Construction and application of fimbriae-free escherichia coli capable of improving production efficiency

A technology of Escherichia coli and recombinant Escherichia coli, applied in the fields of genetic engineering and fermentation engineering, can solve the problems of slow growth of L-threonine fermentation, consumption of large carbon sources, and inability to meet the requirements

Active Publication Date: 2021-04-20
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the substrates acetyl-CoA and oxaloacetate for the synthesis of PHB and L-threonine come from the glycolysis pathway and the citric acid cycle pathway respectively. If high-yield PHB and L-threonine will consume a large amount of carbon sources, it will seriously affect bacterial growth
This makes the improvement of PHB production unable to meet the needs of industrial production, and the growth of L-threonine fermentation is slow

Method used

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  • Construction and application of fimbriae-free escherichia coli capable of improving production efficiency
  • Construction and application of fimbriae-free escherichia coli capable of improving production efficiency
  • Construction and application of fimbriae-free escherichia coli capable of improving production efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of Pili Deletion Engineering Bacteria

[0054] The specific construction process of pili-deficient engineering bacteria is as follows:

[0055] (1) Preparation of MG1655 / pCas competent cells:

[0056] Inoculated with Red recombinant helper plasmid pCas9 (Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S.2015.Multigene editing in the Escherichia coli genome via the CRISPR -Cas9 system. Appl EnvironMicrobiol, 81(7), 2506-14.) Escherichia coli MG1655, in LB liquid medium containing 100 μg / mL kanamycin, 30 ° C, 200 rpm overnight culture. Transfer to 100mL LB liquid medium according to 2% inoculum size, cultivate to OD at 30°C and 200rpm 600 When = 0.2, add L-arabinose (final concentration is 30mmol / L) to induce the expression of recombinase, continue to cultivate to OD 600=0.5, put the culture medium in an ice bath for half an hour, then transfer it to a pre-cooled 50mL centrifuge tube, centrifuge at 8000rpm at 4°C for 10min to collect the ...

Embodiment 2

[0069] Example 2 Knocking out a single pilus gene cluster promotes growth and PHB synthesis

[0070] Escherichia coli contains 12 gene clusters for the synthesis and assembly of pili ( figure 1 ). Pili not only increase the pathogenicity of bacteria, but also consume a large amount of energy and carbon sources during their synthesis and assembly. Therefore, in theory, removing these pili could improve the biosafety and production efficiency of E. coli.

[0071] Twelve pili synthesis and assembly gene clusters were removed from the chromosome of Escherichia coli MG1655 to obtain strains WQM001, WQM002, WQM003, WQM004, WQM005, WQM006, WQM007, WQM008, WQM009, WQM010, WQM011 and WQM012. These strains were cultured in LB and M9 medium, respectively. Detect the OD value of the bacterial solution every 2h and draw the growth curve of the bacterial strain, such as figure 2 Shown: In LB medium, the growth curves of all 12 mutants were similar to the control strain MG1655, indicati...

Embodiment 3

[0075] Example 3 Morphology and metabolite analysis of WQM026

[0076]As can be seen from the results of Example 2, the removal of each pilus synthesis and assembly gene cluster can promote the growth of E. coli cells and the biosynthesis of PHA, so all 12 pilus synthesis and assembly gene clusters on the E. coli MG1655 chromosome was deleted, thereby constructing the pili-deficient bacterial strain WQM026 (see Example 1 for the construction method). Detect the OD value of the bacterial solution every 2h and draw the growth curve of the bacterial strain, such as Figure 5 Shown in A: the growth of WQM026 in LB medium is slightly better than that of MG1655, and the growth in M9 medium is much better than that of MG1655. The OD value of WQM026 is about 3 times that of MG1655 when cultured to 18 hours. This suggests that removing all 12 pili gene clusters in E. coli benefits cell growth, especially in a nutrient-poor environment. Electron microscope observation of MG1655 and WQ...

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Abstract

The invention discloses construction and application of fimbriae-free escherichia coli capable of improving production efficiency, and belongs to the field of gene engineering and fermentation engineering. 64 genes of a fimbriae gene cluster on an escherichia coli genome are knocked out to obtain mutant bacteria WQM026, the strain grows faster in a nutrient-deficient culture medium, and the total amount of thalli is increased. The related genes synthesized by PHB and L-threonine are respectively transformed into the strain WQM026, and the obtained recombinant bacteria WQM026 / pBHR68 and WQM026 / pFW01-thrA*BC-rhtC can be used for respectively and efficiently synthesizing PHB and L-threonine. The synthesized PHB accounts for 87.87% of the dry weight of the cells and is 3.44 times that of wild type control bacteria. The yield of the L-threonine is 2.49 g / L and is 3.66 times that of wild type control bacteria.

Description

technical field [0001] The invention relates to the construction and application of a pili-free Escherichia coli capable of improving production efficiency, and belongs to the fields of genetic engineering and fermentation engineering. Background technique [0002] Bacterial pili are large, long, and thin supramolecular protein appendages that arise on cells and are responsible for biofilm formation, chemotaxis, adhesion, and DNA transmembrane transfer. Pili are also an important component of bacterial biofilms and have multiple adverse effects on industrial activities such as increasing the risk of product spoilage or contamination, causing serious infections, forming dormant cells to increase antibiotic resistance, depleting energy and substrates of bacteria , Block the diffusion of nutrients, form biofouling, reduce heat transfer, increase corrosion, and shorten the service life of fermentation equipment. [0003] Polyhydroxyalkanoates (PHAs) are synthesized from a varie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/62C12P13/08C12R1/19
CPCC12R2001/19C12P13/08C12P7/62C12N15/74
Inventor 王小元乔君檀昕任鸿宇吴铮胡晓清李烨
Owner JIANGNAN UNIV
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