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Method for fermentative production of menaquinone-7 using escherichia coli

A technology of Escherichia coli and menadione, applied in the field of fermentative production of menadione-7, can solve the problems of description, no identification, no biochemical angle, etc.

Inactive Publication Date: 2011-08-31
WACKER CHEM GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although heptaprenyltransferase is a key enzyme that transfers heptaprenyl units to DHNA, this enzyme is neither genetically identified nor biochemically described

Method used

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  • Method for fermentative production of menaquinone-7 using escherichia coli
  • Method for fermentative production of menaquinone-7 using escherichia coli
  • Method for fermentative production of menaquinone-7 using escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: Construction of pKG82 plasmid

[0063] a) Amplification of the hepS gene:

[0064] The Bacillus subtilis (DSM 1088) hepS gene can be amplified by polymerase chain reaction (PCR) using Taq DNA polymerase (Roche, Mannheim, Germany) according to general techniques well known to those skilled in the art. The template used was the chromosomal DNA of Bacillus subtilis strain DSM1088. The primers used were M7-operon-hepS-forward oligonucleotide (SEQ ID NO 4) and M7-operon-hepS-reverse oligonucleotide (SEQ ID NO 5).

[0065] According to the information provided by the manufacturer, the DNA fragment of 783 base pairs obtained in PCR can be purified with DNA absorption column QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany).

[0066] b) Amplification of the hepT gene:

[0067] The Bacillus subtilis (DSM 1088) hepT gene can be amplified by polymerase chain reaction (PCR) using Taq DNA polymerase (Roche, Mannheim, Germany) according to techniques well known to ...

Embodiment 2

[0083] Example 2: Preparation of Menaquinone-7 Production Strain

[0084] by CaCl 2 The pKG82 plasmid in Example 1 can be used for the transformation of E. coli strain W3110 (ATCC 27325). After screening on LB agar plates containing 20 mg / L tetracycline, the plasmid was re-isolated from a transformant, cleaved with restriction endonuclease and detected. This strain, called W3110 / pKG82, can be used to produce menaquinone-7.

Embodiment 3

[0085] Example 3: Initial pre-culture of menaquinone-7 producing strain for fermentation (day culture)

[0086] Mix 20 ml of glucose-containing LB broth (10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L glucose; high pressure treatment) with tetracycline HCl in a 100 ml sterile cone. (final tetracycline hydrochloride concentration: 15 mg / L; stock solution of tetracycline hydrochloride: 10 mg / ml in sterile filtered 50% ethanol).

[0087] The cells of the producer strain were removed from the agar plate with an inoculating loop before the broth began to appear slightly cloudy. Pre-cultures were performed independently at 32°C (strain W3110 and strain W3110 / pKG82) and 30°C (strain DSM 1088), respectively, while spinning at 150 rpm for 8 h.

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Abstract

The invention relates to a method of producing menaquinone-7, characterized in that cells of an E. coli strain comprising the B. subtilis DSM 1088 hepS gene, the B. subtilis DSM 1088 hepT gene and the putative B. subtilis DSM 1088 heptaprenyl transferase gene are fermented in a fermentation medium, with menaquinone-7 being accumulated in the cells of the fermented E. coli strain.

Description

technical field [0001] The invention relates to a method for fermenting and producing menadione-7 by using Escherichia coli. Background technique [0002] Menadione-7 is part of the vitamin K family. Menaquinones in human nutrition are mainly derived from intestinal bacteria such as Lactobacillus acidophilus or Escherichia coli. Biochemically, menadiones are involved in the carboxylation of specific glutamic acid residues to produce γ-carboxyglutamic acid (GLA). A major protein containing three GLA residues is osteocalcin. Osteocalcin is formed in osteoblasts and makes up 15-20% of the non-collagenous proteins in bone. Vitamin K deficiency can lead to non-carboxylated osteocalcin entering the plasma, which becomes an important indicator of bone metabolism disorders. [0003] Studies have shown that menadione-7 supplements have both the effect of promoting bone formation and the effect of preventing atherosclerosis in humans. [0004] For example, one study showed that r...

Claims

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Application Information

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IPC IPC(8): C12P7/66C12N15/70C12N1/21C12R1/19
CPCC12P7/66C12N9/1085
Inventor 托马斯·施勒泽尔
Owner WACKER CHEM GMBH
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