Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
2001-12-21
2004-10-26
Rao, Manjunath N. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S004000, C435S006120, C435S028000, C435S069100, C435S183000, C435S200000, C435S252300, C435S252320, C435S320100, C435S041000, C435S106000, C435S107000, C435S108000, C435S109000, C435S110000, C435S113000, C435S115000, C435S116000, C536S023200, C536S023500, C536S023700
Reexamination Certificate
active
06808914
ABSTRACT:
The invention provides nucleotide sequences coding for the accDA gene and a process for the preparation of L-amino acids, especially L-lysine, by fermentation using corynebacteria in which the accDA gene is amplified.
STATE OF THE ART
L-Amino acids, especially L-lysine, are used in animal nutrition, in human medicine and in the pharmaceutical industry.
It is known that these amino acids are prepared by the fermentation of strains of corynebacteria, especially
Corynebacterium glutamicum
. Because of their great importance, attempts are constantly being made to improve the preparative processes. Improvements to the processes may relate to measures involving the fermentation technology, e.g. stirring and oxygen supply, or the composition of the nutrient media, e.g. the sugar concentration during fermentation, or the work-up to the product form, e.g. by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself.
The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains which are resistant to antimetabolites, e.g. the lysine analog S-(2-aminoethyl) cysteine, or auxotrophic for amino acids of regulatory significance, and produce L-amino acids.
Methods of recombinant DNA technology have also been used for some years in order to improve L-amino acid-producing strains of
Corynebacterium
by amplifying individual amino acid biosynthesis genes and studying the effect on L-lysine production. Surveys of this subject have been published inter alia by 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)).
The enzyme acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. The enzyme from
Escherichia coli
consists of four subunits. The accB gene codes for biotin carboxyl carrier protein, the accC gene for biotin carboxylase and the accA and accD genes for transcarboxylase (Cronan and Rock, Biosynthesis of Membrane Lipids, in:
Escherichia coli
and
Salmonella typhimurium
(ed. F. C. Neidhardt), 1996, pp. 612-636, American Society for Microbiology). Because of the property of the enzyme to carboxylate acyl groups in the form of acyl-CoA, it is also called acyl-CoA carboxylase.
The nucleotide sequence of the accBC gene from
Corynebacterium glutamicum
has been determined by J{overscore (a)}ger et al. (Archives of Microbiology 166, 76-82 (1996)) and is generally available from the data bank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) under accession number U35023. The accBC gene codes for a subunit of acetyl-CoA carboxylase which carries a biotin carboxyl carrier protein domain and a biotin carboxylase domain.
OBJECT OF THE INVENTION
The object which the inventors set themselves was to provide novel procedures for the improved preparation of L-amino acids, especially L-lysine, by fermentation.
REFERENCES:
patent: 197 335 (1986-10-01), None
patent: 358 940 (1986-10-01), None
patent: 752 472 (1997-01-01), None
Eikmanns, Bernhard et al., “Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase,” Microbiology (1994), 140, Nr. 8, pp. 1817-1828, XP-000957470.
Jager, Wolfgang et al., “A Corynebacterium Glutamicum gene encoding a two-domain protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins,” Arch. Microbiology (1996), 166, Nr. 2, pp. 76-82, XP-00957474.
Cremer, Josef et al., “Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes,” Applied and Environmental Microbiology, Jun. 1991, pp. 1746-1752, XP-000616281.
Eggeling, L. et al, “Improved l-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation,” Applied Microbiology and Biotechnology, Springer Verlag, Berlin, Germany (1998), 49, Nr. 1, pp. 24-30, XP-000918549.
Eikmans, Bernhard et al., “Molecular aspects of lysine, threonine, and isoleucine biosynthesis on Corynebacterium glutamicum,” Antonie van Leeuwenhoek, Dordrecht, Netherlands (1993), 634, Nr. 2, pp. 145-163, XP-000918559.
Patek, M., et al., “Identification and transcriptional analysis of the dapB-ORF2-dap-A-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes involved in L-lysine synthesis,” Biotechnology Letters (Nov. 1997) 19, Nr. 11, pp. 1113-1117, XP-000956453.
Eggeling Lothar
Eikmanns Bernhard
Möckel Bettina
Sahm Hermann
Tilg Yvonne
Degussa - AG
Pillsbury & Winthrop LLP
Rao Manjunath N.
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