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A kind of Pseudomonas aeruginosa for improving rhamnolipid production and construction method thereof

A technology of Pseudomonas aeruginosa and rhamnolipid, which is applied in the biological field, can solve problems such as difficulty in rhamnolipid, and achieves the effects of being easy to handle and improving yield

Active Publication Date: 2021-06-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Just like this, on the basis of the existing technology, it is very difficult to further improve the rhamnolipid

Method used

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  • A kind of Pseudomonas aeruginosa for improving rhamnolipid production and construction method thereof
  • A kind of Pseudomonas aeruginosa for improving rhamnolipid production and construction method thereof
  • A kind of Pseudomonas aeruginosa for improving rhamnolipid production and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, have the construction of the high copy plasmid vector pUCP18 of gentamicin (Gm) resistance

[0062] Primers were designed by analyzing the sequences of the high-copy plasmid vector pUCP18 and the gentamicin resistance cassette, and the primers used to amplify the gentamicin resistance were:

[0063] Gm(NdeI)-F:GTGC CATATG AAGTTCCTATACTTTC (SEQ ID NO. 1)

[0064] Gm(NdeI)-R:CGCTTACT CATATG GAATTCTAGATC (SEQ ID NO. 2)

[0065] The Gm resistance gene was amplified from the pPS856 plasmid as a template, then digested with NdeI, and cloned into the pUCP18 vector that was digested with NdeI and dephosphorylated to obtain the plasmid vector pUCP18Gm with gentamicin (Gm) resistance .

Embodiment 2

[0066] Example 2. Construction of rmlACBD and rmlBDAC overexpression strains PAO1 / rmlACBD, PAO1 / rmlBDAC and comparison strain PAO1 / pUCP18Gm and determination of rhamnolipid synthesis.

[0067] The rmlBDAC operon in Pseudomonas aeruginosa encodes the enzyme for dTDP-L-rhamnose synthesis. The order of the genes on the chromosome is rmlBDAC, and the promoter before the rmlB gene is affected by the clustering effect and σ s However, in the synthesis of dTDP-L-rhamnose, the sequence of action of the enzyme is RmlA, RmlB, RmlC and RmlD, and RmlA is responsible for transferring thymonucleotide to glucose-1-phosphate to generate dTDP-1 - Glucose, the rate-limiting enzyme for this series of reactions. RmlABCD plays a catalytic role in the dimer form of a dimer, so we also constructed a rmlACBD arrangement under the constitutive promoter, and compared the effects of these two arrangements on the production of rhamnolipids .

[0068] First, the rmlBDAC gene was cloned into the pUCP18Gm...

Embodiment 3

[0077] Example 3, construction of rhlYZ and algC overexpression strains PAO1 / algC, PAO1 / rhlYZ and determination of rhamnolipid synthesis

[0078] RhlYZ, enoyl-CoA hydratase / isomerase, is a group of enzymes recently discovered in Pseudomonas aeruginosa with a transition function in fatty acid degradation, responsible for the direct conversion of fatty acid β-oxidation intermediates into hydroxyacyl- COA (Abdel-Mawgoud, 2014). AlgC protein has two functions of phosphoglucomutase and phosphomannose mutase at the same time, which can convert glucose-6-phosphate and mannose-6-phosphate into glucose-1-phosphate and mannose-1-phosphate respectively, Glucose-1-phosphate generates dTDP-L-rhamnose under the action of RmlABCD. Therefore, it is speculated that the expression of RhlYZ and AlgC has an effect on the synthesis of rhamnolipids.

[0079] First, the rhlYZ and algC genes were cloned into the pUCP18Gm vector, respectively. Using Pseudomonas aeruginosa PAO1 chromosomal DNA as a ...

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Abstract

The invention relates to a Pseudomonas aeruginosa capable of improving rhamnolipid production and a construction method thereof. The gene expression in group A of the strain is reduced or lost while the gene expression in group B is increased; the genes in group A are selected from pslABCDEFGHIJKLMNO extracellular polysaccharide synthesis gene cluster, pelABCDEFG polysaccharide synthesis gene cluster, alginate synthesis gene Cluster algD-alg8-alg44-algKEGXLIJF and polyhydroxy fatty acid PHA synthesis key gene cluster phaC1-D-C2 in one or a combination of any several genes; the genes in group B are selected from rmlACBD, rmlBDAC, rhlYZ, algC, fadD4, One gene or any combination of several genes in lipC and estA. The invention has the advantages of significantly increasing the yield of rhamnolipid, stabilizing heredity, being beneficial to the separation and purification of downstream products, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pseudomonas aeruginosa for improving rhamnolipid production, a construction method and a method for producing rhamnolipid by using the same. Background technique [0002] Rhamnolipid is a kind of glycolipid biosurfactant, which not only has the physical and chemical properties of chemical surfactants, such as detergent, emulsification, deemulsification, foaming and wetting, but also has the most important Environmentally friendly, easy to degrade, and will not cause secondary pollution to the environment, it is one of the most widely studied biosurfactants at present. It has a wide range of potential applications in the fields of environmental governance, agricultural production, food and medicine, but its high cost limits its large-scale production and application. [0003] At present, the main rhamnolipid producing strain is Pseudomonas aeruginosa, which mainly has p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/78C12P19/44C12R1/385
CPCC07K14/21C12P19/44
Inventor 朱坤于海英
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI