Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

High-yield engineering strain kmust-SQS for ganoderic acid

A technology for engineering strains and ganoderma acid, applied in the fields of genetic engineering and metabolic engineering, can solve the problem of low production of ganoderma acid, and achieve the effects of saving labor, shortening production cycle, and wide application prospects.

Inactive Publication Date: 2014-09-03
KUNMING UNIV OF SCI & TECH
View PDF1 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The object of the present invention is to solve the problem that the wild-type ganoderma lucidum strain itself produces relatively low ganoderma acid, and provide a high-yield ganoderma acid engineering strain, which is a high-yield ganoderma acid Ganoderma lucidum ( Ganoderma lucidum ) engineered bacteria kmust-SQS has been preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms on April 14, 2014, address: No. 1 Beichen West Road, Chaoyang District, Beijing Institute No. 3, Institute of Microbiology, Chinese Academy of Sciences, deposit number is CGMCC NO.9059

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • High-yield engineering strain kmust-SQS for ganoderic acid
  • High-yield engineering strain kmust-SQS for ganoderic acid
  • High-yield engineering strain kmust-SQS for ganoderic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Construction of pJW-EXP vector

[0050] 1. Extraction of Ganoderma lucidum genomic DNA

[0051] Weigh about 0.2 g of mycelium of freeze-dried wild-type Ganoderma lucidum (CCGMC 5.0616), grind it into powder in liquid nitrogen, and transfer the powder into 1.5 mL of CTAB (cetyltrimethylammonium bromide) preheated at 65 °C ) in the extraction buffer, incubate at 65°C for 30 minutes, then centrifuge at 10,000 g for 20 minutes at 4°C, add an equal volume of chloroform:isoamyl alcohol (24:1) mixture to the supernatant, and shake gently for 30 minutes Centrifuge at 10,000 g for 20 min at 4°C; transfer the supernatant into a 1.5 mL centrifuge tube, add 2 / 3 volume of isopropanol pre-cooled at -20°C, shake gently for 5 min, and remove with a glass rod After extracting the DNA, wash 2-3 times with 75% ethanol, dry at room temperature, dissolve in an appropriate amount of TE containing 20 μg / mL RNase, digest the RNA at 37 °C for 30 min, and then obtain the genomic D...

Embodiment 2

[0075] Embodiment 2: Construction of pJW-EXP-tSQS vector

[0076] 1. Cloning of SQS gene

[0077] Genomic DNA of Ganoderma lucidum was used as a template, and primers

[0078] SQS-Nhe-F: 5'-GCTAGCATGGGCGCGACGTCTATGCT-3'

[0079] SQS-Sma-R: 5'-GGGCCCTCACCCGAAAAAGTGGATGAGGAC-3'

[0080] Perform PCR to obtain the SQS gene; the PCR conditions are: 95°C for 10 min, 95°C for 30 s, 61°C for 30 s, 72°C for 2 min for 10 s, and 72°C for 10 min (see image 3 ).

[0081] 2. Insert the SQS gene into the pJW-EXP vector

[0082] The pJW-EXP vector was double-digested with SmaI and NheI, and the digested fragment was recovered. Using T4 ligase at 16°C, the SQS gene was inserted between the NheI and SmaI of the pJW-EXP vector to obtain pJW-EXP- tSQS vector (see Figure 4 ).

Embodiment 3

[0083] Example 3: Transformation of pJW-EXP-tSQS into wild-type Ganoderma lucidum cells by PEG-mediated protoplast fusion

[0084] 1. Preparation and transformation of Ganoderma lucidum protoplasts

[0085] The wild-type Ganoderma lucidum mycelium was first prepared into protoplasts with lysozyme, and then the Ganoderma lucidum protoplasts were suspended in 100 μL of STC (0.55 M sorbitol, 10 mM CaCl 2 , 10 mM Tris-HCl buffer, pH 7.5), then add 1 μg of plasmid DNA and PTC buffer (60% PEG4000 (W / V), 10 mM Tris-HCl buffer, pH 7.5 , 50 mM CaCl 2 ); incubate on ice for 10 min, then add 1 mL of PTC buffer to mix well and incubate at room temperature for 20 min; use 10 mL of melted CYM solid medium to mix the transformed protoplasts, and add carboxin to make carboxin The final concentration is 2 mg / L; several single colonies can grow after culturing at 30°C for 10 days.

[0086] 2. Subculture on plates containing carboxin resistance

[0087] Transfer a single colony to a CYM pla...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a high-yield engineering strain kmust-SQS for a ganoderic acid. The preservation number of the high-yield engineering strain kmust-SQS in the China General Microbiological Culture Collection Center (CGMCC) of the China Committee for Culture Collection of Microorganisms (CCCCM) is CGMCC NO.9059. The ganoderic high-yield engineering strain kmust-SQS is obtained through the overexpression coding of squalenesynthase (SQS) via the selection of a strong ganoderic promoter P-gpd. The shake flask fermentation experiment shows that compared with the content of the ganoderic acid in a wild-type strain (WT strain), according to the high-yield engineering strain kmust-SQS, the content of the ganoderic acid in an SQS converter strain is improved by 23.8% under the condition that the cell growth of the high-yield engineering strain kmust-SQS is not influenced, wherein the contents of ganoderic acid monomers GA-Me, GA-T, GA-Mk and GA-S in the SQS converter strain are respectively improved by 82.96%, 49.84%, 174.51% and 31.80% as compared with the WT strain. Thus, the high-yield strain kmust-SQS can serve as an engineering strain used for producing the ganoderic acid. As a result, the high-yield strain kmust-SQS has a wide application prospect.

Description

technical field [0001] The present invention belongs to the field of genetic engineering and metabolic engineering, and specifically relates to a method for overexpressing the squalene synthase (SQS) gene related to the synthesis of ganoderma acid in the ganoderma lucidum metabolic pathway through genetic engineering, and constructing a high-yielding engineering strain of ganoderma acid . Background technique [0002] Ganoderma lucidum ( Ganoderma lucidum ) are Basidiomycetes, Polyporaceae, Ganoderma lucidum fungi. 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, prote...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/15C12N15/80C12R1/645
Inventor 徐军伟任梦飞周降生纪森林贺宜龙
Owner KUNMING UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com