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Genetically engineered bacteria and application of genetically engineered bacteria in production of 1,5-pentanediamine

A technology of genetically engineered bacteria and uses, applied in the field of biocatalysis, can solve problems such as low synthesis efficiency and inability to meet, and achieve the effects of high bacterial activity, normal growth, and efficient synthesis.

Inactive Publication Date: 2016-11-23
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, it also shows that the PLP synthesized by the strain itself cannot meet the needs of lysine decarboxylase to efficiently convert L-lysine into 1,5-pentanediamine
Part of the reason is that the synthesis of PLP in Escherichia coli mainly relies on the 1-deoxy-5-phosphate xylulose pathway, which requires seven enzymes to participate, and the synthesis efficiency is low; in comparison, there is another ribose-5-phosphate-dependent pathway in organisms The PLP synthesis pathway of phosphate, which only needs two enzymes to participate in the one-step synthesis of PLP by utilizing ribose-5-phosphate, glyceraldehyde-3-phosphate and glutamine that are abundantly present in cells, and this pathway is widely distributed in including In fungi, plants and some prokaryotic cells, the enzymes involved in PLP synthesis are YaaD (also known as PdxS, Pdx1 or SnzP) and YaaE (also known as PdxT, Pdx2, or SnoP)

Method used

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  • Genetically engineered bacteria and application of genetically engineered bacteria in production of 1,5-pentanediamine
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  • Genetically engineered bacteria and application of genetically engineered bacteria in production of 1,5-pentanediamine

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Construction of 1,5-pentanediamine-producing strain

[0037] Bacillus subtilis Bacillus subtilis (GenBank: AL009126.3)

[0038] Primers designed for the pyridoxal phosphate synthase gene sequence:

[0039] YaaDE-NcoI-F: CATGccATGGCTCAAACAGGTACTGAACG;

[0040] YaaDE-SalI-R: ACGCGTCGACTTATACAAGTGCCTTTTGCTTATATTCCTCAACC.

[0041] The yaaDE gene obtained by PCR was cloned into the plasmid pRSF plasmid, and the plasmid pRSF-yaaDE ( figure 1 ). The yaaDE nucleotide sequence is as SEQ ID NO: 1, the plasmid pETDuet-pelB-CadB-CadA (see the inventor's published patent application literature: the title of the invention is an expression recombinant vector and its application, application publication number CN104498519A) and pRSF-yaaDE was introduced into Escherichia coli BL21 (DE3) by conventional methods, and the strain was named BL-BADE, and was preserved in the form of glycerol bacteria or freeze-dried strains.

Embodiment 2

[0042] Example 2 Effect of exogenous addition of pyridoxal phosphate (PLP) on the synthesis of 1,5-pentanediamine

[0043] Pick BL-DAB (see the inventor's published patent application literature: the title of the invention is an expression recombinant vector and its application, application publication number CN104498519A) single colony in 5ml LB liquid medium containing 100 ug / ml ampicillin , 37°C, 200 rpm overnight to activate the strain. The activated strains were transferred to 50 ml liquid fermentation medium containing corresponding antibiotics (100 ug / ml ampicillin) at 1% inoculum, and cultured at 37°C and 200 rpm until the OD600 was about 0.5. Add IPTG to a final concentration of 0.5 mM, induce culture at 30 °C, 200 rpm for 12 hours, and then collect the bacterial cells by centrifugation at 4000 x g, wash twice with 0.9% sodium chloride solution, and resuspend with the transformation reaction solution to a cell concentration of 4 g ( DCW) / l, the conversion reaction so...

Embodiment 3

[0046] Example 3 Transformation of genetically engineered bacteria to produce 1,5-pentanediamine

[0047] Pick a single colony of BL-BADE and place it in 5ml LB liquid medium containing 100 ug / ml ampicillin, activate the strain overnight at 37°C and 200 rpm. The activated strains were transferred to 50 ml liquid fermentation medium containing corresponding antibiotics (100 ug / ml ampicillin, 170 μg / ml chloramphenicol) at 1% inoculum, and cultivated at 37°C and 200 rpm until the OD600 was about 0.5. Add IPTG to a final concentration of 0.5 mM, induce culture at 30 °C, 200 rpm for 12 hours, and then collect the bacterial cells by centrifugation at 4000 x g. Take 10 mg (dry weight) of bacterial cells to detect the intracellular PLP content. The remaining bacteria were washed twice with 0.9% sodium chloride solution, and then resuspended with the transformation reaction solution to a cell concentration of 2 g (DCW) / l. The transformation reaction solution contained L-lysine hydroch...

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Abstract

The invention relates to the technical field of microorganisms, in particular to genetically engineered bacteria for increasing synthesis of PLP (pyridoxal phosphate) in escherichia coli mycetocyte and a method for producing 1,5-pentanediamine from the genetically engineered bacteria. A gene engineering technique is utilized, a key gene for synthesis of PLP through ribose-5-phosphate is subjected to heterologous expression in escherichia coli, and an efficient conversion production strain of 1,5-pentanediamine is established. Under the condition that PLP is not externally added, the genetically engineered bacteria can be converted within 4 h, and the content of produced 1,5-pentanediamine reaches 250 g / l and is far higher than that reported in the prior art.

Description

technical field [0001] The invention relates to the technical field of biocatalysis, in particular to a genetically engineered bacterium that increases the synthesis of pyridoxal phosphate (PLP) in Escherichia coli and its use in the production of 1,5-pentanediamine. Background technique [0002] 1,5-Pentanediamine (pentamethylenediamine for short), that is, cadaverine, is a nitrogenous base with biological activity that exists widely in organisms. It is produced when lysine undergoes decarboxylation under the action of decarboxylase when protein decays. product. In agriculture, 1,5-pentanediamine can be used to regulate the aging process of plants, promote the development of pistils, improve the development of plant fruits, and increase fruit yield; in medicine, it can also be used as an effective drug ingredient for treating dysentery; in industry It is an extremely important chemical raw material. It can be polymerized with dibasic acids such as adipic acid, succinic aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N15/70C12P13/001C12N2800/101
Inventor 陈可泉马伟超齐雁斌何育美沈金山欧阳平凯
Owner NANJING UNIV OF TECH
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