Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-11-23
2004-03-09
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S252330, C435S320100, C435S091500, C435S350000, C435S091500, C435S023000
Reexamination Certificate
active
06703223
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides nucleotide sequences from Coryneform bacteria which code for the MtrA and/or MtrB proteins and processes for the fermentative preparation of amino acids using bacteria in which the mtrA and/or mtrB genes are attenuated.
2. Discussion of the Background
L-Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
It is known that amino acids are prepared by fermentation from strains of Coryneform bacteria, in particular
Corynebacterium glutamicum
. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce amino acids are obtained in this manner.
Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
However, there remains a critical need for improved methods of producing L-amino acids and thus for the provision of strains of bacteria producing higher amounts of L-amino acids. On a commercial or industrial scale even small improvements in the yield of L-amino acids, or the efficiency of their production, are economically significant. Prior to the present invention, it was not recognized that attenuating the mtrA and/or mtrB genes encoding response regulator and histidine protein kinase proteins, respectively, would improve L-amino acid yields.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel measures for the improved production of L-amino acids or amino acid, where these amino acids include L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine and the salts (monohydrochloride or sulfate) thereof.
One object of the present invention is providing a novel process for improving the fermentative production of said L-amino acids, particularly L-lysine. Such a process includes attenuated bacteria, preferably attenuated Coryneform bacteria, which express attenuated amounts MtrA and/or MtrB proteins or proteins that have response regulator or histidine protein kinase activities.
Thus, another object of the present invention is providing such a bacterium, which expresses an enhanced amount of the MtrA and/or MtrB proteins or gene products of the mtrA and/or mtrB genes.
Another object of the present invention is providing a bacterium, preferably a Coryneform bacterium, which expresses a polypeptide that has an attenuated MtrA response regulator and/or histidine protein kinase activities.
Another object of the invention is to provide a nucleotide sequence encoding a polypeptide having the MtrA and/or MtrB sequences. One embodiment of such a sequence providing both mtrA and mtrB is the nucleotide sequence of SEQ ID NO: 1. Additionally, nucleotides 542 to 1219 of SEQ ID NO:1 comprise the MtrA coding region and nucleotides 1310 to 2818 comprise the MtrB coding region.
A further object of the invention is a method of making proteins or isolated polypeptides having MtrA response regulator and/or histidine protein kinase activities, as well as use of such isolated polypeptides in the production of amino acids. One embodiment of the MtrA polypeptide is the polypeptide having the amino acid sequence of SEQ ID NO: 2. One embodiment of the MtrB polypeptide is the polypeptide having the amino acid sequence of SEQ ID NO:3.
Other objects of the invention include methods of detecting nucleic acid sequences homologous to SEQ ID NO: 1, particularly nucleic acid sequences encoding polypeptides that have MtrA response regulator and/or histidine protein kinase activities, and methods of making nucleic acids encoding such polypeptides. The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention.
REFERENCES:
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Bathe Brigitte
Bott Michael
Schaffer Steffen
Schischka Natalie
Achutamurthy Ponnathapu
Degussa - AG
Fronda Christian L.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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