A method for adjusting the content of amino acids in a turkey tail mushroom

Abstract

The application discloses a method for adjusting the content of amino acids in chicken mushroom, and belongs to the field of genetic engineering. The content of amino acids in the chicken mushroom genetic engineering bacteria is improved by overexpressing glutamate dehydrogenase in the chicken mushroom. The chicken mushroom obtained by the application is genetically stable and contains high content of umami and sweet amino acids. The chicken mushroom overexpressing TcGDH gene constructed by the application can be genetically stable; the content of sodium glutamate reaches 3.41 mg / kg, which is increased by 90.5% compared with the wild type; the contents of umami amino acids aspartic acid and glutamic acid reach 0.67 mg / kg and 2.75 mg / kg respectively; the content of sweet amino acid alanine reaches 3.20 mg / kg; the content of cysteine as a precursor of meat flavor flavor synthesis reaches 1.15 mg / kg; and the content of amino acids reaches 23.489 mu mol / g fresh weight, which is increased by 49.4% compared with the wild type.

Description

Technical Field

[0001] The invention relates to a method for adjusting the amino acid content in Albizia albuminosa, and belongs to the field of genetic engineering. Background Art

[0002] Termitomyces albuminosus, a nutritious edible mushroom, is widely popular for its unique flavor and texture. It contains several essential amino acids, including lysine, leucine, and tryptophan, which play important roles in human health. However, the amino acid content in Termitomyces albuminosus is relatively low, limiting its nutritional value and potential for commercial application. Increasing the amino acid content of Termitomyces albuminosus could not only enhance its nutritional value but also improve its market competitiveness and economic benefits.

[0003] Methods for increasing the amino acid content of chicken mushroom mainly include optimizing culture medium formula and adjusting growth conditions. However, these methods have certain technical bottlenecks and applications. The document "Screening of nutrient components of chicken mushroom mother culture medium and optimization of culture conditions; Heilongjiang Agricultural Science. 2018 (11)" discloses the use of six indicators, namely carbon source, nitrogen source, amino acid, vitamin, temperature and pH, to explore the suitable culture components and culture conditions of chicken mushroom mother culture through experiments to meet the rapid growth of chicken mushroom. The document "Artificial cultivation technology of chicken mushroom; China Agricultural Technology Extension. 2024, 40 (02)" discloses the optimal temperature, light intensity, and pH of chicken mushroom, realizing artificial cultivation. Although the above methods of optimizing culture medium formula and adjusting growth conditions are relatively simple, their effects are limited and their stability is insufficient.

[0004] Therefore, exploring and developing more effective, stable and feasible technical means to significantly increase the amino acid content in Albizia albuminosa has important research value and application prospects. Summary of the Invention

[0005] To address the above issues, the present invention increases the amino acid content in the genetically engineered strain of Albizia albuminosa by overexpressing glutamate dehydrogenase in the strain. The Albizia albuminosa constructed by the present invention is genetically stable and contains high levels of umami and sweet amino acids.

[0006] The first object of the present invention is to provide a genetically engineered strain of Albizia albuminosa, which overexpresses glutamate dehydrogenase (TcGDH). The amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.1.

[0007] In one embodiment, the glutamate dehydrogenase nucleotide sequence is shown as SEQ ID NO.2.

[0008] In one embodiment, TcGDH is derived from (Termitomyces clypeatus) Tc01, classified and named Termitomyces clypeatus Tc01, and was deposited in the China Center for Type Culture Collection on December 29, 2021, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20211695.

[0009] In one embodiment, the preparation method comprises:

[0010] The glutamate dehydrogenase nucleotide sequence is amplified and connected with a plasmid vector to obtain a recombinant plasmid that overexpresses glutamate dehydrogenase; the recombinant plasmid is transferred into Agrobacterium tumefaciens to induce and obtain induced cultured Agrobacterium tumefaciens; the induced cultured Agrobacterium tumefaciens is mixed with mycelium of Albizia albuminosa, cultured and verified to obtain genetically engineered Albizia albuminosa bacteria.

[0011] In one embodiment, the plasmid vector is plasmid 1.

[0012] In one embodiment, the Agrobacterium tumefaciens is Agrobacterium tumefaciens GV3101.

[0013] The second object of the present invention is to provide a method for increasing the total free amino acid content in Albizia albuminosa, constructing an engineered Albizia albuminosa bacterium, wherein the engineered Albizia albuminosa bacterium overexpresses glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.1.

[0014] The third object of the present invention is to provide a method for simultaneously increasing the content of monosodium glutamate, umami amino acids, and sweet amino acids in Albizia albuminosa, and reducing the content of bitter amino acids in Albizia albuminosa, and constructing a genetically engineered strain of Albizia albuminosa, wherein the genetically engineered strain overexpresses glutamate dehydrogenase, and the amino acid sequence of glutamate dehydrogenase is shown in SEQ ID NO.1.

[0015] In one embodiment, the umami amino acids include one or more of glutamic acid and aspartic acid;

[0016] Sweet amino acids include one or more of alanine, glycine, serine, proline, and threonine;

[0017] Bitter amino acids include one or more of arginine, histidine, isoleucine, leucine, methionine, phenylalanine, tyrosine and valine.

[0018] The fourth object of the present invention is to provide a method for increasing the cysteine ​​content in Albizia albuminosa, constructing a genetically engineered strain of Albizia albuminosa, wherein the genetically engineered strain overexpresses glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.1.

[0019] A fifth object of the present invention is to provide the use of any of the above-mentioned genetically engineered strains of Alpinia albuminosa or any of the above-mentioned methods in the preparation of amino acids or amino acid salts.

[0020] In one embodiment, the amino acids include one or more of glutamic acid, aspartic acid, alanine, glycine, serine, proline, and threonine.

[0021] The sixth object of the present invention is to provide a method for increasing the glutamic acid content in Albizia albuminosa, using an improved PDA medium to culture the genetically engineered Albizia albuminosa.

[0022] The genetically engineered strain of Alternaria albuminosa overexpresses glutamate dehydrogenase, and the amino acid sequence of glutamate dehydrogenase is shown in SEQ ID NO.1;

[0023] The improved PDA culture medium is prepared by adding 10-20 g / L maltose, 1.0-2.0 g / L urea, 0.02-0.1 g / L VB1, 0.5-0.75 g / L magnesium sulfate, 0.25-1 g / L potassium dihydrogen phosphate and 3-5 g / L ant nest to the PDA culture medium.

[0024] In one embodiment, the improved PDA medium is based on the PDA medium, and additionally adds 10-15 g / L maltose, 1.0-2.0 g / L urea, 0.05-0.1 g / L VB1, 0.5-0.75 g / L magnesium sulfate, 0.25-0.5 g / L potassium dihydrogen phosphate and 4-5 g / L ant nest.

[0025] In one embodiment, the culture conditions of the genetically engineered strain of Albizia albuminosa are:

[0026] The genetically engineered strain seed liquid of Alternaria albuminosa is inoculated into an improved PDA culture medium at a rate of 1-3%, and cultured at 28-30°C for 10-14 days.

[0027] Beneficial effects of the present invention

[0028] The invention improves the amino acid content in the genetically engineered strain of Albizia albuminosa by overexpressing glutamate dehydrogenase in the strain. The Albizia albuminosa obtained by the invention is genetically stable and contains high umami and sweet amino acid contents.

[0029] Specifically:

[0030] (1) The TcGDH gene-overexpressing transformants of Albizia albuminosa constructed by the present invention can be stably inherited;

[0031] (2) The TcGDH gene-overexpressing transformants of the present invention produced a monosodium glutamate content of 3.41 mg / kg, which was 90.5% higher than that of the wild type; the umami amino acids aspartic acid and glutamic acid reached 0.67 mg / kg and 2.75 mg / kg, respectively; the sweet amino acid alanine reached 3.20 mg / kg; and the cysteine, a precursor for the synthesis of meaty flavor, reached 1.15 mg / kg.

[0032] (3) The amino acid content of the TcGDH gene overexpressing transformant of the Termitomyces cerevisiae constructed by the present invention reached 23.489 μmol / g fresh weight, which was 49.4% higher than that of WT.

[0033] Biological deposit materials

[0034] Termitomyces clypeatus Tc01, classified as Termitomyces clypeatus Tc01, was deposited in the China Center for Type Culture Collection on December 29, 2021, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCCNO: M 20211695. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the overexpression vector plasmid1-TcGDH;

[0036] Figure 2 The PCR verification results of plasmid 1-TcGDH are shown in the figure on the left. The left figure shows M: DL2000 DNA Marker, 1-5: hyg; 6-10: TcGDH; the right figure shows M: DL8000 DNA Marker, 1-2: double enzyme digestion of recombinant plasmid 1-TcGDH, 3-4: recombinant plasmid 1-TcGDH.

[0037] Figure 3 The PCR verification results of the overexpression transformants of Alpinia oxysporum are shown in Figure 1. The left image shows M:DL2000 DNA Marker, 1: wild-type strain, 2-9: pseudo-transformants; the right image shows M:DL2000 DNA Marker, 2-9: hyg;

[0038] Figure 4 is the expression level of TcGDH in the overexpression transformants of Alpinia oxysporum;

[0039] Figure 5 These are the results of amino acid content detection of the overexpression transformants of Alpinia oxysporum. DETAILED DESCRIPTION

[0040] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0041] Test method:

[0042] High-performance liquid chromatography:

[0043] The free amino acid content in the test sample is extracted, and the specific steps are as follows:

[0044] (1) Accurately weigh 0.1g of dry sample and place it in a 10ml centrifuge tube;

[0045] (2) Add 4-10 ml (5.5 mL) of 4% sulfosalicylic acid;

[0046] (3) Place in an ultrasonicator and ultrasonically extract for 30-40 minutes, mixing by inverting every 5 minutes;

[0047] (4) After ultrasonic extraction, let the mixture stand for 10 min and take 1.5 ml of the supernatant into a 2 ml centrifuge tube;

[0048] (5) 12000 rpm, centrifugation for 30-40 min;

[0049] (6) After centrifugation, carefully remove the centrifuge tube and remove the supernatant (no less than 800 μl) using a 1 ml syringe;

[0050] (7) Pass through a 0.22 μm disposable water membrane and place in a sample bottle for amino acid analysis.

[0051] The water is Watsons distilled water; the grade of sulfosalicylic acid is GR; the preparation of 3.4% sulfosalicylic acid is as follows: 4 g sulfosalicylic acid + 100 ml water, completely dissolved.

[0052] The amino acid analyzer L-8900 (Hitachi, Japan) was used for detection. The constant volume of the supernatant (20 μL) was centrifuged at 13,680 g for 30 min. An ion exchange column (4.6 mm × 60.0 mm, 2622, Hitachi, Japan) and a reaction column (4.6 mm × 40.0 mm, 855-3523, Hitachi, Japan) were used. The temperature of the reaction column was set to 57° C., and the temperature of the ion exchange column was set to 135° C. The detection wavelength of 570 nm, the flow rate of 0.4 mL / min (the mobile phase was methanol (chromatographic grade)) and the buffer solution for elution were all included in the amino acid analyzer. The reaction time was 10 min and the cycle time was 20 min. The raw materials used in the examples were:

[0053] SpeI and ApaI enzymes were purchased from Beijing NEB; Star Marker (D2000) was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.; FastRNARed Kit was purchased from MP Biomedical Company; StarScriptⅡFirst-strand cDNA Synthesis Kit-Ⅱ, 2×Taq PCR StarMix with Loading Dye, 2×SuperStar Plus Mixwith Loading Dye, EZ-Blunt Simple Zero pTOPO Clone Kit, StarPrep Gel Extraction Kit, StarPrep Plasmid Miniprep Kit, T4DNA Ligase, and 2×RealStar Green Fast Mixture were all purchased from GenStar Company; Plant Genomic DNA Kit was purchased from Tiangen Biotechnology Co., Ltd.; M5 Superlight Mix was purchased from Beijing Polymer Biotechnology Co., Ltd.; ceftriaxone sodium (Cef), hygromycin (Hyg), rifampicin (Rif), ampicillin (Arp), kanamycin (Kan), acetosyringone (AS), and 2-(N-morpholino)ethanesulfonic acid (MES) were all purchased from Solebao Company.

[0054] Amino acid content detection kit and glutamic acid content detection kit were purchased from Beijing BOXBIO Technology Co., Ltd.

[0055] Ant nests were collected from Jiangyin City, Jiangsu Province;

[0056] PDA medium formula: 200g potatoes, 20g glucose (D-Glucose), add water to 1000mL, natural pH; autoclave at 115℃ for 30min. Add 1.3% agar powder to obtain PDA medium, or omit agar to obtain PDY medium.

[0057] Other reagents were of domestic analytical grade.

[0058] Shake flask culture method

[0059] Take the Alpinia tertiorrhiza (3-5 mm, about 3 pieces per bottle) on the PDA solid culture medium, inoculate it into 100 mL of PDY culture medium, and culture it at 28°C and 200 r / min for 7 days. Then use a homogenizer to break the mycelial ball three times, each time for 5 seconds to obtain the seed liquid; take 1 mL of the seed liquid and inoculate it into 100 mL of PDY culture medium, and expand the culture at 28°C and 200 r / min for 12 days.

[0060] Example 1: Construction of TcGDH overexpression vector

[0061] 1. Total RNA Extraction

[0062] The strain Tc01 of Alternaria albuminosa (CCTCC NO: M 20211695; hereinafter referred to as Alternaria albuminosa Tc01) was inoculated on a PDA plate and cultured at 28° C. for 20 days. Mycelia were collected and total RNA was extracted according to the instructions of the FastRNARed Kit.

[0063] The total RNA was reverse transcribed into cDNA according to the instructions of StarScriptⅡFirst-strand cDNA Synthesis Kit-Ⅱ for use in subsequent experiments.

[0064] 2. TcGDH amplification

[0065] The cDNA prepared in step 1 was taken and sequenced to obtain the nucleotide sequence. Specific primers TcGDH-F and TcGDH-R for the gene TcGDH were designed. The cDNA of Termitomyces cerevisiae Tc01 was used as a template to amplify the gene TcGDH (amino acid sequence as shown in SEQ ID NO.1), and the nucleotide sequence was shown in SEQ ID NO.2. Restriction primers SpeⅠ-TcGDH-F and ApaⅠ-TcGDH-R for the TcGDH gene were designed. Restriction sites were added at both ends of the gene using TcGDH as a template. PCR was performed and the SpeⅠ-TcGDH-ApaⅠ was obtained after purification and recovery. The primers are shown in Table 1.

[0066] Table 1 Primers and sequences

[0067]

[0068]

[0069] 3. TcGDH transformation verification

[0070] The recovered product, SpeⅠ-TcGDH-ApaⅠ, prepared in step 2 was ligated into a T vector (i.e., the pTOPO-Blunt II Vector in the EZ-Blunt Simple Zero Background pTOPO II Cloning Kit) using the EZ-Blunt Simple Zero Background pTOPO Blunt End Cloning Kit to obtain the vector T-TcGDH. The vector T-TcGDH was transformed into E. coli DH5α competent cells by heat stimulation and verified using primers M13-F and M13-R. Products with the correct band were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Plasmids were extracted from the bacterial culture medium with the correct sequencing results to obtain T-TcGDH.

[0071] 4. Enzyme Digestion and Enzyme Ligation

[0072] The plasmid 1 was kindly donated by Professor Xie Baogui and Professor Chen Bingzhi of Fujian Agriculture and Forestry University (Chen Bingzhi, Li Ling, Chen Tianci, Qiu Mingmeng, Wu Minwen, Xie Baogui, Jiang Yuji. Construction of transformant strains overexpressing the MADS-box transcription factor Vvrin1 gene in Volvariella volvacea and preliminary analysis of their growth rate differences [J / OL]. Acta Mycologica Sinica. https: / / doi.org / 10.13346 / j.mycosystema.200044).

[0073] Plasmids plasmid1 and T-TcGDH were digested with restriction endonucleases Spe I and Apa I, respectively. The large fragment of the former and the small fragment of the latter (Spe I-TcGDH-Apa I) were recovered by agarose gel electrophoresis. The large fragment (linearized plasmid) and the small fragment (Spe I-TcGDH-Apa I) were ligated separately using 0.5 μL of T4 DNA ligase and incubated overnight at 16°C. An additional 0.5 μL of T4 DNA ligase was added to the system and the reaction was continued at room temperature for 10 minutes to obtain the recombinant plasmid plasmid1-TcGDH. PCR amplification was performed using upstream and downstream primers hyg-F and hyg-R for the resistance gene hyg on plasmid1 and upstream and downstream primers Spe I-TcGDH-F and Apa I-TcGDH-R for TcGDH, confirming the correct overexpression plasmid plasmid1-TcGDH.

[0074] plasmid1 plasmid Figure 1 The PCR verification results of overexpression plasmid plasmid1-TcGDH are shown in Figure 2 As shown, the results showed that both hyg and TcGDH had bands ( Figure 2 Left side), after double enzyme digestion, there are two obvious bands and the band sizes are consistent with ( Figure 2 The overexpression vector plasmid1-TcGDH was successfully constructed.

[0075] Example 2: Transformation of Agrobacterium tumefaciens GV3101 with overexpression plasmid and induction culture

[0076] 1. Transformation of Agrobacterium tumefaciens GV3101

[0077] Take the overexpression plasmid plasmid1-TcGDH prepared in Example 1 and transform the plasmid into Agrobacterium tumefaciens GV3101 competent cells using the freeze-thaw method. The specific steps are as follows:

[0078] (1) Thaw the competent strain of Agrobacterium tumefaciens GV3101 stored at -80°C at room temperature and insert it into ice when it is in an ice-water mixture state.

[0079] (2) Add 1 μg of recombinant plasmid plasmid1-TcGDH to every 100 μL competent cells, gently stir the bottom of the tube to mix, and then place on ice for 5 minutes, quick-freeze in liquid nitrogen for 5 minutes, bath in 37°C water for 5 minutes, and bath on ice for 5 minutes.

[0080] (3) Add 700 μL of antibiotic-free LB liquid medium and culture at 28°C with shaking for 2-3 h.

[0081] (4) Collect the bacteria by centrifugation at 6000 rpm for 1 min, take about 100 μL of supernatant and gently pipette to resuspend the bacteria, spread it on an LB plate containing 50 μg / mL Rif and 50 μg / mL Kan, and culture it upright in a 28°C incubator for 30 min, then invert and culture it for 2-3 days.

[0082] (5) Pick a single colony and expand it in LB liquid medium containing 50 μg / mL Rif and 50 μg / mL Kan, shake it at 28°C, 200 rpm, and culture it in the dark for 2 days.

[0083] (6) Using primers plasmid1-TcGDH-F (located on the vector), plasmid1-TcGDH-R (located on TcGDH) and the upstream and downstream primers hyg-F and hyg-R of hyg, PCR verification was performed to obtain positive Agrobacterium, which was the Agrobacterium tumefaciens that had been transformed with the recombinant plasmid plasmid1-TcGDH.

[0084] 2. Induction culture of Agrobacterium tumefaciens

[0085] 1 mL of the Agrobacterium tumefaciens culture medium carrying the recombinant plasmid plasmid1-TcGDH prepared in step 1 was added to 100 mL of LB liquid culture medium (Kan 50 μg / mL and Rif 50 μg / mL) at 200 rpm and 28° C. for 12 h of expansion culture.

[0086] (2) Collect the bacterial suspension, centrifuge at 4000 rpm for 10 min, remove the supernatant, resuspend with an equal volume of IM medium, and dilute to OD 600 Acetosyringone (AS) was added to a final concentration of 200 μmol / L and cultured in the dark at 200 rpm and 28°C for 6 h until the OD 600 The concentration of Agrobacterium tumefaciens was about 0.7, and the induced cultured Agrobacterium tumefaciens liquid was obtained.

[0087] Example 3: Genetic transformation of Aconitum paniculate mycelium mediated by Agrobacterium tumefaciens

[0088] The induced cultured Agrobacterium tumefaciens culture prepared in Example 2 and the mycelium Tc01 of the Termitomyces cerevisiae cultured at 28° C. for 20 days were used for genetic transformation in the following steps:

[0089] (1) Take the 20-day-old PDA culture medium-grown G. applanatum mycelium into the induction culture Agrobacterium tumefaciens liquid and mix well (about 20 mycelium pieces per 100 ml of Agrobacterium tumefaciens liquid, the diameter of each G. applanatum mycelium is about 0.5-1 cm; the range of parameters does not affect the subsequent experimental results), 28°C, static for 30 min;

[0090] (2) Take a single mycelium piece with sterile forceps, flash dry the surface liquid with sterile filter paper, and then place it in Co-IM medium (containing 200 μmol / L AS) for 3 days of co-culture in the dark;

[0091] (3) After the mycelium piece germinates, pick it up with sterile forceps, rinse it twice in sterile water containing 200 μg / mL Cef, and then rinse it once in sterile water without antibiotics, flash dry the surface moisture with sterile filter paper, and then place it in the primary screening medium (PDA medium containing 400 μg / mL Cef and 20 μg / mL Hyg), and culture at 28°C; after the primary screening mycelium germinates, pick up the new mycelium and transfer it to the secondary screening medium (PDA medium containing 400 μg / mL Cef and 30 μg / mL Hyg), and culture at 28°C for about 20 days; use the TcGDH overexpression verification primers plasmid1-TcGDH-F, plasmid1-TcGDH-R, and the resistance gene hyg upstream and downstream primers hyg-F and hyg-R to perform PCR verification, and obtain G. applanatum overexpression pseudo-transformants.

[0092] Randomly select 11 G. applanatum overexpression pseudo-transformants, extract the mycelium genomic DNA, and perform PCR amplification detection with the primers plasmid1-TcGDH-F, plasmid1-TcGDH-R, and the hygromycin gene hyg upstream and downstream primers hyg-F and hyg-R.

[0093] The PCR verification results are shown in Table 1. Figure 3 Table 1: PCR verification results of G. applanatum overexpression pseudo-transformants

[0094] Example 4: Performance detection of overexpression transformants

[0095] Take the G. applanatum overexpression transformants H1, H2, H3, H4, H5, H6, H7, and H8 prepared in Example 3, and take H2 therefrom to perform performance detection.

[0096] 1. Detection of TcGDH expression level

[0097] H2 and the wild-type strain WT (i.e., Tc01 without overexpression, hereinafter referred to as WT) were inoculated on PDA medium (mycelium of the same size, 3-5 mm) and cultured at 28°C for 20 days. The mycelium was collected and total RNA was extracted from the positive transformants and wild-type mycelium. The total RNA was reverse transcribed into cDNA using a reverse transcription kit. EF1 was used as an internal reference gene, and the relative expression levels of the gene TcGDH in the transformants and wild-type strain were detected by qRT-PCR. The primers were q-TcGDH-F, q-TcGDH-R, EF1-F, and EF1-R (primers are shown in Table 1).

[0098] The results are as follows Figure 4 As shown, the expression level of TcGDH in transformant H2 was upregulated by 3.444 times compared with WT.

[0099] 2. Mitotic stability assay

[0100] The overexpression transformant H2 of Alpinia thunbergii prepared in Example 3 was inoculated onto PDA medium without selection pressure, cultured continuously at 28°C for 5 generations, and then transferred to PDA medium containing 30 μg / mL hygromycin. The transformants with mycelial germination were selected, and the mycelial genomic DNA was extracted for PCR detection.

[0101] The results showed that PCR detection of the transformant H2 of Alstonia aculeatus could amplify the specific fragment of the target gene overexpression vector and hyg, proving that the overexpressed TcGDH gene can be stably inherited in the transformants of Alstonia aculeatus.

[0102] 3. Amino acid analysis

[0103] The amino acid composition of the wild-type strain compared to the overexpression transformants was determined by shake flask culture. Mushrooms of the transformant H2 and the wild-type strain (3-5 mm in diameter) were inoculated into 100 ml of PDY medium (3 slabs per flask) and cultured at 28°C with shaking at 200 rpm. Samples were collected and filtered on the 12th day, and the filtered mycelia were freeze-dried in a vacuum freeze-dried state for 72 hours. Free amino acids in the mycelia were analyzed using high-performance liquid chromatography.

[0104] The results are as follows Figure 5 The results showed that H2 had the highest monosodium glutamate (MSG) content (reaching 3.41 mg / kg), which was much higher than the wild type WT (1.79 mg / kg). The monosodium glutamate content of H2 was 90.5% higher than that of WT. The sweet and bitter amino acid contents of H2 were also significantly better than those of the wild type WT.

[0105] Among them, the content of aspartic acid (Asp) and glutamic acid (Glu), which are umami amino acids in H2, reached 0.67 mg / kg and 2.75 mg / kg respectively; the content of alanine (Ala), which is a sweet amino acid, reached 3.20 mg / kg; the content of cysteine ​​(Cys), which is a precursor for the synthesis of meaty flavor, reached 1.15 mg / kg; the content of bitter amino acids arginine, leucine and isoleucine was significantly lower than that of WT.

[0106] The above results show that the overexpression transformant H2 of Alpinia oxysporum has excellent flavor, which is significantly better than the wild type WT.

[0107] Example 5: Culture medium is optimized

[0108] The overexpression transformant H2 of Alpinia oxysporum was taken, and the culture medium formula was further optimized based on PDA.

[0109] 1. Carbon source optimization

[0110] On the basis of PDA, different carbon sources (20g / L) were added and the glutamate content after H2 growth was measured. The results showed that maltose was the best carbon source. The addition amount was further optimized based on maltose, and the results are shown in Table 2.

[0111] Table 2 Carbon source optimization

[0112] Carbon source 20g / L Glutamic acid content (μmol / g fresh weight) sucrose 3.416609701 maltose 5.351687205 corn flour 2.439383355 starch 2.66405658 Carbon crystalline cellulose 3.00152374 Maltose concentration Glutamic acid content (μmol / g fresh weight) 5g / L 4.297451662 10g / L 8.200933568 15g / L 8.449651184 20g / L 7.468959099 25g / L 5.338107826

[0113] The results showed that the optimal addition amount of maltose was 15g / L.

[0114] 2. Nitrogen source optimization

[0115] On the basis of PDA, different nitrogen sources (2 g / L) were added and the glutamate content after H2 growth was measured. The results showed that urea was the optimal nitrogen source. The addition amount was further optimized based on urea, and the results are shown in Table 3.

[0116] Table 3 Nitrogen source optimization

[0117]

[0118]

[0119] The results showed that the optimal addition amount of urea was 2g / L.

[0120] 3. Growth factor optimization

[0121] Based on the optimal addition levels of maltose and urea confirmed in 1 and 2, a PDA medium containing 15 g / L maltose and 2 g / L urea was prepared. Furthermore, the growth factor (0.1 g / L) was optimized. The glutamate content after H2 growth was measured, and the results showed that VB1 was the optimal growth factor. The addition levels were further optimized based on VB1, and the results are shown in Table 4.

[0122] Table 4 Growth factor optimization

[0123] Growth factors Glutamic acid content (μmol / g fresh weight) VB1 9.231448207 VB2 8.014266544 VB6 5.296326734 VB12 6.739192264 VC 5.833563155 folic acid 4.107165517 VB1 concentration Glutamic acid content (μmol / g fresh weight) 0.02g / L 7.650581741 0.05g / L 8.139348848 0.1g / L 9.006448564 0.15g / L 6.441849438 0.2g / L 5.315857865 0.02g / L 7.650581741

[0124] The results showed that the optimal addition amount of VB1 was 0.1 g / L.

[0125] 4. Optimization of magnesium sulfate concentration

[0126] The optimal addition amounts of maltose and urea were confirmed in 1 and 2, and a PDA culture medium with 15 g / L maltose and 2 g / L urea was prepared. On this basis, the addition amount of magnesium sulfate was further optimized. The results are shown in Table 5.

[0127] Table 5 Magnesium sulfate addition amount

[0128] Magnesium sulfate concentration Glutamic acid content (μmol / g fresh weight) 0.25g / L 4.885747333 0.5g / L 7.264266667 0.75g / L 11.6134 1.0g / L 6.284899667

[0129] The results showed that the optimal addition amount of magnesium sulfate was 0.75 g / L.

[0130] 5. Optimization of potassium dihydrogen phosphate concentration

[0131] The optimal addition amounts of maltose and urea were confirmed in 1 and 2, and a PDA culture medium with 15 g / L maltose and 2 g / L urea was prepared. On this basis, the addition amount of potassium dihydrogen phosphate was further optimized. The results are shown in Table 6.

[0132] Table 6 Amount of potassium dihydrogen phosphate added

[0133] Potassium dihydrogen phosphate concentration Glutamic acid content (μmol / g fresh weight) 0.25g / L 5.872827333 0.5g / L 7.899196333 0.75g / L 5.793641333 1.0g / L 5.727521333

[0134] The results showed that the optimal addition amount of potassium dihydrogen phosphate was 0.5 g / L.

[0135] 6. Ant Nest Usage Optimization

[0136] The optimal addition amounts of maltose and urea were confirmed in 1 and 2, and a PDA culture medium with 15 g / L maltose and 2 g / L urea was prepared. On this basis, the addition amount of the ant nest was further optimized. The results are shown in Table 7.

[0137] Table 7 Ant nest addition amount

[0138] Ant nest concentration Glutamic acid content (μmol / g fresh weight) 2.0g / L 5.577042 3.0g / L 6.198281667 4.0g / L 6.695569333 5g / L 6.425149667

[0139] The results showed that the optimal addition amount of ant nest was 4.0g / L.

[0140] The above results show that the optimal culture medium formula for H2 is to add 15g / L maltose, 2g / L urea, 0.1g / L VB1, 0.75g / L magnesium sulfate, 0.5g / L potassium dihydrogen phosphate and 4.0g / L ant nest to PDA.

[0141] Comparative Example 1: Using different TcGDH sequences

[0142] On the basis of Example 1, TcGDH with another amino acid sequence was used, whose amino acid sequence is shown in SEQ ID NO.17 and nucleotide sequence is shown in SEQ ID NO.18. The remaining steps were consistent with Example 1, and the prepared Alpinia lanceolata overexpression transformant was named G1.

[0143] The wild type Alpinia hortensis WT and the Alpinia hortensis overexpression transformants H2 and G1 prepared in Example 3 were taken, and the amino acid content and glutamate content in the Alpinia hortensis overexpression transformants were detected using an amino acid content detection kit and a glutamate content detection kit. The results are shown in Table 8.

[0144] Table 8 Amino acid and glutamic acid content

[0145] Sample No. Amino acid content (μmol / g fresh weight) Glutamic acid content (μmol / g fresh weight) WT 15.719 6.1 H2 23.489 7.343 G1 17.819 6.561

[0146] The results showed that the amino acid content and glutamate content of the overexpression transformant H2 of Alstonia aculeatus were significantly better than those of the overexpression transformant G1 of Alstonia aculeatus, and the amino acid content of H2 was increased by 49.4% compared with WT.

[0147] Sequences used in the present invention:

[0148] Amino acid sequence of TcGDH (SEQ ID NO. 1)

[0149] MAIMTSALVQMIAKSCPEFSLNLPSTIVAISGSGNVSQFTALKVIELGATVKSLSDSKGSLIAPQGFTKADILSIGQLKLKGGTLESWVSQQPGNRFVYHAGKRPWTLLPVIHHALPGATQNEVS GEEAKALVAAGVRIVAEGSNMGCTEEAIEVFENARKAGKGVWYAPGKASNCGGVAVSGLEMAQNSQRLAWTTQEVDGKLKNIMAEAYSQCLNAGTKWCLARKWLTVSSPLSSLAPMLLVSSRSLML

[0150] Nucleotide sequence of TcGDH (SEQ ID NO. 2)

[0151] ATGGCAATAATGACATCCGCACTTGTACAGATGATCGCCAAGTCCTGTCCTGAATTCTCCCTGAACCTTCCTTCCACTATAGTCGCAATCTCGGGATCTGGTAACGTTTCTCAGTTTACCGCGCTCAAGGTCATCGAGCTTGGCGCGACCGTCAAGTCGCTCTCTGATTCCAAAGGTTCCCTCATCGCCCCTCAGGGCTTCACCAAGGCGGATATTCTGAGCATTGGTCAACTCAAGCTCAAGGGTGGAACCCTCGAGTCATGGGTATCTCAGCAGCCTGGAAACCGCTTCGTCTACCACGCTGGCAAGCGCCCTTGGACTCTCCTCCCCGTCATCCACCATGCTCTTCCCGGCGCGACCCAGAACGAAGTTTCCGGAGAGGAAGCCAAGGCTCTTGTTGCCGCTGGTGTTCGCATTGTTGCCGAGGGTTCCAACATGGGTTGTACAGAGGAGGCCATTGAAGTCTTCGAGAATGCCCGCAAGGCCGGCAAGGGCGTATGGTACGCTCCCGGAAAGGCCTCCAACTGTGGTGGTGTCGCCGTTTCCGGTCTCGAGATGGCTCAAAACAGCCAGCGTCTTGCATGGACGACTCAGGAGGTCGACGGCAAGCTCAAAAACATCATGGCTGAGGCTTACTCCCAGTGTCTCAATGCTGGTACCAAATGGTGTCTGGCGAGAAAATGGCTGACGGTGTCCTCCCCTCTCTCCTCGCTGGCGCCAATGTTGCTGGTTTCATCAAGGTCGCTGATGCTATGA

[0152] Amino acid sequence of TcGDH (SEQ ID NO. 17)

[0153] MVLPVEPEFEQALNELTQSLQPFLAANPRYQKALDIVQVPERVLQFRVVWEDDRGVPQVNRGFRVQYNSALGPYKGGLRLHPSVNLSILKFLGFEQTFKNALTGLSMGGGKGGSDFDPKGKSDSEIRRFCTAFMSELYRHIGQDTDVPAGDIGTGAREIGFLFGAYKKLKNEFVGMLFSTGKGLDWGALS

[0154] Comparative Example 1: Nucleotide sequence of TcGDH (SEQ ID NO.18)

[0155] ATGGTCCTCCCAGTTGAACCCGAATTTGAGCAGGCATTGAATGAGCTTACCCAAAGCTTGCAACCTTTCTTGGCTGCCAATCCTCGCTACCAAAAGGCCCTCGACATTGTCCAGGTCCCAGAGCGTGTCCTCCAGTTCCGTGT CGTCTGGGAGGACGACAGGGGTGTTCCACAAGTCAACCGAGGCTTCCGTGTCCAGTACAACTCAGCCCTCGGTCCCTACAAAGGCGGTCTCCGACTTCACCCTTCCGTCAATCTTCTATTCTCAAGTTCCTTGGTTTTGAGC AAACCTTTAAAAATGCTCTTACCGGACTCAGCATGGGTGGTTGGAAAGGGTGGCCTCTGATTTCGACCCAAAGGGCAAATCTGACTCCGAGATCAGGCGCTTCTGCACTGCATTCATGTCCGAGTCTCACAGGCATATCGGTCAG GACACTGATGTACCTGCCGGTGACATTGGAACTGGTGCTAGGGAGATTGGCTTCCTCTTCGGTGCTTACAAGAAATTGAAGAATGAATTCGTCGGCATGCTCACCGGCAAGGGTCTCGACTGGGGAGCTCTTTCATTCTCCTGA

[0156] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered strain of Albizia albuminosa, characterized in that: The genetically engineered strain of Alternaria albuminosa overexpresses glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.

1.

2. The genetically engineered strain of Albizia albuminosa according to claim 1, wherein The preparation method comprises: The glutamate dehydrogenase nucleotide sequence is amplified and connected with a plasmid vector to obtain a recombinant plasmid that overexpresses glutamate dehydrogenase; the recombinant plasmid is transferred into Agrobacterium tumefaciens to induce and obtain induced cultured Agrobacterium tumefaciens; the induced cultured Agrobacterium tumefaciens is mixed with mycelium of Albizia albuminosa, cultured and verified to obtain genetically engineered Albizia albuminosa bacteria.

3. The genetically engineered strain of Albizia albuminosa according to claim 2, wherein The nucleotide sequence of glutamate dehydrogenase is shown in SEQ ID NO.

2.

4. A method for increasing the total free amino acid content in Albizia albuminosa, characterized in that: A genetically engineered strain of Albizia albuminosa was constructed, wherein the genetically engineered strain overexpressed glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase was shown in SEQ ID NO.

1.

5. A method for simultaneously increasing the content of monosodium glutamate, umami amino acids, and sweet amino acids in Albizia albuminosa, and reducing the content of bitter amino acids in Albizia albuminosa, characterized in that: A genetically engineered strain of Albizia albuminosa was constructed, wherein the genetically engineered strain overexpressed glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase was shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that Umami amino acids include one or more of glutamic acid and aspartic acid; Sweet amino acids include alanine, glycine, serine, proline, and threonine; Bitter amino acids include arginine, histidine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine.

7. A method for increasing the cysteine ​​content in Albizia albuminosa, characterized in that: A genetically engineered strain of Albizia albuminosa was constructed, wherein the genetically engineered strain overexpressed glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase was shown in SEQ ID NO.

1.

8. Use of the genetically engineered strain of Albizia albuminosa according to any one of claims 1 to 3 or the method according to any one of claims 5 to 7 in the preparation of amino acids or amino acid salts, characterized in that: The amino acids include one or more of glutamic acid, aspartic acid, alanine, and proline.

9. A method for increasing the glutamic acid content in Albizia albuminosa, characterized in that: Improved PDA medium was used to culture genetically engineered strains of Alternaria albuminosa; The genetically engineered strain of Alternaria albuminosa overexpresses glutamate dehydrogenase, and the amino acid sequence of glutamate dehydrogenase is shown in SEQ ID NO.1; The improved PDA culture medium is prepared by adding 10-20 g / L maltose, 1.0-2.0 g / L urea, 0.02-0.1 g / L VB1, 0.5-0.75 g / L magnesium sulfate, 0.25-1 g / L potassium dihydrogen phosphate and 3-5 g / L ant nest to the PDA culture medium.

Images