Nucleotide sequences encoding the ptsH gene

Details Nucleotide sequences encoding the ptsH gene Nucleotide sequences encoding the ptsH gene

2001-03-29

2004-11-16

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S069100, C435S252320, C435S320100, C536S023200

Reexamination Certificate

active

06818432

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides nucleotide sequences coding for ptsH and processes for the fermentative preparation of L-amino acids, particularly L-lysine, in which the ptsH gene is enhanced, using coryneform bacteria.
2. Background Information
L-amino acids, particularly L-lysine, are used in human medicine and in the pharmaceutical industry, and particularly in animal nutrition.
It is known to prepare L-amino acids by fermentation of strains of coryneform bacteria, particularly
Corynebacterium glutamicum
. In view of the great importance, work is constantly being carried out to improve the preparation processes. Process improvements may relate to measures involving the fermentation technique, such as, e.g., agitation and oxygen supply, or the composition of the nutrient media such as, e.g., the sugar concentration during fermentation, or the work up to the product form by, e.g., ion exchange chromatography, or the intrinsic performance properties of the microorganism itself.
In order to improve the performance properties of said microorganisms, methods of mutagenesis, selection and mutant selection are employed. Strains thereby obtained are resistant to antimetabolites such as, e.g., the lysine analogue S-(2-aminoethyl) cysteine, or auxotrophic for metabolites of regulatory importance and produce L-lysine.
For some years, methods of recombinant DNA technology have also been used to improve strains of coryneform bacteria producing L-amino acids by amplifying individual biosynthesis genes for L-amino acids and examining the effect on L-amino acid production. Review articles on this subject may be found inter alia in Kinoshita (“Glutamic Acid Bacteria”, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
SUMMARY OF THE INVENTION
Object of the Invention
The inventors set themselves the task of providing new measures for the improved fermentative preparation of L-amino acids, particularly L-lysine.
DESCRIPTION OF THE INVENTION
L-amino acids, particularly L-lysine, are used in human medicine, in the pharmaceutical industry and particularly in animal nutrition. It is of general interest, therefore, to provide new improved processes for the preparation of L-amino acids, particularly L-lysine.
Where the terms L-lysine or lysine are mentioned below, they refer not only to the base but also to the salts such as, e.g., lysine monohydrochloride or lysine sulfate.
The invention provides an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group comprising
a) polynucleotide which is at least 70% identical to a polynucleotide coding for a polypeptide which contains the amino acid sequence of SEQ ID no. 2,
b) polynucleotide which codes for a polypeptide containing an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no.2,
c) polynucleotide which is complementary to the polynucleotides of a) or b), and
d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c).
The invention also provides a polynucleotide which is a DNA, preferably recombinant, which can be replicated in coryneform bacteria.
The invention also provides a polynucleotide which is an RNA.
The invention also provides a polynucleotide which is preferably a replicable DNA containing:
(i) the nucleotide sequence shown in SEQ ID no.1, or
(ii) at least one sequence which corresponds to the sequence (i) within the degeneracy region of the genetic code, or
(iii) at least one sequence which hybridises with the sequence complementary to sequence (i) or (ii), and optionally
(iv) functionally neutral sense mutations in (i).
The invention also provides
a vector containing one of the polynucleotides mentioned, and coryneform bacteria acting as host cell which contain the vector.
The invention also provides polynucleotides comprising substantially a polynucleotide sequence which may be obtained by screening by hybridising an appropriate gene bank containing the complete gene with the polynucleotide sequence corresponding to SEQ ID no. 1, with a probe which contains the sequence of the above-mentioned polynucleotide according to SEQ ID no. 1 or a fragment thereof, and isolating the DNA sequence mentioned.
Polynucleotide sequences according to the invention are suitable as hybridisation probes for RNA, cDNA and DNA, for isolating full-length cDNA which code for component H of the phosphotransferase system (ptsH) and for isolating those cDNA or genes which have great similarity of sequence with that of the gene for component H of the phosphotransferase system.
Polynucleotide sequences according to the invention are also suitable as primers for the preparation of DNA of genes which code for component H of the phosphotransferase system by the polymerase chain reaction (PCR).
The oligonucleotides acting as probes or primers contain at least 30, preferably at least 20, more particularly preferably at least 15 successive nucleotides. Oligonucleotides with a length of at least 40 or 50 nucleotides are also suitable.
“Isolated” means separated from its natural surroundings.
“Polynucleotide” refers generally to polyribonucleotides and polydeoxyribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.
The term “polypeptides” means peptides or proteins which contain two or more amino acids bound by way of peptide bonds.
The polypeptides according to the invention include a polypeptide according to SEQ ID no. 2, and also those with the biological activity of component H of the phosphotransferase system and also those which are at least 70% identical to the polypeptide according to SEQ ID no. 2, preferably at least 80% and in particular those which are 90% to 95% identical to the polypeptide according to SEQ ID no. 2 and have the activity mentioned.
The invention also relates to a process for the fermentative preparation of L-amino acids, particularly L-lysine, using coryneform bacteria which in particular already produce an L-amino acid and in which the nucleotide sequences coding for the ptsH gene are enhanced, particularly overexpressed.
The term “enhancement” describes in this context the increase in intracellular activity of one or more enzymes in a microorganism which are coded for by the corresponding DNA, by, for example, increasing the copy number of the gene or genes or alleles, using a strong promotor or using a gene or allele which codes for a corresponding enzyme with a high activity and optionally combining said measures.
The microorganisms which are the subject of the present invention may produce L-amino acids, particularly L-lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may be representatives of coryneform bacteria, particularly of the Corynebacterium genus. A particular example of the Corynebacterium genus is the
Corynebacterium glutamicum
type which is known by experts to have the ability to produce L-amino acids.
Examples of suitable strains of the
Corynebacterium
genus, particularly of the
Corynebacterium glutamicum
type include the well known wild-type strains
Corynebacterium glutamicum
ATCC13032
Corynebacterium acetoglutamicum
ATCC15806
Corynebacterium acetoacidophilum
ATCC13870
Corynebacterium thermoaminogenes
FERM BP-1539
Corynebacterium melassecola
ATCC17965
Brevibacterium flavum
ATCC14067
Brevibacterium lactofermentum
ATCC13869 and
Brevibacterium divaricatum
ATCC14020
and L-lysine-producing mutants and strains prepared therefrom, such as, for example
Corynebacterium glutamicum
FERM-P 1709
Brevibacterium flavum
FERM-P 1708
Brevibacterium lactofermentum
FERM-P 1712
Corynebacterium glutamicum
FERM-P 6463
Corynebacterium glutamicum
FERM-P 64

Affiliated with

Also associated with

Rating

Nucleotide sequences encoding the ptsH gene does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Nucleotide sequences encoding the ptsH gene, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleotide sequences encoding the ptsH gene will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3302639