Method for simultaneously increasing production of fusarium mycelium protein and betalain

Abstract

The application discloses a method for simultaneously improving the production of fusarium mycelial protein and betalain, comprising the following steps: transforming an overexpression vector loaded with a cytochrome P450 enzyme gene fragment, a dopa 4,5-dioxygenase gene fragment and a glucosyltransferase gene fragment into fusarium to obtain new fusarium, wherein the cytochrome P450 enzyme gene fragment, the dopa 4,5-dioxygenase gene fragment and the glucosyltransferase gene fragment are constructed as linked genes; inoculating the new fusarium into a liquid culture medium for culture, obtaining fermentation liquor and mycelium respectively, and harvesting betalain from the fermentation liquor and mycelial protein from the mycelium. The application achieves the purpose of improving the comprehensive economic benefits in the production of fusarium mycelial protein by directionally overexpressing a betalain synthesis gene RUBY in fusarium venenatum to obtain a fusarium venenatum strain for efficiently synthesizing betalain.

Description

Technical Field

[0001] This invention relates to the field of Fusarium vesicae genetic engineering, specifically to the construction of expression vectors for betaine-producing Fusarium vesicae and the creation of betaine-producing Fusarium vesicae strains, and more specifically to a method for simultaneously increasing the production of Fusarium mycelial protein and betaine. Background Technology

[0002] Mycelial protein produced by fermentation of Fusarium venetum is safe and provides balanced nutrition, showing potential to replace dietary protein from animal and plant sources. However, the high cost of producing mycelial protein from natural strains is a key factor limiting its development. There are two main ways to overcome this limitation: one is to directly reduce costs by modifying the strain to improve the synthesis efficiency of mycelial protein; the other is to synthesize value-added products within the strain to improve overall economic benefits.

[0003] Betalains are a natural red food coloring extracted from red beet roots, possessing antioxidant, anti-inflammatory, lipid-lowering, and anti-tumor effects. However, considering the low betalain content in beet roots, the relatively low product recovery rate during extraction, and the ever-growing global market demand, a new betalain supply model is urgently needed. Currently, from the perspectives of land use, production cycle, and sustainable large-scale production, microbial fermentation for betalain production offers the most significant advantages. Therefore, creating a high-betalain-producing *Fusarium venetum* strain can not only improve the overall economic efficiency of mycelial protein production and alleviate the current betalain shortage, but also expand the application scope of *Fusarium venetum* in the future food sector. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0005] In view of the needs of the prior art, the purpose of this invention is to provide an expression vector and strain of Fusarium venetum that produces betaine.

[0006] Another objective of this invention is to provide a method for simultaneously increasing the yield of Fusarium mycelium protein and betaine.

[0007] Therefore, the present invention provides the following technical solution:

[0008] The method to simultaneously increase the yield of Fusarium mycelium protein and betaine includes the following steps:

[0009] Overexpression vectors loaded with cytochrome P450 enzyme gene fragments, dopa 4,5-dioxygenase gene fragments, and glucosyltransferase gene fragments were transformed into Fusarium to obtain new Fusarium strains. Among them, the cytochrome P450 enzyme gene fragments, dopa 4,5-dioxygenase gene fragments, and glucosyltransferase gene fragments were constructed as linked genes.

[0010] The new Fusarium was inoculated into a liquid culture medium and cultured to obtain fermentation broth and mycelial cells, respectively. Betalain was harvested from the fermentation broth and mycelial protein was harvested from the mycelial cells.

[0011] Preferably, in the method for simultaneously increasing the production of Fusarium mycelial protein and betaine, the open reading frame linking the cytochrome P450 enzyme gene fragment, the dopa 4,5-dioxygenase gene fragment, and the glucosyltransferase gene fragment together via a 2A peptide is named the RUBY gene.

[0012] Preferably, in the method for simultaneously increasing the production of Fusarium mycelium protein and betaine, the method for obtaining the new Fusarium includes the following steps:

[0013] Using the expression vector addgene id:160908 containing the RUBY gene as a template and the primer pair shown in SEQ ID NO:1 and 2 as primers, the RUBY fragment was amplified. Subsequently, the fragment was ligated to the linearized pK2-PgpdA-Ttef1 vector via homologous recombination to obtain the expression vector pK2-PgpdA-RUBY-Ttef1.

[0014] Using the expression vector pK2-PgpdA-RUBY-Ttef1 as a template and a set of primers shown in SEQ ID NO:3 and 4 as primers, the donor DNA fragment PgpdA-RUBY-Ttef1 for CRISPR / Cas9-mediated homologous recombination insertion was amplified.

[0015] The 5SrRNA promoter sequence fragment shown in SEQ ID NO:13 and the sgRNA shown in SEQ ID NO:15 were combined. GFP The fragments were amplified twice by fusion PCR to obtain 5SrRNA-sgRNA. GFP The fused fragment was inserted into the backbone vector pFC322-Cas9 via homologous recombinase to obtain a CRISPR / Cas9 expression vector that mediates RUBY site-directed integration of the Fusarium vesicaria genome.

[0016] The CRISPR / Cas9 expression vector mediating the site-directed integration of RUBY into the genome of Fusarium venetum and the donor DNA fragment PgpdA-RUBY-Ttef1 were sequentially added to a Fusarium venetum protoplast suspension, cultured, and screened to obtain positive transformants that overexpress the betaine synthesis gene RUBY, thus obtaining the new Fusarium.

[0017] Preferably, in the method for simultaneously increasing the production of Fusarium mycelial protein and betaine, during protoplast transformation, the amount of the CRISPR / Cas9 expression vector mediating RUBY site-directed integration into the Fusarium venetum genome and the donor DNA fragment PgpdA-RUBY-Ttef1 added is both 800 ng / μl.

[0018] Preferably, in the method for simultaneously increasing the production of Fusarium mycelium protein and betaine, the endogenous 5S rRNA promoter fragment is amplified from the DNA genome of Fusarium venetum by PCR using a set of primer pairs as shown in SEQ ID NO:5 and 6, and the sgRNA is amplified using a set of primer pairs as shown in SEQ ID NO:7 and 8 as a template on the gRNAscaffold fragment shown in SEQ ID NO:14. GFP The sequence was then amplified twice via fusion PCR, combining the 5S rRNA promoter fragment with the sgRNA. GFP The fragments are merged.

[0019] Preferably, in the method for simultaneously increasing the yield of Fusarium mycelium protein and betaine, the liquid culture medium comprises the following components: glucose 40 g / L, yeast extract 0.5 g / L, ammonium sulfate 12 g / L, magnesium sulfate 1.5 g / L, potassium chloride 0.7 g / L, sodium sulfate 0.5 g / L, potassium dihydrogen phosphate 2 g / L, and calcium carbonate 0.5 g / L.

[0020] Preferably, in the method for simultaneously increasing the production of Fusarium mycelium protein and betaine, the method of inoculating the new Fusarium in a liquid culture medium for cultivation includes:

[0021] The new Fusarium was inoculated on CMC-Na solid medium to produce sporulation, and then a sporulation suspension was prepared. The sporulation suspension was inoculated on CMC-Na liquid medium and cultured on a shaker for several days, and then the fermentation broth was collected.

[0022] The present invention has at least the following beneficial effects:

[0023] This invention obtains a *Fusarium vesicatoria* strain that efficiently synthesizes betaine by directionally overexpressing the betaine synthesis gene RUBY in *Fusarium vesicatoria*, thereby improving the overall economic benefits of mycelial protein production.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0025] Figure 1 This illustration shows the construction of an overexpression vector for the *Fusarium vesicae* betaine synthase gene *RUBY* in this embodiment of the invention. A is a schematic diagram of the pK2-PgpdA-RUBY-Ttef1 expression vector, and B is a schematic diagram of the donor fragment PgpdA-RUBY-Ttef1. CYP76AD1: cytochrome P450 enzyme; DODA: dopa 4,5-dioxygenase; Glucosyl transferase: glucosyltransferase. The above three enzyme editing genes are named *RUBY* through an open reading frame linked together by a 2A peptide. C shows the enzyme digestion electrophoresis results of the pK2-PgpdA-RUBY-Ttef1 expression vector.

[0026] Figure 2 In this embodiment of the invention, a CRISPR / Cas9 expression vector is constructed to mediate the site-specific integration of RUBY into the genome of Fusarium vesicae. A is a Cas9 and sgRNA expression vector (pFC322-Cas9-5srRNA::sgRNA) targeting a previously introduced GFP expression cassette in the genome. GFP (Diagram, B represents pFC322-Cas9-5srRNA::sgRNA) GFP The expression vector digestion electrophoresis results show that C represents pFC322-Cas9-5srRNA::sgRNA. GFP 5S rRNA-sgRNA in expression vector GFP Sequencing alignment results of the fragments.

[0027] Figure 3 The colony morphology of wild-type and betaine-producing strains of Fusarium venetum in embodiments of the present invention is shown on solid fermentation medium.

[0028] Figure 4 This image shows a shake-flask fermentation diagram of the *Fusarium vesicatoria* strain that produces betaine red pigment in an embodiment of the present invention.

[0029] Figure 5The following are the results of biomass and betalain determination of the betalain-producing Fusarium vesicator strain in this embodiment of the invention. A is the peak diagram of betalain standard detected by UPLC-Q-TOF-MS, B is the peak diagram of fermentation supernatant of engineered strain TBruby detected by UPLC-Q-TOF-MS, C is the standard curve plotted after detection of betalain standard by UPLC-Q-TOF-MS, and D is the biomass and betalain concentration in fermentation supernatant of engineered strain TBruby after fermentation. Detailed Implementation

[0030] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0031] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that the experimental methods in the following embodiments, unless otherwise specified, are conventional methods; and the raw materials, reagents, and materials used in the following embodiments, unless otherwise specified, are commercially available products.

[0032] The present invention will be further illustrated below with specific embodiments to provide a better understanding of the invention, but these embodiments do not constitute a limitation thereof.

[0033] This invention provides an expression vector and strain of Fusarium vesicae that produces betaine, including the cloning of the betaine synthesis gene RUBY (OL411875.1), the construction of a vector for overexpression of the RUBY gene and an expression vector for editing specific sites of Fusarium vesicae, and the acquisition of a betaine-producing strain of Fusarium vesicae.

[0034] Specifically, it includes the following steps:

[0035] 1) Construction of an overexpression vector for the betaine synthase gene RUBY from Fusarium venetum.

[0036] Using the expression vector containing the RUBY gene (addgene id:160908) as a template, the RUBY fragment was amplified using RUBY-1 / 2 primers. Subsequently, the fragment was ligated to the linearized pK2-PgpdA-Ttef1 vector using homologous recombinase to obtain the Fusarium vesileum expression vector pK2-PgpdA-RUBY-Ttef1. Further, using this vector as a template and gpdA-1 / Ttef1-2 primers, the donor fragment PgpdA-RUBY-Ttef1 for CRISPR / Cas9-mediated homologous directional recombination insertion was amplified.

[0037] 2) A CRISPR / Cas9 expression vector mediating RUBY site-directed integration into the *Fusarium vesicae* genome was constructed. The endogenous 5S rRNA promoter sequence (FVRRES_5S_rRNA_393) was amplified from the *Fusarium vesicae* DNA genome using primer pair 5S-1 / 5S-2. The sgRNA was amplified from a synthetically produced gRNA scaffold fragment using primer pair sg-1 / sg-2. GFP Sequence. Subsequently, two rounds of amplification were performed using fusion PCR to combine the 5S rRNA fragment with sgRNA. GFP Fragment fusion yields 5SrRNA-sgRNA GFP The fused fragment was ligated to the backbone vector pFC322-Cas9 via homologous recombinase. After enzyme digestion and sequencing verification, the fragment was found to target the 4 sites on the genome (FvPDC6: Tong, S., Chen, W., Hong, R., Chai, M., Sun, Y., Wang, Q., & Li, D. (2024). Efficient mycoprotein production with low CO2 emissions through metabolic engineering and fermentation optimization in Fusarium venenatum. Journal of Agricultural and Food Chemistry, 72, 604-612; Site 1: Tong, S., An, K., Chen, W., Chai, M., Sun, Y., Wang, Q., & Li, D. (2023). Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01. Synthetic and SystemsBiotechnology, 8, 141-147. (FvdLDH: FVRRES_12081; FvlLDH: FVRRES_12506) The imported GFP expression cassettes are Cas9 and sgRNA expression vectors targeting specific sites.

[0038] 3) Obtaining the betaine-producing Fusarium vesicant strain

[0039] The Cas9 and sgRNA expression vectors and donor DNA were introduced into Fusarium vesicae TB01 using protoplast transformation. Positive transformants that showed site-specific integration and overexpression of RUBY were screened by strain color observation and named TBruby.

[0040] 4) Shake-flask fermentation of betaine-producing Fusarium venetianis strain

[0041] The strain was inoculated onto CMC-Na solid medium and cultured for 10 days, followed by preparation of 5×10⁻⁶ samples. 6 Spore suspension at a concentration of spores / mL. Take 100 μL of the above spore suspension and inoculate it into a 250 mL shake flask containing 50 mL of fermentation medium. Incubate at 28 °C and 180 rpm on a shaker for 4 days.

[0042] 5) Determination of biomass and betalain content of betalain-producing Fusarium vesicanthizobium strains

[0043] The fermentation broth from step 4) was filtered to collect the bacterial cells and cell-free supernatant. The collected bacterial cells were dried in an oven to constant weight, and their biomass (g / L) was calculated. The collected supernatant was filtered and analyzed using ultra-high pressure liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) with an ACQUITY UPLC HSS column. The column was T3 (2.1 x 100 mm, 1.8 μm), with a mobile phase of 0.1% formic acid (phase A) - acetonitrile (phase B), gradient elution: 0–15 min, 1%–99% B; 15–20 min, 99% B; 20–30 min, 99%–1% B; column temperature 30℃; flow rate 0.3 mL / min; injection volume 5 μL; mass spectrometry conditions: ESI electrospray ionization source, IDA mode, scan range m / z 50–1000; spray voltage 5500 V; ion source temperature 550℃; declustering voltage 80 V; collision energy 35 V; collision energy superposition 15 V; curtain gas pressure 35 psi; nebulizer (GS1) and auxiliary gas (GS1) both 55 psi; data acquisition time 30 min. Finally, the concentration of betalain in the sample was calculated based on the standard curve prepared from betalain standards.

[0044] Example 1: Construction of an overexpression vector for the betaine synthase gene RUBY from Fusarium venetum.

[0045] 1. Culture medium

[0046] LB: 10g sodium chloride, 10g peptone, 5g yeast extract, bring to a final volume of 1L. (For solids, add an additional 15g of agarose.)

[0047] 2. Primers

[0048]

[0049]

[0050] 3. Fragment amplification and homologous recombination procedures

[0051] RUBY clip 98℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 2 minutes; 35 cycles of Primestar, TaKaRa gpdA-RUBY-Ttef1 fragment 98℃ for 10 seconds, 55℃ for 15 seconds, 72℃ for 3 minutes; 35 cycles of Primestar, TaKaRa Homologous recombination 50℃30min Minerva Super Fusion Cloning Kit, US Everbright

[0052] 4. Experimental Methods

[0053] Using an expression vector containing the RUBY gene (addgene id:160908) as a template, the RUBY fragment was amplified using primers RUBY-1 / 2 (SEQ ID NO:1 and 2), recovered after electrophoresis, and then ligated to a linearized pK2-PgpdA-Ttef1 vector using homologous recombinase to obtain the *Fusarium vesicae* expression vector pK2-PgpdA-RUBY-Ttef1. This vector was then transformed into *E. coli* DH5α, and single colonies were selected for activation, plasmid extraction, and enzyme digestion verification. Transformants with correct enzyme digestion were further sequenced for confirmation. Using the plasmid extracted from the confirmed transformants as a template, and gpdA-1 / Ttef1-2 (SEQ ID NO:3 and 4) as primers, the donor DNA fragment (PgpdA-RUBY-Ttef1) for CRISPR / Cas9-mediated homologous recombination insertion was amplified.

[0054] 5. Results

[0055] Experimental results are as follows Figure 1 As shown, the electrophoresis results after enzyme digestion revealed the target band, indicating that the pK2-PgpdA-RUBY-Ttef1 expression vector had been successfully constructed, and subsequent sequencing results further confirmed its correctness.

[0056] Example 2: Construction of a CRISPR / Cas9 expression vector mediating site-specific integration of RUBY into the genome of Fusarium vesicatoria 1. Culture medium

[0057] LB: 10g sodium chloride, 10g peptone, 5g yeast extract, bring to a final volume of 1L. (For solids, add an additional 15g of agarose).

[0058] 2. Primers

[0059] 5S-1 TAGCTGTTTCGCTGAGGGTTTAATTAAACATACGACCAAAGGTAGTGG 5S-2 TGATATAGACGTTGTGGCTGAACATACAACAGCGGGGATTCG sg-1 CAGCCACAACGTCTATATCAGTTTTAGAGCTAGAAATAGC sg-2 CTGCTGTCTCGGCTGAGGTCTTAATTAAAAAAAAAGCACCGACTCGGTGCC

[0060] 3. Fragment amplification and homologous recombination procedures

[0061]

[0062] 4. Experimental Methods

[0063] The endogenous 5S rRNA promoter sequence (FVRRES_5S_rRNA_393) was amplified from the DNA genome of Fusarium vesicae using primer pairs 5S-1 / 5S-2 (SEQ ID NO: 5 and 6), and sgRNA was amplified from an artificially synthesized gRNA scaffold fragment using primer pairs sg-1 / sg-2 (SEQ ID NO: 7 and 8). GFP Sequence. Subsequently, two rounds of amplification were performed using fusion PCR to combine the 5S rRNA fragment with sgRNA. GFP Fragment fusion yields 5SrRNA-sgRNA GFP The fused fragment was ligated to the backbone vector pFC322-Cas9 via homologous recombinase. After enzyme digestion and sequencing verification, the fragment was found to target the 4 sites on the genome (FvPDC6: Tong, S., Chen, W., Hong, R., Chai, M., Sun, Y., Wang, Q., & Li, D. (2024). Efficient mycoprotein production with low CO2 emissions through metabolic engineering and fermentation optimization in Fusarium venenatum. Journal of Agricultural and Food Chemistry, 72, 604-612; Site 1: Tong, S., An, K., Chen, W., Chai, M., Sun, Y., Wang, Q., & Li, D. (2023). Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01. Synthetic and Systems Biotechnology, 8, 141-147. (FvdLDH: FVRRES_12081; FvlLDH: FVRRES_12506) The imported GFP expression cassettes are Cas9 and sgRNA expression vectors targeting specific sites.

[0064] 5. Results

[0065] Experimental results are as follows Figure 2As shown, the enzyme digestion and sequencing results indicate that the Cas9 and sgRNA expression vector pFC322-Cas9-5srRNA::sgRNA, which targets the previously introduced GFP expression cassette in the genome, has been successfully constructed. GFP .

[0066] Example 3: Obtaining the betaine-producing Fusarium vesicatoria strain

[0067] 1. Culture medium

[0068] CMC-Na solid culture medium: 10g CMC-Na, 5g ammonium sulfate, 1g yeast extract, 1.5g magnesium sulfate, 2g potassium dihydrogen phosphate, 0.7g potassium chloride, 0.5g sodium sulfate, 15g agarose, bring to a final volume of 1L.

[0069] YEPD: 3g yeast powder, 10g peptone, 20g glucose, bring to a final volume of 1L.

[0070] Buffer for dissolving enzyme: 0.7M sodium chloride

[0071] STC: 0.8M sorbitol; 50mM CaCl2, 50mM Tris-HCl (Ph 8.0)

[0072] SPTC: STC containing 40% PEG6000

[0073] Regeneration medium: 1g yeast extract, 1g tryptone, 274g sucrose, 10g agarose, bring to a final volume of 1L. LGY solid medium: 30g glucose, 6g yeast extract, 15g agarose, bring to a final volume of 1L.

[0074] Solid fermentation medium: 40g glucose, 0.5g yeast extract, 12g ammonium sulfate, 1.5g magnesium sulfate, 0.7g potassium chloride, 0.5g sodium sulfate, 2g potassium dihydrogen phosphate, 0.5g calcium carbonate, 15g agarose, bring the volume to 1L.

[0075] 2. Experimental Methods

[0076] 1) Prepare a suspension of Fusarium venetum spores using Tween 80, spread it on CMC-Na solid medium, and incubate at 28℃ for about 10 days to produce spores;

[0077] 2) Prepare spore suspension and inoculate it into YEPD liquid medium (with glass beads), and culture at 28℃ and 200 rpm until the germinating hyphae are 3-4 times the length of the spores (16h);

[0078] 3) Collect spores at 4℃, 13000rpm, and 15min, and wash once with sterile 0.7M sodium chloride;

[0079] 4) Preparation of protoplasts using enzyme lysis buffer (20 mg of lysis enzyme + 40 mg of snail enzyme dissolved in 10 ml of 0.7 M sodium chloride and filtered for sterilization), lysed at 32℃ and 130 rpm for 2 h;

[0080] 5) After filtering with three layers of lens paper, incubate at 4℃, 7000rpm for 10min;

[0081] 6) Wash twice with STC, centrifuge as described above, resuspend the protoplasts with STC, and store on ice for later use;

[0082] 7) Take 80 μl of the above protoplast suspension, add 20 μl of SPTC, mix gently, then add 20 μl (800 ng / μl) of gene editing expression vector and donor DNA fragment in sequence, mix gently and place on ice for 30 min;

[0083] 8) Add 1 ml SPTC and mix gently. Let stand at room temperature for 20 minutes.

[0084] 9) Add the mixture to the regeneration medium at about 40℃, shake well, pour into a plate, and incubate at 28℃ overnight (about 12 hours);

[0085] 10) Pour on GY solid screening medium and incubate at 28°C until colonies grow.

[0086] 11) Select the above colonies and transfer them to solid fermentation medium for growth until the colonies turn red; and use this as the screening basis to select positive transformants that overexpress the betaine synthesis gene RUBY.

[0087] 3. Results

[0088] The results are as follows Figure 3 As shown, compared with the wild-type Fusarium venetum strain, the transformant strain exhibited a distinct red color on the solid fermentation medium, indicating successful overexpression of the beet red synthesis gene RUBY in Fusarium venetum TB01. Subsequent fermentation of the transformant strain followed by collection of the fermentation supernatant was performed for identification and concentration determination of the red beet red pigment.

[0089] Example 4: Shake-flask fermentation of betaine-producing Fusarium venetianis strain

[0090] 1. Culture medium

[0091] CMC-Na solid culture medium: 10g CMC-Na, 5g ammonium sulfate, 1g yeast extract, 1.5g magnesium sulfate, 2g potassium dihydrogen phosphate, 0.7g potassium chloride, 0.5g sodium sulfate, 15g agarose, bring to a final volume of 1L.

[0092] Liquid fermentation medium: 40g glucose, 0.5g yeast powder, 12g ammonium sulfate, 1.5g magnesium sulfate, 0.7g potassium chloride, 0.5g sodium sulfate, 2g potassium dihydrogen phosphate, 0.5g calcium carbonate, bring to a final volume of 1L.

[0093] 2. Experimental Methods

[0094] The *Fusarium vesicae* strain was inoculated onto CMC-Na solid medium and cultured for 10 days to induce sporulation, followed by preparation of 5×10⁻⁶ sporulation media. 6 A spore suspension with a concentration of conidia / mL was prepared. 100 μL of the above spore suspension was inoculated into a 250 mL shake flask containing 50 mL of fermentation medium and cultured on a shaker at 28 °C and 180 rpm for 4 days. Subsequently, the bacterial cells and fermentation broth were collected separately by vacuum filtration.

[0095] 3. Results

[0096] Experimental results are as follows Figure 4 As shown, the bacterial broth from the strain overexpressing the RUBY gene exhibited a distinctly red color after fermentation in liquid fermentation medium. The broth was then filtered, and the supernatant and bacterial cells were collected separately. A bright dark red color was observed in the supernatant, indicating that the betalain synthesized in the engineered strain was primarily secreted extraspores. Further analysis will utilize ultra-high pressure liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to identify whether the red substance in the fermentation supernatant is betalain, and a standard curve will be plotted using standards for quantification of the sample.

[0097] Example 5: Determination of biomass and betalain content of *Fusarium vesicatoria* strain producing betalains

[0098] 1. Experimental Methods

[0099] The bacterial cells from Example 4, fermented for 4 days, were filtered to harvest both the bacterial cells and the cell-free supernatant. The collected bacterial cells were dried in an oven to constant weight, and their biomass (g / L) was calculated. The collected supernatant was filtered and analyzed using ultra-high pressure liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The chromatographic column used was ACQUITYUPLC HSS T3 (2.1 x 100 mm, 1.8 μm), the mobile phase was 0.1% formic acid (phase A) - acetonitrile (phase B), with gradient elution: 0–15 min, 1%–99% B; 15–20 min, 99% B; 20–30 min, 99%–1% B; column temperature 30℃; flow rate 0.3 mL / min; injection volume 5 μL; mass spectrometry conditions: ESI electrospray ionization source, IDA mode, scan range m / z. 50-1000; spray voltage 5500V; ion source temperature 550℃; declusivation voltage 80V; collision energy 35V; collision energy superposition 15V; curtain gas pressure 35psi; nebulizer (GS1) and auxiliary gas (GS1) both 55psi; data acquisition time 30min. Finally, the concentration of betalain in the sample was calculated based on the standard curve prepared from the betalain standard.

[0100] 2. Experimental Results

[0101] The results are as follows Figure 5 As shown, the elution times of the fermentation supernatant and the betalain standard were consistent when detected by UPLC-Q-TOF-MS, indicating that the red substance in the fermentation supernatant was indeed betalain. Subsequently, a standard curve established using the betalain standard was used to further quantify the betalain concentration in the fermentation broth, which was found to be 1.16 g / L; at this point, the bacterial biomass was 8.57 g / L, indicating that our engineered strain TBruby possesses the potential to simultaneously and efficiently synthesize both mycelial protein and betalain.

[0102] RUBY nucleotide sequence (SEQ ID NO:9)

[0103] ATGGATCATGCGACCCTCGCCATGATCCTCGCGATCTGGTTCATCAGCTTCCACTTCATCAAGCTGCTGTTCTCCAGCAGACCACCAAGC

[0104] TGCTTCCGCCAGGACCAAAGCCGCTTCCGATCATCGGCAACATCCTTGAGGTGGGCAAGAAGCCGCATCGGTCCTTCGCCAACCTCGCCA

[0105] AGATTCACGGCCCACTCATTTCCCTCAGACTCGGCTCTGTGACCACCATCGTTGTGTCCTCTGCCGACGTGGCCAAAGAGATGTTCCTCAA

[0106] GAAGGATCACCCGCTCTCCAACCGCACGATCCCGAATAGTGTTACAGCCGGCGACCACCACAAGCTCACCATGTCTTGGCTCCCGGTGTCT

[0107] CCGAAGTGGCGCAACTTCCGCAAGATTACCGCCGTGCATCTGCTCTCCCCACAGAGACTCGATGCCTGCCAGACATTCAGGCACGCCAAG

[0108] GTGCAGCAGCTCTACGAGTACGTTCAAGAGTGCGCCCAGAAAGGCCAGGCCGTGGATATTGGCAAGGCCGCCTTTACGACCAGCCTCAAC

[0109] CTCCTCAGCAAGCTGTTCTTCAGCGTCGAGCTGGCGCACCACAAGTCCCATACCAGCCAAGAGTTCAAAGAGCTGATCTGGAACATCATG

[0110] GAAGATATAGGCAAGCCGAACTACGCCGACTACTTCCCGATTCTCGGCTGCGTTGACCCATCTGGCATTAGAAGAAGGCTCGCCTGCTCCT

[0111] TCGACAAGCTGATCGCCGTGTTCCAGGGCATCATCTGCGAGAGACTCGCCCCAGATTCCTCCACCACAACTACCACCACCACCGACGACG

[0112] TGCTCGATGTGCTCCTCCAGCTGTTCAAGCAGAACGAGCTGACGATGGGCGAGATCAACCACCTCCTCGTGGACATCTTCGACGCCGGCA

[0113] CCGATACCACATCCTCCACATTCGAGTGGGTGATGACCGAGCTGATCCGCAATCCAGAGATGATGGAAAAGGCCCAAGAGGAAATCAAG

[0114] CAGGTCCTCGGCAAGGACAAGCAGATCCAAGAGTCCGACATCATCAACCTGCCGTACCTCCAGGCGATCATCAAAGAGACACTCCGCCTC

[0115] CATCCGCCGACCGTGTTCTTGCTCCCAAGAAAGGCCGACACCGATGTCGAGCTGTACGGCTACATCGTGCCGAAGGATGCCCAGATCCTC

[0116] GTGAACCTCTGGGCCATTGGCAGGGACCCAAACGCCTGGCAGAACGCCGATATTTTCAGCCCAGAGCGCTTCATCGGCTGCGAGATCGAT

[0117] GTTAAGGGCCGCGATTTCGGCCTCCTTCCATTTGGCGCTGGCCGCAGAATTTGCCCAGGCATGAATCTCGCCATCAGGATGCTCACCCTCA

[0118] TGCTCGCCACACTCCTCCAGTTCTTCAACTGGAAGCTCGAAGGCGACATCTCCCCGAAGGACCTCGACATGGACGAGAAGTTCGGCATTG

[0119] CGCTCCAAAAGACCAAGCCGCTCAAGCTCATCCCGATTCCGCGCTACGGTAGCGGAGCTACCAATTTTAGCCTCCTTAAGCAGGCAGGTG

[0120] ATGTAGAAGAGAACCCCGGGCCTATGAAGATGATGAACGGCGAGGACGCCAACGACCAGATGATCAAAGAGTCCTTCTTCATCACCCAC

[0121] GGCAACCCGATCCTCACCGTCGAGGATACACATCCGCTCAGGCCGTTCTTCGAGACATGGCGCGAGAAGATTTTCTCCAAGAAGCCGAAG

[0122] GCCATCCTCATCATCTCCGGCCACTGGGAGACAGTGAAGCCAACCGTGAACGCCGTGCACATCAACGACACCATCCACGACTTCGACGAC

[0123] TACCCAGCCGCCATGTACCAGTTCAAGTACCCAGCTCCAGGCGAGCCAGAGCTTGCGAGAAAGGTGGAAGAGATCCTCAAGAAGTCCGG

[0124] GTTCGAGACAGCCGAGACAGACCAAAAGAGGGGCCTTGATCACGGCGCCTGGGTTCCACTCATGCTCATGTATCCAGAGGCGGACATCCC

[0125] GGTGTGCCAGCTCTCAGTTCAGCCACATCTCGACGGCACCTACCACTACAATCTCGGCAGAGCCCTCGCGCCGCTCAAGAATGATGGCGT

[0126] GCTCATTATTGGCTCCGGCAGCGCCACACATCCACTCGATGAGACACCGCACTACTTCGATGGTGTTGCCCCTTGGGCCGCTGCCTTCGAT

[0127] TCTTGGCTTAGGAAGGCCCTCATCAACGGCCGCTTCGAGGAAGTGAACATCTACGAGAGCAAGGCCCCGAACTGGAAGCTCGCCCATCCA

[0128] TTTCCAGAGCACTTCTACCCGCTCCACGTTGTGCTCGGCGCTGCTGGTGAAAAGTGGAAGGCCGAGCTGATCCACTCCTCCTGGGATCATG

[0129] GCACACTTTGCCACGGCTCCTACAAGTTCACCTCCGCCGGATCCGGAGCAACCAACTTTAGCCTGCTCAAGCAAGCAGGAGATGTTGAGG

[0130] AAAATCCTGGCCCCATGACCGCCATCAAGATGAACACCAACGGCGAGGGCGAGACACAGCACATCCTCATGATCCCGTTCATGGCGCAG

[0131] GGCCACCTCAGGCCATTTCTCGAACTCGCCATGTTCCTCTACAAGCGCTCCCACGTGATCATCACCCTGCTCACAACTCCGCTCAATGCCG

[0132] GCTTCCTCAGGCACCTCCTTCACCACCATTCCTACTCCTCCAGCGGCATCAGGATCGTCGAGCTGCCATTCAACTCCACCAACCACGGACT

[0133] CCCACCGGGCATCGAGAACACCGATAAGCTCACACTCCCGCTCGTGGTGTCCCTCTTCCATTCCACCATCAGCCTCGATCCGCACCTCCGC

[0134] GATTACATCTCCAGGCATTTCAGCCCAGCCAGGCCACCACTCTGCGTGATCCATGATGTGTTCCTCGGCTGGGTTGACCAGGTGGCCAAGG

[0135] ATGTGGGCTCTACAGGCGTGGTGTTCACAACAGGCGGCGCTTATGGCACATCCGCCTACGTGTCCATCTGGAACGATCTCCCGCACCAGA

[0136] ACTACTCCGACGACCAAGAGTTCCCGCTGCCAGGCTTCCCAGAGAACCATAAGTTCCGCAGGTCCCAGCTCCATCGGTTCCTCAGATATGC

[0137] CGACGGCTCCGACGATTGGTCCAAGTATTTCCAGCCGCAGCTCCGCCAGTCCATGAAGTCTTTTGGCTGGCTCTGCAACTCCGTGGAAGAG

[0138] ATCGAGACACTCGGCTTCTCCATCCTCCGCAACTACACCAAGCTGCCGATCTGGGGCATCGGCCCACTTATTGCTTCCCCAGTGCAGCACT

[0139] CCTCCTCCGACAACAATTCAACAGGCGCCGAGTTCGTGCAGTGGCTCAGCCTCAAAGAGCCGGACTCCGTCCTCTACATCTCCTTCGGCTC

[0140] CCAGAACACGATCAGCCCGACGCAGATGATGGAACTCGCTGCTGGCCTTGAGTCCTCCGAGAAGCCATTCCTCTGGGTGATCAGAGCCCC

[0141] GTTCGGCTTCGACATCAACGAAGAGATGCGCCCAGAGTGGCTGCCAGAGGGCTTTGAGGAACGCATGAAGGTGAAGAAACAGGGCAAGC

[0142] TCGTGTACAAGCTCGGCCCGCAGCTTGAGATCCTCAACCATGAATCCATCGGCGGCTTTCTCACCCACTGCGGATGGAACAGCATCCTTGA

[0143] GTCTCTTCGCGAGGGCGTTCCGATGCTTGGATGGCCACTTGCTGCCGAGCAGGCCTACAACCTCAAGTACCTCGAAGATGAGATGGGCGT

[0144] CGCGGTTGAGCTTGCTAGAGGCCTCGAAGGCGAGATCTCCAAAGAGAAGGTCAAGCGCATCGTCGAGATGATCCTTGAGCGCAACGAGG

[0145] GCTCCAAAGGCTGGGAGATGAAGAATCGCGCCGTGGAAATGGGCAAAAAGCTCAAGGACGCCGTGAACGAGGAAAAAGAGCTGAAGGG

[0146] CTCCTCCGTGAAGGCGATCGACGATTTCCTCGACGCCGTCATGCAGGCCAAACTTGAGCCAAGCCTCCAGTGA

[0147] Amino acid sequence of RUBY (SEQ ID NO: 10)

[0148] MDHATLAMILAIWFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNILEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSN

[0149] RTIPNSVTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECAQKGQAVDIGKAAFTTSLNLLSKLFFSVEL

[0150] AHHKSHTSQEFKELIWNIMEDIGKPNYADYFPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFKQNELTMGE

[0151] INHLLVDIFDAGTDTTSSTFEWVMTELIRNPEMMEKAQEEIKQVLGKDKQIQESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPK

[0152] DAQILVNLWAIGRDPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQFFNWKLEGDISPKDLDMDEKFGI

[0153] ALQKTKPLKLIPIPRYGSGATNFSLLKQAGDVEENPGPMKMMNGEDANDQMIKESFFITHGNPILTVEDTHPLRPFFETWREKIFSKKPKAILIISG

[0154] HWETVKPTVNAVHINDTIHDFDDYPAAMYQFKYPAPGEPELARKVEEILKKSGFETAETDQKRGLDHGAWVPLMLMYPEADIPVCQLSVQPHL

[0155] DGTYHYNLGRALAPLKNDGVLIIGSGSATHPLDETPHYFDGVAPWAAAFDSWLRKALINGRFEEVNIYESKAPNWKLAHPFPEHFYPLHVVLG

[0156] AAGEKWKAELIHSSWDHGTLCHGSYKFTSAGSGATNFSLLKQAGDVEENPGPMTAIKMNTNGEGETQHILMIPFMAQGHLRPFLELAMFLYKR

[0157] SHVIITLLTTPLNAGFLRHLLHHHSYSSSGIRIVELPFNSTNHGLPPGIENTDKLTLPLVVSLFHSTISLDPHLRDYISRHFSPARPPLCVIHDVFLGW

[0158] VDQVAKDVGSTGVVFTTGGAYGTSAYVSIWNDLPHQNYSDDQEFPLPGFPENHKFRRSQLHRFLRYADGSDDWSKYFQPQLRQSMKSFGWLC

[0159] NSVEEIETLGFSILRNYTKLPIWGIGPLIASPVQHSSSDNNSTGAEFVQWLSLKEPDSVLYISFGSQNTISPTQMMELAAGLESSEKPFLWVIRAPFG

[0160] FDINEEMRPEWLPEGFEERMKVKKQGKLVYKLGPQLEILNHESIGGFLTHCGWNSILESLREGVPMLGWPLAAEQAYNLKYLEDEMGVAVELA

[0161] RGLEGEISKEKVKRIVEMILERNEGSKGWEMKNRAVEMGKKLKDAVNEEKELKGSSVKAIDDFLDAVMQAKLEPSLQ

[0162] PgpdA promoter sequence (SEQ ID NO:11):

[0163] GGTGTTGATCGTCAACCAAGTCCGTTCACCGGAGCTTCCGGAGAGGAAGAAAAAAAAAACATCTGATATGTTTGTAACAAATTTGCTGAA

[0164] TCCATTGAACTGAATTGAAAGACATCACGGAGAGTTGCACGGTAGAATACGGTGTCTTATTCGGCGGCACCTACAGAAAAAACAGTATCC

[0165] ATCCATTTCGTCTCTGTGAGGAGGAGGATGGATGGACAGAAATGGAATCAACTGAGGTGGCATGGCATGGCATGAGGGGTAGAAAAGGT

[0166] ACTTTTTAATAGAGTGAGGCGTGCATAATGGGACATGTATCTTTTTCAGGGCATCAATCATCACCCCACCATAATGTATTGTAGCTATTGT

[0167] CAGTAGTGGGGACCTTAGCTCACCGAGCTGTGGAAAGTGAGCATGATATGCTCTGTACCTAAGGACATTAACTTGTAAAGCTGTAAATA

[0168] TATTAGTTAGTTCTTATAAAACATTGAAAGGTTAAGTATTACCAAGTCATGGCAACGGGATTCTATGTCCAGCTATCAAGGTACTAGACAG

[0169] CCGTTGAGAGGGGAAGGAAAAAAAAAAAGTCTCCCACCCTCTCACCGATATTCCATGTTCCATTGGCCCCTGTTATGATACGCTACCTAC

[0170] ACTACGATTACCTACCTACCTACTATACTGTACGCCCCCTTGTCTGGCCTGGCCTAGCACCCTGGCAGTCTGGGTCTGGTCTGGGGTGTGTG

[0171] TTCGTGGGATGATGAATGATGTGGTGGGTGGGTTACTAAACTAAACATGAGCTTGATGAGAGAGTCCATGGTTTTCGATTATTAGTATTTG

[0172] TACTCCCACCCTTTCTCCTCGACCTTTTTTCTTTTTCCTCTCTTTTCTTTTCATTCTCATCATCCTCAACAACACAACAACTACTTCTTTCTTC

[0173] ACCAAAAGGTATGTGTCTTACCTGTCTAGCTTTAGCTCTCTAGCTTTGCCTGTCTCTGCCCCTCCATTCTCAGGATTGCTTGGCTGGTTCTAC

[0174] CCCTCCATTCCGCGCGACTACATCACCATCATAACATCACAACATGATCTCGTTGCTTCCATGCTACATCATGTCATATCATACCTGCTTCT

[0175] TATCACCACTCTGTGATGATACGTCAACTATTGGCATTCATCCATTGAGCGGATAACAAGGGGAGGGGAGCAATTGGAAGGCAAAAAAA

[0176] AAAAGGGACAAGAAAAAAGACAGAGCTTCAGAGCTCATATCCTGCCCCTCCTTTGCACCAACGTTGTCCCCTCCCCTCCACTTTCTTCTAC

[0177] TGTGGCAACACCCGTCCTCACCAAAAAAAGCTCTCTTTACATACCATACCATACCATACCATACCATACCATACCTGCCTCTGATCTGATC

[0178] TGTCCCACTCCGTCATACAGACGGCATATAAAATACCCCACTCCCTCGCCTCAACCTTTGCTCTTCCTCCTCATCTTCTTCTCTATCACCATC

[0179] AATCCTAAAGGATAAACAAAACATCTCATCTAACAATCTTTCCTCAAACAGAACCTCCTTAACAAA

[0180] Ttef1 terminator sequence (SEQ ID NO:12):

[0181] ACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATGTCAGCTCCGGAGTTGAGACAAATGG

[0182] TGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATAGCTCCATGTCAACAAGAATAAAA

[0183] CGCGTTTTCGGGTTTACCTCTTCCAGATACAGCTCATCTGCAATGCATTAATGCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGC

[0184] GAAGAGAAGAATAGCTTAGCAGAGCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACTGA

[0185] GGAATCCGCTCTTGGCTCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTTACTCTGATAGCTTGACTATGAA

[0186] AATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTTCCTTGAACTCTCAAGCCTACAGGACACACATTCATCGTAGGTA

[0187] TAAACCTCGAAATCANTTCCTACTAAGATGGTATACAATAGTAACCATGCATGGTTGCCTAGTGAATGCTCCGTAACACCCAATACGCCG

[0188] GCCGAAACTTTTTTACAACTCTCCTATGAGTCGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGG

[0189] 5S rRNA promoter nucleotide sequence (SEQ ID NO:13):

[0190] ACATACGACCAAAGGTAGTGGAAAATACGGGATCCCGTCCGCTCTCCCATAGTCAAGCCACTAACCGGCGGATTAG

[0191] TAGTTGGGTCGGTGACGACCAGCGAATCCCCGCTGTTGTATGTT gRNA scaffold nucleotide sequence (SEQ ID NO:14):

[0192] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG

[0193] GCACCGAGTCGGTGCTTTTTTT sgRNAGFP nucleotide sequence (SEQ ID NO:15):

[0194] CAGCCACAACGTCTATATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC

[0195] GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0196] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0197] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for simultaneously increasing the yield of Fusarium mycelium protein and betaine, characterized in that, Includes the following steps: Overexpression vectors loaded with cytochrome P450 enzyme gene fragments, dopa 4,5-dioxygenase gene fragments, and glucosyltransferase gene fragments were transformed into *Fusarium vesicae* TB01 to obtain new *Fusarium* strains. The cytochrome P450 enzyme gene fragment, dopa 4,5-dioxygenase gene fragment, and glucosyltransferase gene fragment were constructed as linked genes. The open reading frame linking the cytochrome P450 enzyme gene fragment, dopa 4,5-dioxygenase gene fragment, and glucosyltransferase gene fragment through a 2A peptide was named... RUBY The gene, whose nucleotide sequence is shown in SEQ ID NO:9; The new Fusarium was inoculated into a liquid culture medium and cultured to obtain fermentation broth and mycelial cells, respectively. Betalain was harvested from the fermentation broth and mycelial protein was harvested from the mycelial cells.

2. The method for simultaneously increasing the yield of Fusarium mycelium protein and betaine as described in claim 1, characterized in that, The method for obtaining the new Fusarium includes the following steps: To include RUBY Using the gene expression vector addgene id: 160908 as a template, and the primer pair shown in SEQ ID NO: 1 and 2 as primers, the gene was amplified. RUBY The fragment was then linked to the linearized pK2-PgpdA-Ttef1 vector via homologous recombination to obtain the expression vector pK2-PgpdA-RUBY-Ttef1. Using the expression vector pK2-PgpdA-RUBY-Ttef1 as a template and a set of primers shown in SEQ ID NO:3 and 4 as primers, the donor DNA fragment PgpdA-RUBY-Ttef1 for CRISPR / Cas9-mediated homologous recombination insertion was amplified. The 5SrRNA promoter sequence fragment shown in SEQ ID NO:13 and the sgRNA shown in SEQ ID NO:15 were combined. GFP The fragments were amplified twice by fusion PCR to obtain 5SrRNA-sgRNA. GFP The fused fragment was inserted into the backbone vector pFC322-Cas9 via homologous recombinase to obtain a CRISPR / Cas9 expression vector that mediates RUBY site-directed integration of the Fusarium vesicaria genome. Using a protoplast transformation method, the CRISPR / Cas9 expression vector mediating RUBY site-directed integration into the *Fusarium vesicae* genome and the donor DNA fragment PgpdA-RUBY-Ttef1 were sequentially added to a *Fusarium vesicae* protoplast suspension, cultured, and screened to obtain overexpression of the betaine synthesis gene. RUBY The positive transformants were used to obtain the new Fusarium.

3. The method for simultaneously increasing the yield of Fusarium mycelium protein and betaine as described in claim 2, characterized in that, During protoplast transformation, the amount of both the CRISPR / Cas9 expression vector mediating RUBY site-directed integration into the Fusarium vesicator genome and the donor DNA fragment PgpdA-RUBY-Ttef1 added was 800 ng / μl.

4. The method for simultaneously increasing the yield of Fusarium mycelium protein and betaine as described in claim 2, characterized in that, Using the primer pairs shown in SEQ ID NO:5 and 6, the endogenous 5S rRNA promoter fragment was amplified from the DNA genome of Fusarium vesicae by PCR, and the sgRNA was amplified using the gRNA scaffold fragment shown in SEQ ID NO:14 as a template with the primer pairs shown in SEQ ID NO:7 and 8. GFP The sequence was then amplified twice via fusion PCR, combining the 5S rRNA promoter fragment with the sgRNA. GFP The fragments are merged.

5. The method for simultaneously increasing the yield of Fusarium mycelium protein and betaine as described in claim 1, characterized in that, The liquid culture medium contains the following components: 40 g / L glucose, 0.5 g / L yeast extract, 12 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.

6. The method for simultaneously increasing the yield of Fusarium mycelium protein and betaine as described in claim 1, characterized in that, The method of inoculating the novel Fusarium in a liquid culture medium for cultivation includes: The new Fusarium was inoculated on CMC-Na solid medium to produce sporulation, and then a sporulation suspension was prepared. The sporulation suspension was inoculated into the liquid medium and cultured on a shaker for several days, and then the fermentation broth was collected.

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