CN116640753B - Pyruvate decarboxylase gene FvPDC6 and its application in improving mycelial protein production of Fusarium venetiensis - 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 improving mycelium protein of Fusarium veniceris Applications in production

Technical field

The invention belongs to the technical field of Fusarium veniceae genetic engineering technology and relates to the pyruvate decarboxylase gene FvPDC6 and its application in increasing the production of Fusarium venicelis mycelium protein.

Background technique

my country is a country with a large population and has a huge demand for protein resources, with a gap of more than 100 million tons. Coupled with the effects of population growth, environmental pollution, climate change and other factors, the protein supply of traditional agriculture and animal husbandry is insufficient. In addition, with the improvement of consumers' economic level, people's demand for meat protein has also shifted from basic "guaranteed supply" to "nutritional and healthy". Therefore, there is an urgent need for a new protein supply model to ensure the nutritional value, safety and sustainability of meat products. Fusarium venicelis is an industrial strain selected from more than 3,000 strains of fungi that can be used for fermentation to produce mycelium protein. It has good safety and has obtained marketing authorization for food raw materials in 18 countries around the world. The mycelium protein produced by fermentation of this strain has a meat-like tissue structure, low fat content, complete types of amino acids, rich in trace elements and vitamins, and is also rich in edible crude fiber. It is a kind of nutrition that can satisfy modern people. Meat substitutes in demand.

Currently, the production of mycelin by Fusarium veniceae fermentation mainly uses glucose as the carbon source. However, due to the production of by-products during the fermentation process, the carbon source conversion efficiency is low, resulting in excessive loss of glucose, and the acquisition of glucose mainly relies on on food crops. Therefore, whether it is to control the production cost of mycelin or to maintain national food security, it is necessary to improve the carbon source conversion rate when glucose is used as a carbon source to ferment mycelin to produce mycelin.

Contents of the invention

It is an object of the present invention to solve at least the above problems and/or disadvantages and to provide at least the advantages to be explained below.

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 production of mycelium protein of Fusarium venicelis.

Another object of the present invention is to provide the application of the pyruvate decarboxylase gene FvPDC6 or the protein or the strain Fusarium veniceris TB6050 in increasing the production of mycelium protein of Fusarium venicelis.

To this end, the technical solution provided by the present invention is:

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

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 containing the pyruvate decarboxylase gene FvPDC6 and a sequence for expression operably connected to the pyruvate decarboxylase gene FvPDC6 .

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

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

A strain of Fusarium venezali, which is Fusarium venezali TB6050, its classification name is: Fusarium venenatum ( Fusarium venenatum ), Fusarium venenatum ( Fusarium venenatum ) TB6050 is deposited in the General Microorganisms Committee of China Microorganism Culture Collection and Management Committee Center (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 1, Beichen West Road, Chaoyang District, Beijing.

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

1) Knock out the pyruvate decarboxylase gene FvPDC6 in Fusarium and obtain a new Fusarium with the pyruvate decarboxylase gene FvPDC6 knocked out;

2) Inoculate the new Fusarium into a liquid medium for culture, obtain fermentation broth, and harvest mycelium protein from the growing fermentation broth.

Preferably, in the method for improving the mycelium production of Fusarium venicelis, the method for obtaining the new Fusarium includes the following steps:

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

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

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

The FvPDC6 gene editing expression vector is transformed into Fusarium venicelis through protoplast transformation to obtain the new Fusarium.

Preferably, the new Fusarium described in the method for improving mycelium protein production of Fusarium veniceris is strain Fusarium veniceris TB6050, and its classification name is: Fusarium venenatum ( Fusarium venenatum ). venenatum ) TB6050 is deposited in the General Microbiology Center of the China Microbial Culture Collection Committee (CGMCC). The deposit number is: CGMCC NO. 40527. The deposit date is: March 17, 2023. The depository address is: Chaoyang District, Beijing. No. 3, Courtyard 1, Beichen West Road.

Preferably, in the method for increasing the production of mycelium protein of Fusarium vulgaris, in step 2), the liquid medium is a fermentation medium, and during the culture, the culture temperature is 26-30°C, and the culture time is 3- day, 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, 0.7 g/L potassium chloride, 0.5 g/L sodium sulfate , 2 g/L potassium dihydrogen phosphate and 0.5 g/L calcium carbonate.

Application of the pyruvate decarboxylase gene FvPDC6 or the protein or the strain Fusarium veniceris TB6050 in increasing the production of mycelium protein of Fusarium venicelis.

The present invention at least includes the following beneficial effects:

The present invention can completely remove the synthesis of by-product ethanol during the fermentation process by knocking out an endogenous pyruvate decarboxylase of Fusarium venicelis, thereby significantly increasing the flow of carbon sources to mycelial protein synthesis, effectively improving the utilization of glucose, and reducing mycelium Production costs of protein fermentation.

Other advantages, objects, and features of the present invention will be apparent in part from the description below, and in part will be understood by those skilled in the art through study and practice of the present invention.

Description of the drawings

Figure 1 shows the mining of the endogenous main pyruvate decarboxylase gene FvPDC6 of Fusarium venicelis in the embodiment of the present invention: Figure A, a heat map of the expression levels of each pyruvate decarboxylase gene at different fermentation stages; Figure B, each pyruvate decarboxylation Sequence evolutionary 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, a schematic diagram of the gene editing expression vector of FvPDC6 ; Figure B, the result of PacI digestion electrophoresis of the gene editing expression vector of FvPDC6 ; Figure C, the gene editing of FvPDC6 Sequencing comparison results of the 5SrRNA-sgRNA _FvPDC fragment in the expression vector.

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

Figure 4 is a detection chart of the ethanol content in the fermentation supernatant of Fusarium venicelis in the embodiment of the present invention, WT, wild-type Fusarium venicelis TB01 (CGMCC NO. 20740); 1-5, FvPDC6 gene editing transformant.

Figure 5 is a graph showing the detection of the conversion rate of biomass and glucose into mycelium protein synthesis after 4 days of fermentation by F. veniceris in the embodiment of the present invention: WT, wild-type F. veniceris TB01; 1-5, FvPDC6 gene editing transformant.

Fusarium veniceae is Fusarium venezalicum TB6050, and its classification name is: Fusarium venenatum ( Fusarium venenatum ). The strain Fusarium venenatum ( Fusarium venenatum ) TB6050 is deposited in the General Microbiology Center of the Chinese Microbiological Culture Collection Committee (CGMCC), and is preserved The number is: CGMCC NO. 40527, the preservation time is: March 17, 2023, and the address of the preservation unit is: No. 3, Yard 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 text of the description.

It should be understood that terms such as "having," "comprising," and "including" as 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 all conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

The invention provides an endogenous main pyruvate decarboxylase gene FvPDC6 of Fusarium venicelis TB01 and a strain Fusarium veniceris TB6050 that efficiently produces mycelial protein obtained after knocking out the gene, including the endogenous pyruvate decarboxylase gene FvPDC6 (FVRRES_12865). Excavation, construction of FvPDC6 gene editing expression vector and acquisition of the gene editing transformant of Fusarium, wherein the filamentous fungus is Fusarium venetiensis TB6050, its classification is named: Fusarium venenatum ( Fusarium venenatum ), strain Fusarium veniceris ( Fusarium venenatum ) TB6050 is deposited in the General Microbiology Center of the China Council for the Collection of Microbial Cultures (CGMCC for short). The preservation number is: CGMCC NO. 40527. The preservation time is: March 17, 2023. The address of the preservation unit is: Beijing. No. 3, Courtyard 1, Beichen West Road, Chaoyang District.

Specifically, it includes the following steps:

1) Excavation of the main pyruvate decarboxylase gene FvPDC6 of Fusarium venicelis

Through transcriptomic analysis of bacteria in different fermentation stages (not starting to produce ethanol - logarithmic phase of ethanol production - saturation phase of ethanol production), a control gene was discovered from the six endogenous pyruvate decarboxylase genes of Fusarium veniceris The main pyruvate decarboxylase gene for ethanol synthesis. Phylogenetic tree and sequence analysis of six endogenous pyruvate decarboxylase genes found that compared to the other five pyruvate decarboxylase genes ( FvPDC1-5 ), the FvPDC6 gene is in a separate branch evolutionarily, and only the sequence of this gene contains Introns.

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

The primer pair 5SrRNA-1/2 was used to amplify the endogenous 5SrRNA promoter sequence (FVRRES_5S_rRNA_393) from the DNA genome of Fusarium Venetiae TB01, and the primer pair sgRNA-1/2 was used to amplify 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 and the sgRNA _FvPDC fragment were fused to obtain 5SrRNA-sgRNA _FvPDC , and the fused fragment was connected to the backbone vector pFC322-Cas9 by homologous recombinase (Wilson FM, Harrison RJ (Wilson FM, Harrison RJ ( 2021) CRISPR-Cas9 mediated editing of the Quornfungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAstargeting 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 transforming E. coli DH5α, single colonies were selected for PCR verification and positive transformants were obtained.

3) Obtaining editing mutants of FvPDC6 pyruvate decarboxylase gene from Fusarium venicelis

The above-mentioned positive E. coli plasmid was extracted and introduced into Fusarium veniceris TB01 through PEG-mediated protoplast transformation to obtain candidate FvPDC6 gene editing transformants. A simple DNA template of the candidate transformant was prepared by the lysis method, and the target gene sequence was amplified from the simple template using the primer pair PDFyz-1/2, and then sequenced and compared to identify the positive FvPDC6 gene editing mutant.

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 fermentation medium, and culture it on a shaker at 28°C and 180 rpm for 4 days.

5) Determination of ethanol content of FvPDC6 gene-edited mutants

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

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

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

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

Example 1: Excavation of the main pyruvate decarboxylase gene FvPDC6 of Fusarium venicelis

1.Experimental method:

Six pyruvate decarboxylase genes, FvPDC1-6 , were retrieved from the transcriptome of Fusarium veniceris bacteria at different fermentation stages (ethanol production has not begun - logarithmic phase of ethanol production - saturation phase of ethanol production), and the nucleotide sequences are They are shown in SEQ ID No:1-6 respectively, and the amino acid sequences are SEQ ID No:7-12 respectively, and according to their expression abundance, 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 biologicaldata. 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 relationships and sequence characteristics of the six retrieved endogenous pyruvate decarboxylase genes were plotted and displayed using TBtools software.

The analysis results are shown in Figure 1. The pyruvate decarboxylase gene FvPDC6 is expressed at a high level during the fermentation process, especially in the middle and late stages of fermentation (high ethanol production). Its expression is further enhanced. Phylogenetic tree and gene sequence analysis showed 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.

Example 2: Construction of a gene-edited expression vector for the F. veniceris pyruvate decarboxylase gene FvPDC6

1. Primers

2. Fragment amplification and homologous recombination procedures

3. Experimental methods

The endogenous 5SrRNA promoter sequence (FVRRES_5S_rRNA_393, SEQ ID NO:17) was amplified from the DNA genome of Fusarium venicelis TB01 with the primer pair 5SrRNA-1/2 (SEQ ID No:13 and 14), and the primer pair sgRNA-1/ 2 (SEQ ID NO:15 and 16) amplified the sgRNA _FvPDC sequence (SEQ ID NO:19) based on the artificially synthesized gRNAscaffold fragment (SEQ ID NO:18). Subsequently, two rounds of amplification were performed through fusion PCR to fuse the 5SrRNA fragment with the sgRNA _FvPDC fragment, and the fused fragment was inserted into the PacI site of the backbone vector pFC322-Cas9 using 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 perform PacI digestion verification, and the transformants that are correct after digestion verification will be further sequenced for confirmation.

4.Results

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

Example 3: Obtaining editing mutants of the FvPDC6 pyruvate decarboxylase gene of Fusarium venicelis

1. Culture medium

YEPD: 3g yeast powder, 10g peptone, 20g glucose, adjust the volume to 1L

Buffer for solubilizing enzyme: 0.7 M sodium chloride

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

SPTC: STC containing 40% PEG6000

Regeneration medium: 1 g yeast extract, 1 g tryptone, 274 g sucrose, 10 g agarose, adjusted to 1 L

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

Mycelium lysis solution: Dissolve 1.2 gNaOH in deionized water and dilute to 100 mL.

Mycelial neutralization 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 methods

1. Protoplast transformation

1) Prepare Fusarium Venetiae TB01 spore suspension in Tween 80, apply it on GY solid medium, culture at 28°C for 7-10 days to produce spores.

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

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

4) Prepare protoplasts from enzyme lysis solution (20 mg collapsin + 40 mg helicase dissolved in 10 ml 0.7 M sodium chloride, and filtered and sterilized), lyse at 30°C for 2 hours at 100 rpm

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

6) Wash twice with STC, centrifuge as above, use STC to reselect protoplasts (concentration 106), and place on ice for later use.

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

8) Add 1 ml SPTC and mix gently, then leave it at room temperature for 20 minutes.

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

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

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

The amplified fragments were sequenced and compared to identify the target gene editing transformants.

5. Results

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

In view of the consistency of each transformant in terms of ethanol synthesis and glucose conversion efficiency compared with the wild-type strain (not edited), one of the transformants, No. 2, was selected and named strain Fusarium veniceris TB6050 and preserved. Its classification name is : Fusarium venenatum , strain Fusarium venenatum TB6050 is deposited in the General Microbiology Center of the Chinese Microbial Culture Collection Committee (CGMCC), with the preservation number: CGMCC NO. 40527, and the preservation time is: 2023 On March 17, 2017, the address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Example 4: Fermentation of FvPDC6 gene editing mutant in 1 L shake flask

1. Culture medium

Fermentation medium: 40 g/L glucose, 0.5 g/L yeast powder, 6 g/L ammonium sulfate, 1.5g/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 methods

The Fusarium vulgaris strain was inoculated onto 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 fermentation medium, and culture it on a shaker at 28°C and 180 rpm for 4 days.

Example 5: Determination of ethanol content of FvPDC6 gene-edited mutants

1. Reagents

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

2. Experimental methods

The bacterial cells after fermentation for 4 days in Example 3 were centrifuged, and the cell-free supernatant was harvested. The supernatant was then mixed with the internal standard solution isobutanol at a ratio of 4:1 and filtered with a 0.22 μm filter. The ethanol concentration of the above mixed samples was detected using a gas chromatograph (Tianmei). The separation column used is HP-InnoWax 30 m × 0.32 mm × 0.25µm chromatographic column (Agilent). The detection conditions are that the temperatures of the inlet and detector are 200 ℃ and 250 ℃ respectively; the initial column temperature of the column oven is 60 ℃ and maintained 1 min, the first step was raised to 100°C at a rate of 10°C/min and held for 2 min, and the second step was raised to 200°C at a rate of 30°C/min and held 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 almost did not synthesize ethanol, indicating that the synthesis pathway of by-product ethanol has been completely blocked, so the conversion of glucose to mycelium protein synthesis Transformation rates are expected to be significantly improved in the mutant strains.

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

1. Experimental methods

Take 100 mL of the bacterial cells after fermentation for 4 days in Example 3 and perform suction filtration to harvest the bacterial cells and cell-free supernatant respectively. The collected bacterial cells 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 in it was measured using an online biochemical analyzer, and the transfer of glucose to mycelium was finally calculated. The conversion efficiency (g/g) is calculated as: 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 venetiensis, the biomass of the FvPDC6 gene-edited transformant was slightly increased. However, knocking out the FvPDC6 gene blocks the synthesis of by-product ethanol, reduces the loss of carbon sources, and significantly improves glucose utilization. Therefore, compared with the wild type, the carbon source conversion rate of FvPDC6 gene-edited transformants during fermentation increased by about 87% (0.194 vs 0.361). This improvement in conversion efficiency is expected to reduce the production cost of mycelium protein by at least 9,000 yuan per ton (glucose is calculated as 4,000 yuan per ton).

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

Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and embodiments. They can be applied to various fields suitable for the present invention. For those familiar with the art, they can easily Additional modifications may be made, and the invention is therefore not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the claims and equivalent scope.

Claims (6)

1. A strain of Fusarium venetiensis, characterized in that the Fusarium veniceae is Fusarium veniceris TB6050, and its classification name is: Fusarium venenatum ( Fusarium venenatum ). Fusarium venenatum ( Fusarium venenatum ) TB6050 is preserved in China Microbiology General Microbiology Center of the Species Preservation and 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 1, Beichen West Road, Chaoyang District, Beijing. 2. A method for increasing the production of mycelium protein from Fusarium veniceris, which is characterized by comprising the following steps: 1) Knock out the pyruvate decarboxylase gene FvPDC6 in Fusarium to obtain a new Fusarium with the pyruvate decarboxylase gene FvPDC6 knocked out. The pyruvate decarboxylase gene FvPDC6 is the base sequence of a) or b) as follows: a) As shown in SEQ ID NO:6; b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:12; 2) Inoculate the new Fusarium into a liquid medium for culture, obtain fermentation broth, and harvest mycelium protein from the growing fermentation broth. 3. The method for improving the mycelium production of Fusarium venicelis as claimed in claim 2, wherein the method for obtaining the new Fusarium includes the following steps: The endogenous 5SrRNA promoter sequence was obtained by PCR amplification using the DNA genome of Fusarium venetiensis TB01 as a template and a set of primer pairs as shown in SEQ ID NO: 13 and 14 as primers; Using the gRNA scaffold fragment shown in SEQ ID No: 18 as a template and a set of primer pairs shown in SEQ ID NO: 15 and 16 as primers, the sgRNA _FvPDC sequence is obtained through PCR amplification; Subsequently, two rounds of amplification were performed by fusion PCR to fuse the endogenous 5SrRNA promoter sequence with the sgRNA _FvPDC sequence, and the fused fragment was inserted into the PacI site of the backbone vector pFC322-Cas9 by homologous recombinase. The FvPDC6 gene editing expression vector was obtained; The FvPDC6 gene editing expression vector is transformed into Fusarium venicelis through protoplast transformation to obtain the new Fusarium. 4. The method for improving mycelium production of Fusarium venetiensis as claimed in claim 3, characterized in that the new Fusarium is strain Fusarium venetiensis TB6050, and its classification is named: Fusarium venenatum , strain venezia Fusarium venenatum TB6050 is deposited in the General Microbiology Center of the China Council for the Collection of Microbial Cultures (CGMCC). The deposit number is: CGMCC NO. 40527. The deposit date is: March 17, 2023. The depository address is: Beijing. No. 3, Courtyard 1, Beichen West Road, Chaoyang District. 5. The method for improving mycelium production of Fusarium venicelis as claimed in claim 2, characterized in that in step 2), the liquid culture medium is a fermentation culture medium, and during culture, the culture temperature is 26-30°C. The culture time is 3 days. 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. 6. The application of the strain Fusarium vulgaris TB6050 or the knockout pyruvate decarboxylase gene as claimed in claim 1 in improving the mycelial protein production of Fusarium vulgaris, characterized in that the deletion of the pyruvate decarboxylase gene FvPDC6 is in Fusarium veniceae provides the function of promoting mycelium protein production, and the pyruvate decarboxylase gene FvPDC6 is the base sequence of a) or b) as follows: a) As shown in SEQ ID NO:6; b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:12.