Transformation microbe generating 3-hydracrylic acid, construction method and application thereof

A hydroxypropionic acid and microorganism technology, which is applied in the field of transforming microorganisms producing 3-hydroxypropionic acid and their construction to achieve the effects of high stability, inhibiting miscellaneous bacteria and reducing the phenomenon of miscellaneous bacteria

Inactive Publication Date: 2009-09-02
NANJING UNIV OF TECH
View PDF0 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no microorganisms that can directly utilize sugary raw materials or glycerol to efficiently transform and produce 3-HPA have been found in nature, but the key enzymes involved in the 3-hydroxypropionic acid cycle pathway are crucial for the future construction of model genetically engineered bacteria to produce 3-HPA. Acid will be of great guiding significance

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
  • Transformation microbe generating 3-hydracrylic acid, construction method and application thereof
  • Transformation microbe generating 3-hydracrylic acid, construction method and application thereof
  • Transformation microbe generating 3-hydracrylic acid, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Klebsiella pneumoniae ATCC25955-3-pUC18-aldH-Tet R build

[0032] (1) Cloning of the gene aldA: According to the sequence (SEQ ID No.2) of the aldA gene in Escherichia coli K12 published by Genebank, the primers required for synthetic PCR were designed:

[0033] P1 (SEQ ID No. 4): 5 1 -GAATTCATGGGTAAGCTGGAACGCATTGC-3 1

[0034] P2 (SEQ ID No. 5): 5 1 -TCTAGATCAGGCGTTCCCTTTTTCTTTTA-3 1

[0035] Introduce EcoR I and Xba I restriction sites at both ends of the primers respectively, and use the genomic DNA of Acetobacter acetobacter as a template to complete the PCR reaction: PCR reaction conditions: denaturation at 95°C for 5min, 30 cycles at 94°C for 30sec, 52°C for 30sec, and 72°C for 1min40sec , and then extended at 72°C for 3 minutes, the obtained PCR product was confirmed by electrophoresis analysis, purified by the PCR product purification kit, connected to the pMD18-simple-T vector, and sequenced, and the recombinant pMD18-simple was digested with E...

Embodiment 2

[0040] Example 2: Recombinant plasmid pUC18-aldH-Tet R Transform Klebsiella pneumoniae25955 (obtained from ATCC, strain number: Klebsiella pneumoniae ATCC25955)

[0041] The recombinant plasmid pUC18-aldH-Tet was R To transform Klebsiella pneumoniae Klebsiellapneumoniae 25955, the electroporation condition parameters are: voltage 2.5kV, resistance value 200Ω, pulse time 4.5msec. After the transformation by electric shock, spread the electric shock-treated Klebsiella pneumoniae ATCC25955 on the LB medium containing 40 μg / mL tetracycline to obtain a positive clone Klebsiella pneumoniaeATCC25955-3-pUC18-aldH-Tet R .

Embodiment 3

[0042] Example 3: Engineering bacteria Klebsiella pneumoniae 25955-3-pUC18-aldH-Tet R Plasmid stability study

[0043] Inspection method: Pick a single colony from a fresh transformation plate and inoculate it into 3 mL of tetracycline-free LB medium, cultivate the seeds at 37°C for 12 hours as seeds, and replant for 24 hours, that is, 20 generations of seeding, 5 times of replanting, That is 100 generations, spread the above bacterial solution on the LB plate without antibiotics, select 100 single colonies from it, and place them on the plate with and without tetracycline, culture at 37°C for 12-16h, and compare the effect of adding and not adding tetracycline. The stability was determined by the number of colonies on the plate to which tetracycline was added.

[0044] Investigation results: after 100 generations of breeding, the stability of the plasmid is 99%.

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

PropertyMeasurementUnit
tensile strengthaaaaaaaaaa
Login to view more

Abstract

The invention relates to a transformation microbe containing a recombinant vector, wherein the recombinant vector contains protein expressed by a nucleotide sequence shown by a gene code SEQ ID NO.1. A method for preparing the transformation microbe comprises introducing the nucleotide sequence shown by the gene code SEQ ID NO.1 into a host. The transformation microbe containing the recombinant vector is applied to generating 3-hydracrylic acid. A plasmid has high stability in a host strain, and is quite suitable for generating a strain; the final concentration of a product has competitiveness; in addition, trace tetracycline can be added during fermentation so as to achieve the aim of inhibiting part hybrid bacteria, greatly reduce the phenomenon of easily dyeing the hybrid bacteria during industrialization, shorten the fermentation time and lay a good foundation for industrialization production of the 3-hydracrylic acid by a microbe fermentation method.

Description

1. Technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a transformed microorganism producing 3-hydroxypropionic acid and its construction method and application. 2. Background technology [0002] Prior Art: 3-hydroxypropanoate (3-HP) is an important functional organic acid. It is easily soluble in water, ethanol, and ether, and can be converted into commercially important chemicals through a series of mature catalytic technologies in the chemical industry, such as dehydration to generate acrylic acid, oxidation to generate malonic acid, and esterification with alcohol to generate esters , and generate 3-hydroxypropionic acid through reduction reaction; 3-HP also has potential biological applications: for example, in the nutrition industry, 3-HP can be used as a food and feed additive or preservative, endogenous in some plants There is a small amount of 3-HP in fungi, and it is found that it has the abilit...

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): C12N1/00C12N1/21C12N15/09C12N15/74C12P7/42C12R1/00C12R1/22
Inventor 黄和朱建国纪晓俊李霜杜军丁月月
Owner NANJING UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap