Nucleotide sequences which code for the rpoB gene

Details Nucleotide sequences which code for the rpoB gene Nucleotide sequences which code for the rpoB gene

2001-06-25

2004-08-31

Hutson, Richard (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S194000, C435S183000, C435S320100, C435S252300, C435S252320, C536S023100, C536S023200, C536S023700, C536S024300

Reexamination Certificate

active

06783967

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATION
The present application claims priority to German Application No. DE10107229.5 filed Feb. 16, 2001, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to polynucleotides corresponding to the rpoB gene and which encode the &bgr;-subunit of RNA polymerase B, methods of producing L-amino acids, and methods of screening for polynucleotides which encode proteins having activity of the &bgr;-subunit of RNA polymerase B.
2. Discussion of the Background
L-amino acids, especially L-lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and, very especially, in the feeding of animals.
It is known that amino acids are produced by fermentation of strains of coryneform bacteria, especially
Corynebacterium glutamicum.
Because of their great importance, attempts are continuously being made to improve the production processes. Improvements to the processes may concern measures relating to the fermentation, such as, for example, stirring and oxygen supply, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or working up to the product form by, for example, ion-exchange chromatography, or the intrinsic performance properties of the microorganism itself.
In order to improve the performance properties of such microorganisms, methods of mutagenesis, selection and mutant selection are employed. Such methods yield strains which are resistant to antimetabolites or are auxotrophic for metabolites that are important in terms of regulation, and which produce amino acids.
For a number of years, methods of recombinant DNA technology have also been used for improving the strain of L-amino acid-producing strains of Corynebacterium, by amplifying individual amino acid biosynthesis genes and studying the effect on 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 enhancement of the rpoB gene encoding the &bgr;-subunit of RNA polymerase B would improve L-amino acid yields.
SUMMARY OF THE INVENTION
One object of the present invention, is providing a new process adjuvant for improving the fermentative production of L-amino acids, particularly L-lysine and L-glutamate. Such process adjuvants include enhanced bacteria, preferably enhanced Coryneform bacteria which express enhanced levels of the &bgr;-subunit of RNA polymerase B which is encoded by the rpoB gene.
Thus, another object of the present invention is providing such a bacterium, which expresses enhanced amounts of the &bgr;-subunit of RNA polymerase B or gene products of the rpoB gene.
Another object of the present invention is providing a bacterium, preferably a Coryneform bacterium, which expresses a polypeptide that has an enhanced &bgr;-subunit of RNA polymerase B activity.
Another object of the invention is to provide a nucleotide sequence encoding a polypeptide which has a &bgr;-subunit of RNA polymerase B sequence. One embodiment of such a sequence is the nucleotide sequence of SEQ ID NO: 1. Other embodiments of such a sequence is the nucleotide sequences of SEQ ID NOS:3 and 5.
A further object of the invention is a method of making a &bgr;-subunit of RNA polymerase B or an isolated polypeptide having a &bgr;-subunit of RNA polymerase B activity, as well as use of such isolated polypeptides in the production of amino acids. One embodiment of such a polypeptide is the polypeptide having the amino acid sequence of SEQ ID NO: 2. Other embodiments of such a sequence is the amino acid sequence of SEQ ID NOS:4 and 6.
In one embodiment the invention provides isolated polypeptides comprising the amino acid sequences in SEQ ID NOS:2, 4 and/or 6.
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 the activity of a &bgr;-subunit of RNA polymerase B, 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.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and the various references cited therein.
Where L-amino acids or amino acids are mentioned hereinbelow, they are to be understood as meaning one or more amino acids, including their salts, selected from the group 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 and L-arginine. L-lysine is especially preferred.
Where L-lysine or lysine is mentioned hereinbelow, it is to be understood as meaning not only the bases but also the salts, such as, for example, lysine monohydrochloride or lysine sulfate.
The invention provides an isolated polynucleotide from coryneform bacteria, containing a polynucleotide sequence coding for the rpoB gene, selected from the group
a) polynucleotide that is at least 70% identical with a polynucleotide that codes for a polypeptide containing the amino acid sequence of SEQ ID No. 2,
b) polynucleotide that codes for a polypeptide containing an amino acid sequence that is at least 70% identical with the amino acid sequence of SEQ ID No. 2,
c) polynucleotide that is complementary to the polynucleotides of a) or b), and
d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c),
the polypeptide preferably exhibiting the activity of the &bgr;-subunit of RNA polymerase B.
The invention also provides the above-mentioned polynucleotide, it preferably being a replicatable DNA containing:
(i) the nucleotide sequence shown in SEQ ID No. 1, or
(ii) at least one sequence that corresponds to sequence (i) within the region of the degeneracy of the genetic code, or
(iii) at least one sequence that hybridizes with the sequence that is complementary to sequence (i) or (ii), and optionally
(iv) sense mutations in (i) which are neutral in terms of function and which do not change the activity of the protein/polypeptide.
Finally, the invention also provides polynucleotides selected from the group
a) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 1 and 701
b) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 702 and 4199
c) polynucleot

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