Nucleotide sequences encoding the sucC and sucD genes

Details Nucleotide sequences encoding the sucC and sucD genes Nucleotide sequences encoding the sucC and sucD genes

2000-11-27

2003-09-23

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S183000, C435S252300, C435S252320, C435S320100, C536S023200

Reexamination Certificate

active

06623946

ABSTRACT:

This application claims priority from German Application No. 199 56 686.0, filed on Nov. 25, 1999, the subject matter of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides nucleotide sequences of coryneform bacteria coding for the genes sucC and sucD and a process for the fermentative production of amino acids, in particular L-lysine and L-glutamate, using bacteria in which the sucC- and/or sucD-gene is/are attenuated.
2. Background Information
L-amino acids, in particular L-lysine and L-glutamate, are used in human medicine and in the pharmaceutical industry, in the foodstuffs industry, and most particularly in animal nutrition.
It is known that amino acids can be produced by fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum
(
C. glutamicum
). On account of the great importance of amino acids efforts are constantly being made to improve production processes. Improvements in production may involve fermentation technology measures, such as, for example, stirring and provision of oxygen, or altering the composition of the nutrient media, such as for example the sugar concentration during fermentation or the working-up to the product form by, for example, ion exchange chromatography, or improving the intrinsic output properties of the microorganism itself.
Methods involving mutagenesis, selection and choice of mutants are used to improve the output properties. In this way strains are obtained that are resistant to antimetabolites or are auxotrophic for regulatory important metabolites, and that produce amino acids.
For some years recombinant DNA technology methods have also been used to improve Corynebacterium strains producing L-amino acids.
SUMMARY OF THE INVENTION
Object of the Invention
It is an object of the invention to provide new means for improving the fermentative production of amino acids, in particular L-lysine and L-glutamate.
Description of the Invention
Where L-amino acids or amino acids are mentioned hereinafter, it is to be understood that these terms refer to one or more amino acids, including their salts, selected from the group comprising L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine. L-lysine and L-glutamate are particularly preferred.
The present invention provides an isolated polynucleotide containing a polynucleotide sequence selected from the group comprising
a) a polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide, that contains the amino acid sequence of SEQ ID NO:2,
b) a polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide, that contains the amino acid sequence of SEQ ID NO:3,
c) a polynucleotide encoding a polypeptide, that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:2,
d) a polynucleotide encoding a polypeptide, that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:3,
e) a polynucleotide that is complementary to the polynucleotides of a), b), c) or d), and
f) a polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d) or e),
the polypeptide preferably exhibiting the activity of succinyl-CoA synthetase.
The present invention also provides the polynucleotide with the aforementioned features, which is preferably a replicable DNA containing:
(i) the nucleotide sequence shown in SEQ ID NO:l, or
(ii) at least one sequence that corresponds to the sequence (i) within the region of degeneration of the genetic code, or
(iii) at least one sequence that hybridizes with the sequence complementary to the sequence (i) or (ii), and optionally
(iv) functionally neutral sense mutations in (i).
The invention furthermore provides:
a polynucleotide as described above, containing the nucleotide sequence as shown in SEQ ID NO:1,
a polynucleotide according to claim 1, wherein the polynucleotide is a preferably recombinant DNA replicable in coryneform bacteria,
a vector containing parts of the polynucleotide according to the invention, but at least 15 successive nucleotides of the claimed sequence,
and coryneform bacteria in which the sucC- and/or sucD-gene is/are attenuated in particular by an insertion or deletion.
The present invention moreover provides polynucleotides that substantially comprise a polynucleotide sequence, that can be obtained by screening a corresponding gene library by means of hybridization, that contains the complete sucC- and/or sucD-gene with the polynucleotide sequence corresponding to SEQ ID NO:1 with a probe that contains the sequence of the aforementioned polynucleotide according to SEQ ID NO:1 or a fragment thereof, and isolation of the aforementioned DNA sequence.
Polynucleotides that contain the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate cDNA, nucleic acids and/or polynucleotides or genes in their full length that code for succinyl-CoA synthetase, and to isolate such cDNA or genes whose sequence has a high similarity to that of the succinyl-CoA synthetase genes.
Polynucleotides that contain the sequences according to the invention are furthermore suitable as primers, by means of which DNA can be produced by the polymerase chain reaction (PCR) from genes that code for succinyl-CoA synthetase. Such oligonucleotides serving as probes or primers contain at least 30, preferably at least 20, and most particularly preferably at least 15 successive nucleotides. Nucleotides with a length of at least 40 or 50 nucleotides are also suitable.
“Isolated” means separated from its natural environment.
“Polynucleotide” refers in general to polyribonucleotides and polydeoxyribonucleotides, in which connection these terms may refer to unmodified RNA or DNA or modified RNA or DNA.
By the term “polypeptides” are understood peptides or proteins that contain two or more amino acids bound via peptide bonds.
The polypeptides according to the invention include the polypeptides according to SEQ ID NO:2 and SEQ ID NO:3, in particular those having the biological activity of succinyl-CoA synthetase as well as those that are at least 70% identical to the polypeptide according to SEQ ID NO:2 or SEQ ID NO:3, and preferably at least 80% and particularly preferably at least 90% to 95% identical to the polypeptide according to SEQ ID NO:2 or SEQ ID NO:3 and that have the aforementioned activity.
The present invention furthermore relates to a process for the fermentative production of amino acids selected from the group comprising L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine, in particular L-lysine and L-glutamate, using coryneform bacteria that in particular already produce the amino acids, especially L-lysine and/or L-glutamate, and in which the nucleotide sequences coding for the sucC- and/or sucD-gene are attenuated, and in particular are expressed at a low level.
The term “attenuation” describes in this connection the reduction or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism that can be encoded by the corresponding DNA, by for example using a weak promoter or a gene and/or allele that encodes a corresponding enzyme with a low activity and/or inactivates the corresponding gene or enzyme (protein) and optionally combines these features.
The microorganisms that are the subject of the present invention can produce amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may be types of coryneform bacteria, in particular of the genus Corynebacterium. In the genus

Affiliated with

Also associated with

Rating

Nucleotide sequences encoding the sucC and sucD genes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Nucleotide sequences encoding the sucC and sucD genes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleotide sequences encoding the sucC and sucD genes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3057698