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Orynebacterium glutamicum genes encoding phosphoenolpyruvate sugar phosphotransferase system proteins

A technology of phosphoenolpyruvate and Corynebacterium glutamicum, which is applied in the directions of transferase, genetic engineering, plant genetic improvement, etc., can solve problems such as time-consuming and difficult

Inactive Publication Date: 2002-09-25
BASF AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, strain screening for improved production of specific molecules is a time-consuming and difficult process

Method used

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  • Orynebacterium glutamicum genes encoding phosphoenolpyruvate sugar phosphotransferase system proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0198] Example 1: Preparation of all genomic DNA of Corynebacterium glutamicum ATCC 13032

[0199] The culture of Corynebacterium glutamicum (ATCC 13032) was cultured overnight in BHI medium (Difco) with vigorous shaking at 30°C. The cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml of buffer I (5% of the original volume of the culture-all indicated volumes are calculated for 100 ml of the culture volume). The composition of buffer I: 140.34g / l sucrose, 2.46g / l MgSO 4 ×7H 2 O, 10ml / l KH 2 PO 4 Solution (100g / l, KOH adjusted to pH 6.7), 50g / l M12 concentrate (10g / l(NH 4 ) 2 SO 4 , 1g / l NaCl, 2g / l MgSO 4 ×7H 2 O, 0.2g / l CaCl 2 , 0.5g / l yeast extract (Difco)), 10ml / l trace element mixture (200mg / l FeSO 4 ×H 2 O, 10mg / l ZnSO 4 ×7H 2 O, 3mg / l MnCl 2 ×4H 2 O, 30mg / l H 3 BO 3 , 20mg / l CoCl 2 ×6H 2 O, 1mg / l NiCl 2 ×6H 2 O, 3mg / l Na 2 MoO 4 ×2H 2 O), 500mg / l complexing agent (EDTA or citric acid), 100ml / l vitamin mixture (0.2mg / l ...

Embodiment 2

[0200] Example 2: Construction of the genomic library of Corynebacterium glutamicum ATCC13032 in E. coli

[0201] Using DNA prepared as described in Example 1, following a known and well established method (see, for example, Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory Manual" Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or Ausubel, FM et al. (1994) "Current Protocols in Molecular Bilogy", John Wiley & Sons.), cosmid libraries and plasmid libraries can be constructed.

[0202] Any plasmid and cosmid can be used. Plasmid pBR322 (Sutcliffe, JG (1979) Proc. Natl. Acad. Sci. USA, 75: 3737-3741); pACY177 (Change & Cohen (1978) J. Bacteriol 134: 1141-1156), pBS series plasmids (pBSSK+, pBSSK- and other plasmids; Stratagene, LaJolla, USA), cosmid SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, TJ, Rosenthal A. and Waterson, RH (1987) Gene 53:283-286) can be used Special Purpose. A gene library specifically used in Corynebacterium glutamicum ...

Embodiment 3

[0203] Example 3: DNA sequencing and computer function analysis

[0204] According to standard methods, using the genomic library as described in Example 2, DNA sequencing can be performed, especially by the chain termination method using the ABI377 sequencer (see, for example, Fleischman, RD et al. (1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269: 496-512). Sequencing primers with the following nucleotide sequence are used: 5'-GGAAACAGTATGACCATG-3' or 5'-GTAAAACGACGGCCAGT-3'.

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Abstract

The present invention describes isolated nucleic acid molecules encoding novel PTS proteins of Corynebacterium glutamicum, which are referred to as PTS nucleic acid molecules. The present invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PTS nucleic acid molecules, and host cells into which the expression vectors have been introduced. The present invention also further provides isolated PTS proteins, PTS muteins, fusion proteins, antigenic peptides, and methods based on Corynebacterium glutamicum PTS genetic engineering to improve the production of desired compounds by this organism.

Description

[0001] Related application [0002] This application requires priority of the U.S. Provisional Patent Application No. 60 / 142,691 filed on July 1, 1999 and the U.S. Provisional Patent Application No. 60 / 150,310 filed on August 23, 1999. Right, the full text of this application is incorporated by reference. This application also claims the priority of the German patent application No.: 19942095.5 filed on September 3, 1999 and the German patent application No.: 19942097.1 filed on September 3, 1999. This application is hereby The full text is cited as a reference. Background of the invention [0003] Specific products and by-products of metabolic processes that naturally occur in cells have applications in many industries, including food, feed, cosmetics and pharmaceutical industries. These molecules are collectively referred to as "fine chemicals" and include organic acids, protein-derived and non-protein-derived amino acids, nucleotides and nucleosides, lipids and fatty acids, glyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/31C12N9/00C12N9/12C12N15/77C12P1/04G01N33/53C12N1/15C12N1/19C12N1/21C12N5/10C12N9/10C12N15/09C12P13/04C12Q1/04C12Q1/68C12R1/13C12R1/16G01N33/566G01N33/569
CPCC12N9/12C12N9/1223C12Y207/03009C12N15/52
Inventor M·波姆佩朱斯B·克雷格尔H·施雷德尔O·策尔德G·哈贝豪尔
Owner BASF AG
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