Patent Grant
7332310
The present application claims benefit of Japanese Patent Application Nos. Hei. 11-377484 (filed Dec. 16, 1999), 2000-159162 (filed Apr. 7, 2000) and 2000-280988 (filed Aug. 3, 2000), the entire contents of each of which is incorporated herein by reference.
The contents of the attached 3 CD-R compact discs (COPY 1 REPLACEMENT Jun. 12, 2006, COPY 2 REPLACEMENT Jun. 21, 2006 and COPY 3 REPLACEMENT Jun. 12, 2006) are incorporated herein by reference in their entirety. The attached discs contain an identical copy of a file “249-125.txt” which were created Jun. 8, 2001, and are each 25904 KB. The Sequence Listings filed in the parent application Ser. No. 09/738,626 on Dec. 18, 2000 and Jun. 29, 2001, are incorporated herein by reference. The Sequence Listing contained on the attached discs are the same as the “paper” and computer readable copies of the Sequence Listing filed in the parent application Ser. No. 09/738,626 on Dec. 18, 2000 and Jun. 29, 2001.
1. Field of the Invention
The present invention relates to novel polynucleotides derived from microorganisms belonging to coryneform bacteria and fragments thereof, polypeptides encoded by the polynucleotides and fragments thereof, polynucleotide arrays comprising the polynucleotides and fragments thereof, computer readable recording media in which the nucteotide sequences of the polynucleotide and fragments thereof have been recorded, and use of them as well as a method of using the polynucleotide and/or polypeptide sequence information to make comparisons.
2. Brief Description of the Background Art
Coryneform bacteria are used in producing various useful substances, such as amino acids, nucleic acids, vitamins, saccharides (for example, ribulose), organic acids (for example, pyruvic acid), and analogues of the above-described substances (for example, N-acetylamino acids) and are very useful microorganisms industrially. Many mutants thereof are known.
For example, Corynebacterium glutamicum is a Gram-positive bacterium identified as a glutamic acid-producing bacterium, and many amino acids are produced by mutants thereof. For example, 1,000,000 ton/year of L-glutamic acid which is useful as a seasoning for umami (delicious taste), 250,000 ton/year of L-lysine which is a valuable additive for livestock feeds and the like, and several hundred ton/year or more of other amino acids, such as L-arginine, L-proline, L-glutamine, L-tryptophan, and the like, have been produced in the world (Nikkei Bio Yearbook 99, published by Nikkei BP (1998)).
The production of amino acids by Corynebacterium glutamicum is mainly carried out by its mutants (metabolic mutants) which have a mutated metabolic pathway and regulatory systems. In general, an organism is provided with various metabolic regulatory systems so as not to produce more amino acids than it needs. In the biosynthesis of L-lysine, for example, a microorganism belonging to the genus Corynebacterium is under such regulation as preventing the excessive production by concerted inhibition by lysine and threonine against the activity of a biosynthesis enzyme common to lysine, threonine and methionine, i.e., an aspartokinase, (J. Biochem., 65: 849-859 (1969)). The biosynthesis of arginine is controlled by, repressing the expression of its biosynthesis gene by arginine so as not to biosynthesize an excessive amount of arginine (Microbiology, 142: 99-108 (1996)). It is considered that these metabolic regulatory mechanisms are deregulated in amino acid-producing mutants. Similarly, the metabolic regulation is deregulated in mutants producing nucleic acids, vitamins, saccharides, organic acids and analogues of the above-described substances so as to improve the productivity of the objective product.
However, accumulation of basic genetic, biochemical and molecular biological data on coryneform bacteria is insufficient in comparison with Escherichia coli, Bacillus subtilis, and the like. Also, few findings have been obtained on mutated genes in amino acid-producing mutants. Thus, there are various mechanisms, which are still unknown, of regulating the growth and metabolism of these microorganisms.
A chromosomal physical map of Corynebacterium glutamicum ATCC 13032 is reported and it is known that its genome size is about 3,100 kb (Mol. Gen. Genet., 252: 255-265 (1996)). Calculating on the basis of the usual gene density of bacteria, it is presumed that about 3,000 genes are present in this genome of about 3,100 kb. However, only about 100 genes mainly concerning amino acid biosynthesis genes are known in Corynebacterium glutamicum, and the nucleotide sequences of most genes have not been clarified hitherto.
In recent years, the full nucleotide sequence of the genomes of several microorganisms, such as Escherichia coli, Mycobacterium tuberculosis, yeast, and the like, have been determined (Science, 277: 1453-62 (1997); Nature, 393: 537-544 (1998); Nature, 387: 5-105 (1997)). Based on the thus determined full nucleotide sequences, assumption of gene regions and prediction of their function by comparison with the nucleotide sequences of known genes have been carried out. Thus, the functions of a great number of genes have been presumed, without genetic, biochemical or molecular biological experiments.
In recent years, moreover, techniques for monitoring expression levels of a great number of genes simultaneously or detecting mutations, using DNA chips, DNA arrays or the like in which a partial nucleic acid fragment of a gene or a partial nucleic acid fragment in genomic DNA other than a gene is fixed to a solid support, have been developed. The techniques contribute to the analysis of microorganisms, such as yeasts, Mycobacterium tuberculosis, Mycobacterium bovis used in BCG vaccines, and the like (Science, 278: 680-686 (1997); Proc. Natl. Acad. Sci. USA, 96: 12833-38 (1999); Science, 284: 1520-23 (1999)).
An object of the present invention is to provide a polynucleotide and a polypeptide derived from a microorganism of coryneform bacteria which are industrially useful, sequence information of the polynucleotide and the polypeptide, a method for analyzing the microorganism, an apparatus and a system for use in the analysis, and a method for breeding the microorganism.
The present invention provides a polynucleotide and an oligonucleotide derived from a microorganism belonging to coryneform bacteria, oligonucleotide arrays to which the polynucleotides and the oligonucleotides are fixed, a polypeptide encoded by the polynucleotide, an antibody which recognizes the polypeptide, polypeptide arrays to which the polypeptides or the antibodies are fixed, a computer readable recording medium in which the nucleotide sequences of the polynucleotide and the oligonucleotide and the amino acid sequence of the polypeptide have been recorded, and a system based on the computer using the recording medium as well as a method of using the polynucleotide and/or polypeptide sequence information to make comparisons.
This application is based on Japanese applications No. Hei. 11-377484 filed on Dec. 16, 1999, No. 2000-159162 filed on Apr. 7, 2000 and No. 2000-280988 filed on Aug. 3, 2000, the entire contents of which are incorporated hereinto by reference.
From the viewpoint that the determination of the full nucleotide sequence of Corynebacterium glutamicum would make it possible to specify gene regions which had not been previously identified, to determine the function of an unknown gene derived from the microorganism through comparison with nucleotide sequences of known genes and amino acid sequences of known genes, and to obtain a useful mutant based on the presumption of the metabolic regulatory mechanism of a useful product by the microorganism, the inventors conducted intensive studies and, as a result, found that the complete genome sequence of Corynebacterium glutamicum can be determined by applying the whole genome shotgun method.
Specifically, the present invention relates to the following (1) to (65):
As used herein, for example, the at least two polynucleotides can be at least two of the first polynucleotides, at least two of the second polynucleotides, at least two of the third polynucleotides, or at least two of the first, second and third polynucleotides.
As used herein, for example, the at least two polynucleotides can be at least two of the first polynucleotides, at least two of the second polynucleotides, at least two of the third polynucleotides, or at least two of the first, second and third polynucleotides.
(8) A polynucleotide comprising any one of the nucleotide sequences represented by SEQ ID NOS:2 to 3431, or a polynucleotide which hybridizes with the polynucleotide under stringent conditions.
As used herein, the term “proteome”, which is a coined word by combining “protein” with “genome”, refers to a method for examining of a gene at the polypeptide level.
The present invention will be described below in more detail, based on the determination of the full nucleotide sequence of coryneform bacteria.
1. Determination of Full Nucleotide Sequence of Coryneform Bacteria
The term “coryneform bacteria” as used herein means a microorganism belonging to the genus Corynebacterium, the genus Brevibacterium or the genus Microbacterium as defined in Bergeys Manual of Determinative Bacteriology, 8: 599 (1974).
Examples include Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Brevibacterium saccharolyticum, Brevibacterium immariophilum, Brevibacterium roseum, Brevibacterium thiogenitalis, Microbacterium ammoniaphilum, and the like.
Specific examples include Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium callunae ATCC 15991, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13060, Corynebacterium glutamicum ATCC 13826 (prior genus and species: Brevibacterium flavum, or Corynebacterium lactofermentum) Corynebacterium glutamicum ATCC 14020 (prior genus and species: Brevibacterium divaricatum), Corynebacterium glutamicum ATCC 13869 (prior genus and species: Brevibacterium lactofermentum), Corynebacterium herculis ATCC 13868, Corynebacterium lilium ATCC 15990, Corynebacterium melassecola ATCC 17965, Corynebacterium thermoaminogenes FERM 9244, Brevibacterium saccharolyticum ATCC 4066, Brevibacterium immariophilum ATCC 14068, Brevibacterium roseum ATCC 13825, Brevibacterium thiogenitalis ATCC 19240, Microbacterium ammoniaphilum ATCC 15354, and the like.
(1) Preparation of Genome DNA of Coryneform Bacteria
Coryneform bacteria can be cultured by a conventional method.
Any of a natural medium and a synthetic medium can be used, so long as it is a medium suitable for efficient culturing of the microorganism, and it contains a carbon source, a nitrogen source, an inorganic salt, and the like which can be assimilated by the microorganism.
In Corynebacterium glutamicum, for example, a BY medium (7 g/l meat extract, 10 g/l peptone, 3 g/l sodium chloride, 5 g/l yeast extract, pH 7.2) containing 1% of glycine and the like can be used. The culturing is carried out at 25 to 35° C. overnight.
After the completion of the culture, the cells are recovered from the culture by centrifugation. The resulting cells are washed with a washing solution.
Examples of the washing solution include STE buffer (10.3% sucrose, 25 mmol/l Tris hydrochloride, 25 mmol/l ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”), pH 8.0), and the like.
Genome DNA can be obtained from the washed cells according to a conventional method for obtaining genome DNA, namely, lysing the cell wall of the cells using a lysozyme and a surfactant (SDS, etc.), eliminating proteins and the like using a phenol solution and a phenol/chloroform solution, and then precipitating the genome DNA with ethanol or the like. Specifically, the following method can be illustrated.
The washed cells are suspended in a washing solution containing 5 to 20 mg/l lysozyme. After shaking, 5 to 20% SDS is added to lyse the cells. In usual, shaking is gently performed at 25 to 40° C. for 30 minutes to 2 hours. After shaking, the suspension is maintained at 60 to 70° C. for 5 to 15 minutes for the lysis.
After the lysis, the suspension is cooled to ordinary temperature, and 5 to 20 ml of Tris-neutralized phenol is added thereto, followed by gently shaking at room temperature for 15 to 45 minutes.
After shaking, centrifugation (15,000×g, 20 minutes, 20° C.) is carried out to fractionate the aqueous layer.
After performing extraction with phenol/chloroform and extraction with chloroform (twice) in the same manner, 3 mol/l sodium acetate solution (pH 5.2) and isopropanol are added to the aqueous layer at 1/10 times volume and 2 times volume, of the aqueous layer, respectively, followed by gently stirring to precipitate the genome DNA.
The genome DNA is dissolved again in a buffer containing 0.01 to 0.04 mg/ml RNase. As an example of the buffer, TE buffer (10 mmol/l Tris hydrochloride, 1 mol/l EDTA, pH 8.0) can be used. After dissolving, the resultant solution is maintained at 25 to 40° C. for 20 to 50 minutes and then extracted successively with phenol, phenol/chloroform and chloroform as in the above case.
After the extraction, isopropanol precipitation is carried out and the resulting DNA precipitate is washed with 70% ethanol, followed by air drying, and then dissolved in TE buffer to obtain a genome DNA solution.
(2) Production of Shotgun Library
A method for produce a genome DNA library using the genome DNA of the coryneform bacteria prepared in the above (1) include a method described in Molecular Cloning, A laboratory Manual, Second Edition (1989) (hereinafter referred to as “Molecular Cloning, 2nd ed.”). In particular, the following method can be exemplified to prepare a genome DNA library appropriately usable in determining the full nucleotide sequence by the shotgun method.
To 0.01 mg of the genome DNA of the coryneform bacteria prepared in the above (1) a buffer, such as TE buffer or the like, is added to give a total volume of 0.4 ml. Then, the genome DNA is digested into fragments of 1 to 10 kb with a sonicator (Yamato Powersonic Model 50). The treatment with the sonicator is performed at an output off 20 continuously for 5 seconds.
The resulting genome DNA fragments are blunt-ended using DNA blunting kit (manufactured by Takara Shuzo) or the like.
The blunt-ended genome fragments are fractionated by agarose gel or polyacrylamide gel electrophoresis and genome fragments of 1 to 2 kb are Cut out from the gel.
To the gel, 0.2 to 0.5 ml of a buffer for eluting DNA, such as MG elution buffer (0.5 mol/l ammonium acetate, 10 mmol/l magnesium acetate, 1 mmol/l EDTA, 0.1% SDS) or the like, is added, followed by shaking at 25 to 40° C. overnight to elute DNA.
The resulting DNA eluate is treated with phenol/chloroform and then precipitated with ethanol to obtain a genome library insert.
This insert is ligated into a suitable vector, such as pUC18 SmaI/BAP (manufactured by Amersham Pharmacia Biotech) or the like, using T4 ligase (manufactured by Takara Shuzo) or the like. The ligation can be carried out by allowing a mixture to stand at 10 to 20° C. for 20 to 50 hours.
The resulting ligation product is precipitated with ethanol and dissolved in 5 to 20 μl of TE buffer.
Escherichia coli is transformed in accordance with a conventional method using 0.5 to 2 μl of the ligation solution. Examples of the transformation method include the electroporation method using ELECTRO MAX DH10B (manufactured by Life Technologies) for Escherichia coli. The electroporation method can be carved out under the conditions as described in the manufacturer's instructions.
The transformed Escherichia coli is spread on a suitable selection medium containing agar, for example, LB plate medium containing 10 to 100 mg/l ampicillin (LB medium (10 g/l bactotrypton, 5 g/l yeast extract, 10 g/l sodium chloride, pH 7.0) containing 1.6% of agar) when pUC18 is used as the cloning vector, and cultured therein.
The transformant can be obtained as colonies formed on the plate medium. In this step, it is possible to select the transformant having the recombinant DNA containing the genome DNA as white colonies by adding X-gal and IPTG (isopropyl-β-thiogalactopyranoside) to the plate medium.
The transformant is allowed to stand for culturing in a 96-well titer plate to which 0.05 ml of the LB medium containing 0.1 mg/ml of ampicillin has been added in each well. The resulting culture can be used in an experiment of (4) described below. Also, the culture solution can be stored at −80° C. by adding 0.05 ml per well of the LB medium containing 20% glycerol to the culture solution, followed by mixing, and the stored culture solution can be used at any time.
(3) Production of Cosmid Library
The genome DNA (0.1 mg) of the coryneform bacteria prepared in the above (1) is partially digested with a restriction enzyme, such as Sau3AI or the like, and then ultracentrifuged (26,000 rpm, 18 hours, 20° C.) under a 10 to 40% sucrose density gradient using a 10% sucrose buffer (1 mol/l NaCl, 20 mmol/l Tris hydrochloride, 5 mmol/l EDTA, 10% sucrose, pH 8.0) and a 40% sucrose buffer (elevating the concentration of the 10% sucrose buffer to 40%).
After the centrifugation, the thus separated solution is fractionated into tubes in 1 ml per each tube. After confirming the DNA fragment size of each fraction by agarose gel electrophoresis, a fraction rich in DNA fragments of about 40 kb is precipitated with ethanol.
The resulting DNA fragment is ligated to a cosmid vector having a cohesive end which can be ligated to the fragment. When the genome DNA is partially digested with Sau3AI, the partially digested product can be ligated to, for example, the BamHI site of superCos1 (manufactured by Stratagene) in accordance with the manufacture's instructions.
The resulting ligation product is packaged using a packaging extract which can be prepared by a method described in Molecular Cloning, 2nd ed. and then used in transforming Escherichia coli. More specifically, the ligation product is packaged using, for example, a commercially available packaging extract, Gigapack III Gold Packaging Extract (manufactured by Stratagene) in accordance with the manufacture's instructions and then introduced into Escherichia coli XL-1-BlueMR (manufactured by Stratagene) or the like.
The thus transformed Escherichia coli is spread on an LB plate medium containing ampicillin, and cultured therein.
The transformant can be obtained as colonies formed on the plate medium.
The transformant is subjected to standing culture in a 96-well titer plate to which 0.05 ml of the LB medium containing 0.1 mg/ml ampicillin has been added.
The resulting culture can be employed in an experiment of (4) described below. Also, the culture solution can be stored at −80° C. by adding 0.05 ml per well of the LB medium containing 20% glycerol to the culture solution, followed by mixing, and the stored culture solution can be used at any time.
(4) Determination of Nucleotide Sequence
(4-1) Preparation of Template
The full nucleotide sequence of genome DNA of coryneform bacteria can be determined basically according to the whole genome shotgun method (Science, 269: 496-512 (1995) ).
The template used in the whole genome shotgun method can be prepared by PCR using the library prepared in the above (2) (DNA Research, 5: 1-9 (1998)).
Specifically, the template can be prepared as follows.
The clone derived from the whole genome shotgun library is inoculated by using a replicator (manufactured by GENETIX) into each well of a 96-well plate to which 0.08 ml per well of the LB medium containing 0.1 mg/ml ampicillin has been added, followed by stationarily culturing at 37° C. overnight.
Next, the culture solution is transported, using a copy plate (manufactured by Tokken), into each well of a 96-well reaction plate (manufactured by PE Biosystems) to which 0.025 ml per well of a PCR reaction solution has been added using TaKaRa Ex Taq (manufactured by Takara Shuzo). Then, PCR is carried out in accordance with the protocol by Makino et al. (DNA Research, 5: 1-9 (1998)) using GeneAmp PCR System 9700 (manufactured by PE Biosystems) to amplify the inserted fragments.
The excessive primers and nucleotides are eliminated using a kit for purifying a PCR product, and the product is used as the template in the sequencing reaction.
It is also possible to determine the nucleotide sequence using a double-stranded DNA plasmid as a template.
The double-stranded DNA plasmid used as the template can be obtained by the following method.
The clone derived from the whole genome shotgun library is inoculated into each well of a 24- or 96-well plate to which 1.5 ml per well of a 2×YT medium (16 g/l bactotrypton, 10 g/l yeast extract, 5 g/l sodium chloride, pH 7.0) containing 0.05 mg/ml ampicillin has been added, followed by culturing under shaking at 37° C. overnight.
The double-stranded DNA plasmid can be prepared from the culture solution using an automatic plasmid preparing machine KURABO PI-50 (manufactured by Kurabo Industries), a multiscreen (manufactured by Millipore) or the like, according to each protocol.
To purify the plasmid, Biomek 2000 manufactured by Beckman Coulter and the like can be used.
The resulting purified double-stranded DNA plasmid is dissolved in water to give a concentration of about 0.1 mg/ml. Then, it can be used as the template in sequencing.
(4-2) Sequencing Reaction
The sequencing reaction can be carried out according to a commercially available sequence kit or the like. A specific method is exemplified below.
To 6 μl of a solution of ABI PRISM BigDye Terminator Cycle Sequencing, Ready Reaction Kit (manufactured by PE Biosystems), 1 to 2 pmol of an M13 regular direction primer (M13-21) or an M13 reverse direction primer (M13REV) (DNA Research, 5: 1-9 (1998)) and 50 to 200 ng of the template prepared in the above (4-1) (the PCR product or plasmid) to give 10 μl of a sequencing reaction solution.
A dye terminator sequencing reaction (35 to 55 cycles) is carried out using this reaction solution and Gene PCR System 9700 (manufactured by PE Biosystems) or the like. The cycle parameter can be determined in accordance with a commercially available kit, for example, the manufacture's instructions attached with ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit.
The sample can be purified using a commercially available product, such as Multi Screen HV plate (manufactured by Millipore) or the like, according to the manufacture's instructions.
The thus purified reaction product is precipitated with ethanol, dried and then used for the analysis. The dried reaction product can be stored in the dark at −30° C. and the stored reaction product can be used at any time.
The dried reaction product can be analyzed using a commercially available sequencer and an analyze according to the manufacture's instructions.
Examples of the commercially available sequencer include ABI PRISM 377 DNA Sequencer (manufactured by PE Biosystems) Example of the analyzer include ABI PRISM 3700 DNA Analyzer (manufactured by PE Biosystems).
(5) Assembly
A software, such as phred (The University of Washington) or the like, can be used as base call for use in analyzing the sequence information obtained in the above (4). A software, such as Cross_Match (The University of Washington) or SPS Cross_Match (manufactured by Southwest Parallel Software) or the like, can be used to mass the vector sequence information.
For the assembly, a software, such as phrap (The University of Washington), SPS phrap (manufactured by Southwest Parallel Software) or the like, can be used.
In the above, analysis and output of the results thereof, a computer such as UNIX, PC, Macintosh, and the like can be used.
Contig obtained by the assembly can be analyzed using a graphical editor such as consed (The University of Washington) or the like.
It is also possible to perform a series of the operations from the base call to the assembly in a lump using a script phredPhrap attached to the consed.
As used herein, software will be understood to also be referred to as a comparator.
(6) Determination of Nucleotide Sequence in Gap Part
Each of the cosmids in the cosmid library constructed in the above (3) is prepared in the same manner as in the preparation of the double-stranded DNA plasmid described in the above (4-1). The nucleotide sequence at the end of the insert fragment of the cosmid is determined using a commercially available kit, such as ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (manufactured by PE Biosystems) according to the manufacture's instructions.
About 800 cosmid clones are sequenced at both ends of the inserted fragment to detect a nucleotide sequence in the contig derived from the shotgun sequencing obtained in (5) which is coincident with the sequence. Thus, the chain linkage between respective cosmid clones and respective contigs are clarified, and mutual alignment is carried out. Furthermore, the results are compared with known physical maps to map the cosmids and the contigs. In case of Corynebacterium glutamicum ATCC 13032, a physical map of Mol. Gen. Genet., 252: 255-265 (1996) can be used.
The sequence in the region which cannot be covered with the contigs (gap part) can be determined by the following method.
Clones containing sequences positioned at the ends of the contigs are selected. Among these, a clone wherein only one end of the inserted fragment has been determined is selected and the sequence at the opposite end of the inserted fragment is determined.
A shotgun library clone or a cosmid clone derived therefrom containing the sequences at the respective ends of the inserted fragments in the two contigs is identified and the full nucleotide sequence of the inserted fragment of the clone is determined.
According to this method, the nucleotide sequence of the gap part can be determined.
When no shotgun library clone or cosmid clone covering the gap part is available, primers complementary to the end sequences of the two different contigs are prepared and the DNA fragment in the gap part is amplified. Then, sequencing is performed by the primer walking method using the amplified DNA fragment as a template or by the shotgun method in which the sequence of a shotgun clone prepared from the amplified DNA fragment is determined. Thus, the nucleotide sequence of the above-described region can be determined.
In a region showing a low sequence accuracy, primers are synthesized using AUTOFINISH function and NAVIGATING function of consed (The University of Washington), and the sequence is determined by the primer walking method to improve the sequence accuracy.
Examples of the thus determined nucleotide sequence of the full genome include the full nucleotide sequence of genome of Corynebacterium glutamicum ATCC 13032 represented by SEQ ID NO:1.
(7) Determination of Nucleotide Sequence of Microorganism Genome DNA Using the Nucleotide Sequence Represented by SEQ ID NO:1
A nucleotide sequence of a polynucleotide having a homology of 80% or more with. the full nucleotide sequence of Corynebacterium glutamicum ATCC 13032 represented by SEQ ID NO:1 as determined above can also be determined using the nucleotide sequence represented by SEQ ID NO:1, and the polynucleotide having a nucleotide sequence having a homology of 80% or more with the nucleotide sequence represented by SEQ ID NO:1 of the present invention is within the scope of the present invention. The term “polynucleotide having a nucleotide sequence having a homology of 80% or more with the nucleotide sequence represented by SEQ ID NO:1 of the present invention” is a polynucleotide in which a full nucleotide sequence of the chromosome DNA can be determined using as a primer an oligonucleotide composed of continuous 5 to 50 nucleotides in the nucleotide sequence represented by SEQ ID NO:1, for example, according to PCR using the chromosome DNA as a template. A particularly preferred primer in determination of the full nucleotide sequence is an oligonucleotide having nucleotide sequences which are positioned at the interval of about 300 to 500 bp, and among such oligonucleotides, an oligonucleotide having a nucleotide sequence selected from DNAs encoding a protein relating to a main metabolic pathway is particularly preferred. The polynucleotide in which the full nucleotide sequence of the chromosome DNA can be determined using the oligonucleotide includes polynucleotides constituting a chromosome DNA derived from a microorganism belonging to coryneform bacteria. Such a polynucleotide is preferably a polynucleotide constituting chromosome DNA derived from a microorganism belonging to the genus Corynebacterium, more preferably a polynucleotide constituting a chromosome DNA of Corynebacterium glutamicum.
2. Identification of ORF (Open Reading Frame) and Expression Regulatory Fragment and Determination of the Function of ORF
Based on the full nucleotide sequence data of the genome derived from coryneform bacteria determined in the above item 1, an ORF and an expression modulating fragment can be identified. Furthermore, the function of the thus determined ORF can be determined.
The ORF means a continuous region in the nucleotide sequence of mRNA which can be translated as an amino acid sequence to mature to a protein. A region of the DNA coding for the ORF of mRNA is also called ORF.
The expression modulating fragment (hereinafter referred to as “EMF”) is used herein to define a series of polynucleotide fragments which modulate the expression of the ORF or another sequence ligated operatably thereto. The expression “modulate the expression of a sequence ligated operatably” is used herein to refer to changes in the expression of a sequence due to the presence of the EMF. Examples of the EMF include a promoter, an operator, an enhancer, a silencer, a ribosome-binding sequence, a transcriptional termination sequence, and the like. In coryneform bacteria, an EMF is usually present in an intergenic segment (a fragment positioned between two genes; about 10 to 200 nucleotides in length). Accordingly, an EMF is frequently present in an intergenic segment of 10 nucleotides or longer. It is also possible to determine or discover the presence of an EMF by using known EMF sequences as a target sequence or a target structural motif (or a target motif) using an appropriate software or comparator, such as FASTA (Proc. Natl. Acad. Sci. USA, 85: 2444-48 (1988)), BLAST (J. Mol. Biol., 215: 403-410 (1990)) or the like. Also, it can be identified and evaluated using a known EMF-capturing vector (for example, pKK232-8; manufactured by Amersham Pharmacia Biotech).
The term “target sequence” is used herein to refer to a nucleotide sequence composed of 6 or more nucleotides, an amino acid sequence composed of 2 or more amino acids, or a nucleotide sequence encoding this amino acid sequence composed of 2 or more amino acids. A longer target sequence appears at random in a data base at the lower possibility. The target sequence is preferably about 10 to 100 amino acid residues or about 30 to 300 nucleotide residues.
The term “target structural motif” or “target motif” is used herein to refer to a sequence or a combination of sequences selected optionally and reasonably. Such a motif is selected on the basis of the three-dimensional structure formed by the folding of a polypeptide by means known to one of ordinary skill in the art. Various motives are known.
Examples of the target motif of a polypeptide include, but are not limited to, an enzyme activity site, a protein-protein interaction site, a signal sequence, and the like. Examples of the target motif of a nucleic acid include a promoter sequence, a transcriptional regulatory factor binding sequence, a hair pin structure, and the like.
Examples of highly useful EMF include a high-expression promoter, an inducible-expression promoter, and the like. Such an EMF can be obtained by positionally determining the nucleotide sequence of a gene which is known or expected as achieving high expression (for example, ribosomal RNA gene: GenBank Accession No. M16175 or Z46753) or a gene showing a desired induction pattern (for example, isocitrate lyase gene induced by acetic acid: Japanese Published Unexamined Patent Application No. 56782/93) via the alignment with the full genome nucleotide sequence determined in the above item 1, and isolating the genome fragment in the upstream part (usually 200 to 500 nucleotides from the translation initiation site). It is also possible to obtain a highly useful EMF by selecting an EMF showing a high expression efficiency or a desired induction pattern from among promoters captured by the EMF-capturing vector as described above.
The ORF can be identified by extracting characteristics common to individual ORFs, constructing a general model based on these characteristics, and measuring the conformity of the subject sequence with the model. In the identification, a software, such as GeneMark (Nuc. Acids. Res., 22: 4756-67 (1994): manufactured by GenePro)), GeneMark.hmm (manufactured by GenePro), GeneHacker (Protein, Nucleic Acid and Enzyme, 42: 3001-07 (1997)), Glimmer (Nuc. Acids. Res., 26: 544-548 (1998): manufactured by The Institute of Genomic Research), or the like, can be used. In using the software, the default (initial setting) parameters are usually used, though the parameters can be optionally changed.
In the above-described comparisons, a computer, such as UNIX, PC, Macintosh, or the like, can be used.
Examples of the ORF determined by the method of the present invention include ORFs having the nucleotide sequences represented by SEQ ID NOS:2 to 3501 present in the genome of Corynebacterium glutamicum as represented by SEQ ID NO:1. In these ORFs, polypeptides having the amino acid sequences represented by SEQ ID NOS:3502 to 7001 are encoded.
The function of an ORF can be determined by comparing the identified amino acid sequence of the ORF with known homologous sequences using a homology searching software or comparator, such as BLAST, FAST, Smith & Waterman (Meth. Enzym., 164: 765 (1988)) or the like on an amino acid data base, such as Swith-Prot, PIR, GenBank-nr-aa, GenPept constituted by protein-encoding domains derived from GenBank data base, OWL or the like.
Furthermore, by the homology searching, the identity and similarity with the amino acid sequences of known proteins can also be analyzed.
With respect of the term “identity” used herein, where two polypeptides each having 10 amino acids are different in the positions of 3 amino acids, these polypeptides have an identity of 70% with each other. In case wherein one of the different 3 amino acids is analogue (for example, leucine and isoleucine), these polypeptides have a similarity of 80%.
As a specific example, Table 1 shows the registration numbers in known data bases of sequences which are judged as having the highest similarity with the nucleotide sequence of the ORF derived from Corynebacterium glutamicum ATCC 13032, genes of these sequences, functions of these genes, and identities thereof compared with known amino acid translation sequences.
Thus, a great number of novel genes derived from coryneform bacteria can be identified by determining the full nucleotide sequence of the genome derived from coryneform bacterium by the means of the present invention. Moreover, the function of the proteins encoded by these genes can be determined. Since coryneform bacteria are industrially highly useful microorganisms, many of the identified genes are industrially useful.
Moreover, the characteristics of respective microorganisms can be clarified by classifying the functions thus determined. As a result, valuable information in breeding is obtained.
Furthermore, from the ORF information derived from coryneform bacteria, the ORF corresponding to the microorganism is prepared and obtained according to the general method as disclosed in Molecular Cloning, 2nd ed. or the like. Specifically, an oligonucleotide having a nucleotide sequence adjacent to the ORF is synthesized, and the ORF can be isolated and obtained using the oligonucleotide as a primer and a chromosome DNA derived from coryneform bacteria as a template according to the general PCR cloning technique. Thus obtained ORF sequences include polynucleotides comprising the nucleotide sequence represented by any one of SEQ ID NOS:2 to 3501.
The ORF or primer can be prepared using a polypeptide synthesizer based on the above sequence information.
Examples of the polynucleotide of the present invention include a polynucleotide containing the nucleotide sequence of the ORF obtained in the above, and a polynucleotide which hybridizes with the polynucleotide under stringent conditions.
The polynucleotide of the present invention can be a single-stranded DNA, a double-stranded DNA and a single-stranded RNA, though it is not limited thereto.
The polynucleotide which hybridizes with the polynucleotide containing the nucleotide sequence of the ORF obtained in the above under stringent conditions includes a degenerated mutant of the ORF. A degenerated mutant is a polynucleotide fragment having a nucleotide sequence which is different from the sequence of the ORF of the present invention which encodes the same amino acid sequence by degeneracy of a gene code.
Specific examples include a polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOS:2 to 3431, and a polynucleotide which hybridizes with the polynucleotide under stringent conditions.
A polynucleotide which hybridizes under stringent conditions is a polynucleotide obtained by colony hybridization, plaque hybridization, Southern blot hybridization or the like using, as a probe, the polynucleotide having the nucleotide sequence of the ORF identified in the above. Specific examples include a polynucleotide which can be identified by carrying out hybridization at 65° C. in the presence of 0.7-1.0 M NaCl using a filter on which a polynucleotide prepared from colonies or plaques is immobilized, and then washing the filter with 0.1× to 2×SSC solution (the composition of 1×SSC contains 150 mM sodium chloride and 15 mM sodium citrate) at 65° C.
The hybridization can be carried out in accordance with known methods described in, for example, Molecular Cloning, 2nd ed., Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995) or the like. Specific examples of the polynucleotide which can be hybridized include a DNA having a homology of 60% or more, preferably 80% or more, and particularly preferably 95% or more, with the nucleotide sequence represented by any one of SEQ ID NO:2 to 3431 when calculated using default (initial setting) parameters of a homology searching software, such as BLAST, FASTA, Smith-Waterman or the like.
Also, the polynucleotide of the present invention includes a polynucleotide encoding a polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOS:3502 to 6931 and a polynucleotide which hybridizes with the polynucleotide under stringent conditions.
Furthermore, the polynucleotide of the present invention includes a polynucleotide which is present in the 5′ upstream or 3′ downstream region of a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOS:2 to 3431 in a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO:1, and has an activity of regulating an, expression of a polypeptide encoded by the polynucleotide. Specific examples of the polynucleotide having an activity of regulating an expression of a polypeptide encoded by the polynucleotide includes a polynucleotide encoding the above described EMF, such as a promoter, an operator, an enhancer, a silencer, a ribosome-binding sequence, a transcriptional termination sequence, and the like.
The primer used for obtaining the ORF according to the above PCR cloning technique includes an oligonucleotide comprising a sequence which is the same as a sequence of 10 to 200 continuous nucleotides in the nucleotide sequence of the ORF and an adjacent region or an oligonucleotide comprising a sequence which is complementary to the oligonucleotide. Specific examples include an oligonucleotide comprising a sequence which is the same as a sequence of 10 to 200 continuous nucleotides of the nucleotide sequence represented by any one of SEQ ID NOS:1 to 3431, and an oligonucleotide comprising a sequence complementary to the oligonucleotide comprising a sequence of at least 10 to 20 continuous nucleotide of any one of SEQ ID NOS:1 to 3431. When the primers are used as a sense primer and an antisense primer, the above-described oligonucleotides in which melting temperature (Tm) and the number of nucleotides are not significantly different from each other are preferred.
The oligonucleotide of the present invention includes an oligonucleotide comprising a sequence which is the same as 10 to 200 continuous nucleotides of the nucleotide sequence represented by any one of SEQ ID NOS:1 to 3431 or an oligonucleotide comprising a sequence complementary to the oligonucleotide.
Also, analogues of these oligonucleotides (hereinafter also referred to as “analogous oligonucleotides”) are also provided by the present invention and are useful in the methods described herein.
Examples of the analogous oligonucleotides include analogous oligonucleotides in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, analogous oligonucleotides in which a phosphodiester bond in an oligonucleotide is converted to an N3′-P5′ phosphoamidate bond, analogous oligonucleotides in which ribose and a phosphodiester bond in an oligonucleotide is converted to a peptide nucleic acid bond, analogous oligonucleotides in which uracil in an oligonucleotide is replaced with C-5 propynyluracil, analogous oligonucleotides in which uracil in an oligonucleotide is replaced with C-5 thiazoluracil, analogous oligonucleotides in which cytosine in an oligonucleotide is replaced with C-5 propynylcytosine, analogous oligonucleotides in which cytosine in an oligonucleotide is replaced with phenoxazine-modified cytosine, analogous oligonucleotides in which ribose in an oligonucleotide is replaced with 2′-O-propylribose, analogous oligonucleotides in which ribose in an oligonucleotide is replaced with 2′-methoxyethoxyribose, and the like (Cell Engineering, 16: 1463 (1997)).
The above oligonucleotides and analogous oligonucleotides of the present invention can be used as probes for hybridization and antisense nucleic acids described below in addition to as primers.
Examples of a primer for the antisense nucleic acid techniques known in the art include an oligonucleotide which hybridizes the oligonucleotide of the present invention under stringent conditions and has an activity regulating expression of the polypeptide encoded by the polynucleotide, in addition to the above oligonucleotide.
3. Determination of Isozymes
Many mutants of coryneform bacteria which are useful in th production of useful substances, such as amino acids, nucleic acids, vitamins, saccharides, organic acids, and the like, are obtained by the present invention.
However, since the gene sequence data of the microorganism has been, to date, insufficient, useful mutants have been obtained by mutagenic techniques using a mutagen, such as nitrosoguanidine (NTG) or the like.
Although genes can be mutated randomly by the mutagenic method using the above-described mutagen, all genes encoding respective isozymes having similar properties relating to the metabolism of intermediates cannot be mutated. In the mutagenic method using a mutagen, genes are mutated randomly. Accordingly, harmful mutations worsening culture characteristics, such as delay in growth, accelerated foaming, and the like, might be imparted at a great frequency, in a random manner.
However, if gene sequence information is available, such as is provided by the present invention, it is possible to mutate all of the genes encoding target isozymes. In this case, harmful mutations may be avoided and the target mutation can be incorporated.
Namely, an accurate number and sequence information of the target isozymes in coryneform bacteria can be obtained based on the ORF data obtained in the above item 2. By using the sequence information, all of the target isozyme genes can be mutated into genes having the desired properties by, for example, the site-specific mutagenesis method described in Molecular Cloning, 2nd ed. to obtain useful mutants having elevated productivity of useful substances.
4. Clarification or Determination of Biosynthesis Pathway and Signal Transmission Pathway
Attempts have been made to elucidate biosynthesis pathways and signal transmission pathways in a number of organisms, and many findings have been reported. However, there are many unknown aspects of coryneform bacteria since a number of genes have not been identified so far.
These unknown points can be clarified by the following method.
The functional information of ORF derived from coryneform bacteria as identified by the method of above item 2 is arranged. The term “arranged” means that the ORF is classified based on the biosynthesis pathway of a substance or the signal transmission pathway to which the ORF belongs using known information according to the functional information. Next, the arranged ORF sequence information is compared with enzymes on the biosynthesis pathways or signal transmission pathways of other known organisms. The resulting information is combined with known data on coryneform bacteria. Thus, the biosynthesis pathways and signal transmission pathways in coryneform bacteria, which have been unknown so far, can be determined.
As a result that these pathways which have been unknown or unclear hitherto are clarified, a useful mutant for producing a target useful substance can be efficiently obtained.
When the thus clarified pathway is judged as important in the synthesis of a useful product, a useful mutant can be obtained by selecting a mutant wherein this pathway has been strengthened. Also, when the thus clarified pathway is judged as not important in the biosynthesis of the target useful product, a useful mutant can be obtained by selecting a mutant wherein the utilization frequency of this pathway is lowered.
5. Clarification or Determination of Useful Mutation Point
Many useful mutants of coryneform bacteria which are suitable for the production of useful substances, such as amino acids, nucleic acids, vitamins, saccharides, organic acids, and the like, have been obtained. However, it is hardly known which mutation point is imparted to a gene to improve the productivity.
However, mutation points contained in production strains can be identified by comparing desired sequences of the genome. DNA of the production strains obtained from coryneform bacteria by the mutagenic technique with the nucleotide sequences of the corresponding genome DNA and ORF derived from coryneform bacteria determined by the methods of the above items 1 and 2 and analyzing them
Moreover, effective mutation points contributing to the production can be easily specified from among these mutation points on the basis of known information relating to the metabolic pathways, the metabolic regulatory mechanisms, the structure activity correlation of enzymes, and the like.
When any efficient mutation can be hardly specified based on known data, the mutation points thus identified can be introduced into a wild strain of coryneform bacteria or a production strain free of the mutation. Then, it is examined whether or not any positive effect can be achieved on the production.
For example, by comparing the nucleotide sequence of homoserine dehydrogenase gene hom of a lysine-producing B-6-strain of Corynebacterium glutamicum (Appl. Microbiol. Biotechnol., 32: 269-273 (1989)) with the nucleotide sequence corresponding to the genome of Corynebacterium glutamicum ATCC 13032 according to the present invention, a mutation of amino acid replacement in which valine at the 59-position is replaced with alanine (Val59Ala) was identified. A strain obtained by introducing this mutation into the ATCC 13032 strain by the gene replacement method can produce lysine, which indicates that this mutation is an effective mutation contributing to the production of lysine.
Similarly, by comparing the nucleotide sequence of pyruvate carboxylase gene pyc of the B-6 strain with the nucleotide sequence corresponding to the ATCC 13032 genome, a mutation of amino acid replacement in which proline at the 458-position was replaced with serine (Pro458Ser) was identified. A strain obtained by introducing this mutation into a lysine-producing strain of No. 58 (FERM BP-7134) of Corynebacterium glutamicum free of this mutation shows an improved lysine productivity in comparison with the No. 58 strain, which indicates that this mutation is an effective mutation contributing to the production of lysine.
In addition, a mutation Ala213Thr in glucose-6-phosphate dehydrogenase was specified as an effective mutation relating to the production of lysine by detecting glucose-6-phosphate dehydrogenase gene zwf of the B-6 strain.
Furthermore, the lysine-productivity of Corynebacterium glutamicum was improved by replacing the base at the 932-position of aspartokinase gene lysC of the Corynebacterium glutamicum ATCC 13032 genome with cytosine to thereby replace threonine at the 311-position by isoleucine, which indicates that this mutation is an effective mutation contributing to the production of lysine.
Also, as another method to examine whether or not the identified mutation point is an effective mutation, there is a method in which the mutation possessed by the lysine-producing strain is returned to the sequence of a wild type strain by the gene replacement method and whether or not it has a negative influence on the lysine productivity. For example, when the amino acid replacement mutation Val59Ala possessed by hom of the lysine-producing B-6 strain was returned to a wild type amino acid sequence, the lysine productivity was lowered in comparison with the B-6 strain. Thus, it was found that this mutation is an effective mutation contributing to the production of lysine.
Effective mutation points can be more efficiently and comprehensively extracted by combining, if needed, the DNA array analysis or proteome analysis described below.
6. Method of Breeding Industrially Advantageous Production Strain
It has been a general practice to construct production strains, which are used industrially in the fermentation production of the target useful substances, such as amino acids, nucleic acids, vitamins, saccharides, organic acids, and the like, by repeating mutagenesis and breeding based on random mutagenesis using mutagens, such as NTG or the like, and screening.
In recent years, many examples of improved production strains have been made through the use of recombinant DNA techniques. In breeding, however, most of the parent production strains to be improved are mutants obtained by a conventional mutagenic procedure (W. Leuchtenberger, Amino Acids—Technical Production and Use. In: Roehr (ed) Biotechnology, second edition, vol. 6, products of primary metabolism. VCH Verlagsgesellschaft mbH, Weinheim, P 465 (1996)).
Although mutagenesis methods have largely contributed to the progress of the fermentation industry, they suffer from a serious problem of multiple, random introduction of mutations into every part of the chromosome. Since many mutations are accumulated in a single chromosome each time a strain is improved, a production strain obtained by the random mutation and selecting is generally inferior in properties (for example, showing poor growth, delayed consumption of saccharides, and poor resistance to stresses such as temperature and oxygen) to a wild type strain, which brings about troubles such as failing to establish a sufficiently elevated productivity, being frequently contaminated with miscellaneous bacteria, requiring troublesome procedures in culture maintenance, and the like, and, in its turn, elevating the production cost in practice. In addition, the improvement in the productivity is based on random mutations and thus the mechanism thereof is unclear. Therefore, it is very difficult to plan a rational breeding strategy for the subsequent improvement in the productivity.
According to the present invention, effective mutation points contributing to the production can be efficiently specified from among many mutation points accumulated in the chromosome of a production strain which has been bred from coryneform bacteria and, therefore, a novel breeding method of assembling these effective mutations in the coryneform bacteria can be established. Thus, a useful production strain can be reconstructed. It is also possible to construct a useful production strain from a wild type strain.
Specifically, a useful mutant can be constructed in the following manner.
One of the mutation points is incorporated into a wild type strain of coryneform bacteria. Then, it is examined whether or not a positive effect is established on the production. When a positive effect is obtained, the mutation point is saved. When no effect is obtained, the mutation point is removed. Subsequently, only a strain having the effective mutation point is used as the parent strain, and the same procedure is repeated. In general, the effectiveness of a mutation positioned upstream cannot be clearly evaluated in some cases when there is a rate-determining point in the downstream of a biosynthesis pathway. It is therefore preferred to successively evaluate mutation points upward from downstream.
By reconstituting effective mutations by the method as described above in a wild type strain or a strain which has a high growth speed or the same ability to consume saccharides as the wild type strain, it is possible to construct an industrially advantageous strain which is free of troubles in the previous methods as described above and to conduct fermentation production using such strains within a short time or at a higher temperature.
For example, a lysine-producing mutant B-6 (Appl. Microbiol. Biotechnol., 32: 262-273 (1989)), which is obtained by multiple rounds of random mutagenesis from a wild type strain Corynebacterium glutamicum ATCC 13032, enables lysine fermentation to be performed at a temperature between 30 and 34° C. but shows lowered growth and lysine productivity at a temperature exceeding 34° C. Therefore, the fermentation temperature should be maintained at 34° C. or lower. In contrast thereto, the production strain described in the above item 5, which is obtained by reconstituting effective mutations relating to lysine production, can achieve a productivity at 40 to 42° C. equal or superior to the result obtained by culturing at 30 to 34° C. Therefore, this strain is industrially advantageous since it can save the load of cooling during the fermentation.
When culture should be carried out at a high temperature exceeding 43° C., a production strain capable of conducting fermentation production at a high temperature exceeding 43° C. can be obtained by reconstituting useful mutations in a, microorganism belonging to the genus Corynebacterium which can grow at high temperature exceeding 43° C. Examples of the microorganism capable of growing at a high temperature exceeding 43° C. include Corynebacterium thermoaminogenes, such as Corynebacterium thermoaminogenes FERM 9244, FERM 9245, FERM 9246 and FERM 9247.
A strain having a further improved productivity of the target product can be obtained using the thus reconstructed strain as the parent strain and further breeding it using the conventional mutagenesis method, the gene amplification method, the gene replacement method using the recombinant DNA technique, the transduction method or the cell fusion method. Accordingly, the microorganism of the present invention includes, but is not limited to, a mutant, a cell fusion strain, a transformant, a transductant or a recombinant strain constructed by using recombinant DNA techniques, so long as it is a producing strain obtained via the step of accumulating at least two effective mutations in a coryneform bacteria in the course of breeding.
When a mutation point judged as being harmful to the growth or production is specified, on the other hand, it is examined whether or not the producing strain used at present contains the mutation point. When it has the mutation, it can be returned to the wild type gene and thus a further useful production strain can be bred.
The breeding method as described above is applicable to microorganisms, other than coryneform bacteria, which have industrially advantageous properties (for example, microorganisms capable of quickly utilizing less expensive carbon sources, microorganisms capable of growing at higher temperatures).
7. Production and Utilization of Polynucleotide Array
(1) Production of Polynucleotide Array
A polynucleotide array can be produced using the polynucleotide or oligonucleotide of the present invention obtained in the above items 1 and 2.
Examples include a polynucleotide array comprising a, solid support to which at least one of a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOS:2 to 3501, a polynucleotide which hybridizes with the polynucleotide under stringent conditions, and a polynucleotide comprising 10 to 200 continuous nucleotides in the nucleotide sequence of the polynucleotide is adhered; and a polynucleotide array comprising a solid support to which at least one of a polynucleotide encoding a polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOS:3502 to 7001, a polynucleotide which hybridizes with the polynucleotide under stringent conditions, and a polynucleotide comprising 10 to 200 continuous bases in the nucleotide sequences of the polynucleotides is adhered.
Polynucleotide arrays of the present invention include substrates known in the art, such as a DNA chip, a DNA microarray and a DNA macroarray, and the like, and comprises a solid support and plural polynucleotides or fragments thereof which are adhered to the surface of the solid support.
Examples of the solid support include a glass plate, a nylon membrane, and the like.
The polynucleotides or fragments thereof adhered to the surface of the solid support can be adhered to the surface of the solid support using the general technique for preparing arrays. Namely, a method in which they are adhered to a chemically surface-treated solid support, for example, to which a polycation such as polylysine or the like has been adhered (Nat. Genet., 21: 15-19 (1999)). The chemically surface-treated supports are commercially available and the commercially available solid product can be used, as the solid support of the polynucleotide array according to the present invention.
As the polynucleotides or oligonucleotides adhered to the solid support, the polynucleotides and oligonucleotides of the present invention obtained in the above items 1 and 2 can be used.
The analysis described below can be efficiently performed by adhering the polynucleotides or oligonucleotides to the solid support at a high density, though a high fixation density is not always necessary.
Apparatus for achieving a high fixation density, such as an arrayer robot or the like, is commercially available from Takara Shuzo (GMS417 Arrayer), and the commercially available product can be used.
Also, the oligonucleotides of the present invention can be synthesized directly on the solid support by the photolithography method or the like (Nat. Genet., 21: 20-24 (1999)). In this method, a linker having a protective group which can be removed by light irradiation is first adhered to a solid support, such as a slide glass or the like. Then, it is irradiated with light through a mask (a photolithograph mask) permeating light exclusively at a definite part of the adhesion part. Next, an oligonucleotide having a protective group which can be removed by light irradiation is added to the part. Thus, a ligation reaction with the nucleotide arises exclusively at the irradiated part. By repeating this procedure, oligonucleotides, each having a desired sequence, different from each other can be synthesized in respective parts. Usually, the oligonucleotides to be synthesized have a length of 10 to 30 nucleotides.
(2) Use of Polynucleotide Array
The following procedures (a) and (b) can be carried out using the polynucleotide array prepared in the above (1).
(a) Identification of Mutation Point of Coryneform Bacterium Mutant and Analysis of Expression Amount and Expression Profile of Gene Encoded by Genome
By subjecting a gene derived from a mutant of coryneform bacteria or an examined gene to the following steps (i) to (iv), the mutation point of the gene can be identified or the expression amount and expression profile of the gene can be analyzed:
The gene derived from a mutant of coryneform bacteria or the examined gene include a gene relating to biosynthesis of at least one selected from amino acids, nucleic acids, vitamins, saccharides, organic acids, and analogues thereof.
The method will be described in detail.
A single nucleotide polymorphism (SNP) in a human region of 2,300 kb has been identified using polynucleotide arrays (Science, 280: 1077-82 (1998)). In accordance with the method of identifying SNP and methods described in Science, 278: 680-686 (1997); Proc. Natl. Acad. Sci. USA, 96: 12833-38 (1999); Science, 284: 1520-23 (1999), and the like using the polynucleotide array produced in the above (1) and a nucleic acid molecule (DNA, RNA) derived from coryneform bacteria in the method of the hybridization, a mutation point of a useful mutant, which is useful in producing an amino acid, a nucleic acid, a vitamin, a saccharide, an organic acid, or the like can be identified and the gene expression amount and the expression profile thereof can be analyzed.
The nucleic acid molecule (DNA, RNA) derived from the coryneform bacteria can be obtained according to the general method described in Molecular Cloning, 2nd ed. or the like. mRNA derived from Corynebacterium glutamicum can also be obtained by the method of Bormann et al. (Molecular Microbiology, 6: 317-326 (1992)) or the like.
Although ribosomal RNA (rRNA) is usually obtained in large excess in addition to the target mRNA, the analysis is not seriously disturbed thereby.
The resulting nucleic acid molecule derived from coryneform bacteria is labeled. Labeling can be carried out according to a method using a fluorescent dye, a method using a radioisotope or the like.
Specific examples include a labeling method in which psoralen-biotin is crosslinked with RNA extracted from a microorganism and, after hybridization reaction, a fluorescent dye having streptoavidin bound thereto is bound to the biotin moiety (Nat. Biotechnol., 16: 45-48 (1998)); a labeling method in which a reverse transcription reaction is carried out using RNA extracted from a microorganism as a template and random primers as primers, and dUTP having a fluorescent dye (for example, Cy3, Cy5) (manufactured by Amersham Pharmacia Biotech) is incorporated into cDNA (Proc. Natl. Acad. Sci. USA, 96: 12833-38 (1999)); and the like.
The labeling specificity can be improved by replacing the random primers by sequences complementary to the 3′-end of ORF (J. Bacteriol., 181: 6425-40 (1999)).
In the hybridization method, the hybridization and subsequent washing can be carried out by the general method (Nat. Biotechnol., 14: 1675-80 (1996), or the like).
Subsequently, the hybridization intensity is measured depending on the hybridization amount of the nucleic acid molecule used in the labeling. Thus, the mutation point can be identified and the expression amount of the gene can be calculated.
The hybridization intensity can be measured by visualizing the fluorescent signal, radioactivity, luminescence dose, and the like, using a laser confocal microscope, a CCD camera, a radiation imaging device (for example, STORM manufactured by Amersham Pharmacia Biotech), and the like, and then quantifying the thus visualized data.
A polynucleotide array on a solid support can also be analyzed and quantified using a commercially available apparatus, such as GMS418 Array Scanner (manufactured by Takara Shuzo) or the like.
The gene expression amount can be analyzed using a commercially available software (for example, ImaGene manufactured by Takara Shuzo; Array Gauge manufactured by Fuji Photo Film; ImageQuant manufactured by Amersham Pharmacia Biotech, or the like).
A fluctuation in the expression amount of a specific gene can be monitored using a nucleic acid molecule obtained in the time course of culture as the nucleic acid molecule derived from coryneform bacteria. The culture conditions can be optimized by analyzing the fluctuation.
The expression profile of the microorganism at the total gene level (namely, which genes among a great number of genes encoded by the genome have been expressed and the expression ratio thereof) can be determined using a nucleic acid molecule having the sequences of many genes determined from the full genome sequence of the microorganism. Thus, the expression amount of the genes determined by the full genome sequence can be analyzed and, in its turn, the biological conditions of the microorganism can be recognized as the expression pattern at the full gene level.
(b) Confirmation of the Presence of Gene Homologous to Examined Gene in Coryneform Bacteria
Whether or not a gene homologous to the examined gene, which is present in an organism other than coryneform bacteria, is present in coryneform bacteria can be detected using the polynucleotide array prepared in the above (1).
This detection can be carried out by a method in which an examined gene which is present in an organism other than coryneform bacteria is used instead of the nucleic acid molecule derived from coryneform bacteria used in the above identification/analysis method of (1).
8. Recording Medium Storing Full Genome Nucleotide Sequence and ORF Data and Being Readable by a Computer and Methods for Using the Same
The term “recording medium or storage device which is readable by a computer” means a recording medium or storage medium which can be directly readout and accessed with a computer. Examples include magnetic recording media, such as a floppy disk, a hard disk, a magnetic tape, and the like; optical recording media, such as CD-ROM, CD-R, CD-RW, DVD-ROM, DVD-RAM, DVD-RW, and the like; electric recording media, such as RAM, ROM, and the like; and hybrids in these categories (for example, magnetic/optical recording media, such as MO and the like).
Instruments for recording or inputting in or on the recording medium or instruments or devices for reading out the information in the recording medium can be appropriately selected, depending on the type of the recording medium and the access device utilized. Also, various data processing programs, software, comparator and formats are used for recording and utilizing the polynucleotide sequence information or the like of the present invention in the recording medium. The information can be expressed in the form of a binary file, a text file or an ASCII file formatted with commercially available software, for example. Moreover, software for accessing the sequence information is available and known to one of ordinary skill in the art.
Examples of the information to be recorded in the above-described medium include the full genome nucleotide sequence information of coryneform bacteria as obtained in the above item 2, the nucleotide sequence information of ORF, the amino acid sequence information encoded by the ORF, and the functional information of polynucleotides coding for the amino acid sequences.
The recording medium or storage device which is readable by a computer according to the present invention refers to a medium in which the information of the present invention has been recorded. Examples include recording media or storage devices which are readable by a computer storing the nucleotide sequence information represented by SEQ ID NOS:1 to 3501, the amino acid sequence information represented by SEQ ID NOS:3502 to 7001, the functional information of the nucleotide sequences represented by SEQ ID NOS:1 to 3501, the functional information of the amino acid sequences represented by SEQ ID NOS:3502 to 7001, and the information listed in Table 1 below and the like.
9. System Based on a Computer Using the Recording Medium of the Present Invention which is Readable by a Computer
The term “system based on a computer” as used herein refers a system composed of hardware device(s) software device(s), and data recording device(s) which are used for analyzing the data recorded in the recording medium of the present invention which is readable by a computer.
The hardware device(s) are, for example, composed of an input unit, a data recording unit, a central processing unit and an output unit collectively or individually.
By the software device(s), the data recorded in the recording medium of the present invention are searched or analyzed using the recorded data and the hardware device(s) as described herein. Specifically, the software device(s) contain at least one program which acts on or with the system in order to screen, analyze or compare biologically meaningful structures or information from the nucleotide sequences, amino acid sequences and the like recorded in the recording medium according to the present invention.
Examples of the software device(s) for identifying ORF and EMF domains include GeneMark (Nuc. Acids. Res., 22: 4756-67 (1994)), GeneHacker (Protein, Nucleic Acid and Enzyme, 42: 3001-07 (1997)), Glimmer (The Institute of Genomic Research; Nuc. Acids. Res., 26: 544-548 (1998)) and the like. In the process of using such a software device, the default (initial setting) parameters are usually used, although the parameters can be changed, if necessary, in a manner known to one of ordinary skill in the art.
Examples of the software device(s) for identifying a genome domain or a polypeptide domain analogous to the target sequence or the target structural motif (homology searching) include FASTA, BLAST, Smith-Waterman, GenetyxMac (manufactured by Software Development), GCG Package (manufactured by Genetic Computer Group), GenCore (manufactured by Compugen), and the like. In the process of using such a software device, the default (initial setting) parameters are usually used, although the parameters can be changed, if necessary, in a manner known to one of ordinary skill in the art.
Such a recording medium storing the full genome sequence data is useful in preparing a polynucleotide array by which the expression amount of a gene encoded by the genome DNA of coryneform bacteria and the expression profile at the total gene level of the microorganism, namely, which genes among many genes encoded by the genome have been expressed and the expression ratio thereof, can be determined.
The data recording device(s) provided by the present invention are, for example, memory device(s) for recording the data recorded in the recording medium of the present invention and target sequence or target structural motif data, or the like, and a memory accessing device(s) for accessing the same.
Namely, the system based on a computer according to the present invention comprises the following:
This system is usable in the methods in items 2 to 5 as described above for searching and analyzing the ORF and EMF domains, target sequence, target structural motif, etc. of a coryneform bacterium, searching homologs, searching and analyzing isozymes, determining the biosynthesis pathway and the signal transmission pathway, and identifying spots which have been found in the proteome analysis. The term “homologs” as used herein includes both of orthologs and paralogs.
10. Production of Polypeptide Using ORF Derived from Coryneform Bacteria
The polypeptide of the present invention can be produced using a polynucleotide comprising the ORF obtained in the above item 2. Specifically, the polypeptide of the present invention can be produced by expressing the polynucleotide of the present invention or a fragment thereof in a host cell, using the method described in Molecular Cloning, 2nd ed., Current Protocols in Molecular Biology, and the like, for example, according to the following method.
A DNA fragment having a suitable length containing a part encoding the polypeptide is prepared from the full length ORF sequence, if necessary.
Also, DNA in which nucleotides in a nucleotide sequence at a part encoding the polypeptide of the present invention are replaced to give a codon suitable for expression of the host cell, if necessary. The DNA is useful for efficiently producing the polypeptide of the present invention.
A recombinant vector is prepared by inserting the DNA fragment into the downstream of a promoter in a suitable expression vector.
The recombinant vector is introduced to a host cell suitable for the expression vector.
Any of bacteria, yeasts, animal cells, insect cells, plant cells, and the like can be used as the host cell so long as it can be expressed in the gene of interest.
Examples of the expression vector include those which can replicate autonomously in the above-described host cell or can be integrated into chromosome and have a promoter at such a position that the DNA encoding the polypeptide of the present invention can be transcribed.
When a procaryote cell, such as a bacterium or the like, is used as the host cell, it is preferred that the recombinant vector containing the DNA encoding the polypeptide of the present invention can replicate autonomously in the bacterium and is a recombinant vector constituted by, at least a promoter, a ribosome binding sequence, the DNA of the present invention and a transcription termination sequence. A promoter controlling gene can also be contained therewith in operable combination.
Examples of the expression vectors include a vector plasmid which is replicable in Corynebacterium glutamicum, such as pCG1 (Japanese Published Unexamined Patent Application No. 134500/82), pCG2 (Japanese Published Unexamined Patent Application No. 35197/83), pCG4 (Japanese Published Unexamined Patent Application No. 183799/82), pCG11 (Japanese Published Unexamined Patent Application No. 134500/82), pCG116, pCE54 and pCB101 (Japanese Published Unexamined Patent Application No. 105999/83), pCE51, pCE52 and pCE53 (Mol. Gen. Genet., 196: 175-178 (1984)), and the like; a vector plasmid which is replicable in Escherichia coli, such as pET3 and pET11 (manufactured by Stratagene), pBAD, pThioHis and pTrcHis (manufactured by Invitrogen), pKK223-3 and pGEX2T (manufactured by Amersham Pharmacia Biotech), and the like; and pBTrp2, pBTac1 and pBTac2 (manufactured by Boehringer Mannheim Co.), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese Published Unexamined Patent Application No. 110600/83), pKYP200 (Agric. Biol. Chem., 48: 669 (1984)), pLSA1 (Agric. Biol. Chem., 53: 277 (1989)), pGEL1 (Proc. Natl. Acad. Sci. USA, 82: 4306 (1985)), pBluescript II SK(−) (manufactured by Stratagene), pTrs30 (prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)), pTrs32 (prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)), pGHA2 (prepared from Escherichia coli IGHA2 (FERM B-400), Japanese Published Unexamined Patent Application No. 221091/85), pGKA2 (prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Published Unexamined Patent Application No. 221091/85), pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094 and 5,160,735), pSupex, pUB110, pTP5, pC194 and pEG400 (J. Bacteriol., 172: 2392 (1990)), pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), and the like.
Any promoter can be used so long as it can function in the host cell. Examples include promoters derived from Escherichia coli, phage and the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, T7 promoter and the like. Also, artificially designed and modified promoters, such as a promoter in which two Ptrp are linked in series (Ptrp×2), tac promoter, lacT7 promoter letI promoter and the like, can be used.
It is preferred to use a plasmid in which the space between Shine-Dalgarno sequence which is the ribosome binding sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 nucleotides).
The transcription termination sequence is not always necessary for the expression of the DNA of the present invention. However, it is preferred to arrange the transcription terminating sequence at just downstream of the structural gene.
One of ordinary skill in the art will appreciate that the codons of the above-described elements may be optimized, in a known manner, depending on the host cells and environmental conditions utilized.
Examples of the host cell include microorganisms belonging to the genus Escherichia, the genus Serratia, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, the genus Microbacterium, the genus Pseudomonas, and the like. Specific examples include Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485; Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli G1698, Escherichia coli TB1, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Corynebacterium ammoniagenes, Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 14067 (prior genus and species: Brevibacterium flavum), Corynebacterium glutamicum ATCC 13869 (prior genus and species: Brevibacterium lactofermentum, or Corynebacterium lactofermentum), Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium thermoaminogenes FERM 9244, Microbacterium ammoniaphilum ATCC 15354, Pseudomonas putida, Pseudomonas sp. D-0110, and the like.
When Corynebacterium glutamicum or an analogous microorganism is used as a host, an EMF necessary for expressing the polypeptide is not always contained in the vector so long as the polynucleotide of the present invention contains an EMF. When the EMF is not contained in the polynucleotide, it is necessary to prepare the EMF separately and ligate it so as to be in operable combination. Also, when a higher expression amount or specific expression regulation is necessary, it is necessary to ligate the EMF corresponding thereto so as to put the EMF in operable combination with the polynucleotide. Examples of using an externally ligated EMF are disclosed in Microbiology, 142: 1297-1309 (1996).
With regard to the method for the introduction of the recombinant vector, any method for introducing DNA into the above-described host cells, such as a method in which a calcium ion is used (Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)), a protoplast method (Japanese Published Unexamined Patent Application No. 2483942/88), the methods described in Gene, 17: 107 (1982) and Molecular & General Genetics, 168: 111 (1979) and the like, can be used.
When yeast is used as the host cell, examples of the expression vector include pYES2 (manufactured by Invitrogen), YEp13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), pHS19, pHS15, and the like.
Any promoter can be used so long as it can be expressed in yeast. Examples include a promoter of a gene in the glycolytic pathway, such as hexose kinase and the like, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, a heat shock protein promoter, MF α1 promoter, CUP 1 promoter, and the like.
Examples of the host cell include microorganisms belonging to the genus Saccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Schwanniomyces, the genus Pichia, the genus Candida and the like. Specific examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Candida utilis and the like.
With regard to the method for the introduction of the recombinant vector, any method for introducing DNA into yeast, such as an electroporation method (Methods. Enzymol., 194: 182 (1990)), a spheroplast method (Proc. Natl. Acad. Sci. USA, 75: 1929 (1978)), a lithium acetate method (J. Bacteriol., 153: 163 (1983)), a method described in Proc. Natl. Acad. Sci. USA, 75: 1929 (1978) and the like, can be used.
When animal cells are used as the host cells, examples of the expression vector include pcDNA3.1, pSinRep5 and pCEP4 (manufactured by Invitorogen), pRev-Tre (manufactured by Clontech), pAxCAwt (manufactured by Takara Shuzo), pcDNAI and pcDM8 (manufactured by Funakoshi), pAGE107 (Japanese Published Unexamined Patent Application No. 22979/91; Cytotechnology, 3:133 (1990)), pAS3-3 (Japanese Published Unexamined Patent Application No. 227075/90), pcDM8 (Nature, 329: 840 (1987)), pcDNAI/Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 (J. Biochem., 101: 1307 (1987)), pAGE210, and the like.
Any promoter can be used so long as it can function in animal cells. Examples include a promoter of IE (immediate early) gene of cytomegalovirus (CMV), an early promoter of SV40, a promoter of retrovirus, a metallothionein promoter, a heat shock promoter, SRα promoter, and the like. Also, the enhancer of the IE gene of human CMV can be used together with the promoter.
Examples of the host cell include human Namalwa cell, monkey COS cell, Chinese hamster CHO cell, HST5637 (Japanese Published Unexamined Patent Application No. 299/88), and the like.
The method for introduction of the recombinant vector into animal cells is not particularly limited, so long as it is the general method for introducing DNA into animal cells, such as an electroporation method (Cytotechnology, 3: 133 (1990)), a calcium phosphate method (Japanese Published Unexamined Patent Application No. 227075/90), a lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), the method described in Virology, 52: 456 (1973), and the like.
When insect cells are used as the host cells, the polypeptide can be expressed, for example, by the method described in Bacurovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992), Bio/Technology, 6: 47 (1988), or the like.
Specifically, a recombinant gene transfer vector and bacurovirus are simultaneously inserted into insect cells to obtain a recombinant virus in an insect cell culture supernatant, and then the insect cells are infected with the resulting recombinant virus to express the polypeptide.
Examples of the gene introducing vector used in the method include pBlueBac4.5, pVL1392, pVL1393 and pBlueBacIII (manufactured by Invitrogen), and the like.
Examples of the bacurovirus include Autographa californica nuclear polyhedrosis virus with which insects of the family Barathra are infected, and the like.
Examples of the insect cells include Spodoptera frugiperda oocytes Sf9 and Sf21 (Bacurovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)), Trichoplusia ni oocyte High 5 (manufactured by Invitrogen) and the like.
The method for simultaneously incorporating the above-described recombinant gene transfer vector and the above-described bacurovirus for the preparation of the recombinant virus include calcium phosphate method (Japanese Published Unexamined Patent Application No. 227075/90), lipofection method (Proc. Natl. Acad. Sci. USA, 84: 7413 (1987)) and the like.
When plant cells are used as the host cells, examples of expression vector include a Ti plasmid, a tobacco mosaic virus vector, and the like.
Any promoter can be used so long as it can be expressed in plant cells. Examples include 35S promoter of cauliflower mosaic virus (CaMV), rice actin 1 promoter, and the like.
Examples of the host cells include plant cells and the like, such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley, and the like.
The method for introducing the recombinant vector is not particularly limited, so long as it is the general method for introducing DNA into plant cells, such as the Agrobacterium method (Japanese Published Unexamined Patent Application No. 140885/84, Japanese Published Unexamined Patent Application No. 70080/85, WO 94/00977), the electroporation method (Japanese Published Unexamined Patent Application, No. 251887/85), the particle gun method (Japanese Patents 2606856 and 2517813), and the like.
The transformant of the present invention includes a transformant containing the polypeptide of the present invention per se rather than as a recombinant vector, that is, a transformant containing the polypeptide of the present invention which is integrated into a chromosome of the host, in addition to the transformant containing the above recombinant vector.
When expressed in yeasts, animal cells, insect cells or plant cells, a glycopolypeptide or glycosylated polypeptide can be obtained.
The polypeptide can be produced by culturing the thus obtained transformant of the present invention in a culture medium to produce and accumulate the polypeptide of the present invention or any polypeptide expressed under the control of an EMF of the present invention, and recovering the polypeptide from the culture.
Culturing of the transformant of the present invention in a culture medium is carried out according to the conventional method as used in culturing of the host.
When the transformant of the present invention is obtained using a prokaryote, such as Escherichia coli or the like, or a eukaryote, such as yeast or the like, as the host, the transformant is cultured.
Any of a natural medium and a synthetic medium can be used, so long as it contains a carbon source, a nitrogen source, an inorganic salt and the like which can be assimilated by the transformant and can perform culturing of the transformant efficiently.
Examples of the carbon source include those which can be assimilated by the transformant, such as carbohydrates (for example, glucose, fructose, sucrose, molasses containing them, starch, starch hydrolysate, and the like), organic acids (for example, acetic acid, propionic acid, and the like), and alcohols (for example, ethanol, propanol, and the like).
Examples of the nitrogen source include ammonia, various ammonium salts of inorganic acids or organic acids (for example, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and the like), other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermented cells and hydrolysates thereof, and the like.
Examples of inorganic salt include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like.
The culturing is carried out under aerobic conditions by shaking culture, submerged-aeration stirring culture or the like. The culturing temperature is preferably from 15 to 40° C., and the culturing time is generally from 16 hours to 7 days. The pH of the medium is preferably maintained at 3.0 to 9.0 during the culturing. The pH can be adjusted using an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like.
Also, antibiotics, such as ampicillin, tetracycline, and the like, can be added to the medium during the culturing, if necessary.
When a microorganism transformed with a recombinant vector containing an inducible promoter is cultured, an inducer can be added to the medium, if necessary.
For example, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like can be added to the medium when a microorganism transformed with a recombinant vector containing lac promoter is cultured, or indoleacrylic acid (IAA) or the like can by added thereto when a microorganism transformed with an expression vector containing trp promoter is cultured.
Examples of the medium used in culturing a transformant obtained using animal cells as the host cells include RPMI 1640 medium (The Journal of the American Medical Association, 199: 519 (1967)), Eagle's MEM medium (Science, 122: 501 (1952)), Dulbecco's modified MEM medium (Virology, 8, 396 (1959)), 199 Medium (Proceeding of the Society for the Biological Medicine, 73:1 (1950)), the above-described media to which fetal calf serum has been added, and the like.
The culturing is carried out generally at a pH of 6 to 8 and a temperature of 30 to 40° C. in the presence of 5% CO2 for 1 to 7 days.
Also, if necessary, antibiotics, such as kanamycin, penicillin, and the like, can be added to the medium during the culturing.
Examples of the medium used in culturing a transformant obtained using insect cells as the host cells include TNM-FH medium (manufactured by Pharmingen), Sf-900 II SFM (manufactured by Life Technologies), ExCell 400 and ExCell 405 (manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195: 788 (1962)), and the like.
The culturing is carried out generally at a pH of 6 to 7 and a temperature of 25 to 30° C. for 1 to 5 days.
Additionally, antibiotics, such as gentamicin and the like, can be added to the medium during the culturing, if necessary.
A transformant obtained by using a plant cell as the host cell can be used as the cell or after differentiating to a plant cell or organ. Examples of the medium used in the culturing of the transformant include Murashige and Skoog (MS) medium, White medium, media to which a plant hormone, such as auxin, cytokinine, or the like has been added, and the like.
The culturing is carried out generally at a pH of 5 to 9 and a temperature of 20 to 40° C. for 3 to 60 days.
Also, antibiotics, such as kanamycin, hygromycin and the like, can be added to the medium during the culturing, if necessary.
As described above, the polypeptide can be produced by culturing a transformant derived from a microorganism, animal cell or plant cell containing a recombinant vector to which a DNA encoding the polypeptide of the present invention has been inserted according to the general culturing method to produce and accumulate the polypeptide, and recovering the polypeptide from the culture.
The process of gene expression may include secretion of the encoded protein production or fusion protein expression and the like in accordance with the methods described in Molecular Cloning, 2nd ed., in addition to direct expression.
The method for producing the polypeptide of the present invention includes a method of intracellular expression in a host cell, a method of extracellular secretion from a host cell, or a method of production on a host cell membrane outer envelope. The method can be selected by changing the host cell employed or the structure of the polypeptide produced.
When the polypeptide of the present invention is produced in a host cell or on a host cell membrane outer envelope, the polypeptide can be positively secreted extracellularly according to, for example, the method of Paulson et al. (J. Biol. Chem., 264: 17619 (1989)), the method of Lowe et al. (Proc. Natl. Acad. Sci. USA, 86: 8227 (1989); Genes Develop., 4: 1288 (1990)), and/or the methods described in Japanese Published Unexamined Patent Application No. 336963/93, WO 94/23021, and the like.
Specifically, the polypeptide of the present invention can be positively secreted extracellularly by expressing it in the form that a signal peptide has been added to the foreground of a polypeptide containing an active site of the polypeptide of the present invention according to the recombinant DNA technique.
Furthermore, the amount produced can be increased using a gene amplification system, such as by use of a dihydrofolate reductase gene or the like according to the method described in Japanese Published Unexamined Patent Application No. 227075/90.
Moreover, the polypeptide of the present invention can be produced by a transgenic animal individual (transgenic nonhuman animal) or plant individual (transgenic plant).
When the transformant is the animal individual or plant individual, the polypeptide of the present invention can be produced by breeding or cultivating it so as to produce and accumulate the polypeptide, and recovering the polypeptide from the animal individual or plant individual.
Examples of the method for producing the polypeptide of the present invention using the animal individual include a method for producing the polypeptide of the present invention in an animal developed by inserting a gene according to methods known to those of ordinary skill in the art (American Journal of Clinical Nutrition, 63: 639S (1996), American Journal of Clinical Nutrition, 63: 627S (1996), Bio/Technology, 9: 830 (1991)).
In the animal individual, the polypeptide can be produced by breeding a transgenic nonhuman animal to which the DNA encoding the polypeptide of the present invention has been inserted to produce and accumulate the polypeptide in the animal, and recovering the polypeptide from the animal. Examples of the production and accumulation place in the animal include milk (Japanese Published Unexamined Patent Application No. 309192/88), egg and the like of the animal. Any promoter can be used, so long as it can be expressed in the animal. Suitable examples include an α-casein promoter, a β-casein promoter, a β-lactoglobulin promoter, a whey acidic protein promoter, and the like, which are specific for mammary glandular cells.
Examples of the method for producing the polypeptide of the present invention using the plant individual include a method for producing the polypeptide of the present invention by cultivating a transgenic plant to which the DNA encoding the protein of the present invention by a known method (Tissue Culture, 20 (1994), Tissue Culture, 21 (1994), Trends in Biotechnology, 15: 45 (1997)) to produce and accumulate the polypeptide in the plant, and recovering the polypeptide from the plant.
The polypeptide according to the present invention can also be obtained by translation in vitro.
The polypeptide of the present invention can be produced by a translation system in vitro. There are, for example, two in vitro translation methods which may be used, namely, a method using RNA as a template and another method using DNA as a template. The template RNA includes the whole RNA, mRNA, an in vitro transcription product, and the like. The template DNA includes a plasmid containing a transcriptional promoter and a target gene integrated therein and downstream of the initiation site, a PCR/RT-PCR product and the like. To select the most suitable system for the in vitro translation, the origin of the gene encoding the protein to be synthesized (prokaryotic cell/eucaryotic cell), the type of the template (DNA/RNA), the purpose of using the synthesized protein and the like should be considered. In vitro translation kits having various characteristics are commercially available from many companies (Boehringer Mannheim, Promega, Stratagene, or the like), and every kit can be used in producing the polypeptide according to the present invention.
Transcription/translation of a DNA nucleotide sequence cloned into a plasmid containing a T7 promoter can be carried out using an in vitro transcription/translation system E. coli T7 S30 Extract System for Circular DNA (manufactured by Promega, catalogue No. L1130). Also, transcription/translation using, as a template, a linear prokaryotic DNA of a supercoil non-sensitive promoter, such as lacUV5, tac, λPL(con), λPL, or the like, can be carried out using an in vitro transcription/translation system E. coli S30 Extract System for Linear Templates (manufactured by Promega, catalogue No. L1030). Examples of the linear prokaryotic DNA used as a template include a DNA fragment, a PCR-amplified DNA product, a duplicated oligonucleotide ligation, an in vitro transcriptional RNA, a prokaryotic RNA, and the like.
In addition to the production of the polypeptide according to the present invention, synthesis of a radioactive labeled protein, confirmation of the expression capability of a cloned gene, analysis of the function of transcriptional reaction or translation reaction, and the like can be carried out using this system.
The polypeptide produced by the transformant of the present invention can be isolated and purified using the general method for isolating and purifying an enzyme. For example, when the polypeptide of the present invention is expressed as a soluble product in the host cells, the cells are collected by centrifugation after cultivation, suspended in an aqueous buffer, and disrupted using an ultrasonicator, a French press, a Manton Gaulin homogenizer, a Dynomill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a purified product can be obtained by the general method used for isolating and purifying an enzyme, for example, solvent extraction, salting out using ammonium sulfate or the like, desalting, precipitation using an organic solvent, anion exchange chromatography using a resin, such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical) or the like, cation exchange chromatography using a resin, such as S-Sepharose FF (manufactured by Pharmacia) or the like, hydrophobic chromatography using a resin, such as butyl sepharose, phenyl sepharose or the like, gel filtration using a molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis, such as isoelectronic focusing or the like, alone or in combination thereof.
When the polypeptide is expressed as an insoluble product in the host cells, the cells are collected in the same manner, disrupted and centrifuged to recover the insoluble product of the polypeptide as the precipitate fraction. Next, the insoluble product of the polypeptide is solubilized with a protein denaturing agent. The solubilized solution is diluted or dialyzed to lower the concentration of the protein denaturing agent in the solution. Thus, the normal configuration of the polypeptide is reconstituted. After the procedure, a purified product of the polypeptide can be obtained by a purification/isolation method similar to the above.
When the polypeptide of the present invention or its derivative (for example, a polypeptide formed by adding a sugar chain thereto) is secreted out of cells, the polypeptide or its derivative can be collected in the culture supernatant. Namely, the culture supernatant is obtained by treating the culture medium in a treatment similar to the above (for example, centrifugation). Then, a purified product can be obtained from the culture medium using a purification/isolation method similar to the above.
The polypeptide obtained by the above method is within the scope of the polypeptide of the present invention, and examples include a polypeptide encoded by a polynucleotide comprising the nucleotide sequence selected from SEQ ID NOS:2 to 3431, and a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOS:3502 to 6931.
Furthermore, a polypeptide comprising an amino acid sequence in which at least one amino acids is deleted, replaced, inserted or added in the amino acid sequence of the polypeptide and having substantially the same activity as that of the polypeptide is included in the scope of the present invention. The term “substantially the same activity as that of the polypeptide” means the same activity represented by the inherent function, enzyme activity or the like possessed by the polypeptide which has not been deleted, replaced, inserted or added. The polypeptide can be obtained using a method for introducing part-specific mutation(s) described in, for example, Molecular Cloning, 2nd ed., Current Protocols in Molecular Biology, Nuc. Acids. Res., 10: 6487 (1982), Proc. Natl. Acad. Sci. USA, 79: 6409 (1982), Gene, 34: 315 (1985), Nuc. Acids. Res., 13: 4431 (1985), Proc. Natl. Acad. Sci. USA, 82: 488 (1985) and the like. For example, the polypeptide can be obtained by introducing mutation(s) to DNA encoding a polypeptide having the amino acid sequence represented by any one of SEQ ID NOS:3502 to 6931. The number of the amino acids which are deleted, replaced, inserted or added is not particularly limited; however, it is usually 1 to the order of tens, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5, amino acids.
The at least one amino acid deletion, replacement, insertion or addition in the amino acid sequence of the polypeptide of the present invention is used herein to refer to that at least one amino acid is deleted, replaced, inserted or added to at one or plural positions in the amino acid sequence. The deletion, replacement, insertion or addition may be caused in the same amino acid sequence simultaneously. Also, the amino acid residue replaced, inserted or added can be natural or non-natural. Examples of the natural amino acid residue include L-alanine, L-asparagine, L-asparatic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine, and the like.
Herein, examples of amino acid residues which are replaced with each other are shown below. The amino acid residues in the same group can be replaced with each other.
Group A:
leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
Group B:
asparatic acid, glutamic acid, isoasparatic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
Group C:
asparagine, glutamine;
Group D:
lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
Group E:
proline, 3-hydroxyproline, 4-hydroxyproline;
Group F:
serine, threonine, homoserine;
Group G:
phenylalanine, tyrosine.
Also, in order that the resulting mutant polypeptide has substantially the same activity as that of the polypeptide which has not been mutated, it is preferred that the mutant polypeptide has a homology of 60% or more, preferably 80% or more, and particularly preferably 95% or more, with the polypeptide which has not been mutated, when calculated, for example, using default (initial setting) parameters by a homology searching software, such as BLAST, FASTA, or the like.
Also, the polypeptide of the present invention can be produced by a chemical synthesis method, such as Fmoc (fluorenylmethyloxycarbonyl) method, tBoc (t-butyloxycarbonyl) method, or the like. It can also be synthesized using a peptide synthesizer manufactured by Advanced ChemTech, Perkin-Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, or the like.
The transformant of the present invention can be used for objects other than the production of the polypeptide of the present invention.
Specifically, at least one component selected from an amino acid, a nucleic acid, a vitamin, a saccharide, an organic acid, and analogues thereof can be produced by culturing the transformant containing the polynucleotide or recombinant vector of the present invention in a medium to produce and accumulate at least one component selected from amino acids, nucleic acids, vitamins, saccharides, organic acids, and analogues thereof, and recovering the same from the medium.
The biosynthesis pathways, decomposition pathways and regulatory mechanisms of physiologically active substances such as amino acids, nucleic acids, vitamins, saccharides, organic acids and analogues thereof differ from organism to organism. The productivity of such a physiologically active substance can be improved using these differences, specifically by introducing a heterogeneous gene relating to the biosynthesis thereof. For example, the content of lysine, which is one of the essential amino acids, in a plant seed was improved by introducing a synthase gene derived from a bacterium (WO 93/19190). Also, arginine is excessively produced in a culture by introducing an arginine synthase gene derived from Escherichia coli (Japanese Examined Patent Publication 23750/93).
To produce such a physiologically active substance, the transformant according to the present invention can be cultured by the same method as employed in culturing the transformant for producing the polypeptide of the present invention as described above. Also, the physiologically active substance can be recovered from the culture medium in combination with, for example, the ion exchange resin method, the precipitation method and other known methods.
Examples of methods known to one of ordinary skill in the art include electroporation, calcium transfection, the protoplast method, the method using a phage, and the like, when the host is a bacterium; and microinjection, calcium phosphate transfection, the positively charged lipid-mediated method and the method using a virus, and the like, when the host is a eukaryote (Molecular Cloning, 2nd. ed.; Spector et al., Cells/a laboratory manual, Cold Spring Harbour Laboratory Press, 1998)). Examples of the host include prokaryotes, lower eukaryotes (for example, yeasts), higher eukaryotes (for example, mammals), and cells isolated therefrom. As the state of a recombinant polynucleotide fragment present in the host cells, it can be integrated into the chromosome of the host. Alternatively, it can be integrated into a factor (for example, a plasmid) having an independent replication unit outside the chromosome. These transformants are usable in producing the polypeptides of the present invention encoded by the ORF of the genome of Corynebacterium glutamicum, the polynucleotides of the present invention and fragments thereof. Alternatively, they can be used in producing arbitrary polypeptides under the regulation by an EMF of the present invention.
11. Preparation of Antibody Recognizing the Polypeptide of the Present Invention
An antibody which recognizes the polypeptide of the present invention, such as a polyclonal antibody, a monoclonal antibody, or the like, can be produced using, as an antigen, a purified product of the polypeptide of the present invention or a partial fragment polypeptide of the polypeptide or a peptide having a partial amino acid sequence of the polypeptide of the present invention.
(1) Production of Polyclonal Antibody
A polyclonal antibody can be produced using, as an antigen, a purified product of the polypeptide of the present invention, a partial fragment polypeptide of the polypeptide, or a peptide having a partial amino acid sequence of the polypeptide of the present invention, and immunizing an animal with the same.
Examples of the animal to be immunized include rabbits, goats, rats, mice, hamsters, chickens and the like.
A dosage of the antigen is preferably 50 to 100 μg per animal.
When the peptide is used as the antigen, it is preferably a peptide covalently bonded to a carrier protein, such as keyhole limpet haemocyanin, bovine thyroglobulin, or the like. The peptide used as the antigen can be synthesized by a peptide synthesizer.
The administration of the antigen is, for example, carried out 3 to 10 times at the intervals of 1 or 2 weeks after the first administration. On the 3rd to 7th day after each administration, a blood sample is collected from the venous plexus of the eyeground, and it is confirmed that the serum reacts with the antigen by the enzyme immunoassay (Enzyme-linked Immunosorbent Assay (ELISA), Igaku Shoin (1976); Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)) or the like.
Serum is obtained from the immunized non-human mammal with a sufficient antibody titer against the antigen used for the immunization, and the serum is isolated and purified to obtain a polyclonal antibody.
Examples of the method for the isolation and purification include centrifugation, salting out by 40-50% saturated ammonium sulfate, caprylic acid precipitation (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory (1988)), or chromatography using a DEAE-Sepharose column, an anion exchange column, a protein A- or G-column, a gel filtration column, and the like, alone or in combination thereof, by methods known to those of ordinary skill in the art.
(2) Production of Monoclonal Antibody
(a) Preparation of Antibody-Producing Cell
A rat having a serum showing an enough antibody titer against a partial fragment polypeptide of the polypeptide of the present invention used for immunization is used as a supply source of an antibody-producing cell.
On the 3rd to 7th day after the antigen substance is finally administered the rat showing the antibody titer, the spleen is excised.
The spleen is cut to pieces in MEM medium (manufactured by Nissui Pharmaceutical), loosened using a pair of forceps, followed by centrifugation at 1,200 rpm for 5 minutes, and the resulting supernatant is discarded.
The spleen in the precipitated fraction is treated with a Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to eliminate erythrocytes and washed three times with MEM medium, and the resulting spleen cells are used as antibody-producing cells.
(b) Preparation of Myeloma Cells
As myeloma cells, an established cell line obtained from mouse or rat is used. Examples of useful cell lines include those derived from a mouse, such as P3-X63Ag8-U1 (hereinafter referred to as “UP3-U1”) (Curr. Topics in Microbiol. Immunol., 81: 1 (1978); Europ. J. Immunol., 6: 511 (1976)); SP2/O-Ag14 (SP-2) (Nature, 276: 269 (1978)): P3-X63-Ag8653 (653) (J. Immunol, 123: 1548 (1979)); P3-X63-Ag8 (X63) cell line (Nature, 256: 495 (1975)), and the like, which are 8-azaguanine-resistant mouse (BALB/c) myeloma cell lines. These cell lines are subcultured in 8-azaguanine medium (medium in which, to a medium obtained by adding. 1.5 mmol/l glutamine, 5×10−5 mol/l 2-mercaptoethanol, 10 μg/ml gentamicin and 10% fetal calf serum (FCS) (manufactured by CSL) to RPMI-1640 medium (hereinafter referred to as the “normal medium”), 8-azaguanine is further added at 15 μg/ml) and cultured in the normal medium 3 or 4 days before cell fusion, and 2×107 or more of the cells are used for the fusion.
(c) Production of Hybridoma
The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are washed with MEM medium or PBS (disodium hydrogen phosphate: 1.83 g, sodium dihydrogen phosphate: 0.21 g, sodium chloride: 7.65 g, distilled water: 1 liter, pH: 7.2) and mixed to give a ratio of antibody-producing cells:myeloma cells=5:1 to 10:1, followed by centrifugation at 1,200 rpm for 5 minutes, and the supernatant is discarded.
The cells in the resulting precipitated fraction were thoroughly loosened, 0.2 to 1 ml of a mixed solution of 2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM medium and 0.7 ml of dimethylsulfoxide (DMSO) per 108 antibody-producing cells is added to the cells under stirring at 37° C., and then 1 to 2 ml of MEM medium is further added thereto several times at 1 to 2 minute intervals.
After the addition, MEM medium is added to give a total amount of 50 ml. The resulting prepared solution is centrifuged at 900 rpm for 5 minutes, and then the supernatant is discarded. The cells in the resulting precipitated fraction were gently loosened and then gently suspended in 100 ml of HAT medium (the normal medium to which 10−4 mol/l hypoxanthine, 1.5×10−5 mol/l thymidine and 4×10−7 mol/l aminopterin have been added) by repeated drawing up into and discharging from a measuring pipette.
The suspension is poured into a 96 well culture plate at 100 μl/well and cultured at 37° C. for 7 to 14 days in a 5% CO2 incubator.
After culturing, a part of the culture supernatant is recovered, and a hybridoma which specifically reacts with a partial fragment polypeptide of the polypeptide of the present invention is selected according to the enzyme immunoassay described in Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Chapter 14 (1998) and the like.
A specific example of the enzyme immunoassay is described below.
The partial fragment polypeptide of the polypeptide of the present invention used as the antigen in the immunization is spread on a suitable plate, is allowed to react with a hybridoma culturing supernatant or a purified antibody obtained in (d) described below as a first antibody, and is further allowed to react with an anti-rat or anti-mouse immunoglobulin antibody labeled with an enzyme, a chemical luminous substance, a radioactive substance or the like as a second antibody for reaction suitable for the labeled substance. A hybridoma which specifically reacts with the polypeptide of the present invention is selected as a hybridoma capable of producing a monoclonal antibody of the present invention.
Cloning is repeated using the hybridoma twice by limiting dilution analysis (HT medium (a medium in which aminopterin has been removed from HAT medium) is firstly used, and the normal medium is secondly used), and a hybridoma which is stable and contains a sufficient amount of antibody titer is selected as a hybridoma capable of producing a monoclonal antibody of the present invention.
(d) Preparation of Monoclonal Antibody
The monoclonal antibody-producing hybridoma cells obtained in (c) are injected intraperitoneally into 8- to 10-week-old mice or nude mice treated with pristane (intraperitoneal administration of 0.5 ml of 2,6,10,14-tetramethylpentadecane (pristane) followed by 2 weeks of feeding) at 5×106 to 20×106 cells/animal. The hybridoma causes ascites tumor in 10 to 21 days.
The ascitic fluid is collected from the mice or nude mice, and centrifuged to remove solid contents at 3000 rpm for 5 minutes.
A monoclonal antibody can be purified and isolated from the resulting supernatant according to the method similar to that used in the polyclonal antibody.
The subclass of the antibody can be determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The polypeptide amount can be determined by the Lowry method or by calculation based on the absorbance at 280 nm.
The antibody obtained in the above is within the scope of the antibody of the present invention.
The antibody can be used for the general assay using an antibody, such as a radioactive material labeled immunoassay (RIA), competitive binding assay, an immunotissue chemical staining method (ABC method, CSA method, etc.), immunoprecipitation, Western blotting, ELISA assay, and the like (An introduction to Radioimmunoassay and Related Techniques, Elsevier Science (1986); Techniques in Immunocytochemistry, Academic Press, Vol. 1 (1982), Vol. 2 (1983) & Vol. 3 (1985); Practice and Theory of Enzyme Immunoassays, Elsevier Science (1985); Enzyme-linked Immunosorbent Assay (ELISA), Igaku Shoin (1976); Antibodies—A Laboratory Manual, Cold Spring Harbor laboratory (1988); Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987); Second Series Biochemical Experiment Course, Vol. 5, Immunobiochemistry Research Method, Tokyo Kagaku Dojin (1986)).
The antibody of the present invention can be used as it is or after being labeled with a label.
Examples of the label include radioisotope, an affinity label (e.g., biotin, avidin, or the like), an enzyme label (e.g., horseradish peroxidase, alkaline phosphatase, or the like), a fluorescence label (e.g., FITC, rhodamine, or the like), a label using a rhodamine atom, (J. Histochem. Cytochem., 18: 315 (1970); Meth. Enzym., 62: 308 (1979); Immunol., 109: 129 (1972); J. Immunol., Meth., 13: 215 (1979)), and the like.
Expression of the polypeptide of the present invention, fluctuation of the expression, the presence or absence of structural change of the polypeptide, and the presence or absence in an organism other than coryneform bacteria of a polypeptide corresponding to the polypeptide can be analyzed using the antibody or the labeled antibody by the above assay, or a polypeptide array or proteome analysis described below.
Furthermore, the polypeptide recognized by the antibody can be purified by immunoaffinity chromatography using the antibody of the present invention.
12. Production and Use of Polypeptide Array
(1) Production of Polypeptide Array
A polypeptide array can be produced using the polypeptide of the present invention obtained in the above item 10 or the antibody of the present invention obtained in the above item 11.
The polypeptide array of the present invention includes protein chips, and comprises a solid support and the polypeptide or antibody of the present invention adhered to the surface of the solid support.
Examples of the solid support include plastic such as polycarbonate or the like; an acrylic resin, such as polyacrylamide or the like; complex carbohydrates, such as agarose, sepharose, or the like; silica; a silica-based material, carbon, a metal, inorganic glass, latex beads, and the like.
The polypeptides or antibodies according to the present invention can be adhered to the surface of the solid support according to the method described in Biotechniques, 27: 1258-61 (1999); Molecular Medicine Today, 5: 326-7 (1999); Handbook of Experimental Immunology, 4th edition, Blackwell Scientific Publications, Chapter 10 (1986); Meth. Enzym., 34 (1974); Advances in Experimental Medicine and Biology, 42 (1974); U.S. Pat. No. 4,681,870; U.S. Pat. No. 4,282,287; U.S. Pat. No. 4,762,881, or the like.
The analysis described herein can be efficiently performed by adhering the polypeptide or antibody of the present invention to the solid support at a high density, though a high fixation density is not always necessary.
(2) Use of Polypeptide Array
A polypeptide or a compound capable of binding to and interacting with the polypeptides of the present invention adhered to the array can be identified using the polypeptide array to which the polypeptides of the present invention have been adhered thereto as described in the above (1).
Specifically, a polypeptide or a compound capable of binding to and interacting with the polypeptides of the present invention can be identified by subjecting the polypeptides of the present invention to the following steps (i) to (iv):
Specific examples of the polypeptide array to which the polypeptide of the present invention has been adhered include a polypeptide array containing a solid support to which at least one of a polypeptide containing an amino acid sequence selected from SEQ ID NOS:3502 to 7001, a polypeptide containing an amino acid sequence in which at least one amino acids is deleted, replaced, inserted or added in the amino acid sequence of the polypeptide and having substantially the same activity as that of the polypeptide, a polypeptide containing an amino acid sequence having a homology of 60% or more with the amino acid sequences of the polypeptide and having substantially the same activity as that of the polypeptides, a partial fragment polypeptide, and a peptide comprising an amino acid sequence of a part of a polypeptide.
The amount of production of a polypeptide derived from coryneform bacteria can be analyzed using a polypeptide array to which the antibody of the present invention has been adhered in the above (1).
Specifically, the expression amount of a gene derived from a mutant of coryneform bacteria can be analyzed by subjecting the gene to the following steps (i) to (iv):
Specific examples of the polypeptide array to which the antibody of the present invention is adhered include a polypeptide array comprising a solid support to which at least one of an antibody which recognizes a polypeptide comprising an amino acid sequence selected from SEQ ID NOS:3502 to 7001, a polypeptide comprising an amino acid sequence in which at least one amino acids is deleted, replaced, inserted or added in the amino acid sequence of the polypeptide and having substantially the same activity as that of the polypeptide, a polypeptide comprising an amino acid sequence having a homology of 60% or more with the amino acid sequences of the polypeptide and having substantially the same activity as that of the polypeptides, a partial fragment polypeptide, or a peptide comprising an amino acid sequence of a part of a polypeptide.
A fluctuation in an expression amount of a specific polypeptide can be monitored using a polypeptide obtained in the time course of culture as the polypeptide derived from coryneform bacteria. The culturing conditions can be optimized by analyzing the fluctuation.
When a polypeptide derived from a mutant of coryneform bacteria is used, a mutated polypeptide can be detected.
13. Identification of Useful Mutation in Mutant by Proteome Analysis
Usually, the proteome is used herein to refer to a method wherein a polypeptide is separated by two-dimensional electrophoresis and the separated polypeptide is digested with an enzyme, followed by identification of the polypeptide using a mass spectrometer (MS) and searching a data base.
The two dimensional electrophoresis means an electrophoretic method which is performed by combining two electrophoretic procedures having different principles. For example, polypeptides are separated depending on molecular weight in the primary electrophoresis. Next, the gel is rotated by 90° or 180° and the secondary electrophoresis is carried out depending on isoelectric point. Thus, various separation patterns can be achieved (JIS K 3600 2474).
In searching the data base, the amino acid sequence information of the polypeptides of the present invention and the recording medium of the present invention provide for in the above items 2 and 8 can be used.
The proteome analysis of a coryneform bacterium and its mutant makes it possible to identify a polypeptide showing a fluctuation therebetween.
The proteome analysis of a wild type strain of coryneform bacteria and a production strain showing an improved productivity of a target product makes it possible to efficiently identify a mutation protein which is useful in breeding for improving the productivity of a target product or a protein of which expression amount is fluctuated.
Specifically, a wild type strain of coryneform bacteria and a lysine-producing strain thereof are each subjected to the proteome analysis. Then, a spot increased in the lysine-producing strain, compared with the wild type strain, is found and a data base is searched so that a polypeptide showing an increase in yield in accordance with an increase in the lysine productivity can be identified. For example, as a result of the proteome analysis on a wild type strain and a lysine-producing strain, the productivity of the catalase having the amino acid sequence represented by SEQ ID NO:3785 is increased in the lysine-producing mutant.
As a result that a protein having a high expression level is identified by proteome analysis using the nucleotide sequence information and the amino acid sequence information, of the genome of the coryneform bacteria of the present invention, and a recording medium storing the sequences, the nucleotide sequence of the gene encoding this protein and the nucleotide sequence in the upstream thereof can be searched at the same time, and thus, a nucleotide sequence having a high expression promoter can be efficiently selected.
In the proteome analysis, a spot on the two-dimentional electrophoresis gel showing a fluctuation is sometimes derived from a modified protein. However, the modified protein can be efficiently identified using the recording medium storing the nucleotide sequence information, the amino acid sequence information, of the genome of coryneform bacteria, and the recording medium storing the sequences, according to the present invention.
Moreover, a useful mutation point in a useful mutant can be easily specified by searching a nucleotide sequence (nucleotide sequence of promoters, ORF, or the like) relating to the thus identified protein using a recording medium storing the nucleotide sequence information and the amino acid sequence information, of the genome of coryneform bacteria of the present invention, and a recording medium storing the sequences and using a primer designed on the basis of the detected nucleotide sequence. As a result that the useful mutation point is specified, an industrially useful mutant having the useful mutation or other useful mutation derived therefrom can be easily bred.
The present invention will be explained in detail below based on Examples. However, the present invention is not limited thereto.
Determination of the Full Nucleotide Sequence of Genome of Corynebacterium glutamicum
The full nucleotide sequence of the genome of Corynebacterium glutamicum was determined based on the whole genome shotgun method (Science, 269: 496-512 (1995)). In this method, a genome library was prepared and the terminal sequences were determined at random. Subsequently, these sequences were ligated on a computer to cover the full genome. Specifically, the following procedure was carried out.
(1) Preparation of Genome DNA of Corynebacterium glutamicum ATCC 13032
Corynebacterium glutamicum ATCC 13032 was cultured in BY medium (7 g/l meat extract, 10 g/l peptone, 3 g/l sodium chloride, 5 g/l yeast extract, pH 7.2) containing 1% of glycine at 30° C. overnight and the cells were collected by centrifugation. After washing with STE buffer (10.3% sucrose, 25 mmol/l Tris hydrochloride, 25 mmol/l EDTA, pH 8.0), the cells were suspended in 10 ml of STE buffer containing 10 mg/ml lysozyme, followed by gently shaking at 37° C. for 1 hour. Then, 2 ml of 10% SDS was added thereto to lyse the cells, and the resultant mixture was maintained at 65° C. for 10 minutes and then cooled to room temperature. Then, 10 ml of Tris-neutralized phenol was added thereto, followed by gently shaking at room temperature for 30 minutes and centrifugation (15,000×g, 20 minutes, 20° C.). The aqueous layer was separated and subjected to extraction with phenol/chloroform and extraction with chloroform (twice) in the same manner. To the aqueous layer, 3 mol/l sodium acetate solution (pH 5.2) and isopropanol were added at 1/10 times volume and twice volume, respectively, followed by gently stirring to precipitate the genome DNA. The genome DNA was dissolved again in 3 ml of TE buffer (10 mmol/l Tris hydrochloride, 1 mmol/l EDTA, pH 8.0) containing 0.02 mg/ml of RNase and maintained at 37° C. for 45 minutes. The extractions with phenol, phenol/chloroform and chloroform were carried out successively in the same manner as the above. The genome DNA was subjected to isopropanol precipitation. The thus formed genome DNA precipitate was washed with 70% ethanol three times, followed by air-drying, and dissolved in 1.25 ml of TE buffer to give a genome DNA solution (concentration: 0.1 mg/ml).
(2) Construction of a Shotgun Library
TE buffer was added to 0.01 mg of the thus prepared genome DNA of Corynebacterium glutamicum ATCC 13032 to give a total volume of 0.4 ml, and the mixture was treated with a sonicator (Yamato Powersonic Model 150) at an output of 20 continuously for 5 seconds to obtain fragments of 1 to 10 kb. The genome fragments were blunt-ended using a DNA blunting kit (manufactured by Takara Shuzo) and then fractionated by 6% polyacrylamide gel electrophoresis. Genome fragments of 1 to 2 kb were cut out from the gel, and 0.3 ml MG elution buffer (0.5 mol/l ammonium acetate, 10 mmol/l magnesium acetate, 1 mmol/l EDTA, 0.1% SDS) was added thereto, followed by shaking at 37° C. overnight to elute DNA. The DNA eluate was treated with phenol/chloroform, and then precipitated with ethanol to obtain a genome library insert. The total insert and 500 ng of pUC18 SmaI/BAP (manufactured by Amersham Pharmacia Biotech) were ligated at 16° C. for 40 hours.
The ligation product was precipitated with ethanol and dissolved in 0.01 ml of TE buffer. The ligation solution (0.001 ml) was introduced into 0.04 ml of E. coli ELECTRO MAX DH10B (manufactured by Life Technologies) by the electroporation under conditions according to the manufacture's instructions. The mixture was spread on LB plate medium (LB medium (10 g/l bactotrypton, 5 g/l yeast extract, 10 g/l sodium chloride, pH 7.0) containing 1.6% of agar) containing 0.1 mg/ml ampicillin, 0.1 mg/ml X-gal and 1 mmol/l isopropyl-β-D-thiogalactopyranoside (IPTG) and cultured at 37° C. overnight.
The transformant obtained from colonies formed on the plate medium was stationarily cultured in a 96-well titer plate having 0.05 ml of LB medium containing 0.1 mg/ml ampicillin at 37° C. overnight. Then, 0.05 ml of LB medium containing 20% glycerol was added thereto, followed by stirring to obtain a glycerol stock.
(3) Construction of Cosmid Library
About 0.1 mg of the genome DNA of Corynebacterium glutamicum ATCC 13032 was partially digested with Sau3AI (manufactured by Takara Shuzo) and then ultracentrifuged (26,000 rpm, 18 hours, 20° C.) under 10 to 40% sucrose density gradient obtained using 10% and 40% sucrose buffers (1 mol/l NaCl, 20 mmol/l Tris hydrochloride, 5 mmol/l EDTA, 10% or 40% sucrose, pH 8.0). After the centrifugation, the solution thus separated was fractionated into tubes at 1 ml in each tube. After confirming the DNA fragment length of each fraction by agarose gel electrophoresis, a fraction containing a large amount of DNA fragment of about 40 kb was precipitated with ethanol.
The DNA fragment was ligated to the BamHI site of superCos1 (manufactured by Stratagene) in accordance with the manufacture's instructions. The ligation product was incorporated into Escherichia coli XL-1-BlueMR strain (manufactured by Stratagene) using Gigapack III Gold Packaging Extract (manufactured by Stratagene) in accordance with the manufacture's instructions. The Escherichia coli was spread on LB plate medium containing 0.1 mg/ml ampicillin and cultured therein at 37° C. overnight to isolate colonies. The resulting colonies were stationarily cultured at 37° C. overnight in a 96-well titer plate containing 0.05 ml of the LB medium containing 0.1 mg/ml ampicillin in each well. LB medium containing 20% glycerol (0.05 ml) was added thereto, followed by stirring to obtain a glycerol stock.
(4) Determination of Nucleotide Sequence
(4-1) Preparation of Template
The full nucleotide sequence of Corynebacterium glutamicum ATCC 13032 was determined mainly based on the whole genome shotgun method. The template used in the whole genome shotgun method was prepared by the PCR-method using the library prepared in the above (2).
Specifically, the clone derived from the whole genome shotgun library was inoculated using a replicator (manufactured by GENETIX) into each well of a 96-well plate containing the LB medium containing 0.1 mg/ml of ampicillin at 0.08 ml per each well and then stationarily cultured at 37° C. overnight.
Next, the culturing solution was transported using a copy plate (manufactured by Tokken) into a 96-well reaction plate (manufactured by PE Biosystems) containing a PCR reaction solution (TaKaRa Ex Taq (manufactured by Takara Shuzo)) at 0.08 ml per each well. Then, PCR was carried out in accordance with the protocol by Makino et. al. (DNA Research, 5: 1-9 (1998)) using GeneAmp PCR System 9700 (manufactured by PE Biosystems) to amplify the inserted fragment.
The excessive primers and nucleotides were eliminated using a kit for purifying a PCR production (manufactured by Amersham Pharmacia Biotech) and the residue was used as the template in the sequencing reaction.
Some nucleotide sequences were determined using a double-stranded DNA plasmid as a template.
The double-stranded DNA plasmid as the template was obtained by the following method.
The clone derived from the whole genome shotgun library was inoculated into a 24- or 96-well plate containing a 2× YT medium (16 g/l bactotrypton, 10 g/l yeast extract, 5 g/l sodium chloride, pH 7.0) containing 0.05 mg/ml ampicillin at 1.5 ml per each well and then cultured under shaking at 37° C. overnight.
The double-stranded DNA plasmid was prepared from the culturing solution using an automatic plasmid preparing machine, KURABO PI-50 (manufactured by Kurabo Industries) or a multiscreen (manufactured by Millipore) in accordance with the protocol provided by the manufacturer.
To purify the double-stranded DNA plasmid using the multiscreen, Biomek 2000 (manufactured by Beckman Coulter) or the like was employed.
The thus obtained double-stranded DNA plasmid was dissolved in water to give a concentration of about 0.1 mg/ml and used as the template in sequencing.
(4-2) Sequencing Reaction
To 6 μl of a solution of ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (manufactured by PE Biosystems), an M13 regular direction primer (M13-21) or an M13 reverse direction primer (M13REV) (DNA Research, 5: 1-9 (1998) and the template prepared in the above (4-1) (the PCR product or the plasmid) were added to give 10 μl of a sequencing reaction solution. The primers and the templates were used in an amount of 1.6 pmol and an amount of 50 to 200 ng, respectively.
Dye terminator sequencing reaction of 45 cycles was carried out with GeneAmp PCR System 9700 (manufactured by PE Biosystems) using the reaction solution. The cycle parameter was determined in accordance with the manufacturer's instruction accompanying ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit. The sample was purified using MultiScreen HV plate (manufactured by Millipore) according to the manufacture's instructions. The thus purified reaction product was precipitated with ethanol, followed by drying, and then stored in the dark at −30° C.
The dry reaction product was analyzed by ABI PRISM 377 DNA Sequencer and ABI PRISM 3700 DNA Analyzer (both manufactured by PE Biosystems) each in accordance with the manufacture's instructions.
The data of about 50,000 sequences in total (i.e., about 42,000 sequences obtained using 377 DNA Sequencer and about 8,000 reactions obtained by 3700 DNA Analyser) were transferred to a server (Alpha Server 4100: manufactured by COMPAQ) and stored. The data of these about 50,000 sequences corresponded to 6 times as much as the genome size.
(5) Assembly
All operations were carried out on the basis of UNIX platform. The analytical data were output in Macintosh platform using X Window System. The base call was carried out using phred (The University of Washington). The vector sequence data was deleted using SPS Cross_Match (manufactured by Southwest Parallel Software). The assembly was carried out using SPS phrap (manufactured by Southwest Parallel Software; a high-speed version of phrap (The University of Washington)). The contig obtained by the assembly was analyzed using a graphical editor, consed (The University of Washington). A series of the operations from the base call to the assembly were carried out simultaneously using a script phredPhrap attached to consed.
(6) Determination of Nucleotide Sequence in Gap Part
Each cosmid in the cosmid library constructed in the above (3) was prepared by a method similar to the preparation of the double-stranded DNA plasmid described in the above (4-1). The nucleotide sequence at the end of the inserted fragment of the cosmid was determined by using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (manufactured by PE Biosystems) according to the manufacture's instructions.
About 800 cosmid clones were sequenced at both ends to search a nucleotide sequence in the contig derived from the shotgun sequencing obtained in the above (5) coincident with the sequence. Thus, the linkage between respective cosmid clones and respective contigs were determined and mutual alignment was carried out. Furthermore, the results were compared with the physical map of Corynebacterium glutamicum ATCC 13032 (Mol. Gen. Genet., 252: 255-265 (1996) to carrying out mapping between the cosmids and the contigs.
The sequence in the region which was not covered with the contigs was determined by the following method.
Clones containing sequences positioned at the ends of contigs were selected. Among these clones, about 1,000 clones wherein only one end of the inserted fragment had been determined were selected and the sequence at the opposite end of the inserted fragment was determined. A shotgun library clone or a cosmid clone containing the sequences at the respective ends of the inserted fragment in two contigs was identified, the full nucleotide sequence of the inserted fragment of this clone was determined, and thus the nucleotide sequence of the gap part was determined. When no shotgun library clone or cosmid clone covering the gap part was available, primers complementary to the end sequences at the two contigs were prepared and the DNA fragment in the gap part was amplified by PCR. Then, sequencing was performed by the primer walking method using the amplified DNA fragment as a template or by the shotgun method in which the sequence of a shotgun clone prepared from the amplified DNA fragment was determined. Thus, the nucleotide sequence of the domain was determined.
In a region showing a low sequence precision, primers were synthesized using AUTOFINISH function and NAVIGATING function of consed (The University of Washington) and the sequence was determined by the primer walking method to improve the sequence precision. The thus determined full nucleotide sequence of the genome of Corynebacterium glutamicum ATCC 13032 strain is shown in SEQ ID NO:1.
(7) Identification of ORF and Presumption of its Function
ORFs in the nucleotide sequence represented by SEQ ID NO:1 were identified according to the following method. First, the ORF regions were determined using software for identifying ORF, i.e., Glimmer, GeneMark and GeneMark.hmm on UNIX platform according to the respective manual attached to the software.
Based on the data thus obtained, ORFs in the nucleotide sequence represented by SEQ ID NO:1 were identified.
The putative function of an ORF was determined by searching the homology of the identified amino acid sequence of the ORF against an amino acid database consisting of protein-encoding domains derived from Swiss-Prot, PIR or Genpept database constituted by protein encoding domains derived from GenBank database, Frame Search (manufactured by Compugen), or by searching the homology of the identified amino acid sequence of the ORF against an amino acid database consisting of protein-encoding domains derived from Swiss-Prot, PIR or Genpept database constituted by protein encoding domains derived from GenBank database, BLAST. The nucleotide sequences of the thus determined ORFs are shown in SEQ ID NOS:2 to 3501, and the amino acid sequences encoded by these ORFs are shown in SEQ ID NOS:3502 to 7001.
In some cases of the sequence listings in the present invention, nucleotide sequences, such as TTG, TGT, GGT, and the like, other than ATG, are read as an initiating codon encoding Met.
Also, the preferred nucleotide sequences are SEQ ID NOS:2 to 355 and 357 to 3501, and the preferred amino acid sequences are shown in SEQ ID NOS:3502 to 3855 and 3957 to 7001
Table 1 shows the registration numbers in the above-described databases of sequences which were judged as having the highest homology with the nucleotide sequences of the ORFs as the results of the homology search in the amino acid sequences using the homology-searching software Frame Search (manufactured by Compugen), names of the genes of these sequences, the functions of the genes, and the matched length, identities and analogies compared with publicly known amino acid translation sequences. Moreover, the corresponding positions were confirmed via the alignment of the nucleotide sequence of an arbitrary ORF with the nucleotide sequence of SEQ ID NO:1. Also, the positions of nucleotide sequences other than the ORFs (for example, ribosomal RNA genes, transfer RNA genes, IS sequences, and the like) on, the genome were determined.
Brevibacterium flavum dnaA
Mycobacterium smegmatis dnaN
Mycobacterium smegmatis recF
Streptomyces coelicolor yreG
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 yeiH
Hydrogenophilus thermoluteolus
Rhodobacter capsulatus ccdA
Coxiella burnetii com1
Mycobacterium tuberculosis
Mycobacterium leprae
Corynebacterium sp. ATCC
Vibrio parahaemolyticus nutA
Deinococcus radiodurans
Corynebacterium striatum ORF1
Xanthomonas campestris
Thiobacillus ferrooxidans recG
Saccharomyces cerevisiae
Erysipelothrix rhusiopathiae
Streptococcus pyogenes SF370
Escherichia coli K12 fecE
Thermotoga maritima MSB8
Escherichia coli K12 rbsC
Bacillus subtilis 168 rbsA
Petromyzon marinus
Mycobacterium leprae H37RV
Bacillus subtilis 168 yqgP
Escherichia coli K12 fepG
Vibrio cholerae viuC
Vibrio vulnificus MO6-24 viuB
Mycobacterium tuberculosis
Mycobacterium leprae pknB
Streptomyces coelicolor pksC
Streptomyces griseus pbpA
Bacillus subtilis 168 spoVE
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Trichosporon cutaneum ATCC
Escherichia coli K12 gabD
Bacillus subtilis yrkH
Methanococcus jannaschii
Bacillus subtilis yrkF
Synechocystis sp. PCC6803
Mycobacterium tuberculosis
Leishmania major L4768.11
Mycobacterium tuberculosis
Zymomonas mobilis ZM4 clcb
Salmonella typhimurium pnuC
Mycobacterium tuberculosis
Bacillus subtilis citM
Escherichia coli K12 dpiB
Escherichia coli K12 criR
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Mycobacterium tuberculosis
Saccharomyces cerevisiae
Chlamydia muridarum Nigg
Chlamydia pneumoniae
Streptomyces virginiae varS
Bacillus sp.
Saccharomyces cerevisiae hst2
Propionibacterium acnes
Propionibacterium acnes
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Agrobacterium radiobacter echA
Streptomyces viridifaciens vlmF
Escherichia coli K12 htpG
Escherichia coli K12 amn
Aeropyrum pernix K1 APE2509
Salmonella typhimurium putA
Phanerochaete chrysosporium
Escherichia coli K12 ydaH
Enterobacter agglomerans
Escherichia coli K12 yidH
Agrobacterium tumefaciens
Bacillus subtilis yurT
Mycobacterium tuberculosis
Pseudomonas fluorescens mtlD
Klebsiella pneumoniae dalT
Escherichia coli K12 gatR
Streptomyces rubiginosus xylB
Corynebacterium glutamicum
Corynebacterium glutamicum
Arabidopsis thaliana mag
Methanosarcina thermophila
Bacillus subtilis W23 xylR
Lactococcus lactis mef214
Agrobacterium tumefaciens celA
Saccharomyces cerevisiae
Pseudomonas aeruginosa rarD
Escherichia coli K12 yadS
Escherichia coli K12 abrB
Escherichia coli K12 yfcA
Escherichia coli K12 hrpB
Rhizobium leguminosarum bv.
Escherichia coli o373#1 alkB
Escherichia coli K12 tag
Escherichia coli K12 rhtC
Bacillus subtilis yaaA
Streptomyces peucetius dnrV
Schizosaccharomyces pombe
Neisseria meningitidis MC58
Mus musculus nl1
Escherichia coli K12 farR
Beta vulgaris
Streptomyces coelicolor A3(2)
Streptomyces coelicolor msdA
Bacillus subtilis iolB
Bacillus subtilis iolD
Rhizobium meliloti mocC
Bacillus subtilis idh or iolG
Bacillus subtilis iolH
Streptomyces glaucescens tcmA
Bacillus subtilis yvaA
Streptomyces reticuli cebR
Rhizobium sp. NGR234 y4hM
Bacillus subtilis yfiH
Streptomyces coelicolor A3(2)
Stellaria longipes
Bacillus subtilis ccpA
Lactobacillus brevis xylT
Corynebacterium glutamicum
Rhizobium meliloti fixL
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium tuberculosis
Mycobacterium avium embB
Mycobacterium tuberculosis
Pseudomonas sp. phbB
Mycobacterium tuberculosis
Leishmania major ppg1
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Agrobacterium tumefaciens
Yersinia enterocolitica rfbE
Yersinia enterocolitica rfbD
Mycobacterium tuberculosis
Homo sapiens pig3
Mycobacterium tuberculosis
Bacillus subtilis alsT
Synechococcus sp. PCC 7942
Arthrobacter nicotinovorans
Synechococcus sp. PCC 7942
Arthrobacter nicotinovorans
Arthrobacter nicotinovorans
Arthrobacter nicotinovorans
Arthrobacter nicotinovorans
Mycobacterium tuberculosis
Thermococcus litoralis malK
Streptomyces coelicolor A3(2)
Zymomonas mobilis hisC
Brucella abortus oxyR
Bacillius stearothermophilus
Micrococcus rubens puo
Borrelia burgdorferi mgtE
Xenopus laevis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bradyrhizobium japonicum
Mycobacterium tuberculosis
Zymomonas mobilis
Bacillus subtilis ypdP
Streptomyces glaucescens strW
Bacillus subtilis gltX
Pseudomonas syringae tnpA
Brevibacterium lactofermentum
Thermus thermophilus dnaX
Bacillus subtilis yaaK
Bacillus subtilis recR
Heliobacillus mobilis cobQ
Heliobacillus mobilis murC
Mycobacterium tuberculosis
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium smegmatis sigE
Bacillus subtilis katA
Klebsiella pneumoniae lrp
Bacillus subtilis 1A1 aziC
Sinorhizobium sp. As4 arsR
Sinorhizobium sp. As4 arsB
Staphylococcus xylosus arsC
Bacillus firmus OF4 mrpD
Staphylococcus aureus mnhC
Bacillus firmus OF4 mrpA
Alcaligenes eutrophus CH34
Mycobacterium tuberculosis
Lactococcus lactis MG1363 apl
Bacillus subtilis ykuE
Bacillus subtilis yqeY
Mycobacterium leprae pon1
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Escherichia coli K12 shiA
Bacillus subtilis lcfA
Streptomyces coelicolor A3(2)
Bacillus subtilis fabG
Emericella nidulans fluG
Arabidopsis thaliana atg6
Rhizobium leguminosarum nodN
Mycobacterium tuberculosis
Vibrio cholerae crp
Micrococcus luteus pdg
Mycobacterium tuberculosis
Escherichia coli K12 yeaB
Mycobacterium tuberculosis
Corynebacterium sp. C12 cEH
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium tuberculosis
Escherichia coli trbB
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus subtilis yprA
Arthrobacter globiformis SI55
Mycobacterium tuberculosis
Stigmatella aurantiaca B17R20
Bacillus subtilis dnaX
Ureaplasma urealyticum uu033
Deinococcus radiodurans
Escherichia coli K12 rluC
Erwinia chrysanthemi D1 bgxA
Azospirillum irakense salB
Amycolatopsis methanolica
Rhodococcus erythropolis orf5
Escherichia coli K12 fabG
Streptomyces viridifaciens vlmF
Actinoplanes sp. acbB
Mycobacterium tuberculosis
Methanococcus jannaschii JAL-
Escherichia coli K12 yefJ
Salmonella typhimurium ushA
Mycobacterium tuberculosis
Salmonella anatum M32 rfbA
Streptococcus mutans rmlC
Streptococcus mutans XC rmlB
Thermus aquaticus HB8 nox
Staphylococcus aureus sirA
Mycobacterium tuberculosis
Streptomyces coelicolor
Sphingomonas capsulata
Streptomyces coelicolor A3(2)
Corynebacterium
ammoniagenes ATCC 6872
Acinetobacter johnsonii ptk
Acinetobacter johnsonii ptp
Staphylococcus aureus M capD
Vibrio cholerae
Campylobacter jejuni wlaK
Neisseria meningitidis pglB
Staphylococcus aureus M capM
Xanthomonas campestris gumJ
Enterobacter cloacae murA
Bacillus subtilis murB
Vibrio cholerae ORF39 × 2
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium tuberculosis
Pseudomonas aeruginosa PAO1
Corynebacterium glutamicum
Escherichia coli ugd
Escherichia coli wbnA
Escherichia coli 0157 wbhH
Corynebacterium glutamicum
Xanthomonas campestris
Pseudomonas aeruginosa PAO1
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Bacillus subtilis sdhA
Paenibacillus macerans sdhB
Streptomyces coelicolor
Escherichia coli K12 yjiN
Streptomyces glaucescens
Streptomyces fradiae T#2717
Streptomyces fradiae T#2717
Corynebacterium sp. P-1 purU
Bacillus subtilis deoC
Mycobacterium avium GIR10
Mycobacterium tuberculosis
Mycobacterium leprae ctpB
Saccharomyces cerevisiae
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Streptomyces coelicolor C75A
Streptomyces coelicolor C75A
Escherichia coli RDD012 murB
Bacillus subtilis lcfA
Streptomyces coelicolor
Streptomyces coelicolor A3(2)
Mycobacterium bovis senX3
Mycobacterium bovis BCG
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Pseudomonas aeruginosa ppx
Mycobacterium tuberculosis
Corynebacterium glutamicum
Equine herpesvirus 1 ORF71
Mycobacterium leprae
Streptomyces coelicolor
Mycobacterium leprae
Mycobacterium tuberculosis
Mycobacterium leprae hemA
Mycobacterium leprae hem3b
Acinetobacter calcoaceticus
Escherichia coli K12 shiA
Neurospora crassa qa4
Corynebacterium glutamicum
Escherichia coli K12 potG
Serratia marcescens sfuB
Brachyspira hyodysenteriae bitA
Mycobacterium leprae cysG
Streptomyces coelicolor A3(2)
Mycobacterium leprae ctpB
Streptomyces coelicolor A3(2)
Bacillus subtilis hemY
Mycobacterium leprae hemL
Escherichia coli K12 gpmB
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Staphylococcus aureus zntR
Mycobacterium tuberculosis
Escherichia coli K12 menA
Bacteroides fragilis wcgB
Rhizobium trifolii matB
Escherichia coli K12 yqjF
Pseudomonas putida
Pseudomonas putida KDGDH
Bacillus subtilis 168 alsR
Mycobacterium tuberculosis
Sphingomonas sp. LB126 fldB
Mycobacterium tuberculosis
Bacillus subtilis menB
Deinococcus radiodurans
Aquifex aeolicus VF5 phhB
Mycobacterium tuberculosis
Bacillus subtilis menD
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 cycA
Escherichia coli K12 ubiE
Mycobacterium tuberculosis
Bacillus stearothermophilus
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces coelicolor
Mycobacterium tuberculosis
Escherichia coli K12 gabD
Azospirillum brasilense carR
Escherichia coli K12 o341#7
Mycobacterium tuberculosis
Streptomyces lividans P49
Streptomyces griseus N2-3-11
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium intracellulare
Mycobacterium smegmatis
Micrococcus luteus fusA
Chlamydia trachomatis
Escherichia coli K12 fepC
Escherichia coli K12 fepG
Escherichia coli K12 fepD
Thermoanaerobacterium
thermosaccharolyticum actA
Planobispora rosea ATCC
Mycobacterium bovis BCG rplC
Mycobacterium bovis BCG rplD
Mycobacterium bovis BCG rplW
Mycobacterium bovis BCG rplB
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium bovis BCG rpsC
Mycobacterium bovis BCG rplP
Mycobacterium bovis BCG rpmC
Mycobacterium bovis BCG rpsQ
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Micrococcus luteus rplE
Corynebacterium sp.
Wolinella succinogenes fdhD
Streptomyces coelicolor A3(2)
Escherichia coli fdfF
Mycobacterium tuberculosis
Archaeoglobus fulgidus AF1398
Deinococcus radiodurans
Micrococcus luteus
Micrococcus luteus
Micrococcus luteus rplR
Micrococcus luteus rpsE
Escherichia coli K12 rpmJ
Micrococcus luteus rplO
Streptomyces coelicolor msdA
Azospirillum brasilense carR
Rhodococcus rhodochrous
Sphingomonas sp. redA2
Rhodobacter capsulatus fdxE
Pseudomonas putida cymB
Aeropyrum pernix K1 APE0029
Pyrococcus furiosus Vc1 DSM
Pyrococcus furiosus Vc1 DSM
Rhodococcus erythropolis thcB
Erwinia carotovora carotovora
Micrococcus luteus adk
Bacillus subtilis 168 map
Bacillus subtilis infA
Thermus thermophilus HB8
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Bacillus subtilis 168 rpoA
Escherichia coli K12 rplQ
Escherichia coli k12 truA
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Arabidopsis thaliana CV DIM
Escherichia coli K12 cfa
Streptomyces coelicolor A3(2)
Bacillus alcalophilus
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Staphylococcus aureus
Synechocystis sp. PCC6803
Mycobacterium leprae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 yidE
Propionibacterium shermanii pip
Mycobacterium tuberculosis
Escherichia coli K12 riml
Pasteurella haemolytica
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium smegmatis
Mycobacterium tuberculosis
Mycobacterium leprae
Corynebacterium
ammoniagenes ATCC 6872
Pyrococcus horikoshii PH0308
Corynebacterium
ammoniagenes ATCC 6872
Escherichia coli K12 ybiF
Bacillus subtilis gltC
Corynebacterium
ammoniagenes guaA
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 degU
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Deinococcus radiodurans
Mycobacterium marinum
Brevibacterium linens ATCC
Brevibacterium linens ATCC
Streptomyces coelicolor A3(2)
Brevibacterium linens crtE
Brevibacterium linens
Citrobacter freundii blc OS60 blc
Brevibacterium linens
Brevibacterium linens ATCC
Streptococcus suis cps1K
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 yvrO
Helicobacter pylori abcD
Escherichia coli TAP90 abc
Haemophilus influenzae
Thermus aquaticus dnaE
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Archaeoglobus fulgidus AF1676
Streptomyces coelicolor A3(2)
Corynebacterium diphtheriae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium leprae
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Leptospira meyeri metY
Escherichia coli K12 cstA
Escherichia coli K12 yjiX
Mycobacterium tuberculosis
Streptomyces hygroscopicus
Mycobacterium smegmatis
Escherichia coli K12 yneC
Methanothermus fervidus V24S
Bacillus stearothermophilus T-6
Vibrio cholerae OGAWA 395
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Streptomyces venezuelae cmlv
Pseudomonas aeruginosa crc
Haemophilus influenzae Rd
Corynebacterium diphtheriae
Yersinia enterocolitica hemU
Escherichia coli K12 trpS
Escherichia coli K12 yhjD
Salmonella typhimurium LT2
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Lactococcus lactis upp
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycoplasma pirum BER manB
Halobacterium volcanii ATCC
Corynebacterium glutamicum
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 yciC
Bacillus subtilis IS58 trxB
Salmonella typhimurium LT2
Streptomyces hygroscopicus
Aeropyrum pernix K1 APE0223
Mycobacterium smegmatis
Mycobacterium tuberculosis
Corynebacterium glutamicum
Campylobacter jejuni Cj0069
Mycobacterium leprae
Mycobacterium tuberculosis
Escherichia coli K12 yceF
Mycobacterium leprae B1308-
Corynebacterium glutamicum
Corynebacterium glutamicum
Escherichia coli K12 birA
Mycobacterium tuberculosis
Corynebacterium
ammoniagenes ATCC 6872
Escherichia coli K12 kup
Corynebacterium
ammoniagenes ATCC 6872
Actinosynnema pretiosum
Streptomyces coelicolor A3(2)
Chelatobacter heintzii ATCC
Archaeoglobus fulgidus
Bacillus megaterium IAM 1030
Thermotoga maritima MSB8
Bacillus subtilis 168 ywjB
Streptomyces coelicolor A3(2)
Thermococcus litoralis malG
Thermococcus litoralis malF
Thermococcus litoralis malE
Streptomyces reticuli msiK
Deinococcus radiodurans R1
Mycobacterium tuberculosis
Helicobacter pylori J99 jhp0462
Escherichia coli K12 uvrD
Streptomyces coelicolor
Halobacterium sp. NRC-1
Escherichia coli K12 hepA
Mycobacterium tuberculosis
Mycobacterium smegmatis
Saccharomyces cerevisiae
Mycobacterium smegmatis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Salmonella montevideo M40
Mycobacterium tuberculosis
Escherichia coli K12 manA
Enterococcus faecalis plasmid
Trichomonas vaginalis WAA38
Archaeoglobus fulgidus VC-16
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Spinacia oleracea CV rps22
Brevibacterium flavum
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Corynebacterium glutamicum
Mycobacterium tuberculosis
Corynebacterium glutamicum
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Klebsiella pneumoniae CG43
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Methanococcus jannaschii JAL-
Mycobacterium tuberculosis
Escherichia coli K12 uvrD
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Deinococcus radiodurans
Hevea brasiliensis laticifer er1
Aeropyrum pernix K1 APE0247
Bacillus subtilis 168 yaaE
Lysobacter enzymogenes ATCC
Neurospora intermedia LaBelle-
Corynebacterium glutamicum
Streptomyces alboniger pur3
Streptomyces flavopersicus
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Aeropyrum pernix K1 APE2061
Mycobacterium tuberculosis
Escherichia coli K12 smpB
Escherichia coli K12 yeaO
Vibrio cholerae OGAWA 395
Staphylococcus aureus sirA
Mycobacterium leprae
Vibrio anguillarum 775 fatB
Bacillus subtilis 168 yclN
Bacillus subtilis 168 yclO
Bacillus subtilis 168 yclP
Chlamydia muridarum Nigg
Chlamydia pneumoniae
Rattus norvegicus (Rat)
Saccharomyces cerevisiae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Micrococcus luteus rpf
Lactococcus lactis cspB
Mycobacterium leprae
Deinococcus radiodurans
Streptomyces coelicolor A3(2)
Streptomyces azureus tsnR
Mycobacterium tuberculosis
Bacillus circulans ATCC 21783
Escherichia coli K12 accD
Streptomyces coelicolor A3(2)
Pseudomonas fluorescens
Mycobacterium tuberculosis
Corynebacterium
ammoniagenes fas
Leptospira meyeri metX
Deinococcus radiodurans
Mycobacterium avium folA
Escherichia coli K12 thyA
Escherichia coli K12 cysQ
Streptomyces coelicolor A3(2)
Synechococcus elongatus
naegeli mutM
Mycobacterium tuberculosis
Lactococcus lactis MG1363 apl
Streptomyces coelicolor A3(2)
Escherichia coli JM101 pgi
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus stearothermophilus
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 yvrO
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Corynebacterium
ammoniagenes purN
Corynebacterium
ammoniagenes purH
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Cyanophora paradoxa rps18
Escherichia coli K12 rpsN
Escherichia coli K12 rpmG
Escherichia coli K12 rpmB
Bacillus subtilis 168 yvdB
Staphylococcus aureus zntR
Haemophilus ducreyi rpmE
Streptomyces coelicolor A3(2)
Pseudomonas syringae copR
Escherichia coli K12 baeS
Escherichia coli K12 htrA
Arabidopsis thaliana CV cnx1
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Homo sapiens MTHFS
Xanthomonas campestris
Arthrobacter nicotinovorans
Escherichia coli K12 rimJ
Mycobacterium tuberculosis
Escherichia coli K12 cynX
Haemophilus influenzae Rd
Mycobacterium tuberculosis
Bacillus sphaericus E-244
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Methanobacterium
thermoautotrophicum Delta H
Escherichia coli recQ
Methanobacterium
thermoautotrophicum Delta H
Bacillus subtilis 168 yxaG
Enterococcus faecium
Escherichia coli K12
Brevibacterium linens tnpA
Escherichia coli dld
Klebsiella pneumoniae OK8
Enterococcus faecium
Escherichia coli K12
Mycobacterium tuberculosis
Staphylococcus aureus cadD
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 ksgA
Mycobacterium tuberculosis
Saccharopolyspora erythraea
Escherichia coli K12 pdxK
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 yxeH
Mycobacterium tuberculosis
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Haemophilus influenzae Rd
Neisseria meningitidis NMA1953
Mycobacterium tuberculosis
Escherichia coli K12 prfC
Methylophilus methylotrophus
Methylophilus methylotrophus
Methylophilus methylotrophus
Pseudomonas aeruginosa PAO
Pseudomonas aeruginosa PAO
Escherichia coli K12 pth
Williopsis mrakii IFO 0895
Streptomyces roseofulvus gap
Neisseria meningitidis
Escherichia coli K12 pth
Mycobacterium tuberculosis
Salmonella typhimurium D21
Bacillus cereus ATCC 10987
Bacillus subtilis prs
Bacillus subtilis gcaD
Escherichia coli K12 sufI
Rhizobium sp. N33 nodI
Streptomyces lividans ORF2
Escherichia coli K12 uhpB
Streptomyces peucetius dnrN
Streptomyces coelicolor A3(2)
Streptomyces glaucescens strV
Mycobacterium smegmatis exiT
Escherichia coli K12 ggt
Corynebacterium glutamicum
Corynebacterium glutamicum
Escherichia coli tetR
Escherichia coli mfd
Neisseria gonorrhoeae
Escherichia coli mdlB
Mycobacterium tuberculosis
Corynebacterium glutamicum
Bacillus subtilis yabN
Mycobacterium tuberculosis
Bacillus subtilis eno
Aeropyrum pernix K1 APE2459
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli gppA
Escherichia coli tdcB
Thermotoga maritima MSB8
Escherichia coli rhaR
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Escherichia coli greA
Mycobacterium tuberculosis
Streptomyces lincolnensis lmbE
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Escherichia coli coaA
Brevibacterium flavum MJ-233
Streptomyces griseus pabS
Alcaligenes faecalis ptcR
Escherichia coli ybgK
Escherichia coli ybgJ
Emericella nidulans lamB
Bacillus subtilis ycsH
Bacillus subtilis ydhC
Rattus norvegicus (Rat) fumH
Rhodococcus erythropolis
Streptomyces coelicolor A3(2)
Rhodococcus sp. IGTS8 soxA
Rhodococcus sp. IGTS8 soxC
Rhodococcus sp. IGTS8 soxC
Escherichia coli K12 ssuD
Escherichia coli K12 glpX
Mycobacterium tuberculosis
Bacillus subtilis ywmD
Streptomyces coelicolor A3(2)
Escherichia coli K12 MG1655
Escherichia coli K12 MG1655
Escherichia coli K12 lytB
Neisseria gonorrhoeae
Escherichia coli K12 perM
Rattus norvegicus (Rat) SLC6A7
Corynebacterium glutamicum
Bacillus subtilis yyaF
Dichelobacter nodosus intA
Pseudomonas aeruginosa argF
Bacillus subtilis 168 ykkB
Mus musculus RDH4
Streptomyces coelicolor
Escherichia coli K12 yegE
Rhizobium meliloti nodC
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Pseudomonas putida M10 norA
Acinetobacter calcoaceticus
Streptomyces roseofulvus frnS
Synechococcus sp. PCC 7942
Mycobacterium tuberculosis
Rhodobacter sphaeroides ATCC
Amycolatopsis methanolica pgm
Mycobacterium tuberculosis
Streptomyces hygroscopicus
Streptomyces fradiae tlrC
Mycobacterium tuberculosis
Escherichia coli K12 MG1655
Bacillus subtilis 168 yxaD
Streptococcus pneumoniae
Corynebacterium glutamicum
Ruminococcus flavefaciens
Mycobacterium tuberculosis
Bacillus subtilis nadA
Streptomyces coelicolor
Deinococcus radiodurans R1
Streptomyces coelicolor
Escherichia coli K12 MG1655
Escherichia coli K12 lplA
Escherichia coli K12 phnB
Pseudomonas putida pcaK
Pseudomonas aeruginosa phhy
Bacillus subtilis 168 ykoE
Escherichia coli yjjK
Bacillus subtilis 168 ykoC
Escherichia coli chaA
Pyrococcus abyssi Orsay
Bacillus subtilis ywaF
Thermus thermophilus unrA
Mycobacterium tuberculosis
Escherichia coli yedL
Streptomyces coelicolor A3(2)
Penaeus vannamei
Escherichia coli
Bacillus subtilis yyaD
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 typA
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces griseus fer
Bacillus sp: strain YM-2 aat
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Mycobacterium leprae u1756I
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Micromonospora griseorubida
Pediococcus pentosaceus scrB
Escherichia coli K12 MG1655
Streptomyces coelicolor A3(2)
Streptomyces mycarofaciens
Escherichia coli rpoE
Mycobacterium tuberculosis
Escherichia coli mrp
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Corynebacterium glutamicum
Cricetulus griseus (Chinese
Mycobacterium tuberculosis
Escherichia coli aroE
Bacillus subtilis pnbA
Escherichia coli transposon
Streptomyces glaucescens tcmA
Catharanthus roseus metE
Nocardia asteroides strain KGB1
Escherichia coli K12 MG1655
Escherichia coli K12 MG1655
Corynebacterium glutamicum
Corynebacterium glutamicum
Escherichia coli K12 MG1655
Proteus vulgaris mutT
Salmonella typhimurium proY
Klebsiella pneumoniae CG43
Mycobacterium leprae
Sphingomonas flava pcpB
Pseudomonas sp. B13 clcE
Acinetobacter calcoaceticus
Mycobacterium tuberculosis
Saccharomyces cerevisiae
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor
Erwinia chrysanthemi recS
Escherichia coli K12 MG1655 fnr
Shewanella putrefaciens merP
Escherichia coli K12 MG1655
Vibrio sp. S14 relA
Streptomyces lividans tap
Corynebacterium glutamicum
Bacillus subtilis narI
Bacillus subtilis narJ
Bacillus subtilis narH
Aeropyrum pernix K1 APE1291
Aeropyrum pernix K1 APE1289
Bacillus subtilis narG
Escherichia coli K12 narK
Arabidopsis thaliana CV cnx1
Serratia marcescens strain IFO-
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Pseudomonas putida mobA
Mycobacterium tuberculosis
Arabidopsis thaliana cnx2
Pseudomonas oleovorans
Micrococcus luteus rho
Escherichia coli K12 RF-1
Escherichia coli K12
Mycobacterium tuberculosis
Escherichia coli K12 rfe
Corynebacterium glutamicum
Escherichia coli K12 atpB
Streptomyces lividans atpL
Streptomyces lividans atpF
Streptomyces lividans atpD
Streptomyces lividans atpA
Streptomyces lividans atpG
Corynebacterium glutamicum
Streptomyces lividans atpE
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Bacillus subtilis yqjC
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 ssuD
Escherichia coli K12 ssuC
Escherichia coli K12 ssuB
Escherichia coli K12 ssuA
Mycobacterium tuberculosis
Dictyoglomus thermophilum
Escherichia coli K12 fepC
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Rhizobium meliloti fixA
Rhizobium meliloti fixB
Azotobacter vinelandii nifS
Rhizobium sp. NGR234 plasmid
Rhizobium sp. NGR234 plasmid
Escherichia coli K12 MG1655
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces glaucescens tcmA
Rhodothermus marinus dnlJ
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Vibrio vulnificus viuB
Streptomyces coelicolor A3(2)
Amycolatopsis methanolica pfp
Bacillus megaterium ccpA
Escherichia coli K12 rbsA
Escherichia coli K12 MG1655
Escherichia coli K12 MG1655
Escherichia coli K12 MG1655
Saccharomyces cerevisiae
Streptomyces coelicolor
Rattus norvegicus (Rat) NTCI
Staphylococcus aureus WHU 29
Methanococcus jannaschii
Escherichia coli K12 yqjG
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Corynebacterium glutamicum
Mycobacterium tuberculosis
Corynebacterium glutamicum
Sulfolobus solfataricus
Synechococcus sp. nrtD
Enterobacter aerogenes
Anabaena sp. strain PCC 7120
Streptomyces coelicolor
Ralstonia eutropha czcD
Methanococcus jannaschii
Brevibacterium flavum serA
Schizosaccharomyces pombe
Rhodobacter capsulatus strain
Escherichia coli C hpcE
Escherichia coli K12
Bacillus subtilis dhbC
Bacillus subtilis gltX
Streptomyces coelicolor A3(2)
Bacillus subtilis thiA or thiC
Chlamydia trachomatis
Rattus norvegicus (Rat)
Bacillus subtilis yrkH
Methanococcus jannaschii Y441
Escherichia coli K12 spoT
Escherichia coli K12 iclR
Actinoplanes teichomyceticus
Salmonella typhimurium
Mycobacterium tuberculosis
Bacillus subtilis gpdA
Escherichia coli K12 MG1655
Escherichia coli K12 thiL
Mus musculus ung
Mycoplasma genitalium (SGC3)
Escherichia coli K12 recG
Neisseria meningitidis
Propionibacterium freudenreichii
Escherichia coli K12 yhhF
Escherichia coli K12 MG1655
Neisseria gonorrhoeae
Bacillus stearothermophilus
Agrobacterium tumefaciens
Escherichia coli K12 MG1655
Methanobacterium
thermoautotrophicum MTH465
Bacteriophage L54a vinT
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium tuberculosis
Streptomyces lactamdurans
Streptomyces coelicolor A3(2)
Pseudomonas putida morA
Streptomyces coelicolor
Escherichia coli K12 rpsA
Brevibacterium lactofermentum
Crithidia fasciculata iunH
Staphylococcus aureus
Escherichia coli K12 rbsK
Escherichia coli K12 ascG
Streptococcus pneumoniae
Methanococcus jannaschii
Escherichia coli K12 ytfH
Escherichia coli K12 ytfG
Bacillus subtilis yvgS
Streptomyces coelicolor A3(2)
Escherichia coli K12 ycbL
Escherichia coli K12 uvrA
Micrococcus luteus
Micrococcus luteus
Rhodobacter sphaeroides infC
Mycoplasma fermentans
Pseudomonas syringae pv.
Escherichia coli K12 MG1655
Escherichia coli K12 MG1655
Escherichia coli K12 MG1655
Escherichia coli K12 MG1655
Aeropyrum pernix K1 APE0042
Bacillus subtilis glpQ
Escherichia coli K12 MG1655
Bacillus subtilis 168 syfA
Escherichia coli K12 MG1655
Streptomyces scabies estA
Streptomyces mycarofaciens
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Escherichia coli K12 ycaR
Bacillus subtilis syy1
Methanococcus jannaschii
Chlamydia muridarum Nigg
Chlamydia pneumoniae
Borrelia burgdorferi IF2
Bacillus subtilis yzgD
Bacillus subtilis yqxC
Mycobacterium tuberculosis
Escherichia coli K12 recN
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 pyrG
Bacillus subtilis yqkG
Staphylococcus aureus xerD
Streptomyces fradiae tlrC
Caulobacter crescentus parA
Bacillus subtilis ypuG
Datisca glomerata tst
Bacillus subtilis ypuH
Bacillus subtilis rluB
Bacillus subtilis cmk
Bacillus subtilis yphC
Mycobacterium tuberculosis
Corynebacterium striatum M82B
Corynebacterium striatum M82B
Escherichia coli K12 ygiE
Bacillus subtilis ATCC 9372
Escherichia coli K12 o249#9
Archaeoglobus fulgidus AF0675
Bacillus subtills secA
Mycobacterium smegmatis garA
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus subtilis yhdP
Bacillus subtilis yhdT
Thermus thermophilus herA
Mycobacterium tuberculosis
Brevibacterium flavum
Mycobacterium tuberculosis
Rhizobium sp. N33 nodl
Mycobacterium tuberculosis
Escherichia coli K12 yfhH
Escherichia coli K12 phnE
Escherichia coli K12 phnE
Escherichia coli K12 phnC
Salmonella typhimurium thiD
Salmonella typhimurium LT2
Mycobacterium tuberculosis
Burkholderia cepacia Pc701
Thermus flavus AT-62 gpt
Escherichia coli K12 yebN
Sinorhizobium sp. As4 arsB
Streptomyces coelicolor A3(2)
Pseudomonas sp. R9 ORFA
Pseudomonas sp. R9 ORFG
Mycobacterium tuberculosis
Schizosaccharomyces pombe
Escherichia coli K12 Int
Candida albicans lip1
Mycobacterium tuberculosis
Pseudomonas denitrificans
Mycobacterium tuberculosis
Streptococcus mutans LT11
Saccharomyces cerevisiae
Escherichia coli K12 tatC
Mycobacterium leprae
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Aeropyrum pernix K1 APE2014
Rhodococcus erythropolis arc
Mycobacterium leprae pimT
Homo sapiens
Mycobacterium tuberculosis
Dichelobacter nodosus A198
Staphylococcus aureus norA23
Corynebacterium glutamicum
Corynebacterium glutamicum
Thermotoga maritima MSB8
Escherichia coli K12 metH
Xanthomonas campestris ahpF
Saccharomyces cerevisiae
Staphylococcus aureus plasmid
Mycobacterium tuberculosis
Escherichia coli K12 cysS
Escherichia coil K12 bacA
Agrobacterium tumefaciens
Mycobacterium tuberculosis
Agrocybe aegerita ura1
Pseudomonas syringae tnpA
Escherichia coli K12 ybhB
Neisseria meningitidis
Corynebacterium striatum M82B
Corynebacterium striatum M82B
Streptomyces anulatus pac
Escherichia coli K12 argK
Streptomyces cinnamonensis
Streptomyces cinnamonensis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Propionibacterium freudenreichii
Streptococcus faecium
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Methanococcus jannaschii
Streptomyces coelicolor A3(2)
Methanococcus jannaschii
Neisseria meningitidis MC58
Neisseria gonorrhoeae ORF24
Neisseria gonorrhoeae
Synechocystis sp. PCC6803
Streptomyces coelicolor A3(2)
Streptococcus thermophilus
Corynephage 304L int
Escherichia coli K12 yjjK
Micromonospora viridifaciens
Corynebacterium glutamicum
Corynebacterium glutamicum
Pyrococcus abyssi Orsay
Mycobacterium leprae
Aeropyrum pernix K1 APE2025
Mycobacterium leprae nifS
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Synechocystis sp. PCC6803
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium leprae
Mycobacterium tuberculosis
Pyrococcus horikoshii PH0450
Escherichia coli K12 qor
Nitrobacter winogradskyi coxC
Corynebacterium glutamicum
Mycobacterium leprae
Brevibacterium flavum
Mycobacterium tuberculosis
Saccharomyces cerevisiae
Bacillus sp. NS-129
Rhodococcus erythropolis
Corynebacterium glutamicum
Corynebacterium glutamicum
Saccharomyces cerevisiae
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Synechocystis sp. PCC6803
Mycobacterium tuberculosis
Escherichia coli K12
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Mycobacterium tuberculosis ribA
Actinobacillus
Escherichia coli K12 ribD
Saccharomyces cerevisiae
Escherichia coli K12 sun
Pseudomonas aeruginosa fmt
Bacillus subtilis 168 def
Escherichia coli priA
Brevibacterium flavum MJ-233
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Saccharomyces cerevisiae guk1
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli carB
Pseudomonas aeruginosa
Bacillus caldolyticus DSM 405
Pseudomonas aeruginosa
Bacillus caldolyticus DSM 405
Mycobacterium tuberculosis
Bacillus subtilis nusB
Brevibacterium lactofermentum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Aeromonas hydrophila tapD
Streptomyces coelicolor A3(2)
Corynebacterium diphtheriae
Pyrococcus abyssi Orsay
Bacillus subtilis 168 fhuC
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Thiobacillus ferrooxidans ATCC
Mycobacterium tuberculosis
Mycobacterium leprae aspS
Mycobacterium tuberculosis
Saccharomyces cerevisiae
Bacillus subtilis yhgE
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Pseudomonas aeruginosa PAO1
Escherichia coli K12 sdaA
Enterococcus casseliflavus glpO
Staphylococcus aureus
Campylobacter jejuni
Streptomyces chrysomallus
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium tuberculosis
Escherichia coli K12 secF
Rhodobacter capsulatus secD
Mycobacterium leprae
Escherichia coli K12 ruvB
Mycobacterium leprae ruvA
Escherichia coli K12 ruvC
Escherichia coli K12 ORF246
Escherichia coli K12 tesB
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Saccharomyces cerevisiae
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus subtilis thrZ
Bacillus subtilis ywbN
Streptomyces anulatus pac
Actinobacillus
pleuropneumoniae afuC
Zymomonas mobilis dfp
Escherichia coli tnpR
Saccharomyces cerevisiae
Streptomyces coelicolor A3(2)
Thermotoga maritima MSB8
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Erwinia chrysanthemi recJ
Streptococcus phage phi-O1205
Mycoplasma pneumoniae ATCC
Bacteriophage N15 gene57
Schizosaccharomyces pombe
Streptomyces coelicolor
Escherichia coli K12 clpA
Staphylococcus aureus SA20
Streptomyces coelicolor A3(2)
Bacteriophage phi-C31 gp52
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Deinococcus radiodurans
Lactobacillus phage phi-gle
Bacillus anthracis pXO2-16
Escherichia coli clpB
Homo sapiens numA
Sus scrofa domestica
Escherichia coli ecoR1
Mycobacterium tuberculosis
Methanococcus jannaschii
Enterococcus faecalis esp
Corynebacterium glutamicum
Escherichia coli topB
Corynebacterium glutamicum
Staphylococcus aureus nuc
Shewanella sp. ssb
Anopheles gambiae AgSP24D
Mycobacterium phage L5 int
Brevibacterium lactofermentum
Brevibacterium lactofermentum
Brevibacterium lactofermentum
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Mycobacterium phage L5 int
Helicobacter pylori 26695
Bacillus subtilis yxaA
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptococcus gordonii msrA
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Haemophilus influenzae Rd
Streptomyces sp. CL190 dxs
Thermotoga maritima MSB8
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 suhB
Mycobacterium tuberculosis
Corynebacterium glutamicum
Bacillus subtilis yrkO
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces aureofaciens
Corynebacterium glutamicum
lactofermentum) galE
Mycobacterium tuberculosis
Saccharomyces cerevisiae
Escherichia coli oxyR
Escherichia coli hrpA
Streptomyces clavuligerus nrdR
Bacillus subtilis dinR
Escherichia coli K12 gatR
Streptomyces coelicolor A3(2)
Bacillus stearothermophilus ptsI
Escherichia coli K12 glpR
Rhodobacter capsulatus fruK
Escherichia coli K12 fruA
Bacillus stearothermophilus XL-
Bacillus caldolyticus pyrP
Streptomyces fradiae orf11*
Haemophilus influenzae Rd
Escherichia coli K12 miaA
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium leprae
Corynebacterium glutamicum
Neisseria gonorrhoeae
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium leprae recX
Mycobacterium tuberculosis
Bacillus sphaericus bioY
Escherichia coli K12 potG
Bacillus subtilis ybaF
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptococcus pneumoniae R6X
Streptococcus pyogenes pgsA
Arabidopsis thaliana
Streptococcus pneumoniae
Escherichia coli terC
Bacillus subtilis 168 spoIIIE
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces antibioticus gpsI
Bacillus subtilis rpsO
Leishmania major
Corynebacterium
ammoniagenes ATCC 6872 ribF
Bacillus subtilis 168 truB
Corynebacterium
ammoniagenes
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus subtilis 168 rbfA
Stigmatella aurantiaca DW4 infB
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 nusA
Mycobacterium tuberculosis
Bacillus subtilis 168 dppE
Escherichia coli K12 dppB
Bacillus subtilis spo0KC
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Rhodobacter sphaeroides ATCC
Heliobacillus mobilis bchI
Propionibacterium freudenreichii
Clostridium perfringens NCIB
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Burkholderia cepacia AC1100
Escherichia coli K12 map
Streptomyces clavuligerus pcbR
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Deinococcus radiodurans
Bacillus subtilis 168 yvrO
Escherichia coli K12 gcpE
Mycobacterium tuberculosis
Chlamydia trachomatis
Escherichia coli K12 dxr
Thermotoga maritima MSB8
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Pseudomonas aeruginosa
Bacillus subtilis 168 frr
Pseudomonas aeruginosa pyrH
Streptomyces coelicolor A3(2)
Bacillus subtilis rpsB
Mycobacterium tuberculosis
Proteus mirabilis xerD
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Haemophilus influenzae Rd
Streptomyces lividans TK21
Staphylococcus aureus sirA
Bacillus stearothermophilus rplS
Bacillus subtilis 168 thiE
Streptomyces coelicolor A3(2)
Escherichia coli K12 thiS
Escherichia coli K12 thiG
Emericella nidulans cnxF
Bordetella pertussis TOHAMA I
Bacillus subtilis 168 degA
Chlamydophila pneumoniae
Spinacia oleracea chloroplast
Pseudomonas putida pcaB
Escherichia coli K12 trmD
Streptomyces coelicolor A3(2)
Mycobacterium leprae
Helicobacter pylori J99 jhp0839
Bacillus subtilis 168 rpsP
Mus musculus inv
Streptococcus agalactiae cylB
Pyrococcus horikoshll OT3 mtrA
Bacillus subtilis 168 ffh
Escherichia coli K12 ftsY
Saccharomyces cerevisiae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 yfeR
Mycobacterium leprae
Dichelobacter nodosus gep
Escherichia coli K12 mutM or
Bacillus subtilis 168 rncS
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces verticillus
Escherichia coli K12 cydC
Streptomyces coelicolor A3(2)
Thermotoga maritima MSB8
Campylobacter jejuni ATCC
Arabidopsis thaliana SUC1
Thermococcus litoralis malP
Bacillus subtilis 168 yfiE
Staphylococcus aureus FDA 485
Emericella nidulans trpC
Mycobacterium tuberculosis
Rhodobacter sphaeroides ATCC
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces lividans 66 cmlR
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Mycobacterium smegmatis
Schizosaccharomyces pombe
Leishmania donovani SAcP-1
Escherichia coli plasmid RP1
Sulfolobus acidocaldarius treX
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Escherichia coli K12 galR
Bacillus subtilis 168 fhuC
Vibrio cholerae hutC
Bacillus subtilis 168 yvrC
Bacillus subtilis 168 yvrC
Escherichia coli K12 ytfH
Streptomyces coelicolor A3(2)
Arthrobacter sp. Q36 treY
Deinococcus radiodurans
Photorhabdus luminescens
Streptomyces coelicolor A3(2)
Arthrobacter sp. Q36 treZ
Bacillus subtilis 168
Corynebacterium glutamicum
Catharanthus roseus metE
Corynebacterium glutamicum AS019
Streptomyces coelicolor A3(2)
Escherichia coli K12 rarD
Campylobacter jejuni DZ72 hisJ
Archaeoglobus fulgidus AF2388
Bacillus subtilis 168 ydaD
Pseudomonas aeruginosa lysA
Alcaligenes eutrophus CH34
Escherichia coli K12 rluD
Pseudomonas fluorescens NCIB
Streptomyces antibioticus oleB
Rhodococcus erythropolis orf17
Bacillus licheniformis
Escherichia coli K12 dinP
Escherichia coli K12 ybiF
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Saccharomyces cerevisiae
Mycobacterium tuberculosis
Brevibacterium lactofermentum
Corynebacterium glutamicum
Brevibacterium lactofermentum
Mus musculus P4(21)n
Brevibacterium lactofermentum
Corynebacterium glutamicum
Corynebacterium glutamicum
Brevibacterium lactofermentum
Brevibacterium lactofermentum
Brevibacterium lactofermentum
Escherichia coli K12 mraY
Escherichia coli K12 murF
Bacillus subtilis 168 murE
Brevibacterium lactofermentum
Pseudomonas aeruginosa pbpB
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium tuberculosis
Mycobacterium leprae
Streptomyces lividans 1326
Myxococcus xanthus DK1050
Mycobacterium leprae
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Mycobacterium leprae
Mycobacterium tuberculosis
Amycolatopsis mediterranei
Mycobacterium leprae
Mycobacterium tuberculosis
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Listeria ivanovii iap
Listeria grayi iap
Heliobacillus mobilis petB
Streptomyces lividans qcrA
Mycobacterium tuberculosis
Synechococcus vulcanus
Mycobacterium tuberculosis
Rhodobacter sphaeroides ctaC
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium leprae
Rhodobacter capsulatus cobP
Pseudomonas denitrificans
Pseudomonas denitrificans cobV
Streptomyces clavuligerus car
Mus musculus BCAT1
Pseudomonas putida ATCC
Saccharopolyspora erythraea
Streptomyces seoulensis pdhB
Arabidopsis thaliana
Pelobacter carbinolicus GRA BD1
Mycobacterium tuberculosis
Escherichia coli K12 yidE
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Thermotoga maritima MSB8
Vibrio harveyi luxA
Thermotoga maritima MSB8
Escherichia coli hpaX
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Corynebacterium diphtheriae C7
Streptomyces coelicolor A3(2)
Thermotoga maritima MSB8
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Homo sapiens galK1
Brucella abortus vacB
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 gph
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Burkholderia cepacia
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Streptomyces seoulensis pdhA
Escherichia coli K12 glnQ
Bacillus subtilis 168 rbsC
Rickettsia prowazekii Madrid E
Dictyostelium discoideum AX2
Streptomyces coelicolor A3(2)
Myxococcus xanthus ATCC
Escherichia coli K12 nagD
Deinococcus radiodurans
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 phoD
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium smegmatis
Streptomyces aureofaciens BMK
Mycobacterium smegmatis
Mycobacterium smegmatis dgt
Neisseria meningitidis NMA0251
Mycobacterium tuberculosis
Drosophila melanogaster
Thermus aquaticus HB8
Mycobacterium tuberculosis
Escherichia coli K12 fur
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Micrococcus luteus B-P 26 uppS
Mycobacterium tuberculosis
Streptococcus pneumoniae era
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Neisseria meningitidis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Streptomyces albus dnaJ2
Streptomyces albus hrcA
Bacillus stearothermophilus
Saccharomyces cerevisiae
Streptomyces coelicolor A3(2)
Escherichia coli K12 malQ
Lactobacillus brevis plasmid
Neisseria gonorrhoeae
Neisseria meningitidis
Salmonella typhimurium dcp
Anisopteromalus calandrae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Chlamydomonas reinhardtii ipi1
Corynebacterium glutamicum
Corynebacterium glutamicum
Vibrio harveyi luxA
Escherichia coli K12 glcD
Escherichia coli K12 ydfH
Salmonella typhimurium ygiK
Haemophilus influenzae Rd
Bacillus subtilis 168 appB
Escherichia coli K12 dppC
Escherichia coli K12 oppD
Aeropyrum pernix K1 APE1580
Aquifex aeolicus VF5 aq_768
Rhizobium etli rbsK
Streptomyces coelicolor A3(2)
Homo sapiens
Chlamydomonas reinhardtii
Corynebacterium glutamicum
Mycobacteriophage D29 66
Corynebacterium glutamicum
Rhodobacter capsulatus dctM
Klebsiella pneumoniae dctQ
Rhodobacter capsulatus B10
Lycopersicon esculentum
Bacillus subtilis 168 lepA
Mycobacterium tuberculosis
Escherichia coli K12 rpsT
Escherichia coli K12 rhtC
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Bacillus subtilis 168 comEC
Bacillus subtilis 168 comEA
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 pbuX
Corynebacterium sp. ATCC
Streptomyces griseus IFO13189
Streptomyces griseus IFO13189
Escherichia coli K12 rne
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Mycobacterium smegmatis ndk
Deinococcus radiodurans R1
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 balS
Bacillus subtilis 168 oppA
Bacillus subtilis 168 dnaK
Eikenella corrodens ATCC
Thermus aquaticus ATCC 33923
Streptomyces coelicolor A3(2)
Vibrio cholerae aphA
Acinetobacter sp. vanA
Sphingomonas flava ATCC
Acinetobacter sp. vanK
Klebsiella pneumoniae mdcF
Bacillus subtilis clpX
Streptomyces coelicolor A3(2)
Streptomyces sp. 2065 pcaJ
Streptomyces sp. 2065 pcal
Rhodococcus opacus 1CP pcaR
Ralstonia eutropha bktB
Rhodococcus opacus pcaL
Streptomyces coelicolor A3(2)
Rhodococcus opacus pcaL
Rhodococcus opacus pcaB
Rhodococcus opacus pcaG
Rhodococcus opacus pcaH
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Rhodococcus opacus 1CP catB
Rhodococcus rhodochrous catA
Pseudomonas putida plasmid
Pseudomonas putida plasmid
Pseudomonas putida plasmid
Pseudomonas putida plasmid
Rhodococcus erythropolis thcG
Acinetobacter calcoaceticus
Acinetobacter calcoaceticus
Streptomyces coelicolor M145
Streptomyces coelicolor M145
Sulfolobus islandicus ORF154
Bacillus subtilis 168 tig
Streptomyces coelicolor A3(2)
Nocardia lactamdurans LC411
Mus musculus Moa1
Corynebacterium striatum ORF1
Corynebacterium striatum ORF1
Corynebacterium striatum ORF1
Staphylococcus aureus NCTC
Bacillus acidopullulyticus ORF2
Mycobacterium tuberculosis
Streptomyces lividans pepN
Borrelia burgdorferi BB0852
Brevibacterium linens ATCC
Myxococcus xanthus DK1050
Streptomyces griseus JA3933
Listeria monocytogenes lltB
Synechococcus elongatus
Bacillus firmus OF4 dppC
Escherichia coli K12 nikB
Corynebacterium glutamicum
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Chromatium vinosum D phbB
Streptomyces coelicolor actII
Neisseria meningitidis
Pseudomonas putida GM73
Mycobacterium leprae
Pseudomonas aeruginosa
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Aeropyrum pernix K1 APE1182
Escherichia coli K12 yjjK
Mycobacterium tuberculosis
Mycobacterium leprae o659
Bacillus subtilis phoB
Streptococcus mutans
Streptococcus mutans
Thermoanaerobacterium
thermosul amyE
Streptomyces reticuli msiK
Schizosaccharomyces pombe
Rhodococcus rhodochrous
Synechococcus sp. PCC7942
Thermotoga maritima MSB8
Escherichia coli K12 gip
Mycobacterium tuberculosis
Escherichia coli K12 orn
Salmonella enterica iroD
Mycobacterium tuberculosis
Corynebacterium glutamicum
Salmonella typhimurium KP1001
Rattus norvegicus SPRAGUE-
Bacillus subtilis 168 degA
Escherichia coli K12 uxaC
Zea diploperennis perennial
Mycobacterium avium pncA
Mycobacterium tuberculosis
Escherichia coli K12 bcp
Streptomyces coelicolor A3(2)
Corynebacterium
ammoniagenes ATCC 6871 ppt1
Corynebacterium glutamicum
Synechocystis sp. PCC6803
Corynebacterium
ammoniagenes fas
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium tuberculosis
Pseudomonas aeruginosa
Mycobacterium tuberculosis
Corynebacterium glutamicum
Mycobacterium leprae ats
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Flavobacterium sp. nylC
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli dinG
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Escherichia coli K12 serB
Mycobacterium tuberculosis
Corynebacterium glutamicum
Escherichia coli K12 ftnA
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Saccharomyces cerevisiae
Archaeoglobus fulgidus AF0251
Corynebacterium glutamicum
Rickettsia prowazekii
Bacillus subtilis 168 nadE
Synechocystis sp. PCC6803
Mycobacterium tuberculosis
Bacillus stearothermophilus
Bacillus subtilis 168 mmgE
Bacillus subtilis mmg (for mother cell
Arabidopsis thaliana T6K22.50
Escherichia coli K12 pgm
Mycobacterium tuberculosis
Helicobacter pylori J99 jhp1146
Bacillus subtilis 168 ycsI
Rhodococcus erythropolis
Corynebacterium glutamicum
Rhodococcus erythropolis
Bacillus subtilis 168
Streptomyces coelicolor A3(2)
Staphylococcus aureus
Chlamydophila pneumoniae
Chlamydia muridarum Nigg
Streptomyces collinus Tu 1892
Mycobacterium tuberculosis
Chlamydia pneumoniae
Chlamydia muridarum Nigg
Acinetobacter calcoaceticus
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 cysK
Azotobacter vinelandii cysE2
Deinococcus radiodurans R1
Coxiella burnetii Nine Mile Ph I
Aeropyrum pernix K1 APE1069
Bacillus subtilis 168 sucC
Streptomyces roseofulvus frnE
Clostridium kluyveri cat1 cat1
Azospirillum brasilense ATCC
Mycobacterium tuberculosis
Pseudomonas aeruginosa pstB
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Bacillus subtilis 168 bmrU
Mycobacterium tuberculosis
Solanum tuberosum BCAT2
Corynebacterium
ammoniagenes ATCC 6872
Mycobacterium tuberculosis
Corynebacterium
ammoniagenes ATCC 6872
Corynebacterium
ammoniagenes ATCC 6872
Mycobacterium tuberculosis
Corynebacterium
ammoniagenes ATCC 6872
Corynebacterium
ammoniagenes ATCC 6872
Sulfolobus solfataricus
Corynebacterium
ammoniagenes ATCC 6872
Corynebacterium
ammoniagenes ATCC 6872
Corynebacterium
ammoniagenes ATCC 6872
Lactococcus lactis gpo
Aeromonas hydrophila JMP636
Mycobacterium tuberculosis
Salmonella typhimurium LT2
Pseudomonas sp. WO24 dapb1
Corynebacterium
ammoniagenes ATCC 6872
Corynebacterium
ammoniagenes ATCC 6872
Sulfolobus solfataricus ATCC
Corynebacterium
ammoniagenes ATCC 6872
Mycobacterium leprae u296a
Methanosarcina barkeri orf3
Lactococcus lactis subsp. lactis
Corynebacterium glutamicum
Corynebacterium glutamicum
Lactococcus lactis M71plasmid
Thermotoga maritima drrA
Streptomyces lividans tipA
Arthrobacter sp. DK-38
Escherichia coli K12 poxB
Staphylococcus aureus plasmid
Escherichia coli K12 ycdC
Mycobacterium tuberculosis
Rhodococcus erythropolis SQ1
Bacillus subtilis 168 alsR
Mycobacterium tuberculosis
Bacillus subtilis 168 ykrA
Oryctolagus cuniculus kidney
Mycobacterium tuberculosis
Streptomyces griseus hrdB
Schizosaccharomyces pombe
Escherichia coli K12 otsB
Bacillus megaterium ccpA
Haemophilus influenzae Rd
Staphylococcus aureus 8325-4
Mycobacterium tuberculosis
Archaeoglobus fulgidus
Rhodococcus erythropolis SQ1
Thermotoga maritima MSB8
Bacillus subtilis 168 idh or iolG
Escherichia coli K12 shiA
Escherichia coli K12 shiA
Streptomyces coelicolor A3(2)
Saccharomyces cerevisiae
Escherichia coli K12 cysS
Lactococcus lactis sacB
Clostridium acetobutylicum
Escherichia coli K12 nagB
Vibrio furnissii SR1514 manD
Escherichia coli K12 dapA
Streptomyces coelicolor A3(2)
Clostridium perfringens NCTC
Micromonospora viridifaciens
Rhizobium etli ansR
Bacillus firmus OF4 dppA
Bacillus firmus OF4 dappB
Bacillus subtilis 168 oppD
Lactococcus lactis oppF
Escherichia coli K12 rhtB
Bradyrhizobium japonicum lrp
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 baeS
Escherichia coli K12 radA
Bacillus subtilis 168 yacK
Mycobacterium tuberculosis
Pseudomonas putida NCIMB
Chlamydomonas reinhardtii ca1
Streptomyces antibioticus IMRU
Brevibacterium saccharolyticum
Mycobacterium tuberculosis
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Bacillus subtilis 168 mecB
Bacillus cereus ts-4 impdh
Rhodococcus rhodochrous nitR
Trichosporon cutaneum ATCC
Corynebacterium glutamicum
Mycobacterium tuberculosis
Bacillus stearothermophilus lysS
Corynebacterium glutamicum
Mycobacterium leprae
Methylobacterium extorquens
Bacillus subtilis 168 folB
Mycobacterium leprae folP
Bacillus subtilis 168 mtrA
Salmonella typhimurium GP660
Mycobacterium tuberculosis
Actinomadura sp. R39 dac
Escherichia coli K12 ppa
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus subtilis 168 bgIP
Nocardioides sp. KP7 phdD
Streptomyces coelicolor A3(2)
Burkholderia pseudomallei ORFE
Streptomyces roseosporus cpsB
Escherichia coli K12 padA
Campylobacter jejuni Cj0604
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Brevibacterium flavum MJ-233
Homo sapiens MUC5B
Mycobacterium tuberculosis
Staphylococcus aureus mnhA
Bacillus firmus OF4 mrpC
Bacillus firmus OF4 mrpD
Bacillus firmus OF4 mrpE
Rhizobium meliloti phaF
Staphylococcus aureus mnhG
Mycobacterium tuberculosis
Escherichia coli K12 ybdK
Bacillus subtilis 168 def
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Salmonella typhimurium LT2
Bacillus firmus OF4 cls
Escherichia coli K12 bcr
Vibrio cholerae JS1569 nptA
Pseudomonas aureofaciens 30-84
Streptomyces coelicolor A3(2)
Bacillus licheniformis ATCC
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus stearothermophilus
Mycobacterium tuberculosis
Bos taurus
Escherichia coli K12 elaA
Bacillus subtills 168 purT
Corynebacterium glutamicum
Corynebacterium glutamicum
Streptomyces thermoviolaceus
Bacillus brevis ALK36 degU
Corynebacterium
ammoniagenes purA
Mycobacterium tuberculosis
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Mycobacterium tuberculosis
Pyrococcus abyssi pyrE
Mycobacterium tuberculosis
Homo sapiens mpsT
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Synechocystis sp. PCC6803
Staphylococcus aureus cadC
Pyrococcus abyssi Orsay
Rhodococcus rhodochrous
Kryptophanaron alfredi symbiont
Escherichia coli K12 metB
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Methanobacterium
thermoautotrophicum Delta H
Streptomyces coelicolor A3(2)
Azospirillum brasilense carR
Rhodococcus erythropolis thcA
Streptomyces albus G hspR
Mycobacterium tuberculosis
Streptomyces coelicolor grpE
Brevibacterium flavum MJ-233
Streptomyces coelicolor A3(2)
Helicobacter pylori HP0089 mtn
Schizosaccharomyces pombe
Bacillus stearothermophilus
Bacillus subtilis ytnM
Streptomyces coelicolor A3(2)
Escherichia coli K12 cysN
Escherichia coli K12 cysD
Bacillus subtilis cysH
Synechococcus sp. PCC 7942
Saccharomyces cerevisiae
Homo sapiens hypE
Escherichia coli K12 phnB
Streptomyces coelicolor A3(2)
Pseudomonas putida DSMZ ID
Agrobacterium vitis ORFZ3
Alcaligenes eutrophus H16
Haemophilus influenzae hmcB
Haemophilus influenzae hmcB
Bacillus subtilis ydeG
Escherichia coli K12 msgB
Daucus carota
Escherichia coli K12 malK
Lactococcus lactis Plasmid
Vibrio harveyi MAV frp
Crithidia fasciculata iunH
Streptomyces coelicolor A3(2)
Escherichia coli K12 tag
Alcaligenes eutrophus H16 fhp
Streptomyces coelicolor A3(2)
Escherichia coli K12 bglC
Clostridium longisporum B6405
Clostridium longisporum B6405
Methylobacillus flagellatus aat
Corynebacterium glutamicum
Streptomyces coelicolor A3(2)
Escherichia coli K12 dcd
Streptomyces coelicolor A3(2)
Streptomyces thermoviolaceus
Mycobacterium leprae
Mycobacterium leprae
Streptomyces sp. acyA
Mycobacterium leprae
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Neocallimastix frontalis pepck
Pyrococcus abyssi Orsay
Escherichia coli K12 yggH
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Streptomyces erythraeus eryA
Mycobacterium bovis BCG
Mycobacterium tuberculosis
Corynebacterium glutamicum
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus licheniformis ATCC
Sus scrofa fmo1
Escherichia coli K12 glf
Mycobacterium tuberculosis
Pseudomonas aeruginosa
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Escherichia coli K12 farR
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Amycolatopsis methanolica pgm
Mycobacterium smegmatis pzaA
Streptomyces coelicolor A3(2)
Streptomyces lavendulae
Saccharomyces cerevisiae
Bacillus subtilis glpQ
Bacillus subtilis gntP
Corynebacterium glutamicum
Brevibacterium flavum lctA
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Brevibacterium linens ORF1
Escherichia coli K12 MG1655
Mycobacterium tuberculosis
Escherichia coli K12 shiA
Neisseria meningitidis lldA
Bacillus phage phi-105 ORF1
Caenorhabditis elegans
Arabidopsis thaliana ill1
Escherichia coli B msrA
Corynebacterium
pseudodiphtheriticum sod
Bacillus subtilis gltC
Corynebacterium glutamicum
Mycobacterium tuberculosis
Streptomyces cyanogenus lanJ
Bacillus subtilis 168 yxaD
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Streptomyces coelicolor A3(2)
Streptomyces coelicolor A3(2)
Bacillus subtilis spolllJ
Mycobacterium tuberculosis
Escherichia coli K12.MG1655
Mycobacterium tuberculosis
Escherichia coli K12 MG1655
Chlorobium vibrioforme ybc5
Chlamydia pneumoniae
Chlamydia muridarum Nigg
Escherichia coli K12 MG1655
Streptomyces coelicolor
Mycobacterium tuberculosis
Streptomyces coelicolor A3(2)
Pseudomonas aeruginosa TNP5
Saccharopolyspora erythraea fer
Streptomyces coelicolor A3(2)
Corynebacterium glutamicum
Corynebacterium glutamicum
Pyrococcus woesei gap
Synechocystis sp. PCC6803
Archaeoglobus fulgidus AF0152
Escherichia coli K12 baeS
Bacillus subtilis phoP
Pseudomonas syringae pv.
Bradyrhizobium japonicum tlpA
Mus musculus qor
Synechocystis sp. PCC6803
Escherichia coli K12 MG1655
Aeropyrum pernix K1 APE2572
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Escherichia coli K12 thi2
Pseudomonas putida pcaK
Escherichia coli K12 yqjI
Escherichia coli K12 dnaB
Escherichia coli K12 RL9
Escherichia coli K12 ssb
Escherichia coli K12 RS6
Mycobacterium smegmatis
Bacillus subtilis ponA
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacillus subtilis yhgC
Escherichia coli K12 yceA
Escherichia coli K12 ybjZ
Escherichia coli K12 MG1655
Campylobacter jejuni Cj0606
Mycobacterium tuberculosis
Escherichia coli K12 dps
Escherichia coli K12 mutM or
Escherichia coli K12 rtcB
Homo sapiens mgmT
Cavia porcellus (Guinea pig) qor
Mycobacterium tuberculosis
Corynebacterium melassecola
Bacillus subtilis gntK
Enterococcus faecium vanZ
Enterococcus faecium vanZ
Staphylococcus aureus merA
Escherichia coli K12 dadA
Thermus thermophilus nox
Bacillus subtilis syl
Escherichia coli K12
Dichelobacter nodosus vapI
Streptomyces coelicolor
Escherichia coli K12 hpcE
Pseudomonas alcaligenes xlnE
Pectobacterium chrysanthemi
Pseudomonas putida pcaK
Pseudomonas putida
Homo sapiens eat2
Corynebacterium glutamicum
Brevibacterium lactofermentum
Brevibacterium lactofermentum
Corynebacterium glutamicum
Brevibacterium lactofermentum
Brevibacterium lactofermentum
Brevibacterium lactofermentum
Streptomyces coelicolor A3(2)
Escherichia coli K12 ptxA
Pseudomonas stutzeri
Streptomyces coelicolor A3(2)
Chlorobium limicola petC
Thermoanaerobacter brockii
Escherichia coli K12 yfeH
Streptomyces coelicolor A3(2)
Streptomyces coelicolor Plasmid
Thermoanaerobacter brockii
Saccharomyces cerevisiae
Klebsiella terrigena budC
Mycobacterium tuberculosis
Lactococcus lactis subsp. lactis
Escherichia coli K12 acrR
Acinetobacter calcoaceticus
Pseudomonas sp. P51
Escherichia coli K12 xylE
Salmonella typhimurium iclR
Escherichia coli K12 ydgJ
Listeria innocua strain 4450
Streptomyces griseus strI
Bacillus subtilis yvnB
Caenorhabditis elegans unc1
Mycobacterium bovis BCG
Mycobacterium leprae u2266k
Bacillus subtilis thiD
Bacillus subtilis yvgY
Corynebacterium glutamicum
Escherichia coli K12 fecB
Schizosaccharomyces pombe
Bacillus subtilis thiD
Bacillus subtilis yvgY
Bacillus subtilis azlD
Bacillus subtilis azlD
Escherichia coli K12 yqgE
Escherichia coli K12 cca
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Pseudomonas aeruginosa algU
Streptomyces clavuligerus trxB
Chlamydomonas reinhardtii thi2
Bacillus subtilis cwlB
Mycobacterium tuberculosis
Pseudomonas putida ygi2
Mycobacterium tuberculosis
Escherichia coli K12 gidB
Mycobacterium tuberculosis
Bacillus subtilis rnpA
Mycobacterium avium rpmH
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Corynebacterium glutamicum
Determination of Effective Mutation Site
(1) Identification of Mutation Site Based on the Comparison of the Gene Nucleotide Sequence of Lysine-Producing B-6 Strain with that of Wild Type Strain ATCC 13032
Corynebacterium glutamicum B-6, which is resistant to S-(2-aminoethyl)cysteine (AEC), rifampicin, streptomycin and 6-azauracil, is a lysine-producing mutant having been mutated and bred by subjecting the wild type ATCC 13032 strain to multiple rounds of random mutagenesis with a mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and screening (Appl. Microbiol. Biotechnol., 32: 269-273 (1989)). First, the nucleotide sequences of genes derived from the B-6 strain and considered to relate to the lysine production were determined by a method similar to the above. The genes relating to the lysine production include lysE and lysG which are lysine-excreting genes; ddh, dapA, hom and lysC (encoding diaminopimelate dehydrogenase, dihydropicolinate synthase, homoserine dehydrogenase and aspartokinase, respectively) which are lysine-biosynthetic genes; and pyc and zwf (encoding pyruvate carboxylase and glucose-6-phosphate dehydrogenase, respectively) which are glucose-metabolizing genes. The nucleotide sequences of the genes derived from the production strain were compared with the corresponding nucleotide sequences of the ATCC 13032 strain genome represented by SEQ ID NOS:1 to 3501 and analyzed. As a result, mutation points were observed in many genes. For example, no mutation site was observed in lysE, lysG, ddh, dapA, and the like, whereas amino acid replacement mutations were found in hom, lysC, pyc, zwf, and the like. Among these mutation points, those which are considered to contribute to the production were extracted on the basis of known biochemical or genetic information. Among the mutation points thus extracted, a mutation, Val59Ala, in hom and a mutation, Pro458Ser, in pyc were evaluated whether or not the mutations were effective according to the following method.
(2) Evaluation of Mutation, Val59Ala, in hom and Mutation, Pro458Ser, in pyc
It is known that a mutation in horn inducing requirement or partial requirement for homoserine imparts lysine productivity to a wild type strain (Amino Acid Fermentation, ed. by Hiroshi Aida et al., Japan Scientific Societies Press). However, the relationship between the mutation, Val59Ala, in hom and lysine production is not known. It can be examined whether or not the mutation, Val59Ala, in hom is an effective mutation by introducing the mutation to the wild type strain and examining the lysine productivity of the resulting strain. On the other hand, it can be examined whether or not the mutation, Pro458Ser, in pyc is effective by introducing this mutation into a lysine-producing strain which has a deregulated lysine-bioxynthetic pathway and is free from the pyc mutation, and comparing the lysine productivity of the resulting strain with the parent strain. As such a lysine-producing bacterium, No. 58 strain (FERM BP-7134) was selected (hereinafter referred to the “lysine-producing No. 58 strain” or the “No. 58 strain”). Based on the above, it was determined that the mutation, Val59Ala, in hom and the mutation, Pro458Ser, in pyc were introduced into the wild type strain of Corynebacterium glutamicum ATCC 13032 (hereinafter referred to as the “wild type ATCC 13032 strain” or the “ATCC 13032 strain”) and the lysine-producing No. 58 strain, respectively, using the gene replacement method. A plasmid vector pCES30 for the gene replacement for the introduction was constructed by the following method.
A plasmid vector pCE53 having a kanamycin-resistant gene and being capable of autonomously replicating in Coryneform bacteria (Mol. Gen. Genet., 196: 175-178 (1984)) and a plasmid pMOB3 (ATCC 77282) containing a levansucrase gene (sacB) of Bacillus subtilis (Molecular Microbiology, 6: 1195-1204 (1992)) were each digested with PstI. Then, after agarose gel electrophoresis, a pCE53 fragment and a 2.6 kb DNA fragment containing sacB were each extracted and purified using GENECLEAN Kit (manufactured by BIO 101). The pCE53 fragment and the 2.6 kb DNA fragment were ligated using Ligation Kit ver. 2 (manufactured by Takara Shuzo), introduced into the ATCC 13032 strain by the electroporation method (FEMS Microbiology Letters, 65: 299 (1989)), and cultured on BYG agar medium (medium prepared by adding 10 g of glucose, 20 g of peptone (manufactured by Kyokuto Pharmaceutical), 5 g of yeast extract (manufactured by Difco), and 16 g of Bactoagar (manufactured by Difco) to 1 liter of water, and adjusting its pH to 7.2) containing 25 μg/ml kanamycin at 30° C. for 2 days to obtain a transformant acquiring kanamycin-resistance. As a result of digestion analysis with restriction enzymes, it was confirmed that a plasmid extracted from the resulting transformant by the alkali SDS method had a structure in which the 2.6 kb DNA fragment had been inserted into the PstI site of pCE53. This plasmid was named pCES30.
Next, two genes having a mutation point, hom and pyc, were amplified by PCR, and inserted into pCES30 according to the TA cloning method (Bio Experiment Illustrated vol. 3, published by Shujunsha). Specifically, pCES30 was digested with BamHI (manufactured by Takara Shuzo), subjected to an agarose gel electrophoresis, and extracted and purified using GENECLEAN Kit (manufactured by BIO 101). The both ends of the resulting pCES30 fragment were blunted with DNA Blunting Kit (manufactured by Takara Shuzo) according to the attached protocol. The blunt-ended pCES30 fragment was concentrated by extraction with phenol/chloroform and precipitation with ethanol, and allowed to react in the presence of Taq polymerase (manufactured by Roche Diagnostics) and dTTP at 70° C. for 2 hours so that a nucleotide, thymine (T), was added to the 3′-end to prepare a T vector of pCES30.
Separately, chromosomal DNA was prepared from the lysine-producing B-6 strain according to the method of Saito et al. (Biochem. Biophys. Acta, 72: 619 (1963)). Using the chromosomal DNA as a template, PCR was carried out with Pfu turbo DNA polymelase (manufactured by Stratagene). In the mutated hom gene, the DNAs having the nucleotide sequences represented by SEQ ID NOS:7002 and 7003 were used as the primer set. In the mutated pyc gene, the DNAs having the nucleotide sequences represented by SEQ ID NOS:7004 and 7005 were used as the primer set. The resulting PCR product was subjected to agarose gel electrophoresis, and extracted and purified using GENEGLEAN Kit (manufactured by BIO 101). Then, the PCR product was allowed to react in the presence of Taq polymerase (manufactured by Roche Diagnostics) and dATP at 72° C. for 10 minutes so that a nucleotide, adenine (A), was added to the 3′-end.
The above pCES30 T vector fragment and the mutated hom gene (1.7 kb) or mutated pyc gene (3.6 kb) to which the nucleotide A had been added of the PCR product were concentrated by extraction with phenol/chloroform and precipitation with ethanol, and then ligated using Ligation Kit ver. 2. The ligation products were introduced into the ATCC 13032 strain according to the electroporation method, and cultured on BYG agar medium containing 25 μg/ml kanamycin at 30° C. for 2 days to obtain kanamycin-resistant transformants. Each of the resulting transformants was cultured overnight in BYG liquid medium containing 25 μg/ml kanamycin, and a plasmid was extracted from the culturing solution medium according to the alkali SDS method. As a result of digestion analysis using restriction enzymes, it was confirmed that the plasmid had a structure in which the 1.7 kb or 3.6 kb DNA fragment had been inserted into pCES30. The plasmids thus constructed were named respectively pChom59 and pCpyc458.
The introduction of the mutations to the wild type ATCC 13032 strain and the lysine-producing No. 58 strain according to the gene replacement method was carried out according to the following method. Specifically, pChom59 and pCpyc458 were introduced to the ATCC 13032 strain and the No. 58 strain, respectively, and strains in which the plasmid is integrated into the chromosomal DNA by homologous recombination were selected using the method of Ikeda et al. (Microbiology 144: 1863 (1998)). Then, the stains in which the second homologous recombination was carried out were selected by a selection method, making use of the fact that the Bacillus subtilis levansucrase encoded by pCES30 produced a suicidal substance (J. of Bacteriol., 174: 5462 (1992)). Among the selected strains, strains in which the wild type hom and pyc genes possessed by the ATCC 13032 strain and the No. 58 strain were replaced with the mutated hom and pyc genes, respectively, were isolated. The method is specifically explained below.
One strain was selected from the transformants containing the plasmid, pChom59 or pCpyc458, and the selected strain was cultured in BYG medium containing 20 μg/ml kanamycin, and pCG11 (Japanese Published Examined Patent Application No. 91827/94) was introduced thereinto by the electroporation method. pCG11 is a plasmid vector having a spectinomycin-resistant gene and a replication origin which is the same as pCE53. After introduction of the pCG11, the strain was cultured on BYG agar medium containing 20 μg/ml kanamycin and 100 μg/ml spectinomycin at 30° C. for 2 days to obtain both the kanamycin- and spectinomycin-resistant transformant. The chromosome of one strain of these transformants was examined by the Southern blotting hybridization according to the method reported by Ikeda et al. (Microbiology, 144: 1863 (1998)). As a result, it was confirmed that pChom59 or pCpyc458 had been integrated into the chromosome by the homologous recombination of the Cambell type. In such a strain, the wild type and mutated hom or pyc genes are present closely on the chromosome, and the second homologous recombination is liable to arise therebetween.
Each of these transformants (having been recombined once) was spread on Suc agar medium (medium prepared by adding 100 g of sucrose, 7 g of meat extract, 10 g of peptone, 3 g of sodium chloride, 5 g of yeast extract (manufactured by Difco), and 18 g of Bactoagar (manufactured by Difco) to 1 liter of water, and adjusting its pH 7.2) and cultured at 30° C. for a day. Then the colonies thus growing were selected in each case. Since a strain in which the sacb gene is present converts sucrose into a suicide substrate, it cannot grow in this medium (J. Bacteriol., 174: 5462 (1992)). On the other hand, a strain in which the sacB gene was deleted due to the second homologous recombination between the wild type and the mutated hom or pyc genes positioned closely to each other forms no suicide substrate and, therefore, can grow in this medium. In the homologous recombination, either the wild type gene or the mutated gene is deleted together with the sacB gene. When the wild type is deleted together with the sacb gene, the gene replacement into the mutated type arises.
Chromosomal DNA of each the thus obtained second recombinants was prepared by the above method of Saito et al. PCR was carried out using Pfu turbo DNA polymerase (manufactured by Stratagene) and the attached buffer. In the hom gene, DNAs having the nucleotide sequences represented by SEQ ID NOS:7002 and 7003 were used as the primer set. Also, in the pyc gene was used, DNAs having the nucleotide sequences represented by SEQ ID NOS:7004 and 7005 were used as the primer set. The nucleotide sequences of the PCR products were determined by the conventional method so that it was judged whether the hom or pyc gene of the second recombinant was a wild type or a mutant. As a result, the second recombinant which were called HD-1 and No. 58pyc were target strains having the mutated hom gene and pyc gene, respectively.
(3) Lysine Production Test of HD-1 and No. 58pyc Strains
The HD-1 strain (strain obtained by incorporating the mutation, Val59Ala, in the hom gene into the ATCC 13032 strain) and the No. 58pyc strain (strain obtained by incorporating the mutation, Pro458Ser, in the pyc gene into the lysine-producing No. 58 strain) were subjected to a culture test in a 5 l jar fermenter by using the ATCC 13032 strain and the lysine-producing No. 58 strain respectively as a control. Thus lysine production was examined.
After culturing on BYG agar medium at 30° C. for 24 hours, each strain was inoculated into 250 ml of a seed medium (medium prepared by adding 50 g of sucrose, 40 g of corn steep liquor, 8.3 g of ammonium sulfate, 1 g of urea, 2 g of potassium dihydrogenphosphate, 0.83 g of magnesium sulfate heptahydrate, 10 mg of iron sulfate heptahydrate, 1 mg of copper sulfate pentahydrate, 10 mg of zinc sulfate hentahydrate, 10 mg of β-alanine, 5 mg of nicotinic acid, 1.5 mg of thiamin hydrochloride, and 0.5 mg of biotin to 1 liter of water, and adjusting its pH to 7.2, then to which 30 g of calcium carbonate had been added) contained in a 2 l buffle-attached Erlenmeyer flask and cultured therein at 30° C. for 12 to 16 hours. A total amount of the seed culturing medium was inoculated into 1,400 ml of a main culture medium (medium prepared by adding 60 g of glucose, 20 g of corn steep liquor, 25 g of ammonium chloride, 2.5 g of potassium dihydrogenphosphate, 0.75 g of magnesium sulfate heptahydrate, 50 mg of iron sulfate heptahydrate, 13 mg of manganese sulfate pentahydrate, 50 mg of calcium chloride, 6.3 mg of copper sulfate pentahydrate, 1.3 mg of zinc sulfate heptahydrate, 5 mg of nickel chloride hexahydrate, 1.3 mg of cobalt chloride hexahydrate, 1.3 mg of ammonium molybdenate tetrahydrate, 14 mg of nicotinic acid, 23 mg of β-alanine, 7 mg of thiamin hydrochloride, and 0.42 mg of biotin to 1 liter of water) contained in a 5 l jar fermenter and cultured therein at 32° C., 1 vvm and 800 rpm while controlling the pH to 7.0 with aqueous ammonia. When glucose in the medium had been consumed, a glucose feeding solution (medium prepared by adding 400 g glucose and 45 g of ammonium chloride to 1 liter of water) was continuously added. The addition of feeding solution was carried out at a controlled speed so as to maintain the dissolved oxygen concentration within a range of 0.5 to 3 ppm After culturing for 29 hours, the culture was terminated. The cells were separated from the culture medium by centrifugation and then L-lysine hydrochloride in the supernatant was quantified by high performance liquid chromatography (HPLC). The results are shown in Table 2 below.
As is apparent from the results shown in Table 2, the lysine productivity was improved by introducing the mutation, Val59Ala, in the hom gene or the mutation, Pro458Ser, in the pyc gene. Accordingly, it was found that the mutations are both effective mutations relating to the production of lysine. Strain, AHP-3, in which the mutation, Val59Ala, in the hom gene and the mutation, Pro458Ser, in the pyc gene have been introduced into the wild type ATCC 13032 strain together with the mutation, Thr331Ile in the lysC gene has been deposited on Dec. 5, 2000, in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba-shi, Ibaraki, Japan) as FERM BP-7382.
Reconstruction of Lysine-Producing Strain Based on Genome Information
The lysine-producing mutant B-6 strain (Appl. Microbiol. Biotechnol., 32: 269-273 (1989)), which has been constructed by multiple round random mutagenesis with NTG and screening from the wild type ATCC 13032 strain, produces-a remarkably large amount of lysine hydrochloride when cultured in a jar at 32° C. using glucose as a carbon source. However, since the fermentation period is long, the production rate is less than 2.1 g/l/h. Breeding to reconstitute only effective mutations relating to the production of lysine among the estimated at least 300 mutations introduced into the B-6 strain in the wild type ATCC 13032 strain was performed.
(1) Identification of Mutation Point and Effective Mutation by Comparing the Gene Nucleotide Sequence of the B-6 Strain with that of the ATCC 13032 Strain
As described above, the nucleotide sequences of genes derived from the B-6 strain were compared with the corresponding nucleotide sequences of the ATCC 13032 strain genome represented by SEQ ID NOS:1 to 3501 and analyzed to identify many mutation points accumulated in the chromosome of the B-6 strain. Among these, a mutation, Val591Ala, in hom, a mutation, Thr311Ile, in lysC, a mutation, Pro458Ser, in pyc and a mutation, Ala213Thr, in zwf were specified as effective mutations relating to the production of lysine. Breeding to reconstitute the 4 mutations in the wild type strain and for constructing of an industrially important lysine-producing strain was carried out according to the method shown below.
(2) Construction of Plasmid for Gene Replacement Having Mutated Gene
The plasmid for gene replacement, pChom59, having the mutated hom gene and the plasmid for gene replacement, pCpyc458, having the mutated pyc gene were prepared in the above Example 2(2). Plasmids for gene replacement having the mutated lysC and zwf were produced as described below.
The lysC and zwf having mutation points were amplified by PCR, and inserted into a plasmid for gene replacement, pCES30, according to the TA cloning method described in Example 2(2) (Bio Experiment Illustrated, Vol. 3).
Separately, chromosomal DNA was prepared from the lysine-producing B-6 strain according to the above method of Saito et al. Using the chromosomal DNA as a template, PCR was carried out with Pfu turbo DNA polymerase (manufactured by Stratagene). In the mutated lysC gene, the DNAs having the nucleotide sequences represented by SEQ ID NOS:7006 and 7007 were used as the primer set. In the mutated zwf gene, the DNAs having the nucleotide sequences represented by SEQ ID NOS:7008 and 7009 as the primer set. The resulting PCR product was subjected to agarose gel electrophoresis, and extracted and purified using GENEGLEAN Kit (manufactured by BIO 101). Then, the PCR product was allowed to react in the presence of Taq DNA polymerase (manufactured by Roche Diagnostics) and dATP at 72° C. for 10 minutes so that a nucleotide, adenine (A), was added to the 3′-end.
The above pCES30 T vector fragment and the mutated lysC gene (1.5 kb) or mutated zwf gene (2.3 kb) to which the nucleotide A had been added of the PCR product were concentrated by extraction with phenol/chloroform and precipitation with ethanol, and then ligated using Ligation Kit ver. 2. The ligation products were introduced into the ATCC 13032 strain according to the electroporation method, and cultured on BYG agar medium containing 25 μg/ml kanamycin at 30° C. for 2 days to obtain kanamycin-resistant transformants. Each of the resulting transformants was cultured overnight in BYG liquid medium containing 25 μg/ml kanamycin, and a plasmid was extracted from the culturing solution medium according to the alkali SDS method. As a result of digestion analysis using restriction enzymes, it was confirmed that the plasmid had a structure in which the 1.5 kb or 2.3 kb DNA fragment had been inserted into pCES30. The plasmids thus constructed were named respectively pClysC311 and pCzwf213.
(3) Introduction of Mutation, Thr311Ile, in lysC into One Point Mutant HD-1
Since the one mutation point mutant HD-1 in which the mutation, Val59Ala, in hom was introduced into the wild type ATCC 13032 strain had been obtained in Example 2(2), the mutation, Thr311Ile, in lysC was introduced into the HD-1 strain using pClysC311 produced in the above (2) according to the gene replacement method described in Example 2(2). PCR was carried out using chromosomal DNA of the resulting strain and, as the primer set, DNAs having the nucleotide sequences represented by SEQ ID NOS:7006 and 7007 in the same manner as in Example 2(2). As a result of the fact that the nucleotide sequence of the PCR product was determined in the usual manner, it was confirmed that the strain which was named AHD-2 was a two point mutant having the mutated lysC gene in addition to the mutated hom gene.
(4) Introduction of Mutation, Pro458Ser, in pyc into Two Point Mutant AHD-2
The mutation, Pro458Ser, in pyc was introduced into the AHD-2 strain using the pCpyc458 produced in Example 2(2) by the gene replacement method described in Example 2(2). PCR was carried out using chromosomal DNA of the resulting strain and, as the primer set, DNAs having the nucleotide sequences represented by SEQ ID NOS:7004 and 7005 in the same manner as in Example 2(2). As a result of the fact that the nucleotide sequence of the PCR product was determined in the usual manner, it was confirmed that the strain which was named AHD-3 was a three point mutant having the mutated pyc gene in addition to the mutated hom gene and lysC gene.
(5) Introduction of Mutation, Ala213Thr, in zwf into Three Point Mutant AHP-3
The mutation, Ala213Thr, in zwf was introduced into the AHP-3 strain using the pCzwf458 produced in the above (2) by the gene replacement method described in Example 2(2). PCR was carried out using chromosomal DNA of the resulting strain and, as the primer set, DNAs having the nucleotide sequences represented by SEQ ID NOS:7008 and 7009 in the same manner as in Example 2(2). As a result of the fact that the nucleotide sequence of the PCR product was determined in the usual manner, it was confirmed that the strain which was named APZ-4 was a four point mutant having the mutated zwf gene in addition to the mutated hom gene, lysC gene and pyc gene.
(6) Lysine Production Test on HD-1, AHD-2, AHP-3 and APZ-4 Strains
The HD-1, AHD-2, AHP-3 and APZ-4 strains obtained above were subjected to a culture test in a 5 l jar fermenter in accordance with the method of Example 2(3).
Table 3 shows the results.
Since the lysine-producing mutant B-6 strain which has been bred based on the random mutation and selection shows a productivity of less than 2.1 g/l/h, the APZ-4 strain showing a high productivity of 3.0 g/l/h is useful in industry.
(7) Lysine Fermentation by APZ-4 Strain at High Temperature
The APZ-4 strain, which had been reconstructed by introducing 4 effective mutations into the wild type strain, was subjected to the culturing test in a 5 l jar fermenter in the same manner as in Example 2(3), except that the culturing temperature was changed to 40° C.
The results are shown in Table 4.
As is apparent from the results shown in Table 4, the lysine hydrochloride titer and productivity in culturing at a high temperature of 40° C. comparable to those at 32° C. were obtained. In the mutated and bred lysine-producing B-6 strain constructed by repeating random mutation and selection, the growth and the lysine productivity are lowered at temperatures exceeding 34° C. so that lysine fermentation cannot be carried out, whereas lysine fermentation can be carried out using the APZ-4 strain at a high temperature of 40° C. so that the load of cooling is greatly reduced and it is industrially useful. The lysine fermentation at high temperatures can be achieved by reflecting the high temperature adaptability inherently possessed by the wild type strain on the APZ-4 strain.
As demonstrated in the reconstruction of the lysine-producing strain, the present invention provides a novel breeding method effective for eliminating the problems in the conventional mutants and acquiring industrially advantageous strains. This methodology which reconstitutes the production strain by reconstituting the effective mutation is an approach which is efficiently carried out using the nucleotide sequence information of the genome disclosed in the present invention, and its effectiveness was found for the first time in the present invention.
Production of DNA Microarray and use thereof
A DNA microarray was produced based on the nucleotide sequence information of the ORF deduced from the full nucleotide sequences of Corynebacterium glutamicum ATCC 13032 using software, and genes of which expression is fluctuated depending on the carbon source during culturing were searched.
(1) Production of DNA Microarray
Chromosomal DNA was prepared from Corynebacterium glutamicum ATCC 13032 by the method of Saito et al. (Biochem. Biophys. Acta, 72: 619 (1963)). Based on 24 genes having the nucleotide sequences represented by SEQ ID NOS:207, 3433, 281, 3435, 3439, 765, 3445, 1226, 1229, 3448, 3451, 3453, 3455, 1743, 3470, 2132, 3476, 3477, 3485, 3488, 3489, 3494, 3496, and 3497 from the ORFs shown in Table 1 deduced from the full genome nucleotide sequence of Corynebacterium glutamicum ATCC 13032 using software and the nucleotide sequence of rabbit globin gene (GenBank Accession No. V00882) used as an internal standard, oligo DNA primers for PCR amplification represented by SEQ ID NOS:7010 to 7059 targeting the nucleotide sequences of the genes were synthesized in a usual manner.
As the oligo DNA primers used for the PCR,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7010 and 7011 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:207,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7012 and 7013 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3433,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7014 and 7015 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:281,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7016 and 7017 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3435,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7018 and 7019 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3439,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7020 and 7021 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:765,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7022 and 7023 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3445,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7024 and 7025 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:1226,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7026 and 7027 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:1229,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7028 and 7029 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3448,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7030 and 7031 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3451,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7032 and 7033 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3453,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7034 and 7035 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3455,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7036 and 7037 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:1743,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7038 and 7039 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3470,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7040 and 7041 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:2132,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7042 and 7043 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3476,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7044 and 7045 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3477,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7046 and 7047 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3485,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7048 and 7049 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3488,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7050 and 7051 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3489,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7052 and 7053 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3494,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7054 and 7055 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3496,
DNAs having the nucleotide sequence represented by SEQ ID NOS:7056 and 7057 were used for the amplification of the DNA having the nucleotide sequence represented by SEQ ID NO:3497, and
DNAs having the nucleotide sequence represented by SEQ ID NOS:7058 and 7059 were used for the amplification of the DNA having the nucleotide sequence of the rabbit globin gene,
The PCR was carried for 30 cycles with each cycle consisting of 15 seconds at 95° C. and 3 minutes at 68° C. using a thermal cycler (GeneAmp PCR system 9600, manufactured by Perkin Elmer), TaKaRa EX-Taq (manufactured by Takara Shuzo), 100 ng of the chromosomal DNA and the buffer attached to the TaKaRa Ex-Taq reagent. In the case of the rabbit globin gene, a single-stranded cDNA which had been synthesized from rabbit globin mRNA (manufactured by Life Technologies) according to the manufacture's instructions using a reverse transcriptase RAV-2 (manufactured by Takara Shuzo). The PCR product of each gene thus amplified was subjected to agarose gel electrophoresis and extracted and purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). The purified PCR product was concentrated by precipitating it with ethanol and adjusted to a concentration of 200 ng/μl. Each PCR product was spotted on a slide glass plate (manufactured by Matsunami Glass) having MAS coating in 2 runs using GTMASS SYSTEM (manufactured by Nippon Laser & Electronics Lab.) according to the manufacture's instructions.
(2) Synthesis of Fluorescence Labeled cDNA
The ATCC 13032 strain was spread on BY agar medium (medium prepared by adding 20 g of peptone (manufactured by Kyokuto Pharmaceutical), 5 g of yeast extract (manufactured by Difco), and 16 g of Bactoagar (manufactured by Difco) to in 1 liter of water and adjusting its pH to 7.2) and cultured at 30° C. for 2 days. Then, the cultured strain was further inoculated into 5 ml of BY liquid medium and cultured at 30° C. overnight. Then, the cultured strain was further inoculated into 30 ml of a minimum medium (medium prepared by adding 5 g of ammonium sulfate, 5 g of urea, 0.5 g of monopotassium dihydrogenphosphate, 0.5 g of dipotassium monohydrogenphosphate, 20.9 g of morpholinopropanesulfonic acid, 0.25 g of magnesium sulfate heptahydrate, 10 mg of calcium chloride dihydrate, 10 mg of manganese sulfate monohydrate, 10 mg of ferrous sulfate heptahydrate, 1 mg of zinc sulfate heptahydrate, 0.2 mg copper sulfate, and 0.2 mg biotin to 1 liter of water, and adjusting its pH to 6.5) containing 110 mmol/l glucose or 200 mmol/l ammonium acetate, and cultured in an Erlenmyer flask at 30° to give 1.0 of absorbance at 660 nm. After the cells were prepared by centrifuging at 4° C. and 5,000 rpm for 10 minutes, total RNA was prepared from the resulting cells according to the method of Bormann et al. (Molecular Microbiology, 6: 317-326 (1992)). To avoid contamination with DNA, the RNA was treated with DnaseI (manufactured by Takara Shuzo) at 37° C. for 30 minutes and then further purified using Qiagen RNeasy MiniKit (manufactured by QIAGEN) according to the manufacture's instructions. To 30 μg of the resulting total RNA, 0.6 μl of rabbit globin mRNA (50 ng/μl, manufactured by Life Technologies) and 1 μl of a random 6 mer primer (500 ng/μl, manufactured by Takara Shuzo) were added for denaturing at 65° C. for 10 minutes, followed by quenching on ice. To the resulting solution, 6 μl of a buffer attached to SuperScript II (manufactured by Lifetechnologies), 3 μl of 0.1 mol/l DTT, 1.5 μl of dNTPs (25 mmol/l dATP, 25 mmol/l dCTP, 25 mmol/l dGTP, 10 mmol/l dTTP), 1.5 μl of Cy5-dUTP or Cy3-dUTP (manufactured by NEN) and 2 μl of SuperScript II were added, and allowed to stand at 25° C. for 10 minutes and then at 42° C. for 110 minutes. The RNA extracted from the cells using glucose as the carbon source and the RNA extracted from the cells using ammonium acetate were labeled with Cy5-dUTP and Cy3-dUTP, respectively. After the fluorescence labeling reaction, the RNA was digested by adding 1.5 μl of 1 mol/l sodium hydroxide-20 mmol/l EDTA solution and 3.0 μl of 10% SDS solution, and allowed to stand at 65° C. for 10 minutes. The two cDNA solutions after the labeling were mixed and purified using Qiagen PCR purification Kit (manufactured by QIAGEN) according to the manufacture's instructions to give a volume of 10 μl.
(3) Hybridization
UltraHyb (110 μl) (manufactured by Ambion) and the fluorescence-labeled cDNA solution (10 μl) were mixed and subjected to hybridization and the subsequent washing of slide glass using GeneTAC Hybridization Station (manufactured by Genomic Solutions) according to the manufacture's instructions. The hybridization was carried out at 50° C., and the washing was carried out at 25° C.
(4) Fluorescence Analysis
The fluorescence amount of each DNA array having the fluorescent cDNA hybridized therewith was measured using ScanArray 4000 (manufactured by GSI Lumonics).
Table 5 shows the Cy3 and Cy5 signal intensities of the genes having been corrected on the basis of the data of the rabbit globin used as the internal standard and the Cy3/Cy5 ratios.
The ORF function data estimated by using software were searched for SEQ ID NOS:3488 and 3489 showing remarkably strong Cy3 signals. As a result, it was found that SEQ ID NOS:3488 and 3489 are a maleate synthase gene and an isocitrate lyase gene, respectively. It is known that these genes are transcriptionally induced by acetic acid in Corynebacterium glutamicum (Archives of Microbiology, 168: 262-269 (1997)).
As described above, a gene of which expression is fluctuates could be discovered by synthesizing appropriate oligo DNA primers based on the ORF nucleotide sequence information deduced from the full genomic nucleotide sequence information of Corynebacterium glutamicum ATCC 13032 using software, amplifying the nucleotide sequences of the gene using the genome DNA of Corynebacterium glutamicum as a template in the PCR reaction, and thus producing and using a DNA microarray.
This Example shows that the expression amount can be analyzed using a DNA microarray in the 24 genes. On the other hand, the present DNA microarray techniques make it possible to prepare DNA microarrays having thereon several thousand gene probes at once. Accordingly, it is also possible to prepare DNA microarrays having thereon all of the ORF gene probes deduced from the full genomic nucleotide sequence of Corynebacterium glutamicum ATCC 13032 determined by the present invention, and analyze the expression profile at the total gene level of Corynebacterium glutamicum using these arrays.
Homology Search Using Corynebacterium glutamicum Genome Sequence
(1) Search of Adenosine Deaminase
The amino acid sequence (ADD—ECOLI) of Escherichia coli adenosine deaminase was obtained from Swiss-prot Database as the amino acid sequence of the protein of which function had been confirmed as adenosine deaminase (EC3.5.4.4). By using the full length of this amino acid sequence as a query, a homology search was carried out on a nucleotide sequence database of the genome sequence of Corynebacterium glutamicum or a database of the amino acids in the ORF region deduced from the genome sequence using FASTA program (Proc. Natl. Acad. Sci. ISA, 85: 2444-2448 (1988)). A case where E-value was le−10 or less was judged as being significantly homologous. As a result, no sequence significantly homologous with the Escherichia coli adenosine deaminase was found in the nucleotide sequence database of the genome sequence of Corynebacterium glutamicum or the database of the amino acid sequences in the ORF region deduced from the genome sequence. Based on these results, it is assumed that Corynebacterium glutamicum contains no ORF having adenosine deaminase activity and thus has no activity of converting adenosine into inosine.
(2) Search of Glycine Cleavage Enzyme
The sequences (GCSP—ECOLI, GCST—ECOLI and GCSH—ECOLI) of glycine decarboxylase, aminomethyl transferase and an aminomethyl group carrier each of which is a component of Escherichia coli glycine cleavage enzyme as the amino acid sequence of the protein, of which function had been confirmed as glycine cleavage enzyme (EC2.1.2.10), were obtained from Swiss-prot Database.
By using these full-length amino acid sequences as a query, a homology search was carried out on a nucleotide sequence database of the genome sequence of Corynebacterium glutamicum or a database of the ORF amino acid sequences deduced from the genome sequence using FASTA program. A case where E-value was le−10 or less was judged as being significantly homologous. As a result, no sequence significantly homologous with the glycine decarboxylase, the aminomethyl transferase or the aminomethyl group carrier each of which is a component of Escherichia coli glycine cleavage enzyme, was found in the nucleotide sequence database of the genome sequence of Corynebacterium glutamicum or the database of the ORF amino acid sequences estimated from the genome sequence. Based on these results, it is assumed that Corynebacterium glutamicum contains no ORF having the activity of glycine decarboxylase, aminomethyl transferase or the aminomethyl group carrier and thus has no activity of the glycine cleavage enzyme.
(3) Search of IMP Dehydrogenase
The amino acid sequence (IMDH ECOLI) of Escherichia coli IMP dehydrogenase as the amino acid sequence of the protein, of which function had been confirmed as IMP dehydrogenase (EC1.1.1.205), was obtained from Swiss-prot Database. By using the full length of this amino acid sequence as a query, a homology search was carried out on a nucleotide sequence database of the genome sequence of Corynebacterium glutamicum or a database of the ORF amino acid sequences predicted from the genome sequence using FASTA program. A case where E-value was le−10 or less was judged as being significantly homologous. As a result, the amino acid sequences encoded by two ORFs, namely, an ORF positioned in the region of the nucleotide sequence No. 615336 to 616853 (or ORF having the nucleotide sequence represented by SEQ ID NO:672) and another ORF positioned in the region of the nucleotide sequence No. 616973 to 618094 (or ORF having the nucleotide sequence represented by SEQ ID NO:674) were significantly homologous with the ORFs of Escherichia coli IMP dehydrogenase. By using the above-described predicted amino acid sequence as a query in order to examine the similarity of the amino acid sequences encoded by the ORFs with IMP dehydrogenases of other organisms in greater detail, a search was carried out on GenBank (http://www.ncbi.nlm.nih.gov/) nr-aa database (amino acid sequence database constructed on the basis of GenBankCDS translation products, PDB database, Swiss-Prot database, PIR database, PRF database by eliminating duplicated registrations) using BLAST program. As a result, both of the two amino acid sequences showed significant homologies with IMP dehdyrogenases of other organisms and clearly higher homologies with IMP dehdyrogenases than with amino acid sequences of other proteins, and thus, it was assumed that the two ORFs would function as IMP dehydrogenase. Based on these results, it was therefore assumed that Corynebacterium glutamicum has two ORFs having the IMP dehydrogenase activity.
Proteome Analysis of Proteins Derived from Corynebacterium glutamicum
(1) Preparations of Proteins Derived from Corynebacterium glutamicum ATCC 13032, FERM BP-7134 and FERM BP-158
Culturing tests of Corynebacterium glutamicum ATCC 13032 (wild type strain), Corynebacterium glutamicum FERM BP-7134 (lysine-producing strain) and Corynebacterium glutamicum (FERM BP-158, lysine-highly producing strain) were carried out in a 5 l jar fermenter according to the method in Example 2(3). The results are shown in Table 6.
After culturing, cells of each strain were recovered by centrifugation. These cells were washed with Tris-HCl buffer (10 mmol/l Tris-HCl, pH 6.5, 1.6 mg/ml protease inhibitor (COMPLETE; manufactured by Boehringer Mannheim)) three times to give washed cells which could be stored under freezing at −80° C. The freeze-stored cells were thawed before use, and used as washed cells.
The washed cells described above were suspended in a disruption buffer (10 mmol/l Tris-HCl, pH 7.4, 5 mmol/l magnesium chloride, 50 mg/l RNase, 1.6 mg/ml protease inhibitor (COMPLETE: manufactured by Boehringer Mannheim)), and disrupted with a disrupter (manufactured by Brown) under cooling. To the resulting disruption solution, DNase was added to give a concentration of 50 mg/l, and allowed to stand on ice for 10 minutes. The solution was centrifuged (5,000×g, 15 minutes, 4° C.) to remove the undisrupted cells as the precipitate, and the supernatant was recovered.
To the supernatant, urea was added to give a concentration of 9 mol/l, and an equivalent amount of a lysis buffer (9.5 mol/l urea, 2% NP-40, 2% Ampholine, 5% mercaptoethanol, 1.6 mg/ml protease inhibitor (COMPLETE; manufactured by Boehringer Mannheim) was added thereto, followed by thoroughly stirring at room temperature for dissolving.
After being dissolved, the solution was centrifuged at 12,000×g for 15 minutes, and the supernatant was recovered.
To the supernatant, ammonium sulfate was added to the extent of 80% saturation, followed by thoroughly stirring for dissolving.
After being dissolved, the solution was centrifuged (16,000×g, 20 minutes, 4° C.), and the precipitate was recovered. This precipitate was dissolved in the lysis buffer again and used in the subsequent procedures as a protein sample. The protein concentration of this sample was determined by the method for quantifying protein of Bradford.
(2) Separation of Protein by Two Dimensional Electrophoresis
The first dimensional electrophoresis was carried out as described below by the isoelectric electrophoresis method.
A molded dry IPG strip gel (pH 4-7, 13 cm, Immobiline DryStrips; manufactured by Amersham Pharmacia Biotech) was set in an electrophoretic apparatus (Multiphor II or IPGphor; manufactured by Amersham Pharmacia Biotech) and a swelling solution (8 mol/l urea, 0.5% Triton X-100, 0.69 dithiothreitol, 0:5% Ampholine, pH 3-10) was packed therein, and the gel was allowed to stand for swelling 12 to 16 hours.
The protein sample prepared above was dissolved in a sample solution (9 mol/l urea, 2% CRAPS, 1% dithiothreitol, 2% Ampholine, pH 3-10), and then about 100 to 500 μg (in terms of protein) portions thereof were taken and added to the swollen IPG strip gel.
The electrophoresis was carried out in the 4 steps as defined below under controlling the temperature to 20° C.:
After the isoelectric electrophoresis, the IPG strip gel was put off from the holder and soaked in an equilibration buffer A (50 mmol/l Tris-HCl, pH 6.8, 30% glycerol, 1% SDS, 0.25% dithiothreitol) for 15 minutes and another equilibration buffer B (50 mmol/l Tris-HCl, pH 6.8, 6 mol/l urea, 30% glycerol, 1% SDS, 0.45% iodo acetamide) for 15 minutes to sufficiently equilibrate the gel.
After the equilibrium, the IPG strip gel was lightly rinsed in an SDS electrophoresis buffer (1.4% glycine, 0.1% SDS, 0.3% Tris-HCl, pH 8.5), and the second dimensional electrophoresis depending on molecular weight was carried out as described below to separate the proteins.
Specifically, the above IPG strip gel was closely placed on 14% polyacrylamide slub gel (14% polyacrylamide, 0.37% bisacrylamide, 37.5 mmol/l Tris-HCl, pH 8.8, 0.1% SDS, 0.1% TEMED, 0.1% ammonium persulfate) and subjected to electrophoresis under a constant voltage of 30 mA at 20° C. for 3 hours to separate the proteins.
(3) Detection of Protein Spot
Coomassie staining was performed by the method of Gorg et al. (Electrophoresis, 9: 531-546 (1988)) for the slub gel after the second dimensional electrophoresis. Specifically, the slub gel was stained under shaking at 25° C. for about 3 hours, the excessive coloration was removed with a decoloring solution, and the gel was thoroughly washed with distilled water.
The results are shown in
(4) In-Gel Digestion of Detected Protein Spot
The detected spots were each cut out from the gel and transferred into siliconized tube, and 400 μl of 100 mmol/l ammonium bicarbonate : acetonitrile solution (1:1, v/v) was added thereto, followed by shaking overnight and freeze-dried as such. To the dried gel, 10 μl of a lysylendopeptidase (LysC) solution (manufactured by WAKO, prepared with 0.1% SDS-containing 50 mmol/l ammonium bicarbonate to give a concentration of 100 ng/μl) was added and the gel was allowed to stand for swelling at 0° C. for 45 minutes, and then allowed to stand at 37° C. for 16 hours. After removing the LysC solution, 20 μl of an extracting solution (a mixture of 60% acetonitrile and 5% formic acid) was added, followed by ultrasonication at room temperature for 5 minutes to disrupt the gel. After the disruption, the extract was recovered by centrifugation (12,000 rpm, 5 minutes, room temperature). This operation was repeated twice to recover the whole extract. The recovered extract was concentrated by centrifugation in vacuo to halve the liquid volume. To the concentrate, 20 μl of 0.1% trifluoroacetic acid was added, followed by thoroughly stirring, and the mixture was subjected to desalting using ZipTip (manufactured by Millipore). The protein absorbed on the carriers of ZipTip was eluted with 5 μl of α-cyano-4-hydroxycinnamic acid for use as a sample solution for analysis.
(5) Mass Spectrometry and Amino Acid Sequence Analysis of Protein Spot with Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometer (MALDI-TOFMS)
The sample solution for analysis was mixed in the equivalent amount with a solution of a peptide mixture for mass calibration (300 nmol/l Angiotensin II, 300 nmol/l Neurotensin, 150 nmol/l ACTHclip 18-39, 2.3 μmol/l bovine insulin B chain), and 1 μl of the obtained solution was spotted on a stainless probe and crystallized by spontaneously drying.
As measurement instruments, REFLEX MALDI-TOF mass spectrometer (manufactured by Bruker) and an N2 laser (337 nm) were used in combination.
The analysis by PMF (peptide-mass finger printing) was carried out using integration spectra data obtained by measuring 30 times at an accelerated voltage of 19.0 kV and a detector voltage of 1.50 kV under reflector mode conditions. Mass calibration was carried out by the internal standard method.
The PSD (post-source decay) analysis was carried out using integration spectra obtained by successively altering the reflection voltage and the detector voltage at an accelerated voltage of 27.5 kV.
The masses and amino acid sequences of the peptide fragments derived from the protein spot after digestion were thus determined.
(6) Identification of Protein Spot
From the amino acid sequence information of the digested peptide fragments derived from the protein spot obtained in the above (5), ORFs corresponding to the protein were searched on the genome sequence database of Corynebacterium glutamicum ATCC 13032 as constructed in Example 1 to identify the protein.
The identification of the protein was carried out using MS-Fit program and MS-Tag program of intranet protein prospector.
(a) Search and Identification of Gene Encoding High-Expression Protein
In the proteins derived from Corynebacterium glutamic ATCC 13032 showing high expression amounts in CBB-staining shown in
As a result, it was found that Spot-1 corresponded to enolase which was a protein having the amino acid sequence of SEQ ID NO:4585; Spot-2 corresponded to phosphoglycelate kinase which was a protein having the amino acid sequence of SEQ ID NO:5254; Spot-3 corresponded to glyceraldehyde-3-phosphate dehydrogenase which was a protein having the amino acid sequence represented by SEQ ID NO:5255; Spot-4 corresponded to fructose bis-phosphate aldolase which was a protein having the amino acid sequence represented by SEQ ID NO:6543; and Spot-5 corresponded to triose phosphate isomerase which was a protein having the amino acid sequence represented by SEQ ID NO:5252.
These genes, represented by SEQ ID NOS:1085, 1754, 1775, 3043 and 1752 encoding the proteins corresponding to Spots-1, 2, 3, 4 and 5, respectively, encoding the known proteins are important in the central metabolic pathway for maintaining the life of the microorganism. Particularly, it is suggested that the genes of Spots-2, 3 and 5 form an operon and a high-expression promoter is encoded in the upstream thereof (J. of Bacteriol., 174: 6067-6086 (1992)).
Also, the protein corresponding to Spot-9 in
Based on these results, the proteins having high expression level were identified by proteome analysis using the genome sequence database of Corynebacterium glutamicum constructed in Example 1. Thus, the nucleotide sequences of the genes encoding the proteins and the nucleotide sequences upstream thereof could be searched simultaneously. Accordingly, it is shown that nucleotide sequences having a function as a high-expression promoter can be efficiently selected.
(b) Search and Identification of Modified Protein
Among the proteins derived from Corynebacterium glutamicum FERM BP-7134 shown in
Accordingly, all of Spots-6, 7 and 8 detected as spots differing in isoelectric mobility were all products derived from a catalase gene having the nucleotide sequence represented by SEQ ID NO:285. Accordingly, it is shown that the catalase derived from Corynebacterium glutamicum FERM BP-7134 was modified after the translation.
Based on these results, it is confirmed that various modified proteins can be efficiently searched by proteome analysis using the genome sequence database of Corynebacterium glutamicum constructed in Example 1.
(c) Search and Identification of Expressed Protein Effective in Lysine Production
It was found out that in
Based on these results, it was found that hopeful mutated proteins can be efficiently searched and identified in breeding aiming at strengthening the productivity of a target product by the proteome analysis using the genome sequence database of Corynebacterium glutamicum constructed in Example 1.
Moreover, useful mutation points of useful mutants can be easily specified by searching the nucleotide sequences (nucleotide sequences of promoter, ORF, or the like) relating to the identified proteins, using the above database and using primers designed on the basis of the sequences. As a result of the fact that the mutation points are specified, industrially useful mutants which have the useful mutations or other useful mutations derived therefrom can be easily bred.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. All references cited herein are incorporated in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| P. 11-377484 | Dec 1999 | JP | national |
| P. 2000-159162 | Apr 2000 | JP | national |
| P. 2000-280988 | Aug 2000 | JP | national |
The present application is a divisional of application Ser. No. 09/738,626, filed Monday, Dec. 18, 2000 now abandoned, the entire contents of which is hereby incorporated by reference.
| Number | Date | Country |
|---|---|---|
| 0358940 | Mar 1990 | EP |
| 0387527 | Sep 1990 | EP |
| 0555 661 | Aug 1993 | EP |
| 0974647 | Jan 2000 | EP |
| WO 8809819 | Dec 1988 | WO |
| WO 9309225 | May 1993 | WO |
| WO 0100843 | Jan 2001 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060228712 A1 | Oct 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09738626 | Dec 2000 | US |
| Child | 10805394 | US |
