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The method for increasing the production of rhamnolipid and its specific Pseudomonas aeruginosa

A technology of Pseudomonas aeruginosa and rhamnolipid, which can be applied in the biological field and can solve problems such as unclear chemical structure

Active Publication Date: 2016-09-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pel (Pellicle formation) is an operon (pel operon) composed of 7 genes ( figure 1 ) encoded protein synthesis, rich in glucose residues, but not cellulosic polysaccharides, its specific chemical structure is not clear

Method used

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  • The method for increasing the production of rhamnolipid and its specific Pseudomonas aeruginosa
  • The method for increasing the production of rhamnolipid and its specific Pseudomonas aeruginosa
  • The method for increasing the production of rhamnolipid and its specific Pseudomonas aeruginosa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The preparation of embodiment 1, PAO1-psl and PAO1-pslpel

[0060] The nucleotide sequence of the promoter of the psl polysaccharide synthesis gene cluster of Pseudomonas aeruginosa PAO1 refers to the following website: http: / / www.pseudomonas.com, the promoter of the Pel polysaccharide synthesis gene cluster of Pseudomonas aeruginosa PAO1 See the following website for the nucleotide sequence of : http: / / www.pseudomonas.com. The promoter of the psl polysaccharide synthesis gene cluster of Pseudomonas aeruginosa PAO1 was knocked out to obtain a mutant of Pseudomonas aeruginosa PAO1, and the mutant was named recombinant Pseudomonas aeruginosa PAO1-psl, referred to as PAO1-psl ; The promoter of the Pel polysaccharide synthesis gene cluster in PAO1-psl was knocked out to obtain a mutant of PAO1-psl, and the mutant was named recombinant Pseudomonas aeruginosa PAO1-pslpel, referred to as PAO1-pslpel.

[0061] The preparation method of PAO1-psl and PAO1-pslpel is as follows: ...

Embodiment 2

[0071] Example 2, PAO1-psl and PAO1-pslpel and the exopolysaccharide synthesis of the starting strain PAO1

[0072] The experiment was repeated three times, and the steps of each repeated experiment were as follows:

[0073] The single clones of PAO1-psl, PAO1-pslpel and the starting strain PAO1 were respectively placed in LBNS liquid medium (the solvent of LBNS liquid medium is water, and its solute and concentration are respectively yeast powder 5g / L, peptone 10g / L) in Shake culture at 37° C. and 200 rpm for 12 hours to obtain PAO1-psl culture solution, PAO1-pslpel culture solution and PAO1 culture solution, respectively. Take PAO1-psl culture solution, PAO1-pslpel culture solution and PAO1 culture solution, centrifuge at 12000rpm for 2min, discard the supernatant, and obtain PAO1-psl cell pellet, PAO1-pslpel cell pellet and PAO1 cell pellet respectively. Add 200 μl of 0.5M EDTA to the PAO1-psl cell pellet, PAO1-pslpel cell pellet, and PAO1 cell pellet with the same cell co...

Embodiment 3

[0075] Example 3, PAO1-psl and PAO1-pslpel and the rhamnolipid synthesis and detection of the starting strain PAO1

[0076] The experiment was repeated three times, and the steps of each repeated experiment were as follows:

[0077] 1. Rhamnolipid synthesis of PAO1-psl and PAO1-pslpel and the starting strain PAO1

[0078]Inoculate the single bacterium colony of PAO1-psl, PAO1-pslpel and starting bacterial strain PAO1 in 50mL nutrient broth medium respectively (the solvent of nutrient broth medium is water, and its solute and concentration are respectively nutrient broth powder 8g / L (Oxoid company), glucose 5g / L), shake culture at 37°C and 200rpm for 2 days to obtain PAO1-psl culture fluid, PAO1-pslpel culture fluid and PAO1 culture fluid respectively. Take 40mL of PAO1-psl culture solution, PAO1-pslpel culture solution and PAO1 culture solution respectively, and centrifuge at 5000rpm for 10min to obtain PAO1-psl supernatant, PAO1-pslpel supernatant and PAO1 supernatant respec...

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Abstract

The invention discloses a method for preparing rhamnolipid and its special Pseudomonas aeruginosa. The method for preparing rhamnolipids includes 1) and 2): 1) constructing a recombinant Pseudomonas aeruginosa with reduced Psl polysaccharide synthesis ability and / or reduced Pel polysaccharide synthesis ability compared with Pseudomonas aeruginosa as the starting bacterium 2) fermenting the recombinant Pseudomonas aeruginosa to obtain rhamnolipids. The experiment of the present application proves that compared with Pseudomonas aeruginosa as the starting bacterium, the rhamnolipid synthesis ability of recombinant Pseudomonas aeruginosa with reduced Psl polysaccharide synthesis ability and / or reduced Pel polysaccharide synthesis ability is improved.

Description

technical field [0001] The invention relates to a method for increasing the yield of rhamnolipid in the field of biotechnology and the special Pseudomonas aeruginosa. Background technique [0002] Rhamnolipid is a glycolipid anionic surfactant. It has good biocompatibility and efficient emulsification, solubilization and surface tension reduction capabilities, and has broad application prospects in petroleum exploration, biomedicine, environmental protection and food and other fields. [0003] At present, the production strain of rhamnolipid is mainly Pseudomonas aeruginosa, and the improvement of its production performance is generally through various physical and chemical mutagenesis techniques to improve the production capacity of the strain rhamnolipid, and physical and chemical mutagenesis is very easy to reverse the mutation , resulting in unstable production performance of the strain, and a new method for constructing a rhamnolipid high-yielding strain is urgently ne...

Claims

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Application Information

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IPC IPC(8): C12P19/44C12N1/20C12R1/385
Inventor 马旅雁王世伟
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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