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Oscillating gene expression system, construction method and application of oscillating gene expression system in rhamnolipid fermentation

A gene expression and rhamnolipid technology, applied in the field of genetic engineering, can solve the problems of low yield, restrict the industrialization development of rhamnolipid, affect bacterial growth and metabolism, etc., and achieve the effect of increasing yield

Pending Publication Date: 2021-07-16
NANJING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above method introduced rhamnolipid-producing related genes into Escherichia coli and verified the feasibility of heterologous synthesis of rhamnolipids, the yield of rhamnolipids in Escherichia coli was low, and due to the synthesis of rhamnose Lipid precursors mostly come from the central metabolic pathway of Escherichia coli, and overexpression of rhamnolipids in bacteria will affect the growth and metabolism of the bacteria themselves
[0005] In summary, the safety requirements of rhamnolipid fermentation limit the fermentation application of Pseudomonas aeruginosa, and the low yield of rhamnolipid expressed by heterologous microorganisms also limits the industrial development of rhamnolipid

Method used

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  • Oscillating gene expression system, construction method and application of oscillating gene expression system in rhamnolipid fermentation
  • Oscillating gene expression system, construction method and application of oscillating gene expression system in rhamnolipid fermentation
  • Oscillating gene expression system, construction method and application of oscillating gene expression system in rhamnolipid fermentation

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Embodiment 1

[0041] Example 1: Construction of oscillatory gene expression plasmids pOSC and pOSC-rh1AB.

[0042] 1. The construction of recombinant plasmid pOSC comprises the following steps:

[0043] (1) Using the plasmid pTD103 as a DNA template, using primers sfGFPFor and PtetRev to amplify the 1Kb T1-sfGFP-PluxI-Ptet sequence,

[0044] sfGFPFor (SEQ ID: NO.10): 5'-gtacctttctcctctttaatgcggcggatttgtcctactca-3',

[0045] PtetRev (SEQ ID: NO.11):

[0046] 5'-tggtttgttatagtcgaaTgcaagcagcagtatctctatcactgatagggatgtcaagcacctgtaggatcgtac-3'.

[0047] (2) Digest the plasmid pTD103 with the restriction endonuclease EcoRI, recover and purify the 3.6Kb vector fragment luxR-kan-ColE1-luxI after digestion.

[0048] (3) Ligate step (1) and the fragment purified and recovered in step (2) by Gibson assembly to form a new plasmid vector, namely to obtain plasmid pTD-Ptet. The Gibson assembly system is T1-sfGFP-PluxI-Ptet fragment, luxR-kan-ColE1-luxI fragment 1.5μl each, 2×Gibson assembly Mix 3μl, t...

Embodiment 2

[0089] Example 2: Expression of rhamnolipids using Escherichia coli oscillating gene expression system

[0090] The constructed recombinant plasmids pOSC-rh1AB and pTD-rh1AB were transformed into DH10B to obtain engineering bacteria DH10B / pOSC-rh1AB and DH10B / pTD-rh1AB. The engineered bacteria DH10B / pOSC-rh1AB and the control group DH10B / pTD-rh1AB were inoculated in LB liquid medium (containing 50 μg / ml kanamycin), and cultured at 37°C and 220 rpm / min for 16 hours to make seed solution. Inoculate in a fermentation medium (LB medium formula + 3% glycerol + 3% glucose) at a volume ratio of 1%, culture at 30° C. and shake at 220 rpm for 24 to 48 hours, and obtain a fermentation broth containing rhamnolipids.

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Abstract

The invention discloses an oscillating gene expression system, a construction method and application of the oscillating gene expression system in rhamnolipid fermentation. The oscillating gene expression system comprises a promoter PluxI, a repressor gene tetR, a promoter Pet, response regulatory factor genes LuxI and LuxR, and a protein degradation tag gene ssrA. By regulating the periodic switching process of gene expression, the oscillating gene expression system can relieve the supply pressure of metabolic intermediate products and energy of escherichia coli in a stable growth period, so that the gene expression and substrate supply are balanced, and the yield of secondary metabolites is further improved. According to the invention, by introducing a rhamnolipid biosynthetic pathway related gene into an oscillating gene expression regulatory plasmid, heterologous synthesis of rhamnolipid in Escherichia coli is realized, and the fermentation yield of the engineering bacteria of the oscillating gene expression system for high yield of rhamnolipid is up to 838mg / L.

Description

technical field [0001] The invention relates to an oscillatory gene expression system, a construction method and its application in rhamnolipid fermentation, belonging to the technical field of genetic engineering. Background technique [0002] As an important Gram-negative model strain, Escherichia coli has the characteristics of non-pathogenicity, relatively simple structure, clear genetic background, and easy isolation and culture. It is often used as a model organism to participate in biological experiments or as an object of genetic engineering. It is an ideal host for heterologous expression and secretion of secondary metabolites. [0003] Rhamnolipid is a crystalline acid, which belongs to anionic surfactant, and is an amphiphilic molecule composed of one or two rhamnose and 3-hydroxy fatty acid connected by glycosidic bonds. There are many types of rhamnolipids and various chemical structures. Up to now, as many as 60 homologues of rhamnolipids have been found. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N1/21C12P19/44C12R1/19
CPCC12N15/70C07K14/245C12P19/44Y02A50/30
Inventor 汪洋程鑫茹李晓波王玉琪
Owner NANJING UNIV OF SCI & TECH
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