Construction and application of monascus over-expression GAD genetically engineered bacterium

Abstract

The invention relates to the technical field of biology, and particularly discloses construction and application of monascus overexpression GAD genetically engineered bacterium, and the construction of the monascus overexpression GAD genetically engineered bacterium comprises the following steps: constructing an expression vector pNeo-GAD recombinant plasmid, transforming the recombinant plasmid into agrobacterium, transforming monascus by an agrobacterium-mediated method, and carrying out PCR identification on monascus transformants. The application method comprises the following steps: culturing to obtain a seed solution of the monascus overexpression GAD genetically engineered bacterium, inoculating the seed solution into a fermentation culture medium, and carrying out fermentation culture to obtain a fermentation product of the monascus overexpression GAD genetically engineered bacterium; a fermentation product of the obtained monascus overexpression GAD genetic engineering bacterium contains high-yield monacolin K and high-yield gamma-aminobutyric acid, and meanwhile citrinin is not detected in the fermentation product of the monascus overexpression GAD genetic engineering bacterium.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the construction and application of a Monascus purpureus overexpressing the GAD gene. Background Technology

[0002] Monascus purpureus, belonging to the kingdom Fungi, phylum Ascomycota, class Ascomycota, order Monascoales, family Monasceae, and genus Monascus, is an acid-resistant, aerobic, and saprophytic filamentous fungus that can grow at temperatures ranging from 25 to 45°C and pH values ​​from 2.5 to 8.

[0003] Monascus purpureus fermentation can produce various functionally active substances, such as monacolin K (MK) and gamma-aminobutyric acid (GABA). Monacolin K has significant effects in inhibiting cholesterol synthesis, as well as antibacterial, anti-inflammatory, breast cancer reduction, antioxidant, and hepatoprotective effects. Monacolin K produced by Monascus purpureus has fewer side effects than that produced by Aspergillus terreus and has better application prospects. GABA has the effects of regulating blood pressure, improving brain function, promoting mental stability, and also has various physiological activities such as antidepressant, anti-anxiety, and hepatoprotective and nephroprotective effects. However, a few Monascus purpureus strains may produce citrinin during fermentation. Citrinin is a nephrotoxic fungal toxin with teratogenic effects.

[0004] Currently, Monascus purpureus fermentation produces relatively low levels of Monacolin K, and there is a lack of high-yielding strains. Therefore, it is of great significance to obtain high-yielding Monascus purpureus strains by overexpressing or knocking out key genes involved in Monacolin K production using genetic engineering techniques. However, most existing techniques for constructing high-yielding Monascus purpureus strains focus on manipulating the mokA-mokI gene clusters in the Monascus purpureus biosynthesis gene chain, while research on other genes that can affect Monascus accumulation is limited. Based on the above, this invention provides the construction and application of a Monascus purpureus strain overexpressing the GAD gene. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the construction and application of an engineered strain of Monascus purpureus overexpressing the GAD gene.

[0006] In a first aspect, the present invention provides a method for constructing an engineered strain of Monascus purpureus overexpressing the GAD gene, using the following technical solution:

[0007] The construction of a Monascus purpureus overexpressing the GAD gene includes the following steps:

[0008] A1. Obtain the full-length cDNA sequence of Monascus purpureus GAD gene, add restriction enzyme sites to both ends of the GAD gene, synthesize the GAD gene, clone the GAD gene into a vector, and obtain a recombinant plasmid.

[0009] A2. Transform the recombinant plasmid into Agrobacterium to obtain recombinant Agrobacterium;

[0010] A3. Induced culture of recombinant Agrobacterium to obtain Agrobacterium bacterial solution, enrichment culture of Monascus purpureus to obtain Monascus purpureus spore solution, and co-culture Agrobacterium bacterial solution and Monascus purpureus spore solution at a mass ratio of 1:0.9-1.1.

[0011] A4. After co-culturing the Monascus spore liquid and Agrobacterium tumefaciens liquid, pour the mixture into the screening medium for the first screening culture. Select the transformants that grow normally on the first screening medium and transfer them to a new screening medium for the second screening culture.

[0012] A5. Transformants that grow normally on the second screening medium are cultured, and genomic DNA is extracted from the mycelium of the transformants after culture. Specific primers GAD-W1C and PgpdA-F3 are designed to verify the transformants by PCR, and engineered bacteria of Monascus purpureus overexpressing the GAD gene are obtained.

[0013] Preferably, the Monascus pilosus strain used in step A1 is Monascus pilosus strain CICC5045.

[0014] Preferably, adding restriction sites to both ends of the GAD gene in step A1 specifically refers to adding HindIII restriction site to the 5' end of the GAD gene and SacI restriction site to the 3' end of the gene; the vector in step A1 is pNeo-0380.

[0015] Preferably, the OD of the Agrobacterium tumefaciens bacterial solution in step A3 is... 600 The value was 0.5-0.7, and the spore concentration of Monascus purpureus spore solution was 0.9 × 10⁻⁶. 6 -1.1×10 6 per mL.

[0016] Preferably, the screening medium in step A4 is a malt extract agar medium containing 75-85 μg / mL of genimycin, 190-210 μmol / L of cephalosporin, and 0.15-0.25% Triton X-100.

[0017] Secondly, this invention provides an application of a Monascus purpureus overexpressing the GAD gene in an engineered bacterium, employing the following technical solution:

[0018] A genetically engineered strain of Monascus purpureus overexpressing the GAD gene has been used to produce Monacoline K and γ-aminobutyric acid.

[0019] Preferably, the application method of the *Monascus purpureus* overexpressing GAD gene engineered strain includes the following steps:

[0020] S1. Inoculate the engineered strain of Monascus purpureus overexpressing the GAD gene into the seed culture medium and culture it in a shaker at 26-30℃ and 160-200rpm for 46-50h. After the culture is completed, filter it in a clean bench using sterilized three-layer lens paper to obtain the seed culture.

[0021] S2. The seed culture obtained in step S1 is inoculated onto the fermentation medium at an inoculation rate of 8-12% (v / v) to obtain the fermentation product of the Monascus purpureus overexpressing the GAD gene.

[0022] Preferably, the spore concentration in the seed solution in step S1 is 0.9 × 10⁻⁶. 6 -1.1×10 6 per mL.

[0023] Preferably, the fermentation medium in step S2 is liquid rice culture medium or solid buckwheat culture medium.

[0024] Preferably, the culture conditions for the liquid rice culture medium are: shaking culture at 26-30℃ and 160-200 rpm for 13-17 days.

[0025] Preferably, the culture conditions for the solid buckwheat culture medium are: cultured in an incubator at 26-30℃ for 10-14 days.

[0026] In summary, the present invention has the following beneficial effects:

[0027] 1. This invention constructs the expression vector pNeo-GAD recombinant plasmid, transforms the recombinant plasmid into Agrobacterium, transforms Monascus purpureus using Agrobacterium-mediated transformation, and identifies the Monascus purpureus transformants by PCR, successfully obtaining an engineered strain of Monascus purpureus overexpressing the GAD gene; the Monascus purpureus engineered strain overexpressing the GAD gene obtained by this invention can be used for fermentation to produce high yields of Monacoline K and γ-aminobutyric acid, and will not produce citrinin during fermentation.

[0028] 2. The total MK yield in the fermentation product of the Monascus purpureus overexpressing the GAD gene obtained in this invention is increased by 52.5% compared with the original strain Monascus purpureus CICC5045.

[0029] 3. The total GABA yield in the fermentation product of the Monascus purpureus overexpressing the GAD gene obtained in this invention is increased by 134.14% compared with the original strain Monascus purpureus CICC5045.

[0030] 4. No citrinin was detected in the fermentation products of the Monascus purpureus overexpressing the GAD gene engineered strain obtained in this invention, that is, its citrinin production was below the detection limit or it did not produce citrinin. Attached Figure Description

[0031] Figure 1 Electrophoresis diagram for PCR identification of Escherichia coli plasmid DNA.

[0032] Figure 2 This is a plasmid map of the eukaryotic expression vector pNeo-GAD.

[0033] Figure 3 Electrophoresis diagram for PCR identification of Agrobacterium transformants.

[0034] Figure 4 Electrophoresis diagram for PCR identification of Monascus purpureus resistant transformants.

[0035] Figure 5 High-performance liquid chromatography (HPLC) chromatograms of MK standard, starting strain Monascus purpureus CICC5045, and transformed strain in MK content detection.

[0036] Figure 6 The MK yield of the starting strain Monascus purpureus CICC5045 and the transformed strain.

[0037] Figure 7 This is a high-performance liquid chromatogram of different samples for GABA content detection.

[0038] Figure 8 This is the GABA standard curve.

[0039] Figure 9 The GABA production of the starting strain Monascus purpureus CICC5045 and the transformed strain was measured.

[0040] Figure 10 The high-performance liquid chromatograms are of citrinin standard, the starting strain Monascus purpureus CICC5045, the transformed strain, and commercially available red yeast rice. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the embodiments.

[0042] The strains and vectors used in the embodiments of this invention are as follows:

[0043] Monascus pilosus strain CICC5045 was purchased from the China Industrial Microbial Culture Collection Center and used as the starting strain. Biological Preservation Information: Monascus pilosus TG08 was deposited on October 28, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 41583. It is classified and named Monascus pilosus as the starting strain.

[0044] Agrobacterium strain AGL-1, preserved in our laboratory, is used to mediate the genetic transformation of Monascus purpureus;

[0045] pNeo-GAD plasmid, synthesized by Shanghai Platinum Biotech Co., Ltd., is used as a recombinant plasmid.

[0046] The vector pNeo-0380, preserved in our laboratory, is used for the construction of recombinant plasmids.

[0047] The main reagents used in the embodiments of this invention are as follows:

[0048] Taq DNA polymerase was purchased from Beijing TransGen Biotech Co., Ltd.; restriction endonucleases Sac I and HindIII, and DNA Markers DL2000 and DL15000 were purchased from (TAKARA) Biotech Co., Ltd.

[0049] Plasmid extraction kit PlasmidKit I was purchased from Omega in the USA;

[0050] The specific primers GAD-W1C and PgpdA-F3 were both synthesized by Shanghai Platinum Biotech Co., Ltd.

[0051] Kanamycin, Genomycin, and Triton X-100 were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0052] 50×TAE buffer, rifampin, and Agrobacterium rhizogenes liquid culture medium (YEB) were all purchased from Shanghai Sobo Biotechnology Co., Ltd.; cephalosporin, LB medium (powder), and ethidium bromide were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0053] Agrobacterium rhizogenes culture medium (YEB) was purchased from Shanghai Suoqiao Biotechnology Co., Ltd.; malt extract agar medium and malt extract culture medium were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0054] Agar powder was purchased from Shanghai Zhihua Chemical Technology Co., Ltd.

[0055] All other chemical reagents involved in the embodiments of this invention were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0056] The culture medium and solution preparation process used in the embodiments of the present invention are as follows:

[0057] LB medium: Prepare according to the LB medium instructions. When preparing solid medium, add an additional 1.5% agar powder, heat to completely dissolve before dispensing, sterilize at 121℃ for 20 minutes, and set aside.

[0058] Liquid YEB medium: Weigh 16g of Agrobacterium rhizogenes liquid medium powder, dissolve in 1000mL of distilled water, heat to boiling until completely dissolved, dispense an appropriate amount of medium into Erlenmeyer flasks, and sterilize at 121℃ for 20min. Cool to 50℃, add filtered and sterilized antibiotic solution under aseptic conditions, mix well, and dispense for use.

[0059] Solid YEB medium: Weigh 31.5g of Agrobacterium rhizogenes solid medium powder, dissolve it in 1000mL of distilled water, stir and heat until completely dissolved, dispense the medium into Erlenmeyer flasks, sterilize at 121℃ for 20min, cool to 50℃, add filtered and sterilized antibiotic solution under aseptic conditions, mix well, pour into plates, and set aside.

[0060] Malt extract agar medium: Weigh 145.1g of this product, heat and dissolve it in 1000mL of distilled water, dispense an appropriate amount of medium into an Erlenmeyer flask, autoclave at 121℃ for 15min, cool to 50℃, pour into plates, and set aside.

[0061] Malt extract culture medium: Weigh 130.1g of this product, heat and dissolve it in 1000mL of distilled water, dispense an appropriate amount of culture medium into an Erlenmeyer flask, autoclave at 121℃ for 15min, and set aside.

[0062] Phosphate buffer solution: Weigh 17g of potassium dihydrogen phosphate and 21.8g of dipotassium hydrogen phosphate, dissolve them separately in distilled water, and then bring each solution to a final volume of 100mL. Adjust the pH of the dipotassium hydrogen phosphate solution to 4.8 using the potassium dihydrogen phosphate solution. Autoclave at 121℃ for 15min and store at 4℃ for later use.

[0063] MN buffer: Weigh 3g magnesium sulfate heptahydrate and 1.5g sodium chloride, dissolve them in distilled water, bring the volume up to 100mL, autoclave at 121℃ for 15min, and store at 4℃ for later use.

[0064] IM Trace Element Storage Solution: Weigh 0.01 g each of zinc sulfate heptahydrate, boric acid, copper sulfate pentahydrate, manganese sulfate monohydrate, and sodium molybdate dihydrate, dissolve them in distilled water, and finally adjust the volume to 100 mL. Autoclave at 121 °C for 15 min and store at 4 °C for later use.

[0065] Acetyleugenol (AS) solution: Weigh 78.5 mg acetyleugenol and dissolve it in 2 mL of dimethyl sulfoxide solution to prepare a 0.2 mol / L stock solution. Sterilize by filtration through a sterile filter membrane in a clean bench and store at 4 °C for later use.

[0066] 1M Morpholine Ethylene Sulfate (MES) Solution: Weigh 19.5g of MES and dissolve it in 70mL of distilled water (stirring in a 50℃ water bath). Adjust the pH to 5.5 with 5M sodium hydroxide solution, and finally bring the volume to 100mL. Filter the solution through a sterile membrane in a laminar flow hood for sterilization, and store at 4℃ for later use.

[0067] Add the sample and mix according to the formula in Table 1, add distilled water to a final volume of 100 mL, and sterilize at 121°C for 20 min to obtain liquid AIM medium. When preparing solid AIM medium, add an additional 2% agar.

[0068] Table 1 AIM culture medium formulation

[0069] Element content MN buffer solution 2mL IM solution 500μL 50% glycerol aqueous solution 1mL Phosphate buffer solution 80μL <![CDATA[1% CaCl2 solution]]> 100μL <![CDATA[(NH4)SO4 solution]]> 250μL 20% glucose solution 500μL (250μL for solid-state applications) AS solution 100μL (filtered and sterilized, add before use) <![CDATA[0.01% FeSO4 solution]]> 1mL (filtered and sterilized, add before use) MES solution 4mL (filtered and sterilized, add before use)

[0070] Seed culture medium: Weigh 30g glucose, 70g glycerol, 10g peptone, 15g soybean meal powder, 1g magnesium sulfate heptahydrate, 2g dipotassium hydrogen phosphate, and 2g sodium nitrate. Dissolve them in 1000mL distilled water by heating. Then dispense 100mL into 250mL Erlenmeyer flasks (each containing 5 glass beads). Autoclave at 121℃ for 20min.

[0071] Solid buckwheat culture medium: Add 30g of buckwheat to a 300mL PE fermentation flask, add 20mL of a glycerol and water mixture with a mass ratio of 1:3, mix well, and then autoclave at 121℃ for 20min.

[0072] Liquid rice culture medium: Rice is pulverized and passed through a 60-mesh sieve to obtain rice flour. Weigh out 20g of rice flour, 90g of glycerol, 2g of zinc sulfate heptahydrate, 2.5g of dipotassium hydrogen phosphate, 5g of sodium nitrate, 10g of peptone, and 1g of magnesium sulfate heptahydrate. Dissolve the rice flour in 100mL of distilled water. Dispense the solution into 250mL Erlenmeyer flasks (100mL each) and autoclave at 121℃ for 20min.

[0073] The main instruments used in the embodiments of this invention are shown in Table 2 below:

[0074] Table 2 Main Experimental Instruments

[0075] instrument model Manufacturer Smart City Constant Temperature Incubator Shaker ZWY-2102C Shanghai Zhicheng Analytical Instruments Manufacturing Co., Ltd. Vertical shaking incubator MQL-61R Shanghai Minquan Instrument Co., Ltd. Biochemical incubator LRH-250 Shanghai Yiheng Scientific Instruments Co., Ltd. Biological microscope BM1800 Nanjing Jiangnan Yongxin Optics Co., Ltd. Vertical automatic pressure steam sterilizer GI54DS Zhiwei (Xiamen) Instruments Co., Ltd. Dual-frequency CNC ultrasonic cleaner KQ-500VDE Kunshan Ultrasonic Instruments Co., Ltd. Electric heating drying oven DHG-9005 Shanghai Yiheng Scientific Instruments Co., Ltd. Circulating water multi-purpose vacuum pump SHB-III Zhengzhou Great Wall Science & Industry Trade Co., Ltd. High-speed multi-functional pulverizer WJX-100 Shanghai Yuanwo Industry & Trade Co., Ltd. High Performance Liquid Chromatography Waters e2695 Waters Technology (Shanghai) Co., Ltd. High Performance Liquid Chromatography 1260 Infinity II Agilent Technologies Clean bench VS-1300L-U Suzhou Antai Air Technology Co., Ltd. pH meter FE28 Mettler Toledo Instruments (Shanghai) Co., Ltd. Electronic balance BSA2202S-CW Sartorius Scientific Instruments (Beijing) Co., Ltd. Electric constant temperature water bath HWS-26 Shanghai Yiheng Scientific Instruments Co., Ltd.

[0076] Example 1: Obtaining recombinant plasmids

[0077] The full-length cDNA sequence of the Monascus tuber GAD gene was obtained from the USDA JGI database (Monascus tuber NRRL 1597 v1.0). Hind III restriction enzyme site was added to the 5' end of the GAD gene, and Sac I restriction enzyme site was added to the 3' end of the GAD gene to synthesize a 1657bp GAD gene. The GAD gene was then cloned into the pNeo-0380 vector to obtain the recombinant plasmid pNeo-GAD.

[0078] The recombinant plasmid pNeo-GAD was transformed into competent E. coli Top10 cells using a heat shock method and evenly spread on solid LB medium (containing 50 mg / L Kan) and incubated upside down at 37°C for 14 h. Single colonies were picked and transferred to liquid LB medium (containing 50 mg / L Kan) and incubated in a shaker at 37°C and 180 rpm for 14 h. E. coli plasmid DNA was then extracted using a plasmid extraction kit.

[0079] Based on the full-length cDNA sequence of the *Monascus purpureus* GAD gene and the promoter sequence of the PgpdA gene, specific primers GAD-W1C and PgpdA-F3 were designed. PCR identification of *E. coli* plasmid DNA was performed using primers GAD-W1C and PgpdA-F3. The reaction system is shown in Table 3, and the reaction conditions are shown in Table 4. The *E. coli* plasmid DNA was then identified by double digestion with Sac I and Hind III. The reaction system is shown in Table 5, and the reaction conditions were incubation at 37℃ for 15 min. The PCR products and enzyme digestion products were loaded onto a 0.8% agarose gel and electrophoresed for 40 min. The target bands were then detected using a gel imaging system. The results are shown below. Figure 1 As shown in the figure, left M represents DNA Marker DL 15000; 1 represents Sac I and Hind III digestion products; 2 represents PCR products; right M represents DNA Marker DL 2000. Figure 1 The target band identified by double digestion with Sac I and Hind III was approximately 12kb and 1.6kb, respectively. The target band identified by PCR was approximately 600bp. The electrophoresis results were consistent with the expected results, indicating that the vector was successfully constructed and the recombinant plasmid pNeo-GAD was obtained. The map of the recombinant plasmid is shown below. Figure 2 As shown.

[0080] Table 3 Reaction System

[0081] Composition Dosage (μL) 2×Fine TaqTM PCR SuperMix(+dye) 5.0 GAD-W1C (20μM) 0.2 PgpdA-F3 (20μM) 0.2 pNeo-GAD plasmid DNA 0.8 <![CDATA[ddH2O]]> 3.8

[0082] Table 4 Reaction conditions

[0083]

[0084] Table 5. Reaction system for double enzyme digestion identification of pNeo-GAD expression vector

[0085] Composition Dosage (μL) 10×QuickCut Buffer 1.0 QuickCut™ Hind III 0.2 QuickCut™ Sac I 0.2 pNeo-GAD plasmid DNA (≤0.2ug) 1.5 <![CDATA[ddH2O]]> 7.1

[0086] Example 2: Obtaining recombinant Agrobacterium

[0087] 1. Transformation of Agrobacterium with recombinant plasmid pNeo-GAD: ​​Take two tubes of AGL-1 Agrobacterium competent cells from the laboratory and place them at room temperature until slightly thawed. While in an ice-water mixture, insert them into ice. Add 1 μg of recombinant plasmid pNeo-GAD to one tube, and use the other tube as a blank control. Gently pipette and mix each tube thoroughly. First, place them on ice for 30 min, then flash freeze in liquid nitrogen for 5 min, then incubate in water at 37°C for 5 min, and finally incubate on ice for 5 min. Add 800 μL of preheated (28°C) liquid YEB medium to a clean bench and incubate at 28°C and 180 rpm for 2 h with shaking. Centrifuge the tubes at 5000g for 5 min to collect the cells. After removing 800 μL of supernatant, mix the remaining 200 μL of bacterial culture and spread 100 μL onto solid YEB medium (containing 50 mg / L Rif and 50 mg / L Kan). Incubate at 28°C upside down for 2 days until single colonies grow.

[0088] 2. Identification of Agrobacterium transformants: Single colonies of Agrobacterium were picked and cultured in liquid YEB medium (containing 50 mg / L Rif and 50 mg / L Kan) at 28°C and 180 rpm for 48 h in a shaker. Agrobacterium plasmids were extracted according to the plasmid extraction kit. Using recombinant plasmid DNA as a template, the PCR identification reaction system is shown in Table 6, and the reaction conditions are shown in Table 4. The PCR products were analyzed by 1% agarose gel electrophoresis to determine their size. The PCR identification results are shown in Table 4. Figure 3 As shown in the figure, M represents DNA Marker DL 2000; 1 represents Agrobacterium transformant; and 2 represents positive control (plasmid pNeo-GAD). The target band identified by PCR was approximately 600 bp, and the electrophoresis results were consistent with the expected results, indicating that the expression vector pNeo-GAD of the Monascus purpureus GAD gene was successfully transformed into Agrobacterium. Recombinant Agrobacterium AGL-1 / pNeo-GAD was obtained.

[0089] Table 6 Reaction System

[0090] Composition Dosage (μL) 2×Fine TaqTM PCR SuperMix(+dye) 5.0 GAD-W1C (20μM) 0.2 PgpdA-F3 (20μM) 0.2 Recombinant plasmid DNA 0.8 <![CDATA[ddH2O]]> 3.8

[0091] Example 3: Obtaining a genetically engineered strain of Monascus purpureus overexpressing the GAD gene.

[0092] 1. Induction culture of Agrobacterium: Recombinant Agrobacterium AGL-1 / pNeo-GAD was streaked onto solid YEB medium (containing 50 mg / L Rif and 50 mg / L Kan) at 28°C for 42 h until single colonies appeared. A single Agrobacterium colony was picked and inoculated into 20 mL of liquid YEB medium (containing 50 mg / L Rif and 50 mg / L Kan), and cultured at 28°C and 180 rpm in a shaker for 48 h. The OD of the bacterial culture was then measured. 600 The bacterial culture was diluted to OD using liquid AIM induction medium (containing 200 μmol / L acetylsyleugenone). 600 The value was 0.15, and the culture was continued for another 6 hours until the OD value was 0.15. 600 The value is 0.6.

[0093] 2. Enrichment of Monascus purpureus conidia: The starting strain Monascus purpureus CICC5045 was inoculated onto malt extract agar medium and cultured at 28°C for 7 days. The spore suspension was collected in a clean bench, centrifuged at 5000g for 5 minutes to obtain a concentrated spore solution. Spores were counted using a hemocytometer under an optical microscope, and the spore concentration was adjusted to 1×10⁻⁶. 6 per mL.

[0094] 3. Mix the Agrobacterium tumefaciens bacterial suspension from step 1 and the Monascus purpureus spore suspension from step 2 at a mass ratio of 1:1, spread the mixture onto solid AIM medium, and incubate upright at 28°C for 2 hours, then invert for 48 hours. After co-culturing the Agrobacterium tumefaciens bacterial suspension and Monascus purpureus spore suspension for 48 hours, pour the mixture into screening medium (malt extract agar + 80 μg / mL G418 + 200 μmol / L Cef + 0.2% X-100), and incubate inverted at 28°C for 6 days for the first screening culture. Select the transformants that grow normally on the first screening medium and transfer them to a new screening medium (malt extract agar + 80 μg / mL G418 + 200 μmol / L Cef + 0.2% X-100) for a second screening culture to obtain resistant transformants.

[0095] 4. The resistant transformants were inoculated onto malt extract agar medium and cultured normally, then transferred to liquid malt extract medium and cultured for 3 days at 28℃ and 220 rpm in a shaker. Genomic DNA was extracted from the mycelium of the transformants using a novel rapid genomic DNA extraction kit. Using the starting strain *Monascus purpureus* CICC5045 as a negative control, and the genomic DNA from the transformant mycelium as a template, PCR verification of the resistant transformants was performed using specific primers GAD-W1C and PgpdA-F3. The reaction system is shown in Table 7, and the reaction conditions are shown in Table 4. After the reaction, the PCR products were loaded onto a 1.2% agarose gel for electrophoresis for 30 min. Finally, the target band was detected using a gel imaging system. The results are shown in Table 4. Figure 4As shown in the figure, M represents DNA Marker DL 2000; 1-9 represent Monascus purpureus resistance transformants; 10 represents the positive control (pNeo-GAD plasmid); and 11 represents the negative control (starting strain Monascus purpureus CICC5045). The target band identified by PCR was approximately 600 bp, and the electrophoresis results were consistent with expectations. A total of 20 Monascus purpureus strains overexpressing the GAD gene were obtained, designated as TG01-TG20.

[0096] Table 7 Reaction System

[0097] Composition Dosage (μL) 2×Fine TaqTM PCR SuperMix(+dye) 5.0 GAD-W1C (20μM) 0.2 PgpdA-F3 (20μM) 0.2 Genomic DNA of transformant mycelium 0.8 <![CDATA[ddH2O]]> 3.8

[0098] Example 4: Application of Monascus purpureus overexpressing GAD gene-engineered bacteria

[0099] 1. Activation of Monascus purpureus strains: The GAD-overexpressing engineered strain TG01-TG20 and the starting strain Monascus purpureus CICC5045 were inoculated onto malt extract agar medium using an inoculation loop in a clean bench and cultured at 28°C for 7 days to obtain activated strains TG01-TG20 and CICC5045.

[0100] 2. Preparation of Monascus purpureus seed culture: In a clean bench, activated strains TG01-TG20 and CICC5045 were separately inoculated into seed culture medium using a sterile punch. The culture was then incubated at 28℃ and 180 rpm for 48 hours. After incubation, the culture was filtered through sterile triple-layer lens paper in a clean bench to obtain seed cultures of engineered strains TG01-TG20 and CICC5045. The spore concentration in the seed cultures was controlled to be 1×10⁻⁶. 6 per mL.

[0101] 3. Solid-state fermentation culture: Seed cultures of engineered strains TG01-TG20 and Monascus purpureus CICC5045 were inoculated into solid buckwheat culture medium at a rate of 10% (v / v), stirred thoroughly with a sterile glass rod, and incubated at 28℃ for 12 days. Four replicates were set up for each strain to obtain fermentation products of engineered strains TG01-TG20 and Monascus purpureus CICC5045, respectively.

[0102] In another embodiment, the solid-state fermentation culture in Example 4 can be replaced with liquid-state fermentation culture, as follows:

[0103] Liquid fermentation culture: Seed cultures of engineered strains TG01-TG20 and Monascus purpureus CICC5045 were inoculated into liquid rice culture medium at a rate of 10% (v / v), and then cultured in a shaker at 28℃ and 180 rpm for 15 days. Four replicates were set up for each strain to obtain the fermentation products of engineered strains TG01-TG20 and Monascus purpureus CICC5045, respectively.

[0104] Performance testing

[0105] The contents of Monacolin K, γ-aminobutyric acid and citrinin in the fermentation products of engineered bacteria TG01-TG20 and Monascus purpureus CICC5045 in Example 4 of the present invention were tested respectively.

[0106] I. Detection of MK content

[0107] The fermentation products of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045 were dried separately in a 55℃ forced-air drying oven, then pulverized and passed through a 60-mesh sieve to obtain the test samples of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045. 1g of each test sample was weighed and placed in a 10mL centrifuge tube, and 75% ethanol solution was added to bring the volume to the 10mL mark. After mixing thoroughly, the mixture was extracted ultrasonically for 30min, shaking every 15min. After extraction, the mixture was shaken again and allowed to stand. The supernatant was filtered through a 0.22μm filter membrane for instrumental analysis of MK content.

[0108] The MK content in the sample was detected by high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 column (4.6 mm × 150 mm, 5 μm); mobile phase: acetonitrile: 0.1% phosphoric acid aqueous solution = 55:44 (v / v); flow rate: 1 mL / min; column temperature: 25℃; injection volume: 20 μL; and UV detection wavelength: 238 nm. The formula for calculating the MK content in the sample is as follows:

[0109]

[0110] HPLC detection results of MK content are as follows Figure 5 As shown in the figure, A represents the high-performance liquid chromatography (HPLC) chromatogram of the acidic MK standard, B represents the HPLC chromatogram of the lactone MK standard, C represents the HPLC chromatogram of the test sample from the starting strain Monascus purpureus CICC5045, and D represents the HPLC chromatogram of the test sample from the transformed strain. The retention time R of the acidic MK standard is... t =8.915min ( Figure 5 -A), Retention time R of lactone-type standards t =17.559min ( Figure 5-B), the retention times of the acidic MK and lactone MK of the test sample from the starting strain Monascus purpureus CICC5045 were R, respectively. t =9.027min and R t =17.566min ( Figure 5 -C), the retention times of the acidic MK and lactone MK in the transformed strain sample were R t =9.068min and R t =17.451min ( Figure 5 -D).

[0111] The MK content calculation results are as follows Figure 6 As shown, the different lowercase letters among the different strains indicate that the differences in MK yield reached a significant level (p < 0.05). Among the transformed strains of Monascus purpureus overexpressing the GAD gene, 13 strains (TG05, TG06, TG07, TG08, TG09, TG10, TG11, TG12, TG13, TG15, TG18, TG19, TG20) had significantly higher total MK yields than the starting strain Monascus purpureus CICC5045, with an increase of 12.84%-52.50%. The strains with the largest increases were TG12 (1935.21 mg / kg) and TG08 (1871.99 mg / L), which increased MK yields by 52.5% and 47.51% respectively compared to the starting strain Monascus purpureus CICC5045 (1269.03 mg / kg). The remaining transformed strains did not show significant differences from the starting strain.

[0112] II. Detection of GABA content

[0113] 1. Preparation of test samples: The fermentation products of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045 were dried in a 55℃ forced-air drying oven, pulverized, and passed through a 60-mesh sieve to obtain the test samples of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045. The detection method for GABA content in the test samples refers to the national standard NY / T2890-2016, specifically: 1g of the above test sample was weighed and added to a 50mL centrifuge tube, 10mL of 80% ethanol was added, and ultrasonic extraction was performed for 30min. After shaking, the mixture was allowed to stand for 5min, and the supernatant was transferred to a 25mL volumetric flask. The filter residue was repeated, and the two extracts were combined. The volume was adjusted to 25mL with 80% ethanol solution, and the mixture was shaken to obtain the sample solutions of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045, which were then subjected to derivatization treatment.

[0114] 2. Preparation of γ-aminobutyric acid stock solution.

[0115] Accurately weigh 10 mg of γ-aminobutyric acid (GABA) standard, dissolve it in acetonitrile, and dilute to 10 mL to obtain a 1000 mg / L standard solution. Prepare working standard solutions of 1 mg / L, 5 mg / L, 25 mg / L, 60 mg / L, and 100 mg / L using the standard solution. Prepare working standard solutions fresh each time you need to use them.

[0116] 3. Derivatization test of sample solution.

[0117] Pipette 1 mL of the sample solution of engineered bacteria TG01-TG20 and Monascus purpureus CICC5045 into stoppered test tubes. Add 1 mL of standard working solution, 1 mL of 80% ethanol solution, 0.2 mL of sodium bicarbonate solution, and 0.4 mL of 4-(N,N-dimethylamino)azobenzene-4'-sulfonic acid chloride (CAS: 56512-49-3) derivatizing reagent to the stoppered test tubes. Mix well and react in a water bath at 70℃ for 20 min. Cool to room temperature and filter through a 0.22 μm organic filter membrane to obtain the samples of engineered bacteria TG01-TG20 and Monascus purpureus CICC5045 for HPLC detection.

[0118] 4. HPLC detection conditions for GABA content

[0119] The chromatographic column was a C18 column (4.6 mm × 150 mm, 5 μm), the mobile phase was V(acetonitrile) : V(sodium acetate trihydrate solution) = 35 : 65, the flow rate was 1 mL / min, the column temperature was 28 ℃, the injection volume was 10 μL, and the UV detection wavelength was 438 nm.

[0120] The formula for calculating the γ-aminobutyric acid content in the sample to be tested is as follows:

[0121]

[0122] ρ: The mass concentration (mg / L) of γ-aminobutyric acid in the sample measured by the instrument;

[0123] V: The final volume (mL) of the sample used in the test chamber;

[0124] m: Mass of the sample used in the test (g).

[0125] The HPLC detection results of GABA content are as follows: Figure 7As shown in the figure, A represents the high-performance liquid chromatography (HPLC) chromatogram of the derivatizing reagent 4-(N,N-dimethylamino)azobenzene-4'-sulfonic acid chloride; B represents the HPLC chromatogram of the γ-aminobutyric acid standard; C represents the HPLC chromatogram of the test sample from the starting strain Monascus purpureus CICC5045; and D represents the HPLC chromatogram of the test sample from the transformed strain. The retention time R of the derivatizing reagent 4-(N,N-dimethylamino)azobenzene-4'-sulfonic acid chloride (CAS: 56512-49-3) is shown in the figure. t =3.572min ( Figure 7 -A), Retention time R of γ-aminobutyric acid standard t =5.208min( Figure 7 -B). The γ-aminobutyric acid retention times of the starting strain Monascus purpureus CICC5045 and the transformed strain in the test samples were R. t =5.310min ( Figure 7 -C) and R t =5.279min( Figure 7 -D).

[0126] After derivatizing the prepared standard working solutions, 10 μL of each solution was injected. Based on the high-performance liquid chromatography (HPLC) results, a standard curve was plotted with the mass concentration of γ-aminobutyric acid (GABA) on the x-axis and the peak area on the y-axis, as shown below. Figure 8 As shown.

[0127] Based on the HPLC detection results and the GABA standard curve, the GABA content in the starting strain *Monascus purpureus* CICC5045 and the transformed strain in the test samples was calculated. The results are as follows: Figure 9 As shown in the figure, the different lowercase letters among different strains indicate that the differences in their GABA production reached a significant level (p < 0.05).

[0128] Among the Monascus purpureus strains overexpressing the GAD gene, 17 strains (TG02, TG03, TG04, TG05, TG06, TG07, TG08, TG09, TG10, TG12, TG13, TG14, TG16, TG17, TG18, TG19, and TG20) showed significantly higher GABA production than the original strain, Monascus purpureus CICC5045, with increases ranging from 17.47% to 134.14%. Among these, strain TG18 (1700.5%) showed the highest GABA production. The GABA production of the TG08 strain (TG01, TG02, TG03, TG04, TG19, TG16, TG08, TG08, TG09, TG08 ...

[0129] III. Detection of Citrulline Content

[0130] The fermentation products of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045 were dried separately in a 55℃ forced-air drying oven, then pulverized and passed through a 60-mesh sieve to obtain the test samples of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045. 1g of each test sample was weighed and placed in a 10mL centrifuge tube, and 80% methanol solution was added to a final volume of 6mL. The tube was shaken well, and the mixture was extracted ultrasonically for 30min. Afterward, the tube was shaken again and placed in a constant-temperature shaker for 60min. After extraction, the tubes were allowed to stand, and the supernatant was filtered through a 0.22μm filter membrane to obtain the HPLC samples of engineered bacteria TG01-TG20 and *Monascus purpureus* CICC5045 for analysis.

[0131] The content of citrinin in the sample was detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (4.6 mm × 150 mm, 5 μm); mobile phase: V (ultrapure water, pH adjusted to 2.5 with phosphoric acid): V (acetonitrile) = 50:50; flow rate: 1 mL / min; column temperature: 30 °C; injection volume: 20 μL; fluorescence detection wavelength: λex = 331 nm, λem = 500 nm.

[0132] The formula for calculating the citrinin content in the sample to be tested is as follows:

[0133]

[0134] The HPLC detection results of citrinin content are as follows: Figure 10As shown in the figure, A represents the high-performance liquid chromatography (HPLC) chromatogram of citrinin standard, B represents the HPLC chromatogram of the test sample from the starting strain *Monascus purpureus* CICC5045, C represents the HPLC chromatogram of the test sample from the transformed strain, and D represents the HPLC chromatogram of the test sample from commercially available red yeast rice. The retention time R of the citrinin standard is shown in the figure. t =15.243min ( Figure 10 -A), Retention time R of citrinin in commercially available red yeast rice samples t =15.246min ( Figure 10 -D), while citrinin was not detected in the test samples of the starting strain Monascus purpureus CICC5045 and the transformed strain. Figure 10 -B and Figure 10 -C). This indicates that the citrinin production of Monascus purpureus strains overexpressing GAD is below the detection limit or that they do not produce citrinin.

[0135] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Construction of a genetically engineered strain of Monascus purpureus overexpressing the GAD gene, characterized in that, Includes the following steps: A1. Obtain the full-length cDNA sequence of Monascus purpureus GAD gene, add restriction enzyme sites to both ends of the GAD gene, synthesize the GAD gene, clone the GAD gene into a vector, and obtain a recombinant plasmid. A2. Transform the recombinant plasmid into Agrobacterium to obtain recombinant Agrobacterium; A3. Induced culture of recombinant Agrobacterium to obtain Agrobacterium bacterial solution, enrichment culture of Monascus purpureus to obtain Monascus purpureus spore solution, and co-culture Agrobacterium bacterial solution and Monascus purpureus spore solution at a mass ratio of 1:0.9-1.

1. A4. After co-culturing the Monascus spore liquid and Agrobacterium tumefaciens liquid, pour the mixture into the screening medium for the first screening culture. Select the transformants that grow normally on the first screening medium and transfer them to a new screening medium for the second screening culture. A5. Transformants that grow normally on the second screening medium are cultured, and genomic DNA is extracted from the mycelium of the transformants after culture. Specific primers GAD-W1C and PgpdA-F3 are designed to verify the transformants by PCR, and engineered bacteria of Monascus purpureus overexpressing the GAD gene are obtained.

2. The construction of the engineered strain of Monascus purpureus overexpressing the GAD gene according to claim 1, characterized in that, In step A1, the Monascus purpureus is Monascus purpureus CICC5045.

3. The construction of the engineered strain of Monascus purpureus overexpressing the GAD gene according to claim 1, characterized in that, In step A1, adding restriction sites to both ends of the GAD gene specifically refers to adding HindIII restriction site to the 5' end of the GAD gene and SacI restriction site to the 3' end of the gene; the vector in step A1 is pNeo-0380.

4. The construction of the engineered strain of Monascus purpureus overexpressing the GAD gene according to claim 1, characterized in that, The OD of the Agrobacterium tumefaciens bacterial solution in step A3 600 The value was 0.5-0.7, and the spore concentration of Monascus purpureus spore solution was 0.9 × 10⁻⁶. 6 -1.1×10 6 per mL.

5. The construction of the engineered strain of Monascus purpureus overexpressing the GAD gene according to claim 1, characterized in that, The screening medium in step A4 is malt extract agar medium containing 75-85 μg / mL of genimycin, 190-210 μmol / L of cephalosporin, and 0.15-0.25% Triton X-100.

6. The application of a Monascus purpureus-overexpressing GAD gene-engineered bacterium, characterized in that, The Monascus purpureus overexpressing GAD gene engineered strain is obtained by constructing the Monascus purpureus overexpressing GAD gene engineered strain according to any one of claims 1-5, and the Monascus purpureus overexpressing GAD gene engineered strain is used to produce Monacoline K and γ-aminobutyric acid.

7. The application of the Monascus purpureus overexpressing the GAD gene engineered strain according to claim 6, characterized in that, The application method includes the following steps: S1. Inoculate the GAD-overexpressing engineered strain of Monascus purpureus into the seed culture medium and culture it in a shaker at 26-30℃ and 160-200rpm for 46-50h. After the culture is completed, filter it in a clean bench using sterilized three-layer lens paper to obtain the seed culture. S2. The seed culture obtained in step S1 is inoculated onto the fermentation medium at an inoculation rate of 8-12% to obtain the fermentation product of the Monascus purpureus overexpressing the GAD gene.

8. The application of the Monascus purpureus overexpressing the GAD gene engineered strain according to claim 7, characterized in that, In step S1, the spore concentration in the seed solution is 0.9 × 10⁻⁶. 6 -1.1×10 6 per mL.

9. The application of the Monascus purpureus overexpressing the GAD gene engineered strain according to claim 7, characterized in that, In step S2, the fermentation medium is either liquid rice culture medium or solid buckwheat culture medium.

10. The application of the Monascus purpureus overexpressing the GAD gene engineered strain according to claim 9, characterized in that, The culture conditions for the liquid rice culture medium are: shaking culture in a shaker at 26-30℃ and 160-200rpm for 13-17 days; the culture conditions for the solid buckwheat culture medium are: culture in an incubator at 26-30℃ for 10-14 days.

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