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
1999-07-14
2001-03-13
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252320, C435S320100
Reexamination Certificate
active
06200785
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to L-lysine-producing strains of corynebacteria with amplified lysE gene (lysine export carrier gene), in which strains additional genes, chosen from the group comprising the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the dapB gene (dihydrodipicolinate reductase gene) and the pyc gene, but especially the dapA gene and the lysC gene (aspartate kinase gene), are amplified and, in particular, overexpressed, and to a process for the preparation of L-lysine.
2. Background Information
L-Lysine is a commercially important L-amino acid which is used especially as a feed additive in animal nutrition. The need has been steadily increasing in recent years.
L-Lysine is prepared by a fermentation process with L-lysine-producing strains of corynebacteria, especially Corynebacterium glutamicum. Because of the great importance of this product, attempts are constantly being made to improve the preparative process. Improvements to the process 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 Microrganism 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. S-(2-aminoethyl)cysteine, or auxotrophic for amino acids, e.g. L-leucine, and produce L-lysine.
Methods of recombinant DNA technology have also been used for some years in order to improve L-lysine-producing strains of Corynebacterium glutamicum by amplifying individual biosynthesis genes and studying the effect on L-lysine production.
Thus EP-A-0 088 166 reports the increase in productivity, after amplification, of a DNA fragment conferring resistance to aminoethylcysteine. EP-B-0 387 527 reports the increase in productivity, after amplification, of an lysC allele coding for a feedback-resistant aspartate kinase. EP-B-0 197 335 reports the increase in productivity, after amplification, of the dapA gene coding for dihydrodipicolinate synthase. EP-A-0 219 027 reports the increase in productivity, after amplification, of the asd gene coding for aspartate semialdehyde dehydrogenase. Pisabarro et al. (Journal of Bacteriology 175(9), 2743-2749 (1993)) describe the dapB gene coding for dihydrodipicolinate reductase.
The effect of the amplification of primary metabolism genes on L-lysine production has also been studied. Thus EP-A-0 219 027 reports the increase in productivity, after amplification, of the aspc gene coding for aspartate aminotransferase. EP-B-0 143 195 and EP-B-0 358 940 report the increase in productivity, after amplification, of the ppc gene coding for phosphoenolpyruvate carboxylase. Offenlegungsschrift DE-A-198 31 609 reports the increase in productivity, after amplification, of the pyc gene coding for pyruvate carboxylase.
Finally, Offenlegungsschrift DE-A-195 48 222 describes that an increased activity of the L-lysine export carrier coded for by the lysE gene promotes lysine production.
In addition to these attempts to amplify an individual gene, attempts have also been made to amplify two or more genes simultaneously and thereby to improve L-lysine production in corynebacteria. Thus Offenlegungsschrift DE-A-38 23 451 reports the increase in productivity, after simultaneous amplification, of the asd gene and the dapA gene from
Escherichia coli.
Offenlegungsschrift DE-A-39 43 117 discloses the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistance and of the dapA gene by means of plasmid pJC50. EP-A-0 841 395 particularly reports the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant (sic) and of the dapB gene; further improvements could be achieved by additional amplification of the dapB, lysA and ddh genes. EP-A-0 854 189 describes the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistance and of the dapA, dapB, lysA and aspc genes. EP-A-0 857 784 particularly reports the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistance and of the lysA gene; a further improvement could be achieved by additional amplification of the ppc gene.
It is clear from the many processes described in the state of the art that there is a need for the development of novel approaches and for the improvement of existing processes for lysine production with corynebacteria.
SUMMARY OF THE INVENTION
Object of the Invention
The object of the invention consists in using novel measures to provide improved L-lysine-producing strains of corynebacteria.
DESCRIPTION OF THE INVENTION
L-Lysine is a commercially important L-amino acid which is used especially as a feed additive in animal nutrition.
When L-lysine or lysine is mentioned in the following text, it is understood as meaning not only the base but also the appropriate salts, e.g. lysine hydrochloride or lysine sulfate.
The invention provides L-lysine-producing strains of corynebacteria amplified lysE gene (lysine export carrier gene), wherein they additionally contain genes chosen from the group comprising the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the dapb gene (dihydrodipicolinate reductase gene) and the pyc gene (pyruvate carboxylase gene), but especially the dapA gene and the lysC gene, which, individually or together, are amplified and, preferably, overexpressed.
The novel DNA sequence located upstream (5′ end) from the dapB gene has also been found which carries the −35 region of the dapB promoter and is advantageous for the expression of the dapB gene. It is shown as SEQ ID No. 1.
A corresponding DNA capable of replication, with the nucleotide sequence shown in SEQ ID No. 1, is therefore claimed as well.
The invention also provides the MC20 or MA16 mutations of the dapA promoter shown in SEQ ID No. 5 and SEQ ID No. 6, deposited under DSM12868 and DSM12867 respectively.
The invention also provides L-lysine-producing strains of corynebacteria with amplified lysE gene, wherein additionally the dapA and dapB genes are simultaneously amplified and, in particular, overexpressed.
Finally, the invention also provides L-lysine-producing strains of corynebacteria with amplified lysE gene, wherein additionally the dapA and lysc genes are simultaneously amplified and, in particular, overexpressed.
In this context the term “amplification” describes the increase in the intracellular activity, in a microorganism, of one or more enzymes which are coded for by the appropriate DNA, by increasing the copy number of the gene(s), using a strong promoter or using a gene coding for an appropriate enzyme with a high activity, and optionally combining these measures.
A process for the preparation of L-lysine by the fermentation of these corynebacteria is also claimed.
The microorganisms which the present invention provides can prepare L-lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch or cellulose or from glycerol and ethanol, especially from glucose or sucrose. Said microorganisms are corynebacteria, especially of the genus Corynebacterium. The species
Corynebacterium glutamicum
may be mentioned in particular in the genus Corynebacterium, being known to those skilled in the art for its ability to produce amino acids. This species includes wild-type strains such as
Corynebacterium glutamicum
ATCC13032, Brevibacterium flavum ATCC14067, Corynebacterium melassecola ATCC17965 and strains or mutants derived therefrom. Examples of L-lysine-producing mutants of corynebacteria are for example:
Corynebacterium glutamicum
FERM-P 1709
Brevibacterium flavum
FERM-P 1708
Brevibacterium lactofermentum
FE
Eggeling Lothar
Hans Stephan
Kreutzer Caroline
Mockel Bettina
Patek Miroslav
Degussa-Huls Aktiengesellschaft
Ketter James
Pillsbury Madison & Sutro LLP
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