WO2000037647A1 - Transporteur abc et gene le codant - Google Patents

Transporteur abc et gene le codant Download PDF

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Publication number
WO2000037647A1
WO2000037647A1 PCT/JP1999/007079 JP9907079W WO0037647A1 WO 2000037647 A1 WO2000037647 A1 WO 2000037647A1 JP 9907079 W JP9907079 W JP 9907079W WO 0037647 A1 WO0037647 A1 WO 0037647A1
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WO
WIPO (PCT)
Prior art keywords
sequence
amino acid
seq
protein
dna
Prior art date
Application number
PCT/JP1999/007079
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English (en)
Japanese (ja)
Inventor
Sohei Kanno
Eiichiro Kimura
Kazuhiko Matsui
Tsuyoshi Nakamatsu
Original Assignee
Ajinomoto Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ajinomoto Co., Inc. filed Critical Ajinomoto Co., Inc.
Priority to AU16878/00A priority Critical patent/AU1687800A/en
Publication of WO2000037647A1 publication Critical patent/WO2000037647A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/345—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Brevibacterium (G)

Definitions

  • the present invention relates to a novel ABC transporter and a gene encoding a protein which is a component thereof.
  • the gene can be used for breeding microorganisms having altered cell membrane transport of amino acids.
  • One of them is the ATP binding cassette, a superfamily one (ABC transporter). It is known (CF Higgins, Ann. Rev. Cell Biol., 8, 67 (1992)).
  • the ATP-binding cassette is a group of proteins having an ATP-binding domain including a transmembrane domain, and its physiological function is mainly the uptake of substances into cells, but it is also involved to some extent in the excretion of substances.
  • Is believed to be Bacteria often contain membrane proteins (membrane components), proteins that are inside the membrane and have ATPase activity, and binding proteins that are outside the membrane and bind to transported substances.
  • the membrane protein and the protein having ATase activity form a multimeric complex. It is said that the substance emission system lacks a binding protein that binds to the substance to be transported (Reizer, J. et al., Prot. Sci. 1, 1326 (1992)).
  • the present invention relates to proteins that are components of the ABC transporter, and DNAs that encode them.
  • the first component of the ABC transporter of the present invention is a protein represented by the following (A) or (B).
  • the amino acid sequence comprises one or several amino acid substitutions, deletions, insertions, additions, or inversions, and comprises an ABC transporter.
  • the second component of the ABC transporter of the present invention is a protein represented by the following (C) or (D).
  • the third component of the ABC transporter of the present invention is a protein represented by the following (E) or (F).
  • the present invention also provides a DNA encoding a protein that is a component of each of the above ABC transporters.
  • the invention further provides an operon encoding the ABC transporter.
  • the present invention will be described in detail.
  • the DNA of the present invention has been found as an ORF existing in the vicinity of the g1tBD gene from Brevibacterium and Lactophamentum, and can be obtained as follows.
  • Brevibacterium lactofermentum such as Brevibacterium lactobacillus ATCC13869 chromosomal DNA
  • Escherichia coli K — 12 Gene, Vol. 60, 1: L: 1 1987
  • yeast Sacharomyces' Celepiche, GenBank accession No. X89221.
  • a DNA fragment of about 1.4 kb is obtained by PCR (polymerase chain reaction) using a primer having the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.
  • Pre-Bacterium 'Lacto Amentum ATCC13869 is available from the ATCC (American Type Culture Collection: 20852, United States, Maryland 20852, Rockville, Park Live 10301) be able to.
  • the PCR-amplified fragment obtained as described above was used as a probe, and a colony hybridization of the chromosomal DNA library of Brevibacterium 'lactofermentum ATCC13869 was performed.
  • the DNA fragment to be hybridized to the probe was subjected to colony hybridization.
  • the DNA of the present invention can be obtained together with the gitBD gene.
  • the above-mentioned DNA fragment can be obtained as a fragment having a size of about 14 kb.
  • the above DNA fragment contains the g1tBD gene, downstream of which g There are two open reading frames (ORFs) opposite to the 1 t BD gene. These ORFs correspond to the second and third ORFs in the base sequence shown in SEQ ID NO: 7.
  • the two ORFs form an operon together with another ORF existing upstream of these two ORFs.
  • This ORF corresponds to the first ORF in ⁇ RF contained in the nucleotide sequence shown in SEQ ID NO: 7.
  • the first ORF has a chromosomal DNA of Brevibacterium lactofamentum, for example, Brevibacterium lactofamentum ATCC13869, and has a base sequence having the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing and the sequence in the sequence listing. It can be obtained as a DNA fragment of about 1.8 kb by PCR using a primer having the nucleotide sequence shown in No. 6. This DNA fragment has a region presumed to be a promoter region upstream of the target ORF.
  • the nucleotide sequence shown in SEQ ID NO: 7 links the above-described nucleotide sequence (1.3 kb) in the approximately 14 kb DNA fragment with the nucleotide sequence (1.1 kb) in the approximately 1.8 kb DNA fragment. It was done.
  • nucleotide sequence and the nucleotide sequence of the adjacent region of each ORF have been clarified, it can also be obtained by PCR using oligonucleotides prepared based on those nucleotide sequences as primers. .
  • the second ORF and the amino acid sequence encoded thereby were compared with a known sequence for homology.
  • the databases used were EMBL and SWISS-PR0T. As a result, these sequences were found to have homology to the previously reported ATPase protein constituting the ABC transporter responsible for amino acid transport and the gene encoding the same, as shown in Table 1.
  • the three ORFs including this are operons May form, Table 1
  • genes encoding the components of the ABC transporter of the present invention may be substituted or deleted by one or several amino acids at one or more positions, as long as the properties of each encoded protein are not impaired.
  • ATP binding tan with insertion, addition or inversion It may be one that encodes protein.
  • "several" differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. It is derived from the fact that some amino acids, such as isocyanate isine and palin, have highly related amino acids, and such amino acid differences do not significantly affect the three-dimensional structure of the protein.
  • DNA encoding a protein substantially the same as the components of the ABC transporter as described above can be obtained by substituting, deleting, inserting, adding, or substituting an amino acid residue at a specific site by, for example, site-directed mutagenesis. Alternatively, it can be obtained by modifying the nucleotide sequence to include an inversion. The modified DNA as described above can also be obtained by conventionally known mutation treatment.
  • Examples of the mutation treatment include a method in which DNA encoding each protein is treated in vitro with hydroxylamine, etc., and a method in which a microorganism having a DNA encoding each protein, such as a bacterium belonging to the genus Escherichia, is irradiated with ultraviolet light or Examples of the method include treatment with a mutagen that is commonly used for mutagenesis, such as N, -nitro-N-nitrosoguanidine (NTG) or nitrous acid.
  • NTG -nitro-N-nitrosoguanidine
  • substitutions, deletions, insertions, additions, or inversions of bases as described above include mutations that occur naturally, such as those based on individual differences of microorganisms carrying each component and differences in species and genera. mutant or variant) is also included.
  • a DNA encoding a protein substantially identical to the components of the ABC transporter can be obtained.
  • stringent conditions refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Say. Although it is difficult to quantify these conditions clearly, one example is that DNAs with high homology, for example, DNAs with homology of 40% or more, hybridize and have lower homology 60 ° C, 1 XSSC, 0.1% SDS, preferably 0.1 lxSSC, 0.1% SDS, which is the condition under which DNA does not hybridize with each other, or the conditions for washing ordinary Southern hybridizations. Conditions for hybridization at the corresponding salt concentration are mentioned.
  • genes that hybridize under these conditions include those with a stop codon in the middle and those that have lost their activity due to mutations in the active center, but these are expressed using a commercially available activity expression vector. It can be easily removed by characterizing the product.
  • the DNA encoding the components of the ABC transporter of the present invention and the operon of the ABC transpouse are used for breeding coryneform bacteria. Can be used for That is, since the ABC transport protein of the present invention or its component is considered to be involved in amino acid transport, the expression of these genes is modified to modify the properties of cells for amino acid transport. It is thought that it can be done.
  • Coryneform bacteria to which the present invention can be applied include bacteria that were previously classified into the genus Brevipacterium, but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 1, 255 ( 1981)), and also includes bacteria of the genus Brevibacterium closely related to the genus Corynebacterium. Examples of such coryneform bacteria include the following.
  • Corynebacterium lilium Corynebacterium .gulpus micam
  • Corynebacterium Jumiumium (Corynebacterium glutamicum) AT C C 15990
  • Previbacterium divaricatum (Corynebacterium glutamicum) AT CC 14020
  • Brevipacterium 'Flavum (Corynebacterium' glutamicum) AT CC 13826, ATCC 14067 Brevipacterium immariophyllum ATCC 14068 Brevipacterium lactofermentum (corynebacterium glutamicum): AT CC 13665, AT CC 13869,
  • Methods for modifying the genes encoding the ABC transporter or its components include amplification or disruption of these genes.
  • Amplification of a gene or the like can be performed by transforming a coryneform bacterium with a recombinant vector obtained by ligating a gene to a vector such as a plasmid. At that time, the efficiency of amplification can be increased by using a multi-copy type vector. Examples of such vectors include plasmids that can autonomously replicate in coryneform bacteria, such as the following.
  • Transformation of a coryneform bacterium can be performed by an electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791).
  • Gene amplification can also be achieved by allowing the gene of the present invention to exist in multiple copies on the chromosomal DNA of the host.
  • Target on chromosome DNA of coryneform bacteria In order to introduce a gene in multiple copies, homologous recombination is performed by utilizing a sequence present in multiple copies on chromosomal DNA (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory press (1972); Matsuyama, S and Mizushima, S., J. BacterioL, 162, 1196 (1985)).
  • a sequence present in multiple copies on chromosomal DNA it is possible to use reactive DNA and inverted repeats present at the end of a transposable element.
  • a gene of interest can be mounted on a transposon, transferred and transfected into chromosomal DNA in multiple copies.
  • the expression of a gene that is originally present on a chromosome can also be modified by replacing an expression control sequence such as a promoter with a strong or weak function.
  • chromosomal DNA of Brevipacterium 'lactofermentum AT CC 13869 was prepared using the Bacterial Genomic DNA Purification Kit (manufactured by Advanced Genetic Technologies Corp.). Using this chromosomal DNA as type I, PCR was performed under standard reaction conditions described on page 8 of PCR Technology (Henry and Erich, Stockton Press, 1989) using the oligonucleotide as a primer. Was done. Agarose gel electrophoresis of the PCR product revealed that a DNA fragment of approximately 1.4 kb had been amplified. Was.
  • the nucleotide sequences at both ends of the obtained DNA were determined using the oligonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • the agarose gel electrophoresis of the Hindi II fragment of brevipacterium 'lactofermentum ATCC 13869 chromosomal DNA prepared by a standard method was performed, and a DNA fragment of about 10 kilobases or more was recovered using glass powder, and the recovered DNA fragment was recovered.
  • PMW219 (manufactured by Takara Shuzo) digested with restriction enzyme Hindlll (manufactured by Takara Shuzo) was ligated using a ligation kit (manufactured by Takara Shuzo) to obtain Escherichia coli KM.
  • the obtained transformant was prepared by the alkaline method (Biotechnology Experiment Book, edited by the Society of Biotechnology, Japan, 1
  • SEQ ID No. 3 and SEQ ID No. 3 were prepared based on the portion of the DNA sequence used as the probe whose base sequence was determined. PCR was performed under the above conditions using the above-mentioned plasmid as a primer and a synthetic oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 4 as a primer. Using this primer, Brevipacterium, Lactofamentum ATCC 13869 A transformant having a plasmid capable of obtaining an amplified fragment having a size of about 1.3 kilobases, which is the same as the DNA fragment amplified when PCR was performed using the chromosome as type I, was selected. (2) Determination of the entire nucleotide sequence of the DNA fragment containing the Brevibacterium lactofermentum ATCC 13869 1 tBD gene and isolation of the ABC transporter overnight gene
  • Plasmid DNA prepared from the transformant obtained in the above (1) by an alkaline method contained a DNA fragment of about 14 kilobases derived from the chromosome of Brevibacterium lactofermentum ATCC 13869.
  • the entire nucleotide sequence of a DNA fragment of about 14 kilobases derived from the chromosome of Brevipacterium lactofermentum ATCC 13869 of the obtained plasmid was determined.
  • the obtained DNA fragment contained the entire length of the g1 tBD gene, but an open reading frame of 500 bp or more was directed downstream from the end of the g1 tBD gene. It was found that there was a sequence that was presumed to be one minute and one minute downstream of these open reading frames. However, since the open reading frame lacked the promoter region, the upstream portion was cloned as follows.
  • these three open reading 'frames could be operons.
  • the nucleotide sequence of these open reading frames is as shown in SEQ ID NO: 7 in the sequence listing.
  • the sequence of SEQ ID NO: 7 also shows the amino acid sequence of the product deduced from the nucleotide sequence.
  • base numbers 1 to 1101 are the first open reading frame
  • 1117 to 1725 are the second open reading frame
  • 1759 to 2367 are the third open reading frame.
  • the methionine residue at the N-terminus of the protein encoded by each open 'reading' frame is derived from the initiation codon ATG, and is often unrelated to the original function of the protein.
  • the homology was compared with the known sequence for each of the nucleotide sequence and the amino acid sequence.
  • the data bases used were EMBL and SWISS-PR0T.
  • the DNA shown in SEQ ID No. in the sequence listing and each protein encoded therewith were novel genes and proteins in the genus Corynebacterium.
  • the second open reading frame and the protein encoded by it have high homology to the previously reported ATP-binding protein of ABC transporter and the gene encoding it, and coryneforms.
  • Pacterium bacteria it was found to be a gene encoding a novel ATP binding protein.
  • INDUSTRIAL APPLICABILITY The present invention provides the components of the ABC transporter of Brevipacterium lactofarmentum and the DNA encoding them.
  • the gene of the present invention can be used for breeding coryneform bacteria.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur des constituants d'un transporteur ABC de Brevibacterium lactofermentum qui possède les séquences d'acides aminés représentée par les NOs ID SEQ 8, 9 et 10 et sur des ADN le codant. Ces ADN peuvent être utilisés dans la sélection de corynébactérium.
PCT/JP1999/007079 1998-12-18 1999-12-16 Transporteur abc et gene le codant WO2000037647A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU16878/00A AU1687800A (en) 1998-12-18 1999-12-16 Abc transporter and genes encoding the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP10/360621 1998-12-18
JP36062198A JP2002293797A (ja) 1998-12-18 1998-12-18 Ａｂｃトランスポーター及びそれをコードする遺伝子

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WO2000037647A1 true WO2000037647A1 (fr) 2000-06-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7335496B2 (en) 2003-06-05 2008-02-26 Ajinomoto Co., Inc. Method for producing target substance
WO2008133161A1 (fr) 2007-04-17 2008-11-06 Ajinomoto Co., Inc. Procédé de fabrication d'une substance acide ayant un groupe carboxyle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIMMELREICH ET AL.: "Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae", NUCLEIC ACIDS RESEARCH, vol. 24, no. 22, 1996, pages 4420 - 4449, XP002924582 *
NORBERT PEEKHAUS ET AL.: "The gluEMP operon from Zymomonas mobilis encodes a high-affinity glutamate carrier with similarity to binding-protein-dependent transport systems", ARCHIVES OF MICROBIOLOGY, vol. 165, no. 5, 1996, pages 325 - 332, XP002924583 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7335496B2 (en) 2003-06-05 2008-02-26 Ajinomoto Co., Inc. Method for producing target substance
WO2008133161A1 (fr) 2007-04-17 2008-11-06 Ajinomoto Co., Inc. Procédé de fabrication d'une substance acide ayant un groupe carboxyle
US9822385B2 (en) 2007-04-17 2017-11-21 Ajinomoto Co., Inc. Method for producing an L-glutamic acid and L-aspartic acid using a recombinant microorganism having enhanced expression of a ybjL protein

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JP2002293797A (ja) 2002-10-09
AU1687800A (en) 2000-07-12

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