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Mutant microorganism with high ability of producing putrescine and preparation of putrescine using same

A technology of microorganisms and putrescine, applied in biochemical equipment and methods, botany equipment and methods, applications, etc., can solve problems such as complex materials, slowing down of putrescine biosynthesis, and induction of putrescine degradation

Active Publication Date: 2010-03-24
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the disclosed material is very complex, there is a problem that many purification steps must be carried out in order to obtain putrescine and cadaverine products
However, when the putrescine content increases due to the increase of ornithine decarboxylase, there is a problem in that the biosynthesis of putrescine is slowed down, and the degradation of putrescine is induced (Igarashi and Kashiwagi et al., Biochem.J., 347: 297-303, 2000)

Method used

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  • Mutant microorganism with high ability of producing putrescine and preparation of putrescine using same
  • Mutant microorganism with high ability of producing putrescine and preparation of putrescine using same
  • Mutant microorganism with high ability of producing putrescine and preparation of putrescine using same

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1: Preparation of mutant microorganisms in which genes involved in putrescine degradation or utilization pathways are removed

[0051] In the present invention, genes (puuA, puuP, ygjG, speE, speG, argF and argI) were removed. The lox71-chloramphenicol marker (CmR)-lox66 cassette was prepared by PCR using a primer containing 50 nucleotides homologous to the upstream and downstream of the target gene. pECmulox (Kim, J.M., Lee, K.H. & Lee, S.Y., FEMS Microbiol. Lett., 278:78-85, 2008) including the lox71-CmR-lox66 cassette was used as template in the PCR reaction. The PCR product was transformed into electrocompetent cells containing λ recombinase. Colonies were selected on Luria-Bertani (LB) agar plates containing 34 μg / ml chloramphenicol (Cm) (Sambrook, J., Fritsch E.F., & Maniatis, T., Molecular cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2000). use cm RSuccessful gene replacement was confirmed by direct cloning PCR. An...

Embodiment 2

[0089] Embodiment 2: the replacement of promoter

[0090] In order to improve the ability to produce putrescine, the promoter of the mutant strain (XQ26) prepared in Example 1 was replaced with a strong promoter (trc).

[0091] 2-1: Preparation of XQ33 strain

[0092]Replacement of the promoter of the argECBH operon itself with the trc promoter was performed in the following manner. The fused lox71-chloramphenicol marker antibiotic marker-lox66 DNA fragment was generated by the first PCR reaction using SEQ ID NOs 18 and 19 as primers and pECmulox as template.

[0093] Sequence 18: 5'-TATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACAGCTGACACTATAGAACGCGGCCG-3'

[0094] Sequence 19: 5'-TATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACAGCTCCGCATAGGCCACTAGTGGA-3'

[0095] In order to induce the trc promoter, use sequence numbers: 20 and 21 as primers and use the first PCR product as a template for the second PCR reaction. To introduce homologous regions into the final PCR ...

Embodiment 3

[0128] Example 3: Preparation of putrescine using mutant microorganisms

[0129]Each of the mutant strains (E.coli K12 WL3110 mutant) in Table 1 prepared in Examples 1 and 2 contained 4g / L (NH 4 ) 2 HPO 4 , 13.5g / L KH 2 PO 4 , 1.7g / L citric acid, 0.7g / LMgSO 4 ·7H 2 O and 0.5% (v / v) trace metal solutions were cultured in a small amount of R medium (Lee, S.Y. & Chang, H.N., Biotechnol. Lett., 15:971-974, 1993). Trace metal solution contains (per liter): 5M HCl, 10g FeSO 4 ·7H 2 O, 2.25g ZnSO 4 ·7H 2 O, 1gCuSO 4 ·5H 2 O, 0.5g MnSO 4 ·5H 2 O, 0.23g Na 2 B 4 o 7 10H 2 O, 2g CaCl 2 2H 2 O and 0.1g (NH 4 ) 6 Mo 7 o 24 . Solutions containing glucose (100 g / l) were sterilized separately and added to the sterile medium to a final concentration of 10 g / l.

[0130] Inoculate 100 μL of each cell culture activated in LB medium into minimal medium, then culture at 220 rpm at 30 °C for 24 h until the maximum OD 600 reach 5. Then, 1 ml of the culture solution was add...

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Abstract

The present invention relates to a mutant microorganism having a high ability of producing putrescine in which genes associated with the decomposition or use pathway of putrescine are weakened or deleted, in a microorganism having a putrescine production metabolic pathway, and to a method for preparing putrescine with a high yield rate by culturing the mutant microorganism under an anaerobic condition. The mutant microorganism having a high ability of producing putrescine according to the present invention is useful for the high-yield production of putrescine and has broad industrial application.

Description

technical field [0001] The present invention relates to a mutant microorganism with high putrescine production capacity and a method for using the microorganism to produce putrescine, in particular to a mutant microorganism with high putrescine production capacity, wherein the genes involved in the putrescine degradation or utilization pathway are inactivated or removal, and a method for cultivating the mutant microorganism to produce putrescine in high yield under anaerobic conditions. Background technique [0002] Putrescine (or 1,4-butylene diamine), is an important raw material for the production of polyamine-4,6 including nylon-4,6, acrylonitrile produced mainly by adding hydrocyanic acid to acrylonitrile produced on an industrial scale by hydrogenation. The known chemical synthesis processes of this compound require non-renewable petrochemical products as raw materials, and rather severe reaction conditions of temperature and pressure in multiple steps and multiple re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00
CPCC12P13/001
Inventor 李相烨钱志刚夏晓霞全勇宰
Owner KOREA ADVANCED INST OF SCI & TECH
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