Method for efficiently synthesizing PHB by knocking out flagellum and pilus genes

A flagella and gene technology, applied in the field of efficient synthesis of PHB, can solve the problems such as the inability of PHB to meet the needs of industrial production

Active Publication Date: 2020-01-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods make the improvement of PHB outpu

Method used

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  • Method for efficiently synthesizing PHB by knocking out flagellum and pilus genes
  • Method for efficiently synthesizing PHB by knocking out flagellum and pilus genes
  • Method for efficiently synthesizing PHB by knocking out flagellum and pilus genes

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Embodiment 1

[0050] Construction of embodiment 1 knockout plasmid

[0051] Using the CRISPR / Cas9 knockout system to knock out flagella and pilus synthesis and transport gene clusters, four knockout plasmids need to be constructed: pTargetF-flhD-motA, pTargetF-fliY-fliR, pTargetF-flgN-flgL, pTargetF-fimB-fimH , the construction process of the plasmid is as follows:

[0052] (1) Through the analysis of the website http: / / crispr.mit.edu / , select a 20nt N that is complementary to the target sequence of the target gene 20 sequence, specific N 20 The sequences are the underlined sequences of the primers pTargetF-flhE-motA-F, pTargetF-fliY-fliR-F, pTargetF-flgN-flgL-F, and pTargetF-fimB-fimH-F, and these sequences are modified into the plasmid pTargetF forward primer At the 5' end, forward primers pTargetF-flhD-motA-F, pTargetF-fliY-fliR-F, pTargetF-flgN-flgL-F, and pTargetF-fimB-fimH-F were respectively obtained. Using the plasmid pTargetF as a template, the forward primers pTargetF-flhD-motA...

Embodiment 2

[0059] The construction of embodiment 2 genetically engineered bacteria WJW010 and WJW011

[0060] The CRISPR / Cas9 knockout system was used to knock out the flagella synthesis and transport gene cluster of wild-type Escherichia coli W3110 to obtain engineering strain WJW010; the pili gene cluster was knocked out to get WJW011. The specific knockout process is as follows:

[0061] (1) Preparation of Escherichia coli electroporation knockout competent

[0062] The plasmid pCas is transformed into Escherichia coli W3110 to obtain recombinant Escherichia coli W3110 / pCas containing the pCas plasmid, and the Escherichia coli W3110 / pCas is activated on the LB solid plate adding 30mg / L Kanamycin (Kan), and inoculated into LB +Kan test tube cultured overnight to obtain seed liquid; transfer the seed liquid to 25mL LB+Kan medium at 1%, and cultivate to OD at 30°C and 200rpm 600 =0.2, add 500 μL L-arabinose solution to induce, continue to culture to OD 600 =0.5, ice bath for 30min; 4°...

Embodiment 3

[0075] Example 3 Growth status of flagella-reduced strain WJW010 and pilus-reduced strain WJW011

[0076] The streamlined strains WJW010 and WJW011 were 20, 6440bp and 8753bp streamlined respectively compared with the wild type W3110, and the streamlined genome proportions were about 1.13% and 0.19% respectively. W3110, WJW010 and WJW011 were cultured, and their growth curves in LB medium were determined.

[0077] The results showed that the growth curve as Figure 5 As shown, all three entered the stable phase at 10 h; in the early logarithmic period, the growth of the strains WJW010 and WJW011 was slightly slower than that of the wild type; It was basically the same as the wild type W3110; after the stable period, the OD of the streamlined strains WJW010 and WJW011 600 Taller, highest OD of the flagellar-reduced strain WJW010 600 Compared with W3110, it increased by 12.4%, and finally increased by 10.7%; the pili reduced strain WJW011 had the highest OD 600 A 6.6% improv...

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Abstract

The invention discloses a method for efficiently synthesizing PHB by knocking out flagellum and pilus genes, which belongs to the field of gene engineering and fermentation engineering. According to the invention, three flagellum gene clusters flhE-motA, fliY-fliR and flgN-flgL on an escherichia coli genome are knocked out to obtain a flagellum simplified strain WJW010, and a fimbriae gene clusterfimB-fimH is knocked out to obtain a fimbriae simplified strain WJW011, the three genes synthesized by PHB can be transformed into strains WJW010 and WJW011, and the recombinant bacteria WJW010/pBHR68 and the recombinant bacteria WJW011/pBHR68 are obtained; pHB with the cell dry weight of 19.2% and 2.8% can be synthesized under the normal fermentation condition, and the volume yield of PHB is 9.6times and 1.4 times that of wild control bacteria W3110/pBHR68 (2%).

Description

technical field [0001] The invention relates to a method for efficiently synthesizing PHB by knocking out flagella and pilus genes, and belongs to the fields of genetic engineering and fermentation engineering. Background technique [0002] Polyhydroxyalkanoates (PHA) are a class of renewable and degradable polymers with multiple material properties synthesized by microorganisms, and have broad application prospects in the fields of medicine, materials and environmental protection. Polyhydroxyalkanoate is widely present in microbial cells, mainly as a carbon source and energy storage carrier. The higher the ratio of C:N in the growth environment, the more favorable the synthesis of PHA. PHA exists in the form of hydrophobic particles in cells, and its content can exceed 90% of the dry weight of cells under certain conditions. According to different types of monomers and polymerization methods, PHA has a series of diverse material properties ranging from hard and brittle ha...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N9/88C12N15/52C12P7/625
Inventor 王小元王建莉马文渐王甜忆周晴方宇李烨
Owner JIANGNAN UNIV
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