An engineered bacterium with high substrate utilization efficiency and a construction method and application thereof

Abstract

This invention relates to the field of genetic engineering technology, and more particularly to an engineered bacterium with high substrate utilization efficiency, its construction method, and its applications. The method includes overexpressing laccase and lignin peroxidase in two monokaryotic Ganoderma lucidum strains, respectively, and then hybridizing the two monokaryotic strains to form a new Ganoderma lucidum. Ganoderma lingzhi MKLGE253252, depositary 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. 42594. It synergistically enhances lignin degradation capacity, mycelial utilization efficiency, and growth capacity, and significantly improves the strength of mycelial fiber sheath. This invention fundamentally solves the problems of low lignin utilization efficiency, slow mycelial growth, and insufficient mycelial sheath strength in existing technologies, providing a completely new technical path for the production of bio-based leather.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an engineered bacterium with high substrate utilization efficiency, its construction method, and its application. Background Technology

[0002] Ganoderma lucidum Ganoderma lingzhi Ganoderma lucidum is a rare medicinal fungus whose mycelia intertwine and form a dense mycelial skin under solid culture conditions. The biomass accumulation rate and mechanical strength of the mycelial skin directly determine the yield and quality of Ganoderma lucidum in industrial applications such as mycelial substrate, biofuel, active substance extraction, and medicinal raw materials. Lignin is the main carbon source component in Ganoderma lucidum solid culture media (such as sawdust, straw, corn cobs, and other agricultural and forestry waste). Its complex structure consists of highly cross-linked phenylpropane units through COC ether bonds and CCC carbon-carbon bonds, forming a three-dimensional network macromolecule that is difficult for most microorganisms to depolymerize. Ganoderma lucidum mycelia must secrete a set of lignin-degrading enzymes extracellularly, mainly including laccase, lignin peroxidase, and manganese peroxidase, to gradually depolymerize lignin into small aromatic compounds and further metabolize them into absorbable carbon sources, thereby supporting rapid mycelial growth, mycelial bundle extension, and the formation of a dense mycelial skin.

[0003] However, the ability of wild-type and conventionally cultivated Ganoderma lucidum strains to secrete lignin-degrading enzymes is generally low. This low endogenous secretion level leads to low lignin degradation efficiency, and a large amount of lignin in the culture medium cannot be effectively utilized. The mycelium is in a state of carbon source starvation for a long time, which is directly manifested as: slow mycelial growth on solid culture medium, loose mycelial layer, insufficient thickness and poor mechanical strength, which cannot meet the requirements of industrial fermentation production or mycelial material preparation.

[0004] Currently, there are no reports of efficiently overexpressing both laccase and lignin peroxidase genes and then fusing two highly expressed mononuclear strains through hybridization breeding to synergistically enhance lignin degradation capacity. Therefore, there is an urgent need for a method to construct engineered bacteria that can systematically improve the secretion level of lignin-degrading enzyme systems in Ganoderma lucidum while simultaneously improving mycelial growth rate and mycelial skin mechanical strength, in order to overcome the technical bottlenecks of low lignin utilization efficiency, slow mycelial growth, and insufficient mycelial skin strength in existing technologies. Summary of the Invention

[0005] Therefore, this invention provides an engineered microorganism with high substrate utilization efficiency, its construction method, and its application, in order to overcome the problems of low lignin utilization efficiency, slow mycelial growth, and insufficient mycelial strength caused by the limited secretion of lignin-degrading enzymes in the prior art.

[0006] To achieve the above objectives, the present invention provides engineered bacteria with high substrate utilization efficiency, wherein the engineered bacteria are derived from Ganoderma lucidum. Ganoderma lingzhi The strain was obtained through genetic modification and hybridization breeding. The gene was modified to overexpress laccase (LacC) and lignin peroxidase (LiP). The amino acid sequence of laccase (LacC) is shown in SEQ ID NO:1; the amino acid sequence of lignin peroxidase (LiP) is shown in SEQ ID NO:2; the engineered strain was named Ganoderma lucidum. Ganoderma lingzhi MKLGE253252; Depository Institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Deposit Date: March 11, 2026; Deposit Number: CGMCC No. 42594.

[0007] Furthermore, the nucleotide sequence of the laccase LacC is shown in SEQ ID NO:3, and the nucleotide sequence of the lignin peroxidase LiP is shown in SEQ ID NO:4.

[0008] This invention provides a method for constructing engineered bacteria with high substrate utilization efficiency, the steps of which include: The laccase LacC and the lignin peroxidase LiP were cloned into the pUC57-EXP vector to construct the overexpression plasmids pUC57-EXP-lacc and pUC57-EXP-lip, respectively. The starting strain was monokaryized to obtain monokaryotic strains RE-2K.12 and RE-4.61; Protoplasts were prepared using the monokaryotic strain RE-2K.12 and the monokaryotic strain RE-4.61, respectively. The overexpression plasmid pUC57-EXP-lacc was transformed into the protoplasts of the monokaryotic strain RE-2K.12, and the overexpression plasmid pUC57-EXP-lip was transformed into the protoplasts of the monokaryotic strain RE-4.61. The protoplasts of the transformed monokaryotic strain RE-2K.12 and the protoplasts of the monokaryotic strain RE-4.61 were regenerated, initially screened, and then screened again to obtain the lacc monokaryotic strain overexpressing laccase and the lip monokaryotic strain overexpressing lignin peroxidase, respectively. The LACC mononuclear strain and the lip mononuclear strain were hybridized to obtain the engineered strain with high substrate utilization efficiency.

[0009] Furthermore, the method for uninucleation is as follows: the mycelium is washed with citrate buffer, enzymatically digested with lysozyme, the enzyme solution is removed and then spread on regeneration medium, single colonies without clamp connections are picked, and strains RE-2K.12 and RE-4.61 are obtained after hybridization.

[0010] Furthermore, the conversion was carried out using a PEG chemical-mediated method.

[0011] Furthermore, the initial screening is for the antibiotic oxychloride.

[0012] Furthermore, the method of hybridization breeding is as follows: [The text abruptly ends here, so the translation stops.] lacc Mononuclear strains and the lip Mononuclear strains were co-inoculated on the same solid culture medium plate and co-cultured until the hyphae came into contact. Then, hyphae from the hybridization area were picked for isolation and purification.

[0013] Furthermore, the engineered bacteria are applied to the production of mycelial fiber skin.

[0014] Furthermore, the mycelial fiber skin is obtained through solid-state fermentation of the engineered bacteria.

[0015] Furthermore, the specific method for producing mycelial fiber sheath 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 to obtain the mycelium fiber skin.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly increases mycelial biomass: The engineered Ganoderma lucidum strain constructed in this invention... Ganoderma lingzhi MKLGE253252 significantly improved lignin utilization efficiency through synergistic overexpression of laccase and lignin peroxidase, resulting in vigorous mycelial vegetative growth. Experimental results showed that the average biomass of the engineered strain was 32.3% higher than that of the wild-type strain, providing a sufficient raw material basis for the large-scale production of mycelial fiber sheaths.

[0017] 2. Significantly improves the mechanical strength of mycelial fiber leather: The mycelial fiber leather formed by engineered fungi is denser and thicker. Testing showed that the strength of the raw mycelial leather was 105.8% higher than that of the wild type, and the strength of the finished leather was 96.5% higher, significantly improving the strength of mycelial leather. Attached Figure Description

[0018] Figure 1 Expanding upon embodiments of the present invention lacc Genes (Figure a) and lip Electrophoresis diagram of the gene (Figure b), where M in Figure a represents the DL5000 DNA marker and P represents... lacc The gene amplification product, water represents the negative control, in figure b M represents the DL2000 DNA marker, and Q represents... lip The amplified product of the gene; water represents the negative control. Figure 2 The overexpression plasmid pUC57-EXP- is an example of this invention. lacc (Figure a) and the overexpression plasmid pUC57-EXP- lip (Figure b) shows the plasmid map; Figure 3 Candidate for embodiments of the present invention lacc Single-core transformants (Figure a) and candidates lip Electrophoresis diagram of PCR verification of single-nuclear transformants (Figure b), where M represents DL5000 DNA marker, 1-8 represent candidate transformants, and WT represents wild-type strain; Figure 4 The mononuclear strains RE-2K.12 and RE-4.61 of this invention were overexpressed in the embodiments of the present invention. lacc and lip Bar chart showing the relative gene expression levels in gene-positive transformants; Figure 5 The engineered Ganoderma lucidum strain used in this embodiment of the invention Ganoderma lingzhi Electron micrograph of clamp connections of mycelium in MKLGE253252 under a 40×10x electron microscope; Figure 6 The wild-type strain WT and the engineered strain Ganoderma lucidum are examples of strains used in this invention. Ganoderma lingzhi Image showing the culture results of MKLGE253252.

[0019] The preservation information is as follows: Classification and naming: Ganoderma lucidum Ganoderma lingzhi ; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: March 11, 2026; Accession number: CGMCC No.42594. Detailed Implementation

[0020] This invention discloses an engineered bacterium with high substrate utilization efficiency, 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.

[0021] The culture medium components used in the following examples are: PDA medium: 40g of potato dextrose agar powder, diluted to 1L with deionized water; 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; 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; 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. 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 B1 0.01%wt.

[0022] The reagent components used in the following examples are: 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. 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; Lesion enzyme solution: 1g of lysion enzyme from Guangdong Institute of Microbiology, diluted to 50mL with deionized water; 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; Example 1

[0023] In this embodiment, the Ganoderma lucidum strain was isolated from Ganoderma lucidum fruiting bodies collected in a broad-leaved forest in Changsha, Hunan Province in July 2025.

[0024] The strain was identified by gene sequencing; the sequence of the internal transcribed spacers (ITS) located between the 3' end of the 18S rDNA and the 5' end of the 28S rDNA was determined; the internal transcribed spacers were determined using primers ITS1F and ITS4 from Table 1, and the internal transcribed spacer region sequence is shown in SEQ ID NO:5. The internal transcription spacer region sequence was compared with the GenBank nucleic acid sequence database for homology. The results showed that the ITS sequence of Ganoderma lucidum had a similarity of more than 99%, and it was identified as Ganoderma lucidum and named MKL25101.

[0025] Table 1 Primers and their sequences required for this experiment

[0026] Example 2

[0027] This embodiment provides an engineered bacteria with high substrate utilization efficiency and its construction method.

[0028] 1. Overexpression plasmid pUC57-EXP- lacc and overexpression plasmid pUC57-EXP- lip Build 1.1 lacc Genes and lip Gene amplification and enzyme digestion The Ganoderma lucidum genome was extracted using the CTAB method. Using the extracted genome as a template, the upstream primers listed in Table 1 were employed. lacc -F and downstream primers lacc -R pairs lacc Gene fragment amplification, upstream primer lip -F and downstream primers lip -R pairs lip Gene fragments were amplified. The PCR amplification reaction system is shown in Table 2. The PCR amplification program was as follows: 98℃ pre-denaturation for 5 min, 94℃ denaturation for 10 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, it is an amplification. lacc Genes and lip Electrophoresis diagram of genes, lacc The full-length gene is 2096 bp, as shown in SEQ ID NO:3. lip The full-length gene is 1322 bp, as shown in SEQ ID NO:4. The target fragment was recovered using the OMEGA gel recovery kit.

[0029] Using restriction endonucleases Nhe I-HF and Sma I pair lacc Target fragment and lip The target fragment was digested with enzymes, and 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 then subjected to 1.5% agarose gel electrophoresis. After verifying the correctness of the bands, the fragments were recovered using an OMEGA gel extraction kit. lacc Target fragment and lip Target segment.

[0030] Table 2. PCR amplification reaction system

[0031] Table 3 Enzyme digestion reaction system reagents Volume (μL) Target fragment 8 Ⅰ-HF 1 SⅠ 1 rCutSmart™ Buffer 10 Total volume 20 1.2 Linearization of the pUC57-EXP vector Using restriction endonucleases Nhe I-HF and S ma I. The pUC57-EXP vector was linearized. The reaction system is shown in Table 3. The enzyme digestion reaction was incubated at 37℃ for 15 min. 3 μL of the enzyme digestion product was taken for 1.5% agarose gel electrophoresis. After verifying that the bands were correct, the linear vector pUC57-EXP was recovered using the OMEGA gel recovery kit. Its full length was 6.8 kb.

[0032] 1.3 Connection Transformation Using T4 DNA Ligase to digest the enzyme lacc Target fragment and lip The target fragments were ligated into the linear vector pUC57-EXP, and the ligation system is shown in Table 4. The ligation products were transformed into *E. coli* DH-5α competent cells, and single colonies were selected for PCR verification and sequencing re-verification to finally obtain the overexpression plasmid pUC57-EXP-. lacc and overexpression plasmid pUC57-EXP- lip ,like Figure 2 As shown, it is an overexpression plasmid pUC57-EXP- lacc (Figure a) and the overexpression plasmid pUC57-EXP- lip The plasmid maps in (b) are 8.9 kb and 8.2 kb in length, respectively.

[0033] Table 4 Connection System reagents Volume (μL) T4 DNA Ligase 5 T4 DNA Ligase Reaction Buffer 10 Target fragment 3.5 Linear vector pUC57-EXP 1.5 Total volume 20 2. Conversion and Screening 2.1 Preparation of monokaryotic strains Wash the mycelial precipitate with an appropriate amount of citrate buffer, centrifuge at 8500g for 8 min, and discard the supernatant. Add a small amount of citrate buffer to the mycelium, aliquot into 2.0mL centrifuge tubes, aspirate a large amount of aggregated impurities and mycelium, centrifuge at 12000g for 5 min, and discard the supernatant. Add 2% (w / v) lysozyme solution to the mycelium at a ratio of 0.04g lysozyme per 300mg wet weight cells, and incubate at 100rpm and 30℃ on a shaker for 2.5h, followed by centrifugation at 1700g and 4℃ for 5 min, and discard the supernatant. Resuspend the mycelium in an appropriate amount of citrate buffer, centrifuge at 1700g and 4℃ for 5 min, and discard the supernatant to obtain the cell precipitate. Resuspend the cell precipitate in citrate buffer, count the cells using a hemocytometer, and prepare a final concentration of 10. 6 A bacterial suspension of 10 cells / mL was spread on CYM regeneration medium and cultured in the dark. After colonies grew, single colonies were picked and transferred to PDA plates. Microscopic examination revealed no clamping junctions in the hyphae, thus obtaining a monokaryotic strain. Ten monokaryotic strains were selected for hybridization experiments to determine the karyotype, resulting in monokaryotic strains RE-2K.12 and RE-4.61.

[0034] 2.2 Protoplast Preparation Monokaryotic strains RE-2K.12 and RE-4.61 were inoculated separately into test tubes containing PDA slant medium and incubated 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 incubated at 25°C for 5 days to obtain mycelial culture. During this period, the Erlenmeyer flask was shaken several times to break up the mycelia. 50 mL of the prepared mycelial culture was then poured into 200 mL of fresh seed culture medium and transferred to a 500 mL Erlenmeyer flask containing 2-3 layers of glass beads. The mixture was incubated for another 2 days to obtain seed culture, during which the Erlenmeyer flask was shaken several times to break up the mycelia.

[0035] 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 twice 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.

[0036] 2.3 Protoplast transformation and screening The overexpression plasmid pUC57-EXP- was expressed using a PEG chemical-mediated method. lacc Transformed into protoplasts of the mononuclear strain RE-2K.12, the overexpression plasmid pUC57-EXP- lip Transformation into protoplasts of mononuclear strain RE-4.61: Add 5 μL heparin sodium, 5 μL spermidine, and 30 ng overexpression plasmid to 100 μL of protoplasts, mix by inversion, and incubate on ice for 10 min; add 200 μL fresh PTC buffer, incubate on ice for 10 min, then add another 200 μL fresh PTC buffer, incubate on ice for 10 min, then add 800 μL fresh PTC buffer, and incubate at 30°C for 30 min; add the reacted sample to 15 mL of CYM regeneration medium, pour into a plate, cool, and seal; after incubation at 25°C for 2 days, pour 15 mL of CYM medium containing 2 mg / L carboxin into the plate, incubate at 30°C for approximately 9-12 days, pick 60 single colonies that grow on the surface of the upper medium, and subculture them onto new CYM medium containing 2 mg / L carboxin. After 5 generations of screening, 8 stable candidate strains were obtained. lacc Single-core transformants and 8 candidates lip Mononuclear transformant; Scrape each candidate lacc Single-core transformants and candidates lip A suitable amount of hyphae from mononuclear transformants were placed in 50 μL of 1×TEBuffer and heated in a microwave oven on medium heat for 2 min to release the hyphal genome, which served as a template for PCR verification. Sequencing primers F and R1 were used to sequence candidate transformants. lacc Mononuclear transformants were validated by PCR using sequencing primers F and R2. lip PCR verification of single-nuclear transformants, such as Figure 3 As shown, it is a candidate. lacc Single-core transformants and candidates lip Electrophoresis image of single-nuclear transformant PCR verification, yielding 7 positive results. lacc Single transformant and 8 positive lip The mononuclear transformants are named OE- lacc With OE- lip .

[0037] RNA was extracted from the wild-type strain and the above-mentioned positive transformants using the TGuide Smart Universal Total RNA Extraction Kit, followed by PrimeScript. TM The RT reagent kit (Takara) was used to prepare cDNA. The reaction system and operation 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 10 μL in a nuclease-free PCR tube. The mixture was incubated at 42℃ 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 mixture was reacted at 37℃ for 15 min. Finally, the mixture was incubated at 85℃ for 5 s to obtain cDNA. The cDNA was then stored at -20℃ for later use.

[0038] The 18S gene was used as an internal reference gene and synthesized by Beijing Qingke Biotechnology Co., Ltd. lacc Target gene 、 lip Validation primers for the target gene and the 18S gene lacc -F2、 lacc -R2、 lip -F2、 lip -R2, 18S-F and 18S-R.

[0039] The specific reaction system and operation procedure for qRT-PCR are as follows: Add 10 μL TB GreenPremix Ex Taq, 1 μL of the upstream verification primer, 1 μL of the downstream verification primer, 2 μL cDNA, and 6 μL ddH2O to each nuclease-free PCR tube; vortex the PCR tubes to mix thoroughly, then place them in a real-time PCR instrument. The reaction program is as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, annealing at 57℃ for 30 s, extension at 72℃ for 30 s, with a cycle number of 40. This invention uses the Pfaffl method for data processing, and the calculation formula is as follows: Relative expression level of the target gene = (1 + E...) / ( ... 目的基因 ∆CT （对照—实验） / (1+E 内参基因 ∆CT （对照—实验） .like Figure 4 As shown, it is overexpressed in mononuclear strains RE-2K.12 and RE-4.61, respectively. lacc and lip A bar chart showing the relative gene expression levels of gene-positive transformants. The mononuclear strains with the highest relative gene expression levels are OE- lacc -3 and OE- lip -6.

[0040] 3. Hybrid breeding The mononuclear strain OE- with the highest expression level mentioned above lacc -3 and OE- lip -6 Take 1cm each 3 Mycelial blocks were placed 1 cm apart on PDA medium and cultured until the two types of hyphae came into contact. The contacting hyphae were then transferred to 50 mL of liquid seed culture medium for expansion. Protoplasts were then prepared according to the method described in section 2.2 above. These protoplasts were inoculated into CYM medium containing 2 mg / L carboxin and cultured for 5 days. Single colonies of varying sizes were randomly selected and passaged three times onto PDA plates containing 2 mg / L carboxin to obtain stably expressed plasmids containing the overexpression plasmid pUC57-EXP-. lacc and overexpression plasmid pUC57-EXP- lip The binucleate strain OE- lacc & lip It was named Ganoderma lucidum. Ganoderma lingzhi MKLGE253252, such as Figure 5 As shown, it is an engineered fungus, Ganoderma lucidum. Ganoderma lingzhi Electron micrograph of MKLGE253252 mycelium clamp junctions under a 40×10x electron microscope. Microscopic observation shows that clamp junctions have been formed.

[0041] Example 3

[0042] This embodiment provides both wild-type strains and the engineered strain of Ganoderma lucidum constructed in Example 2. Ganoderma lingzhi Application of MKLGE253252 in the production of mycelial fiber skin.

[0043] Wild-type strain WT, Ganoderma lucidum Ganoderma lingzhi The liquid culture of MKLGE253252 (mycelium cultured in shake flasks under identical conditions, filtered to obtain 5g of mycelium) was fermented in a solid-state fermentation medium to obtain mycelial fibrous skin. The specific fermentation method is as follows: 1. Add water to the solid-state fermentation medium to adjust the moisture content to 65% wt, and use it as the fermentation substrate; 2. Dispense the above base material into 2400mL culture boxes according to a 50% filling coefficient, and cover the culture box with the lid; 3. Place the culture boxes evenly into the sterilizer and sterilize at 121℃ for 90 minutes; 4. After sterilization, cool the incubator to room temperature in a clean room; 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. 6. Place the incubation room in a constant temperature room and incubate for 15 days at 28-30℃ and RH 60%-70%; 7. After the cultivation cycle is completed, open the lid and separate and remove the mycelial fiber skin on top of the substrate.

[0044] Cut a 2.5cm × 10cm piece of mycelial fiber raw material and fix it on a tensile testing machine to test the strength of the mycelial sheet. Figure 6 As shown, it is a wild-type strain WT and an engineered strain of Ganoderma lucidum. Ganoderma lingzhi Image showing the culture results of MKLGE253252 In addition, the mycelial fiber skin was soaked in a 50% glycerol solution for 24 hours and dried on a stretcher for 24 hours to obtain the finished mycelial skin. A sample of 2.5cm×10cm was cut and fixed on a DL-5000 electronic universal testing machine to test the tensile strength at a speed of 100±20 mm / min.

[0045] The results are shown in Table 5. Compared with the wild-type strain WT, the strain Ganoderma lucidum... Ganoderma lingzhi The biomass of MKLGE253252 was increased, and the strength of both mycelial raw and finished skins obtained by the same method for both strains was improved. Among them, the strain *Ganoderma lucidum* showed improved biomass. Ganoderma lingzhi The original skin strength of MKLGE253252 strain was increased by 105.8% compared to the wild-type strain. (Strain Ganoderma lucidum) Ganoderma lingzhi The finished leather strength of MKLGE253252 strain was increased by 96.5% compared to the wild type strain.

[0046] Table 5 Wild-type strain WT and engineered Ganoderma lucidum strain Ganoderma lingzhi Mean biomass and mean intensity of MKLGE253241 strain Average biomass (g) Average strength of mycelial protocoel (MPa) Average strength of finished mycelial skin (MPa) WT 83.2 0.86 1.13 Lingzhi MKLGE253252 110.1 1.77 2.22 In summary, this invention successfully constructed an engineered strain of Ganoderma lucidum with high substrate utilization efficiency by simultaneously overexpressing laccase and lignin peroxidase in Ganoderma lucidum through a combination of genetic engineering and hybridization breeding. Ganoderma lingzhi MKLGE253252. Compared with the wild-type strain, this engineered strain has a significantly enhanced ability to degrade lignin. Under solid-state fermentation conditions, the mycelium grows rapidly, and the resulting mycelial fiber skin has higher biomass and better mechanical strength, showing broad prospects for industrial application. It provides an excellent strain resource for fields such as mycelial leather, biofuels, and pharmaceutical raw materials.

[0047] The relevant sequences used in this invention are as follows: SEQ ID NO:1 (Amino acid sequence of LacC) MAKFQSLLSYITLLFAASAYAGIGPTTDLTISNADISPDGFSRAAVVVNGVFPGPLITGNMGDRFQLNVVDQMTNHSMLKTTSIHWHGFFQKGTNWADGPAFVNQCPIASGNSFLYDFQVPDQSGTYWYH SHLSTQYCDGLRGPFVVYDPNDPHKSLYDVDDDSTVITLADWYHVAARLGPRFPLGSDSTLINGLGRSPATPTADLAVISVTKGKRYRFRLVSLSCDPNYVFSIDGHDLSVIEADGIETQPVTVNAIQIF AAQRYSFVLNANQTADNYWIRANPNFGNVGFTDGINSAILRYDTADPVEPVTSQQSTQNLLKETDLHPLVARATPGNPTQGGVDKAINMVFNFNGSSFFINDASFVPPTVPVLLQILSGAQAAQDLLPSG SVYELPINSSIELTFPATTNAPGAPHPFHLHGHAFAVVRSAGSTVYNYDNPVWRDVVSTGTPAAGDNVTIRFQTDNPGPWFLHCHIDFHLEAGFAVVFAEDSPDASAANPVPQAWSDLCPTYNALSPDDQ* SEQ ID NO:2 (Amino acid sequence of LiP) MAFKALLSIVTLVAALQVADAALTRRVACPDGKNTATNAACCQLFAIADDLQQNLFDNGGCGEDVHESLRLTFHDAIAFSPSMNARGQNGGGGADGSIALFESIETNFHASLGLDEIVNAQRPIVQRHNITTADFIMFAAAVGVANCPGAPQLDVFLGRADATQPSPDGLIPEPFDPPDMLLARMADAGFDPIETVWLLSSHTIAAADLVDPTIPGTPFDSTPELFDTQFFIETQLRGTLFPGTGGNQGEVESPLRGEIRLQSDHLLARDSRTACEWQSFVNNQPKIQGRFHDAFHDLSLLGHDINDLIDCSEVIQTPPPPASNAHFPAGLSNADVEQACADTPFPTLPTDPGPVTSVAPV* SEQ ID NO:3( lacc nucleotide sequence) SEQ ID NO:4 ( lip (nucleotide sequence) SEQ ID NO:5 (nucleotide sequence of the internal transcribed spacer region) CCGAGGCATGTGCACGCCCTGCTCATCCACTCTACACCTGTGCACTTACTGTGGGCTTCAGATTGCGATGCACGCTCTTTACCGGGCTTGCGGAGCATATCTGTGCCTGCGTTTATCACAAACTCTATATAGTAACAGAA TGTGTATTGCGATGTAACACATCTATATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCT TGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATGAAATCTTCAACCTACAAGCTTTTGTGGTTTGTAGGCTTGGACTTGGAGGCTTGTCGGCCGTTATCGGTCGGCTCCTCTTAAATGCATTAGCTTGGT TCCTTGCGGATCGGCTCTCGGTGTGATAACGTCTACGCCGCGACCGTGAAGCGTTTGGCGAGCTTCTAACCGTCTTATAAGACAGCTTTATGACCTCTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATC 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 with high substrate utilization efficiency, characterized in that, The engineered bacteria are Ganoderma lucidum. Ganoderma lingzhi The strain was obtained through genetic modification and hybridization breeding. The gene was modified to overexpress laccase LacC and lignin peroxidase LiP. The amino acid sequence of laccase LacC is shown in SEQ ID NO:1; the amino acid sequence of lignin peroxidase LiP is shown in SEQ ID NO:

2. The hybridization breeding involves overexpressing laccase (LacC). lacc Mononuclear strains and strains overexpressing lignin peroxidase LiP lip Hybridization of monokaryotic strains; The engineered bacteria was named Ganoderma lucidum. Ganoderma lingzhi MKLGE253252; Depository Institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Deposit Date: March 11, 2026; Deposit Number: CGMCC No. 42594.

2. The engineered bacteria with high substrate utilization efficiency according to claim 1, characterized in that, The nucleotide sequence of the laccase LacC is shown in SEQ ID NO:3, and the nucleotide sequence of the lignin peroxidase LiP is shown in SEQ ID NO:

4.

3. The method for constructing engineered bacteria with high substrate utilization efficiency according to claim 2, characterized in that, step include: The laccase LacC and the lignin peroxidase LiP were cloned into the pUC57-EXP vector, respectively, to construct the overexpression plasmid pUC57-EXP- lacc and overexpression plasmid pUC57-EXP- lip ; The starting strain was monokaryized to obtain monokaryotic strains RE-2K.12 and RE-4.61; Protoplasts were prepared using the monokaryotic strain RE-2K.12 and the monokaryotic strain RE-4.61, respectively. The overexpression plasmid pUC57-EXP- lacc Transformed into protoplasts of the mononuclear strain RE-2K.12, the overexpression plasmid pUC57-EXP- lip Transformed into protoplasts of the monokaryotic strain RE-4.61; The protoplasts of the transformed monokaryotic strain RE-2K.12 and the monokaryotic strain RE-4.61 were regenerated, initially screened, and then re-screened to obtain the protoplasts overexpressing laccase. lacc The mononuclear strain and the lignin peroxidase overexpression lip Mononuclear strains; The lacc Mononuclear strains and the lip The mononuclear strain was hybridized to obtain the engineered strain with high substrate utilization efficiency.

4. The method for constructing engineered bacteria with high substrate utilization efficiency according to claim 3, characterized in that, The method for uninucleation is as follows: the mycelium is washed with citrate buffer, enzymatically digested with lysozyme, the enzyme solution is removed and then spread on regeneration medium, single colonies without clamp connections are picked, and the uninucleate strains RE-2K.12 and RE-4.61 are obtained after hybridization.

5. The method for constructing engineered bacteria with high substrate utilization efficiency according to claim 4, characterized in that, The conversion was performed using a PEG chemical-mediated method.

6. The method for constructing engineered bacteria with high substrate utilization efficiency according to claim 5, characterized in that, The initial screening was for the antibiotic oxychloride.

7. The method for constructing engineered bacteria with high substrate utilization efficiency according to claim 6, characterized in that, The hybridization breeding method is as follows: [The method is described in the original text]. lacc Mononuclear strains and the lip Mononuclear strains were co-inoculated on the same solid culture medium plate and co-cultured until the hyphae came into contact. Then, hyphae from the hybridization area were picked for isolation and purification.

8. The application of the engineered bacteria with high substrate utilization efficiency according to any one of claims 1-2, characterized in that, The engineered bacteria are used to produce mycelial fiber skin.

9. The application of the engineered bacteria with high substrate utilization efficiency according to claim 8 in the production of mycelial fiber skin, characterized in that, The mycelial fiber skin is obtained through solid-state fermentation of the engineered bacteria.

10. The application of the engineered bacteria with high substrate utilization efficiency according to claim 9 in the production of mycelial fiber skin, characterized in that, The specific method for producing mycelial fiber sheath 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 to obtain the mycelium fiber skin.

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