Corynebacterium glutamicum gene encoding hpr of phosphoenolpyruvate:sugar phosphotransferase system

A coding and allelic technology, applied in the direction of transferase, genetic engineering, plant genetic improvement, etc., can solve problems such as time-consuming and difficult

Inactive Publication Date: 2007-10-17
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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] Example 1: Preparation of all genomic DNA of Corynebacterium glutamicum ATCC 13032

[0156] 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, 2.46 g / 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 / lCoCl 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

[0157] Example 2: Construction of the genomic library of Corynebacterium glutamicum ATCC13032 in Escherichia coli

[0158] 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.

[0159] 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) Gene53:283-286) can be used for special use. A gene library specifically used in Corynebacterium glu...

Embodiment 3

[0160] Example 3: DNA sequencing and computer function analysis

[0161] 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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PUM

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Abstract

Isolated nucleic acid molecules, designated PTS nucleic acid molecules, which encode novel PTS proteins from Corynebacterium glutamicum are described. The 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 invention still further provides isolated PTS proteins, mutated PTS proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C. glutamicum based on genetic engineering of PTS genes in this organism.

Description

[0001] This application is a divisional application of Chinese patent application 00812165.6 "Corynebacterium glutamicum gene encoding phosphoenolpyruvate: carbohydrate phosphotransferase system protein" dated June 27, 2000. Technical field [0002] The present invention relates to an isolated nucleic acid molecule encoding a new Corynebacterium glutamicum PTS protein. The present invention also relates to antisense nucleic acid molecules, recombinant expression vectors containing PTS nucleic acid molecules, and host cells into which expression vectors have been introduced. The present invention also further relates to isolated PTS proteins, PTS mutant proteins, fusion proteins, antigen peptides, and methods for improving the production of desired compounds by the organism based on the genetic engineering of Corynebacterium glutamicum PTS genes. Background technique [0003] Specific products and by-products of metabolic processes that naturally occur in cells have applications in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12P21/02C12P1/04C12NC12P
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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