Method for adjusting content of amino acid in termitomyces albuminosus

Abstract

The invention discloses a method for adjusting the content of amino acid in termitomyces albuminosus, and belongs to the field of genetic engineering. According to the invention, glutamate dehydrogenase is overexpressed in termitomyces albuminosus, so that the content of amino acid in the termitomyces albuminosus genetically engineered bacteria is increased. The termitomyces albuminosus constructed by the method is stable in heredity and high in delicate flavor and sweet amino acid content. The constructed termitomyces albuminosus overexpression TcGDH gene transformant can be stably inherited; the sodium glutamate content reaches 3.41 mg / kg and is increased by 90.5% compared with that of a wild type; aspartic acid and glutamic acid of the delicious amino acids respectively reach 0.67 mg / kg and 2.75 mg / kg; the content of alanine in sweet amino acid reaches 3.20 mg / kg; cysteine used as a meat flavor synthesis precursor reaches 1.15 mg / kg; and the amino acid content reaches 23.489 [mu] mol / g fresh weight, which is increased by 49.4% compared with WT.

Description

Technical Field

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

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

[0003] Methods for increasing the amino acid content of Alternaria albuminosa mainly include optimizing the culture medium formula and adjusting the growth conditions. However, these methods have certain technical bottlenecks and applications. The document "Screening of nutrient components of the culture medium of Alternaria albuminosa mother strain 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 Alternaria albuminosa mother strain through experiments to meet the rapid growth of Alternaria albuminosa. The document "Artificial cultivation technology of Alternaria albuminosa; China Agricultural Technology Extension. 2024, 40 (02)" discloses the optimum temperature, light intensity, and pH of Alternaria albuminosa, realizing artificial cultivation. Although the above methods of optimizing the culture medium formula and adjusting the 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 Alpinia terrestris has important research value and application prospects. Summary of the invention

[0005] To solve the above problems, the present invention increases the amino acid content in the genetically engineered strain of Alpinia terrestris by overexpressing glutamate dehydrogenase in the Alpinia terrestris. The Alpinia terrestris constructed by the present invention is genetically stable and contains a high content of umami and sweet amino acids.

[0006] The first object of the present invention is to provide a genetically engineered strain of Albion fungus, which overexpresses glutamate dehydrogenase (TcGDH), and 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, which 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 Alternaria albuminosa, cultured and verified to obtain genetically engineered Alternaria 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 Albion, constructing a genetically engineered strain of Albion, wherein the genetically engineered strain of Albion 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 sodium glutamate, umami amino acids and sweet amino acids in Album albuminus and reducing the content of bitter amino acids in Album albuminus, constructing a genetically engineered strain of Album albuminus, wherein the genetically engineered strain of Album albuminus 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 Albion, constructing a genetically engineered strain of Albion, wherein the genetically engineered strain of Albion overexpresses glutamate dehydrogenase, and the amino acid sequence of 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 bacteria of Alpinia terrestris 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 Album albuminum, using an improved PDA medium to culture the genetically engineered bacteria of Album albuminum;

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

[0023] The improved PDA culture medium is based on the PDA culture medium, and 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 are additionally added.

[0024] In one embodiment, the improved PDA medium is based on the PDA medium, and additionally contains 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 bacteria of Albizia albuminosa are:

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

[0027] Beneficial Effects of the Invention

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

[0029] Specifically:

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

[0031] (2) The TcGDH gene overexpressing transformant of Alpinia terrestris constructed by the present invention has a sodium glutamate content of 3.41 mg / kg, which is 90.5% higher than that of the wild type; the umami amino acids aspartic acid and glutamic acid reach 0.67 mg / kg and 2.75 mg / kg respectively; the sweet amino acid alanine reaches 3.20 mg / kg; and the cysteine, which is a precursor for the synthesis of meaty flavor, reaches 1.15 mg / kg;

[0032] (3) The amino acid content of the transformants of the TcGDH gene overexpressing Convolvulus albuminus 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 at Wuhan University, Wuhan, China, and the deposit number is 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 plasmid1-TcGDH; the left picture shows M:DL2000 DNA Marker, 1-5:hyg; 6-10:TcGDH; the right picture shows M:DL8000 DNA Marker, 1-2: double restriction 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; the left picture is M:DL2000 DNA Marker, 1: wild-type strain, 2-9: pseudo-transformants; the right picture is 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 overexpression transformants of Alpinia oxyphylla. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0041] Test method:

[0042] HPLC:

[0043] The free amino acid content is extracted from the test sample. The specific steps are as follows:

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

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

[0046] (3) Place in an ultrasonicator for 30-40 min and mix by inverting every 5 min;

[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 800ul) using a 1ml syringe;

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

[0051] The water is Watsons distilled water; the grade of sulfosalicylic acid is superior grade (GR); the configuration of 3.4% sulfosalicylic acid is: 4g sulfosalicylic acid + 100ml 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 used for elution were all included in the amino acid analyzer. The reaction time was 10 min and the cycle time was 20 min. Raw materials used in the embodiment:

[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 cDNASynthesis Kit-Ⅱ, 2×Taq PCR StarMix with Loading Dye, 2×SuperStar Plus Mixwith Loading Dye, EZ-Blunt Simple Zero pTOPO Clone Kit, StarPrep Gel ExtractionKit, StarPrep Plasmid Miniprep Kit, T4DNA Ligase, and 2×RealStar Green Fast Mixture were all purchased from GenStar; Plant Genomic DNA Kit was purchased from Tiangen Biotechnology Co., Ltd.; M5 Super Light Mix was purchased from Beijing Juhemei 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] The 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; sterilize at 115℃ high pressure steam for 30min, add 1.3% agar powder to get PDA medium, and PDY medium can be obtained without adding agar;

[0057] Other reagents were of domestic analytical grade.

[0058] Shake flask culture method

[0059] Take the Alpinia terrestris (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 balls three times, each time for 5 seconds, to obtain the seed solution; take 1 mL of the seed solution and inoculate it into 100 mL of PDY culture medium, and expand it 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 of Alternaria japonica Tc01 (CCTCC NO: M 20211695; hereinafter referred to as Alternaria japonica 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 FastRNARed Kit.

[0063] According to the instructions of StarScriptⅡFirst-strand cDNASynthesis Kit-Ⅱ, the total RNA was reverse transcribed into cDNA for subsequent experiments.

[0064] 2. TcGDH amplification

[0065] Take the cDNA prepared in step 1, sequence to obtain the nucleotide sequence, design specific primers TcGDH-F and TcGDH-R for the gene TcGDH, and use the cDNA of Termitomyces cerevisiae Tc01 as a template to amplify the gene TcGDH (the amino acid sequence is shown in SEQ ID NO.1), and the nucleotide sequence is shown in SEQ ID NO.2; design restriction primers SpeⅠ-TcGDH-F and ApaⅠ-TcGDH-R for the TcGDH gene, use TcGDH as a template to add restriction sites at both ends of the gene, PCR and purification to obtain SpeⅠ-TcGDH-ApaⅠ, and the primers are shown in Table 1.

[0066] Table 1 Primers and sequences

[0067]

[0068]

[0069] 3. TcGDH transformation verification

[0070] Take the recovered product SpeⅠ-TcGDH-ApaⅠ prepared in step 2, and connect the recovered product to the T vector (i.e., the pTOPO-Blunt II Vector in the EZ-Blunt Simple zero background pTOPO II cloning kit) according to the EZ-Blunt Simple zero background pTOPO flat end cloning kit to obtain the vector T-TcGDH. The vector T-TcGDH was transformed into Escherichia coli DH5α competent cells by thermal stimulation, and primers M13-F and M13-R were used for verification. The product with the correct band was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The plasmid was extracted from the bacterial solution with the correct sequencing result to obtain T-TcGDH.

[0071] 4. Enzyme Digestion and Enzyme Ligation

[0072] The plasmid1 plasmid was kindly donated by Professor Xie Baogui and Teacher Chen Bingzhi from 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 of Volvariella volvacea and preliminary analysis of their growth rate differences [J / OL]. Journal of Mycology. https: / / doi.org / 10.13346 / j.mycosystema.200044).

[0073] Plasmid plasmid1 and T-TcGDH were digested with restriction endonucleases SpeⅠ and ApaⅠ, respectively. The large fragment of the former and the small fragment of the latter (SpeⅠ-TcGDH-ApaⅠ) were recovered by agarose gel electrophoresis. The large fragment (linear plasmid) and the small fragment (SpeⅠ-TcGDH-ApaⅠ) were ligated with 0.5μL T4 DNA ligase, respectively, and left to stand overnight at 16℃. 0.5μL T4 DNA ligase was added to the above system at room temperature and reacted for 10min to obtain the recombinant plasmid plasmid1-TcGDH. PCR amplification was performed using the upstream and downstream primers hyg-F and hyg-R of the resistance gene hyg on plasmid1 and the upstream and downstream primers SpeⅠ-TcGDH-F and ApaⅠ-TcGDH-R of TcGDH, and the correct overexpression plasmid plasmid1-TcGDH was obtained.

[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 Right figure), that is, 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) Take the competent strain of Agrobacterium GV3101 stored at -80°C and thaw it at room temperature. When it is in an ice-water mixture state, put it into ice.

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

[0080] (3) Add 700 μL of LB liquid medium without antibiotics 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 the supernatant and gently pipette to resuspend the bacteria. Spread the suspension on an LB plate containing 50 μg / mL Rif and 50 μg / mL Kan. Incubate the plate upright at 28°C for 30 min and then invert 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, and culture it in the dark at 28°C, 200 rpm 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 obtained a positive Agrobacterium, which is the Agrobacterium tumefaciens that has been transformed with the recombinant plasmid plasmid1-TcGDH.

[0084] 2. Induced culture of Agrobacterium tumefaciens

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

[0086] (2) Collect the bacterial suspension, centrifuge at 4000 rpm for 10 min, remove the supernatant, resuspend in an equal volume of IM medium, and dilute to OD 600 The value was 0.2, and acetosyringone (AS) was added to a final concentration of 200 μmol / L. The cells were cultured in the dark at 200 r / min and 28°C for 6 h until the OD 600 is about 0.7, and the induced cultured Agrobacterium tumefaciens bacterial solution is obtained.

[0087] Example 3: Genetic transformation of Alpinia tumefaciens hyphae mediated by Agrobacterium tumefaciens

[0088] The induced cultured Agrobacterium tumefaciens culture liquid prepared in Example 2 and the mycelium Tc01 of Alternaria japonica cultured at 28° C. for 20 days were taken for genetic transformation, and the steps were as follows:

[0089] (1) The mycelium of Altissima scabra growing for 20 days on PDA medium was inoculated into the induced culture of Agrobacterium tumefaciens and mixed evenly (about 20 mycelium were inoculated into every 100 ml of Agrobacterium tumefaciens culture, and the diameter of each mycelium of Altissima scabra was about 0.5-1 cm; the parameters within the range did not affect the subsequent experimental results), and the mixture was allowed to stand at 28°C for 30 minutes;

[0090] (2) Use sterile tweezers to pick up a single mycelium block, blot the surface bacterial liquid on sterile filter paper, and then place it in Co-IM medium (containing 200 μmol / L AS) and co-culture it in the dark for 3 days;

[0091] (3) After the mycelium blocks germinate, use sterile tweezers to pick up the mycelium blocks and rinse them twice in sterile water containing 200 μg / mL Cef, then rinse them once in sterile water without antibiotics, use sterile filter paper to slightly dry the surface moisture, and then put them into the primary screening medium (PDA medium containing 400 μg / mL Cef and 20 μg / mL Hyg) and culture them 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), culture them at 28°C for about 20 days, and use the TcGDH overexpression verification primers plasmid1-TcGDH-F and plasmid1-TcGDH-R and the upstream and downstream primers hyg-F and hyg-R of the resistance gene hyg to perform PCR verification to obtain the overexpression pseudo-transformants of Alpinia oxyphylla.

[0092] Eleven overexpression pseudo-transformants of Alternaria obliquus were randomly selected, and mycelial genomic DNA was extracted respectively. PCR amplification was performed using plasmid1-TcGDH-F, plasmid1-TcGDH-R and the upstream and downstream primers hyg-F and hyg-R of the hygromycin gene hyg.

[0093] PCR verification results Figure 3 As shown, the amplified products of the eight proposed transformants contained a specific sequence of the TcGDH overexpression vector of about 756 bp and a hyg sequence of about 800 bp. The 756 bp band was sent to the company for sequencing, and the results were consistent with the target sequence, indicating that the recombinant plasmid had been successfully transferred into the eight transformants, which were named H1, H2, H3, H4, H5, H6, H7, and H8 respectively.

[0094] Example 4: Performance testing of overexpression transformants

[0095] The Alpinia scabra overexpression transformants H1, H2, H3, H4, H5, H6, H7, and H8 prepared in Example 3 were taken, and H2 was taken for performance testing.

[0096] 1. Detection of TcGDH expression level

[0097] H2 and 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), respectively. After culturing at 28°C for 20 days, the mycelium was collected, and the total RNA of the positive transformants and wild-type mycelium was extracted, respectively. 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 level of the gene TcGDH in the transformants and the wild-type strain was 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 test

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

[0101] The results showed that the specific fragments of the target gene overexpression vector and hyg were amplified by PCR detection of the transformant H2 of Alternaria obliquus, proving that the overexpressed TcGDH gene can be stably inherited in the transformants of Alternaria obliquus.

[0102] 3. Amino acid analysis

[0103] The amino acid composition of the wild-type strain compared with the overexpression transformant was detected by shaking flask culture. The blocks of transformant H2 and wild-type strain of the same diameter (3-5 mm) were inoculated in 100 ml PDY medium (3 blocks per bottle), and cultured at 28 ° C and 200 r / min. The samples were filtered on the 12th day, and the filtered mycelium was vacuum freeze-dried for 72 hours. The free amino acids in the mycelium were analyzed by high performance liquid chromatography.

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

[0105] Among them, 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; alanine (Ala), which is a sweet amino acid, reached 3.20 mg / kg; cysteine ​​(Cys), a precursor for the synthesis of meaty flavor, reached 1.15 mg / kg; and the bitter amino acids arginine, leucine, and isoleucine were significantly lower than WT.

[0106] The above results show that the overexpression transformant H2 of Alpinia oxyphylla 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 oxyphylla 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 (20 g / L) were added, and the glutamate content after H2 growth was detected. The results showed that maltose was the best carbon source. The addition amount was further optimized on the basis of maltose, and the results are shown in Table 2.

[0111] Table 2 Carbon source optimization

[0112] Carbon source 20g / L Glutamate 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 Glutamate 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 detected. The results showed that urea was the optimal nitrogen source. The addition amount was further optimized on the basis of 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] The optimal addition amount of maltose and urea was confirmed in 1 and 2, and PDA medium with 15g / L maltose and 2g / L urea was prepared. On this basis, the growth factor (0.1g / L) was further optimized, and the glutamate content after H2 growth was detected. The results showed that VB1 was the optimal growth factor. The addition amount was further optimized on the basis of VB1, and the results are shown in Table 4.

[0122] Table 4 Growth factor optimization

[0123] Growth Factors Glutamate 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 Glutamate 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 0.1g / 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 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 Glutamate 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 amount of magnesium sulfate added 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 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 Addition amount of potassium dihydrogen phosphate

[0133] Potassium dihydrogen phosphate concentration Glutamate 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 medium with 15 g / L maltose and 2 g / L urea was prepared. On this basis, the addition amount of ant nest was further optimized. The results are shown in Table 7.

[0137] Table 7 Ant nest addition amount

[0138] Ant nest concentration Glutamate 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 amount of ant nest added 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 other amino acid sequences was used, whose amino acid sequence was shown in SEQ ID NO.17 and nucleotide sequence was 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 oxysporum WT and the Alpinia oxysporum overexpression transformants H2 and G1 prepared in Example 3 were taken, and the amino acid content and glutamate content in the Alpinia oxysporum 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) Glutamate 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 Alpinia oxysporum were significantly better than those of the overexpression transformant G1 of Alpinia oxysporum, and the amino acid content of H2 was increased by 49.4% compared with WT.

[0147] The sequences used in the present invention are:

[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 in Comparative Example 1 (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 as above in the form of a preferred embodiment, 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 bacteria of Alternaria albuminosa overexpress glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.

1.

2. The genetically engineered bacteria of Alpinia terrestris according to claim 1, characterized in that 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 Alternaria albuminosa, cultured and verified to obtain genetically engineered Alternaria albuminosa bacteria.

3. The genetically engineered bacteria of Alpinia terrestris according to claim 2, characterized in that 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 Albion fungus is constructed. The genetically engineered strain of Albion fungus overexpresses glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.

1.

5. A method for simultaneously increasing the content of sodium 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 Albion fungus is constructed. The genetically engineered strain of Albion fungus overexpresses glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase is 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 one or more of alanine, glycine, serine, proline and threonine; Bitter amino acids include one or more of arginine, histidine, isoleucine, leucine, methionine, phenylalanine, tyrosine and valine.

7. A method for increasing the cysteine ​​content in Alpinia terrestris, characterized in that: A genetically engineered strain of Albion fungus is constructed. The genetically engineered strain of Albion fungus overexpresses glutamate dehydrogenase, and the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO.

1.

8. Use of the genetically engineered bacteria of Alpinia terrestris 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.

9. The use according to claim 8, characterized in that: The amino acids include one or more of glutamic acid, aspartic acid, alanine, glycine, serine, proline, and threonine.

10. A method for increasing the glutamic acid content in Albizia albuminosa, characterized in that: Using improved PDA medium to culture genetically engineered bacteria of Alternaria albuminosa; The genetically engineered bacteria of Alternaria albuminosa overexpress glutamate dehydrogenase, and the amino acid sequence of glutamate dehydrogenase is shown in SEQ ID NO.1; The improved PDA culture medium is based on the PDA culture medium, and 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 are additionally added.

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