Fusarium venenatum engineered strain with aspartic protease gene knocked out, construction method and application thereof

Abstract

The application discloses an engineered Fusarium venenatum strain with aspartic protease gene knocked out, a construction method and application thereof, and belongs to the technical field of microbial genetic engineering and fermentation engineering. The technical problem to be solved by the application is to improve the biomass and glucose conversion rate of Fusarium venenatum. To solve the problem, the application obtains a mutant strain of ΔFVRRES_10343 or a mutant strain of ΔFVRRES_12522 by knocking out aspartic protease genes FVRRES_10343 or FVRRES_12522 in Fusarium venenatum. The application further provides a method for fermenting and producing mycelial protein by using the engineered strain. The engineered strain can significantly improve the biomass and glucose conversion rate when used for producing mycelial protein.

Description

Technical Field

[0001] This invention relates to the fields of microbial genetic engineering and fermentation engineering. More specifically, this invention relates to an engineered strain of *Fusarium vesicae* with the aspartic protease gene knocked out, its construction method, and its applications. Background Technology

[0002] Venetian Fusarium ( Fusarium venenatum Fusarium venetum is an industrial filamentous fungus that has been widely studied and applied. The mycelial protein produced by its fermentation has a meat-like texture, is low in fat, contains a complete range of amino acids, and is rich in trace elements and dietary fiber, making it a high-quality meat substitute. Currently, the production of mycelial protein from Fusarium venetum fermentation has obtained food ingredient marketing authorization in many countries around the world, demonstrating huge market potential (see Chinese Patent CN116640753B, Pyruvate Decarboxylase Gene FvPDC6 and its Application in Improving Fusarium venetum Mycelial Protein Yield, Authorization Announcement Date: October 13, 2023).

[0003] In the genetic engineering of *Fusarium vesicatoria*, CRISPR-Cas9 technology has become a powerful tool for achieving precise genome editing. For example, Chinese patent CN114410635B, "Endogenous U6 Promoter of *Fusarium vesicatoria* and its CRISPR / Cas9-based Gene Editing Method" (authorization announcement date June 14, 2022), has successfully extracted and identified endogenous U6 promoters (such as U6_374, whose nucleotide sequence is shown in SEQ ID NO:2) from *Fusarium vesicatoria*, and established an efficient CRISPR-Cas9 gene editing system based on this, achieving targeted knockout of target genes (such as the GFP reporter gene) with an editing efficiency of up to 67%. This lays a technical foundation for systematic functional genomics research and metabolic engineering modification of this strain.

[0004] In optimizing *Fusarium vesicae* as a host, existing technologies mainly focus on two aspects: first, reducing carbon loss from fermentation byproducts to improve conversion rates; and second, knocking out enzymes that may degrade the target product to simplify downstream processing. For example, the aforementioned Chinese patent CN116640753B discloses that by knocking out the pyruvate decarboxylase gene FvPDC6, the synthesis of the byproduct ethanol can be blocked, increasing the conversion rate of glucose to mycelial protein by approximately 87% (i.e., from 0.194 g / g to 0.361 g / g). However, this patent... Figure 5 The results clearly show that the biomass (mycelial dry weight) of the FvPDC6 knockout strain was only "slightly increased," with neither the wild-type nor the mutant strain exceeding 11 g / L. This indicates that simply blocking the byproduct metabolic pathway cannot effectively drive significant mycelial proliferation. On the other hand, US Patent US8647856B2, in enzyme-deficient strains... Fusarium venenatum The patent, published on February 11, 2014, discloses methods for producing polypeptides in mutant strains. These methods involve modifying genes such as pyrG, amyA, and alpA in *Fusarium venetum* to remove orotate nucleoside-5'-phosphate decarboxylase, α-amylase, and alkaline protease, thereby constructing enzyme-deficient mutant strains for the production of heterologous polypeptides. The core objective of this patent is to reduce enzymes produced by the host that may interfere with the recovery and downstream processing of the target product; its focus is not on increasing the host strain's biomass or glucose conversion rate.

[0005] Aspartic proteases are a class of proteolytic enzymes widely found in fungi, participating in various physiological processes. However, the specific biological functions of each member of the aspartic protease family in *Fusarium venetum*, particularly their effects on mycelial growth, biomass accumulation, and substrate (such as glucose) conversion efficiency, remain unclear. It is noteworthy that although US Patent 9567596B2, "Protease-Deficient Filamentous Fungal Cells and Methods of Use Thereof" (authorization date February 14, 2017), discloses a technique for increasing heterologous antibody production by knocking out multiple proteases (including aspartic proteases pep1, pep2, pep3, etc.) in *Trichoderma reesei*, it explicitly teaches that knocking out certain proteases (such as slp2) negatively impacts strain growth, leading to "reduced spores and slower growth." The core objective of this patent is to "improve the yield and stability of heterologous proteins (such as antibodies)," addressing the problem of secretory protein degradation rather than increasing the biomass and substrate conversion rate of the strain itself.

[0006] Therefore, existing technologies have neither revealed the negative regulatory role of specific aspartic protease genes in *Fusarium venetum* on strain growth and carbon source metabolism, nor provided a technical solution to simultaneously achieve a significant increase in biomass and glucose conversion efficiency by knocking out these genes. More importantly, based on the negative teachings of the aforementioned US9567596B2 (i.e., knocking out certain proteases inhibits growth), those skilled in the art find it difficult to anticipate that knocking out specific aspartic protease genes might actually promote the growth and metabolism of *Fusarium venetum*. Therefore, how to screen for specific genes with negative growth-regulating effects from the *Fusarium venetum* aspartic protease family and synergistically improve biomass and carbon source conversion efficiency by knocking out these genes is a pressing technical challenge in this field. Summary of the Invention

[0007] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0008] To achieve these objectives and other advantages according to the present invention, a *Fusarium venetum* strain with the aspartic protease gene knocked out is provided. This strain is a mutant obtained by knocking out the aspartic protease gene FVRRES_10343 or FVRRES_12522 in *Fusarium venetum*, and is classified as *Fusarium venetum*. Fusarium venenatum .

[0009] Preferably, the mutant strain is the ΔFVRRES_10343 mutant strain or the ΔFVRRES_12522 mutant strain; Among them, the ΔFVRRES_10343 mutant strain, with the preservation name TB01.GEA10343, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC No. 42671 and the deposit date is March 30, 2026; The ΔFVRRES_12522 mutant strain, with the accession name TB01.GEA12522, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42672 and accession date of March 30, 2026.

[0010] This invention provides the application of the *Fusarium vesicae* strain with the aspartic protease gene knocked out in the production of mycelial protein.

[0011] A method is provided for producing mycelial protein by fermentation using an engineered strain of Fusarium venetum with the aspartic protease gene knocked out. The engineered strain is cultured in a fermentation medium, the mycelium is collected, and the mycelial protein is obtained by drying.

[0012] Preferably, the fermentation conditions are: temperature 25℃~30℃, stirring speed 100~350rpm, dissolved oxygen concentration ≥30%, and pH value 5.5~6.5; The fermentation medium contains the following components: yeast extract 0.3–0.8 g / L, MgSO4·7H2O 1.0–2.0 g / L, KCl 0.5–1.0 g / L, Na2SO4 0.3–0.8 g / L, KH2PO4 1.5–2.5 g / L, CaCO3 0.3–0.8 g / L, (NH4)2SO4 8–12 g / L, glucose 50–70 g / L, and ZnSO4·7H2O 0.01–0.03 g / L.

[0013] A method for constructing an engineered strain of *Fusarium vesicae* with the aspartic protease gene knocked out is provided, comprising: Knock out the aspartic protease gene FVRRES_10343 or FVRRES_12522 in Fusarium vesicerii; The knockout includes: constructing an sgRNA expression vector targeting the FVRRES_10343 gene, introducing the strain through PEG-mediated protoplast transformation, and screening to obtain ΔFVRRES_10343 mutant strains; And / or construct an sgRNA expression vector targeting the FVRRES_12522 gene, introduce the strain through PEG-mediated protoplast transformation, and screen to obtain ΔFVRRES_12522 mutant strains.

[0014] A mycelial protein is provided, which is produced by fermentation of the engineered strain.

[0015] A target gene for improving Fusarium vesicatoria biomass and / or glucose conversion rate is provided, the nucleotide sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0016] A sgRNA for knocking out the aspartic protease gene of Fusarium vesicatoria is provided, the sgRNA comprising a guide sequence capable of targeting the gene sequence shown in SEQ ID NO:1 or SEQ ID NO:2, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0017] An expression vector or CRISPR-Cas9 system comprising the sgRNA described above for knocking out the aspartic protease gene of Fusarium vesicatoria is provided.

[0018] The present invention has at least the following beneficial effects: First, in existing technologies, strategies for improving mycelial protein yield by genetically engineering *Fusarium venetum* mainly focus on reducing byproduct synthesis to improve carbon source conversion (e.g., knocking out the pyruvate decarboxylase gene) or knocking out specific enzyme genes to simplify product recovery (e.g., knocking out amylase and protease genes). However, none of these approaches have achieved a significant increase in the biomass of the host strain itself. This invention, by knocking out specific aspartic protease genes FVRRES_10343 or FVRRES_12522, yielded engineered strains whose mycelial biomass and glucose-to-mycelial protein conversion rates were significantly higher than those of the wild-type strain during fermentation. Notably, the biomass of the wild-type and mutant strains reached 9.45 g / L and 12.87 g / L, respectively, with the latter successfully breaking through the 11 g / L bottleneck that is difficult to overcome in existing technologies for *Fusarium venetum* biomass. This technological achievement overcomes the limitations of existing technologies that can only improve conversion rates or eliminate interfering enzymes, achieving a synergistic improvement in biomass accumulation and substrate utilization efficiency. Furthermore, while the biomass of the mutant strain increased significantly, the mycelial protein content remained stable or even slightly increased, resulting in a doubling of the total protein yield at the fermentation endpoint, demonstrating excellent potential for industrial application.

[0019] Secondly, existing research indicates that knocking out certain protease genes (such as the slp2 gene in *Trichoderma reesei*) in filamentous fungi leads to negative effects such as slower growth and decreased spore production. Based on this, those skilled in the art generally believe that the deletion of protease genes may adversely affect the normal physiological metabolism of the fungus. Through systematic functional screening, this invention unexpectedly discovered that knocking out the aspartic protease genes FVRRES_10343 or FVRRES_12522 in *Fusarium venetum* not only did not inhibit the growth of the strain, but also showed a significant growth advantage, with colony diameters significantly larger than the wild type.

[0020] Third, through systematic functional screening of multiple members of the aspartic protease family, this invention found that only the knockout of the FVRRES_10343 and FVRRES_12522 genes could produce the aforementioned beneficial effects, while the knockout of other members of the family did not show a significant increase in biomass or conversion rate. This indicates that the technical effect of this invention is not a common function of this type of protease, but rather stems from the precise selection of specific target genes, and cannot be directly derived or expected from existing technologies.

[0021] Fourth, the engineered strains provided by this invention can achieve higher mycelial yield and better carbon source utilization without changing existing fermentation process conditions. This helps reduce the raw material consumption cost per unit of mycelial protein product, improve the production intensity of fermentation equipment, and provides a new strain resource and technical solution with practical value for the industrial production of mycelial protein from *Fusarium venetum*. Furthermore, this invention systematically characterized the processing functional characteristics of the mutant mycelial protein, finding that ΔFVRRES_12522 has significantly improved water absorption and stable emulsifying properties, making it suitable for succulent imitation meat products; while ΔFVRRES_10343 has significantly improved oil absorption and foaming properties, making it suitable for high-fat imitation meat and aerated foods. These targeted improvements in functional characteristics further broaden the application scenarios of *Fusarium venetum* mycelial protein in the plant-based food industry and enhance its market competitiveness.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0023] Figure 1 This is a bioinformatics analysis diagram of the aspartic protease family of the present invention.

[0024] Figure 2 This is a statistical chart of fermentation biomass of various mutant strains of the aspartic protease family of the present invention.

[0025] Figure 3 This is a statistical chart showing the fermentation protein content of various mutant strains of the aspartic protease family of this invention.

[0026] Figure 4 The phenotype analysis diagrams for ΔFVRRES_10343 and ΔFVRRES_12522 of this invention are shown.

[0027] Figure 5 The stress phenotypes of strains ΔFVRRES_10343 and ΔFVRRES_12522 of this invention are shown in the diagram.

[0028] Figure 6 The results show the water absorbency of mycelial proteins from strains WT, ΔFVRRES_10343, and ΔFVRRES_12522 of this invention.

[0029] Figure 7 The results show the oil absorption properties of mycelial proteins from strains WT, ΔFVRRES_10343, and ΔFVRRES_12522 of this invention.

[0030] Figure 8The results show the foaming properties and foam stability of mycelial proteins from strains WT, ΔFVRRES_10343, and ΔFVRRES_12522 of this invention.

[0031] Figure 9 The results are the emulsifying properties of mycelial proteins from strains WT, ΔFVRRES_10343, and ΔFVRRES_12522 of this invention.

[0032] Figure 10 The results are for the solubility determination of mycelial proteins of strains WT, ΔFVRRES_10343, and ΔFVRRES_12522 of this invention.

[0033] Biological material deposit: *Fusarium venetum* ΔFVRRES_10343 (deposit name: TB01.GEA10343) has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 30, 2026, the accession number is CGMCC No. 42671, and the taxonomic name is *Fusarium venetum*. Fusarium venenatum ; Fusarium venetum ΔFVRRES_12522 (accession name: TB01.GEA12522) has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 30, 2026, and the accession number is CGMCC No. 42672. The taxonomic name is Fusarium venetum. Fusarium venenatum . Detailed Implementation

[0034] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0035] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0036] First, the materials, strains, culture media, and experimental methods used in the embodiments of the present invention will be described.

[0037] 1. Materials and Methods 1.1 Strains and Culture Conditions The wild-type strain of *Fusarium venetum*, TB01 (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 20740, isolated and deposited by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences), was inoculated with 2 μL of wild-type conidial suspension onto YEPD solid medium and cultured for 3–4 days to achieve colony activation. Gene cloning and vector construction were performed using *Escherichia coli* DH5α strain, cultured at 37°C in LB medium, and the taxonomic name was *Fusarium venetum*. Fusarium venenatum Among them, the wild strain TB01 is described in Chinese patent publication number CN115786384A, entitled "Integration site of Fusarium venetum TB01 and its application", with an application publication date of March 14, 2023.

[0038] The culture medium used in this invention is as follows: LB medium (NaCl 10 g / L, yeast extract 5 g / L, tryptone 10 g / L) was used for Escherichia coli culture.

[0039] YEPD medium (yeast extract 3g / L, tryptone 10g / L, glucose 20g / L, agar 15g / L): used for strain activation and mycelial culture.

[0040] Sodium carboxymethyl cellulose (CMC-Na) medium (CMC-Na 10 g / L, KH2PO4 2 g / L, yeast extract 1 g / L, MgSO4 1.5 g / L, CaCl2 0.2 g / L, KCl 0.7 g / L, (NH4)2SO4 5 g / L, Na2SO4 0.5 g / L): used for the induction and preparation of conidia.

[0041] PDA medium (potato 200g / L, glucose 20g / L): used for strain culture and phenotypic observation.

[0042] GMM medium (10 g / L glucose, 1.5 g / L MgSO4・7H2O, 0.7 g / L KCl, 0.5 g / L Na2SO4, 2 g / L KH2PO4, 0.5 g / L CaCO3, 10 g / L (NH4)2SO4): used for strain phenotypic identification.

[0043] Fermentation (FM) medium (yeast extract 0.5 g / L, MgSO4・7H2O 1.5 g / L, KCl 0.7 g / L, Na2SO4 0.5 g / L, KH2PO4 2 g / L, CaCO3 0.5 g / L, (NH4)2SO4 10 g / L, glucose 60 g / L, ZnSO4・7H2O 0.02 g / L): used for the fermentation culture of the strain. The routine culture conditions are 28℃ and shaking culture at 180 rpm.

[0044] Regeneration medium (yeast extract 1g / L, tryptone 1g / L, sucrose 274g / L, agar powder 10g / L): used for the regeneration culture of strains during genetic transformation.

[0045] 1.2 Bioinformatics Analysis Aspartic protease family gene sequences, including the nucleotide sequences and encoded amino acid sequences of FVRRES_10343 (SEQ ID NO.1) and FVRRES_12522 (SEQ ID NO.2), were obtained from the NCBI database. The amino acid sequences of the proteins encoded by the FVRRES_10343 and FVRRES_12522 genes are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively (see Table 3). The conserved motifs and functional domains of the aspartic proteases were analyzed using the MEME online tool and the NCBI Batch-CD-Search tool, and a phylogenetic tree was constructed using MEGA-X software.

[0046] 1.3 Obtaining gene knockout transformants 1.3.1 Construction of gene editing vectors Using the sgRNACas9 tool and an editing efficiency prediction website, sgRNA guide sequences were designed at the genome level targeting the genes FVRRES_10343 and FVRRES_12522, respectively, resulting in sgRNA-10343 (SEQ ID NO.3) and sgRNA-12522 (SEQ ID NO.4). Using the pFC332 plasmid containing the Fusarium vesicae 5S rRNA sequence as a template, sgRNA expression elements were constructed by PCR amplification. For FVRRES_10343, the 5S promoter, the upstream fragment of sgRNA, and the downstream fragment of sgRNA-scaffold were amplified using primers 332-1F / 343-1R and 343-2F / 332-2R, respectively. For FVRRES_12522, the same fragment was amplified using primers 332-1F / 522-1R and 522-2F / 332-2R.

[0047] Simultaneously, the empty pFC332 vector was linearized and purified using BglII. The 5S rRNA::sgRNA fusion fragment was ligated to the linearized pFC332 vector using ClonExpress® II one-step cloning enzyme to construct the gene editing expression vectors pFC332-5S rRNA::sgRNA targeting FVRRES_10343 and FVRRES_12522, respectively. The recombinant vectors were heat-shocked and transformed into *E. coli* DH5α, and the correctness was verified by colony PCR and sequencing. All PCR amplifications were performed using 2×Es TaqMaster Mix.

[0048] Table 1. PCR Amplification Primer Information 1.3.2 PEG-mediated protoplast transformation Take a concentration of 1×10 6 Inoculate 2 mL of spore suspension per mL into 50 mL of YEPD medium and culture at 28 °C and 180 rpm for 12–14 h with shaking until the spores germinate and the buds reach 3–4 times the diameter of the spores.

[0049] Collect bacterial cells by centrifugation at 12,000 rpm for 10 min. After discarding the supernatant, resuspend the bacterial cells in 5 mL of 0.7 mol / L NaCl solution and wash them. Centrifuge again at 10,000 rpm and discard the supernatant completely. The bacterial cells can be used immediately or temporarily stored at 4°C.

[0050] Prepare a 0.7 mol / L NaCl sterile solution containing snailase (4 mg / mL) and catabolase (2 mg / mL). Add 1 mL of bacterial suspension to the solution, wrap it with aluminum foil to protect it from light, and incubate it in a shaker at 37°C and 100 rpm for 1.5 h. After the enzymatic hydrolysis is complete, collect the filtrate containing protoplasts.

[0051] After aliquoting the filtrate, centrifuge at 4000g for 5 min at 4℃, discard the supernatant, gently resuspend the precipitate in 700 μL STC buffer (containing 1.2 mol / L sorbitol, 1.47 g / L calcium chloride, and 10 mL / L Tris-HCl (pH 7.5)), centrifuge again at 4000g for 2 min, and discard the supernatant.

[0052] Add 50 μL of STC buffer and 20 μL of vector DNA to the precipitate, mix gently, and incubate on ice for 50 min. Transfer the entire transformation system after ice incubation to a 10 mL centrifuge tube, add 1.25 mL of 60% PEG solution (containing 60 g / 100 mL PEG 6000, 0.735 g / 100 mL calcium chloride, and 5 mL / L Tris-HCl (pH 7.5)), mix thoroughly, incubate the centrifuge tube at an angle at room temperature for 30 min, then add 4 mL of pre-chilled STC buffer and mix gently to terminate the PEG-mediated transformation reaction.

[0053] Mix all the transformation solution thoroughly with 50 mL of preheated regeneration medium, pour it into a plate immediately, cover the top with YEPD medium containing hygromycin (35 μL / 100 mL), invert the plate and place it in a 28℃ incubator for static culture for 2-3 days. Starting from the second day of culture, observe the growth of transformants daily.

[0054] 1.3.3 Transformer Verification Single colonies grown on the plates were picked and inoculated into selection medium for rescreening to obtain highly pure suspected positive transformants. Genomic DNA was extracted from the suspected positive transformants using standard DNA extraction methods for subsequent validation.

[0055] Validation primers were designed for the target genes FVRRES_10343 and FVRRES_12522 respectively: the validation primers for FVRRES_10343 were 343YZ-F (SEQ ID NO.11) and 343YZ-R (SEQ ID NO.12), and the validation primers for FVRRES_12522 were 522YZ-F (SEQ ID NO.13) and 522YZ-R (SEQ ID NO.14). The Tm values ​​of all validation primers were controlled at 55-60℃, the primer length was 40-60 bp, and the number of bases in a single primer was 18-22 bp.

[0056] Using extracted genomic DNA as a template, PCR amplification was performed using 2×Es Taq Master Mix reagent, and the amplification products were verified by sequencing. By comparing the PCR amplification results with the sequencing sequence, the success of gene knockout was confirmed, and correct transformants were obtained.

[0057] Among them, *Fusarium vesicae* ΔFVRRES_10343 (strain preservation name: TB01.GEA10343) has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42671 and deposit date of March 30, 2026; *Fusarium vesicae* ΔFVRRES_12522 (strain preservation name: TB01.GEA12522) has been deposited at CGMCC with accession number CGMCC No. 42672 and deposit date of March 30, 2026.

[0058] 1.4 Phenotypic Identification 1.4.1 Plate Phenotypic Identification The activated engineered strain was inoculated into CMC-Na liquid medium and cultured with shaking for 3-4 days. 2 μL of spore solution was then inoculated onto FM, PDA, and GMM solid plates, respectively, and cultured upside down at 28℃ for 5 days. The growth phenotype of the strain was observed and recorded.

[0059] 1.4.2 Stress Sensitivity Analysis PDA plate culture media containing different stress factors were prepared, namely 1.5 mol / L sorbitol, 0.8 mol / L NaCl, 250 μg / mL Congo red, and 25 μg / mL calcium fluoride; a concentration of 1×10⁻⁶ was used. 6 Two μL of a conidial suspension of 1 conidium / mL was inoculated onto the above-mentioned stress culture medium plates. The plates were incubated in the dark at 28°C for 3 days. Subsequently, the colony growth status was observed and the colony diameter was measured. The effect of different stress conditions on the growth of the strain was evaluated by the colony growth.

[0060] 1.4.3 Observation of mycelial morphology The bacterial culture was inoculated into the center of a PDA plate and incubated at 28℃ for 3–5 days. Fresh hyphae were then picked from the plate for microscopic observation. The septation characteristics and branching of the hyphae were observed at ×400 and ×1000 magnifications, and the hyphal diameter was measured. The presence and attachment mode (intercalary, terminal, or chain-like) of chlamydospores were also systematically observed. In each biological replicate, each observation index was measured three times and the average value was calculated. The experiment was conducted in triplicate, and data were statistically analyzed using GraphPadPrism software.

[0061] 1.4.4 Analysis of conidia yield and morphology The preserved strain was plate-activated and then inoculated into a liquid medium containing sodium carboxymethyl cellulose (CMC-Na). The medium was then shaken and cultured at 28°C for 3–4 days to obtain a uniformly dispersed conidial suspension.

[0062] Conidium production determination: The above spore suspension was counted using a hemocytometer. Each sample was counted three times. After removing outliers, the average value was calculated. The sporulation capacity of the strain was characterized by the number of spores per unit volume (spores / mL).

[0063] Conidial morphology observation: 10 μL of fresh spore suspension was dropped onto a glass slide, covered with a coverslip to make a temporary mounting, and the morphological characteristics of the spores were systematically observed and recorded under an optical microscope at 400 magnification.

[0064] 1.5 Fermentation Assessment 1.5.1 Fermentation Culture Take a 1×10⁻⁶ dose from CMC-Na liquid culture medium that has been cultured at 28℃ with shaking for 3 days. 6 One mL of conidial suspension (1 conidia / mL) was inoculated into a 500 mL shake flask containing 100 mL of fermentation broth and cultured at 28 °C and 180 rpm for 24 h with shaking to obtain the seed culture. The entire seed culture was then transferred to a 5 L fermenter containing 3.7 L of fermentation broth for fermentation. During the fermentation process, the following parameters were strictly controlled: dissolved oxygen concentration ≥30%, maximum stirring speed 350 rpm, and pH maintained at 6.0.

[0065] 1.5.2 Biomass Measurement Take the fungal fermentation broth cultured for 2 days in a 5L fermenter. During fermentation, the stirring speed starts at 100 rpm and gradually increases as the dissolved oxygen concentration decreases, with a maximum speed not exceeding 350 rpm. The fermentation broth is filtered using an SHZ-D(III) type vacuum filtration device (Baoling, Shanghai, China), and the mycelium is collected and washed three times with distilled water to remove residual culture medium. The washed mycelium is placed in a 101-00B type electric thermostatic drying oven (Lichen, Zhejiang, China) and dried to constant weight. The dried mycelium sample is then removed, accurately weighed using an analytical balance, and the data is recorded.

[0066] 1.5.3 Protein content determination The dried mycelium was ground into powder. 3 mg of the mycelium powder was accurately weighed and placed in a Flashsmart elemental analyzer (Thermo Scientific, Waltham, USA) to determine the total nitrogen content (%). The protein content in the mycelium was calculated by multiplying the measured total nitrogen content by a protein conversion factor of 6.25.

[0067] 1.5.4 Glucose Conversion Rate Determination Glucose concentration determination: The fermentation broth after fermentation was completed was filtered through a 0.22μm filter (Jinlong, Tianjin, China) for sterilization; the filtered sample was diluted with distilled water to a suitable concentration, and the glucose concentration in the sample was determined using an M-online biochemical analyzer (Siemans, Shenzhen, China).

[0068] Glucose conversion rate calculation: Glucose conversion rate (g / g) is calculated according to the following formula: Glucose conversion rate = dry mycelium weight (g / L fermentation broth) / (initial glucose concentration (g / L) - final glucose concentration (g / L)).

[0069] All experiments were performed in triplicate. Results are expressed as mean ± standard deviation, with error bars representing the standard deviation of triplicate.

[0070] 1.5.5 Water Absorption Test Water Absorption Capacity (WAC) of the sample was determined by centrifugation: The fermentation mycelium was filtered, washed three times, freeze-dried to constant weight, and homogenized into a homogenized protein powder. 0.5 g of the mycelial protein sample was accurately weighed and placed in a pre-dried and weighed 15 mL centrifuge tube. The total mass of the centrifuge tube and sample was recorded. 5 mL of deionized water (ddH2O) was added. The mixture was vortexed for 1 min to ensure full contact between the sample and water. The mixture was then allowed to stand at room temperature for 30 min to allow the sample to fully absorb water and swell. After standing, the sample was centrifuged at 5000 rpm for 30 min. After centrifugation, the supernatant was carefully discarded, and the centrifuge tube was inverted on filter paper and allowed to drain for 10 min to remove residual moisture from the tube wall. The total mass of the centrifuge tube and precipitate was weighed. Water absorption capacity is expressed as the mass of water adsorbed per gram of sample (g / g), calculated using the following formula: In the formula: m0 represents the dry mass of the sample, g; m1 represents the total mass of the centrifuge tube and the dry sample, g; m2 represents the total mass of the centrifuge tube and the wet precipitate after centrifugation and draining, g.

[0071] 1.5.6 Oil Absorption Test Oil Absorption Capacity (OAC) of the sample was determined by centrifugation: The fermentation mycelium was filtered, washed three times, freeze-dried to constant weight, and homogenized into a homogenized protein powder. 0.5 g of the mycelial protein sample was accurately weighed and placed in a pre-dried and weighed 15 mL centrifuge tube. The total mass of the centrifuge tube and sample was recorded. 5 mL of corn germ oil was added, and the mixture was vortexed for 1 min to ensure thorough contact between the sample and oil. The mixture was allowed to stand at room temperature for 30 min to allow for complete oil adsorption. After standing, the sample was centrifuged at 5000 rpm for 30 min. After centrifugation, the supernatant oil and its residue were carefully discarded, and the centrifuge tube was inverted onto filter paper and allowed to drain for 10 min to remove any remaining free oil from the tube wall. The total mass of the centrifuge tube and wet precipitate was weighed. Oil absorption capacity is expressed as the mass of oil adsorbed per gram of sample (g / g), calculated using the following formula: In the formula: m0 represents the dry mass of the sample, g (i.e., 0.50 g); m1 represents the total mass of the centrifuge tube and the dry sample, g; m2 represents the total mass of the centrifuge tube and the wet precipitate after centrifugation and draining, g.

[0072] 1.5.7 Foaming property test Accurately weigh an appropriate amount of mycelial protein sample and prepare a 5 mg / mL suspension with deionized water. Homogenize using a tissue disperser for 2 min to ensure thorough protein dispersion and foaming. Immediately after homogenization, record the foam volume as the initial foam volume V0. After standing at room temperature for 30 min, record the foam volume again as V1. Each sample is measured in triplicate. Foaming Capacity (FC) and Foaming Stability (FS) are calculated using the following formulas: In the formula: V0 represents the foam volume measured immediately after homogenization, mL; V1 represents the foam volume after standing for 30 min, mL.

[0073] 1.5.8 Emulsification test The methods for determining the emulsifying activity index (EAI) and emulsifying stability index (ESI) of mycelial protein are as follows: Fermentation mycelium was filtered and washed three times, freeze-dried to constant weight, and homogenized into homogenized protein powder using a homogenizer. A mycelial protein suspension with a concentration of 5 mg / mL was mixed with corn germ oil at a volume ratio of 5:2 (i.e., 5 mL protein suspension + 2 mL oil). The mixture was homogenized at 10000 r / min for 2 min using a tissue disperser to obtain an emulsion. Immediately after homogenization (0 min) and after standing for 10 min, 50 μL of the emulsion was taken from the bottom of the container and added to 5 mL of 0.1% sodium dodecyl sulfate (SDS) solution. The absorbance was measured at a wavelength of 500 nm and recorded as A0 and A1, respectively. 10 The calculation formula is as follows: In the formula: A0, A 10 The absorbance values ​​are 0 min and 10 min, respectively; C is the protein concentration in the suspension (g / mL); Φ is the volume fraction of the oil phase in the emulsion, which is 0.25 in this experiment (i.e., the volume of oil / total volume of the emulsion); and N is the dilution factor, which is 100 in this experiment.

[0074] 1.5.9 Solubility Determination The fermentation mycelium was filtered, washed three times, freeze-dried to constant weight, and homogenized into homogenized protein powder. An appropriate amount of mycelial protein sample was accurately weighed and prepared into a suspension with a concentration of 5.0 mg / mL (based on sample dry weight) using deionized water. 5 mL of the suspension was transferred to a 15 mL centrifuge tube and vortexed at 2000 rpm for 30 min at room temperature to ensure thorough dispersion and dissolution. Then, the sample was centrifuged at 10000 rpm for 15 min. After centrifugation, the supernatant was carefully aspirated and transferred to a new centrifuge tube for later use. The protein concentration in the supernatant was determined using the BCA method: the supernatant was diluted to an appropriate factor based on preliminary experimental results. Bovine serum albumin (BSA) was used as a standard, and the absorbance was measured at 562 nm to plot a standard curve. Three replicates were set up for each sample, and the protein mass in the supernatant was calculated. Protein solubility (PS) was calculated using the following formula: .

[0075] 2. Experimental verification 2.1 Bioinformatics analysis of FVRRES_10343 and FVRRES_12522 like Figure 1 As shown, whole-genome search results revealed 20 aspartic protease family genes identified in the *Fusarium vesicae* genome. Bioinformatics analysis indicated that the proteins encoded by the FVRRES_10343 and FVRRES_12522 genes share the same conserved motifs and typical conserved domains as other members of this family, suggesting a possible synergistic function. However, due to differences in their domains and expression regulation patterns, functional differences still exist among the proteins in this family.

[0076] 2.2, FVRRES_10343, and FVRRES_12522 gene knockout transformants significantly increased biomass. like Figure 2 and Figure 3 As shown, in order to explore the function of each gene in the aspartic protease family of Fusarium vesicatoria, this invention uses CRISPR-Cas9 gene editing technology to target and knock out 20 genes in this family, and successfully constructs 20 corresponding gene-edited transformants.

[0077] Fermentation results in a 5L fermenter showed that the wild-type strain had a biomass of 9.45 g / L, a glucose conversion rate of 0.20 g / g, and a cell protein content of 52.64%. The mutant strains ΔFVRRES_10343 and ΔFVRRES_12522 exhibited significant growth advantages. ΔFVRRES_10343 had a biomass of 12 g / L, a 27% increase compared to the wild-type, a glucose conversion rate of 0.24 g / g, a 20% increase compared to the wild-type, and a cell protein content of 53.28%, which remained stable and slightly increased compared to the wild-type (52.64%). ΔFVRRES_12522 had a biomass of 12.87 g / L, a 36% increase compared to the wild-type, a glucose conversion rate of 0.25 g / g, a 25% increase compared to the wild-type, and a cell protein content of 54.29%. The above results show that the mutant strain constructed in this invention not only achieved a significant leap in biomass but also maintained or slightly optimized the protein content of the mycelium, thereby achieving a synergistic doubling of total protein yield at the fermentation endpoint.

[0078] 2.3 Phenotypic Analysis of ΔFVRRES_10343 and ΔFVRRES_12522 like Figure 4As shown in the plate phenotypic identification results, on GMM solid medium, the colony diameter of the ΔFVRRES_12522 mutant was 4.23 cm, and the colony diameter of the ΔFVRRES_10343 mutant was 4.33 cm, both significantly larger than the wild-type strain (3.68 cm). On FM solid medium, the colony diameter of the ΔFVRRES_12522 mutant was 6.45 cm, and the colony diameter of the ΔFVRRES_10343 mutant was 6.33 cm, also significantly larger than the wild-type strain (5.35 cm). These results indicate that knockout of both the FVRRES_10343 and FVRRES_12522 genes significantly promotes the growth of *Fusarium venetum*.

[0079] 2.4 Transformant Stress Sensitivity Experiment like Figure 5 As shown, to further investigate the growth adaptability of ΔFVRRES_10343 and ΔFVRRES_12522 mutant strains under different stress environments, 2 μL of spore suspensions of wild-type (WT), ΔFVRRES_10343 and ΔFVRRES_12522 strains were inoculated into PDA plates supplemented with different stress factors (1.5 mol / L sorbitol, 0.8 mol / L NaCl, 250 μg / mL Congo red (CR), 25 μg / mL calcium fluoride (CFW)). The growth of the strains was observed after incubation at 28℃ for 3 days. The results showed that the growth of the ΔFVRRES_10343 and ΔFVRRES_12522 mutant strains was not significantly inhibited on the stress medium containing Congo red (CR); under other stress conditions of 1.5 mol / L sorbitol, 0.8 mol / L NaCl, 25 μg / mL calcium fluoride (CFW), the growth rate of the two mutant strains was not significantly different from that of the wild-type strain.

[0080] 2.5 Protein performance testing Water absorbency (WAC) is used to characterize the ability of proteins to bind water, expressed as the mass of water adsorbed per unit mass of sample (g / g). The measurement results are as follows... Figure 6 As shown, the water absorption of the mycelial protein samples ranged from 2 to 3 g / g. Compared with the wild-type strain, the mutant strain ΔFVRRES_12522 exhibited significantly increased water absorption, indicating that this mutant strain possesses a strong hydration capacity. This characteristic may be related to its higher content of hydrophilic amino acids or its more porous microstructure. This property enables it to more effectively retain moisture and reduce juice loss during processing in applications such as meat products, baked goods, and plant-based protein beverages, thereby improving the juiciness, tenderness, and overall yield of the final product, demonstrating excellent processing adaptability.

[0081] Oil absorbency (OAC) is used to characterize the ability of proteins to bind lipids, expressed as the mass of oil adsorbed per unit mass of sample (g / g). Results are as follows... Figure 7 As shown, the oil absorption of the mycelial protein samples ranged from 2.8 to 4.2 g / g. Compared with the wild-type strain, the mutant strain ΔFVRRES_10343 exhibited significantly increased oil absorption, indicating that this mutant strain possesses a stronger oil-binding capacity. This may be attributed to its higher surface hydrophobicity or the exposure of more hydrophobic groups. This advantage is particularly important in emulsified minced meat products, imitation fat fillings, and fried meat substitutes, not only contributing to the formation of a delicate and stable texture but also effectively reducing oil precipitation, thus imparting a richer, more aromatic flavor and a smoother mouthfeel to the products.

[0082] Foaming capacity (FC) was expressed as the percentage (%) of the initial foam volume to the total suspension volume, while foam stability (FS) was expressed as the percentage (%) of the remaining foam volume to the initial foam volume after standing for 30 minutes. Results are as follows: Figure 8 As shown, the foaming ability of the samples ranged from 6% to 14%, and the foam stability ranged from 70% to 100%. Compared with the wild-type strain, both mutant strains showed significantly improved foaming ability. Although foam stability decreased slightly, it is worth noting that the wild-type strain had extremely low foaming ability (very small initial foam volume), thus its foam stability values ​​have limited reference value. In this context, the significant improvement in foaming ability of the mutant strains is a more important functional improvement. The differences in foaming performance among different samples may be related to their surface hydrophobicity and gas-liquid interface film-forming ability. The significant improvement in foaming ability of the mutant strains provides new possibilities for the application of mycelial proteins in aerated foods (such as mousse, cakes, ice cream, or meringue), and can serve as a natural foaming agent to replace part of the egg white protein, meeting the dual requirements of clean labeling and vegan ingredients.

[0083] Emulsifying property refers to the surface property of proteins adsorbing at the oil-water interface and stabilizing the emulsion system. The results are as follows: Figure 9 As shown, there were no significant differences in emulsifying activity between the mutant strains and the wild-type strains. Notably, the emulsifying stability of the mutant strain ΔFVRRES_12522 remained consistent with the wild-type, without any adverse changes. Combined with its significantly enhanced oil absorption, ΔFVRRES_12522 exhibits stronger oil-loading capacity while maintaining good emulsifying stability. This gives it a unique processing advantage in products such as plant-based mayonnaise, salad dressings, and high-fat dairy beverages, as it can stabilize the emulsion system and carry richer oils to achieve better flavor. The reasons for this phenomenon may include factors such as excessively high surface hydrophobicity leading to enhanced attraction between oil droplets and insufficient mechanical strength of the interfacial protein film.

[0084] Solubility (PS) is expressed as the percentage (%) of total protein dissolved in the supernatant. Results are as follows: Figure 10 As shown, none of the mutant strains differed significantly from the wild-type strain, indicating that gene editing did not disrupt the basic dispersion properties of the protein in solvent systems. The unchanged solubility is a crucial prerequisite for the aforementioned functional property improvements: it ensures that while maintaining good aqueous dispersion, the enhanced water absorption, oil absorption, and foaming properties of the mutant strains are entirely attributable to higher-order structural changes such as the exposure of hydrophobic groups on the protein surface and increased conformational flexibility, rather than simple changes in solubility. This characteristic of "directed functional enhancement without change in basic solubility" provides a stable processing window for food processing.

[0085] Table 2 Nucleotide Sequence List Table 3 Nucleotide sequence list Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A *Fusarium vesicae* strain with the aspartic protease gene knocked out, characterized in that, The engineered strain is a mutant strain obtained by knocking out the aspartic protease gene FVRRES_10343 or FVRRES_12522 in Fusarium venenatum, and its taxonomic name is Fusarium venenatum. The mutant strain is either the ΔFVRRES_10343 mutant strain or the ΔFVRRES_12522 mutant strain; Among them, the ΔFVRRES_10343 mutant strain, with the preservation name TB01.GEA10343, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC No. 42671 and the deposit date is March 30, 2026. The ΔFVRRES_12522 mutant strain, with the accession name TB01.GEA12522, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42672, on March 30, 2026.

2. The application of the *Fusarium vesicae* strain with the aspartic protease gene knocked out as described in claim 1 in the production of mycelial protein.

3. A method for producing mycelial protein by fermentation using an engineered strain of *Fusarium vesicae* with the aspartic protease gene knocked out, characterized in that, The engineered strain described in claim 1 was cultured in a fermentation medium, mycelium was collected, and dried to obtain mycelial protein.

4. The method for producing mycelial protein by fermentation using an engineered strain of *Fusarium vesicae* with the aspartic protease gene knocked out, as described in claim 3, is characterized in that... The fermentation conditions are: temperature 25℃~30℃, stirring speed 100~350rpm, dissolved oxygen concentration ≥30%, pH value 5.5~6.5; The fermentation medium contains the following components: yeast extract 0.3–0.8 g / L, MgSO4·7H2O 1.0–2.0 g / L, KCl 0.5–1.0 g / L, Na2SO4 0.3–0.8 g / L, KH2PO4 1.5–2.5 g / L, CaCO3 0.3–0.8 g / L, (NH4)2SO4 8–12 g / L, glucose 50–70 g / L, and ZnSO4·7H2O 0.01–0.03 g / L.

5. A method for constructing an engineered strain of *Fusarium vesicae* with the aspartic protease gene knocked out, characterized in that, include: Using the wild-type strain TB01 of Fusarium venetum as the substrate bacteria, the aspartic protease gene FVRRES_10343 or FVRRES_12522 in Fusarium venetum was knocked out. The knockout includes: constructing an sgRNA expression vector targeting the FVRRES_10343 gene, introducing the strain through PEG-mediated protoplast transformation, and screening to obtain ΔFVRRES_10343 mutant strains; And / or construct an sgRNA expression vector targeting the FVRRES_12522 gene, introduce the strain through PEG-mediated protoplast transformation, and screen to obtain ΔFVRRES_12522 mutant strains.

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