Monorhamnolipid production strain and application thereof

A technology for the production of bacterial strains and rhamnolipids, which is applied in the direction of bacteria, microorganisms, and methods based on microorganisms, can solve the problems of high cost, complex chemical synthesis process, and restrictions on the application of ATCC9027 strains, and reduce the separation and purification process. Effect

Pending Publication Date: 2021-04-30
上海恒什生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the chemical synthesis process is complex and the cost remains high
[0005] Although as early as 1980, researchers isolated a strain of Pseudomonas aeruginosa ATCC 9027 (DSM 1128) from patients with external ear infections, which can specifically produce mono-rhamnolipids, but at 37°C, its The level of monorhamnolipid produced is much lower than that of Pseudomonas aeruginosa PAO1 strain (Complete genome sequence of Pseudomonasaeruginosa PAO1, an opportunistic pathogen. Nature 2000,406:959-964.); The personnel verified that the ATCC9027 strain does not have infectivity, but at 30°C, the level of pyocyanin produced by the ATCC9027 strain is much higher than that of the PAO1 strain, which is still a problem that cannot be ignored; these problems greatly limit the development of the ATCC9027 strain. Application (María-Victoria Grosso-Becerra et al., Pseudomonasaeruginosa ATCC

Method used

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  • Monorhamnolipid production strain and application thereof
  • Monorhamnolipid production strain and application thereof
  • Monorhamnolipid production strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 The construction of the genetically engineered bacterial strain that efficiently produces monorhamnolipid

[0034] Using the genomic DNA of Pseudomonas aeruginosa PAO1 strain as a template, the primers rhlC-UF (SEQ IDNO: 5) / rhlC-UR (SEQ ID NO: 6) were used for PCR amplification respectively, and the primers rhlC-DF (SEQ ID NO: 6) were used for PCR amplification. ID NO: 7) / rhlC-DR (SEQ ID NO: 8) combined for PCR amplification, and the DNA fragment rhlC-U (SEQ ID NO: 1) upstream and downstream of the rhlC gene (KEGG accession no. PA1130) was obtained after gel cutting and recovery and rhlC-D (SEQ ID NO: 2), the length is 700bp; with the genome of Pseudomonas aeruginosa PAO1 as template, using primer rhlA-F (SEQ ID NO: 9) / rhlR-R (SEQ ID NO: 10) PCR amplification was carried out in combination and the DNA fragment rhlAB-R (SEQ ID NO: 3) including the rhlAB-R gene cluster and the promoter upstream of rhlA and the terminator sequence downstream of rhlR (SEQ ID NO...

Embodiment 2

[0038] Example 2 Fermentative production of rhamnolipid by recombinant strain P2

[0039] Fermentation verification is carried out to the genetically engineered recombinant strain P2 obtained in Example 1. First, the glycerol tube bacterium liquid that draws 100 μ L of recombinant bacterial strain P2 is inoculated into 25 mL LB (10 g / L tryptone, 5 g / L yeast powder) in a 100 mL shake flask. , 10g / L sodium chloride) liquid medium, cultured at 37°C, 120rpm for 24h. Transfer 5 mL of overnight cultured LB culture broth to 200 mL seed medium in a 1 L shake flask, and cultivate at 37 °C, 120 rpm for 24 h. The composition of the seed medium is: 125g / L sunflower oil (commercially available Arowana sunflower oil), 1.5g / L NaNO 3 ,0.05g / L MgSO4 7H 2 O, 0.1g / L KCl, 0.1M NaH 2 PO 4 -Na 2 HPO 4 Buffer, pH 6.5, 1mL / L trace element solution (2.0g / L sodium citrate dihydrate, 0.28g / L FeCl 3 ·6H 2 O,1.4g / L ZnSO 4 ·7H 2 O,1.2g / L CoCl 2 ·6H 2 O,1.2g / L CuSO 4 ·5H 2 O,0.8g / L MnSO 4 ·H ...

Embodiment 3

[0040] Example 3 Determination of concentration and purity of recombinant strain P2 fermented to produce monorhamnolipid

[0041] Centrifuge the fermentation broth at 5000rpm for 10min, take the supernatant, add concentrated hydrochloric acid to adjust the pH to 2.0, add an equal volume of chloroform / methanol (v:v=2:1) ​​solution, vortex at high speed for 1min, extract twice, and collect The obtained organic phases were combined and volatilized in vacuo to finally obtain the rhamnolipid product, and the total rhamnolipid concentration was detected by the sulfate anthrone method. The specific measurement method is: add 100 μL rhamnolipid solution diluted with methanol to 1 mL 0.1% anthrone solution (prepared with 70% sulfuric acid), treat at 80°C for 30 min, then cool to room temperature, and detect 625nm At the same time, different concentrations of rhamnose were used to make a standard curve, and finally the concentration of the measured liquid rhamnose was obtained through t...

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Abstract

The invention discloses a monorhamnolipid production strain and an application thereof, and belongs to the technical field of biology. The pseudomonas aeruginosa PAO1 strain is used as an original strain, the rhlC gene of the pseudomonas aeruginosa PAO1 strain is replaced with an rhlAB-R gene cluster through a homologous recombination method, and the pseudomonas aeruginosa strain capable of specifically producing monorhamnolipid is obtained. The random mutation is further combined, and a mutant strain with the single rhamnolipid production level remarkably improved is screened out. According to the mutant strain, commercially available goldfish sunflower seed oil is used as a substrate, fermentation is carried out in a 5L bioreactor at 37 DEG C for 90 hours, the concentration of monorhamnolipid reaches 62.7 g/L, the purity reaches 95.16%, and the ratio of Rha-C10-C10 is the maximum and accounts for 68.59%.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology. 【Background technique】 [0002] Rhamnolipids are a class of surface active molecules secreted by Pseudomonas aeruginosa or Burkholderia and belong to biosurfactants. It has the characteristics of biodegradable, low toxicity, and effective in extreme environments. In addition, it also has the ability to inhibit microorganisms, skin compatibility and chelate heavy metal ions to deal with heavy metal pollution, and promote the dissolution and biodegradation of slightly soluble organic compounds. These characteristics of rhamnolipids make it widely used in fields such as biological control, cosmetics, medicine, detergent, environmental cleaning and mining. [0003] The commonly referred to as "rhamnolipid" is not a single structure, but a mixture of many homologous structures. Up to 28 rhamnolipid structures with different structures have been found in known reports. It has the basic characteristics...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N1/20C12P19/44C12R1/385
CPCC07K14/21C12P19/44
Inventor 陈玲汪江林
Owner 上海恒什生物科技有限公司
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