A transformation strain of Monascus rubrum TS-18 and its application

Abstract

The invention discloses a Monascus pubescens transformation strain TS-18 and an application thereof, belonging to the technical field of microorganisms. The Monascus pubescens transformation strain TS-18 is classified and named as Monascus pubescens, and its Latin name is: Monascus pilosus , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC NO.41081. Its total MK production is significantly improved compared with the starting strain CICC 5045.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Monascus rubrum transformation strain TS-18 and an application thereof. Background Art

[0002] Monascus, also known as red yeast rice, red mold rice, Danqu and Anka, is named beni-koji in Japan. It is a specialty of East Asian countries and belongs to the Aspergillus family in the Ascomycetes class. Studies have found that red yeast can produce a variety of biologically active metabolites, including pigments, monacolin K, γ-aminobutyric acid, antibiotics, and polysaccharides. Its application in medicine and health products is also popular.

[0003] Monacolin K (MK) is a secondary metabolite produced by Monascus with important application value. It can mainly treat hypercholesterolemia, and also has antibacterial, colon cancer reduction, breast cancer reduction, antioxidant, prostate reduction, liver protection, and accelerated nerve regeneration effects, and has good application prospects. As a substance that inhibits cholesterol, MK inhibits cholesterol synthesis by competitively inhibiting the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (3-Hydroxy-3-methylglutaryl-coenzyme A reductase, HMG-CoA), the rate-limiting enzyme in cholesterol synthesis, thereby reducing cholesterol levels in human and animal blood. In order to obtain higher MK production, there have been many studies and reports on increasing the production of Monascus MK in recent years. Commonly used methods include screening of high-yield strains, optimization of Monascus fermentation conditions and addition of exogenous substances, physical and chemical mutagenesis, etc. However, these methods have certain limitations and the improvement effect is also limited. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to obtain a high-yield MK Monascus rubrum strain.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a transformation strain TS-18 of Monascus pilosus, classified and named: Monascus pilosus, Latin name: Monascus pilosus, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit date of January 12, 2024, and a deposit number: CGMCC NO.41081.

[0006] The above-mentioned Monascus rubrum transformed strain TS-18 is used in the preparation of monacolin K.

[0007] The beneficial effect of the present invention is that the total MK yield of the Monascus rubrum transformed strain TS-18 provided by the present invention is significantly improved compared with the starting strain CICC 5045. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Shown is the PCR identification result of the Escherichia coli transformant in Example 1 of the present invention;

[0009] Figure 2 The figure shows the double enzyme digestion identification result of the recombinant plasmid pNeo-ST in Example 1 of the present invention;

[0010] Figure 3 Shown is the PCR identification result of the transformed strain of Monascus pubescens in Example 1 of the present invention;

[0011] Figure 4 The results are shown for detecting the MK yield of the transformed strain and the starting strain of Monascus pubescens in Example 2 of the present invention;

[0012] Figure 5 The biomass detection results of the Monascus rubrum transformation strain and the starting strain in Example 2 of the present invention are shown;

[0013] Figure 6 Shown are scanning electron micrographs of the mycelium morphology of the Monascus rubrum transformed strain TS-18 and the starting strain CCIC 5045 in Example 2 of the present invention. DETAILED DESCRIPTION

[0014] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0015] A transformed strain of Monascus pilosus TS-18, classified and named: Monascus pilosus, Latin name: Monascuspilosus, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit date of January 12, 2024, and a deposit number of CGMCC NO.41081.

[0016] From the above description, it can be seen that the beneficial effects of the present invention are: (technical effect of the unique technical solution)

[0017] Furthermore, the preparation method of the above-mentioned Monascus pubescens transformation strain TS-18 comprises the following steps:

[0018] S1. Construction of the eukaryotic expression vector pNeo-ST of the MpST gene

[0019] The MpST gene was amplified using specific primers ST-FH (5'-CCCAAGCTTATGAGTCAGGCTCGTTCATACC-3') and ST-R2-S (5'-CGAGCTCTTGCTCGACTCTTCATCTTCCT-3') with recognition sites for restriction endonucleases Hind III and Sac I, the PCR product was recovered and digested to obtain the digestion product of the MpST gene fragment, the digestion product of the MpST gene fragment was connected to the expression vector pNeo-0380 to obtain a ligation product, the ligation product was transformed into competent cells of Escherichia coli Top10, and after culture, plasmid DNA was extracted using a plasmid extraction kit to obtain the eukaryotic expression vector pNeo-ST;

[0020] S2. Agrobacterium-mediated transformation of Monascus pilosula

[0021] The eukaryotic expression vector pNeo-ST was transformed into Agrobacterium tumefaciens AGL-1 by the freeze-thaw method to obtain recombinant Agrobacterium AGL-1 / pNeo-ST; the recombinant Agrobacterium AGL-1 / pNeo-ST was then cultured to obtain AGL-1 / pNeo-ST bacterial solution, the AGL-1 / pNeo-ST bacterial solution was mixed with a conidia suspension of Monascus pubescens in a volume ratio of 1:1, and then screening culture was carried out using a screening medium.

[0022] Furthermore, the screening medium consisted of malt extract medium, 20.0 μL / mL G418 (Geneticin), 20.0 μL / mL X-100, and 20.0 μL / mL Cef (Cefoxim).

[0023] Furthermore, according to the full-length cDNA sequence of the MpST gene of M. ruber NRRL 1597v1.0 in the JGI database of the United States Department of Agriculture (https: / / mycocosm.jgi.doe.gov / Monru1 / Monru1.home.html), the online program Primer3web version 4.1.0 (https: / / primer3.ut.ee / ) was used to design specific primers ST-F2 (5′-TTTTAAACCCCTTGCAACCCAT-3′) and

[0024] ST-R2 (5'-TTGCTCGACTCTTCATCTTCCT-3') was constructed using the first-strand cDNA of Monascus rubrum CICC 5045 (stored by the Food Nutrition Laboratory, College of Food Science, Fujian Agriculture and Forestry University) as a template. HSDNAPolymerase was used for PCR amplification. A gene fragment of about 2 kb was obtained by PCR amplification. The sequencing results showed that the coding region of the sugar transporter gene of Monascus rubrum was 1677 bp in length and was named MpST. The nucleotide sequence of MpST was compared online with the GenBank database, and it was found that the homology between MpST and the MFStransporter gene (XM 013472746.1) of Rasamsonia emersonii CBS 393.64 was 73.85%, indicating that the protein encoded by the MpST gene has similar functions to the MFS transporter.

[0025] Furthermore, the sequence characteristics of MpST were analyzed using online software ProtParam and DNAMAN. The open reading frame of MpST contains 558 amino acids. From Tables 1 and 2, it can be seen that leucine, alanine, isoleucine and phenylalanine account for a large proportion of the amino acid composition of MpST, and there are many types of amino acids that make up MpST. The molecular weight of the encoded protein is 62.87 kDa, the isoelectric point is 6.42, and the molecular formula is C 2891 H 4396 N 746 O 785 S 22 , instability coefficient 39.81, fat coefficient 96.31, and total average hydrophilicity 0.255. When the instability coefficient is less than or equal to 40, the protein is called a stable protein. Therefore, MpST belongs to a stable protein. The cDNA sequence of MpST and its encoded amino acid sequence are shown in Table 1, and the specific amino acid composition is shown in Table 2.

[0026] Table 1

[0027]

[0028]

[0029] Table 2

[0030] Amino acid name Number percentage(%) Amino acid name Number percentage(%) Alanine 48 8.6 Leucine 49 8.8 Arginine 32 5.7 Lysine 12 2.2 Asparagine 23 4.1 Methionine 16 2.9 Aspartic acid 19 3.4 Phenylalanine 45 8.1 Cysteine 6 1.1 Proline 21 3.8 Glutamine 12 2.2 Serine 38 6.8 Glutamate 30 5.4 Threonine 34 6.1 Glycine 38 6.8 Tryptophan 9 1.6 Histidine 18 3.2 Tyrosine 21 3.8 Isoleucine 46 8.2 Valine 41 7.3

[0031] Embodiment 1:

[0032] The preparation method of Monascus rubrum transformation strain TS-18 comprises the following steps:

[0033] 1.1 Experimental Materials

[0034] 1.1.1 Vectors and strains

[0035] Strains: Monascus pilous (M. pilous) strain CICC 5045 was purchased from China Industrial Microbiological Culture Collection Center; Escherichia coli Top 10 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; Agrobacterium AGL-1 was preserved by the Food Nutrition Laboratory, College of Food Science, Fujian Agriculture and Forestry University.

[0036] Vector: The vector pNeo-0380 was maintained by the Food Nutrition Laboratory, College of Food Science, Fujian Agriculture and Forestry University.

[0037] 1.1.2 Main reagents and culture medium

[0038] Reagents: restriction endonucleases, DNA Marker DL2 000, DL15000 and DNA ligation kit were purchased from TAKARA Biotechnology Co., Ltd.; 2×Fine TaqPCR SupperMix (+dye) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; plasmid extraction kit ( Gel Extraction Kit) was purchased from Omega Bio-Tek (Omega); phosphate buffer (0.01 M), 50×TAE buffer, and rifampicin were purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio); kanamycin (West Asia Reagent), geneticin (Geneview), and Triton X-100 were purchased from Sinopharm Chemical Reagent Co., Ltd.; LB medium and cephalosporin were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; malt extract medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; YEB medium was purchased from Shanghai Ruichu Biotechnology Co., Ltd.; glycerol, sodium molybdate, boric acid, calcium chloride, zinc sulfate, copper sulfate, manganese sulfate, magnesium sulfate heptahydrate, sodium chloride, calcium chloride, ammonium sulfate, ferric sulfate, and glucose (all analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0039] Main culture medium:

[0040] IM trace element solution: weigh 10.0 mg of sodium molybdate, boric acid, calcium chloride, zinc sulfate, copper sulfate and manganese sulfate respectively, dissolve them in water and make up to 100 mL for later use;

[0041] MN buffer solution: weigh 3.0 g of magnesium sulfate heptahydrate and 1.5 g of sodium chloride, dissolve in water and dilute to 100 mL for later use;

[0042] Morpholineethanesulfonic acid (MES) solution: weigh 19.5 g of MES and dissolve it in a 50°C water bath, stir and dilute to 100 mL, then adjust the pH to 5.5 with NaOH for later use;

[0043] AIM induction medium: Take 78.5 mg of acetosyringone (AS, 0.2 mmol / L) and add it to dimethyl sulfoxide solution (2.0 mL) to prepare a 0.2 mmol / L stock solution.

[0044] AIM liquid culture medium preparation (see Table 3):

[0045] Table 3

[0046]

[0047] Preparation of AIM solid medium: add 2.5 mL of glucose solution (2.0 g / L), 15.0 g of agar powder, and other reagents are the same as those of AIM liquid medium.

[0048] 1.1.3 Main instruments (see Table 4)

[0049] Table 4

[0050] Test equipment model Manufacturer High Pressure Steam Sterilizer MJ-54A Shidukai Instrument Equipment Co., Ltd. Fungal incubator MJX-50 Ningbo Jiangnan Instrument Factory Clean bench VS-1300L-U Suzhou Antai Air Technology Co., Ltd. Double-slot gradient PCR instrument S1000 Bio-Rad Corporation Automatic gel imaging instrument Universal Hood II Beijing Saizhi Venture Technology Co., Ltd. Horizontal electrophoresis apparatus: PowerPacTM Basic Beijing Liuyi Instrument Factory

[0051] 1.2 Experimental methods

[0052] 1.2.1 Construction of the eukaryotic expression vector of MpST gene

[0053] (1) The MpST gene was amplified using specific primers ST-FH (5'-CCCAAGCTTATGAGTCAGGCTCGTTCATACC-3') and ST-R2-S (5'-CGAGCTCTTGCTCGACTCTTCATCTTCCT-3') with restriction endonuclease Hind III and Sac I recognition sites, the PCR product was recovered and digested, and then the digested product of the MpST gene fragment was ligated to the expression vector pNeo-0380. The ligation reaction system is shown in Table 5:

[0054] Table 5

[0055] MpST gene fragment digestion product 4.0μL pNeo-0380 plasmid vector digestion product 1.0μL T4 DNA Ligase(Solution I) 0.2μL Total 10.0μL

[0056] After the reaction system is mixed, it is placed at 16°C for 30 minutes to obtain a ligation product for use.

[0057] (2) The ligation product was transformed into competent cells of Escherichia coli Top10 using the heat shock method, spread on LB plates (containing 50 ng / mL kanamycin), and a single colony was picked and inoculated into LB liquid culture medium (containing 50 ng / mL kanamycin). The culture was shaken at 37°C and 220 rpm in a shaker for 12 to 16 h. The plasmid DNA was extracted using a plasmid extraction kit, and then PCR and enzyme digestion were performed to obtain the eukaryotic expression vector pNeo-ST.

[0058] The reaction system for PCR identification of the eukaryotic expression vector pNeo-ST is shown in Table 6:

[0059] Table 6

[0060] <![CDATA[2×Fine Taq TM PCR Super Mix]]> 5.0μL ST-FH (20 μmol / L) 0.2μL ST-R2-S (20 μmol / L) 0.2μL pNeo-ST plasmid DNA 0.8μL <![CDATA[ddH 2 The]]> 3.8μL Total 10.0μL

[0061] The reaction conditions for PCR identification of the eukaryotic expression vector pNeo-ST are as follows:

[0062]

[0063] After 1% agarose gel electrophoresis of the PCR product, the target gene fragment was recovered and sent to Fuzhou Shangya Biotechnology Co., Ltd. for sequencing.

[0064] The reaction system for restriction enzyme digestion identification of pNeo-ST expression vector is shown in Table 7:

[0065] Table 7

[0066] 10×QuickCut Buffer 5.0μL <![CDATA[QuickCut TM Sac I]]> 1.0μL <![CDATA[QuickCut TM Hind III or BamH I]]> 1.0μL pNeo-ST plasmid DNA 2.8μL <![CDATA[ddH 2 The]]> 40.2μL Total 50.0μL

[0067] The reaction system was mixed and incubated at 37°C for 20 min.

[0068] The PCR identification results of Top10 transformants are shown in Figure 1 As shown, Figure 1 M is DNA Marker DL 2000, and 1 is PCR product. The results of double restriction enzyme digestion of recombinant plasmid pNeo-ST are shown in Figure 2 As shown, Figure 2 M is DNA Marker DL 15000, 1 is the product of Sac I / Hind III digestion, and 2 is the product of Sac I / BamH I digestion. The sequencing results showed that the eukaryotic expression vector pNeo-ST of the MpST gene was successfully constructed.

[0069] 1.2.2 Agrobacterium-mediated transformation of Monascus rubrum

[0070] 1.2.2.1 Transformation of Agrobacterium with recombinant vector

[0071] The constructed eukaryotic expression vector pNeo-ST was transformed into Agrobacterium tumefaciens AGL-1 by freeze-thaw method. Specifically, the competent cells of AGL-1 Agrobacterium stored at -80°C were dissolved on ice. In the clean bench, 1.0 μg of expression vector pNeo-ST was added to one tube, and sterile water was added to one tube as a negative control. After the two tubes were mixed, they were ice-bathed for 30 minutes, and then liquid nitrogen was quickly frozen and 37°C water bathed for 5 minutes each. The operation was repeated once and ice-bathed for 30 minutes. Subsequently, 800.0 μL of LB medium (preheated at 28°C) was added and cultured for 2 hours. The culture conditions were 28°C and 180 rpm. Place the centrifuge tube in a centrifuge and centrifuge at 5000g for 5 minutes to separate the bacteria, remove the supernatant, retain part of the supernatant to resuspend the bacteria, aspirate 100 μL of the resuspended bacterial solution and spread it evenly on a YEB plate (50.0 mg / L kanamycin (Kan) + 50.0 mg / L rifampicin (Lif)), and culture at 28°C for 1 to 2 days.

[0072] 1.2.2.2 Agrobacterium-mediated transformation of Monascus rubrum

[0073] The recombinant Agrobacterium (AGL-1 / pNeo-ST) was inverted and cultured at 28°C for 48h using YEB medium plates (containing 20.0μL / mL Geneticin G418, 20.0μL / mL Triton X-100, and 20.0μL / mL Cef). The AGL-1 / pNeo-ST bacterial solution was diluted to an OD600 of 0.15-0.16 and cultured for 6h. AIM induction medium and a conidia suspension of Monascus pubescens (spore concentration 1×106 / mL) were prepared in advance. The AGL-1 / pNeo-ST bacterial solution and the conidia suspension of Monascus pubescens were mixed in a ratio of 1:1, and then spread on the AIM induction medium plates and inverted and cultured at 28°C for 2d. Then, a layer of screening culture medium (malt extract medium + 20.0 μL / mL G41 + 20.0 μL / mL X-100 + 20.0 μL / mL Cef) was added to the plate for screening and culture for 5 to 7 days to obtain resistant transformants of Monascus rubrum.

[0074] 1.2.3 PCR identification of Monascus rubrum transformants

[0075] The resistant transformants obtained through screening and cultivation were cultured in liquid form in malt extract medium for 2-3 days (with the starting strain CICC 5045 as a negative control) at 28°C and 220rpm. After the cultivation was completed, genomic DNA was extracted from the mycelium of the resistant transformants using a new genomic DNA rapid extraction kit, and PCR identification of the resistant transformants was performed using specific primers PgpdA F3 (5'-ACTTCATCGCAGCTTGACTAAC-3') and ST-W3C (5'-GTGGGCACGG TTGATGTATTTA-3'), and positive transformants containing the MpST gene were screened out.

[0076] The reaction system for PCR identification of Monascus resistant transformants is shown in Table 8:

[0077] Table 8

[0078]

[0079]

[0080] The reaction conditions for PCR identification of Monascus resistant transformants are as follows:

[0081]

[0082] The PCR identification results of the transformed strains of Monascus rubrum are shown in Figure 3 As shown, Figure 3 In the figure, M is DNA Marker DL 2000, 1-10 are transformed strains of Monascus rubrum, 11 is a positive control (plasmid pNeo-ST), and 12 is a negative control (starting strain CICC5045).

[0083] Embodiment 2:

[0084] The fermentation culture of Monascus rubrum transformation strain TS-18 comprises the following steps:

[0085] 2.1 Experimental Materials

[0086] 2.1.1 Test strains

[0087] The transformed strains of Monascus pilous were TS-1 to TS-22; the control strain was the starting strain: Monascus pilous (M. pilous) strain CICC 5045.

[0088] 2.1.2 Main reagents and culture media

[0089] Rice, commercially available; malt extract agar medium: Qingdao Haibo Biotechnology Co., Ltd.; glutaraldehyde solution, peptone and fermentation-specific soybean meal powder were purchased from Beijing Solebow Technology Co., Ltd.; methanol, acetonitrile, phosphoric acid (all chromatographic grade) were purchased from Grace Chemical Company; potassium dihydrogen phosphate, glucose, magnesium sulfate heptahydrate, sodium nitrate, glycerol, ethanol (all analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.; MK standard (lovastatin, HPLC = 98%) was purchased from China Food and Drug Inspection Institutes.

[0090] Malt extract plate culture medium: Weigh 145.1 g of malt extract agar powder, heat with distilled water to dissolve, make up to 1.0 L, divide into glass conical bottles, and sterilize at 121°C for 20 min.

[0091] Seed liquid culture medium: weigh 30.0 g of glucose, 70.0 g of glycerol, 15.0 g of soybean meal, 10.0 g of peptone, 2.0 g of potassium dihydrogen phosphate, 2.0 g of sodium nitrate, and 1.0 g of magnesium sulfate heptahydrate, dissolve in 1.0 L of distilled water by heating, dispense into glass conical bottles at 100 mL per bottle, and sterilize by high pressure at 121°C for 20 min.

[0092] Solid-state fermentation medium: weigh 30 g of rice in a PE fermentation bottle (300 mL), add 30 mL of a mixture of water and glycerol (4:1), seal the bottle, and sterilize it under high pressure at 121°C for 20 min for later use.

[0093] Liquid fermentation medium: Weigh 20.0 g of rice flour (ground from rice and passed through a 60-mesh sieve), 90.0 g of glycerol, 10.0 g of peptone, 2.0 g of zinc sulfate, 2.5 g of potassium dihydrogen phosphate, 5.0 g of sodium nitrate, and 0.1 g of magnesium sulfate heptahydrate, dissolve in 1.0 L of distilled water by heating, dispense into glass conical bottles at 100 mL per bottle, and sterilize by high pressure at 121°C for 20 min.

[0094] 2.1.3 Main instruments (see Table 9)

[0095] Table 9

[0096]

[0097]

[0098] 2.2 Fermentation culture of MpST transformed strains

[0099] Seed liquid culture: After activation, the transformed strains of Monascus rubrum TS-1~TS-22 and CICC 5045 were inoculated into slant wort plate medium and cultured at 28℃ for 7 days. After the culture was completed, a hole was punched with a hole puncher in the clean bench and inoculated into the prepared seed liquid medium at 28℃ and 180r / min for 48h.

[0100] Solid-state fermentation culture: After the seed solution culture is completed, the spore concentration is diluted to 1×10 6 / mL, inoculate the seed liquid into the solid culture medium at a 10% inoculation rate (set 4 replicates per group), stir the culture medium and seed liquid evenly with a glass rod, and culture in a constant temperature incubator at 28°C for 15 days. After the fermentation is completed, place the sample in an oven to dry to constant weight, grind it into powder and pass it through a 60-mesh sieve for later use.

[0101] Liquid fermentation culture: After the seed liquid culture is completed, the spore concentration is diluted to 1×10 6 The seed solution was inoculated into the liquid culture medium at a 10% inoculation rate (4 replicates were set for each group) and cultured in a shaker at 28°C and 180 rpm until the fermentation was completed.

[0102] 1.3 Detection of MK production of MpST-transformed strains by HPLC

[0103] Instrument: Waters high performance liquid chromatograph.

[0104] Chromatographic conditions for HPLC detection: chromatographic column is Sunfire C18 (5μmol / L, 250×4.6mm); mobile phase is V(A:B)=V(acetonitrile:0.1% phosphoric acid aqueous solution)=55:45; flow rate is 1mL / min; detector wavelength is 238nm; column temperature is 30°C; injection volume is 20μL.

[0105] The calculation formula of MK content is:

[0106] MK content (mg / kg) = MK standard sample concentration × (sample chromatographic peak area / standard sample peak area) × sample volume / sample mass

[0107] According to the elution time of the acid and lactone forms of the MK standard, calculate the MK production of the MpST transformed strain and the starting strain to be tested. The results are shown in Figure 4 The results showed that the MK production (the sum of acid MK and lactone MK) of 13 transformed strains (TS-1, TS-4, TS-5, TS-9, TS-10, TS-11, TS-12, TS-13, TS-14, TS-15, TS-16, TS-18, and TS-20) was significantly higher than that of the starting strain, with an increase of 17.23% to 51.37%, among which TS-18 had the highest MK production of 51.37%.

[0108] 1.4 Detection of mycelial biomass of MpST-transformed strains

[0109] The seed liquid of the MpST transformed strains TS-1 to TS-22 and the starting strain CICC 5045 was inoculated into the liquid fermentation medium. After 15 days of culture, the cells were filtered with filter paper and washed with pure water. They were placed in a 55°C oven and dried to constant weight. Then the dry weight of the mycelium of the transformed strains and the starting strains was weighed, the data was recorded and the biomass was calculated. The experiment was carried out in two batches. The first batch was for 12 transformed strains with an increase of more than 20% in MK production, and the remaining 10 transformed strains were fermented as the second batch. CICC 5045 was used as the control strain in both batches. The results are shown in Figure 5 The results showed that the biomass of 9 transformed strains in the first batch was significantly higher than that of the starting strain, namely TS-4, TS-5, TS-10, TS-11, TS-12, TS-13, TS-14, TS-18, and TS-20. In the second batch of fermentation samples, except for TS-3, whose biomass was significantly higher than that of the starting strain, the other strains had no significant difference from the starting strain.

[0110] 1.5 Electron microscopic analysis of mycelial morphology of transformed strains

[0111] The transformed strain TS-18 and the starting strain CICC 5045 were cultured in liquid culture medium until the 8th day, filtered by funnel and rinsed with PBS, fresh mycelium of the transformed strain and the starting strain was taken into a 2.0mL centrifuge tube, and a pre-cooled 2.5% glutaraldehyde solution was added, and fixed overnight in a 4°C refrigerator. Then the mycelium was rinsed three times with phosphate buffer (PB) for 15 minutes, and then fixed with 1% osmium acid solution for 1-2 hours, and then the mycelium was rinsed with PB three times after the end, each time for 15 minutes. The two Monascus mycelium were dehydrated with gradient concentrations of ethanol solution, 30%, 50%, 70%, 80%, 90% and 95% for 15 minutes respectively, and then the mycelium was treated with 100% ethanol for 20 minutes, and finally the two samples were placed in 100% ethanol for storage and waiting for machine detection.

[0112] Instrument: Biological scanning electron microscope (Hitachi Reguius8100).

[0113] Observation on the machine: After drying in a critical dryer and gold spraying, the morphology of the mycelium of the transformed strain and the starting strain was observed using a scanning electron microscope, and the image results were collected. Figure 6 As shown in the figure, the mycelium of the starting strain CCIC 5045 is fuller and smoother under the microscope, while the mycelium of the transformed strain of Monascus rubrum has many wrinkles on the surface, the spores are not full, and the spores are shriveled as a whole. This indicates that the overexpression of the MpST gene has an effect on the mycelium of Monascus rubrum.

[0114] In summary, the total MK yield (the sum of acidic MK and lactone MK) of the Monascus pubescens transformed strain TS-18 provided by the present invention is significantly improved compared with the starting strain, with an increase of up to 51.37%, and the biomass is also higher than that of the starting strain. The preparation method provided by the present invention successfully utilizes the sugar transporter gene MpST of Monascus pubescens to transform Monascus to obtain a high-yield MK transformed strain.

[0115] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A Monascus rubrum transformation strain TS-18, characterized in that: Classification and naming: Monascus rubrum, Latin name: Monascus pilosus , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC NO. 41081.

2. Use of the Monascus pubescens transformed strain TS-18 as claimed in claim 1 in the preparation of monacolin K.

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