Pseudomonas aeruginosa for increasing yield of rhamnolipid, and construction method thereof

A technology of Pseudomonas aeruginosa and rhamnolipids, which is applied in the biological field, can solve problems such as rhamnolipid difficulties, and achieve the effects of increasing oil utilization capacity, increasing yield, and increasing yield

Active Publication Date: 2018-05-22
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Pseudomonas aeruginosa for increasing yield of rhamnolipid, and construction method thereof
  • Pseudomonas aeruginosa for increasing yield of rhamnolipid, and construction method thereof
  • Pseudomonas aeruginosa for increasing yield of rhamnolipid, 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 for improving the yield of rhamnolipid, and a construction method thereof. The gene expression level of group A of the above strain is decreased or lost while the gene expression level of group B of the strain is increased; gene in the group A is one selected from a pslABCDEFGHIJKLMNO extracellular polysaccharide synthetic gene cluster, a pelABCDEFG polysaccharide synthetic gene cluster, an alginate synthetic gene cluster algD-alg8-alg44-algKEGXLIJF and a polyhydroxy fatty acid PHA synthetic key gene cluster phaC1-D-C2, or an arbitrary combination of the genes; and gene in the group B is one selected from rmlACBD, rmlBDAC, rhlYZ, algC, fadD4, lipC and estA, or an arbitrary combination of the genes. The Pseudomonas aeruginosa has the advantages of significant improvement of the yield of the rhamnolipid, stability in inheriting, and facilitation of the separation and purification of downstream products.

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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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
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