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Microorganism producing O-acetyl-homoserine and the method of producing O-acetyl-homoserine using the microorganism

An acetyl and amino acid technology, applied in the field of microbial strains, can solve the problems of reduced catalytic activity and achieve high environmental friendliness

Active Publication Date: 2011-04-06
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It was found that in the presence of 40 mM CoA, the wild-type protein retained only about 20% of its catalytic activity, whereas no reduction in the catalytic activity of the R106A mutant protein was detected at the same concentration of CoA

Method used

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  • Microorganism producing O-acetyl-homoserine and the method of producing O-acetyl-homoserine using the microorganism
  • Microorganism producing O-acetyl-homoserine and the method of producing O-acetyl-homoserine using the microorganism
  • Microorganism producing O-acetyl-homoserine and the method of producing O-acetyl-homoserine using the microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Preparation of O-acetylhomoserine-producing strain

[0058] Cloning of acs gene

[0059] For the cloning of the acs gene, the acs gene encoding acetyl-CoA synthase was amplified by PCR using the genomic DNA of E. coli W3110 (ATCC 27325) as a template. The base sequence of the acs gene was obtained from the GenBank database of NIH (NCBI-gi: 89110790), and represented as SEQ ID NO.7. On the basis of the base sequence, primer pairs (SEQ ID NOS. 1 and 2) containing selective restriction sites EcoRV and HindIII were used, and the genomic DNA of E. PfuUltra TM The ORF from ATG to TAA was amplified by PCR in the presence of (Stratagene). The PCR included 30 cycles of denaturation at 96°C for 30 seconds, annealing at 50°C for 30 seconds and extension at 72°C for 2 minutes to synthesize an approximately 2.0 kb acs gene containing EcoRV and HindIII sites.

[0060] After digestion with restriction enzymes EcoRV and HindIII, the amplified acs gene was ligated into...

Embodiment 2

[0069] Example 2: Fermentation to produce O-acetylhomoserine

[0070] In order to examine the ability of the strains prepared in Example 1 to produce the methionine precursor O-acetylhomoserine, these strains were grown in Erlenmeyer flasks.

[0071] For this cultivation, the O-acetylhomoserine titer medium shown in Table 1 was used.

[0072] Table 1

[0073] Composition of the medium for the production of O-acetylhomoserine

[0074] composition

Concentration ( / L)

glucose

60g

Ammonium sulfate

17g

KH 2 PO 4

1.0g

MgSO 4 ·7H 2 O

0.5g

FeSO 4 ·7H 2 O

5mg

[0075] MnSO 4 ·8H 2 O

5mg

ZnSO 4

5mg

CaCO 3

30g

Yeast extract

2g

Methionine

0.15g

Threonine

0.15g

[0076] Single colonies produced on LB plates cultured overnight at 32°C were taken out using platinum rings, inoculated into 25 mL of O-acetylhomoseri...

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Abstract

Disclosed herein are a microorganism strain capable of producing the L-methionine precursor O-acetyl homoserine in high yield and a method of producing O-acetyl homoserine using the same. The microorganism strain is a strain of Escherichia sp. in which an acetyl-CoA synthase gene (acs) and / or a pantothenate kinase gene (coA) encoding a pantothenate kinase gene refractory to feedback inhibition by CoA is introduced and expressed.

Description

technical field [0001] The present invention relates to microbial strains capable of producing O-acetylhomoserine (L-methionine precursor) in high yield. In addition, the present invention also relates to a method for producing O-acetylhomoserine in high yield using the microorganism strain. Background technique [0002] Methionine used in animal feed, food and medicine can be synthesized chemically or biologically. [0003] In chemical synthesis, methionine is basically produced by hydrolysis of 5-(β-methylmercaptoethyl)hydantoin. However, synthetic methionine has the disadvantage of being present as a mixture of L- and D-forms, which requires difficult additional methods to separate them from each other. In order to solve this problem, the inventors of the present invention developed a biological method for selectively synthesizing L-methionine, which is a chemical substance claimed in an existing patent application (WO 2008 / 103432). This method (referred to as "two-ste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/04C12R1/185C12R1/19
CPCC12N9/93C12P13/06C12Y602/01001C12Y207/01033C12N9/1205
Inventor 金素影申容旭许仁庚金贤雅徐昌一金朱恩孙晟光李相穆全成后李汉珍罗光镐
Owner CJ CHEILJEDANG CORP
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