Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-03-22
2004-05-25
Benzion, Gary (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024320, C536S024300, C435S320100, C435S352000
Reexamination Certificate
active
06740742
ABSTRACT:
This application claims priority from German Application No. 100 14 546.9, filed on Mar. 23, 2000, 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 encoding the dapC gene and a process for the fermentative production of L-lysine, using coryneform bacteria in which the dapC gene (N-succinylaminoketopimelate transaminase gene) is enhanced, in particular over-expressed.
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 produced by fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum.
Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-(2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant metabolites and produce L-amino acids, such as for example L-lysine.
For some years, methods of recombinant DNA technology have likewise been used to improve strains of Corynebacterium which produce amino acids by amplifying individual amino acid biosynthesis genes and investigating the effect on 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
It is an object of the invention to provide novel methods for the improved fermentative production of L-lysine.
DESCRIPTION OF THE INVENTION
L-lysine is used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is accordingly general interest in providing novel improved processes for the production of L-lysine.
Any subsequent mention of L-lysine or lysine should be taken to mean not only the base, but also salts, such as for example lysine monohydrochloride or lysine sulfate.
The invention provides an isolated polynucleotide from coryneform bacteria containing at least one polynucleotide sequence selected from the group
a) polynucleotide which is at least 70% identical to a polynucleotide which encodes a polypeptide containing the amino acid sequence of SEQ ID no. 2,
b) polynucleotide which encodes a polypeptide which contains 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), or
d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequences of a), b) or c).
The invention also provides the polynucleotide according to claim 1, wherein it preferably comprises replicable DNA containing:
(i) the nucleotide sequence shown in SEQ ID no. 1, or
(ii) at least one sequence which matches the sequence (i) within the degeneration range of the genetic code, or
(iii) at least one sequence which hybridizes with the complementary sequence to sequence (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
The invention also provides
a polynucleotide according to claim 4, containing the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide which encodes a polypeptide which contains the amino acid sequence as shown in SEQ ID no. 2,
a vector containing the polynucleotide according to claim 1, in particular a shuttle vector or the plasmid vector pXT-dapCexp, which is shown in FIG.
2
and is deposited under number DSM 13254 in DSM 5715.
and coryneform bacteria acting as host cell which contain the vector.
The invention also provides polynucleotides which substantially consist of a polynucleotide sequence, which are obtainable by screening by means of hybridization of a suitable gene library, which contains the complete gene having the polynucleotide sequence according to SEQ ID no. 1, with a probe which contains the sequence of the stated polynucleotide according to SEQ ID no. 1, or a fragment thereof, and isolation of the stated DNA sequence.
Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA in order to isolate full length cDNA which encode N-succinylaminoketopimelate transaminase and to isolate such cDNA or genes, the sequence of which exhibits a high level of similarity with that of the N-succinylaminoketopimelate transaminase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the production of DNA of genes which encode N-succinylaminoketopimelate transaminase by the polymerase chain reaction (PCR).
Such oligonucleotides acting as probes or primers contain at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. oligonucleotides having 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 unmodified or modified.
“Polypeptides” are taken to mean peptides or proteins which contain two or more amino acids connected by peptide bonds.
The polypeptides according to the invention include a polypeptide according to SEQ ID no. 2, in particular those having the biological activity of N-succinylaminoketopimelate transaminase and also those which are at least 70%, preferably at least 80%, identical to the polypeptide according to SEQ ID no. 2 and in particular are at least 90% to 95% identical to the polypeptide according to SEQ ID no. 2 and exhibit the stated activity.
The invention furthermore relates to a process for the fermentative production 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 encode the dapC gene are enhanced, in particular over-expressed.
In this connection, the term “enhancement” describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are encoded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene or allele which encodes a corresponding enzyme having elevated activity and optionally by combining these measures.
The microorganisms, provided by the present invention, may produce L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium, the species
Corynebacterium glutamicum
may in particular be mentioned, which is known in specialist circles 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
Corynebac
Hartmann Michael
Kalinowski Jörn
Mockel Bettina
Pfefferle Walter
Pühler Alfred
Benzion Gary
Degussa - AG
Pillsbury & Winthrop LLP
Switzer Juliet C.
LandOfFree
Nucleotide sequences encoding the dapC gene and process for... 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 dapC gene and process for..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleotide sequences encoding the dapC gene and process for... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3199101