions. When the wild-type strain had fully colonized the fermentation broth, fermentation was stopped, and mycelial balls and fermentation broth were collected for measurement of cell mass, reducing sugar content, and extracellular polysaccharide content.

[0072] 2. Colony status and mycelial growth

[0073] Mycelial blocks were cut from pre-activated PDA plates using a punch and inoculated onto antibiotic-free PDA plates, then incubated at 30°C. The mycelial diameter was measured every 24 hours using the cross-hatching method to calculate the mycelial growth rate. Incubation was stopped when the mycelium of a particular strain completely covered the plate, and the colony morphology was photographed and recorded.

[0074] Using transformants 2 (PTR-2697OE-2) and 4 (PTR-2697OE-4) as experimental groups, and wild-type Tiger Milk Mushroom strain (WT) and strain introduced with empty vector p19T-msdhb (MS) as control groups, the effects of GME2697 overexpression on colony morphology and growth rate were studied.

[0075] The growth of mycelium is as follows Figure 8 As shown, transformants 2 and 4 exhibited rapid hyphal growth, covering the entire plate by day 5, while WT and MS showed very slow growth in the early stages. However, the hyphal growth of the transformants was not vigorous, appearing sparse and uneven, whereas the hyphal growth of the control groups WT and MS strains was vigorous and relatively uniform. These results indicate that GME2697 overexpression can significantly affect hyphal morphology and growth rate.

[0076] 3. Morphology and diameter distribution of fungal balls

[0077] Liquid fermentation was performed on different strains of *Lactarius deliciosus*. Mycelia cultured for days 2, 4, 6, 8, and 10 were collected, rinsed thoroughly with water, and a suitable amount of mycelial pellets were placed in clean plastic petri dishes. Water was added to the petri dishes to cover the pellets, and photographs were taken. Mycelial pellets were counted and their diameters calculated using ImageJ-1.54g software. The results were statistically analyzed, and a diameter distribution map of the mycelial pellets was plotted using Origin 2024. Differences in mycelial pellet diameter and distribution among wild-type strains, empty vector strains, and transformed strains were compared.

[0078] Take a random sample of the homogeneous fermentation broth from day 8 and photograph it on a plate for observation. Figure 9 It can be seen that the surface of the WT and MS control group mycelial balls is relatively rough with obvious mycelial protrusions, but the transformant mycelial balls are very smooth. This indicates that GME2697 overexpression significantly affects the morphology of Tiger Milk Mushroom mycelial balls.

[0079] Based on the measured diameter of the mycelial pellets, they were classified into four types: S-type pellets with a diameter less than 1 mm, M-type pellets with a diameter greater than 1 mm and less than 2 mm, L-type pellets with a diameter greater than 2 mm and less than 3 mm, and XL-type pellets with a diameter greater than 3 mm. The final results are as follows: Figure 9 As shown, most of the bacterial cocci in WT and MS were S-shaped, with S-shaped cocci accounting for 52.26% in WT and a high proportion of 75.19% in MS. PTR-2697OE-2 and PTR-2697OE-4 had fewer S-shaped cocci and a higher proportion of M-shaped cocci, at 71.51% and 44.85%, respectively. During the measurement process, some very large cocci were found in WT and MS, with the largest WT cocci reaching a diameter of 4.12 mm. However, because their cocci were mainly S-shaped, the average diameters of WT and MS remained relatively small, at 1.30 mm and 0.94 mm, respectively. Compared to the control group, the average diameters of PTR-2697OE-2 and PTR-2697OE-4 increased significantly, reaching 1.62 mm and 1.50 mm, respectively. Figure 10 Based on the combined results of coli diameter, biomass, and extracellular polysaccharide production, overexpression of GME2697 decreased biomass but increased the average coli diameter and simultaneously enhanced extracellular polysaccharide production. Preliminary speculation suggests that the increase in coli diameter is related to carbon source consumption and the large-scale production of extracellular polysaccharides.

[0080] 4. Determination of mycelial biomass and fermentation residual sugar

[0081] Tiger Milk Mushrooms were collected for testing on days 2, 4, 6, 8, and 10 of liquid fermentation, with three replicates for each strain. The mycelium was filtered through a 100-mesh filter and repeatedly washed with distilled water until the filtrate was colorless. The collected mycelium was then frozen at -80°C for at least 24 hours, freeze-dried, and finally weighed and its biomass recorded.

[0082] The supernatant was collected by centrifugation, and the reducing sugar content was determined using the DNS colorimetric method. This method requires the prior preparation of a standard curve. A stock solution with a glucose concentration of 1 mg / mL was prepared, and then diluted to concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / mL to obtain standards. 0.5 mL of each standard was mixed with 1 mL of DNS, boiled in a water bath for 5 min, cooled to room temperature, and then 2.5 mL of ultrapure water was added and mixed thoroughly before measuring the OD. 540 And draw a standard curve.

[0083] Mycelial biomass, residual fermentation sugar, and extracellular polysaccharide production of *Gymnocladus orientalis* were measured at days 2, 4, 6, 8, and 10 of liquid fermentation. The effect of GME2697 overexpression on mycelial growth was analyzed. The results are as follows: Figure 11As shown in the figure, the transformants exhibited rapid cell growth during the early fermentation phase (days 2 to 4), generally exceeding that of the WT and MS controls. The cell count reached its maximum on day 6, with PTR-2697OE-2 at 10.01 ± 0.32 g / L and PTR-2697OE-4 at 8.48 ± 0.31 g / L. During the later fermentation phase, the cell count of the transformants decreased, while WT and MS remained in the logarithmic growth phase from day 6 to day 8, showing rapid cell growth, reaching its maximum on day 8, and then slightly decreasing. At the end of fermentation on day 10, the cell counts of the two transformants were only 67.60% and 57.26% of the wild type, respectively. Therefore, overexpression of GME2697 downregulated the growth of the *Lactarius deliciosus* strain.

[0084] The residual reducing sugars in the fermentation broth showed the same trend across different strains, being continuously consumed with increasing fermentation time. However, the transformants consumed the reducing sugars in the fermentation broth significantly faster. At the end of fermentation, the residual sugars in PTR-2697OE-2 and PTR-2697OE-4 were 1.08±0.10 g / L and 1.30±0.13 g / L, respectively, which were lower than those in WT (5.56±0.43 g / L) and MS (7.68±1.27 g / L). Furthermore, the cell mass of the transformants reached its maximum on day 6, while the extracellular polysaccharide production peaked on day 8. By day 8, the extracellular polysaccharide production of PTR-2697OE-2 was as high as 2.63±0.22 g / L, 2.16 times that of WT (1.22±0.03 g / L). The extracellular polysaccharide production of PTR-2697OE-4 was lower, but still reached 2.05±0.21 g / L. However, from day 8 to day 10 of fermentation, the production of extracellular polysaccharides decreased significantly. This may be because the reducing sugars in the fermentation broth could not support the normal metabolism of the cells in the last two days, causing the transformants to begin to decompose the extracellular polysaccharides to produce the carbon source needed for their own growth and development.

[0085] Example 4: Effects of GME2697 overexpression on cell wall and extracellular polysaccharide structure

[0086] 1. Effects on cell wall structure

[0087] (1) Cell wall thickness

[0088] On day 8 of liquid fermentation, the fermentation product was rinsed with deionized water and then placed in a 1.5 mL centrifuge tube. Cells approximately the size of soybeans were collected by centrifugation. The cells were washed 2-3 times with 4% glutaraldehyde, and then fixed by slowly adding 4% glutaraldehyde along the tube wall. After standing at room temperature for 1-2 hours, the cells were placed in a 4°C refrigerator overnight. The fixed samples were then sent to the Scientific Compass testing platform for transmission electron microscopy (TEM) observation, and cell wall thickness was measured using an ImageJ-1.54g microscope to compare the differences in cell wall thickness between wild-type and transformants.

[0089] (2) Cell wall content

[0090] Cell walls were extracted from freeze-dried bacterial cells fermented for 6, 8, and 10 days. The bacterial cells were then pulverized using a mortar and pestle and passed through a 40-mesh sieve.

[0091] Take an appropriate amount into a 5 mL centrifuge tube, wash it successively with 5% NaCl, 2% NaCl and 1% NaCl solutions, washing 3 times with each solution, then wash it at least 5 times with ultrapure water, centrifuge and collect the precipitate, freeze dry and weigh.

[0092] β-glucan is a core component of the cell wall in *Lactarius deliciosus*. The role of this enzyme can be inferred by analyzing the effect of GME2697 on cell wall synthesis. Based on the changes in biomass and extracellular polysaccharide production obtained in the early stages, cell wall content was measured during the later stages of fermentation when there was a significant increase. The cell wall content of each strain on days 6, 8, and 10 is as follows: Figure 12 As shown, overexpression of GME2697 effectively increased cell wall content, and the changes in cell wall content and extracellular polysaccharide content in transformants showed the same trend. PTR-2697OE-2 reached its highest cell wall content of 38.15% on day 8, which was 1.62 times that of the WT strain (23.50%). The cell wall content of PTR-2697OE-4 also reached its maximum of 34.28% on day 8, and decreased slightly on day 10, but the decrease was significantly less than that of PTR-2697OE-2, which is consistent with the changes in extracellular polysaccharide content.

[0093] 2. Effects on extracellular polysaccharides

[0094] (1) Molecular weight determination

[0095] 10 mg of lyophilized polysaccharide sample was dissolved in 2 mL of 0.1 mol / L NaNO3 solution. Dissolution was promoted by heating in an 80°C metal bath and sonication. If complete dissolution was not achieved, the supernatant was collected after centrifugation and filtered through a 0.45 μm aqueous filter membrane before being sent to the laboratory. Size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) was used to determine the weight-average molecular weight (Mw), root mean square radius (RMS radius), and polydispersity index (Mw / Mn) of the extracellular polysaccharide. Gel column type: Ultrahydrogel. TMColumn size: 2000 (30cm × 7.8mm); Mobile phase: 0.1mol / L NaNO3 solution; Flow rate: 0.5mL / min; Column temperature: 25℃; MALLS light source: 690nm helium-neon laser. 500μL samples were injected for each analysis, and data acquisition and result analysis were performed using Astra-7.0 software.

[0096] Based on previous results, water-insoluble polysaccharide (WIP) and 50% ethanol-precipitated polysaccharide (50% EP), which have a high proportion in the extracellular polysaccharides of *Lactarius deliciosus*, were selected for gel size exclusion chromatography analysis. Figure 7 ). Figure 14 The chromatographic results show that WIP-WT and 50% EP-WT have a main peak and a smaller peak preceding the main peak, WIP-2 has two distinct peaks, and the results of other samples all show a single symmetrical elution peak. This indicates that the polysaccharide samples other than WIP-2 have good homogeneity.

[0097] The static light scattering model Zimm was used for analysis, and data from the main elution peaks were selected for analysis. The results are shown in Table 3. Compared with WT and MS, overexpression of the GME2697 gene increased the molecular weight of water-insoluble polysaccharides and 50% alcohol-precipitated polysaccharides of PTR-2697OE-2 and PTR-2697OE-4. In addition, overexpression of GME2697 also led to a decrease in the root mean square radius (RMS radius).

[0098] Table 3. SEC-MALLS analysis results of the two polysaccharides

[0099]

[0100]

[0101] Note: Mw is the weight-average molecular weight, RMSradius is the root mean square radius, and Mw / Mn is the polydispersity index.

[0102] (2) Cell wall thickness measurement

[0103] Bacterial cells collected on day 8 were analyzed using transmission electron microscopy (TEM). Cell wall thickness was measured from images with clear cell walls. Five TEM images were taken from each strain as parallel samples, and five points were randomly selected from each image. The measurement results are as follows: Figure 13As shown, the average cell wall thicknesses of WT and MS were 72.6 nm and 69.6 nm, respectively, while the average cell wall thickness of PTR-2697OE-2 was 91.6 nm, and that of PTR-2697OE-4 was 78.2 nm. Through observation... Figure 13 C and Figure 13 D found that PTR-2697OE-2 and PTR-2697OE-4 contained a large amount of flocculent polysaccharides that detached from the cell wall, which may be the reason why the cell wall thickening was not obvious, but the content and yield of extracellular polysaccharides in the fermentation broth were significantly increased.

[0104] (2) Monosaccharide composition analysis

[0105] Take 3 mg of lyophilized polysaccharide, add 100 μL of 12 mol / L sulfuric acid solution, and hydrolyze at 35 °C for 1 h. Then add 0.5 mL of ultrapure water and mix well. Transfer the solution to a 98 °C water bath and continue hydrolysis for 1 h. After the sample cools to room temperature, adjust it to neutral with sodium hydroxide solution and dilute appropriately. Finally, filter the sample through a 0.45 μm aqueous filter membrane and send it for qualitative and quantitative analysis of monosaccharide composition using ion chromatography.

[0106] The monosaccharide composition of each sample was determined using ion chromatography, and the monosaccharide composition and proportion in the samples were obtained (Table 4). Glucose was the main component in all samples. In water-insoluble polysaccharides (WIP), a new monosaccharide, glucosamine, appeared in the GME2697 overexpression strain, and its proportion was relatively high, at 30.474% and 25.806%, respectively, while the proportions of glucose and galacturonic acid decreased significantly. In 50% alcohol-precipitated polysaccharides (50% EP), the proportion of glucose in the polysaccharides of the GME2697 overexpression strain decreased, while the proportions of mannose and glutarose increased significantly. Arabinose and galacturonic acid also appeared, while these components were not found in 50% EP-WT.

[0107] Table 4. Monosaccharide composition and proportion

[0108]

[0109]

[0110] Fermentation results have shown that GME2697 overexpression leads to improved carbon source utilization, with more carbon being diverted to cell wall thickening and extracellular polysaccharide synthesis. Preliminary screening results of transformants showed that by day 8 of fermentation, the extracellular polysaccharide yields of PTR-2697OE-2 and PTR-2697OE-4 were 2.59±0.30 g / L and 2.14±0.21 g / L, respectively, significantly higher than the wild-type's 0.69±0.11 g / L. In PTR-2697OE-2, the WIP, 50% EP, and 75% EP components were 1.28±0.19 g / L, 0.68±0.12 g / L, and 0.62±0.09 g / L, respectively; while in PTR-2697OE-4, they were 1.02±0.18 g / L, 0.63±0.02 g / L, and 0.49±0.05 g / L, respectively. In the transformants, the WIP component was the most abundant component, accounting for 49.53% and 47.66% of the total extracellular polysaccharides, respectively. However, in the WT, WIP accounted for only 32.25% of the total extracellular polysaccharides, indicating that GME2697 overexpression led to an increase in the proportion of water-insoluble polysaccharides. Combined with monosaccharide composition analysis, the glucosamine content in the WIP of the transformants was significantly increased, indicating the presence of a large amount of chitin. Therefore, it is inferred that GME2697 overexpression leads to cell wall thickening, and when the cell wall thickness reaches a certain level, polysaccharide components will dissociate from the cell wall. Figure 15 A) refers to the insoluble polysaccharides (WIP) in the fermentation broth. Figure 15 B).

[0111] 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 β-1,4-glucosyltransferase GME2697, characterized in that, The amino acid sequence of the β-1,4-glucosyltransferase GME2697 is shown in SEQ ID NO.

1.

2. The gene encoding the β-1,4-glucosyltransferase GME2697 as described in claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. A recombinant cell expressing the β-1,4-glucosyltransferase GME2697 of claim 1, characterized in that, The host of the recombinant cells includes bacteria or fungi.

5. A method for preparing a transformant expressing the β-1,4-glucosyltransferase GME2697 of claim 1, characterized in that, Includes the following steps: S1. Add lysozyme to Tiger Milk Mushroom to obtain Tiger Milk Mushroom protoplasts; S2. Under the condition of the presence of the transfection reagent, the recombinant plasmid of claim 3 is mixed with the protoplast of *Lactarius deliciosus* and subjected to an ice bath to obtain the transformant, wherein the transfection reagent includes polyethylene glycol.

6. The transformant prepared by the method of claim 5.

7. The use of the β-1,4-glucosyltransferase GME2697 of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, the recombinant cell of claim 4, or the transformant of claim 6 in the synthesis of β-glucan.

8. The application of the transformant according to claim 6 in the production of high molecular weight polysaccharides.

9. The application according to claim 8, characterized in that, The weight-average molecular weight of the high molecular weight polysaccharide is 4.621 × 10⁻⁶. 6 -2.242×10 8 g / mol.

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