CN116640753B - 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 application thereof in improving yield of fusarium venenatum hypha protein

Technical Field

The invention belongs toIn the technical field of genetic engineering of Fusarium Veneticum, relates to a pyruvate decarboxylase geneFvPDC6And the application thereof in improving the yield of fusarium venenatum hypha protein.

Background

China is a large population country, the protein resource demand is huge, and the gap exceeds 1 hundred million tons; and the influence of factors such as population growth, environmental pollution, climate change and the like, leads to insufficient protein supply of the traditional agriculture and animal husbandry. In addition, as consumer economics increase, the demand for meat proteins has also shifted from a basic "guaranteed supply" to "nutritional health". Therefore, a new protein supply mode is urgently needed to ensure the nutritional value, safety and sustainability of meat products. Fusarium Veneticum is an industrial strain which is screened from more than 3000 fungi and can be used for fermenting and producing hypha protein, has good safety, and has been licensed in 18 countries worldwide. The mycelium protein produced by the strain fermentation has a tissue structure similar to meat quality, has low fat content and complete amino acid variety, is rich in microelements and vitamins, and also contains rich edible crude fibers, so that the mycelium protein is a meat substitute capable of meeting the nutritional requirements of modern people.

Currently, the fermentation production of hypha protein by fusarium venenatum mainly adopts a production process using glucose as a carbon source, but the conversion efficiency of the carbon source is low due to the generation of byproducts in the fermentation process, so that excessive loss of glucose is caused, and the acquisition of glucose mainly depends on food crops. Therefore, it is necessary to increase the conversion rate of carbon source when glucose is used as carbon source for fermentation production of mycelium proteins, both from the standpoint of controlling the production cost of mycelium proteins and maintaining national food safety.

Disclosure of Invention

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

It is still another object of the present invention to provide a pyruvate decarboxylase geneFvPDC6。

Another object of the present invention is to provide a method for increasing the yield of Fusarium Veneti hypha protein.

It is still another object of the present invention to provide said pyruvate decarboxylase geneFvPDC6Or the protein or the application of the strain fusarium venenatum TB6050 in improving the yield of fusarium venenatum hypha protein.

For this purpose, the technical scheme provided by the invention is as follows:

pyruvate decarboxylase geneFvPDC6The pyruvate decarboxylase geneFvPDC6The deletion of (a) provides a function of promoting the increase of the yield of hyphal protein in Fusarium Veneti, said pyruvate decarboxylase geneFvPDC6The base sequence of a), b) or c) is 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.

A recombinant vector comprising said pyruvate decarboxylase geneFvPDC6And a pyruvate decarboxylase geneFvPDC6Operably linked sequences for expression.

A host cell comprising said pyruvate decarboxylase geneFvPDC6Or a recombinant vector as described.

And the amino acid sequence of the protein is shown as SEQ ID NO. 12.

A strain of fusarium venenatum, which is fusarium venenatum TB6050, classified under the name: fusarium VeneticumFusarium venenatum) Fusarium VeneticumFusarium venenatum) TB6050 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No. 40527, the preservation time is as follows: 2023, 03 and 17, the deposit unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

A method for increasing the yield of fusarium venenatum hypha protein comprising the steps of:

1) Knocking out pyruvate decarboxylase gene in fusariumFvPDC6Obtaining the pyruvate decarboxylase geneFvPDC6Knocked out new fusarium;

2) Inoculating the novel fusarium into a liquid culture medium for culturing to obtain fermentation liquor, and harvesting hypha protein from the grown fermentation liquor.

Preferably, in the method for improving the yield of fusarium venenatum hypha protein, the method for obtaining the novel fusarium venenatum comprises the following steps:

taking the DNA genome of fusarium venenatum TB01 as a template, taking a group of primer pairs shown as SEQ ID NO. 13 and 14 as primers, and obtaining an endogenous 5SrRNA promoter sequence through PCR amplification;

the gRNA is obtained by PCR amplification by using the gRNA scaffold fragment shown as SEQ ID No. 18 as a template and a group of primer pairs shown as SEQ ID No. 15 and 16 as primers _FvPDC A sequence;

two rounds of amplification were then performed by fusion PCR, the endogenous 5SrRNA promoter sequence was amplified with the sgRNA _FvPDC The sequences are fused, and the fused fragment is inserted into PacI site of a framework vector pFC322-Cas9 by homologous recombinase to obtainFvPDC6A gene editing expression vector;

the saidFvPDC6The gene editing expression vector is transformed into fusarium venenatum by a protoplast transformation mode to obtain the novel fusarium venenatum.

Preferably, in the method for improving the yield of fusarium hypha protein, the novel fusarium is strain fusarium venenatum TB6050, which is classified as: fusarium VeneticumFusarium venenatum) Fusarium venenatum strainFusarium venenatum) TB6050 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No. 40527, the preservation time is as follows: 2023, 03 and 17, the deposit unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Preferably, in the method for improving the yield of fusarium venenatum mycelium protein, in the step 2), the liquid culture medium is a fermentation culture medium, and in the culture, the culture temperature is 26-30 ℃ and the culture time is 3-days, and the fermentation culture medium comprises: 40 g/L glucose, 0.5g/L yeast powder, 6 g/L ammonium sulfate, 1.5g/L magnesium sulfate, 0.7 g/L potassium chloride, 0.5g/L sodium sulfate, 2 g/L potassium dihydrogen phosphate and 0.5g/L calcium carbonate.

The pyruvate decarboxylase geneFvPDC6Or the protein or the application of the strain fusarium venenatum TB6050 in improving the yield of fusarium venenatum hypha protein.

The invention at least comprises the following beneficial effects:

according to the invention, the synthesis of the byproduct ethanol in the fermentation process can be completely cut off by knocking out the endogenous pyruvate decarboxylase of the fusarium venenatum, so that the flow direction of a carbon source to the synthesis of hypha protein is obviously improved, the utilization rate of glucose is effectively improved, and the production cost of hypha protein fermentation is reduced.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows the excavation of the endogenous major pyruvate decarboxylase gene FvPDC6 of Fusarium Veneti in an example of the invention: FIG. A is a heat map of the expression level of each pyruvate decarboxylase gene at different fermentation stages; and B, analyzing sequence evolutionary trees and sequence structures of the pyruvate decarboxylase genes.

FIG. 2 shows construction of a gene editing expression vector for FvPDC6 in the example of the present invention: a drawing is a drawing of a graph,FvPDC6schematic representation of a gene editing expression vector; in the view of the drawing B,FvPDC6the result of PacI enzyme digestion electrophoresis of the gene editing expression vector; a graph C, wherein the graph C is a graph,FvPDC65SrRNA-sgRNA in the gene editing expression vector of (2) _FvPDC Sequencing and comparison results of fragments.

FIG. 3 shows an embodiment of the present inventionFvPDC6PCR amplification and sequencing of Gene editing transformants: a drawing is a drawing of a graph,FvPDC6electrophoresis result after PCR amplification of the gene editing transformant, M, DNA marker; in the view of the drawing B,FvPDC6PCR amplification of Gene editing transformants after sequencingAnd (5) comparing results.

FIG. 4 is a graph showing the measurement of ethanol content in fermentation supernatant of Fusarium venenatum, WT, wild type Fusarium venenatum TB01 (CGMCC No. 20740); 1-5,FvPDC6a gene editing transformant.

FIG. 5 is a graph showing the measurement of the conversion rate of glucose into hyphal protein synthesis after 4 days of fermentation by Fusarium Venetii in the examples of the present invention: WT, wild-type fusarium venenatum TB01;1-5,FvPDC6a gene editing transformant.

Fusarium venenatum is Fusarium venenatum TB6050, which is classified as: fusarium VeneticumFusarium venenatum) Fusarium venenatum strainFusarium venenatum) TB6050 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No. 40527, the preservation time is as follows: 2023, 03 and 17, the deposit unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

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

The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.

The invention provides a fusarium venenatum TB01 endogenous major pyruvate decarboxylase geneFvPDC6And the strain fusarium venenatum TB6050 which is obtained after the gene knockout and is used for efficiently producing hypha protein, comprising endogenous pyruvate decarboxylase genesFvPDC6(fvres 12865),FvPDC6construction of a Gene editing expression vector and the acquisition of Fusarium this Gene editing transformant, wherein the filamentous fungus is Fusarium Veneticum TB6050, its class designation: wini' sFusarium roseum (S. Sp.) SiebFusarium venenatum) Fusarium venenatum strainFusarium venenatum) TB6050 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No. 40527, the preservation time is as follows: 2023, 03 and 17, the deposit unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Specifically, the method comprises the following steps:

1) Fusarium Veneticum major pyruvate decarboxylase geneFvPDC6Is excavated of (2)

By transcriptomic analysis of the cells from the different fermentation stages (not started to produce ethanol- -producing ethanol logarithmic phase- -producing ethanol saturation phase), a major pyruvate decarboxylase gene controlling ethanol synthesis was mined from 6 endogenous pyruvate decarboxylase genes of F.Veneti. The analysis of the evolution tree and the sequence of six endogenous pyruvate decarboxylases shows that compared with other 5 pyruvate decarboxylase genes @ the gene @FvPDC1-5），FvPDC6The gene is evolutionarily in separate branches and only the sequence of the gene contains introns within it.

2) Fusarium Veneticum major pyruvate decarboxylase geneFvPDC6Gene editing expression vector construction of (2)

Endogenous 5SrRNA promoter sequence (FVRRES_5S_rRNA_393) was amplified from the DNA genome of F.Venetian TB01 with primer pair 5SrRNA-1/2, and sgRNA was amplified from primer pair 1/2 on the basis of the artificially synthesized fragment of the gRNA scaffold _FvPDC Sequence. Two rounds of amplification were then performed by fusion PCR, the 5SrRNA fragment was amplified with sgRNA _FvPDC The fragments are fused to obtain 5SrRNA-sgRNA _FvPDC And ligating the fused fragment to a backbone vector pFC322-Cas9 (Wilson FM, harrison RJ (2021) CRISPR-Cas9 mediated editing of the Quorn fungus) by homologous recombinaseFusarium venenatumA3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker genePKS12Fungal Biol Biotechnol 8:15 https:// doi.org/10.1186/s 40694-021-00121-8)FvPDC6Gene editing expression vector. After the E.coli DH5 alpha is transformed, single colony is selected for PCR testPositive transformants were obtained.

3) Fusarium Venetian pyruvate decarboxylase geneFvPDC6Obtaining of editing mutants

Extracting the positive escherichia coli plasmid, and introducing the positive escherichia coli plasmid into fusarium venenatum TB01 by a PEG-mediated protoplast transformation method to obtain a candidateFvPDC6 geneEditing the transformant. Preparing DNA simple template of candidate transformant by cleavage method, amplifying target gene sequence from simple template by primer pair PDFyz-1/2, and sequencing and comparing to identify positiveFvPDC6A gene editing mutant.

4）FvPDC6Gene editing mutant 1L shake flask fermentation

The strain was inoculated on CMC-Na solid medium and cultured for 10 days, followed by preparation of 5X 10 ⁶ A spore suspension at concentration of conidia/mL. 400. Mu.L of the above spore suspension was inoculated into 300 mL fermentation medium and cultured on a 180 rmp shaker at 28℃for 4 days.

5）FvPDC6Determination of ethanol content of Gene editing mutant

And centrifuging the thalli after 4 days of fermentation, and obtaining cell-free supernatant. The ethanol concentration in the sample was then measured using a gas chromatograph using isobutanol as an internal standard. The separation column is an HP-InnoWax 30 m multiplied by 0.32 mm multiplied by 0.25 mu m chromatographic column, and the detection conditions are that the temperature of a sample inlet and the temperature of a detector are 200 ℃ and 250 ℃ respectively; the initial column temperature of the column box is 60 ℃, the column is kept for 1 min, the first stage is raised to 100 ℃ at the speed of 10 ℃/min, the column is kept for 2 min, and the second stage is raised to 200 ℃ at the speed of 30 ℃/min, and the column is kept for 6 min.

6）FvPDC6Evaluation of carbon source conversion efficiency of the gene editing mutant.

And (3) taking 100 mL of the thalli after 4 days of fermentation, and carrying out suction filtration to respectively obtain thalli and cell-free supernatant. Drying the thalli to constant weight by using an oven, and calculating the biomass (g/L) of the thalli; the concentration of residual glucose in the supernatant is measured by an online biochemical analyzer, and the conversion efficiency (g/g) of glucose to hypha protein is finally calculated, wherein the calculation formula is as follows: biomass/(initial sugar concentration-residual sugar concentration)

For a better understanding of the technical solution of the present invention, the following examples are now provided for illustration:

example 1: fusarium Veneticum major pyruvate decarboxylase geneFvPDC6Is excavated of (2)

1. The experimental method comprises the following steps:

6 pyruvate decarboxylase genes were retrieved from the transcriptome of the cells of Fusarium Veneticum at different fermentation stages (not started to produce ethanol- -ethanol production logarithmic phase- -ethanol production saturation phase)FvPDC1-6The nucleotide sequences are shown as SEQ ID No. 1-6, the amino acid sequences are shown as SEQ ID No. 7-12, and according to the expression abundance, TBtools software (ChenC, chen H, zhang Y, thomas HR, frank MH, he Y, xia R (2020) Tbtools: an integrative toolkit developed for interactive analyses of big biological data.mol Plant 13 (8): 1194-1202. Https:// doi.org/10.1016/j.mol p.2020.06.009) is used for drawing a thermal map. Meanwhile, the evolutionary relationship and sequence characteristics of the 6 retrieved endogenous pyruvate decarboxylase genes are plotted and displayed through TBtools software.

The analysis results are shown in FIG. 1, in which the pyruvate decarboxylase geneFvPDC6High levels of expression during fermentation, especially during the mid-to late-fermentation (high ethanol production) stage, are further enhanced. Analysis of the evolutionary tree and the gene sequence shows that compared with other 5 pyruvate decarboxylase genes @ theFvPDC1-5），FvPDC6The gene is evolutionarily in separate branches and only the sequence of the gene contains introns within it.

Example 2: fusarium Venetian pyruvate decarboxylase geneFvPDC6Gene editing expression vector construction of (2)

1. Primer(s)

2. Fragment amplification and homologous recombination procedure

3. Experimental method

Amplification of endogenous 5SrRNA promoter sequence (FVRRES_ 5S_rRNA_393, SEQID NO: 17) from the DNA genome of Fusarium Veneticum TB01 with primer pair 5SrRNA-1/2 (SEQ ID NOS: 13 and 14) and amplification of sgRNA with primer pair 1/2 (SEQ ID NOS: 15 and 16) on the basis of the artificially synthesized gRNAscaffold fragment (SEQ ID NO: 18) _FvPDC Sequence (SEQ ID NO: 19). Two rounds of amplification were then performed by fusion PCR, the 5SrRNA fragment was amplified with sgRNA _FvPDC The fragments are fused, and the fused fragments are inserted into PacI sites of a framework vector pFC322-Cas9 by homologous recombination enzymes to obtainFvPDC6Gene editing expression vector. After the escherichia coli DH5 alpha is transformed, single colony activation is selected to extract plasmids, pacI digestion verification is carried out, and further sequencing and confirmation are carried out on the transformants with correct digestion verification.

4. Results

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

Example 3: fusarium Venetian pyruvate decarboxylase geneFvPDC6Obtaining of editing mutants

1. Culture medium

Yeast powder 3g, peptone 10g, glucose 20g, constant volume 1L

Buffer for dissolving enzyme: 0.7 M sodium chloride

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

SPTC STC with 40% PEG6000

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

Screening the culture medium: glucose 30 g, yeast powder 6 g, agar powder 15 g, constant volume 1L

Mycelium lysate: 1.2 g NaOH was dissolved in deionized water and then the volume was set to 100 mL.

Mycelium neutralization solution: 10 mL 1M Tris-HCl (pH 8.0), 40 mL 0.3M HCl, was sized to 800 mL with deionized water.

2. Primer(s)

3. Fragment amplification program

4. Experimental method

1. Protoplast transformation

1) Tween 80 is used for preparing fusarium venenatum TB01 spore suspension, the suspension is coated on GY solid culture medium, and 7-10 d spores are cultivated at 28 ℃.

2) The spore suspension was prepared and inoculated into YEPD liquid medium (glass beads added), cultured at 28℃and 200 rmp until the sprouting hypha length was 3-4 times that of spores (16 h).

3) Spores were collected at 4℃at 13000 rpm for 15 min and washed 1 time with sterile 0.7. 0.7M sodium chloride.

4) Preparation of protoplasts from enzyme lysate (20 mg crashase+40 mg snailase in 10 ml 0.7M sodium chloride and filter sterilized), lysis 2h at 30℃100 rpm

5) Three layers of mirror cleaning paper are filtered at 4 ℃,7000 rpm and 10 min

6) STC was washed 2 times, centrifuged as above, and protoplasts (concentration 106) were re-selected with STC and placed on ice for further use.

7) Taking 80. Mu.l of the protoplast suspension, adding 20. Mu.l of SPTC, gently mixing, adding 20. Mu.l of FvPDC6 gene editing expression vector (800 ng/. Mu.l), gently mixing, and standing on ice for 30 min

8) 1 ml SPTC was added and gently mixed, and left at room temperature for 20 min

9) Adding the mixed solution into regeneration culture medium at 40deg.C, shaking, pouring into plate, and culturing at 28deg.C overnight (about 12 hr)

10 Pouring the screening medium, and culturing the grown transformant (3-4 d) at 28 DEG C

11 For transformants grown on the selection medium, a simple template was prepared using mycelium lysate and neutralization solution (a small amount of mycelium was taken in 8. Mu.l mycelium lysate, treated at 98℃for 2 min, followed by addition of 170. Mu.l mycelium neutralization solution as a simple template), and FvPDC6 gene fragment containing the editing site was amplified by primer pair PDCyz-1/2 (SEQ ID NOS: 20 and 21).

Sequencing and comparing the amplified fragments to identify the target gene editing transformant.

5. Results

The experimental results are shown in figure 3,FvPDC6PCR amplification and sequencing comparison results of the gene editing transformant show that the gene editing transformant is introducedFvPDC6After editing the expression vectorFvPDC6The gene undergoes base insertion at a specific sgRNA binding site, resulting in frame shift mutation of the entire gene.

In view of the consistency that each transformant exhibits in terms of ethanol synthesis and glucose conversion efficiency compared to the wild-type strain (not edited), one of them, designated as strain fusarium venenatum TB6050, was selected and deposited, and its classification was designated as: fusarium VeneticumFusarium venenatum) Fusarium venenatum strainFusarium venenatum) TB6050 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No. 40527, the preservation time is as follows: 2023, 03 and 17, the deposit unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Example 4: fvPDC6 gene editing mutant 1L shake flask fermentation

1. Culture medium

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

2. experimental method

Fusarium Veneti strain is inoculated on CMC-Na solid culture medium for 10 days to produce spore, and then 5×10 is prepared ⁶ A spore suspension at concentration of conidia/mL. 400. Mu.L of the above spore suspension was inoculated into 300 mL fermentation medium and cultured on a 180 rmp shaker at 28℃for 4 days.

Example 5:FvPDC6determination of ethanol content of Gene editing mutant

1. Reagent(s)

Preparing an internal standard solution: 3.5 g of isobutanol is dissolved in 1M of hydrochloric acid, evenly mixed and filtered for sterilization.

2. Experimental method

The cells obtained after 4 days of fermentation in example 3 were centrifuged to obtain a cell-free supernatant. The supernatant was then mixed with the internal standard isobutanol at 4:1 and filtered through a 0.22 μm filter. The above mixed sample was subjected to detection of ethanol concentration by a gas chromatograph (Tianmei). The separation column is an HP-InnoWax 30 m multiplied by 0.32 multiplied by mm multiplied by 0.25 mu m chromatographic column (Agilent), and the detection conditions are that the temperature of a sample inlet and the temperature of a detector are 200 ℃ and 250 ℃ respectively; the initial column temperature of the column box is 60 ℃, the column is kept for 1 min, the first stage is raised to 100 ℃ at the speed of 10 ℃/min, the column is kept for 2 min, and the second stage is raised to 200 ℃ at the speed of 30 ℃/min, and the column is kept for 6 min.

3. Experimental results

The experimental results are shown in FIG. 4, compared with the wild type strain, the knockout was performedFvPDC6The mutant transformant after the gene almost has no synthetic ethanol, which shows that the synthetic pathway of the byproduct ethanol is completely blocked, so that the conversion rate of glucose to hypha protein synthesis is expected to be obviously improved in the mutant strain.

Example 6:FvPDC6evaluation of carbon source conversion efficiency of Gene editing mutant

1. Experimental method

The cells obtained after fermentation in example 3 of 100 mL for 4 days were collected by suction filtration, and cell-free supernatants were obtained. The collected thalli are dried to constant weight by a baking oven, and the biomass (g/L) is calculated; the concentration of residual glucose in the collected supernatant is measured by an online biochemical analyzer after the collected supernatant is diluted by 100 times, and finally, the conversion efficiency (g/g) of glucose to hypha protein is calculated by the following calculation formula: biomass/(initial sugar concentration-residual sugar concentration).

2. Experimental results

The experimental results are shown in FIG. 5, which shows that, compared with the wild-type strain of Fusarium Veneticum,FvPDC6gene editingThe biomass of the transformant is slightly improved. However, due to the knockoutFvPDC6The synthesis of byproduct ethanol is blocked after the gene, the loss of carbon sources is reduced, and the utilization rate of glucose is obviously improved. Thus, compared to the wild-type,FvPDC6the conversion rate of the carbon source in the fermentation process of the gene editing transformant is improved by about 87 percent (0.194 vs 0.361). This increase in conversion efficiency is expected to reduce the production cost per ton of hyphal protein by at least 9000 yuan (glucose calculated as 4000 yuan per ton).

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

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims (6)

1. A strain of fusarium venenatum, characterized in that said fusarium venenatum is fusarium venenatum TB6050, classified under the following names: fusarium VeneticumFusarium venenatum) Fusarium VeneticumFusarium venenatum) TB6050 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No. 40527, the preservation time is as follows: 2023, 03 and 17, the deposit unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

2. A method for increasing the yield of fusarium venenatum hypha protein, comprising the steps of:

1) Knocking out pyruvate decarboxylase gene in fusariumFvPDC6Obtaining the pyruvate decarboxylase geneFvPDC6Novel Fusarium knocked out, the pyruvate decarboxylase geneFvPDC6For example asThe base sequence of a) or b) below:

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;

2) Inoculating the novel fusarium into a liquid culture medium for culturing to obtain fermentation liquor, and harvesting hypha protein from the grown fermentation liquor.

3. The method for increasing the yield of fusarium venenatum hypha protein of claim 2, wherein the method for obtaining the novel fusarium venenatum comprises the steps of:

taking the DNA genome of fusarium venenatum TB01 as a template, taking a group of primer pairs shown as SEQ ID NO. 13 and 14 as primers, and obtaining an endogenous 5SrRNA promoter sequence through PCR amplification;

the gRNA is obtained by PCR amplification by using the gRNA scaffold fragment shown as SEQ ID No. 18 as a template and a group of primer pairs shown as SEQ ID No. 15 and 16 as primers _FvPDC A sequence;

two rounds of amplification were then performed by fusion PCR, the endogenous 5SrRNA promoter sequence was amplified with the sgRNA _FvPDC The sequences are fused, and the fused fragment is inserted into PacI site of a framework vector pFC322-Cas9 by homologous recombinase to obtainFvPDC6A gene editing expression vector;

the saidFvPDC6The gene editing expression vector is transformed into fusarium venenatum by a protoplast transformation mode to obtain the novel fusarium venenatum.

4. A method of increasing the yield of fusarium venenatum hypha protein of claim 3, wherein the novel fusarium is strain fusarium venenatum TB6050, classified under the following names: fusarium VeneticumFusarium venenatumFusarium venenatum strainFusarium venenatum TB6050 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No. 40527, the preservation time is as follows: 2023, 03 and 17, the deposit unit address is: beijing Kong yangRegional north cinquefoil hospital No. 1, no. 3.

5. The method for increasing the yield of fusarium venenatum hypha protein of claim 2, wherein in step 2) the liquid medium is a fermentation medium comprising: 40 g/L glucose, 0.5g/L yeast powder, 6 g/L ammonium sulfate, 1.5g/L magnesium sulfate, 0.7 g/L potassium chloride, 0.5g/L sodium sulfate, 2 g/L potassium dihydrogen phosphate and 0.5g/L calcium carbonate.

6. Use of the strain fusarium venenatum TB6050 or the knock-out pyruvate decarboxylase gene of claim 1 for increasing the yield of fusarium venenatum hypha protein, wherein the pyruvate decarboxylase geneFvPDC6The deletion of (a) provides a function of promoting the increase of the yield of hyphal protein in Fusarium Veneti, said pyruvate decarboxylase geneFvPDC6The base sequence of a) or b) is 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.