Nucleotide sequences which encode the pfk gene

Details Nucleotide sequences which encode the pfk gene Nucleotide sequences which encode the pfk gene

2000-11-20

2004-10-19

Steadman, David (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

C435S115000, C435S320100, C435S252300, C435S252320, C536S023100, C536S023200, C536S023700, C536S024320

Reexamination Certificate

active

06806068

ABSTRACT:

This application claims priority from German Application No. 199 56 131.1, filed on Nov. 23, 1999, the subject matter of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides nucleotide sequences which encode the pfk gene and a process for the fermentative preparation of amino acids, in particular L-lysine, using coryneform bacteria in which the pfk gene is enhanced.
2. Background Information
Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but in particular 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 processes 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, such as, for example, the lysine analogue S-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as, for example, L-lysine, are obtained in this manner.
Recombinant DNA techniques have also been employed for some years for improving Corynebacterium strains which produce amino acids, by amplifying individual amino acid biosynthesis genes and investigating the effects of such changes on the amino acid production. Review articles on this subject are to 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
It is an object of the invention to provide new means for improved fermentative preparation of amino acids, in particular L-lysine.
Description of the Invention
Amino acids, in particular L-lysine, are used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.
When L-lysine or lysine are mentioned in the following, not only the base but also the salts, such as, for example, lysine monohydrochloride or lysine sulfate, are also meant.
The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of
a) a polynucleotide which is at least 70% identical to a polynucleotide which encodes a polypeptide which comprises the amino acid sequence of SEQ ID NO:2,
b) a polynucleotide which encodes a polypeptide which comprises an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2,
c) a polynucleotide which is complementary to the polynucleotides of a) or b), and
d) a polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c).
In a preferred embodiment, the invention also provides the polynucleotide with the features described above, preferably being a DNA 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 optionally
(iv) sense mutations of neutral function in (i).
The invention also provides
a polynucleotide with the aforementioned features, comprising the nucleotide sequence as shown in SEQ ID NO:1,
a polynucleotide with the aforementioned features, which encodes a polypeptide which comprises the amino acid sequence as shown in SEQ ID NO:2,
a vector containing the polynucleotide with features a)-d) above, in particular a shuttle vector or plasmid vector
and coryneform bacteria serving as the host cell, which contain the vector.
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 comprises 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 phosphofructokinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphofructokinase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the preparation of DNA of genes which code for phosphofructokinase by the polymerase chain reaction (PCR).
Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. oligonucleotides which have a length of at least 40 or 50 nucleotides are also suitable.
“Isolated” means separated from its natural environment.
“Polynucleotide” generally relates to polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be modified or unmodified.
“Polypeptides” is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
The polypeptides according to the invention include a polypeptide according to SEQ ID NO:2, in particular those with the biological activity of phosphofructokinase, and also those which are at least 70% identical to the polypeptide according to SEQ ID NO:2, and preferably are at least 80% identical, and most preferably 90% to 95% identical 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 amino acids, in particular L-lysine, using coryneform bacteria which in particular already produce an amino acid, and in which the nucleotide sequences which code for the pfk gene are enhanced, in particular over-expressed.
The term “enhancement” in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene which encodes a corresponding enzyme having a high activity, and optionally combining these measures.
The microorganisms provided by the present invention can be used to prepare L-amino acids, in particular L-lysine, 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, the species
Corynebacterium glutamicum
, which is known among experts for its ability to produce L-amino acids, is particularly useful.
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

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