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

Genetically engineered bacterium for producing L-menthol as well as construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and menthol, applied in the field of metabolic engineering, can solve problems such as large quantities, single production method, slow market feedback, etc., and achieve the effects of overcoming limitations, fast market demand, and short cycle.

Active Publication Date: 2021-01-01
JIANGNAN UNIV
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the industrial production method of L-menthol is relatively single, mainly natural extraction and chemical synthesis. The natural extraction method requires a large amount of mint plant raw materials, and the chemical synthesis of L-menthol also requires compounds such as thymol from plant sources as substrates. Perform reaction splitting
However, the growth cycle of plant raw materials is long, the market feedback is slow, and it is easily affected by natural environmental factors such as climate and region, resulting in unstable output
In addition, there are methods for preparing L-menthol by whole cells or enzymatically catalyzing the preparation of L-menthol with long-leaved menthone and the like as substrates, which still cannot solve the limiting factors of plant raw materials

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 for producing L-menthol as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing L-menthol as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Optimizing the endogenous MVA pathway

[0019] (a) Using the genome of Saccharomyces cerevisiae S288C as a template, use primers tHMG1-F and tHMG1-R to amplify gene tHMG1, tHMG1 is formed by removing the first 530 amino acid sequences of gene HMG1, and use primers GPD-F and GPD-R to amplify The promoter gene GPD (SEQ ID NO.11) was obtained, and the terminator gene ADH1-1 (SEQ ID NO.12) was obtained by amplification with primers ADH1-F1 and ADH1-R1; the amplification with primers IDI-F and IDI-R Obtain gene IDI, adopt primer TEF1-F, TEF1-R amplification obtains promoter gene TEF1 (SEQ ID NO.13), adopts primer CYC1-F1, CYC1-R1 amplification obtains terminator gene CYC1-1 (SEQ ID NO.13) .14), use primers 208a-D-F, 208a-D-R to amplify to obtain gene fragment 208a-D, and use primers 208a-U-F, 208a-U-R to amplify to obtain gene fragment 208a-F.

[0020] (b) divide the six fragments tHMG1, GPD, ADH1-1 and IDI, TEF1, CYC1-1 gene fragments obtained in step (a) into t...

Embodiment 2

[0026] Example 2: Reconstruction of L-menthol metabolic synthesis pathway

[0027] (a) Artificially synthesized gene fragment P GAL1 -tLIS -T ADH1 -P GAL10 -L3H-T CYC1 -P GAL7 -CPR-T TDH3 ,P GAL1 -IPDH-T ADH1 -P GAL10 -IPR-T CYC1 -P GAL7 -KSI-T TDH3 ,P GAL1 -PGR-T ADH1 -P GAL10 -MMR-T CYC1 , wherein the tLIS gene is formed by removing the first 56 amino acid sequences of the gene LIS. Using the genome of Saccharomyces cerevisiae S288C as a template, use primers 911b-U-F, 911b-U-R to amplify the gene fragment 911b-U, use primers 911b-D-F, 911b-D-R to amplify the gene fragment 911b-D, use primers 308a-U-F, 308a-U-R amplified to obtain gene fragment 308a-U, using primers 308a-D-F, 308a-D-R to amplify to obtain gene fragment 308a-D, using primers 1622b-U-F, 1622b-U-R to amplify to obtain gene fragment 1622b-U, using primers 1622b-D-F, 1622b-D-R were amplified to obtain the gene fragment 1622b-D.

[0028](b) Using the plasmid pML104 as a template, using primers 308...

Embodiment 3

[0034] Embodiment 3: Utilize genetic engineering bacterial strain WMT4 to ferment and produce L-menthol

[0035] Inoculate a single colony of WMT4 on a solid YPD plate in 2ml of YPD medium, culture at 220rpm for 16-20 hours at 30°C, and then insert 1% of the inoculum into a 250mL round-bottomed shaker flask containing 25mL of YPD liquid medium, at 30°C, Cultivate at 220rpm for 96h. When fermenting to 16h, add 10% galactose aqueous solution. After the fermentation, 600 μL of the bacterial liquid was added to 0.5 mm glass beads and an equal volume of ethyl acetate to break up, and then the upper ethyl acetate layer was taken by high-speed centrifugation for detection by gas chromatography-mass spectrometry. The fermentation yield of L-menthol was 5.03 mg / L.

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 genetically engineered bacterium for producing L-menthol as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of metabolic engineering. According to the genetically engineered bacterium, saccharomycetes are used as starting strains, a metabolic pathway of the L-menthol is constructed in the saccharomycetes, and the L-menthol is produced by a fermentation method for the first time; according to the constructed genetically engineered bacterium for producing the L-menthol, a cheap fermentation culture medium is used for culturing for about 4 days, and the fermentation yield of the L-menthol reaches 5.03 mg / L; and compared with the current production technology of the L-menthol, the fermentation method for producing the L-menthol has the advantages that the period is short, the market demand is fed back quickly, only common fermentation raw materials are needed in the production process, plantsource precursor substances such as menthone and thymol do not need to be added, and the limitation of plant raw materials is overcome.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing L-menthol and its construction method and application, belonging to the technical field of metabolic engineering. Background technique [0002] L-menthol (L-menthol) is a highly volatile monocyclic monoterpene substance, white needle-like crystals, easily soluble in organic solvents such as petroleum ether and chloroform, and slightly soluble in water. L-menthol is one of the most sold spices in the world, with a global annual demand of more than 20,000 tons. L-menthol is widely used in food, daily chemical, medicine, tobacco and other fields because of its cooling, sterilizing, antipruritic and analgesic effects; and it can also be used as a precursor to synthesize other fragrance substances. At present, the industrial production method of L-menthol is relatively simple, mainly natural extraction and chemical synthesis. The natural extraction method requires a large amount of mint pla...

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): C12N1/19C12N15/81C12N15/61C12N15/60C12N15/53C12P7/02C12R1/865
CPCC12N15/52C12N9/88C12N9/0006C12N9/0042C12N9/90C12N9/001C12N9/0073C12N15/81C12Y106/02004C12Y401/01033C12Y101/01088C12Y402/03016C12Y402/0302C12Y503/03001C12Y101/01207C12Y103/01081C12Y101/01223C12Y103/01082C12P7/02
Inventor 刘龙陈坚吕雪芹堵国成李江华于文文马骏房峻
Owner JIANGNAN UNIV
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