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
1999-07-29
2002-04-30
Prouty, Rebecca E. (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...
C435S109000, C435S113000, C435S115000, C435S116000, C435S189000, C435S232000, C435S252300, C435S252310, C435S320100
Reexamination Certificate
active
06379934
ABSTRACT:
The invention provides a process for the fermentative preparation of L-amino acids, in particular lysine, using coryneform bacteria in which the accBC gene is amplified.
Prior Art
L-amino acids, in particular 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 fermentation using strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the high degree of importance of these products, a constant effort is made to improve the method of preparation. Process improvements may be based on fermentation engineering steps such as, for example, stirring and supplying with oxygen, or the composition of the nutrient medium such as, for example, the concentration of sugar during fermentation, or the working up process aimed at obtaining the product itself by, for example, ion-exchange chromatography or the intrinsic power of the microorganism itself.
The methods of mutagenesis, selection and mutant choice are used to improve the power of these microorganisms. Strains which are resistant to antimetabolites such as, for example, the lysine analogon S-(2-aminoethyl)-cysteine or which are auxotrophic for significant regulatory amino acids, and produce L-amino acids, are obtained in this way.
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. Review articles about this topic can 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 Sham et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
Object of the Invention
The inventor has formulated the object as the provision of new steps for the improved fermentative preparation of L-amino acids, in particular L-Lysine.
DESCRIPTION OF THE INVENTION
L-amino acids, in particular L-lysine, are used in animal nutrition, in human medicine and in the pharmaceuticals industry. There is, therefore, general interest in the provision of new, improved methods for preparing these compounds.
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 process for the fermentative preparation of L-amino acids, in particular L-lysine, using coryneform bacteria which in particular already produce the desired amino acid and in which the subunits carrying the biotin-carboxyl carrier protein domain and the biotin-carboxylase domain in the nucleotide sequence encoding the enzyme acetyl-CoA carboxylase is amplified, in particular is overexpressed.
Preferred embodiments are given in the Claims.
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 by using a strong promoter or a gene which encodes for a corresponding enzyme with high activity and optionally combining these measures.
The microorganisms which are the object of the present invention can produce L-amino acids, in particular L-lysine from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They are members of the group of coryneform bacteria, in particular those of the genus Corynebacterium. With regard to the genus Corynebacterium, 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 the recognized wild strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020 and the mutants and strains prepared therefrom which can produce L-amino acids, in particular L-lysine, such as, for example
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Brevibacterium flavum FERM-P 6463 and
Brevibacterium flavum FERM-P 6464.
The accBC gene encodes for a subunit of acetyl-CoA carboxylase which carries a biotin-carboxyl carrier protein domain and a biotin-carboxylase domain. The nucleotide sequence of the accBC gene in Corynebacterium glutamicum was determined by Jäger et al. (Archives of Microbiology 166, 76-82 (1996)) and it is generally available at the Databank of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany) under Accession Number U35023.
The accBC gene of C. glutamicum described by Jäger et al. (Archives of Microbiology 166, 76-82 (1996)) can be used in accordance with the invention. Furthermore, alleles of the accBC gene which are produced as a result of the degenerativeness of the genetic code or by function-neutral sense mutations can also be used.
To produce an overexpression, the copy number of the corresponding gene can be increased or the promoter and regulation region or the ribosome bonding site, which are located upstream of the coding sequence, can be mutated. Expression cassettes, which are incorporated upstream of the coding sequence, operate in the same way. It is also possible to increase expression during the course of fermentative L-lysine production with inducible promoters. Expression is also improved by measures aimed at prolonging the lifetime of m-RNA. Furthermore, enzyme activity can also be amplified by inhibiting degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids with different copy numbers or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes concerned may also be achieved by modifying the composition of the media and management of the culture.
Instructions for these procedures may be found by a person skilled in the art in, inter alia, Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European Patent EP-B 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in Patent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in Japanese Patent JP-A- 10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in well-known textbooks relating to genetics and molecular biology.
An example of a plasmid with the aid of which the accBC gene can be overexpressed is pZ1accBC (FIG.
1
), which is contained within the strain MH20-22B/pZ1accBC. Plasmid pZ1accBC is an
E.coli
—C. glutamicum shuttle vector based on Plasmid pZ1 (Menkel et al., Applied and Environmental Microbiology 55(3), 684-688 (1989)) which carries the accBC gene.
In addition, it may be advantageous for the production of L-amino acids to overexpress one or more enzymes in the corresponding biosynthetic pathway, in addition to the accBC gene. Thus, for example, when preparing L-lysine
the dapA gene encoding for dihydrodipicolinate synthase can be simultaneously overexpressed (EP-B 0 197 335), or
a S-(2-aminoethyl)-cysteine-resistance promoting DNA fragment can be simultaneously a
Eggeling Lothar
Eikmanns Bernd
Mockel Bettina
Pfefferle Walter
Sahm Hermann
Degussa - AG
Prouty Rebecca E.
Rao Manjunath N.
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