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
1999-08-13
2003-05-27
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...
C435S115000, C435S320100, C435S252320, C536S023100, C536S023200, C536S023700
Reexamination Certificate
active
06569650
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides nucleotide sequences encoding for the superoxide dismatase (sod) gene and a process for the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine using coryneform bacteria in which the sod gene is amplified.
2. Background Information
Nucleotides, vitamins and L-amino acids, in particular L-lysine, are used in the foodstuffs industry, in animal nutrition, in human medicine and in the pharmaceutical industry.
It is known that these substances can be prepared by fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum
. Constant efforts are made to improve the method of preparation due to the high degree of importance of these substances. Process improvements may relate to fermentation engineering factors such as e.g. stirring and supplying with oxygen, or the composition of the nutrient medium, such as e.g. the sugar concentration during fermentation, or the working up process aimed at obtaining the product itself by e.g. ion exchange chromatography or the intrinsic power of the microorganism itself.
To improve the power of the microorganisms, the methods of mutagenesis, selection and mutant choice are used. Strains which are resistant to antimetabolites or which are auxotrophic for significant regulatory intermediates are obtained in this way and produce nucleotides, vitamins and amino acids.
For some time now the methods of recombinant DNA engineering have also been used for the strain-improvement of L-amino acid-producing strains of
Corynebacterium glutamicum
, by amplifying individual amino acid biosynthetic genes and investigating the effect on L-amino acid production.
U.S. Pat. No. 5,179,010 describes strains of coryneform bacteria which are e.g. resistant to methylviologen or benzoyl peroxide, have an increased activity to superoxide dismutase and have an improved lysine yield. These strains were produced by non-targeted mutagenesis using the mutagen N-methyl-N-nitro-N-nitrosoguanidine. The increase in the concentration of superoxide dismutase in the strains mentioned there was at most 56%.
U.S. Pat. No. 4,529,697 describes mutants of coryneform bacteria which produce glutamic acid. The increase in the concentration of superoxide dismutase in the strains mentioned there was at most 105%.
SUMMARY OF THE INVENTION
The inventor has formulated the object as the provision of new steps for the improved handling of superoxide dismutase from coryneform bacteria. These steps can be used during the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine.
DETAILED DESCRIPTION OF THE INVENTION
Nucleotides, vitamins and L-amino acids, in particular L-lysine, are used in the foodstuffs industry, in animal nutrition, in human medicine and in the pharmaceutical industry. Lysine-producing strains of coryneform bacteria are known from the prior art, in which the concentration of superoxide dismutase is increased by 27 to 56% and which liberate amplified lysine. These strains were obtained by non-targeted mutagenesis.
Whenever L-lysine or lysine is mentioned in the following this is intended to mean not only the base but also salts such as, for example, lysine monohydrochloride or lysine sulfate.
The invention provides a preferably recombinant DNA from the Corynebacterium source which can replicate in coryneform microorganisms and which contains at least the nucleotide sequence which encodes for the sod gene represented in SEQ-ID-No.1.
The invention also provides a replicable DNA in accordance with Claim 1 comprising:
(i) the nucleotide sequence, shown in SEQ-ID-No.1, or
(ii) at least one sequence which corresponds to the sequence (i) within the region of degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence which is complementary to sequence (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
Coryneform microorganisms, in particular the strain Corynebacterium, transformed by the introduction of the replicatable DNA mentioned above, are also provided by the invention.
Furthermore the invention provides a process for the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine, using coryneform bacteria which in particular already produce the relevant product and in which the nucleotide sequences encoding for the sod gene are amplified, in particular are overexpressed.
The expression “amplification” 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 copy number of the gene or genes, using a strong promoter or a gene which encodes for a corresponding enzyme with high activity and optionally combining these steps.
The microorganisms which are the subject of the present invention can produce nucleotides, vitamins and L-amino acids, in particular L-lysine, from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may be members of the coryneform bacteria family in particular the genus Corynebacterium. In the case of the Corynebacterium genus, in particular the species
Corynebacterium glutamicum
, it should be mentioned that this is well-known in the specialist field for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular the species
Corynebacterium glutamicum
are for example the known wild-type strains
Corynebacterium glutamicum
ATCC13032
Corynebacterium acetoglutamicum
ATCC15806
Corynebacterium acetoacidophilum
ATCC13870
Corynebacterium ammoniagenes
ATCC6871
Corynebacterium thermoaminogenes
FERM BP-1539
Brevibacterium flavum
ATCC14067
Brevibacterium lactofermentum
ATCC13869 and
Brevibacterium divaricatum
ATCC14020
Corynebacterium melassecola
ATCC17965
Brevibacterium ammoniagenes
IF012072
and mutants or strains prepared therefrom which can produce nucleotides, vitamins and L-amino acids,
such as for example the 5′-inosinic acid-producing strains
Corynebacterium ammoniagenes
ATCC15190
Corynebacterium ammoniagenes
ATCC15454
Corynebacterium glutamicum
ATCC14998 or
such as for example the 5′-guanylic acid-producing strains
Corynebacterium glutamicum
ATCC21171 or
Corynebacterium ammoniagenes
ATCC19216 or
such as for example the L-lysine producers
Corynebacterium glutamicum
FERM-P 1709
Brevibacterium flavum
FERM-P 1708
Brevibacterium lactofermentum
FERM-P 1712
Corynebacterium glutamicum
FERM-P 6463 and
Corynebacterium glutamicum
FERM-P 6464
Corynebacterium glutamicum DSM
5714.
The inventors were able to isolate the new sod gene from
Corynebacterium melassecola
ATCC17965.
Here, the superoxide dismutase enzyme protein was first purified to homogeneity using chromatographic methods. Methods and instructions for protein purification and preparation are fully described e.g. in the textbook by Schleifer and Wensink: Practical Methods in Molecular Biology (Springer Verlag, Berlin, Germany, 1981), in the manual by Harris and Angal: Protein Purification Methods: A Practical approach (IRL Press, Oxford, UK, 1989), in the textbook by Scopes: Protein Purification: Principles and Practice, 3rd ed. (Springer Verlag, New York, USA, 1993) and in generally well-known textbooks and manuals. The pure enzyme protein can then be broken down into peptides by treating with suitable enzymes such as e.g. trypsin or chymotrypsin. The amino acid sequence in these peptides can be determined by the method of N-terminal sequencing described by Edman (Archives of Biochemistry 22, 475, (1949)). Methods and instructions for protein sequencing are given e.g. in Smith: Protein Sequencing Protocols: Methods in Molecular Biology, Vol. 64 and Vol. 112 (Humana Press, Totowa, N.J., USA, 1996) and in Kamp et al.: Protein Structure Analysis: Preparation, Characterization and Microsequencing (Springer Verlag, New York, N.Y., USA, 1997). The amino acid sequence in the superoxide dis
Guyonvarch Armel
Marx Achim
Merkamm Muriel
Achutamurthy Ponnathapu
Degussa - AG
Kerr Kathleen
Pillsbury & Winthrop LLP
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