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Engineered Escherichia coli strain capable of realizing high-yield production of pentamethylene diamine and method for high-yield production of pentamethylene diamine via same

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 low mass transfer efficiency, reduce conversion efficiency, limit industrial scale production, etc., and achieve mass transfer efficiency High, improve production efficiency, solve the effect of low mass transfer efficiency

Active Publication Date: 2020-05-08
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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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 production pentamethylenediamine

[0028] Using the sequences Seq.1 and Seq.2 as primers and the pET-22b empty 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 Escherichia coli genome as a template, the Pcad promoter sequence was obtained by colony PCR amplification, and the sequence was Seq.6. The sequences Seq.7 and Seq.8 were used as primers, and the Escherichia coli genome was used as a template to obtain the L-lysine decarboxylase DNA sequence through colony PCR amplification, and the sequence was Seq.9. The 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 the sequence Seq.10. through CaCl 2 Transformation method The recombinant...

Embodiment 2

[0029] Example 2. Fermentation preparation of Escherichia coli engineering bacteria TJU-cadA-1

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

[0031]Inoculate empty-loaded Escherichia coli BL21(DE3) and Escherichia coli engineering bacteria TJU-cadA-1 into tw...

Embodiment 3

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

[0038] Get 2 bottles of 80 ml of fermented liquid obtained in Example 2 respectively, and centrifuge to obtain Escherichia coli engineering bacterium 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 after treatment at 0° C. and 50 rpm for 1 min, the permeabilized cells rich in L-lysine decarboxylase were collected by centrifugation. The above-mentioned Escherichia coli engineering bacteria TJU-cadA-1 and permeable cells were used Na 2 HPO 4 - Citric acid solution (100mM, pH 5.6) was resuspended to a cell concentration of 1g / L, used for the catalytic reaction in Example 4, and carried out for enzyme activity determination. The results of enzyme activity determination are as follows: the enzyme activity of L-lysine decarboxylas...

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PUM

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Abstract

The invention relates to an engineered Escherichia coli strain capable of realizing high-yield production of pentamethylene diamine and method for high-yield production of pentamethylene diamine via the same. The engineered Escherichia coli strain is Escherichia coli with an accession number of CGMCC No. 17454. The method comprises the following steps: (1) constructing engineered Escherichia colistrain TJU-cadA-1 for exogenous expression of L-lysine decarboxylase, wherein the enzyme activity of the L-lysine decarboxylase is about 100 times the enzyme activity of a no-load strain; (2) carryingout permeability treatment on L-lysine decarboxylase-rich whole cells, wherein the enzyme activity of the L-lysine-rich decarboxylase in permeabilized cells is 3-4.5 times the enzyme activity of thewhole cells; and (3) catalyzing L-lysine hydrochloride to produce pentamethylene diamine by using the permeabilized cells, wherein the yield of pentamethylene diamine is 90%-100%, and the concentration of pentamethylene diamine can reach 220 g / L or above. According to the method, the L-lysine hydrochloride is used for efficiently producing the pentamethylene diamine; and compared with the prior art, an expensive IPTG inducer is not needed in the invention, so the method has the advantages that mass transfer efficiency is high, yield is high, production cost is low, and higher economic feasibility and practicability are obtained.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to an Escherichia coli engineering bacterium with high production of pentamethylenediamine and a method. Background technique [0002] 1,5-Pentanediamine, also known as 1,5-diaminopentane, pentamethylenediamine, cadaverine and cadaverine, is a natural polyamine with various biological activities, which can be decarboxylated by lysine The enzyme (E.C.4.1.1.18) catalyzes the direct decarboxylation formation of L-lysine and is widely distributed in prokaryotes and eukaryotes. 1,5-Pentanediamine has wide applications in agriculture, medicine and industry, especially as an important monomer of bio-based polyamides. Pentamethylenediamine has a similar structure to the non-renewable fossil fuel hexamethylenediamine, which can be substituted for the synthesis of conventional polyamides, especially when polymerized with dicarboxylic acids from renewable resources to produce...

Claims

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Application Information

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Patent Type & Authority Applications(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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