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

Genetically engineered bacterium of high-yield gibberellin GA3, construction method and application

A technology of genetically engineered bacteria and gibberellins, applied in genetic engineering, microorganism-based methods, applications, etc., can solve problems such as difficulty in improving, and achieve the effects of improving production capacity, improving synthesis capacity, and weakening transcriptional inhibition.

Pending Publication Date: 2022-05-20
ZHEJIANG UNIV OF TECH
View PDF5 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese invention patents (publication numbers CN104892554A, CN201910304928.1 and CN201910438160.7) describe GA 3 The preparation method, mutagenesis results and ARTP mutagenesis technology, but the yield of the strain obtained through the traditional mutagenesis scheme reached 2 g / L and then it was difficult to improve

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
  • Genetically engineered bacterium of high-yield gibberellin GA3, construction method and application
  • Genetically engineered bacterium of high-yield gibberellin GA3, construction method and application
  • Genetically engineered bacterium of high-yield gibberellin GA3, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: F. fujikuroi -OE: Acquisition of AreA

[0030] Amplified by PCR using primers 1 and 2 using the pAN7-1 plasmid as a template wxya For the promoter fragment, the PCR reaction conditions are as follows: 98°C for 5min; 98°C for 30s, 60°C for 30s, 72°C for 1min, repeating 30 cycles; 72°C for 5min. Using the same method using primers 3 and 4 to amplify trpC Terminator fragment, amplified using primers 5 and 6 to give hygromycin hyg fragment. The PCR products were detected by 1.0% agarose gel electrophoresis and the gel was cut to recover the purified fragments ( wxya promoter fragment, trpC terminator fragment and hygromycin hyg1 The nucleotide sequences of the fragments are respectively shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.7).

[0031] by PCR using primers 7 and 8 to Gibberella fujikura ( Fusarium fujikuroi ) CCTCC NO: M2019378 (CN110527630A) genome was amplified as a template areA For gene fragments, the PCR reaction conditions are a...

Embodiment 2

[0037] Example 2: F. fujikuroi -OE: Acquisition of Lae1

[0038] Amplified by PCR using primers 9 and 10 using the Aspergillus nidulans genome as a template tef1 For the promoter fragment, the PCR reaction conditions are as follows: 98°C for 5min; 98°C for 30s, 60°C for 30s, 72°C for 1min, repeating 30 cycles; 72°C for 5min. Using the same method using primers 11 and 12 to amplify tef1 The terminator fragment, using the pAN7-1 plasmid as a template, was amplified using primers 13 and 14 to obtain hygromycin hyg2 fragment. The PCR products were detected by 1.0% agarose gel electrophoresis and the gel was cut to recover the purified fragments ( tef1 promoter fragment, tef1 terminator fragment and hygromycin hyg2 The nucleotide sequences of the fragments are respectively shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.8).

[0039] Amplified by PCR using primers 15 and 16 using the genome of Gibberella fujikuroi (Fusarium fujikuroi) CCTCC NO:M2019378 as a template ...

Embodiment 3

[0045] Example 3: F. fujikuroi -OE: AreA OE: Acquisition of Lae1

[0046] Using primers 17 and 18 by PCR, the pUC19-Hyg2- Ptef1 -Lae1- Ttef1 The vector is used as a template to amplify the complete Lae1 expression cassette. The PCR reaction conditions were as follows: 98°C for 8 minutes; 30 cycles of 98°C for 30s, 60°C for 30s, and 72°C for 4 minutes; 72°C for 8 minutes. The PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by cutting the gel.

[0047] With pUC19-Hyg1- PgpA -Are A- TrpC The carrier vector was used as a template, and was connected with the ClonExpress II One Step Cloning Kit (purchased from Nanjing Weizan vazyme) to construct pUC19-Hyg1-P wxya -Are A-T trpC-Ptef1 -Lae1- Ttef1 The vector and the ligated product were transformed into E. coli DH5α recipient bacteria, spread on LB solid plates containing a final concentration of 100 mg / L ampicillin resistance, and cultured at 37°C for 12 hours....

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 relates to a genetically engineered bacterium for producing gibberellin GA3 at high yield, a construction method and application. According to the present invention, by enhancing the gene expressing the nitrogen source regulatory factor AreA and the gene expressing the global regulatory factor Lae1 in the gibberella zeylanica, the positive regulation effect of the gibberella zeylanica on the related gene in the gibberellin anabolism path is enhanced, the transcriptional level of the related gene is improved, and the accumulation of gibberellin GA3 is improved. According to the recombinant gibberellin GA3 high-yield recombinant gibberellin GA3 provided by the invention, compared with wild gibberellin GA3, the transformed recombinant gibberellin GA3 has the advantages that the overall transcriptional level of a GA gene cluster is enhanced, the synthesis capability of a metabolic process is improved, the transcriptional inhibition of GA3 synthesis related genes is weakened, a carbon source substance and a nitrogen source substance can be better utilized for producing gibberellin GA3, and the yield of gibberellin GA3 is improved. The level of the transformed strain with the optimal performance for producing gibberellin GA3 through fermentation is improved from 2 g / L to 3.25 g / L compared with the original strain, the production capacity of gibberellin GA3 is remarkably improved, and the strain can be applied to large-scale production of gibberellin GA3.

Description

technical field [0001] The invention relates to a high-yielding gibberellin GA 3 Genetically engineered bacteria, construction methods and applications. Background technique [0002] Gibberellin (Gibberellin Acid, GA 3 ) is a plant growth hormone synthesized by plants and some fungi. It is widely used in agriculture, horticulture and wine production because of its special ability to stimulate plant growth. Gibberella Fujikura was first discovered by Japanese plant pathologists in the study of rice bakanae disease. Subsequent studies have confirmed that Gibberella Fujikura naturally has a complete GA 3 synthetic ability. GA 3 Is a tetracyclic diterpene carboxylic acid, molecular formula C 19 h 22 o 6 , molecular weight 346.37, melting point 233~235 ℃, easily soluble in alcohols, acetone, ethyl acetate, sodium bicarbonate solution and phosphate buffer solution with pH 6.2, insoluble in water and ether. GA 3 As a secondary metabolite, it is generally obtained through ...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/31C12P27/00C12R1/77
CPCC07K14/37C12P27/00
Inventor 柳志强王浩南柯霞黄良刚郑裕国
Owner ZHEJIANG UNIV OF 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