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
1999-06-03
2002-03-12
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...
Reexamination Certificate
active
06355454
ABSTRACT:
SUMMARY OF THE INVENTION
The inventors set themselves the object of providing novel measures for the improved fermentative production of other L-amino acids.
DESCRIPTION OF THE INVENTION
L-Amino acids are used in animal nutrition, human medicine and the pharmaceuticals industry. There is accordingly general interest in providing improved processes for the production of L-amino acids.
When L-amino acids are mentioned below, they are intended to mean the protein-forming amino acids L-lysine, L-threonine, L-isoleucine, L-valine, L-proline, L-tryptophan and optionally the salts thereof and also L-homoserine, in particular L-lysine, L-threonine and L-tryptophan.
The present invention provides a process for the fermentative production of L-amino acids using coryneform bacteria, which in particular already produce the corresponding L-amino acids and in which the nucleotide sequence coding for the enzyme glutamate dehydrogenase is amplified, in particular overexpressed.
Preferred embodiments are stated in the claims.
In this connection, the term “amplification” describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are coded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene which codes for a corresponding enzyme having elevated activity and optionally by combining these measures.
The microorganisms provided by the present invention are capable of producing L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium,
Corynebacterium glutamicum
may in particular be mentioned, which is known in specialist circles for its ability to produce L-amino acids. Suitable strains of the genus Corynebacterium, in particular of the species
Corynebacterium glutamicum
, are the known wild type 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 mutants or strains produced therefrom, such as for example
the L-lysine producing strains
Corynebacterium glutamicum
FERM-P 1709
Brevibacterium flavum
FERM-P 1708 and
Brevibacterium lactofermentum
FERM-P 1712,
or the L-threonine producing strains
Corynebacterium glutamicum
FERM-P 5835
Brevibacterium flavum
FERM-P 4164 and
Brevibacterium lactofermentum
FERM-P 4180,
or the L-isoleucine producing strains
Corynebacterium glutamicum
FERM-P 756
Brevibacterium flavum
FERM-P 759 and
Brevibacterium lactofermentum
FERM-P 4192
or the L-valine producing strains
Brevibacterium flavum
FERM-P 512 and
Brevibacterium lactofermentum
FERM-P 1845,
and the L-tryptophan producing strains
Corynebacterium glutamicum
FERM-BP 478
Brevibacterium flavum
FERM-BP 475 and
Brevibacterium lactofermentum
FERM-P 7127. It is noted that Corynebacterium and Brevibacterium are both considered to be corynebacteria in the state of the art at the time the invention was made. Furthermore,
Corynebacterium glutamacin
and
Brevibacterium lactofermentum
were considered to be the same species.
The inventors discovered that, after overexpression of L-glutamate dehydrogenase, coryneform bacteria produce L-amino acids in an improved manner, wherein L-glutamic acid is not claimed here.
The glutamate dehydrogenase gene of
C. glutamicum
described by Börmann et al. (
Molecular Microbiology
6, 317-326 (1992)) may be used according to the invention. The glutamate dehydrogenase gene from other microorganisms, such as for example that from
Peptostreptococcus asaccharolyticus
, which has been described by Snedecor et al. (
Journal of Bacteriology
173, 6162-6167 (1991)), is also suitable. Alleles of the stated genes arising from the degeneracy of the genetic code or from functionally neutral sense mutations may also be used.
Overexpression may be achieved by increasing the copy number of the corresponding genes, or the promoter and regulation region located upstream from the structural gene may be mutated. Expression cassettes incorporated upstream from the structural gene act in the same manner. It is additionally possible to increase expression during fermentative L-amino acid production by means of inducible promoters. Expression is also improved by measures to extend the lifetime of the mRNA. Enzyme activity is moreover amplified by preventing degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids in a variable copy number or be integrated in the chromosome and amplified. Alternatively, overexpression of the genes concerned may also be achieved by modifying the composition of the nutrient media and culture conditions.
The person skilled in the art will find guidance in this connection inter alia in 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 EPS 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler (
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 Jensen and Hammer (
Biotechnology and Bioengineering
58, 191-195 (1998)), in Makrides (
Microbiological Reviews
60:512-538 (1996)) and in known textbooks of genetics and molecular biology.
Examples of plasmids by means of which glutamate dehydrogenase may be overexpressed are pEK1.9gdh-1 and pEKExpgdh, which are present in strains ATCC13032/pEK1.9gdh-1 and DH5&agr;/pEKExpgdh. Plasmid pEK1.9gdh-1 is a shuttle vector, which contains the NAD-dependent glutamate dehydrogenase gene of
C. glutamicum
. Plasmid pEKExpgdh is a shuttle vector, which contains the NAD-dependent glutamate dehydrogenase gene of
Peptostreptococcus asaccharolyticus.
It may additionally be advantageous for the production of the corresponding L-amino acids to overexpress one or more enzymes of the particular amino acid biosynthesis pathway as well as glutamate dehydrogenase. Thus, for example
the dapA gene which codes for dihydrodipicolinate synthase may additionally be overexpressed in order to improve L-lysine producing coryneform bacteria (EP-B 0197335),
the gene which codes for acetohydroxy acid synthase may additionally be overexpressed in order to improve L-valine producing coryneform bacteria (EP-B 0356739),
the gene which codes for anthranilic acid phosphoribosyl transferase may additionally be overexpressed in order to improve L-tryptophan producing coryneform bacteria (EP-B 0124048),
the gene which codes for homoserine dehydrogenase may additionally be overexpressed in order to improve coryneform bacteria which produce L-homoserine or L-threonine or L-isoleucine (EP-A 0131171).
It may furthermore be advantageous for the production of the corresponding L-amino acid to switch off unwanted secondary reactions in addition to overexpressing glutamate dehydrogenase (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
For the purposes of L-amino acid production, the microorganisms according to the invention may be cultivated continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process. A summary of known cultivation methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must adequatel
De Graaf Albert
Eggeling Lothar
Marx Achim
Mockel Bettina
Pfefferle Walter
Achutamurthy Ponnathapu
Degussa -Huls AG
Kerr Kathleen
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