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Fermentation process of preparing O-acetyl-L-serine

A technology of acetyl and serine, applied in the field of preparation of O-acetyl-L-serine, which can solve the problems of bacterial growth and damage

Inactive Publication Date: 2002-09-25
WACKER CHEM GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] Another serious problem when preparing O-acetyl-L-serine was reported by Dassler et al. (2000, Mol. Microbiol. 36: 1101-1112), namely the overproduction of Orf299 leading to severe impairment of bacterial growth

Method used

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  • Fermentation process of preparing O-acetyl-L-serine

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Experimental program
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Embodiment 1

[0043] To get a more precise idea of ​​the isomerization reaction under conditions close to the fermentation conditions, 0.9 g of O-acetyl-L-serine was introduced into 100 ml of fermentation medium (see Example 3). The pH was then adjusted to 7.0 with 25% ammonia and samples were taken at various times while the reaction temperature was maintained at 32°C. These samples were analyzed by reverse phase HPLC on a LUNA 5[mu] C18(2) column (Phenomenex, Aschaffenburg, Germany). The eluent used was dilute phosphoric acid (0.1 ml concentrated phosphoric acid / l) at a flow rate of 0.5 ml / min. The result is as figure 1 shown. Example 2: Pre-culture of production strains

Embodiment 2

[0044] As a fermented preculture, 20 ml of LB medium (10 g tryptone / l, 5 g yeast extract / l, 10 g NaCl / l), which additionally contained 15 mg Tetracycline / l, cultured with strain w3110 / pACYC184-cysEX-GAPDH-ORF306 (in EP 0885962 A1 corresponding to US Patent Application Serial No. SN 09 / 097759 (which is incorporated herein by reference)). Seven hours later, the entire mixture was transferred to 100 ml SM1 medium (12 g K 2 HPO 4 / l; 3g KH 2 PO 4 / l; 5g (NH 4 ) 2 SO 4 / l; 0.3gMgSO 4 ×7H 2 O / l; 0.015g CaCl 2 ×2H 2 O / l; 0.002g FeSO 4 ×7H 2 O / l; 1g sodium citrate × 2 H 2 O / l; 0.1g NaCl / l; 1ml trace element solution / l, this solution consists of 0.15g NaCl 2 MoO 4 ×2H 2 O / l; 2.5gNa 3 BO 3 / l; 0.7g CoCl 2 ×6H 2 O / l; 0.25g CuSO 4 ×5H 2 O / l; 1.6g MnCl 2 ×4H 2 O / l; 0.3g ZnSO 4 ×7H 2 O / l composition), wherein supplemented with 5g glucose / l; 0.5mg vitamin B1 / l and 15mg tetracycline / l. Subsequent cultivation was performed at 30° C. and 150 rpm for 17 hours. Example ...

Embodiment 3

[0045] The fermentor used was a Biostat M unit supplied by Braun Biotech (Melsungen, Germany) with a maximum culture volume of 2 l. The fermenter contains 900ml fermentation medium (15g glucose / l; 10g tryptone / l; 5g yeast extract / l; 5g (NH 4 ) 2 SO 4 / l; 1.5g KH 2 PO 4 / l; 0.5g NaCl / l; 0.3g MgSO 4 ×7H 2 O / l; 0.015g CaCl 2 ×2H 2 O / l; 0.075gFeSO 4 ×7H 2 O / l; 1g trisodium citrate × 2 H 2 0 / l and 1 ml trace element solution (see above) / l, 5 mg vitamin B1 / l and 15 mg tetracycline / l, with 25% ammonia to adjust the pH to 6.0), the fermentor was used as described in Example 2 Precultures were inoculated (optical density at 600 nm of approximately 3). During the fermentation, the temperature was set at 32° C. and the pH was kept constant at 6.0 by metering in 25% ammonia. Fill the culture with sterile compressed air at a rate of 1.5 vol / vol / min, and the stirring rate of the stirrer at 200 rpm. After the oxygen saturation decreased to 50%, the speed of the stirrer was incre...

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Abstract

The present invention relates to a method for the fermentative preparation of O-acetyl-L-serine, in which a microbial strain is cultured in a fermentation medium, the microbial strain being derived from a wild type and showing increased O compared to the wild type Endogenous formation of -acetyl-L-serine and increased efflux of O-acetyl-L-serine, the method includes setting the pH in the fermentation medium in the range of 5.1 to 6.5.

Description

technical field [0001] The present invention relates to a method for preparing O-acetyl-L-serine by fermentation. Background technique [0002] Fermentation methods currently used for the production of amino acids are extremely common. In particular, these methods are used to prepare representatives of the twenty proteinogenic amino acids, which are highly relevant from an economic point of view, such as L-glutamic acid, L-lysine and L-threonine. However, there are increasing reports on the production methods of proteinogenic amino acids, such as L-phenylalanine and L-cysteine, which occupy a small market of 1,000 to 10,000 tons per year. [0003] In contrast, hardly any corresponding methods are known for the preparation of the twenty proteinogenic amino acid biosynthetic precursors. However, it is these precursors that can represent interesting products, since they often possess chiral centers and can be used as building blocks for the synthesis of pharmaceutically activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/06C12P13/00C12R1/19
CPCC12P13/06
Inventor 托马斯·迈尔托比亚斯·达斯勒奥古斯特·伯克
Owner WACKER CHEM GMBH
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