Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
2000-08-03
2002-10-08
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
Reexamination Certificate
active
06461852
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to dihydrodipicolinate synthase and dihydrodipicolinate reductase derived thermophilic Bacillus bacteria and genes coding for them.
2. Related Art
In the production of L-lysine by fermentation, strains isolated from nature or artificial mutants thereof have been used in order to improve the productivity. Many artificial mutant strains that produce L-lysine are known, and many of them are aminoethylcysteine (AEC) resistant strains and belong to the genus Brevibacterium, Corynebacterium, Bacillus or Escherichia. Further, various techniques have been disclosed for increasing the amino acid production, for example, use of a transformant obtained by using recombinant DNA (U.S. Pat. No. 4,278,765).
Dihydrodipicolinate synthase (abbreviated as “DDPS” hereinafter) is an enzyme that synthesizes dihydrodipicolinate through dehydration condensation of aspartic acid semialdehyde and pyruvic acid, and this reaction serves as an entrance of the branching into the L-lysine biosynthesis system in the biosynthesis of amino acids of aspartic acid type. Further, dihydrodipicolinate reductase (abbreviated as “DDPR” hereinafter) is known as one of important enzymes of the L-lysine biosynthesis system, which catalyzes the reaction in which the dihydrodipicoliniate generated in the aforementioned reaction is reduced to generate piperidinedicarboxylic acid.
As for microorganisms belonging to the genus Escherichia or Corynebacterium, the gene (dapA) which codes for DDPS has been cloned, and the nucleotide sequence thereof has also be determined. As for the genus Escherichia, methods for producing L-lysine by enhancing DDPS have been disclosed in Japanese Patent Laid-open Publication (Kokai) No. 56-18596/1981, U.S. Pat. No. 4,346,170 and Applied Microbiology and Biotechnology, 15, pp.227-331 (1982). Furthermore, a method for producing L-lysine using an Escherichia bacterium introduced with DDPS derived form Corynebacterium bacteria, which is known not to suffer feedback inhibition by L-lysine, has been disclosed in Korean Patent Publication No. 92-8382.
The gene coding for DDPR (dapB) has also been already obtained from the genus Escherichia (Bouvier, J. et al.,
J. Biol. Chem.,
259, 14829 (1984)) and the genus Corynebacterium (
Journal of Bacteriology,
175 (9), 2743-2749 (1993)). Furthermore, there has also been disclosed a method for improving production rate and productivity of L-lysine by enhancing the dapB gene derived from Corynebacterium bacterium together with the aspartokinase gene (WO96/40934).
The current mainstream of the L-lysine production is the fermentative production by using a coryneform bacterium or an Escherichia bacterium. In this production, however, enzymes required for the fermentation may be inactivated or the production bacteria may be killed due to temperature increase in the medium during the fermentation, and thus it is necessary to cool the medium during the fermentation.
By the way, enzymes and proteins produced by thermophilic bacteria are generally stable at elevated temperatures, and also stable against pH variation or organic solvents. Therefore, applications thereof as diagnostic regents, industrial catalysts and so forth have been highly developed. If it becomes possible to produce L-lysine by fermentation at elevated temperatures by utilizing such stability and durability of enzymes derived from thermophilic bacteria, the cooling of the medium becomes unnecessary, and therefore the cost for cooling during the fermentation can be reduced. Moreover, if fermentation at elevated temperatures is realized, it is expected that the reaction rate may also be improved
SUMMARY OF THE INVENTION
The present invention is accomplished in view of the aforementioned technical aspect, and its object is to obtain genes of the L-lysine biosynthesis system of thermophilic bacteria and thereby provide novel methods for producing L-lysine.
The inventors of the present invention assiduously studied in order to achieve the aforementioned object. As a result, they successfully isolated genes that coded for DDPS and DDPR from
Bacillus methanolicus,
which is one of the thermophilic Bacillus bacteria, and determined the nucleotide sequences of these genes. Thus, they accomplished the present invention.
That is, the present invention provides the followings.
(1) A protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing, or (B) a protein which has an amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate synthase activity.
(2) A DNA which codes for a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing, or
(B) a protein which has an amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate synthase activity.
(3) The DNA according to (2), which is a DNA defined in the following (a) or (b):
(a) a DNA which has a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 924 in SEQ ID NO: 1 shown in Sequence Listing; or
(b) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 924 in SEQ ID NO: 1 shown in Sequence Listing under a stringent condition, and codes for a protein having dihydrodipicolinate synthase activity.
(4) The DNA according to (3), wherein the stringent condition is a condition in which washing is performed at 60° C., 1×SSC and 0.1% SDS.
(5) A protein defined in the following (C) or (D):
(C) a protein which has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing, or
(D) a protein which has an amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate reductase activity.
(6) A DNA which codes for a protein defined in the following (C) or (D):
(C) a protein which has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing, or
(D) a protein which has an amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate reductase activity.
(7) The DNA according to (6), which is a DNA defined in the following (c) or (d):
(c) a DNA which has a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 798 in SEQ ID NO: 3 shown in Sequence Listing; or
(d) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 798 in SEQ ID NO: 3 shown in Sequence Listing under a stringent condition, and codes for a protein having dihydrodipicolinate reductase activity.
(8) The DNA according to (7), wherein the stringent condition is a condition in which washing is performed at 60° C., 1×SSC and 0.1% SDS.
(9) A microorganism which is introduced with the DNA according to (2) or the DNA according to (6) or the both in a form that allows expression of a protein encoded by each DNA.
(10) A method for producing L-lysine, which comprises culturing the microorganism according to (9) in a medium to produce and accumulate L-lysine in the medium, and collecting the L-lysine from the medium.
In the present invention, the expression of “to have dihydrodipicolinate synthase activity” is used to mean to have an activity for catalyzing the reaction of dehydration condensation of aspartic acid semialdehyde and pyruvic acid to generate dihydrodipicolinate. The expression of “to have dihydrodipicolinate reductase activity” is used to mean to have an activity for catalyzing the reaction of reduction of dihydrodipicolinate
Kawahara Yoshio
Sugimoto Shinichi
Tsujimoto Nobuharu
Yasueda Hisashi
Achutamurthy Ponnathapu
Ajinomoto Co. Inc.
Kerr Kathleen M
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
LandOfFree
Dihydrodipicolinate synthase from Bacillus methanolicus does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Dihydrodipicolinate synthase from Bacillus methanolicus, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Dihydrodipicolinate synthase from Bacillus methanolicus will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2932164