Engineering bacterium with high chitin content as well as construction method and application of engineering bacterium

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a high-chitin-content engineering bacterium as well as a construction method and application thereof. According to the invention, enzyme related to filamentous fungus chitin synthesis is subjected to overexpression through a genetic engineering means to construct engineering bacteria, the obtained engineering bacteria have high chitin content, and a high-strength mycelium material can be obtained through conventional solid state fermentation. The invention provides a feasible solution with controllable product quality for the production of the high-strength mycelium material, and has good application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium with high chitin content, a method for constructing the same, and an application thereof. Background Art

[0002] Mycelium material is a material made from fungal mycelium through processes such as extraction, cleaning, and processing. It has a unique three-dimensional structure and various excellent properties, so it has a wide range of applications in environmental protection materials, construction, clothing, and packaging materials. Mycelium, as the basic unit of mycelium material, its own composition jointly endows it with unique physical and chemical properties.

[0003] The cell wall is the outer barrier of the mycelium and is mainly composed of polysaccharides such as chitin and glucan. These polysaccharides form a complex three-dimensional network structure, endowing the mycelium material with a stable form and structure, enabling it to maintain integrity under various environmental conditions. Chitin is one of the main components of the mycelium cell wall and plays important roles in many aspects in the mycelium material: 1. Providing structural support and mechanical strength: Chitin forms a strong network structure in the cell wall, endowing the mycelium material with good mechanical strength and stability, enabling it to withstand certain physical pressures and mechanical loads; 2. Affecting the properties of the material: Chitin not only affects the hardness and strength of the material, but also has an impact on its flexibility, water absorption, and thermal stability. For example, the polysaccharide network structure of chitin helps to improve the water absorption and water retention of the mycelium material, making it have better hydrophilicity and biocompatibility in some applications; 3. Participating in the biodegradability of the material: Chitin has good biodegradability, which enables the mycelium material to degrade naturally after use and will not cause pollution to the environment. This biodegradability conforms to the concept of sustainable development, making the mycelium material an environmentally friendly alternative; 4. Affecting the interaction with other materials. In practical applications, mycelium materials often need to be used in combination with other materials. The presence of chitin will affect the interaction between the mycelium and other materials. For example, the surface properties and chemical composition of chitin will affect the adhesion and compatibility between the mycelium and other materials, thereby affecting the performance and application effect of the composite material; 5. Biological activity and application potential: Chitin has various biological activities such as antibacterial, antioxidant, and immunomodulatory effects. These characteristics make the mycelium material have broad application potential in many fields such as medical treatment, agricultural production increase, and environmental protection management. For example, chitin can be used to make medical materials such as surgical sutures, hemostatic tablets, and membranes, and can also be used as a health care product with the effect of regulating the intestinal environment.

[0004] Currently, most mycelial materials are induced to produce more chitin by adding inducers such as chitin synthesis precursors during cultivation, thereby improving the strength of the mycelial materials. However, this method often requires precise control of the culture medium components to obtain an ideal induction effect, thus increasing the process complexity. In addition, excessive addition of precursors may lead to uneven internal structure of the mycelium, which may instead cause a decrease in the mechanical properties of the mycelial materials. Summary of the Invention

[0005] In view of the above problems, the present invention provides a chitin-rich engineered bacterium, a method for constructing the same, and an application thereof. The engineered bacterium provided by the present invention has a high chitin content in its cell wall, and a mycelial material with high strength can be obtained under conventional solid-state fermentation conditions, having good application prospects.

[0006] To achieve the above invention object, the present invention adopts the following technical solutions: In the first aspect of the present invention, a chitin-rich engineered bacterium is provided. The engineered bacterium is constructed by genetic modification using a filamentous fungus as the starting strain; the genetic modification includes: overexpressing chitin synthase (Chitinsynthases, CHS), glutamine-fructose-6-phosphate aminotransferase (GFAT), and uridine diphosphate-N-acetylglucosamine pyrophosphorylase (UAP).

[0007] By expressing GFAT, UAP, and CHS enzymes in the fungus, more energy metabolism flows towards chitin synthesis, so that the chitin content in the cell wall of the constructed engineered bacterium can be significantly increased under conventional fermentation conditions without the need to externally add inducers such as chitin synthesis precursors, thereby improving the mechanical strength of the mycelial material obtained by its fermentation, providing a new way to obtain a high-strength mycelial material.

[0008] Preferably, the filamentous fungus includes Ganoderma lucidum, Pleurotus ostreatus, Cordyceps militaris, Coriolus versicolor, Flammulina velutipes.

[0009] Preferably, the chitin synthase is chitin synthase CHS-5 or CHS-8.

[0010] Preferably, the uridine diphosphate-N-acetylglucosamine pyrophosphorylase is uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1.

[0011] Preferably, the method for overexpressing the chitin synthase, glutamine-fructose-6-phosphate aminotransferase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase is: increasing the copy number of the encoding genes of each enzyme.

[0012] In a second aspect of the present invention, a method for improving the strength of mycelial materials is provided. The method is to increase chitin, β-glucan in the mycelial cell wall, and proteins that can bind to cell wall components.

[0013] Preferably, the method is to overexpress chitin synthase, glutamine-fructose-6-phosphate aminotransferase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase.

[0014] In a third aspect of the present invention, a Ganoderma engineering bacterium with high chitin content is provided. The Ganoderma engineering bacterium is constructed by genetic modification using Ganoderma as the starting strain; the genetic modification includes: overexpressing chitin synthase CHS-5 or CHS-8, glutamine-fructose-6-phosphate aminotransferase GFAT, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1. The amino acid sequence of chitin synthase CHS-5 is as shown in GenBank: AZQ26802.1, the amino acid sequence of chitin synthase CHS-8 is as shown in GenBank: AZQ26805.1, the amino acid sequence of glutamine-fructose-6-phosphate aminotransferase GFAT is as shown in SEQ ID NO.1, and the amino acid sequence of uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1 is as shown in SEQ ID NO.2.

[0015] Preferably, the nucleotide sequence of the coding gene of chitin synthase CHS-5 is as shown in GenBank: MH463055.1, the nucleotide sequence of the coding gene of chitin synthase CHS-8 is as shown in GenBank: MH463058.1, the nucleotide sequence of the coding gene of glutamine-fructose-6-phosphate aminotransferase GFAT is as shown in SEQID NO.3, and the nucleotide sequence of the coding gene of uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1 is as shown in SEQ ID NO.4.

[0016] Exemplarily, the present invention also provides a method for constructing the above-mentioned Ganoderma engineering bacterium with high chitin content, which specifically includes the following steps: S1. Construct a recombinant plasmid 1303-GLP-chitin of chitin synthase CHS-5 or CHS-8, glutamine-fructose-6-phosphate aminotransferase GFAT, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1; S2. Transform the recombinant plasmid 1303-GLP-chitin into Ganoderma protoplasts, and screen the obtained positive transformants to obtain the Ganoderma engineering bacterium with high chitin content.

[0017] When screening the obtained positive transformants in S2, select the positive transformants with significantly high expression of chitin synthase CHS-5 or CHS-8, glutamine-fructose-6-phosphate aminotransferase GFAT, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1, and the engineered Ganoderma lucidum strain with high chitin content can be obtained. Among them, "significantly high expression" means that the degree of increase in gene expression level has a significant difference as conventionally understood in the art.

[0018] The fourth aspect of the present invention provides an engineered strain GL-chintin-19 with high chitin content, and its taxonomic name is Ganoderma lucidum ( Ganoderma lingzh i), which was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on May 21, 2025, at the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 42012.

[0019] The above-mentioned engineered strain GL-chintin-19 was constructed by overexpressing chitin synthase CHS-5, glutamine-fructose-6-phosphate aminotransferase GFAT, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1 using commercial Ganoderma lucidum as the starting strain. Compared with the chitin content of the starting strain, the chitin content of the engineered strain GL-chintin-19 increased by 548%. Compared with the mycelial fiber skin produced by the starting strain, the strength of the mycelial fiber skin produced by the engineered strain GL-chintin-19 increased by 149%.

[0020] The fifth aspect of the present invention provides the application of the above-mentioned engineered strain, Ganoderma lucidum engineered strain, and engineered strain GL-chintin-19 in the production of mycelial materials.

[0021] The sixth aspect of the present invention provides a method for producing mycelial fiber skin, which uses the above-mentioned engineered strain or engineered strain GL-chintin-19 to obtain mycelial fiber skin through solid-state fermentation.

[0022] The method specifically includes the following operations: spraying the mycelial suspension of the above-mentioned engineered strain or engineered strain GL-chintin-19 on the surface of the solid fermentation substrate, culturing at a constant temperature of 28-30°C and RH60%-70%, and separating and taking out the mycelial skin above the substrate after the end of the culture period to obtain the mycelial fiber skin.

[0023] Preferably, the mycelial fiber skin is obtained by solid-state fermentation using the above-mentioned engineered strain GL-chintin-19.

[0024] The beneficial effects of the present invention are as follows: The present invention provides an innovative method based on genetic engineering technology for improving the performance of filamentous fungal mycelium materials. By genetically modifying filamentous fungi, the chitin synthesis and metabolism pathway of filamentous fungi is precisely regulated, and the engineered bacteria constructed have cell walls with high chitin content. When using this strain to produce mycelium materials, high-strength mycelium materials can be obtained without exogenous addition of chitin precursors, breaking through the various defects faced by traditional induction methods and being applicable to the industrial production of mycelium materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the plasmid map of 1303-GLP-chitin in Example 1 of the present invention; Figure 2 It is each plate 20 days after transforming the 1303-GLP-chitin plasmid in Example 1 of the present invention; Figure 3 It is the standard curve of D-glucosamine content and chitin quality in Example 1 of the present invention; Figure 4 It is the schematic diagram of testing the skin strength of mycelium fibers by a tensile testing machine in Example 1 of the present invention.

[0026] The engineered bacteria GL-chintin-19, taxonomically named Ganoderma lucidum ( Ganoderma lingzhi ), preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC), preservation unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation number: CGMCC No. 42012, preservation date: May 21, 2025. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following further details the present invention in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0028] The mycelium is the basic unit of the mycelium material, and chitin, as one of the main components of the cell wall of fungal mycelium, endows it with unique physical and chemical properties with its own composition. Currently, most mycelium materials induce mycelium to produce more chitin by adding inducers such as chitin synthesis precursors during cultivation, so as to improve the strength of the mycelium material. However, this method increases the process complexity and may also lead to uneven internal structure of the mycelium, resulting in a decline in the mechanical properties of the mycelium material.

[0029] An embodiment of the present invention provides a genetically engineered bacterium with a high chitin content. This genetically engineered bacterium is constructed by genetic modification using a filamentous fungus as the starting strain. The genetic modification includes overexpressing chitin synthase, glutamine-fructose-6-phosphate aminotransferase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase. Under conventional fermentation conditions without the exogenous addition of chitin synthesis precursors and other inducers, this genetically engineered bacterium can contain more chitin in its cell wall, thereby improving the mechanical strength of the mycelial material obtained by its fermentation.

[0030] An embodiment of the present invention also provides a method for improving the strength of mycelial materials, which is to increase the chitin, β-glucan, and proteins that can bind to cell wall components in the mycelial cell wall.

[0031] An embodiment of the present invention also provides a Ganoderma lucidum genetically engineered bacterium with a high chitin content and a method for constructing the same.

[0032] An embodiment of the present invention also provides a genetically engineered bacterium GL-chintin-19 with a high chitin content.

[0033] An embodiment of the present invention also provides the applications of the above-mentioned genetically engineered bacterium, Ganoderma lucidum genetically engineered bacterium, and genetically engineered bacterium GL-chintin-19 in the production of mycelial materials.

[0034] An embodiment of the present invention also provides a method for producing mycelial fiber skin.

[0035] The following illustrates the solution of the present invention through specific examples.

[0036] Ganoderma lucidum G10 used in the following examples is a commercial strain purchased from Wuhan Zhou Yulin Edible Fungi Research Institute.

[0037] The medium components used in the following examples are as follows: PDB medium (potato dextrose liquid medium): purchased from Tianjin Guangfu Technology Development Co., Ltd.; CYM medium: maltose 10 g / L, glucose 20 g / L, tryptone 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen phosphate 4.6 g / L, yeast powder 2 g / L, low melting point agarose 15 g / L, vitamin B1 0.125 mg / L, mannitol 109.56 g / L; Solid state fermentation medium: wood chips 77.84%wt, wheat bran 20%wt, sucrose 1%wt, gypsum 1%wt, potassium dihydrogen phosphate 0.1%wt, magnesium sulfate 0.05%wt, VB 0.01%wt.

[0038] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods in the art, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all obtained from commercial sources unless otherwise specified.

[0039] Example 1 This example provides a method for constructing a Ganoderma engineering bacterium with a high chitin content.

[0040] 1. Plasmid construction 1.1 PCR amplification Use the primers CHS-F / R (shown in SEQ ID NO.5 and SEQ ID NO.6), GFAT-F / R (shown in SEQ ID NO.7 and SEQ ID NO.8), UAP-F / R (shown in SEQ ID NO.9 and SEQ ID NO.10) in Table 1 to perform PCR amplification on the endogenous CHS-5, GFAT, and UAP1 gene fragments of Ganoderma lucidum G10, and use overlap PCR to connect these three gene fragments to form the CHS-linker-GFAT-linker-UAP gene fragment. Use the primers 1303-GLP-F / R (shown in SEQ ID NO.11 and SEQ ID NO.12) to perform PCR amplification on the plasmid backbone 1303-GLP-iEGFP (purchased from Miaoling Biology, originally named pCAMBIA1303-GpdA-3×FLAG-EGFP-TrpC-Hygro).

[0041] Table 1 Primers required for this experiment and their sequences

[0042] The reaction system and amplification conditions for the above PCR amplification are shown in Tables 2 and 3.

[0043] Table 2 Reaction system for PCR amplification

[0044] Table 3 Reaction conditions for PCR amplification

[0045] 1.2 Multi-fragment DNA homologous recombination Use the ClonExpress Ultra One Step Cloning Kit (product of Nanjing Novoprotein Co., Ltd.) to ligate the amplification product of plasmid backbone 1303-GLP-iEGFP with the CHS-linker-GFAT-linker-UAP gene fragment to form the recombinant plasmid 1303-GLP-chitin. The specific operation steps are as follows: (1) Prepare the multi-fragment DNA homologous recombination reaction system as shown in Table 4 in a microcentrifuge tube, with a total volume of 10 μL.

[0046] Table 4 Multi-fragment DNA homologous recombination reaction system

[0047] (2) Mix by flicking the bottom of the centrifuge tube with your finger.

[0048] (3) Incubate at 50 °C for 15 min and then immediately cool on ice.

[0049] The gene expression pattern of the recombinant plasmid 1303-GLP-chitin is as Figure 1 shown.

[0050] 2. Transformation and screening 2.1 Preparation of protoplasts Prepare a 2% (w / v) lysing enzyme solution and filter sterilize it through a 0.22 μm microporous membrane. Inoculate Ganoderma lucidum G10 on PDB medium and incubate statically at 25 °C for 7 d. Pick the mycelium and wash it 3 times with 0.6 M mannitol solution (osmotic stabilizer), centrifuge at 6000 r / min for 10 min, and discard the supernatant. Add 2% (w / v) lysing enzyme solution at a ratio of 0.2 g of wet mycelium to 1 mL of enzyme solution, shake well to disperse the cells, and enzymatically digest at a constant temperature of 31 °C in a water bath for 3.5 h. During the enzymatic digestion process, gently shake every 0.5 h. After the enzymatic digestion is completed, remove the mycelial fragments with a 0.45 µm microporous membrane, centrifuge the filtrate at 4000 r / min for 10 min, discard the supernatant, and obtain the protoplast precipitate. Resuspend the protoplast precipitate in 0.6 M mannitol solution, centrifuge at 4000 r / min for 10 min, and repeat 3 times to completely remove the enzyme solution. Add the protoplasts to an appropriate amount of 0.6 M mannitol solution to prepare a protoplast suspension.

[0051] Set 3 replicates according to the above experimental procedure, count with a hemocytometer, and calculate the number of protoplasts per ml of suspension.

[0052] 2.2 Protoplast transformation Mix the 1303-GLP-chitin recombinant plasmid with the protoplasts of the monokaryotic Ganoderma resinaceum strain (10 7Perform PEG-mediated transformation: Centrifuge the protoplast suspension at 1258×g, 4 °C for 5 min. Discard the supernatant, add STC buffer (218.6 g / L sorbitol, 0.01 M Tris-HCL, 11.1 g / L CaCl2), and gently mix to 100 μL to obtain the STC suspension of protoplasts. Mix 30 μg of the 1303-GLP-chitin recombinant plasmid, 100 μg of heparin sodium, 0.25 mM spermidine with water to 100 μL, add it to the STC suspension of protoplasts, mix quickly, and place on ice for 10 min. Add 200 μL of PTC (75% PEG4000, 1 mM Tris-HCl (pH = 7.5), 2.5 mM CaCl2), gently rotate the centrifuge tube to mix, place on ice for 10 min, then add another 200 μL of PTC, gently rotate the centrifuge tube to mix, place on ice for 10 min, then add 800 μL of PTC, flick to mix, and incubate at 30 °C for 30 min. Dilute the transformation system with 1 mL of STC buffer, and finally add it to 20 mL of CYM medium (containing carboxin with a final mass concentration of 4 mg / L), mix evenly and pour into plates. After the plates solidify, place them in an incubator at 26 °C for 10 - 15 d, and visible colonies will grow. Cultivate until the 20th d ( Figure 2 ), transfer the resistant colonies to fresh CYM medium (containing carboxin with a final mass concentration of 4 mg / L) for secondary screening, and continuously subculture for 5 generations to detect their stability. A total of 35 stable mutants were obtained. Randomly select 9 strains from the screened strains and select the CHS gene for the determination of the relative expression level by qPCR. As shown in Table 5, since Ganoderma lucidum randomly integrates foreign DNA through PEG transformation, the expression levels of the CHS-5 gene in different strains are different.

[0053] Table 5 Relative expression of CHS-5 gene in resistant colonies

[0054] Select strains G10-chitin-15 and G10-chitin-19 with relatively high expression levels of the CHS-5 gene from the positive transformants for subsequent experiments.

[0055] 3. Detection of chitin content in mutants The mononuclear strains G10, G10-chitin-15 and the control strain G10-chitin-19 with different relative expression levels were cultured in a CYM liquid shake flask. After 6 days of culture, the mycelia were collected, washed thoroughly, and then vacuum freeze-dried. Chitin powder of different masses was weighed to plot a standard curve, and 0.01 g of the freeze-dried mycelia of G10, G10-chitin-15 and G10-chitin-19 were weighed to determine the chitin content: 400 μL of 4 M HCl was added to 0.01 g of ground mycelia and heated at 96 °C for 16 h. Then, 400 μL of autoclaved water was added to cool the reaction. 9 mg of ground activated carbon was added to each sample, and the samples were vortexed three times within 1 hour. After that, the samples were centrifuged, and 300 μL of the supernatant was diluted 10-fold with 2.63 mL of autoclaved distilled water and 70 μL of 8 M aqueous NaOH solution to neutralize the samples. Using a D-glucosamine assay kit (Megazyme, Ireland), the relative percentage of chitin after extraction was determined according to the protocol in the instruction manual.

[0056] After obtaining the standard curve of D-glucosamine content versus chitin mass (as Figure 3 shown), the chitin contents of G10, G10-chitin-15 and G10-chitin-19 as shown in Table 6 were calculated. It can be seen that the chitin contents of the mutant strains obtained by the gene editing method of the present invention were increased by 110% and 548% respectively compared with the starting strain. With the increase of the integration copy number and relative expression level, the chitin content of the strains also increased.

[0057] Table 6 Determination of chitin content in mutant strains

[0058] The strain G10-chitin-19 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on May 21, 2025, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 42012 and named GL-chintin-19.

[0059] Example 2 This example provides the application of the mononuclear strain G10 and G10-chitin-15 and G10-chitin-19 constructed in Example 1 in the production of mycelial materials.

[0060] The liquid spawns of mononuclear strains G10, G10-chitin-15, and G10-chitin-19 (5 g of mycelia obtained by culturing mycelia in shake flasks under the same conditions and filtering) were subjected to solid-state fermentation in a solid-state fermentation medium to obtain mycelial fiber skins. The specific fermentation method is as follows: 1. Add water to the solid-state fermentation medium to adjust the moisture content to 65% wt to obtain the fermentation substrate; 2. Divide the above substrate into 2400 ml culture boxes according to a loading coefficient of 50%, and cover the culture box lids; 3. Stack the culture boxes evenly in an autoclave and sterilize at 121 °C for 90 min; 4. After sterilization, cool the culture boxes to room temperature in a clean room; 5. In a laminar flow hood, open the box lid, evenly spray 70 mL of liquid spawn (containing 5 g of mycelia) on the surface of the substrate, and cover the box lid; 6. Place the culture room in a constant temperature chamber and incubate at a constant temperature of 28 - 30 °C and an indoor RH of 60% - 70% for 15 d; 7. After the culture cycle ends, open the box lid and separate and remove the mycelial skin above the substrate.

[0061] Cut out mycelial slices of 2.5 cm × 10 cm in size, fix them on a tensile testing machine to detect the strength of the mycelial slices (as Figure 4 shown). The results are shown in Table 7. Compared with the starting strain G10, the strength of the mycelial materials obtained from G10-chitin-15 and G10-chitin-19 by the same method increased by 78% and 149%, respectively.

[0062] Table 7 Strength test of mycelial fiber skins of mutant strains

[0063] It can be seen that G10-chitin-15 and G10-chitin-19 can obtain high-strength mycelial materials under conventional fermentation conditions without exogenous addition of chitin precursors, and have the potential for industrial application in the production of high-strength mycelial materials.

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. An engineered bacterium with a high chitin content, characterized in that, The engineered bacterium is constructed by genetic modification using filamentous fungi as the starting strain; the genetic modification includes: overexpressing chitin synthase, glutamine-fructose-6-phosphate aminotransferase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase.

2. The engineered bacterium with high chitin content according to claim 1, characterized in that, The filamentous fungi include Ganoderma lucidum, Pleurotus ostreatus, Cordyceps militaris, Coriolus versicolor, Flammulina velutipes; and / or The chitin synthase is chitin synthase CHS-5 or CHS-8; and / or The uridine diphosphate-N-acetylglucosamine pyrophosphorylase is uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1; and / or The method for overexpressing the chitin synthase, glutamine-fructose-6-phosphate aminotransferase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase is: increasing the copy number of the encoding genes of each enzyme.

3. A method for improving the strength of mycelium material, characterized in that, Increase the chitin, β-glucan in the mycelial cell wall, and the proteins that can bind to cell wall components.

4. The method according to claim 3, characterized in that Overexpress chitin synthase, glutamine-fructose-6-phosphate aminotransferase, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase.

5. A Ganoderma engineering bacterium with a high chitin content, characterized in that, The Ganoderma lucidum engineered bacterium is constructed by genetic modification using Ganoderma lucidum as the starting strain; the genetic modification includes: overexpressing chitin synthase CHS-5 or CHS-8, glutamine-fructose-6-phosphate aminotransferase GFAT, and uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1. The amino acid sequence of chitin synthase CHS-5 is shown in GenBank: AZQ26802.1, the amino acid sequence of chitin synthase CHS-8 is shown in GenBank: AZQ26805.1, the amino acid sequence of glutamine-fructose-6-phosphate aminotransferase GFAT is shown in SEQ ID NO.1, and the amino acid sequence of uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1 is shown in SEQ ID NO.

2.

6. The Ganoderma engineering bacteria with high chitin content according to claim 5, characterized in that, The nucleotide sequence of the encoding gene of chitin synthase CHS-5 is shown in GenBank: MH463055.1, the nucleotide sequence of the encoding gene of chitin synthase CHS-8 is shown in GenBank: MH463058.1, the nucleotide sequence of the encoding gene of glutamine-fructose-6-phosphate aminotransferase GFAT is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene of uridine diphosphate-N-acetylglucosamine pyrophosphorylase UAP1 is shown in SEQ ID NO.

4.

7. An engineered bacterium GL-chintin-19 with a high chitin content, characterized in that, Its classification name is Ganoderma lucidum ( Ganoderma lingzhi ), which was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on May 21, 2025. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Deposit number: CGMCC No. 42012.

8. Use of the engineered bacterium according to claim 1 or 2, the Ganoderma lucidum engineered bacterium according to claim 5 or 6, and the engineered bacterium GL-chintin-19 according to claim 7 in the production of mycelial materials.

9. A method for producing a mycelium fiber skin, characterized in that, Obtain mycelial fiber skin by solid-state fermentation using the engineered bacterium according to claim 1 or 2, the Ganoderma lucidum engineered bacterium according to claim 5 or 6, and the engineered bacterium GL-chintin-19 according to claim 7.

10. The method according to claim 9, characterized in that, The mycelial fiber skin is obtained by solid-state fermentation with the engineered bacterium GL-chintin-19 described in claim 7.

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