Method and application for improving biomass of filamentous fungi and substrate utilization of filamentous fungi

Abstract

The invention relates to the technical field of microorganisms and food biology, and particularly discloses application of recombinant filamentous fungi in production of mycelium protein. The recombinant filamentous fungus provided by the invention is obtained by regulating and expressing alpha galactosidase and a mutant thereof, and has the advantages of improving the utilization efficiency of polysaccharide, improving the protein content of recombinant bacteria and accelerating the growth speed. The recombinant filamentous fungus can utilize agricultural and sideline products as a carbon source or a nitrogen source, and the fermentation mycelium protein is complete in amino acid variety, contains various essential amino acids required by human bodies, and has wide application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food biotechnology, and particularly relates to the function of alpha galactosidase gene ScMel1 in improving the biomass and substrate utilization rate of Fusarium venenatum and the application thereof in producing mycelial protein. BACKGROUND

[0002] Filamentous fungi are an important class of eukaryotic microorganisms, widely exist in nature, and play an important role in industry, agriculture and medicine. Filamentous fungi have high protein production capacity, can grow using simple and inexpensive medium, and have attracted attention in industrial fermentation. However, it is worth noting that filamentous fungi generally have low substrate utilization rate and slow growth, which makes the fermentation period of filamentous fungi longer, and the yield and production intensity of target products lower, which becomes the key to solving the problems of high fermentation cost and low utilization rate of production equipment.

[0003] Soybean molasses is a by-product obtained during the production of soybean protein concentrate, which is a collection of various plant components with a sugar content of more than 50%, containing a large amount of sugars (such as raffinose, stachyose, sucrose, etc.) that can be utilized by yeast. Because soybean molasses contains a large amount of ash, pectin, pigment and other impurities and anti-nutritional factors and is difficult to handle, it has not been well utilized, and most manufacturers have discarded it, causing environmental pollution and resource waste. Filamentous fungi can produce mycelial protein using soybean molasses, which not only improves the added value of soybean molasses, effectively protects the environment, but also brings higher economic benefits, extends the industrial chain, and promotes the development of circular economy. However, the ability of Fusarium to utilize raffinose and stachyose is low. In order to improve the utilization efficiency of soybean molasses by Fusarium and improve the conversion of raffinose and stachyose, a heterologous alpha galactosidase gene is expressed. Alpha galactosidase is an important enzyme for the hydrolysis of raffinose and stachyose to form monosaccharides, and increasing the copy number of alpha galactosidase gene can improve the hydrolysis efficiency of raffinose and stachyose in soybean molasses. SUMMARY

[0004] The present application provides a method for improving the substrate utilization and growth rate of filamentous fungi Fusarium venenatum. The present application can significantly improve the growth rate of the strain. In order to achieve this purpose, the present application adopts the following technical solutions.

[0005] In a first aspect, the present invention provides a recombinant filamentous fungus obtained by expressing ScMEL1 and its ScMEL1 mutant (A50S / D123E) or regulating its expression level using genetic engineering methods. Its growth rate and / or carbon source substrate utilization ability are enhanced. Preferably, the carbon source substrate is selected from monosaccharides, polysaccharides, polysaccharides, plant biomass, or combinations thereof. Preferably, the polysaccharide is selected from sucrose, raffinose, stachyose, or combinations thereof; the polysaccharide is selected from cellulose, crystalline cellulose, hemicellulose, starch, or combinations thereof; and the monosaccharide is selected from glucose, xylose, arabinose, or combinations thereof.

[0006] In a more specific embodiment, the ScMEL1 is derived from Saccharomyces cerevisiae and codon-optimized to encode a polypeptide having the amino acid sequence shown in SEQ ID NO.2 and an amino acid sequence with homology of 70%, preferably 80%, 90%, 95%, and 99%, preferably from a fungus, and more preferably from a filamentous fungus.

[0007] More preferably, the ScMEL1 is a polypeptide having the amino acid sequence shown in SEQ ID NO.2, and preferably its encoded nucleotide acid is as shown in SEQ ID NO.1.

[0008] In a preferred embodiment, the expression of ScMEL1 is regulated by a strong promoter derived from PgpdA of Fusarium venetum, a signal peptide derived from SamyA of Aspergillus niger, and a terminator derived from TtrpC of Fusarium venetum.

[0009] In a preferred embodiment, hygromycin is used as a screening marker, and HPH is a resistance gene.

[0010] Secondly, the present invention provides a ScMEL1 mutant, wherein the mutant nucleotide is changed from G to T at position 148 and from T to A at position 369.

[0011] In a preferred embodiment, the amino acid sequence of the ScMEL1 mutant is changed from A (Ala) to S (Ser) at position 50 and from D (Asp) to E (Glu) at position 123.

[0012] In a preferred embodiment, the amino acid sequence of the ScMEL1 mutant is changed from A to S at position 50, which enhances the hydrophilicity of the substrate binding pocket and improves substrate affinity.

[0013] In a preferred embodiment, the amino acid sequence of the ScMEL1 mutant is changed from D to E at position 123, which strengthens the acidic catalytic site of the active center and improves catalytic efficiency.

[0014] In one specific embodiment, the expression of the ScMEL1 mutant is regulated by a strong promoter derived from PgpdA (gpd box) of Fusarium vesicae, with three gpd box enhancer elements tandemly 240 upstream of the promoter.

[0015] Thirdly, this invention provides a method for producing mycelial proteins by fermentation using recombinant filamentous fungi. This method utilizes genetic engineering to overexpress ScMEL1 and its mutants, thereby obtaining filamentous fungi with enhanced growth rate and / or carbon source substrate utilization. Preferably, the carbon source substrate is selected from monosaccharides, polysaccharides, polysaccharides, or combinations thereof. Preferably, the polysaccharide is selected from sucrose, raffinose, stachyose, or combinations thereof; the polysaccharide is selected from oligosaccharides, starch, cellulose, or combinations thereof; and the monosaccharide is selected from glucose, xylose, arabinose, or combinations thereof.

[0016] In a preferred embodiment, the method is implemented through gene editing, where the nonhomologous end-joining (NHEJ) mechanism randomly integrates the overexpressed element into the genome, thereby obtaining a large number of copies.

[0017] In one specific embodiment, after transforming the expression cassettes of ScMEL1 and its mutants into protoplasts of Fusarium vesicans, they were randomly integrated into the genome via non-homologous end joining (NHEJ) to obtain different copy numbers, thereby obtaining recombinant filamentous fungi.

[0018] The present invention also provides the application of the recombinant filamentous fungal mutant in the preparation of mycelial proteins.

[0019] Preferably, the mutant strain is fermented to produce mycelial protein.

[0020] In one specific embodiment, glucose is used as the carbon source for fermentation culture. The glucose fermentation culture medium contains 60 g of glucose, 2.1 g of ammonium dihydrogen phosphate, 6.0 g of ammonium sulfate, 2.1 g of potassium sulfate, 0.87 g of magnesium sulfate, 3.64 mg of ferric chloride, 19.30 mg of zinc sulfate, 15.46 mg of manganese sulfate, and 1.85 mg of copper sulfate per 1000 mL, with the pH adjusted to 6.0.

[0021] In one specific embodiment, raffinose is used as the carbon source for fermentation culture. The glucose fermentation medium contains 60 g of glucose, 2.1 g of ammonium dihydrogen phosphate, 6.0 g of ammonium sulfate, 2.1 g of potassium sulfate, 0.87 g of magnesium sulfate, 3.64 mg of ferric chloride, 19.30 mg of zinc sulfate, 15.46 mg of manganese sulfate, and 1.85 mg of copper sulfate per 1000 mL, with the pH adjusted to 6.0.

[0022] In another embodiment, soybean molasses is used as a carbon source for fermentation. The soybean molasses fermentation medium contains, per 1000 mL: 60 g soybean molasses, 2.1 g ammonium dihydrogen phosphate, 6.0 g ammonium sulfate, 2.1 g potassium sulfate, 0.87 g magnesium sulfate, 3.64 mg ferric chloride, 19.30 mg zinc sulfate, 15.46 mg manganese sulfate, and 1.85 mg copper sulfate, with the pH adjusted to 6.0.

[0023] This invention obtains ScMEL1 mutant filamentous fungi by regulating the ScMEL1 gene and its mutant genes, significantly accelerating substrate utilization efficiency and biomass, enabling the production of mycelial proteins. Therefore, this invention is of great significance for improving the fermentation level of filamentous fungi and the production efficiency of target products. Attached Figure Description

[0024] Figure 1 ScMEL1 expression box.

[0025] Figure 2 The rate of raffinose consumption by Fusarium vesicola expressing ScMEL1 strain.

[0026] Figure 3 The α-galactosidase activity of Fusarium vesicola expressed ScMEL1 mutant strain was compared with that of Fusarium vesicola.

[0027] Figure 4 Biomass of Fusarium vesicola under different carbon source conditions for the expression of ScMEL1 and its mutant strains. Detailed Implementation

[0028] Unless otherwise specified, the methods used in the following examples are conventional methods, such as those described in *Molecular Cloning: A Laboratory Manual* by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available products that can be purchased through legitimate channels.

[0029] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0030] The method for preparing the culture medium in this embodiment of the invention is as follows:

[0031] Sporulation solid culture medium: Weigh 20 g glucose, 2.1 g ammonium dihydrogen phosphate, 6.0 g ammonium sulfate, 2.1 g potassium sulfate, and 0.87 g magnesium sulfate, make up to 1000 mL with water, adjust the pH to 5.5, and sterilize at 121℃ for 15 min.

[0032] Inorganic salt liquid culture medium: Weigh 20 g glucose, 2.1 g ammonium dihydrogen phosphate, 6.0 g ammonium sulfate, 2.1 g potassium sulfate, 0.87 g magnesium sulfate, 3.64 mg ferric chloride, 19.30 mg zinc sulfate, 15.46 mg manganese sulfate, and 1.85 mg copper sulfate. Make up the volume with water to 1000 mL, adjust the pH to 5.5, and sterilize at 121℃ for 15 min.

[0033] Regeneration medium: 1 g yeast extract, 1 g peptone, 274 g sucrose, 10 g agar, bring the volume to 1000 mL with water, adjust the pH to 5.5, and sterilize at 121 °C for 15 min.

[0034] Upper screening medium: 8 g yeast extract, 30 g glucose, 10 g agar, hygromycin. Add water to a final volume of 1000 mL, adjust pH to 5.5, sterilize at 121℃ for 15 min, cool to 60℃, add 12.5 μg / mL hygromycin, and then prepare plates.

[0035] Raffinose fermentation medium: Weigh 60 g raffinose, 2.1 g ammonium dihydrogen phosphate, 6.0 g ammonium sulfate, 2.1 g potassium sulfate, 0.87 g magnesium sulfate, 3.64 mg ferric chloride, 19.30 mg zinc sulfate, 15.46 mg manganese sulfate, and 1.85 mg copper sulfate. Make up the volume with water to 1000 mL, adjust the pH to 5.5, and sterilize at 121℃ for 15 min.

[0036] Soybean molasses fermentation medium: Weigh 60 g soybean molasses, 2.1 g ammonium dihydrogen phosphate, 6.0 g ammonium sulfate, 2.1 g potassium sulfate, 0.87 g magnesium sulfate, 3.64 mg ferric chloride, 19.30 mg zinc sulfate, 15.46 mg manganese sulfate, and 1.85 mg copper sulfate. Make up the volume with water to 1000 mL, adjust the pH to 5.5, and sterilize at 121℃ for 15 min.

[0037] Example 1: Overexpression of the ScMEL1 gene can increase the biomass of the strain.

[0038] 1.1 Construction of the expression box in ScMEL1 ( Figure 1 )

[0039] To improve the utilization efficiency of Fusarium soybean molasses and enhance the conversion of raffinose and stachyose, the α-galactosidase gene (ScMEL1) was heterologously expressed. α-galactosidase is an important enzyme in the hydrolysis of raffinose and stachyose to form monosaccharides, thus improving the hydrolysis efficiency of raffinose and stachyose in soybean molasses.

[0040] The expression cassette, PgpdA-ScMEL1-TtrpC-linkere-HYG-TtrpC, is a double-stranded DNA molecule, as shown in SEQ ID NO. 5. In SEQ ID NO. 5, positions 1-2129 are the promoter PgpdA (2129 bp) from *Fusarium vesicae*, positions 2130-3605 are the artificially synthesized optimized *Saccharomyces cerevisiae* α-galactosidase gene ScMEL1 (1476 bp), positions 3606-3712 are the artificially synthesized linker (116 bp), positions 3722-4091 are the artificially synthesized promoter Phpg (370 bp), positions 4092-5111 are the artificially synthesized selection marker hygromycin gene HYG (1020 bp), and positions 5112-5881 are the terminator TtrpC (770 bp) from *Fusarium vesicae*. Each DNA fragment was amplified using KOD One™ PCRMaster Mix (Toyobo, catalog number KMM201S), expression cassettes were constructed using overlap extension PCR technology, and sequenced at Qingke Biotechnology to obtain the correct expression cassette elements.

[0041] 1.2 Conversion of Expression Frame Components

[0042] Preparation of strain spore suspension:

[0043] In this embodiment, Fusarium venenatum TB04 (patent CN112226373A) was used as a wild strain.

[0044] Fusarium moniliforme TB04, preserved on a slant, was streaked onto a sporulation medium plate and incubated at 28°C for 79 days. Once the plate surface was covered with Fusarium moniliforme cells, spores were gently scraped off using an inoculation loop, washed three times with physiological saline, and the spore suspension was transferred to an Erlenmeyer flask. A small amount of glass beads was added, and the mixture was shaken for 10 min to ensure complete spore dispersion. The suspension was then filtered through absorbent cotton to obtain a Fusarium moniliforme spore suspension. The spore concentration was calculated using a hemocytometer, and the final concentration of the spore suspension was adjusted to 1 x 10⁻⁶. 6 per ml.

[0045] Preparation of protoplasts:

[0046] Fresh spore suspension was prepared at 1×10 6 Inoculate 50 mL of YEPD liquid medium into an Erlenmeyer flask, adding 10 glass beads to prevent the young mycelia from clumping together. Incubate at 28°C and 200 rpm. After approximately 16 h of incubation, centrifuge at 4°C and 12000 rpm for 30 min, discarding the supernatant. Wash twice with 10 mL of sterile 0.7 M NaCl. Add enzyme lysis buffer (20 mg of lysis enzyme and 40 mg of snailase dissolved in 10 mL of sterile 0.7 M NaCl) to the EP tube containing the young mycelia, and gently mix using a pipette. After lysis at 32°C and 150 rpm for 2.5 h, add 10 mL of pre-chilled sterile STC, and gently pipette up and down to release the protoplasts attached to the mycelia into the solution. Centrifuge at 4°C and 5000 rpm for 10 min. Discard the supernatant, wash twice with 10 mL STC, and resuspend the protoplasts in 100-600 μL STC (concentration of 1×10⁶) to obtain a protoplast suspension.

[0047] Transformation:

[0048] Take 100 μL of the above protoplast suspension, add 20 μL of SPTC, and gently mix with a pipette tip. Add more than 10 μg of the fragment, gently mix with a pipette tip, and incubate on ice for 50 min. Add 1 mL of SPTC (in 4 portions), gently mix with a pipette tip, and incubate at room temperature for 30 min. Add the mixture to regeneration medium at approximately 40°C, shake well, pour onto a plate, and incubate upright at 28°C overnight. Heat the selection medium, cool to approximately 60°C, and add hygromycin (final concentration: 12.5 μg / mL). Cool the selection medium to approximately 40°C, pour it directly onto the regeneration medium to cover, and incubate upside down at 28°C. Colonies will grow in approximately 3 days.

[0049] Colony PCR verification:

[0050] Single colonies grown on the plate were transferred to 6 cm selection medium for re-screening. 8 μL of lysis buffer was added, along with some transformant hyphae, and incubated at 98°C for 3 min. After lysis, 170 μL of neutralization buffer was added to obtain the template for PCR amplification. Primers were designed, and the target gene expression cassette was amplified using a 2×Es TaqMasterMix. Sequencing verification was performed by Qingke Biotechnology Co., Ltd., yielding transformant TB0401.

[0051] 1.3 Determination of sugar consumption rate in Fusarium vesicator transformants

[0052] Spore suspensions of wild-type fungus TB04 and transformant TB0401 were prepared according to the spore suspension preparation method, and the spore count was adjusted to 1 x 10⁻⁶. 6 Species / ml were inoculated with 2 ml of spore suspension into 100 ml of raffinose fermentation medium (250 ml Erlenmeyer flask) and incubated at 30℃ and 200 rpm. Samples were collected at 24 h, 36 h, 48 h, 60 h, and 72 h to determine the residual sugar content. (See attached image) Figure 2 As shown, the transformant TB0401 completely consumed raffinose in 60 h, while the wild-type strain TB04 still had raffinose residue after 72 h.

[0053] Example 2: Overexpression of the ScMEL1 mutant gene increases the biomass of Fusarium venetum.

[0054] 2.1 Construction of recombinant Fusarium vesicanthii

[0055] Strain construction and screening: The ScMEL1 gene was analyzed using the NCBI Conserved Domain Database (CDD) (https: / / www.ncbi.nlm.nih.gov / cdd / ). The ScMEL1 mutant gene was obtained by changing G at position 148 to T, T at position 369 to A, A (Ala) at position 50 (Ser), and D (Asp) at position 123 (Glu). The expression of the ScMEL1 mutant gene was regulated using three gpd box elements (nucleotide sequences shown in SEQ ID NO. 4) tandemly within the PgpdA (gpdbox) promoter (nucleotide sequence shown in SEQ ID NO. 4). Following the TB0401 construction method, the elements of the ScMEL1 mutant gene were incorporated into TB04, and the correct transformant TB04113 was screened.

[0056] 2.2 Galactosidase activity analysis

[0057] Fusarium moniliforme TB04, TB0401, and TB04113 were inoculated from plates into 15 mL test tubes with 3 mL of inorganic salt solution and cultured at 30°C for 48 h. 2 mL of the inoculum was then transferred to a 100 mL Erlenmeyer flask with 30 mL of inorganic salt solution and cultured at 30°C for 48 h. 5 mL of the bacterial culture was transferred to a 50 mL sterile centrifuge tube and centrifuged at 12000 g for 10 min, discarding the supernatant. The supernatant was then discarded after mixing with 20 mL of sterile water and centrifuged at 12000 g for 10 min. 5 mL of PBS buffer and an equal volume of acidic glass beads were added. The mixture was vortexed at 3000 rpm for 30 s, then incubated on ice for 30 s, and vortexed for 10 min. Finally, the mixture was centrifuged at 10000 g for 10 min to prepare the crude enzyme solution.

[0058] The protein concentration in the crude enzyme solution was determined using a BCA protein concentration assay kit (Solarbio, catalog number PC002050T), and the α-galactosidase activity was determined using an α-galactosidase activity assay kit (Beijing Box Biotechnology Co., Ltd., catalog number AKSU041C70 tubes / 25 samples). Figure 3 It can be seen that the specific enzyme activity of α-galactosidase in mutant TB040113 is 4.62 U / mg, the specific enzyme activity of α-galactosidase in transformant TB0401 is 1.62 U / mg, and the specific enzyme activity of α-galactosidase in TB04 is 0.34 U / mg. The specific enzyme activities of transformants TB040113 and TB0401 are higher than those of the original strain.

[0059] 2.3 Biomass determination of *Fusarium vesicae* mutant

[0060] Spore suspensions of wild-type fungus TB04, transformant TB0401, and mutant TB040113 were prepared according to the spore suspension preparation method, and the spore count was adjusted to 1 x 10^6 spores. 6 Cells / ml were inoculated with 2 ml of spore suspension into 100 ml of raffinose fermentation medium (250 ml Erlenmeyer flask), and cultured at 30°C and 200 rpm for 48 h. After fermentation, the cells were filtered through a Buchner funnel, collected, and dried at 105°C for 3 h until constant weight was achieved. Figure 4 As shown, the biomass of wild-type TB04, transformant TB0401, and mutant TB040113 were 3.4 g / L, 4.2 g / L, and 6.7 g / L, respectively. In summary, regulating overexpression of the ScMEL1 mutant significantly increases the mutant's biomass.

[0061] Example 3: Regulating the expression of the ScMEL1 mutant gene to promote carbon source utilization in mycelial protein production

[0062] Spore suspensions of wild-type fungus TB04 and mutant fungus TB040113 were prepared according to the spore suspension preparation method, and the spore count was adjusted to 1 x 10^6 spores. 6 Cells / ml were inoculated with 2 ml of spore suspension into 100 ml of fermentation medium (250 ml Erlenmeyer flask). Glucose, xylose, sucrose, raffinose, galactose, stachyose, starch, and cellulose were used as carbon sources at a concentration of 20 g / L. The culture was carried out at 30℃ and 200 rpm for 48 h. After fermentation, the cells were filtered through a Buchner funnel, collected, and dried at 105℃ for 3 h until constant weight. Figure 4 As shown, under the conditions of glucose, xylose, sucrose, raffinose, galactose, stachyose, starch, and cellulose as carbon sources, the biomass of the TB040113 strain with regulated expression of the ScMEL1 gene mutation was higher than that of the wild strain. This indicates that the regulated expression of the ScMEL1 gene mutant in Fusarium venetum can significantly improve the growth of the mutant under different substrate conditions (glucose, xylose, sucrose, raffinose, galactose, stachyose, starch, and cellulose).

[0063] Example 4: Regulating the expression of the ScMEL1 gene mutant to improve the utilization of soybean molasses

[0064] Fusarium moniliformes TB04, TB0401, and TB040113 were inoculated from plates into 30 mL of inorganic salt solution and cultured in 100 mL Erlenmeyer flasks at 30°C for 48 h. 5 mL of the inoculum was then inoculated into 200 mL of inorganic salt solution and cultured in 500 mL Erlenmeyer flasks at 30°C for 24 h. 200 mL of the seed culture was then inoculated into a 5 L fermenter containing 2.8 L of sucrose-molasses fermentation medium. The pH was adjusted to 5.5 with ammonia, the fermentation speed was 300 rpm, the oxygen flow rate was 1 ppm, and the fermentation time was 72 h. After fermentation, the fermentation broth was filtered through a Buchner funnel, washed with 15 L of distilled water, and dried at 105°C for 3 h until constant weight. TB04 yielded 33.69±0.2 g of mycelial dry weight (11.23±0.4 g / L), TB0401 yielded 39.6±0.1 g of mycelial dry weight (13.2±0.5 g / L), and TB040113 yielded 45.96±0.6 g of mycelial dry weight (15.3±0.2 g / L). This demonstrates that regulating the expression of the ScMEL1 gene mutant can improve the utilization efficiency of various sugars in soybean molasses and increase biomass.

Claims

1. The application of a recombinant filamentous fungus in the production of mycelial protein, characterized in that: The recombinant Fusarium vesicatoria is obtained by overexpressing the α-galactosidase gene using genetic engineering methods, and preferably the recombinant filamentous fungus is Fusarium vesicatoria.

2. The application according to claim 1, characterized in that, The recombinant bacteria were obtained by regulating the expression of the gene for α-galactosidase ScMEL1 or its mutant. The overexpression of the gene was regulated by a strong promoter, specifically PgpdA from Fusarium venetum. The overexpression elements also included a signal peptide and a terminator, wherein the signal peptide was SamyA from Aspergillus niger and the terminator was TtrpC from Fusarium venetum.

3. The application according to claim 2, characterized in that, The ScMEL1 gene is derived from Saccharomyces cerevisiae and has undergone codon optimization. Specifically, it encodes a polypeptide having the amino acid sequence shown in SEQ ID NO.2 and an amino acid sequence with homology of 70%, preferably 80%, 90%, 95%, and 99%, preferably from a fungus, and more preferably from a filamentous fungus; the ScMEL1 gene encodes a protein with the amino acid sequence shown in SEQ ID NO.

2.

4. The application according to claim 2, characterized in that, The mutant gene has its nucleotide sequence changing from A (Ala) at position 50 to S (Ser) and D (Asp) at position 123 to E (Glu) compared to the original sequence.

5. The application according to claim 2, characterized in that, The ScMEL1 mutant is characterized in that, relative to the original sequence, the nucleotide sequence of the mutant has the following changes: at position 148 (original G becomes T, position 369 T becomes A).

6. The application according to claim 1, characterized in that, The carbon source substrates for producing mycelial proteins are selected from monosaccharides, polysaccharides, polysaccharides, plant biomass, or combinations thereof.

7. The application according to claim 6, characterized in that, The polysaccharide is selected from sucrose, raffinose, stachyose, or combinations thereof; the polysaccharide is selected from cellulose, crystalline cellulose, hemicellulose, starch, or combinations thereof; and the monosaccharide is selected from glucose, xylose, arabinose, or combinations thereof.

8. The application according to any one of claims 1-7, characterized in that, It ferments the recombinant bacteria to produce mycelial protein.

9. A mutant α-galactosidase ScMEL1, characterized in that, Compared to the original sequence shown in SEQ ID No: 2, its amino acid sequence has the following changes: at position 50, A (Ala) is changed to S (Ser), and at position 123, D (Asp) is changed to E (Glu).

10. A gene encoding the α-galactosidase ScMEL1 mutation as described in claim 9, preferably wherein the nucleotide sequence is altered such that G at position 148 is changed to T and T at position 369 is changed to A, relative to the original sequence shown in SEQ ID No: 1.

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