Escherichia coli for synthesizing monophosphate lipoid A containing only three fatty acid chains

A technology for Escherichia coli and lipids, which is applied in the fields of genetic engineering and bioengineering, and can solve the problems that lipid A has a large molecular weight and cannot be used to identify lipid A acyltransferases and multi-structural components.

Pending Publication Date: 2022-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular weight of intact E. coli lipid A is relatively large, and ESI / MS may produce more structural components when identifying its structure, and wild-type E. coli cannot be used to identify lipid A acyltransferases of other Gram-negative bacteria

Method used

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  • Escherichia coli for synthesizing monophosphate lipoid A containing only three fatty acid chains
  • Escherichia coli for synthesizing monophosphate lipoid A containing only three fatty acid chains
  • Escherichia coli for synthesizing monophosphate lipoid A containing only three fatty acid chains

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Experimental program
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Effect test

Embodiment 1

[0037] Construction of embodiment 1 knockout plasmid

[0038] Using the CRISPR / Cas9 knockout system to knock out three genes related to lipid A secondary acyl chain transferase in E. coli requires the construction of three knockout plasmids: pTargetF-lpxL, pTargetF-lpxP and pTargetF-lpxM, these plasmids The construction process is as follows:

[0039] (1) Select 20nt N complementary to the target sequence of the target gene 20 sequence, specific N 20 The sequences are the underlined sequences of the primers pTargetF-lpxL, pTargetF-lpxP and pTargetF-lpxM respectively, and these sequences are modified to the 5' ends of the forward and reverse primers of the plasmid pTargetF to obtain the forward primers pTargetF-lpxL-F, pTargetF-lpxP- F, pTargetF-lpxM-F and reverse primers pTargetF-lpxL-R, pTargetF-lpxP-R, pTargetF-lpxM-R.

[0040] Using the plasmid pTargetF as a template, the forward and reverse primers of pTargetF-lpxL, pTargetF-lpxP and pTargetF-lpxM were used for PCR ampl...

Embodiment 2

[0044] Example 2 Construction of Escherichia coli lipid A structure-reduced strain HWJ003

[0045] The CRISPR / Cas9 knockout system was used to knock out the lipid A secondary acyl chain transferase-related genes of Escherichia coli HW003. The specific knockout process is as follows ( figure 1 ):

[0046] (1) Preparation of Escherichia coli electroporation knockout competent cells HW003 / pCas

[0047] The plasmid pCas is transformed into Escherichia coli HW003 to obtain recombinant Escherichia coli HW003 / pCas containing the pCas plasmid, and the Escherichia coli HW003 / pCas is activated on the LB solid plate adding 30mg / L Kanamycin (Kan), and inoculated into LB (Kan+) test tube was cultivated overnight to obtain seed liquid; the seed liquid was transferred to 25mL LB (Kan+) medium at 1% (v / v), and cultivated 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°C, centrifuge at 4000rpm for 10min...

Embodiment 3

[0057] Example 3 Extraction and Verification of HWJ003 Lipid A

[0058] (1) Lipid A extraction and purification method: the extraction of lipid A adopts the mixed phase extraction method of chloroform / methanol / water solution. The strain HWJ003 was inoculated in LB liquid medium and cultured overnight at 37°C to obtain the bacterial liquid, and the initial OD of the bacterial liquid was 600 =0.02 Transfer to 1L LB liquid medium, cultivate to the late logarithmic period of the bacteria at 37°C, collect the bacteria by centrifugation at 4000rpm for 20min, and use Bligh-Dyer one-phase system chloroform / H 2 O / methanol (1:0.8:2v / v / v) 76mL mixture to suspend the cell pellet, magnetically stir for 1h, centrifuge at 2000rpm for 30min to collect the cell debris, add 27mL sodium acetate (pH 4.5) to the precipitate, and ultrasonically homogenize for 10min , and heated in a boiling water bath for 30min. The suspension was mixed with 60mL of methanol / chloroform (1:1v / v) to form a Bligh-Dy...

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Abstract

The invention discloses escherichia coli for synthesizing monophosphate lipoid A containing only three fatty acid chains, and belongs to the field of genetic engineering and bioengineering. According to the invention, coding secondary acyl chain transferase genes lpxL, lpxP and lpxM on an escherichia coli HW003 genome are knocked out. The invention provides escherichia coli with a simplified lipoid A structure which lacks 1-site phosphoric acid, 3-site primary acyl chains, 2'site secondary acyl chains and 3 'site secondary acyl chains compared with wild type lipoid A. The lipoid A structure provides a method for identifying Gram-negative fungus lipoid A secondary acyl chain transferase gene functions in escherichia coli.

Description

technical field [0001] The invention relates to an Escherichia coli that synthesizes monophosphoric acid lipid A containing only three fatty acid chains, and belongs to the fields of genetic engineering and bioengineering. Background technique [0002] Escherichia coli is a Gram-negative model bacterium in the study of lipid A biosynthesis, and its lipid A synthesis begins with UDP-glucosamine acetic anhydride (UDP-GlcNAc), which is catalyzed successively by three soluble enzymes LpxA, LpxC and LpxD A 3-OH fatty acid chain is added at C2 and C3 respectively to form UDP-Diacyl-GlcN, which is then hydrolyzed by LpxH to ​​form Lipid X. A molecule of Lipid X and a molecule of UDP-Diacyl-GlcN are condensed by LpxB into 1-phosphate disaccharide (Disaccharide-1-P), and transferred to the inside of the inner membrane of the cell, and then phosphorylated at the C4' position by LpxK to form Lipid IV A . Next, KdtA adds two molecules of Kdo groups to the C6' position, forming Kdo2-L...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/55C12N15/52C12N15/31C12N1/21C12N15/70C12P19/26A61K39/39C12R1/19
CPCC12N9/1029C12N9/16C12N9/78C07K14/245C12N15/52C12N15/70C12P19/26A61K39/39A61K2039/55594
Inventor 王小元季帆檀昕王震郭勇韩雅宁
Owner JIANGNAN UNIV
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