Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
1999-10-27
2003-12-30
Achutamurthy, Ponnathapu (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...
C435S320100, C435S252300, C435S254110, C435S419000, C435S325000, C536S023100, C536S023200
Reexamination Certificate
active
06670156
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides nucleotide sequences which code for the dapF gene and a process for the fermentative preparation of L-lysine using coryneform bacteria in which the dapF gene is amplified.
2. Background Invention
L-Lysine is used in human medicine and in the pharmaceuticals industry, but in particular in animal nutrition.
It is known that L-lysine is 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 process. Improvements to the process 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 for example the lysine analogue S-(2-aminoethyl)-cysteine, or are auxotrophic for amino acids of regulatory importance and produce L-lysine are obtained in this manner.
Methods using recombinant DNA techniques have also been employed for some years for improving Corynebacterium strains which produce L-lysine, by amplifying individual lysine biosynthesis genes and investigating the effect on the L-lysine production. Review articles in this context 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)).
In prokaryotes, three different routes are known for the biosynthesis of D,L-diaminopimelate- or L-lysine. These routes differ in the reaction of L-piperidine-2,6-dicarboxylate (tetrahydrodipicolinate).
In the succinylase route, tetrahydrodipicolinate is converted into D,L diaminopimelate via a succinylation by tetrahydrodipicolinate succinylase, subsequent transamination (N-succinyl-amino-ketopimelate transaminase) of the keto group, desuccinylation (N-succinyl-amino-ketopimelate desuccinylase) and then epimerization (diaminopimelate epimerase) (Gilvarg, 1958, The Journal of Biological Chemistry, 233: 1501-1504).
In the acetylase route, which is present in Bacillus subtilis and Bacillus megaterium, the acylation of tetrahydrodipicolinate is carried out by an acetyl radical (Weinberger & Gilvarg, 1970, Journal of Bacteriology, 101:323-324).
A third biosynthesis route is described for B. sphaericus (Misono et al., 1976, Journal of Bacteriology, 137:22-27). In this biosynthesis step, which is called the dehydrogenase route, direct reductive amination of the tetrahydrodipicolinate to D,L-DAP takes place.
With the aid of genetic and enzymatic studies, Schrumpf et al. (Journal of Bacteriology 173, 4510-4516 (1991)) showed that in
Corynebacterium glutamicum
, lysine biosynthesis takes place both by the dehydrogenase route and by the succinylase route.
In vivo NMR studies by Marx et al., (Biotechnology and Bioengineering 56, 168-180 (1997)) and Sonntag (European Journal of Biochemistry 213: 1325-1331 (1993)) have shown that in
Corynebacterium glutamicum
both the succinylase route and the dehydrogenase route contribute towards the production of L-lysine.
The gene for desuccinylase (dapE) from
Corynebacterium glutamicum
has been cloned and sequenced by Wehrmannn et al. (Journal of Bacteriology 177: 5991-5993 (1995)). It has also be possible to clone and sequence the gene for succinylase (dapD) from
Corynebacterium glutamicum
(Wehrmannn et al,. Journal of Bacteriology 180, 3159-3165 (1998)).
SUMMARY OF THE INVENTION
OBJECT OF THE INVENTION
The inventors had the object of providing new measures for improved fermentative preparation 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 therefore a general interest in providing new improved processes for the preparation of 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) 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 which is expressed by the dapF gene contained on plasmid pEC-XT99A-dapF in the deposited
C. glutamicum
strain DSM 12968,
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 polynucleotide 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 that is capable of replication in coryneform bacteria, which is preferably recombinant DNA, comprising the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide that is capable of replication in coryneform bacteria, which is preferably recombinant DNA, which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No.2,
a vector containing the polynucleotide sequence as described in (i)-(iv) above, in particular pEC-XT99A-dapF, deposited as DSM 12968.
and coryneform bacteria serving as the host cell, which contain the vector a shuttle vector pEC-XT99A-dapF characterized at the restriction map shown in
FIG. 2
, which has been deposited under the designation DSM 12968.
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, full length cDNA which encodes diamindpimelate epimerase and to isolate those cDNA or genes which have a high similarity of sequence with that of the diaminopimelate epimerase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the preparation of DNA of genes which code for diaminopimelate epimerase 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 least 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 polyd
Bathe Brigitte
Hartmann Michael
Kalinowski Jorn
Kirchner Oliver
Möckel Bettina
Achutamurthy Ponnathapu
Degussa - AG
Kerr Kathleen
Pillsbury & Winthrop LLP
LandOfFree
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