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A kind of Escherichia coli engineering bacteria and method for high production of pentamethylenediamine

A technology of Escherichia coli and engineering bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of reducing conversion efficiency, limiting industrial scale production, and low mass transfer efficiency, so as to improve production efficiency , solve the effect of low mass transfer efficiency and broad market prospects

Active Publication Date: 2022-06-28
TIANJIN UNIV
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AI Technical Summary

Problems solved by technology

However, the problem with this method is that the mass transfer efficiency is low: the natural barrier function of the cell wall and cell membrane limits the intracellular transport of the substrate L-lysine and the release of the accumulated pentamethylenediamine product in the cell, thereby reducing the Conversion efficiency, which limits industrial scale production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1. the preparation method of the Escherichia coli engineering bacteria of high-yielding pentamethylenediamine

[0028] Using the sequences Seq.1 and Seq.2 as primers, and using the pET-22b empty vector plasmid as a template, the pET-22b vector DNA sequence was obtained by PCR amplification, and the sequence was Seq.3. Using the sequences Seq.4 and Seq.5 as primers and the E. coli genome as the template, the Pcad promoter sequence was obtained by colony PCR amplification, and the sequence was Seq.6. Using the sequences Seq.7 and Seq.8 as primers, and using the Escherichia coli genome as the template, the L-lysine decarboxylase DNA sequence was obtained by colony PCR amplification, and the sequence was Seq.9. Sequences Seq.3, Seq.6 and seq.9 were connected into a circle by means of pEASY-Uni Seamless Cloning and Assembly Ki technology to form a recombinant plasmid pET-22b-cadA with sequence Seq.10. by CaCl 2 Transformation method The recombinant plasmid pET-2...

Embodiment 2

[0029] Example 2. Preparation of Escherichia coli engineering bacteria TJU-cadA-1 by fermentation

[0030] The empty E. coli BL21 (DE3) and E. coli engineering strain TJU-cadA-1 were inoculated into 2 bottles of 100 mL liquid medium containing 1 g / L yeast powder, 1 g / L peptone, 1 g / L NaCl, and 1 mg / L ampicillin respectively. At 16 °C and 50 rpm, exogenously express L-lysine decarboxylase, after culturing for 1 h, sample 1 ml of fermentation broth, and centrifuge to collect the L-lysine decarboxylase-rich E. coli engineering bacteria TJU-cadA- 1. Carry out enzyme activity assay. The experimental results of fermentation for 1h are as follows: Cell density OD of empty cells 600 is 0.483, and the enzyme activity is 0.032U / mg; the cell density OD of Escherichia coli engineering strain TJU-cadA-1 600 is 0.417, and the enzyme activity is 5.108U / mg;

[0031]The empty E. coli BL21 (DE3) and E. coli engineering strain TJU-cadA-1 were inoculated into two bottles of 100 mL liquid mediu...

Embodiment 3

[0037] Example 3. Permeabilization of Escherichia coli TJU-cadA-1

[0038] Two bottles of 80 ml of the fermentation broth obtained in Example 2 were respectively taken and centrifuged to obtain E. coli engineering bacteria TJU-cadA-1 rich in L-lysine decarboxylase. One is used for permeability treatment and the other is not treated. In the permeabilization treatment group, 15% methanol solution by volume was added to resuspend the cells, and the cells were centrifuged to collect the permeabilized cells rich in L-lysine decarboxylase after treatment at 0°C and 50 rpm for 1 min. The above-mentioned Escherichia coli engineering bacteria TJU-cadA-1 and permeable cells were respectively used Na 2 HPO 4 - The citric acid solution (100 mM, pH 5.6) was resuspended to a bacterial concentration of 1 g / L, used for the catalytic reaction of Example 4, and the enzyme activity was measured. The results of enzyme activity assay are as follows: the enzyme activity of L-lysine decarboxylase...

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Abstract

The present invention relates to an Escherichia coli engineering bacterium with a high production of pentamethylenediamine and a method thereof, the engineering bacterium is Escherichia coli whose preservation number is CGMCC NO.17454; the method is as follows: (1) constructing exogenously expressed L-lysine decarboxylase coli engineering bacterium TJU‑cadA‑1, the enzymatic activity of L‑lysine decarboxylase is about 100 times that of the empty strain; The enzyme activity of L-lysine decarboxylase in sex cells is 3 to 4.5 times that of whole cells; (3) Utilize permeable cells to catalyze L-lysine hydrochloride to produce pentamethylenediamine, and the yield of pentamethylenediamine is 90 %~100%, the concentration can reach more than 220g / L. The present invention utilizes L-lysine hydrochloride to efficiently produce pentamethylenediamine. Compared with the prior art, it does not need expensive IPTG inducers, has high mass transfer efficiency, high yield, low production cost, and is more economically feasible. and practicality.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to an Escherichia coli engineering bacteria and a method for high-yielding pentamethylenediamine. Background technique [0002] 1,5-Pentanediamine, also known as 1,5-diaminopentane, pentamethylenediamine, cadaverine, and cadaverine, is a natural polyamine with various biological activities that can be decarboxylated by lysine Enzymes (E.C. 4.1.1.18) catalyze the direct decarboxylation of L-lysine and are widely distributed in prokaryotes and eukaryotes. 1,5-Pentanediamine has a wide range of applications in agriculture, medicine and industry, especially as an important monomer for bio-based polyamides. Pentamethylene diamine has a similar structure to the non-renewable fossil fuel hexamethylene diamine, and can replace it to synthesize conventional polyamides, especially to produce bio-based polyamides by polymerizing with dicarboxylic acids from renewable resource...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/00C12R1/19
CPCC12N9/88C12N15/70C12P13/001C12Y401/01018
Inventor 齐崴尤生萍张伟芮金秋吕佳佳苏荣欣
Owner TIANJIN UNIV
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