Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-01
2002-06-25
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S252320, C435S193000, C435S115000
Reexamination Certificate
active
06410705
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims priority under 35 U.S.C. §119 to German application 199 41 478.5, filed on Sep. 1, 1999.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to nucleotide sequences coding for the thrE gene and a process for the enzymatic production of L-threonine using coryneform bacteria, in which the thrE gene is amplified.
2. Background Information
L-threonine is used in animal nutrition, in human medicine and in the pharmaceutical industry.
It is known that L-threonine can be produced by fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum.
On account of the great importance of L-threonine, attempts are constantly being made to improve the production processes. Production improvements may relate to fermentation technology measures such as for example stirring and provision of oxygen, or the composition of the nutrient medium such as for example the sugar concentration during fermentation, or the working-up to the product form by for example ion exchange chromatography, or the intrinsic production properties of the microorganism itself.
Methods employing mutagenesis, selection and choice of mutants are used to improve the production properties of these microorganisms. In this way strains are obtained that are resistant to antimetabolites such as for example the threonine analogon &agr;-amino-&bgr;-hydroxyvaleric acid (AHV) or are auxotrophic for regulatory significant amino acids and produce L-threonine.
For some years now recombinant DNA technology methods have also been used for the strain improvement of L-threonine producing strains of Corynebacterium, by amplifying individual threonine biosynthesis genes and investigating the action on L-threonine production.
SUMMARY OF THE INVENTION
OBJECT OF THE INVENTION
The inventors have aimed to provide new measures for the improved enzymatic production of L-threonine.
DESCRIPTION OF THE INVENTION
L-threonine is used in animal nutrition, in human medicine and in the pharmaceutical industry. There is therefore a general interest in providing new improved processes for producing L-threonine.
The object of the invention is a preferably recombinant DNA derived from Corynebacterium and replicable in coryneform microorganisms, which contains at least the nucleotide sequence coding for the thrE gene, represented in the sequences SEQ-ID-No.1 and SEQ-ID-No.3.
The object of the invention is also a replicable DNA according to claim
1
with:
(i) the nucleotide sequences shown in SEQ-ID-No.1 or SEQ-ID-No.3, that code for the thrE gene, or
(ii) at least one sequence that corresponds to the sequences (i) within the degeneration region of the genetic code, or
(iii) at least one sequence that hybridises with the sequence complementary to the sequences (i) or (ii), and/or optionally
(iv) functionally neutral sense mutations in (i).
The object of the invention are also coryneform microorganisms, in particular of the genus Corynebacterium, transformed by the introduction of the aforementioned replicable DNA.
The invention finally relates to a process for the enzymatic production of L-threonine using coryneform bacteria, which in particular already produce L-threonine and in which the nucleotide sequence(s) coding for the thrE gene is/are amplified, in particular overexpressed.
The term “amplification” describes in this connection the enhancement of the intracellular activity of one or more enzymes in a microorganism that are coded by the corresponding DNA, by for example increasing the copy number of the gene or genes or using a strong promoter or a gene that codes for a corresponding enzyme having a high activity, and if necessary using a combination of these measures.
The microorganisms that are the object of the present invention can produce L-threonine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may be representatives of coryneform bacteria, in particular of the genus Corynebacterium. In the genus Corynebacterium the species
Corynebacterium glutamicum
should in particular be mentioned, which is known to those in the specialist field for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species
Corynebacterium glutamicum,
are in particular the known wild type strains
Corynebacterium glutamicum
ATCC13032
Corynebacterium acetoglutamicum
ATCC15806
Corynebacterium acetoacidophilum ATCC
13870
Corynebacterium melassecola
ATCC17965
Corynebacterium thermoaminogenes
FERM BP-1539
Brevibacterium flavum
ATCC14067
Brevibacterium lactofermentum
ATCC13869 and
Brevibacterium divaricatum
ATCC14020
and L-threonine-producing mutants or strains obtained thereform, for example
Corynebacterium glutamicum
ATCC21649
Brevibacterium flavum
BB69
Brevibacterium flavum
DSM5399
Brevibacterium lactofermentum
FERM-BP 269
Brevibacterium lactofermentum
TBB-10
The inventors have successfully managed to isolate the thrE gene of
Corynebacterium glutamicum.
In order to isolate the thrE gene a mutant of
C. glutamicum
defective in the thrE gene is first of all produced. To this end a suitable starting strain such as for example ATCC14752 or ATCC13032 is subjected to a mutagenesis process.
Conventional mutagenesis processes include treatment with chemicals, for example N-methyl-N-nitro-N-nitrosoguanidine, or UV irradiation. Such processes for initiating mutation are generally known and may be consulted in, inter alia, Miller (A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for
Escherichia coli
and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992)) or in the handbook “Manual of Methods for General Bacteriology” The American Society for Bacteriology (Washington D.C., USA, 1981).
Another mutagenesis process is the method of transposon mutagenesis in which the property of a transposon is utilised to “jump” in DNA sequences and thereby interfere with or switch off the function of the relevant gene. Transposons of coryneform bacteria are known in the specialist field. For example, the erythromycin resistance transposon Tn5432 (Tauch et al., Plasmid (1995) 33: 168-179) and the chloramphenicol resistance transposon Tn5546 have been isolated from
Corynebacterium xerosis
strain M82B.
Another transposon is the transposon Tn5531 described by Ankri et al. (Journal of Bacteriology (1996) 178: 4412-4419) and that was used for example in the course of the present invention. The transposon Tn5531 contains the aph3 kanamycin resistance gene and can be delivered for example in the form of the plasmid vector pCGL0040, which is shown in FIG.
1
. The nucleotide sequence of the transposon Tn5531 is freely available under the accession number U53587 from the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA).
After mutagenesis, preferably transposon mutagenesis, has been carried out a search is made for a mutant defective in the thrE gene. A mutant defective in the thrE gene is recognised by the fact that it exhibits good growth on minimal agar, but poor growth on minimal agar that has been supplemented with threonine-containing oligopeptides, for example the tripeptide threonyl-threonyl-threonine.
An example of such a mutant is the strain ATCC14752&Dgr;ilvAthrE::Tn5531.
A strain produced in the described manner may be used to isolate and clone the thrE gene.
To this end a gene bank of the bacterium that is of interest may be established. The establishment of gene banks is recorded in generally known textbooks and manuals. There may be mentioned by way of example the textbook by Winnacker: Gene und Klone, eine Einfuhrung in die Gentechnologie (Gene and Clones, An Introduction to Gene Technology) (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well-known gene bank is that of the
E. coli
K-12 strain W3110, which has been established by Kohara et al. (Cel
Eggeling Lothar
Sahm Hermann
Thierbach Georg
Ziegler Petra
Degussa Huls Aktiengesellschaft
Prouty Rebecca E.
Steadman David J
LandOfFree
Nucleotide sequences coding for the thrE gene and process... 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 coding for the thrE gene and process..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleotide sequences coding for the thrE gene and process... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2942914