An engineering bacterium for suppressing robustness and a construction method and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a fruiting inhibition engineering bacterium and a construction method and application thereof, comprising overexpressing a transcription factor Pcc1 in a ganoderma strain, constructing an engineering bacterium MKLGE253241 with high expression of Pcc1, a preservation unit: China General Microbiological Culture Collection Center, address: No. 1, Xibei Road, No. 3, Chaoyang District, Beijing, preservation date: March 11, 2026, preservation number: CGMCC No. 42592. The present application blocks the formation of fruiting body primordium from the molecular level, maintains the mycelium in vegetative growth, and increases the average biomass by 15.2%, without fruiting body; meanwhile, the leather flatness of the mycelium produced by the engineering bacterium is significantly improved, and the scale qualified rate is obviously improved. The present application fundamentally solves the problem of fruiting body leather, avoids chemical inhibitors, and provides a new path for high-performance bio-based leather.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an engineered bacterium that inhibits fruit setting, its construction method, and its application. Background Technology

[0002] With increasing environmental awareness and escalating scarcity of petrochemical resources, the development of sustainable alternatives to traditional animal leather and petroleum-based synthetic leather has become a research hotspot. Animal leather production has a long cycle and consumes a large amount of resources; petroleum-based synthetic leather relies on fossil raw materials, consumes a lot of energy, and has poor breathability. Against this backdrop, mycelial leather, prepared by solid-state or liquid-state fermentation using fungal mycelium, is considered the most promising next-generation bio-based material due to its lightweight, breathability, feel that mimics animal leather, and low environmental impact.

[0003] Among numerous fungi, Ganoderma lingzhi stands out due to its superior mycelial softness, tensile strength, and toughness, resulting in a dense and uniform mycelial skin structure, making it an ideal raw material for producing high-quality leather-like products. However, its industrialization is limited by the fruiting problem: after the mycelium completes its vegetative growth, it is induced by environmental signals to shift to reproductive growth, forming fruiting bodies. This not only consumes nutrients and reduces biomass but also damages the integrity of the mycelial skin, leading to a decrease in the yield of qualified products.

[0004] Chinese Patent Application Publication No. CN115610036A, published on January 17, 2023, discloses a Ganoderma lucidum-based leather and its preparation method. This invention does not use animal hides as the raw material for leather, but rather utilizes Ganoderma lucidum mycelium as the raw material. A water-soluble culture medium is used to allow the Ganoderma lucidum mycelium to reproduce asexually, facilitating the subsequent acquisition of Ganoderma lucidum composite biomaterials. This effectively shortens the mycelium cultivation cycle, improves production efficiency, and is easier to industrialize. The invention adds a composite adjuvant when the Ganoderma lucidum mycelium develops a growth advantage, which facilitates cross-linking, encapsulation, and adsorption between the mycelium and the added adjuvant, promoting the orderly growth of the Ganoderma lucidum mycelium and forming Ganoderma lucidum composite biomaterials, which is beneficial for further preparation of Ganoderma lucidum-based leather. However, the following problems still exist:

[0005] 1. Failure to fundamentally solve the biological bottleneck of fruiting: This patent promotes orderly mycelial growth and the formation of composite materials by adding compound adjuvants, but it still relies on the inherent growth characteristics of Ganoderma lucidum. During mycelial cultivation, if environmental signals induce fruiting body formation, adding adjuvants cannot block this developmental process. Once fruiting occurs, the mycelial network structure will still be damaged, leading to hardening or pores in the material, thus affecting the yield of the finished product. This invention only optimizes material forming at the physicochemical level and fails to solve the fundamental problem of mycelial transition from vegetative to reproductive growth at the molecular level.

[0006] 2. Additives may affect the natural properties of materials and subsequent processing: While introducing exogenous chemical additives can help mycelial cross-linking and shaping, these additives may remain in the final product, affecting the "pure bio-based" properties of mycelial leather. Furthermore, the type and timing of additive addition need precise control; improper addition may interfere with normal mycelial metabolism or even inhibit mycelial growth. In addition, residual additives may cause unpredictable chemical reactions in subsequent tanning, dyeing, and other processing steps, increasing process complexity and the difficulty of quality control. Summary of the Invention

[0007] Therefore, the present invention provides an engineered bacterium that inhibits fruiting, its construction method, and its application, in order to overcome the problem of low mycelial biomass caused by fruiting body formation in the prior art.

[0008] To achieve the above objectives, the present invention provides engineered bacteria for fruit set inhibition. The engineered bacteria are constructed by genetic modification of Ganoderma lucidum strain. The genetic modification includes overexpression of transcription factor Pcc1, and the amino acid sequence of transcription factor Pcc1 is shown in SEQ ID NO:1.

[0009] Furthermore, the nucleotide sequence of the transcription factor Pcc1 is shown in SEQ ID NO:2.

[0010] The present invention also provides a method for constructing engineered bacteria that inhibits fruit set, comprising: cloning the transcription factor Pcc1 into the pUC57-EXP vector to construct the overexpression plasmid pUC57-EXP-pcc1; preparing protoplasts using the starting strain; transforming the overexpression plasmid pUC57-EXP-pcc1 into the protoplasts; and regenerating, initially screening, and rescreening the transformed protoplasts to obtain the engineered bacteria that overexpress the transcription factor Pcc1.

[0011] Furthermore, the overexpression plasmid pUC57-EXP-pcc1 was transformed into the protoplasts using a PEG chemical-mediated transformation method.

[0012] Furthermore, the initial screening is for the antibiotic oxychloride.

[0013] Furthermore, the concentration of the oxycortisone antibiotic is 2 mg / L.

[0014] Furthermore, the secondary screening is qRT-PCR screening.

[0015] The present invention also provides the application of engineered bacteria with strong inhibition in the production of mycelial leather.

[0016] Furthermore, the mycelial leather is obtained through solid-state fermentation of the engineered bacteria.

[0017] Furthermore, the specific method for producing mycelial leather includes the following steps:

[0018] The engineered bacteria were activated to obtain activated bacterial cells;

[0019] The bacterial cells were inoculated into a liquid culture medium and fermented on a shaker for 7 days. The fermentation broth was then centrifuged to remove the supernatant, and mycelium was obtained.

[0020] The mycelium was inoculated into a solid fermentation medium and cultured at a constant temperature for 15 days. The mycelial skin was then separated and removed to obtain the mycelial leather.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. By overexpressing the transcription factor Pcc1, the formation of fruiting bodies is inhibited at the molecular level, allowing the mycelium to maintain long-term vegetative growth, avoiding biomass loss caused by fruiting, and significantly increasing the mycelium yield.

[0023] 2. Pcc1 overexpression synergistically improves hyphal branching patterns and cell wall structure, promotes the formation of a dense and uniform three-dimensional network, avoids hardening and pore defects, and endows mycelial leather with better smoothness and pass rate. Attached Figure Description

[0024] Figure 1 This is an electrophoresis diagram of the amplified gene pcc1 in an embodiment of the present invention, where M represents the DL5000 DNA marker, P represents the amplification product of gene pcc1, and D represents the negative control.

[0025] Figure 2 This is a plasmid map of the overexpression plasmid pUC57-EXP-pcc1 in an embodiment of the present invention;

[0026] Figure 3 The relative expression levels of the pcc1 gene in the OE-pcc1-1, OE-pcc1-4, and OE-pcc1-6 engineered strains of this invention are shown in the embodiments of the present invention.

[0027] Figure 4 The images show the culture results of wild-type strain WT and engineered strain MKLGE253241 in embodiments of the present invention. Detailed Implementation

[0028] This invention discloses an engineered bacterium for inhibiting fruit set, its construction method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0029] The culture medium components used in the following examples are:

[0030] PDA medium: 40g of potato dextrose agar powder, diluted to 1L with deionized water;

[0031] Seed culture medium: 5g tryptone, 0.5g magnesium sulfate heptahydrate, 2.5g yeast powder, 1g potassium dihydrogen phosphate, 35g glucose, 0.05g vitamin B1, diluted to 1L with deionized water, pH=5.5;

[0032] CYM medium: 10g maltose, 20g glucose, 2g tryptone, 0.5g magnesium sulfate heptahydrate, 4.6g potassium dihydrogen phosphate, 2g yeast extract, 15g low melting point agarose, and diluted to 1L with deionized water;

[0033] CYM regeneration medium: 10g maltose, 20g glucose, 2g tryptone, 0.5g magnesium sulfate heptahydrate, 4.6g potassium dihydrogen phosphate, 2g yeast extract, 15g low-melting-point agarose, 109.56g mannitol, and diluted to 1L with deionized water.

[0034] Solid-state fermentation medium: sawdust 77.84%wt, wheat bran 20%wt, sucrose 1%wt, gypsum 1%wt, potassium dihydrogen phosphate 0.1%wt, magnesium sulfate 0.05%wt, vitamin B 0.01%wt.

[0035] The reagent components used in the following examples are:

[0036] Citrate buffer: Prepare 0.1M citric acid solution and 0.1M sodium citrate solution. Add sodium citrate solution to citric acid solution at a volume ratio of 4:1 and adjust pH to 5.5.

[0037] STC buffer: 218.6g sorbitol, 10mL 1M Tris-HCl (pH=7.5), 11.1g calcium chloride, and bring the volume to 1L with deionized water;

[0038] Lesion enzyme solution: 1g of lysion enzyme from Guangdong Institute of Microbiology, diluted to 50mL with deionized water;

[0039] PTC buffer: PEG4000 15g, 1M Tris-HCl (pH=7.5) 1mL, 1M calcium chloride 2.5mL, bring to a final volume of 20mL with deionized water, filter to sterilize, and prepare fresh before use;

[0040] Example 1

[0041] This embodiment provides a method for constructing engineered bacteria that inhibits fruit set.

[0042] 1. Homology comparison of pcc1 gene

[0043] In this embodiment, the Ganoderma lucidum strain was isolated from fruiting bodies collected in a broad-leaved forest in Nanning, Guangxi Province in June 2025. The coding region sequence and corresponding amino acid sequence of the *Coprinus sarcodactylis* pcc1 gene (GenBank accession number: AB007760) were obtained from the NCBI database. Using the *Coprinus sarcodactylis* pcc1 amino acid sequence as a query probe, a homology search was performed on the *Ganoderma lucidum* genome database using the BLASTp program on the NCBI website, and candidate homologous sequences were initially screened. Multiple sequence alignment of the *Coprinus sarcodactylis* pcc1 amino acid sequence with the candidate homologous sequences of *Ganoderma lucidum* was performed using BioEdit software to determine the coding region of the homologous gene. The analysis results showed that, as shown in SEQ ID NO:1, it is the amino acid sequence encoded by this *Ganoderma lucidum* gene, with a similarity of up to 70.14% to the *Coprinus sarcodactylis* pcc1 protein, proving that the two have a high degree of homology.

[0044] 2. Construction of overexpression plasmid pUC57-EXP-pcc1

[0045] 2.1 pcc1 gene amplification and enzyme digestion

[0046] The PCR genome was extracted from Ganoderma lucidum using the CTAB method. Using the extracted genome as a template, the pcc1 gene fragment was amplified using primers pcc1-F and pcc1-R (Table 1). The PCR amplification reaction system is shown in Table 2. The PCR amplification program was: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 2 min, and 72℃ final extension for 10 min, for a total of 30 cycles. The obtained PCR amplification products were detected by 1.5% agarose gel electrophoresis. Figure 1 As shown in SEQ ID NO:2, the full-length gene is 1941 bp. The target fragment was recovered using an OMEGA gel recovery kit.

[0047] The pcc1 target fragment was digested with restriction endonucleases NheⅠ-HF and SmaⅠ. The reaction system is shown in Table 3. The digestion reaction was incubated at 37℃ for 15 min. 3 μL of the digestion product was subjected to 1.5% agarose gel electrophoresis. After verifying the correctness of the bands, the pcc1 target fragment was recovered using an OMEGA gel recovery kit.

[0048] Table 1 Primers and their sequences required for this experiment

[0049] Table 2. PCR amplification reaction system

[0050] Table 3 Enzyme digestion reaction system

[0051] 2.2 Linearization of the pUC57-EXP vector

[0052] The pUC57-EXP vector was linearized using restriction endonucleases NheⅠ-HF and SmaⅠ. The reaction system is shown in Table 3. The enzyme digestion reaction was incubated at 37℃ for 15 min. 3 μL of the digestion product was subjected to 1.5% agarose gel electrophoresis. After verifying the correctness of the bands, the linear vector pUC57-EXP was recovered using an OMEGA gel recovery kit. Its full length was 6.8 kb.

[0053] 2.3 Connection Conversion

[0054] The digested pcc1 target fragment was ligated into the linear vector pUC57-EXP using T4 DNA Ligase. The ligation system is shown in Table 4. The ligation product was transformed into E. coli DH-5α competent cells, and single colonies were picked for PCR verification and sequencing re-verification. The overexpression plasmid pUC57-EXP-pcc1 was finally obtained, with a full length of 8.7 kb. Figure 2 As shown.

[0055] Table 4 Connection System

[0056] 3. Conversion and Screening

[0057] 3.1 Protoplast Preparation

[0058] The Ganoderma lucidum strain was inoculated into test tubes containing PDA slant medium and cultured at 25°C for 7 days. 50 mL of liquid seed culture medium was added to the test tubes, and mycelia were scraped from the slant using a spatula. The seed culture medium containing mycelia was then transferred to a 250 mL sterile Erlenmeyer flask containing 2-3 layers of glass beads and cultured at 25°C for 5 days to obtain mycelial culture solution. During this period, the Erlenmeyer flask was shaken several times to break up the mycelia. 50 mL of the cultured mycelial culture solution was poured into 200 mL of fresh seed culture solution, and then transferred to a 500 mL Erlenmeyer flask containing 2-3 layers of glass beads. The mixture was further cultured for 2 days to obtain seed solution, during which the Erlenmeyer flask was shaken several times to break up the mycelia.

[0059] Pour the cultured seed culture into 50 mL centrifuge tubes and centrifuge at 8000 g for 10 min. Discard the supernatant and wash the mycelial precipitate with 30 mL of citrate buffer. Centrifuge at 8500 g for 8 min and discard the supernatant. Add 10 mL of citrate buffer to the mycelium and mix by pipetting. Aliquot 1 mL into 2.0 mL centrifuge tubes and aspirate any aggregated impurities and mycelium. Centrifuge at 12000 g for 5 min, discard the supernatant, and weigh the cell count. Add 2% (w / v) lysozyme solution at a ratio of 0.04 g of lysozyme per 300 mg wet weight cells. Incubate at 100 g and 30 °C on a shaker for 2.5 h. Then centrifuge at 1700 g and 4 °C for 5 min and discard the supernatant. Add 1 mL of STC buffer to the protoplast precipitate and mix by pipetting. Centrifuge at 1700 g and 4 °C for 5 min and discard the supernatant. Repeat this process several times to completely remove the enzyme solution. The protoplasts were resuspended in 1 mL of STC buffer and counted using a hemocytometer to prepare a final concentration of 10. 7 Protoplast suspension of 100 μL / mL was aliquoted into 2.0 mL centrifuge tubes.

[0060] 3.2 Determination of the screening concentration of carboxin

[0061] Resistance gradient experiments were conducted using protoplast regeneration. Fresh Ganoderma lucidum protoplasts were uniformly spread on CYM regeneration medium containing 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mg / L of oxychloride. The medium was incubated statically at 28°C for 20 days, and protoplast regeneration and colony formation were observed. The results showed that when the oxychloride concentration was below 2 mg / L, a large number of regenerated colonies appeared on the regeneration medium; however, when the concentration reached 2.5 mg / L or higher, protoplast regeneration was completely inhibited, and no colony formation was observed. Therefore, a final concentration of 2 mg / L of oxychloride was determined to be the optimal screening concentration for Ganoderma lucidum genetic transformation.

[0062] 3.3 Protoplast transformation and screening

[0063] The overexpression plasmid pUC57-EXP-pcc1 was transformed into Ganoderma lucidum protoplasts using PEG-mediated transformation: 5 μL of heparin sodium, 5 μL of spermidine, and 30 ng of the overexpression plasmid pUC57-EXP-pcc1 were added to 100 μL of protoplasts, mixed by inversion, and incubated on ice for 10 min; 200 μL of fresh PTC buffer was added, and the mixture was incubated on ice for 10 min, followed by another 200 μL of fresh PTC buffer, and then incubated on ice for 10 min. Finally, 800 μL of fresh PTC buffer was added, and the mixture was incubated at 30°C for 30 min; the resulting sample was then added to 15 mL of CYM regeneration medium, poured into agar plates, cooled, and capped; after incubation at 25°C for 2 days, 15 mL of a solution containing 2 mg / L carboxin was added to the agar plates. In CYM medium, the bacteria were incubated at 30℃ for about 9-12 days. 67 single colony transformants that grew on the surface of the upper medium were picked and transferred to new CYM medium containing 2 mg / L carboxin for subculture. After 5 generations of screening, 13 stable candidate strains with high expression of the pcc1 gene were obtained.

[0064] A suitable amount of mycelium was scraped from each candidate high-expression pcc1 gene strain and placed in 50 μL of 1×TE Buffer. The mycelium was heated in a microwave oven on medium heat for 2 min to release the mycelial genome, which served as a template for PCR verification. The transformants were verified by PCR using sequencing primers F and R, and three high-expression pcc1 gene strains were obtained, namely OE-pcc1-1, OE-pcc1-4, and OE-pcc1-6.

[0065] RNA was extracted from wild-type strains and engineered strains OE-pcc1-1, OE-pcc1-4, and OE-pcc1-6 using the TGuide Smart Universal Total RNA Extraction Kit. cDNA was then prepared using the Prime Script™ RT reagent Kit (Takara). The reaction system and procedure were as follows: 1 μg Total RNA, 2 μL 5×gDNA Eraser Buffer, 1 μL gDNA Eraser, 2 μL Exnase II, and RNase-Free ddH2O were added to a nuclease-free PCR tube to a final volume of 10 μL. The tube was incubated at 42°C for 2 min. Then, 4 μL 5×Prime Script Buffer II, 4 μL RNase-Free ddH2O, 1 μL RT Prime Mix × 4, and 1 μL Prime Script RT Enzyme Mix I were added to each tube. The reaction was carried out at 37°C for 15 min, followed by incubation at 85°C for 5 s to obtain cDNA. The cDNA was then stored at -20°C for later use.

[0066] Using the 18S gene as an internal reference gene, primers pcc1-F2, pcc1-R2, 18S-F, and 18S-R for the target gene pcc1 and the 18S gene were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0067] The specific reaction system and operation process of qRT-PCR are as follows: Add 10 μL TB GreenPremix Ex Taq, 1 μL upstream primer, 1 μL downstream primer, 2 μL cDNA, and 6 μL ddH2O to each nuclease-free PCR tube; after vortexing the PCR tube, place it in a real-time PCR instrument, and the reaction program is as follows: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, annealing temperature of 57℃ for 30s, extension at 72℃ for 30s, and the number of cycles is 40; the present invention uses the Pfaffl method for data processing, and the calculation formula is as follows: relative expression level of the target gene = (1+E target gene)∆CT(control-experiment) / (1+E internal reference gene)∆CT(control-experiment).

[0068] When the cDNA is the target gene pcc1, the upstream primer is pcc1-F2 and the downstream primer is pcc1-R2; when the cDNA is the 18S gene, the upstream primer is 18S-F and the downstream primer is 18S-R.

[0069] The pcc1 gene was highly expressed in three engineered strains: OE-pcc1-1, OE-pcc1-4, and OE-pcc1-6. Among them, OE-pcc1-1 showed the highest pcc1 expression level. Figure 3 As shown.

[0070] Example 2

[0071] This embodiment provides the application of wild-type strains and the engineered strain OE-pcc1-1 constructed in Example 1 in the production of mycelial leather.

[0072] Mycelial sheets were obtained by solid-state fermentation of wild-type strains WT and OE-pcc1-1 (named MKLGE253241) in a solid-state fermentation medium. The liquid culture was prepared by culturing mycelium in shake flasks under the same conditions, followed by filtration to obtain 5g of mycelium from each strain. The specific solid-state fermentation method was as follows:

[0073] 1. Add water to the solid-state fermentation medium to adjust the moisture content to 65% wt, and use it as the fermentation substrate;

[0074] 2. Dispense the above base material into 2400mL culture boxes according to a 50% filling coefficient, and cover the culture box with the lid;

[0075] 3. Place the culture boxes evenly into the sterilizer and sterilize at 121℃ for 90 minutes;

[0076] 4. After sterilization, cool the incubator to room temperature in a clean room;

[0077] 5. Inside the clean bench, open the lid and evenly spray 70mL of liquid inoculum (containing 5g of mycelium) onto the substrate surface, then close the lid.

[0078] 6. Place the incubation room in a constant temperature room and incubate for 15 days at 28-30℃ and RH 60%-70%;

[0079] 7. After the cultivation cycle is completed, open the lid and separate and remove the mycelium skin on top of the substrate.

[0080] Observe whether primordia or fruiting bodies are produced; the culture results are shown in the following section. Figure 4 As shown, the mycelial biomass and the number of primordia / fruiting bodies formed were recorded, and the pass rate of a single batch was calculated.

[0081] As shown in Table 5, primordium formation in the engineered strain MKLGE253241 was completely blocked throughout the entire culture cycle, and no fruiting body primordia or differentiation structures were observed. In contrast, the wild-type strain WT showed obvious primordia and even small fruiting bodies under the same culture conditions. Because the sexual development of the engineered strain MKLGE253241 was effectively inhibited, its vegetative growth was more uniform, with mycelia spreading evenly on the culture medium surface. The resulting mycelial sheets were smooth, dense, and of uniform thickness, without local bulges or developmental nodules. Further verification of its production stability was conducted on a pilot-scale basis. The mycelial sheets prepared using the engineered strain MKLGE253241 were significantly superior to those prepared using the wild-type strain WT in terms of uniformity. The yield rate of mycelial sheets produced on a large scale using the engineered strain MKLGE253241 increased to 83%, a 144% improvement compared to the wild-type strain WT. The engineered bacterium MKLGE253241, which highly expresses pcc1, avoids sheet structure defects caused by local differentiation, significantly reduces the defect rate, and improves production efficiency and resource utilization.

[0082] Table 5. Average biomass, fruiting body number, and mycelial sheet test results of strain WT and engineered strain MKLGE253241

[0083] In summary, this invention successfully constructed an engineered strain that inhibits fruiting by overexpressing the transcription factor Pcc1 in Ganoderma lucidum using genetic engineering techniques. This engineered strain precisely blocks fruiting body formation at the molecular level, allowing mycelium to maintain long-term vegetative growth, significantly increasing mycelial biomass, and substantially improving the smoothness and yield of mycelial leather. This invention fundamentally solves the fruiting problem in the industrialization of mycelial leather, avoiding the use of chemical inhibitors, and provides a new technical path and application prospect for the large-scale production of high-performance bio-based leather.

[0084] The relevant sequences used in this invention are as follows:

[0085] SEQ ID NO:1 (Amino acid sequence of Pcc1)

[0086] MPKAPPHREADDDSKPDKIPRPPNAWIIYRGDRLTEWKASRSPNDPPVKQADISRLIARRWKEENDVIKLEYEKRAAIAKAEHKKRFPDYKYNPMSKEAKEKMRAEEKEAKKRAREEAKAAKMAPPRPSYVHPPQPSYPSPPSGSNVKLENMESPPAESPAAETESVTLPRMAVEPIERGCGPSPPIDYDPQSPSSSSISSLSASSSSSSSASARSSPLPSSSQLAPSPYDASPAWHPHIPSPLSSSPTQAPPPAYPSTFAHSPVPSRQSSYNLHPSSSSTPSESPPSYSDGENWQPGPPDSAFHPFTPLPDFQSNWNTPSQDPLLLPQNVPDVNFSLPPFDPLLTLPQDDQFNADALSAGAIFDLSAQGPEMGSANSPPTELELDVNAFNQHFATIGQQPAFHPLADQLAPMLDMPELHETMEDRAAEAGLPTPGVAQGSTLEDFAGFVNAHPQDAISLADPDFYATLGRQILAQFPQLAASLIPQAAPPQYMQDPNNVFSPVDFAPMQPQPQLQPEDQAFDASLYQMFMQQQPQLAMPTPMRPIPVTDISAVSTPVQREPSYTYPNAPLAQPAPAPVAAAPMPATESAAPAPPRDSE*

[0087] SEQ ID NO:2 (Nucleotide sequence of Pcc1)

[0088]

[0089] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. An engineered bacterium that inhibits fruit setting, characterized in that, The engineered bacteria were constructed by genetic modification of Ganoderma lingzhi as the starting strain. The genetic modification includes overexpression of transcription factor Pcc1, the amino acid sequence of which is shown in SEQ ID NO:1; the engineered bacterium is named MKLGE253241. Depository Institution: China General Microbiological Culture Collection Center; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit Date: March 11, 2026; Accession number: CGMCC No.42592.

2. The engineered bacteria for inhibiting fruit setting according to claim 1, characterized in that, The nucleotide sequence of the transcription factor Pcc1 is shown in SEQ ID NO:

2.

3. The method for constructing engineered bacteria for fruit set inhibition according to claim 2, characterized in that, include: The transcription factor Pcc1 was cloned into the pUC57-EXP vector to construct the overexpression plasmid pUC57-EXP-pcc1. Protoplasts were prepared using the starting strain; The overexpression plasmid pUC57-EXP-pcc1 was transformed into the protoplasts; The transformed protoplasts were regenerated, initially screened, and then re-screened to obtain the engineered bacteria that overexpressed the transcription factor Pcc1.

4. The method for constructing engineered bacteria for fruit set inhibition according to claim 3, characterized in that, The overexpression plasmid pUC57-EXP-pcc1 was transformed into the protoplasts using a PEG chemical-mediated transformation method.

5. The method for constructing engineered bacteria for fruit set inhibition according to claim 4, characterized in that, The initial screening was for the antibiotic oxychloride.

6. The method for constructing engineered bacteria for fruit set inhibition according to claim 5, characterized in that, The concentration of the chlorothalonil antibiotic is 2 mg / L.

7. The method for constructing engineered bacteria for fruit set inhibition according to claim 6, characterized in that, The secondary screening was performed using qRT-PCR.

8. The application of the engineered bacteria for fruit set inhibition according to any one of claims 1-2, characterized in that, The engineered bacteria are used to produce mycelial leather.

9. The application of the engineered bacteria for inhibiting fruit set according to claim 8 in the production of mycelial leather, characterized in that, The mycelial leather was obtained through solid-state fermentation of the engineered bacteria.

10. The application of the engineered bacteria for inhibiting fruit set according to claim 9 in the production of mycelial leather, characterized in that, The specific method for producing mycelium leather includes the following steps: The engineered bacteria were activated to obtain activated bacterial cells; The bacterial cells were inoculated into a liquid culture medium and fermented on a shaker for 7 days. The fermentation broth was then centrifuged to remove the supernatant, and mycelium was obtained. The mycelium was inoculated into a solid fermentation medium and cultured at a constant temperature for 15 days. The mycelial skin was then separated and removed to obtain the mycelial leather.

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