Process for the preparation of L-amino acids by fermentation...

Details Process for the preparation of L-amino acids by fermentation... Process for the preparation of L-amino acids by fermentation...

1999-07-29

2002-03-26

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S183000, C435S252300, C435S252320, C435S320100, C536S023200

Reexamination Certificate

active

06361986

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims priority under 35 U.S.C. §119 to German application 199 24 365.4, filed on May 27, 1999.
BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Background Information
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 Jäger 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
SUMMARY 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.
DESCRIPTION OF THE INVENTION
L-Amino acids are used in animal nutrition, in human medicine and in the pharmaceutical industry. It is therefore of general interest to provide novel improved processes for the preparation of L-amino acids.
When L-lysine or lysine is mentioned in the following text, it is understood as meaning not only the base but also the salts, e.g. lysine monohydrochloride or lysine sulfate.
The invention provides a preferably recombinant DNA originating from Corynebacterium which is capable of replication in coryneform microorganisms and which at least contains the nucleotide sequence coding for the accDA gene shown in SEQ ID No. 1.
The invention also provides a DNA capable of replication, as claimed in claim 1, with:
(i) the nucleotide sequence shown in SEQ ID No. 1, or
(ii) at least one sequence corresponding to the sequence (i) within the region of degeneracy of the genetic code, or
(iii) at least one sequence hybridizing with the sequence complementary to the sequence (i) or (ii), and optionally
(vi) [sic] neutral sense mutations in (i).
The invention also provides coryneform microorganisms, especially of the genus Corynebacterium, transformed by the introduction of said DNA capable of replication.
The invention further relates to a process for the preparation of L-amino acids by fermentation using corynebacteria which, in particular, already produce the L-amino acids and in which the nucleotide sequences coding for the accDA gene are amplified and, in particular, overexpressed.
Finally, the invention also provides a process for the amplification of acyl-CoA carboxylase in corynebacteria by joint overexpression of the novel accDA gene according to the invention and the known accBC gene.
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, for example 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.
The microorganisms which the present invention provides can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch or cellulose or from glycerol and ethanol. Said microorganisms can be representatives of 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 L-amino acids.
Suitable strains of the genus Corynebacterium, especially of the species
Corynebacterium glutamicum
for example, 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 L-amino acid-producing mutants or strains prepared therefrom, for example:
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
The inventors have succeeded in isolating the novel accDA gene from
C. glutamicum.
The accDA gene or other genes are isolated from
C. glutamicum
by first constructing a gene library of this microrganism [sic] in
E. coli.
The construction of gene libraries is documented in generally well-known textbooks and handbooks. Examples which may be mentioned are the textbook by Winnacker entitled From Genes to Clones, Introduction to Gene Technology (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al. entitled Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well-known gene library is that of the
E. coli
K-12 strain W3110 constructed by Kohara et al. (Cell 50, 495-508 (1987)) in &lgr; vectors. Bathe et al. (Molecular and General Genetics 252, 255-265 (1996)) describe a gene library of
C. glutamicum
ATCC13032 constructed using cosmid vector SuperCos I (Wahl et al., Proceedings of the National Academy of Sciences USA 84, 2160-2164 (1987)) in the
E. coli
K-12 strain NM554 (Raleigh et al., Nucleic Acids Research 16, 1563-1575 (1988)). Börmann et al. (Molecular Microbiology 6(3), 317-326) in turn describe a gene library of
C. glutamicum
ATCC13032 using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). A gene library of
C. glutam

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