Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-12-23
2003-09-02
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S243000, C435S252300, C536S023700
Reexamination Certificate
active
06613545
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims priority under 35 U.S.C. §119 to German Application 199 51 708.8, filed on Oct. 27, 1999.
FIELD OF THE INVENTION
The present invention provides nucleotide sequences coding for the export of branched-chain amino acids, a process for the identification and isolation thereof and a process for the fermentative production of branched-chain amino acids using coryneform bacteria in which genes which code for the export of branched-chain amino acids are amplified.
BACKGROUND INFORMATION
The branched-chain amino acids L-isoleucine, L-valine and L-leucine are used in the pharmaceuticals industry, in human medicine and in animal nutrition.
It is known that branched-chain amino acids may be 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 isoleucine analogue isoleucine hydroxyamate (Kisumi M, Komatsubara S, Sugiura, M, Chibata I (1972) Journal of Bacteriology 110: 761-763), the valine analogue 2-thiazolealanine (Tsuchida T, Yoshinanga F, Kubota K, Momose H (1975) Agricultural and Biological Chemistry; Japan 39: 1319-1322) or the leucine analogue &agr;-aminobutyrates (Ambe-Ono Y, Sato K, Totsuka K, Yoshihara Y, Nakamori S (1996) Bioscience Biotechnology Biochemistry 60: 1386-1387) or which are auxotrophic for regulatorily significant metabolites and produce branched-chain amino acids (Tsuchida T, Yoshinaga F, Kubota K, Momose H, Okumura S (1975) Agricultural and Biological Chemistry; Nakayama K, Kitada S, Kinoshita S (1961) Journal of General and Applied Microbiology, Japan 7: 52-69; Nakayama K, Kitada S, Sato Z, Kinoshita (191) Journal of General and Applied Microbiology, Japan 7: 41-51).
For some years, the methods of recombinant DNA technology have also been used for improvement of strains of Corynebacterium which produce branched-chain amino acids by amplifying individual biosynthesis genes for branched-chain amino acids and investigating the effect on branched-chain 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)), Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)), and Eggeling et al., Journal of Biotechnology 56: 168-180 (1997)).
SUMMARY OF THE INVENTION
Object of the Invention
The inventors set themselves the object of providing novel measures for the improved fermentative production of branched-chain amino acids.
Description of the Invention
Branched-chain amino acids are used in the pharmaceuticals industry, in human medicine and in animal nutrition. There is accordingly general interest in providing novel improved processes for the production of branched-chain amino acids.
Any subsequent mention of branched-chain amino acids should be taken to mean in particular L-isoleucine, L-valine or L-leucine.
The present invention provides isolated polynucleotides containing at least one polynucleotide sequence selected from the group
a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing at least one amino acid sequence SEQ ID no. 3 or 5,
b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no. 3 or 5,
c) polynucleotide which is complementary to the polynucleotides of a) or b) and
d) polynucleotide containing at least 15 successive bases of the polynucleotide sequences of a), b) or c).
The present invention also provides preferably recombinant DNA replicable in coryneform bacteria and originating from Corynebacterium which contains at least the nucleotide sequences which code for the genes brnF and/or brnE, as shown in SEQ ID no. 1 and in SEQ ID no. 6.
The present invention also provides replicable DNA as described in a)-c) above containing:
(i) the nucleotide sequences shown in SEQ ID no. 1 or SEQ ID no. 6 which code for the genes brnE and/or brnF, 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 hybridises with the complementary sequence to sequences (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
The present invention also provides
polynucleotides as in a)-d) above which comprise recombinant DNA replicable in coryneform bacteria and contain containing at least one of the nucleotide sequences selected from those shown in SEQ ID no. 1, 2, 4 or 6
polypeptides as described in a)-d) above which comprimise recombinant DNA replicable in coryneform bacteria which code for polypeptides which contain at least one of the amino acid sequences as shown in SEQ ID no. 3 or 5
a vector containing the polynucleotide or polynucleotides as described in a)-c) above or the DNA sequence shown in SEQ ID no. 1 or SEQ ID no. 6.
and coryneform bacteria acting as host cell which contain the vector.
The present invention also provides polynucleotides which substantially consist of one polynucleotide sequence, which are obtainable by screening by means of hybridisation of a suitable gene library, which contains the complete genes having the polynucleotide sequences according to SEQ ID no. 1, 2, 4 or 6 with a probe which contains the sequence of the stated polynucleotides according to SEQ ID no. 1, 2, 4 or 6 or a fragment thereof and isolation of the stated DNA sequences.
Polynucleotide sequences according to the invention are suitable as hybridisation probes for RNA, cDNA and DNA in order to isolate full length cDNA which codes for isoleucine, leucine or valine export proteins and to isolate such cDNA or genes, the sequences of which exhibit a high level of similarity with that of the brnF and/or brnE gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers, with the assistance of which, usingthe polymerase chain reaction (PCR), DNA of genes whichcode for isoleucine, leucine or valine export proteins may be produced.
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 base pairs 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 aminoacids connected by peptide bonds.
The polypeptides according to the invention include the polypeptides according to SEQ ID no. 3 and/or 5, in particular those having the biological activity of transporting branched-chain amino acids and also those which are at least 70% identical to the polypeptides according to SEQ ID no. 3 and/or 5, preferably at least 80% and in particular 90% to 95% identical to the polypeptides according to SEQ ID no. 3 and/or 5 and exhibit the stated activity.
The present invention also provides coryneform microorganisms, in particular of the genus Corynebacte
Eggeling Lothar
Kennerknecht Nicole
Pfefferle Walter
Sahm Hermann
Degussa-Huls Aktiengesellschaft
Fitch Even Tabin & Flannery
Graser Jennifer E.
Sanzo Michael A.
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