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Corynebacterium glutamicum genes encoding phosphoenolpyruvate: sugar phosphotransferase system proteins

a technology of sugar phosphotransferase and corynebacterium glutamicum, which is applied in the direction of transferases, peptides, bacteria based processes, etc., can solve the problems of time-consuming and difficult process of selecting strains for the production of a particular molecule, and achieve the optimization of activity, increase the quantity of glucose uptake or the rate at which glucose is translocated into the cell, and improve the yield

Inactive Publication Date: 2005-09-01
BASF SE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004]C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The PTS nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g., by fermentation processes. Modulation of the expression of the PTS nucleic acids of the invention, or modification of the sequence of the PTS nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g., to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).
[0008] The PTS molecules of the invention may be modified such that the yield, production, and / or efficiency of production of one or more fine chemicals is improved. For example, by modifying a PTS protein involved in the uptake of glucose such that it is optimized in activity, the quantity of glucose uptake or the rate at which glucose is translocated into the cell may be increased. The breakdown of glucose and other sugars within the cell provides energy that may be used to drive energetically unfavorable biochemical reactions, such as those involved in the biosynthesis of fine chemicals. This breakdown also provides intermediate and precursor molecules necessary for the biosynthesis of certain fine chemicals, such as amino acids, vitamins and cofactors. By increasing the amount of intracellular high-energy carbon molecules through modification of the PTS molecules of the invention, one may therefore increase both the energy available to perform metabolic pathways necessary for the production of one or more fine chemicals, and also the intracellular pools of metabolites necessary for such production.
[0009] Further, the PTS molecules of the invention may be involved in one or more intracellular signal transduction pathways which may affect the yields and / or rate of production of one or more fine chemical from C. glutamicum. For example, proteins necessary for the import of one or more sugars from the extracellular medium (e.g., HPr, Enzyme I, or a member of an Enzyme II complex) are frequently posttranslationally modified upon the presence of a sufficient quantity of the sugar in the cell, such that they are no longer able to import that sugar. While this quantity of sugar at which the transport system is shut off may be sufficient to sustain the normal functioning of the cell, it may be limiting for the overproduction of the desired fine chemical. Thus, it may be desirable to modify the PTS proteins of the invention such that they are no longer responsive to such negative regulation, thereby permitting greater intracellular concentrations of one or more sugars to be achieved, and, by extension, more efficient production or greater yields of one or more fine chemicals from organisms containing such mutant PTS proteins.
[0021] The PTS polypeptide, or a biologically active portion thereof, can be operatively linked to a non-PTS polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of the PTS protein alone. In other preferred embodiments, this fusion protein results in increased yields, production, and / or efficiency of production of a desired fine chemical from C. glutamicum. In particularly preferred embodiments, integration of this fusion protein into a host cell modulates the production of a desired compound from the cell.
[0024] Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates PTS protein activity or PTS nucleic acid expression such that a cell associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for the uptake of one or more sugars, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates PTS protein activity can be an agent which stimulates PTS protein activity or PTS nucleic acid expression. Examples of agents which stimulate PTS protein activity or PTS nucleic acid expression include small molecules, active PTS proteins, and nucleic acids encoding PTS proteins that have been introduced into the cell. Examples of agents which inhibit PTS activity or expression include small molecules, and antisense PTS nucleic acid molecules.

Problems solved by technology

However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Total Genomic DNA of Corynebacterium glutamicum ATCC 13032

[0126] A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30° C. with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I (5% of the original volume of the culture—all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g / l sucrose, 2.46 g / l MgSO4×7H2O, 10 ml / l KH2PO4 solution (100 g / l, adjusted to pH 6.7 with KOH), 50 ml / l M12 concentrate (10 g / l (NH4)2SO4, 1 g / l NaCl, 2 g / l MgSO4×7H2O, 0.2 g / l CaCl2, 0.5 g / l yeast extract (Difco), 10 ml / l trace-elements-mix (200 mg / l FeSO4×H2O, 10 mg / l ZnSO4×7 H2O, 3 mg / l MnCl2×4 H2O, 30 mg / l H3BO3 20 mg / l CoCl2×6 H2O, 1 mg / l NiCl2×6 H2O, 3 mg / l Na2MoO4×2 H2O, 500 mg / l complexing agent (EDTA or critic acid), 100 ml / l vitamins-mix (0.2 mg / l biotin, 0.2 mg / l folic acid, 20 mg / l p-amino benzoic a...

example 2

Construction of Genomic Libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032.

[0127] Using DNA prepared as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see e.g., Sambrook, J. et al. (1 989) “Molecular Cloning : A Laboratory Manual”, Cold Spring Harbor Laboratory Press, or Ausubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley & Sons.)

[0128] Any plasmid or cosmid could be used. Of particular use were the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK− and others; Stratagene, LaJolla, USA), or cosmids as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J., Rosenthal A. and Waterson, R. H. (1987) Gene 53:283-286. Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL109 (Lee, H.-S. and ...

example 3

DNA Sequencing and Computational Functional Analysis

[0129] Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R. D. et al. (1995) “Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-512). Sequencing primers with the following nucleotide sequences were used:

5′-GGAAACAGTATGACCATG-3′or5′-GTAAAACGACGGCCAGT-3′.

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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

RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 60 / 142,691, filed on Jul. 1, 1999, and also to U.S. Provisional Patent Application No. 60 / 150,310, filed on Aug. 23, 1999, incorporated herein in their entirety by this reference. This application also claims priority to German Patent Application No. 19942095.5, filed on Sep. 3, 1999, and also to German Patent Application No. 19942097.1, filed on Sep. 3, 1999, incorporated herein in their entirety by this reference.BACKGROUND OF THE INVENTION [0002] Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and pharmaceutical industries. These molecules, collectively termed ‘fine chemicals’, include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and co...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/04C12N1/12C12N1/20C12N1/21C12N9/00C12N9/10C12N9/12C12N15/00C12N15/74C12P1/04C12P13/04C12P21/06
CPCC07K14/34C12N9/00C12N9/1205C12N9/1223C12P1/04C12Y207/01069C12P13/04C12R1/15C12R2001/15C12N1/205
Inventor POMPEJUS, MARKUSKROGER, BURKHARDSCHRODER, HARTWIGZELDER, OSKARHABERHAUER, GREGOR
Owner BASF SE
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