CN116640753A - Pyruvate decarboxylase gene FvPDC6 and application thereof in improving yield of fusarium venenatum hypha protein - Google Patents

Abstract

The invention discloses a pyruvate decarboxylase geneFvPDC6And the application thereof in improving the yield of fusarium venenatum hypha protein, which belongs to the technical field of fusarium venenatum genetic engineering and pyruvate decarboxylase geneFvPDC6The deletion of (a) provides a function of promoting the increase of the yield of hyphal protein in Fusarium Veneticum, and is a base sequence of a), b) or c) as follows: a) As shown in SEQ ID NO. 6; b) A nucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO. 12; c) Hybridizes to the nucleotide sequence defined in a) under stringent hybridization conditions and encodes a nucleotide sequence having control over ethanol synthesis. According to the invention, the flow direction of a carbon source to hypha protein synthesis can be obviously improved by knocking out fusarium venenatum, the utilization rate of glucose is effectively improved, and the production cost of hypha protein fermentation is reduced.

Description

Pyruvate decarboxylase gene FvPDC6 and its role in enhancing mycelial protein of Fusarium venezia Applications in production

technical field

The invention belongs to the technical field of gene engineering of Fusarium veniceum, and relates to pyruvate decarboxylase gene FvPDC6 and its application in improving the mycelium protein production of Fusarium venezia.

Background technique

my country is a country with a large population, and the demand for protein resources is huge, with a gap of more than 100 million tons. Coupled with the influence of factors such as population growth, environmental pollution, and climate change, the protein supply of traditional agriculture and animal husbandry is insufficient. In addition, with the improvement of the consumer's economic level, people's demand for meat protein has also shifted from the basic "guaranteed supply" to "nutrition and health". Therefore, a new protein supply mode is urgently needed to ensure the nutritional value, safety and sustainability of meat products. Fusarium venezia is an industrial strain screened from more than 3,000 fungi that can be used to ferment and produce mycelium protein. It has good safety and has been approved as a food raw material in 18 countries around the world. The mycelium protein produced by the fermentation of this strain has a tissue structure similar to meat, and is low in fat, complete in amino acids, rich in trace elements and vitamins, and also rich in edible crude fiber. demand for meat substitutes.

Currently, Fusarium venetiza is fermented to produce mycelial protein mainly using glucose as the carbon source. However, due to the production of by-products in the fermentation process, the conversion efficiency of the carbon source is low, resulting in excessive loss of glucose, and the acquisition of glucose mainly depends on in food crops. Therefore, whether it is to control the production cost of mycelial protein or to maintain national food security, it is necessary to increase the conversion rate of carbon source when glucose is used as carbon source to ferment mycelial protein.

Contents of the invention

An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages as will be described hereinafter.

Another object of the present invention is to provide a pyruvate decarboxylase gene FvPDC6.

Another object of the present invention is to provide a method for increasing the mycelial protein production of Fusarium venezia.

Another object of the present invention is to provide the application of the pyruvate decarboxylase gene FvPDC6 or the protein or the strain Fusarium venetica TB6050 in improving the mycelial protein production of Fusarium venezia.

For this reason, the technical scheme provided by the invention is:

The pyruvate decarboxylase gene FvPDC6 , the deletion of the pyruvate decarboxylase gene FvPDC6 provides the function of promoting the production of mycelium protein in Fusarium venetica, and the pyruvate decarboxylase gene FvPDC6 is as follows a), b) or c) base sequence:

a) as shown in SEQ ID NO:6;

b) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 12;

c) hybridizes to the nucleotide sequence defined in a) under stringent hybridization conditions and encodes a nucleotide sequence that controls ethanol synthesis.

A recombinant vector, which contains the pyruvate decarboxylase gene FvPDC6 and a sequence for expression that is operably linked to the pyruvate decarboxylase gene FvPDC6 .

A host cell, the host cell contains the pyruvate decarboxylase gene FvPDC6 or the recombinant vector.

A protein that promotes the increase of mycelium protein production, the amino acid sequence of the protein is shown in SEQ ID NO:12.

A strain of Fusarium venenatum, said Fusarium venenatum is Fusarium venenatum TB6050. Center (CGMCC for short), the deposit number is: CGMCC NO.40527, the deposit date is: March 17, 2023, and the address of the depository unit is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

A method for improving the production of Fusarium venetica mycelium protein, comprising the steps of:

1) Knockout the pyruvate decarboxylase gene FvPDC6 in Fusarium spp., and obtain a new Fusarium spp. with pyruvate decarboxylase gene FvPDC6 knockout;

2) Inoculate the new Fusarium in a liquid medium for cultivation, obtain a fermentation liquid, and harvest mycelial protein from the growing fermentation liquid.

Preferably, in the method for improving the mycelial protein production of Fusarium venetica, the method for obtaining the new Fusarium comprises the following steps:

Using the DNA genome of Fusarium veniceum TB01 as a template, and using a set of primer pairs as shown in SEQ ID NO: 13 and 14 as primers, the endogenous 5SrRNA promoter sequence is obtained by PCR amplification;

Using the gRNA scaffold fragment as shown in SEQ ID No: 18 as a template, and using a set of primer pairs as shown in SEQ ID NO: 15 and 16 as primers, the sgRNA _FvPDC sequence is obtained by PCR amplification;

Then two rounds of amplification were performed by fusion PCR, the endogenous 5SrRNA promoter sequence was fused with the sgRNA _FvPDC sequence, and the fused fragment was inserted into the PacI site of the backbone vector pFC322-Cas9 by homologous recombinase Obtain the FvPDC6 gene editing expression vector;

The FvPDC6 gene editing expression vector was transformed into Fusarium veniceum by means of protoplast transformation to obtain the new Fusarium spp.

Preferably, the new Fusarium described in the method for increasing the production of Fusarium venenatum mycelium protein is strain Fusarium venenatum TB6050, and its classification is named: Fusarium venenatum ( Fusarium venenatum ), strain Fusarium venezium ( Fusarium venenatum ) venenatum ) TB6050 was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short), the preservation number is: CGMCC NO. 40527, the preservation time is: March 17, 2023, and the preservation unit address is: Chaoyang District, Beijing No. 3, No. 1 Courtyard, Beichen West Road.

Preferably, in the method for increasing the production of mycelial protein of Fusarium venetica, in step 2), the liquid medium is a fermentation medium, and during cultivation, the cultivation temperature is 26-30°C, and the cultivation time is 3- day, the fermentation medium contained: 40 g/L glucose, 0.5 g/L yeast powder, 6 g/L ammonium sulfate, 1.5 g/L magnesium sulfate, 0.7 g/L potassium chloride, 0.5 g/L sodium sulfate , 2 g/L potassium dihydrogen phosphate and 0.5 g/L calcium carbonate.

The application of the pyruvate decarboxylase gene FvPDC6 or the protein or the bacterial strain Fusarium venetica TB6050 in increasing the production of Fusarium venezia mycelium protein.

The present invention at least includes the following beneficial effects:

In the present invention, by knocking out an endogenous pyruvate decarboxylase of Fusarium veniceum, the synthesis of by-product ethanol in the fermentation process can be completely cut off, thereby significantly improving the flow direction of carbon source to mycelium protein synthesis, effectively improving the utilization rate of glucose, and reducing mycelium Production costs of protein fermentation.

Other advantages, objectives and features of the present invention will partly be embodied through the following descriptions, and partly will be understood by those skilled in the art through the research and practice of the present invention.

Description of drawings

Figure 1 shows the excavation of the endogenous major pyruvate decarboxylase gene FvPDC6 of Fusarium veniceum in the embodiment of the present invention: Figure A, the heat map of the expression level of each pyruvate decarboxylase gene at different fermentation stages; Figure B, the decarboxylation of each pyruvate Sequence phylogenetic tree and sequence structure analysis of enzyme genes.

Figure 2 shows the construction of the gene editing expression vector of FvPDC6 in the embodiment of the present invention: Figure A, the schematic diagram of the gene editing expression vector of FvPDC6 ; Figure B, the result of PacI digestion and electrophoresis of the gene editing expression vector of FvPDC6 ; Figure C, the gene editing of FvPDC6 Sequencing alignment results of 5SrRNA-sgRNA _FvPDC fragments in the expression vector.

Figure 3 shows the PCR amplification and sequencing of the FvPDC6 gene edited transformant in the embodiment of the present invention: A, the electrophoresis result of the FvPDC6 gene edited transformant after PCR amplification, M, DNA marker; B, the FvPDC6 gene edited transformant Comparison results after PCR amplification and sequencing.

Fig. 4 is a graph showing the detection of ethanol content in the fermentation supernatant of Fusarium venetica in an example of the present invention, WT, wild-type Fusarium venetica TB01 (CGMCC NO.20740); 1-5, FvPDC6 gene-edited transformants.

Fig. 5 is a detection graph of biomass and conversion rate of glucose to mycelial protein synthesis after 4 days of fermentation of Fusarium venetica in an example of the present invention: WT, wild-type Fusarium venetica TB01; 1-5, FvPDC6 gene-edited transformants.

Fusarium venenatum is Fusarium venenatum TB6050, and its classification is named: Fusarium venenatum ( Fusarium venenatum ) . The number is: CGMCC NO. 40527, the preservation time is: March 17, 2023, and the address of the preservation unit is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings, so that those skilled in the art can implement it with reference to the description.

It should be understood that terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

It should be noted that the experimental methods described in the following embodiments, unless otherwise specified, are conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

The present invention provides an endogenous main effect pyruvate decarboxylase gene FvPDC6 of Fusarium veniceum TB01 and a high-efficiency mycelial protein-producing strain Fusarium veniceum TB6050 obtained after knockout of the gene, including the endogenous pyruvate decarboxylase gene FvPDC6 (FVRRES_12865) The excavation, the construction of the FvPDC6 gene editing expression vector and the acquisition of the gene editing transformant of Fusarium, wherein the filamentous fungus is Fusarium venenatum TB6050, and its classification is named: Fusarium venenatum ( Fusarium venenatum ), strain Fusarium venenatum ( Fusarium venenatum ) TB6050 was deposited in the General Microorganism Center of China Committee for Microorganism Culture Collection (CGMCC for short), the preservation number is: CGMCC NO. 40527, the preservation time is: March 17, 2023, and the depository address is: Beijing No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District.

Specifically, include the following steps:

1) Mining of the main pyruvate decarboxylase gene FvPDC6 of Fusarium venezia

Through the transcriptomic analysis of bacteria in different fermentation stages (not starting ethanol production-logarithmic ethanol production-saturation period of ethanol production), a control gene was excavated from six endogenous pyruvate decarboxylase genes of Fusarium venetica. Master pyruvate decarboxylase gene for ethanol synthesis. Phylogenetic tree and sequence analysis of six endogenous pyruvate decarboxylases revealed that, compared with the other five pyruvate decarboxylase genes ( FvPDC1-5 ), the FvPDC6 gene is in a separate branch in evolution, and only the sequence of this gene contains Introns.

2) Construction of the gene editing expression vector of the main pyruvate decarboxylase gene FvPDC6 of Fusarium veniceum

Use the primer pair 5SrRNA-1/2 to amplify the endogenous 5SrRNA promoter sequence (FVRRES_5S_rRNA_393) from the DNA genome of Fusarium veniceum TB01, and use the primer pair sgRNA-1/2 to amplify the sgRNA based on the artificially synthesized gRNA scaffold fragment _FvPDC sequence. Subsequently, two rounds of amplification were performed by fusion PCR, and the 5SrRNA fragment was fused with the sgRNA _FvPDC fragment to obtain 5SrRNA-sgRNA _FvPDC , and the fused fragment was connected to the backbone vector pFC322-Cas9 by homologous recombinase (Wilson FM, Harrison RJ ( 2021) CRISPR-Cas9 mediated editing of the Quornfungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNA targeting endogenous marker gene PKS12 . Fungal Biol Biotechnol 8:15. https://doi.org/10.1186/s40694- 021-00121- 8) Obtain the FvPDC6 gene editing expression vector. After it was transformed into Escherichia coli DH5α, a single colony was selected for PCR verification, and positive transformants were obtained.

3) Acquisition of FvPDC6 editing mutant of Fusarium venezium pyruvate decarboxylase gene

The plasmids of the above-mentioned positive E. coli were extracted and introduced into Fusarium venetica TB01 by PEG-mediated protoplast transformation to obtain candidate FvPDC6 gene- edited transformants. A simple DNA template of candidate transformants was prepared by lysis method, and the target gene sequence was amplified from the simple template with the primer pair PDFyz-1/2, followed by sequencing and comparison to identify positive FvPDC6 gene editing mutants.

4) FvPDC6 gene editing mutant 1L shake flask fermentation

The strain was inoculated on CMC-Na solid medium and cultured for 10 days, and then a spore suspension with a concentration of 5 × 10 ⁶ conidia/mL was prepared. Take 400 μL of the above spore suspension and inoculate it into 300 mL of fermentation medium, and culture it on a shaker at 28°C and 180 rpm for 4 days.

5) Determination of ethanol content in FvPDC6 gene editing mutants

The above-mentioned bacterial cells after 4 days of fermentation were centrifuged, and the cell-free supernatant was harvested. Subsequently, the concentration of ethanol in the samples was detected by gas chromatography using isobutanol as the internal standard. The separation column used was HP-InnoWax 30 m × 0.32 mm × 0.25 µm chromatographic column. The detection conditions were that the temperature of the inlet and the detector were 200 °C and 250 °C, respectively; the initial column temperature of the column oven was 60 °C and kept for 1 min. The first stage was raised to 100°C at a rate of 10°C/min and held for 2 minutes, and the second stage was raised to 200°C at a rate of 30°C/min and held for 6 minutes.

6) Evaluation of carbon source conversion efficiency of FvPDC6 gene editing mutants.

Take 100 mL of the above-mentioned bacteria after 4 days of fermentation for suction filtration, and harvest the bacteria and cell-free supernatant respectively. Use an oven to dry the bacteria to constant weight, calculate its biomass (g/L); measure the concentration of residual glucose in the supernatant by an online biochemical analyzer, and finally calculate the conversion efficiency of glucose to mycelial protein (g/g ), the calculation formula is: biomass/(initial sugar concentration-residual sugar concentration)

In order to make those skilled in the art better understand the technical scheme of the present invention, the following examples are now provided for illustration:

Example 1: Mining of the main pyruvate decarboxylase gene FvPDC6 of Fusarium veniceum

1. Experimental method:

Six pyruvate decarboxylase genes FvPDC1-6 were retrieved from the transcriptomics of different fermentation stages of Fusarium veniceum (not starting to produce ethanol - logarithmic ethanol production - saturated ethanol production) and their nucleotide sequences Shown as SEQ ID No: 1-6 respectively, the amino acid sequence is SEQ ID No: 7-12 respectively, and according to its expression abundance using TBtools software (ChenC, Chen H, Zhang Y, Thomas HR, Frank MH, He Y , Xia R (2020) Tbtools: anintegrative toolkit developed for interactive analyzes of big biological data. Mol Plant 13(8):1194-1202. https://doi.org/10.1016/j.molp.2020.06.009) Drawing heat maps . At the same time, the evolutionary relationship and sequence characteristics of the retrieved 6 endogenous pyruvate decarboxylase genes were drawn and displayed by TBtools software.

The analysis results are shown in Figure 1. The pyruvate decarboxylase gene FvPDC6 was expressed at a high level during the fermentation process, and its expression was further enhanced especially in the middle and late stages of fermentation (high ethanol production). Phylogenetic tree and gene sequence analysis showed that compared with the other five pyruvate decarboxylase genes ( FvPDC1-5 ), the FvPDC6 gene was in a separate branch in evolution, and only the sequence of this gene contained introns.

Example 2: Construction of gene editing expression vector of Fusarium venezium pyruvate decarboxylase gene FvPDC6

1. Primers

2. Fragment amplification and homologous recombination procedures

3. Experimental method

The endogenous 5SrRNA promoter sequence (FVRRES_5S_rRNA_393, SEQ ID NO:17) was amplified from the DNA genome of Fusarium veniceum TB01 with primer pair 5SrRNA-1/2 (SEQ ID No:13 and 14), and the primer pair sgRNA-1/ 2 (SEQ ID NO:15 and 16) Extend the sgRNA _FvPDC sequence (SEQ ID NO:19) based on the artificially synthesized gRNA scaffold fragment (SEQ ID NO:18). Then two rounds of amplification were performed by fusion PCR, the 5SrRNA fragment was fused with the sgRNA _FvPDC fragment, and the fused fragment was inserted into the PacI site of the backbone vector pFC322-Cas9 by homologous recombinase to obtain the FvPDC6 gene editing expression vector. Transform it into Escherichia coli DH5α, select a single colony, activate it, extract the plasmid, and then perform PacI digestion verification, and further sequence confirmation for the correct transformant verified by enzyme digestion.

4. Results

The experimental results are shown in Figure 2. The results of electrophoresis and sequence sequencing showed that the fusion fragment 5SrRNA-sgRNA _FvPDC had been successfully fused to the gene editing expression vector pFC322-Cas9, and then the correct plasmid was stored at -20°C.

Example 3: Acquisition of Fusarium venezium Pyruvate Decarboxylase Gene FvPDC6 Editing Mutant

1. Medium

YEPD: Yeast powder 3g, peptone 10g, glucose 20g, dilute to 1L

Buffer for lysing enzyme: 0.7 M NaCl

STC: 0.8 M sorbitol; 50 mM CaCl ₂ , 50 mM Tris-HCl (pH 8.0)

SPTC: STC with 40% PEG6000

Regeneration medium: 1 g yeast extract, 1 g tryptone, 274 g sucrose, 10 g agarose, constant volume 1 L

Screening medium: glucose 30 g, yeast powder 6 g, agar powder 15 g, dilute to 1 L

Mycelia lysate: Dissolve 1.2 g of NaOH in deionized water and dilute to 100 mL.

Mycelia neutralizing solution: 10 mL 1M Tris-HCl (pH8.0), 40 mL 0.3 M HCl, dilute to 800 mL with deionized water.

2. Primers

3. Fragment amplification procedure

4. Experimental method

1. Protoplast Transformation

1) Tween 80 to prepare Fusarium venezia TB01 spore suspension, spread on GY solid medium, culture at 28°C for 7-10 days to produce spores.

2) Prepare the spore suspension and inoculate it in YEPD liquid medium (with glass beads), culture at 28°C and 200 rpm until the length of germinated hyphae is 3-4 times that of the spore (16 h).

3) Collect spores at 4°C, 13000 rpm, for 15 minutes, and wash once with sterile 0.7 M sodium chloride.

4) Prepare protoplasts from enzyme lysate (dissolve 20 mg collapsing enzyme + 40 mg helicase in 10 ml 0.7 M sodium chloride, filter and sterilize), lyse at 30°C for 2 h at 100 rpm

5) After filtering with three layers of lens cleaning paper, 4°C, 7000 rpm, 10 min

6) Wash twice with STC, re-select protoplasts (concentration 106) with STC after centrifugation as above, and put them on ice for later use.

7) Take 80 μl of the above protoplast suspension, add 20 μl SPTC, mix gently, add FvPDC6 gene editing expression vector (800 ng/μl) 20 μl, mix gently and place on ice for 30 min

8) Add 1 ml SPTC and mix gently, and place at room temperature for 20 min

9) Add the mixture to the regeneration medium at about 40°C, shake well and pour the plate, and culture at 28°C overnight (about 12h)

10) Pour over the selection medium and culture at 28°C to grow transformants (3-4 days)

11) For the transformants grown on the screening medium, use mycelia lysate and neutralization solution to prepare a simple template (take a small amount of mycelia in 8 μl mycelia lysate, treat at 98°C for 2 min, then add 170 μl mycelia neutralization solution is a simple template), and the FvPDC6 gene fragment containing the editing site was amplified by the primer pair PDCyz-1/2 (SEQ ID NO:20 and 21).

The amplified fragments were sequenced and compared to identify target gene-edited transformants.

5. Results

The experimental results are shown in Figure 3. The results of PCR amplification and sequencing comparison of the FvPDC6 gene editing transformants showed that after the introduction of the FvPDC6 gene editing expression vector, a base insertion occurred in the FvPDC6 gene at the specific sgRNA binding position, resulting in a frameshift mutation of the entire gene. .

In view of the consistency of the ethanol synthesis and glucose conversion efficiency of each transformant compared with the wild-type strain (not edited), one of the strains No. 2 was selected and named strain Fusarium venezia TB6050 and preserved, and its classification was named : Fusarium venenatum ( Fusarium venenatum ), strain Fusarium venenatum ( Fusarium venenatum ) TB6050 was deposited in the General Microbiology Center of China Committee for the Collection of Microorganisms (CGMCC for short), the preservation number is: CGMCC NO. 40527, and the preservation time is: 2023 On March 17, 2009, the address of the preservation unit is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Example 4: FvPDC6 gene editing mutant 1 L shake flask fermentation

1. Medium

Fermentation medium: 40 g/L glucose, 0.5 g/L yeast powder, 6 g/L ammonium sulfate, 1.5 g/L magnesium sulfate, 0.7 g/L potassium chloride, 0.5 g/L sodium sulfate, 2 g/L Potassium dihydrogen phosphate, 0.5g/L calcium carbonate

2. Experimental method

The Fusarium venetica strain was inoculated on CMC-Na solid medium and cultured for 10 days to produce spores, and then a spore suspension with a concentration of 5 × 10 ⁶ conidia/mL was prepared. Take 400 μL of the above spore suspension and inoculate it into 300 mL of fermentation medium, and culture it on a shaker at 28°C and 180 rpm for 4 days.

Example 5: Determination of ethanol content in FvPDC6 gene editing mutants

1. Reagents

Preparation of internal standard solution: Dissolve 3.5 g of isobutanol in 1 M hydrochloric acid, mix well, and filter to sterilize.

2. Experimental method

The cells fermented for 4 days in Example 3 were centrifuged to harvest the cell-free supernatant. The supernatant was then mixed with the internal standard isobutanol at a ratio of 4:1 and filtered through a 0.22 μm filter. The above mixed samples were tested for ethanol concentration using a gas chromatograph (Tianmei). The separation column used was HP-InnoWax 30 m × 0.32 mm × 0.25 µm chromatographic column (Agilent), and the detection conditions were that the temperature of the injection port and the detector were 200 °C and 250 °C, respectively; the initial column temperature of the column oven was 60 °C, and kept For 1 min, the first step was raised to 100 °C at a rate of 10 °C/min and kept for 2 min, and the second step was raised to 200 °C at a rate of 30 °C/min and kept for 6 min.

3. Experimental results

The experimental results are shown in Figure 4. Compared with the wild-type strain, the mutant transformant after knocking out the FvPDC6 gene hardly synthesizes ethanol, indicating that the synthesis pathway of by-product ethanol has been completely blocked, so the synthesis of glucose to mycelial protein The transformation rate is expected to be significantly improved in the mutant strain.

Example 6: Evaluation of carbon source conversion efficiency of FvPDC6 gene editing mutants

1. Experimental method

Take 100 mL of the cells fermented for 4 days in Example 3 and perform suction filtration, and harvest the cells and cell-free supernatant respectively. The collected bacteria were dried in an oven to constant weight, and their biomass (g/L) was calculated; the collected supernatant was diluted 100 times and the concentration of residual glucose was measured by an online biochemical analyzer, and finally the glucose to mycelial protein was calculated. The conversion efficiency (g/g), the calculation formula is: biomass / (initial sugar concentration - residual sugar concentration).

2. Experimental results

The experimental results are shown in Figure 5. Compared with the wild-type strain of Fusarium veniceum, the biomass of FvPDC6 gene-edited transformants was slightly increased. However, because the synthesis of by-product ethanol was blocked after knocking out the FvPDC6 gene, the loss of carbon source was reduced, and the utilization rate of glucose was significantly improved. Therefore, compared with the wild type, the conversion rate of carbon source in the fermentation process of the FvPDC6 gene-edited transformants increased by about 87% (0.194 vs 0.361). This improvement in conversion efficiency is expected to reduce the production cost of mycelial protein by at least 9,000 yuan per ton (glucose is calculated at 4,000 yuan per ton).

The number of modules and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations to the present invention will be apparent to those skilled in the art.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details and examples shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims (10)

1. The pyruvate decarboxylase gene FvPDC6 is characterized in that, the deletion of the pyruvate decarboxylase gene FvPDC6 provides the function of promoting the production of mycelium protein in Fusarium veniceum, and the pyruvate decarboxylase gene FvPDC6 is as follows a) , b) or c) base sequence: a) as shown in SEQ ID NO:6; b) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 12; c) hybridizes to the nucleotide sequence defined in a) under stringent hybridization conditions and encodes a nucleotide sequence that controls ethanol synthesis. 2. A recombinant vector, characterized in that it contains the pyruvate decarboxylase gene FvPDC6 according to claim 1 and a sequence for expression operably linked to the pyruvate decarboxylase gene FvPDC6 . 3. The host cell, characterized in that the host cell contains the pyruvate decarboxylase gene FvPDC6 according to claim 1 or the recombinant vector according to claim 2. 4. A protein that promotes the production of mycelium protein, characterized in that the amino acid sequence of the protein is as shown in SEQ ID NO:12. 5. A strain of Fusarium venenatum, characterized in that the Fusarium venezium is Fusarium venenatum TB6050, and its classification is named: Fusarium venenatum ( Fusarium venenatum ), and Fusarium venenatum ( Fusarium venenatum ) TB6050 is preserved in China Microorganism General Microorganism Center of Species Preservation Management Committee (CGMCC for short), the preservation number is: CGMCC NO. 40527, the preservation time is: March 17, 2023, and the address of the preservation unit is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. 6. A method for improving Fusarium venetica mycelium protein output, characterized in that, comprising the steps: 1) Knockout the pyruvate decarboxylase gene FvPDC6 in Fusarium spp., and obtain a new Fusarium spp. with pyruvate decarboxylase gene FvPDC6 knockout; 2) Inoculate the new Fusarium in a liquid medium for cultivation, obtain a fermentation liquid, and harvest mycelial protein from the growing fermentation liquid. 7. the method for improving Fusarium venicei mycelium protein output as claimed in claim 6, is characterized in that, the method for obtaining described new Fusarium comprises the steps: Using the DNA genome of Fusarium veniceum TB01 as a template, and using a set of primer pairs as shown in SEQ ID NO: 13 and 14 as primers, the endogenous 5SrRNA promoter sequence is obtained by PCR amplification; Using the gRNA scaffold fragment as shown in SEQ ID No: 18 as a template, and using a set of primer pairs as shown in SEQ ID NO: 15 and 16 as primers, the sgRNA _FvPDC sequence is obtained by PCR amplification; Then two rounds of amplification were performed by fusion PCR, the endogenous 5SrRNA promoter sequence was fused with the sgRNA _FvPDC sequence, and the fused fragment was inserted into the PacI site of the backbone vector pFC322-Cas9 by homologous recombinase Obtain the FvPDC6 gene editing expression vector; The FvPDC6 gene editing expression vector was transformed into Fusarium veniceum by means of protoplast transformation to obtain the new Fusarium spp. 8. The method for improving the mycelial protein output of Fusarium venezium as claimed in claim 7, wherein said new Fusarium is bacterial strain Fusarium venenatum TB6050, and its classification is named as: Fusarium venenatum ( Fusarium venenatum ), The strain Fusarium venenatum ( Fusarium venenatum ) TB6050 was deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures (CGMCC for short), the preservation number is: CGMCC NO. 40527, the preservation time is: March 17, 2023, and the address of the preservation unit For: No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing. 9. The method for increasing the mycelial protein production of Fusarium veniceum according to claim 6, characterized in that, in step 2), the liquid medium is a fermentation medium, and during cultivation, the cultivation temperature is 26-30°C, The cultivation time is 3-days, and the fermentation medium contains: 40 g/L glucose, 0.5 g/L yeast powder, 6 g/L ammonium sulfate, 1.5 g/L magnesium sulfate, 0.7g/L potassium chloride, 0.5 g/L sodium sulfate, 2 g/L potassium dihydrogen phosphate and 0.5 g/L calcium carbonate. 10. The application of the pyruvate decarboxylase gene FvPDC6 as claimed in claim 1 or the protein as claimed in claim 4 or the Fusarium venetica TB6050 as claimed in claim 5 in increasing the production of mycelial protein of Fusarium venezia.