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Method for catalytically synthesizing rhamnolipid by using exoenzyme

A technology of rhamnolipid and rhamnose, applied in the field of biocatalysis, can solve the problems of foaming, economic loss, and the quality of rhamnolipid affected by defoamer

Pending Publication Date: 2021-08-13
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production strain of rhamnolipids is mainly produced by the fermentation of Pseudomonas aeruginosa monobacteria. A large amount of foam will be generated during the fermentation of monobacteria to produce rhamnolipids, which not only affects the yield of rhamnolipids, And it will use a large amount of antifoaming agents in the fermentation process to affect the quality of rhamnolipids, causing unnecessary economic losses
Therefore, it is urgent to develop a new rhamnolipid production process to solve the foaming problem in the fermentation process of traditional rhamnolipid monobacteria

Method used

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  • Method for catalytically synthesizing rhamnolipid by using exoenzyme
  • Method for catalytically synthesizing rhamnolipid by using exoenzyme
  • Method for catalytically synthesizing rhamnolipid by using exoenzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example is used to illustrate the deletion of the rhlAB gene cluster to obtain a precursor strain PAO1Δrhl that can only synthesize dTDP-L-rhamnose

[0036] The gene rhlA and rhlB deleted in PAO1Δrhl are PA3478-PA3479 on the genome of Pseudomonas aeruginosa PAO1 ( www.pseudomonas.com ), the construction reference of PAO1Δrhl Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nature Protocol, 2015, 10(11): 1820-41. The knockout plasmid is pEX18Gm, and the promoter of the gene cluster and the 987bp fragment of the rhlA gene coding region are knocked out. Primers were designed according to the rhlAB gene sequence and promoter sequence, and its upstream and downstream homologous fragments were amplified. Primers used for homology arm amplification are as follows:

[0037] rhl_KO_UF:5'-tatgaccatgattacgaattcCCCTCGGCGTGAACCT-3'

[0038] rhl_KO_UR:5'-ttgtcgcggctgcggatcgctgtgcg-3'

[0039] rhl_KO_DF:5'-ccgcagccgcgacaagctgctcaag-3' ...

Embodiment 2

[0044] This example is used to illustrate the knockout of the rmlABCD gene cluster to obtain the precursor strain PAO1Δrml that can only synthesize HAAs

[0045] The gene rmlABCD deleted in PAO1Δrml is PA5161-PA5164 on the genome of Pseudomonas aeruginosa PAO1 ( www.pseudomonas.com ), the construction reference of PAO1Δrml Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nature Protocol, 2015,10(11):1820-41. The knockout plasmid is pEX18Gm, and the promoter of the gene cluster, most of the coding regions of the rmlB gene and the rmlD gene are knocked out with a fragment of 1499 bp in total. Primers were designed according to the rmlABCD gene sequence and promoter sequence, and its upstream and downstream homologous fragments were amplified. Primers used for homology arm amplification are as follows:

[0046] rml_KO_UF:5'-tatgaccatgattacgaattcCTCTCCGACCTGCCCCCC-3'

[0047] rml_KO_UR:5'-aatgcaccagAAGTGCCATCACTGGGTAGAAAG-3'

[0048] rml...

Embodiment 3

[0053] Functional validation of precursor synthetic strains PAO1Δrml and PAO1Δrhl

[0054] 1. Detection of rhamnolipid synthesis ability of PAO1Δrml and PAO1Δrhl

[0055] The single bacterium colony of PAO1Δrml and PAO1Δrhl is inoculated 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 L) Shaking culture at 37° C. and 200 rpm for 2 days to obtain PAO1Δrml culture solution and PAO1Δrhl culture solution respectively. Take 40 mL each of PAO1Δrml culture solution and PAO1Δrhl culture solution, and centrifuge at 5000 rpm for 10 minutes to obtain PAO1Δrml supernatant and PAO1Δrhl supernatant respectively, and adjust the supernatant to pH 2 with concentrated hydrochloric acid. Then add an equal volume of chloroform / methanol (v:v=2:1) ​​solution, vortex at high speed for 1 min, extract twice, then combine the collected organic phases, ev...

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Abstract

The invention discloses a method for catalytically synthesizing rhamnolipid by an exoenzyme. The rhamnolipid is synthesized by using rhamnosyltransferase RhlB to extracellularly catalyze rhamnolipid precursors dTDP-L-rhamnose and HAAs. The dTDP-L-rhamnose and the HAAs are produced based on genetically modified strains, one of which can be used for synthesizing the dTDP-L-rhamnose rather than the HAAs, and the other of which can only be used for synthesizing the HAAs rather than the dTDP-L-rhamnose. After the rhamnolipid precursors synthesized by the two strains are mixed, the rhamnolipid can be synthesized by connecting extracellular catalytic precursors dTDP-L-rhamnose and HAAs by extracellularly using rhamnolipid synthetase RhlB. In implementation of the method, temporary shaking is just needed for uniform mixing, thereby solving the problem of substantial foaming in the rhamnolipid fermentation process. The method of the present invention has wide application prospects in the field of rhamnolipid biosynthesis.

Description

technical field [0001] The invention relates to the field of biocatalysis, in particular to a method for synthesizing rhamnolipids catalyzed by extracellular enzymes. 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. In Pseudomonas aeruginosa, the synthesis of rhamnolipids depends on three metabolic pathways, which are lipid precursor β-hydroxy fatty acid (HAAs) synthesis pathway, glycosyl precursor dTDP-L-rhamnose (dTDP- L-rhamnose) synthesis pathway and the polymerization of lipid precursors and glycosyl precursors to form mono-rhamnolipids and dirhamnolipids. dTDP-L-rhamnose precursor is the hydrophilic part of rhamnolipid, which is synthesized from D-glucose-6-phosphate under the action of glucose pho...

Claims

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Application Information

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IPC IPC(8): C12P19/44C12P19/18C12R1/385
CPCC12P19/44C12P19/18
Inventor 徐安明张晓晓董维亮周杰姜岷
Owner NANJING UNIV OF TECH
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