Genetically engineered bacterium for producing pentamethylene diamine and method for preparing pentamethylene diamine

A technology of genetically engineered bacteria and pentamethylenediamine, applied in the field of microorganisms, can solve problems such as no technical innovation research, and achieve the effect of protecting catalyst activity, realizing repeated use and efficient transformation

Active Publication Date: 2016-03-02
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] It can be seen that the prior art has not carried out technological innovation research

Method used

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  • Genetically engineered bacterium for producing pentamethylene diamine and method for preparing pentamethylene diamine
  • Genetically engineered bacterium for producing pentamethylene diamine and method for preparing pentamethylene diamine
  • Genetically engineered bacterium for producing pentamethylene diamine and method for preparing pentamethylene diamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Escherichia coli chromosome lysine decarboxylase temperature-regulated promoter and replacement of signal peptide

[0039] 1. Cloning of partial sequence of ldcC gene

[0040] Using Escherichia coli B0013-070 chromosomal DNA as a template, primers ldc-up1 (SEQ ID NO: 3) and ldc-up2 (SEQ ID NO: 4) were used to amplify the 5' end of ldcC and its upstream sequence (ldcC-up) by PCR. Primers add EcoRI sites. The size of the PCR product is ~920bp; the PCR product is cloned into pMD-18T-simple to obtain the recombinant plasmid pLDC-UP;

[0041] ldc-up1:G GAATTC GCAACCTGCGTGAAATGTC; EcoRI

[0042] ldc-up2:G GAATTC TGAACGGCGGTGTAATGTT; EcoRI

[0043] 2. Obtaining homology arms of ldcC gene recombination

[0044] EcoRI digestion of the recombinant plasmid pLDC-UP releases 915bp and 2.7kb fragments; using the DNA of the recombinant plasmid pLDC-up as a template, reverse PCR amplification was performed with primers ldc-invF (SEQ ID NO: 5) and ldc-invR (SEQ ID NO: 6...

Embodiment 2

[0059] Embodiment 2: p in strain 42# (pT-ldcC) R -p L Determination of promoter activity

[0060] The patented strain 42# (pT-ldcC) and the original strain B0013-070 were cultured at 25-36°C and 37-50°C for 2-10 hours, and the culture medium was (g / L): yeast extract 15, peptone 0.5, anhydrous MgSO 4 0.25, glucose 5. And measure their cell disruption liquid lysine decarboxylase (LDC) specific enzyme activity, typical result is as image 3 shown. The preparation process of the cell disruption solution is as follows: put 30mL fermentation broth in a 50mL centrifuge tube, centrifuge at 6000rpm for 8min; discard the supernatant, add 10mLddH 2 O, vortex to mix, add ddH 2 From 0 to 30 mL, centrifuge at 6000 rpm for 8 min; discard the supernatant, add 10 mL of PBS, vortex to mix, add PBS to 30 mL, and centrifuge at 6000 rpm for 8 min. Repeat washing the cells 1x. Add 10mL PBS to resuspend the bacteria, and ultrasonically disrupt. (Sonication conditions: ultrasonication for 3s...

Embodiment 3

[0062] Example 3 Identification of bacterial strain 42# (pT-ldcC) cell activity and secretion expression process of lysine decarboxylase

[0063] The patented strain 42# (pT-ldcC) and the original strain B0013-070 were cultured at 25-36°C and 37-50°C for 2-10 hours, and the culture medium was (g / L): yeast extract 0-20, peptone 0- 20, anhydrous MgSO 4 0-10, glucose 5. And the expression process of lysine decarboxylase was strengthened by adding lactose, IPTG, etc. The fermentation broth was centrifuged at 6000rpm for 8min, and the supernatant was taken to directly measure the enzyme activity as the enzyme activity in the fermentation broth; the enzyme activity measured after the cells collected by centrifugation were resuspended in the medium to the initial volume was taken as the enzyme activity of the periplasmic space; The measured enzyme activity was taken as the total enzyme activity in the periplasmic space and in the cell. A typical measurement result is as Figure 4...

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Abstract

The invention provides a pentamethylene diamine producing strain and a process for efficient production of the pentamethylene diamine. The strain, when a lysine decarboxylase promoter 1dcCp is replaced as an environmental/nutritional factor-control promoter, is capable of simultaneously expressing a signal peptide; the activity of an enzyme of the strain for the catalytic synthesis of the pentamethylene diamine is greatly improved and is adjusted through ambient temperature; and the finally expressed enzyme falls between an intracellular membrane and a cell wall and is not released in a fermentation system, so that the purpose and the effect of the efficient transformation of the pentamethylene diamine are achieved. In three stages, namely strain fermented culture, variable-temperature efficient enzyme production and target product rapid transformation, at 25-50 DEG C, a pentamethylene diamine production level reaches 106-116.8g/L and a lysine transformation rate is 91-97% of a theoretical transformation rate.

Description

Technical field: [0001] The invention belongs to the technical field of microbes, in particular to a genetically engineered bacterium capable of efficiently producing pentamethylenediamine and its application. Background technique: [0002] 1,5-Pentanediamine, also known as cadaverine, 1,5-diaminopentane, pentamethylenediamine or cadaverine, is an aliphatic biogenic amine (including spermine, putrescine, One of spermine and pentamethylenediamine, etc.). In 1885, Ludwig Brieger, a doctor in Berlin, Germany, first discovered this amine in a decayed corpse, and named it cadaverine. In the cell, pentamethylenediamine is an elongation reaction of the lysine synthesis pathway, which is produced by decarboxylation of lysine under the action of lysine decarboxylase (E.C.4.1.1.18) (see attached figure 1 ), as a kind of botulinum amine exists in spoilage, and like putrescine, the decarboxylation product of ornithine, is a component of the odor produced by the decay of biological cor...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/00C12R1/19
CPCC12P13/00C12N1/205C12R2001/19
Inventor 田康明路福平王正祥
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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