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Ganoderic acid high-producing engineering strain kmust-VGB-1

A technology for engineering strains and ganoderma lucidum acid, applied in the fields of genetic engineering and metabolic engineering, can solve the problem of low production of ganoderma lucidum acid, and achieve the effects of saving labor, shortening production cycle, and reducing production cost

Active Publication Date: 2016-02-03
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to solve the problem that wild-type ganoderma lucidum strain self produces ganoderma acid lower, provide a kind of ganoderma acid high-yield bacterial strain, and this bacterial strain is the ganoderma lucidum of high yield ganoderma acid ( Ganodermal lucidum ) engineering bacteria kmust-VGB-1, which has been preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on September 6, 2015, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences , the deposit number is CGMCCNO.11301

Method used

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  • Ganoderic acid high-producing engineering strain kmust-VGB-1
  • Ganoderic acid high-producing engineering strain kmust-VGB-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Construction of pJW-EXP vector

[0047] 1. Extraction of Ganoderma lucidum genomic DNA

[0048] Weigh about 0.2 g of mycelium of freeze-dried wild-type Ganoderma lucidum (CCGMC5.0616), grind it into powder in liquid nitrogen, and transfer the powder into 1.5 mL of CTAB (cetyltrimethyl bromide) preheated at 65 °C. Ammonium) in the extraction buffer, keep warm at 65°C for 30min, then centrifuge at 10,000g for 20min at 4°C, take the supernatant and add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, shake gently for more than 30min, Centrifuge at 10,000g for 20min at 4°C; transfer the supernatant into a 1.5mL centrifuge tube, add 2 / 3 volume of isopropanol pre-cooled at -20°C, shake gently for 5min, remove the DNA with a glass rod, and use 75 % ethanol was washed 2-3 times, dried at room temperature, dissolved in an appropriate amount of TE containing 20 μg / mL RNase, digested RNA at 37°C for 30 min, and then the genomic DNA of Ganoderma lucidu...

Embodiment 2

[0072] Example 2: Construction of pJW-EXP-tVGB vector

[0073] 1. Cloning of VGB gene

[0074]Using the plasmid PUC8:16 provided by Professor B.C. Stark of the IIT Center Illinois Institute of Technology in Chicago, USA, the gene of Vitiligo hyaline hemoglobin was cloned (the primers used were VGB-Nhe-F and VGB-Sma-R), and the primers

[0075] VGB-Nhe-F: 5'-GCTAGCCTAGCTAGCATGTTAGACCAACAAACCGT-3'

[0076] VGB-Sma-R: 5'-GGGCCCGATTTGTACGCTCAAGACGCTGAATAAGA-3'

[0077] Perform PCR to obtain the VGB gene, the nucleotide sequence of which is shown in SEQ ID NO: 1, encoding Vitreum hyaline hemoglobin shown in SEQ ID NO: 2; the PCR conditions are: 95°C for 10min, 95°C for 30s, 60°C for 30s, and 72°C for 30s , 72℃10min (see image 3 ).

[0078] 2. Insert the VGB gene into the pJW-EXP vector

[0079] The pJW-EXP vector was double-digested with SmaI and NheI, and the digested fragment was recovered. Using T4 ligase at 16°C, the VGB gene was inserted between the NheI and SmaI of th...

Embodiment 3

[0080] Example 3: Transformation of pJW-EXP-tVGB into wild-type Ganoderma lucidum cells by PEG-mediated protoplast fusion

[0081] 1. Preparation and transformation of Ganoderma lucidum protoplasts

[0082] First, wild-type Ganoderma lucidum mycelium was prepared into protoplasts with lysozyme, and then Ganoderma lucidum protoplasts were suspended in 100 μL of STC (0.55M sorbitol, 10 mM CaCl 2 , 10mM Tris-HCl buffer, pH 7.5), then add 1 μg of plasmid DNA and PTC buffer (60% PEG4000 (W / V), 10mM Tris-HCl buffer, pH 7.5, 50mM CaCl 2 ); incubate on ice for 10 min, then add 1 mL of PTC buffer, mix well and incubate at room temperature for 20 min; use 10 mL of melted CYM solid medium to mix with transformed protoplasts, pour the plate and add carboxin to make the final concentration of carboxin 2 mg / L; Several single colonies can grow after cultured at 30°C for 10 days.

[0083] 2. Subculture on plates containing carboxin resistance

[0084] Transfer a single colony to a CYM ...

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Abstract

The invention discloses a ganoderic acid high-producing engineering strain kmust-VGB-1, which is preserved in China General Microbiological Culture Collection Center with preservation number of CGMCC NO. 11301. The lucid ganoderma engineering strain kmust-VGB-1 for high production of ganoderic acid is prepared by expressing a vitreoscilla hemoglobin VGB gene through a lucid ganoderma strong promoter P-gpd; through a fermentation experiment in a shaking flask, under the circumstance of avoiding influence on cell growth, yield of monomer ganoderic acids GA-Me, GA-T, GA-Mk and GA-S produced by the VGB converter strain are respectively 3.1 times, 3.2 times, 2.1 times and 3.6 times that of monomer ganoderic acids produced by a WT strain; therefore, the high-producing strain, as an engineering strain for producing the ganoderic acid, has a broad application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering and metabolic engineering, and specifically relates to a method for expressing a vitreoscillahemoglobin (VGB) gene in ganoderma lucidum through genetic engineering, and constructs a high-yielding engineering strain of ganoderma acid. Background technique [0002] Ganoderma lucidum ( Ganodermal lucidum ) is Basidiomycetes, Polyporaceae, Ganoderma lucidum fungus. There are more than 20 kinds of ganoderma fungus in our country, including red ganoderma, yellow ganoderma, purple ganoderma, black ganoderma, thin-cover ganoderma, tree tongue and so on. Ganoderma lucidum has been used as medicine in my country for more than two thousand years. Ganoderma lucidum has significant curative effects on enhancing human immunity, regulating blood sugar, controlling blood pressure, assisting tumor radiotherapy and chemotherapy, protecting the liver and promoting sleep. Due to its special medicinal value, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12R1/645
Inventor 徐军伟张德怀李焕军韩李梁
Owner KUNMING UNIV OF SCI & TECH
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