Nucleotide sequences which code for the pck gene

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

1999-12-07

2002-07-16

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S069100, C435S183000, C435S252300, C435S320100, C536S023200

Reexamination Certificate

active

06420151

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
The present application is based on German application DE 199 50 409.1, filed on Oct. 20, 1999, which disclosure is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides nucleotide sequences from coryneform bacteria which code for the pck gene and a process for the fermentative preparation of L-amino acids, in particular L-lysine and L-threonine, by attenuation of the pck gene.
2. Prior Art
Amino acids, in particular lysine and threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine.
It is known that these substances are prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of its great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form by e.g. 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 metabolism products of regulatory importance and produce the desired amino acid 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 [sic].
OBJECT OF THE INVENTION
The inventors had the object of providing the general public with new measures for improved fermentative preparation of amino acids.
DESCRIPTION OF THE INVENTION
Amino acids, in particular L-lysine and L-threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine. There is therefore a general interest in providing new improved processes for the preparation of these products.
The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of
a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
b) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for the polypeptide mentioned and is contained on the plasmid pEK-pckA (
FIG. 1
) or pEK-pckB (FIG.
2
),
c) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2,
d) polynucleotide which is complementary to the polynucleotides of a), b) or c), and
e) polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of a), b), c) or d).
The invention also provides a preferably recombinant DNA with Corynebacterium origin which is capable of replication in coryneform microorganisms and contains at least the nucleotide sequence which codes for the pck gene, shown in SEQ ID No. 1.
The invention also provides a DNA according to claim
1
which is capable of replication, comprising
(i) the nucleotide sequence shown in SEQ ID no. 1, or
(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and/or optionally
(iv) sense mutations of neutral function in (i).
The invention also provides
a polynucleotide according to claim
2
, comprising the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide according to claim
2
, which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2,
a vector containing the polynucleotide according to claim
1
, in particular pEK-pckA or pEK-pckB, shown in
FIGS. 1 and 2
and coryneform bacteria serving as the host cell, into which the &Dgr;pck deletion has been incorporated.
The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which comprise the complete gene with the polynucleotide sequence corresponding to SEQ ID no. 1, with a probe which comprises the sequence of the polynucleotide mentioned, according to SEQ ID no. 1 or a fragment thereof, and isolation of the DNA sequence mentioned.
Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for phosphoenol pyruvate carboxykinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphoenol pyruvate carboxykinase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the preparation of DNA of genes which code for phosphoenol pyruvate carboxykinase by the polymerase chain reaction (PCR).
Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, especially preferably at lease 15 successive bases. Oligonucleotides which have a length of at least 40 or 50 base pairs are also suitable.
“Isolated” means separated out of its natural environment.
“Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA and DNA or modified RNA and DNA.
“Polypeptides” is understood as meaning peptides or proteins which obtain [sic] two or more amino acids bonded via peptide bonds.
The polypeptides according to the invention include the polypeptide according to SEQ ID No. 2, in particular those with the biological activity of PEP carboxykinase, and also those which are identical to the extent of at least 70% to the polypeptide according to SEQ ID No. 2, preferably to the extent of at least 80%, and in particular those which are identical to the extent of at least 90% to 95% to the polypeptide according to SEQ ID no. 2, and have the activity mentioned.
The invention also provides a process for the fermentative preparation of L-amino acids, in particular L-lysine and L-threonine, using coryneform bacteria which in particular already produce the L-amino acids and in which the nucleotide sequence(s) which code(s) for the pck gene are attenuated, in particular expressed at a low level.
The term “attenuation” in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding enzyme (protein), and optionally combining these measures.
The microorganisms which the present invention provides can produce L-amino acids, in particular lysine and threonine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species
Corynebacterium glutamicum
, which is known among specialists for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species
Corynebacterium glutamicum
, are, for example, the 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
ATCC

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