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

A method for efficiently utilizing fatty acids to produce mevalonate and the constructed genetically engineered bacteria

A technology for mevalonate and fatty acid, applied in genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as rare raw materials, high cost, and environmental pollution

Active Publication Date: 2016-03-09
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the synthesis of mevalonate mainly includes enzymatic synthesis, combined synthesis of biocatalysis and chemical catalysis, chemical synthesis, etc., but there are problems such as rare raw materials, complicated steps, pollution of the environment, and high cost. Hydroxyvalonic acid can solve the above problems well

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
  • A method for efficiently utilizing fatty acids to produce mevalonate and the constructed genetically engineered bacteria
  • A method for efficiently utilizing fatty acids to produce mevalonate and the constructed genetically engineered bacteria
  • A method for efficiently utilizing fatty acids to produce mevalonate and the constructed genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The construction of long-chain fatty acid transporter gene (fadL) and acyl-CoA synthetase gene (fadD) co-expression vector, the specific process is as follows:

[0036] The long-chain fatty acid transferase ( fadL), and introduced EcoI and HindIII restriction sites at the 5' end and 3' end, respectively, and then cloned the gene into the pET-30a vector with the above restriction sites to obtain the recombinant plasmid pET-fadL( figure 1 ); using oligonucleotides 5'-GGGAATTCCATATGACCGCTCAGGTTACATGCG-3' and 5'-CCGCTCGAGTTAAACCAGACGAACTTCGTGC-3' as primers, using Escherichia coli genomic DNA as a template, acyl-CoA synthesis was amplified by polymerase chain reaction (PCR) enzyme (fadD), and introduced KpnI and BamHI restriction sites at the 5' end and 3' end, respectively, and then cloned the gene into the pET-fadL vector using the above restriction sites to obtain the recombinant plasmid pET-fadL-fadD ( figure 2 ).

Embodiment 2

[0038] The preparation of engineering Escherichia coli strain BL21(DE3) / pET-fadL-fadD / pYJM11 for synthesizing mevalonate, and using the strain to ferment and transform fatty acid into mevalonate, the specific process is as follows:

[0039] The recombinant plasmid pET-fadL-fadD constructed in Example 1 and the recombinant plasmid pYJM11 (Jianming Yang et al., BioresourceTechnology 104, 642-647, 2012) constructed in this laboratory were extracted by alkaline lysis, and 5 μl of recombinant plasmids were recombined by heat shock transformation Transform the plasmid into Escherichia coli BL21 (DE3) competent cells, and then take 50 μl of the transformed bacterial solution and spread it on a medium containing 34 μg·mL -1 Chloramphenicol with 50 μg·mL -1 Positive clones were screened on the ammonia LB plate, and the grown colonies were recombinant Escherichia coli strains co-expressing long-chain fatty acid transferase and fatty acyl-CoA synthetase. Pick a single colony of recombin...

Embodiment 3

[0041] Preparation of engineering Escherichia coli strain W3110 / pET-fadL-fadD / pYJM11 for synthesizing mevalonate, and using this strain to ferment and convert fatty acids into mevalonate, the specific process is as follows:

[0042] The recombinant plasmid pET-fadL-fadD constructed in Example 1 and the recombinant plasmid pYJM11 (Jianming Yang et al., BioresourceTechnology 104, 642-647, 2012) constructed in this laboratory were extracted by alkaline lysis, and 5 μl of recombinant plasmids were recombined by heat shock transformation The plasmid was transformed into Escherichia coli W3110 competent cells, and then 50 μl of the transformed bacteria liquid was applied to the cells containing 34 μg·mL -1 Chloramphenicol and 50 μg·mL -1 Positive clones were screened on the ammonia LB plate, and the grown colonies were recombinant Escherichia coli strains co-expressing long-chain fatty acid transferase and fatty acyl-CoA synthetase. Pick a single colony of recombinant Escherichia c...

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 method for efficiently producing mevalonic acid from fatty acid and a constructed gene engineering bacterium. The method comprises the following steps: overexpressing a fatty acid transport protein gene (fadL) and an acyl-CoA synthetase gene (fadD) in an Escherichia coli, thus obtaining a recombinant Escherichia coli; and using the recombinant Escherichia coli to produce the mevalonic acid through fermentation. According to the method disclosed by the invention, long-chain fatty acid is used as a carbon source for synthesis of the mevalonic acid, the mevalonic acid yield can be up to 230mg / L, and the productivity can be up to 56%. The method disclosed by the invention can also be used for producing isoprene taking mevalonic acid as an intermediate product and terpenoid compounds synthesized by taking isoprene as a unit through fermentation.

Description

technical field [0001] The invention relates to a method for efficiently utilizing fatty acids to produce mevalonate and the constructed genetically engineered bacteria. Background technique [0002] Mevalonate is an intermediate metabolite of isoenzyme pyrophosphate (IPP) produced by the mevalonate pathway, and is an important intermediate in the synthesis of sterols, terpenoids, and isoprenoids. At the same time, it also has important uses in the chemical industry. At present, the synthesis of mevalonate mainly includes enzymatic synthesis, combined synthesis of biocatalysis and chemical catalysis, chemical synthesis, etc., but there are problems such as rare raw materials, complicated steps, pollution of the environment, and high cost. Valeric acid can well solve the above problems. In biological cells, mevalonate is obtained from acetyl coenzyme A through a three-step catalytic reaction. There is only one optically active mevalonate in the synthetic product, which has h...

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 Patents(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/52C12N15/70C12P7/42C12P7/04C12R1/19
Inventor 咸漠程涛曹玉锦邹慧斌
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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