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Bioconversion method for producing 1,5-pentanediamine

A technology for biotransformation and pentamethylenediamine, which is applied in the field of genetic engineering, can solve the problems of long fermentation period, complex metabolic regulation and low efficiency, and achieve the effects of saving production costs, promoting production fermentation, and improving the utilization rate of carbon sources.

Pending Publication Date: 2020-01-17
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of 1,5-pentanediamine by direct fermentation of glucose by microorganisms requires complex metabolic regulation, long fermentation cycle and low efficiency, and 1,5-pentanediamine is highly toxic to microbial cells, and there is still a lot of work to be done
[0004] It can be seen that the prior art has not carried out metabolic engineering research on how to carry out metabolic engineering transformation on the engineering strain of E. coli that produces 1,5-pentanediamine by one-step fermentation

Method used

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  • Bioconversion method for producing 1,5-pentanediamine
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  • Bioconversion method for producing 1,5-pentanediamine

Examples

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Effect test

preparation example Construction

[0045] (3) Preparation of electroporation competent

[0046] The plasmid Pcas was transferred into 1,5-pentanediamine-producing strain DFC1001 competent cells, and a single colony was picked and inoculated in 5 mL of LB liquid medium, cultured at 30°C, 200 rpm, for 8 hours. Transfer to 50mL LB liquid medium containing 1% inoculum and cultivate to OD 600 About 0.2, add 1.5mL 1mM L-arabinose solution, continue to cultivate to OD 600 0.4 to 0.5; then transferred to a 50mL centrifuge tube for 20 minutes on ice to stop growth. 4°C, 4000rpm, centrifuge for 10min, and collect the bacteria. Wash the cells with 40 mL of pre-cooled sterile water, and collect the cells by centrifugation. Repeat the above steps; wash the cells with 20 mL of pre-cooled 10% glycerol, and collect the cells by centrifugation. Finally, 500 μL of pre-cooled 10% glycerol was used to resuspend the bacterial cells, and the obtained NT1003-cas competent cells were allocated for use;

[0047] (4) Gene knockout ...

Embodiment 1

[0052] Example 1 Knockout of the 1,5-pentanediamine degradation pathway of the production strain

[0053] 1. Knockout of degradation genes

[0054] The sgRNA and homology arms of the four degradation genes puuA, speE, speG and patA were constructed respectively. See Table 2 and Table 3 for primers. The plasmid sg-X and X fragments were transferred into DFC1001-cas competent cells by electroporation, cultured at 30°C, 200rpm for 2h, and coated with 40μg·mL -1 Streptomycin resistance and 50 μg mL -1 Culture overnight at 30°C on kanamycin-resistant LB plates. The colonies in the plate were screened by means of colony PCR, and the PCR primers were X-F1 / X-R2. Three of the positive clones were selected for sequencing, and the correctly sequenced strains were stored in a -80°C refrigerator for later use.

[0055] Table 2 sgRNA-puuA, speE, speG and patA primers

[0056]

[0057] Table 3 PuuA, speE, speG and patA homology arm primers

[0058]

[0059] 2. Fermentation verif...

Embodiment 2

[0064] Example 2 The increase of the production strain 1,5-pentanediamine synthesis precursor lysine

[0065] On the basis of the modified strain in Example 1, the transformation was continued, and the production was further increased by increasing the synthesis of lysine, the precursor of 1,5-pentanediamine synthesis. The specific steps are as follows:

[0066] 1. Knockout of lysine synthesis inhibitory pathway

[0067]The sgRNA and homology arms of pepck and rimL genes were constructed respectively. See Table 4 and Table 5 for primers. The plasmid sg-X and X fragments were transferred into DFC1001-cas competent cells by electroporation, cultured at 30°C, 200rpm for 2h, and coated with 40μg·mL -1 Streptomycin resistance and 50 μg mL -1 Culture overnight at 30°C on kanamycin-resistant LB plates. The colonies in the plate were screened by means of colony PCR, and the PCR primers were X-F1 / X-R2. Three of the positive clones were selected for sequencing, and the correctly se...

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Abstract

The invention belongs to the technical field of genetic engineering, and relates to a bioconversion method for producing 1,5-pentanediamine. The method can increase the yield of 1,5-pentanediamine bymodifying a metabolic pathway of Escherichia coli. The method specifically comprises the following steps: selecting an Escherichia coli strain, firstly, knocking out a 1,5-pentanediamine degradation pathway; secondly, adding a synthetic precursor,namely lysine, of 1,5-pentanediamine; thirdly, overexpressing a transporter gene and finally directly fermenting the Escherichia coli strain DFC1002 to produce 1,5-pentanediamine. After the metabolic pathway of Escherichia coli is modified, themethod can produce 1,5-pentanediamine with high yield.The method is simple and easy to implement, has high yield of 1,5-pentanediamine, few by-products, and good repeatability, and is suitable for industrialization.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a biotransformation method for producing 1,5-pentanediamine. Background technique [0002] 1,5-Pentanediamine, namely cadaverine, is a nitrogenous base with biological activity widely present in organisms, which is produced when lysine undergoes decarboxylation reaction under the action of decarboxylase during protein corruption. 1,5-pentanediamine has many important physiological functions. For example, 1,5-pentanediamine is an "iron-affinity system" that regulates the concentration of iron ions in microbial cells and some strictly anaerobic Gram-negative bacteria. A major building block of sugars; 1,5-pentanediamine also plays an important role in closing microporin channels and protecting E. coli from oxygen toxicity. 1,5-Pentanediamine has a wide range of applications in agriculture, medicine and industry: in agriculture, it can be used to regulate the aging process...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N1/21C12N15/70C12R1/19
CPCC12P13/001C12N9/88C12Y401/01018C12N15/70
Inventor 陈可泉郭兴毛静文许亚飞王昕欧阳平凯
Owner NANJING UNIV OF TECH
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