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

A kind of engineering bacteria producing coniferyl alcohol, its construction method and the application of producing coniferyl alcohol

A construction method and technology for engineering bacteria, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, bacteria, etc., to achieve the effects of being beneficial to industrial production, solving source problems, and reducing production costs.

Active Publication Date: 2021-01-15
TIANJIN UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no literature report using engineering microorganisms to synthesize coniferyl alcohol

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 kind of engineering bacteria producing coniferyl alcohol, its construction method and the application of producing coniferyl alcohol
  • A kind of engineering bacteria producing coniferyl alcohol, its construction method and the application of producing coniferyl alcohol
  • A kind of engineering bacteria producing coniferyl alcohol, its construction method and the application of producing coniferyl alcohol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030]Example 1 Design of TAL, COMT, 4CL and CCR genes

[0031]Phanerochaete chrysosporium (Phanerochaete chrysosporium) tyrosine deaminase TAL, Arabidopsis thaliana (Arabidopsis thaliana) O-methyltransferase COMT, coumadin-4-CoA ligase 4CL, cinnamoyl Coenzyme A reductase CCR, using JCAT online codon optimization software (http: / / www.jcat.de) combined with OPTIMIZER codon optimization tool (http: / / genomes.urv.es / OPTIMIZER / ), use E. coli to pair the code The preference of the child is optimized, and the full-length TAL, COMT, 4CL and CCR genes are designed. The nucleotide sequences of the genes TAL, COMT, 4CL and CCR are shown as SEQ ID No. 05, SEQ ID No. 06 and SEQ in the sequence table. ID No.07 and SEQ ID No.08 are shown.

Embodiment 2

[0032]Example 2 The lambda-red homologous recombination method integrates the T7 RNA polymerase gene into the chromosome of the chassis strain SyBE-002447.

[0033]The detailed steps of the construction process of the chassis strain integrating the T7 RNA polymerase gene into the chassis strain SyBE-002447 chromosome are as follows:

[0034]1. Using the E. coli BL21 (DE3) genome as a template, the T7 RNA polymerase gene was cloned using Fastpfu enzyme for PCR reaction.

[0035]2. Use overlap extension PCR to prepare chloramphenicol-resistant T7 RNA polymerase fragments.

[0036]3. Introduce the pKD46 plasmid into the strain SyBE-002447 to obtain SyBE-002447 / pKD46. Inoculate the activated strain SyBE-002447 / pKD46 in 10ml LB liquid medium at 30℃, 200rpm, and cultivate to OD600Is 0.4-0.6. Add L-arabinose to a final concentration of 10mM, and continue to incubate for 3h. Centrifuge at 4000 rpm for 8 min at 4°C to collect the cells. The supernatant was discarded, and ice-cold 10% glycerol was added ...

Embodiment 3

[0039]Example 3 Construction of recombinant expression vector pTHM

[0040]Using the E. coli genome as a template, the Fastpfu enzyme was used for PCR reaction to obtain the hpaBC gene with KpnI and XhoI restriction sites on both ends (the nucleotide sequence uses SEQ ID No. 01). The PCR reaction was used to make the two ends of the TAL gene fragment obtained by chemical total synthesis have HindIII and BamHI restriction sites respectively; the two ends of the COMT gene fragment were equipped with HindIII and NotI restriction sites.

[0041]The full chemical synthesis expression vector backbone pBPE001, its sequence is shown in SEQ ID No.09 in the sequence table.

[0042]Use FastDigest endonuclease HindIII and BamHI to digest the TAL gene and pBPE001. The reaction system is: 5μL 10*FD buffer, 2.5μL HindIII, 2.5μLBamHI, 30μL TAL gene fragment and 10μL ultrapure water. The reaction conditions are: 37°C, 1h. Use PCR purification kit to recover the digestion system, add 250μL Binding Buffer solu...

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 an engineering bacterium for producing coniferyl alcohol, a construction method and an application of coniferyl alcohol production. The construction method comprises the following steps: fully chemically compounding TAL, COMT, 4CL and CCR genes; performing PCR cloning on hpaBC, yahK, metK and pntAB genes; fully chemical compounding pBPE001, pBPE002 and pBPE003; linking the genes TAL, hpaBC and COMT with pBPE001, thereby acquiring pTHM; linking the genes 4CL, CCR and yahK with pBPE002, thereby acquiring pLRH; linking the genes pntAB and metK with pBPE003, thereby acquiring pNAM; integrating the T7RNA polymerase gene into SyBE-002447 base strain, thereby acquiring a SyBE-002447(DE3) strain; converting pTHM, pLRH and pNAM into SyBE-002447(DE3), thereby acquiring the engineering bacterium SyBE-CNF; taking glucose as a carbon source and fermenting, thereby producing coniferyl alcohol. According to the invention, the engineering escherichia coli are used for producing coniferyl alcohol, so that the source problem of the coniferyl alcohol can be solved and the cost can be lowered.

Description

Technical field[0001]The technical field of biomedicine to which the present invention belongs relates to an engineered bacteria producing coniferyl alcohol, a construction method, and a use for producing coniferyl alcohol.Background technique[0002]Coniferyl alcohol, the molecular formula is C10H12O3, Molecular weight is 180.20, flake crystal, easily soluble in ether, soluble in ethanol, insoluble in water. As an important pharmaceutical intermediate, coniferyl alcohol is used in medicine as an important intermediate for the synthesis of anticancer drugs podophyllotoxin and antihepatitis drugs silybin.[0003]There are currently three main methods for the synthesis of coniferyl alcohol.[0004](1) Using acetylferulic acid as the raw material, the acid chloride is then esterified and finally reduced to obtain coniferyl alcohol, but the total yield is only 24.4%; (2) Using ferulic acid as the raw material, after esterification, acetyl The total yield is 70.2%; both methods need to use lit...

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): C12N15/70C12N1/21C12P7/22C12R1/19
CPCC12N9/0006C12N9/0008C12N9/0036C12N9/0071C12N9/1007C12N9/1085C12N9/88C12N9/93C12N15/70C12N2800/101C12P7/22C12Y101/01001C12Y102/01044C12Y106/01001C12Y114/14009C12Y205/01006C12Y403/01023C12Y602/01012
Inventor 赵广荣刘津马雅婷
Owner TIANJIN 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