JP2004321169A - Microorganism strain improved in growth - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism strain improved in growth, and to provide a method for producing a useful substance using the microorganism. <P>SOLUTION: This microorganism has such a chromosome DNA as a part or the whole of a DNA homologous to a DNA comprising a specific base sequence derived form Escherichia coil to an extent of ≥ 80% is deleted, and is improved in the growth, when compared with that of a parent strain having such a chromosome DNA as any part of the DNA is not deleted. Further, the microorganism has such a DNA on a chromosome DNA that the DNA encodes a protein comprising an amino acid sequence in which one or more amino acids are subjected to deletion, substitution or addition in an amino acid sequence homologous to a specific amino acid sequence to an extent of ≥ 60%, and is improved in the growth, when compared with that of a parent strain having such a DNA on the chromosome DNA that the DNA encodes a protein comprising an amino acid sequence in which any amino acid is not subjected to the deletion, nor the substitution, nor the addition. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a microorganism having improved growth, and a method for producing a useful substance using the microorganism.

  Wild-type strains of microorganisms grow well on various media. A variety of mutant strains have been produced so far, but in general, these mutant strains can obtain a smaller amount of cells in a given culture time, have a slower growth rate, and can grow at a higher temperature than wild-type strains. In many cases, the growth characteristics are poor, such as a narrow range, nutrient requirement, and low resistance to various stresses. This is because common mutation treatment introduces multiple mutations on the chromosome, and it is highly possible that harmful mutations are introduced into any of the many genes that govern the complex process of growth. This is probably because it is difficult to obtain a well-growing strain from a mutant library prepared by general mutation treatment.

Therefore, a mutant strain of a microorganism whose growth has been improved due to a mutation in a specific gene is not known.
On the other hand, in many microorganisms, the entire base sequence of the chromosomal DNA has been clarified (Non-Patent Document 1). In addition, a method for producing a microorganism in which a specific gene or a chromosomal DNA region is deleted as intended using a homologous recombination technique is known (Non-Patent Document 2). A library in which each gene on the chromosomal DNA of a microorganism is comprehensively destroyed using the above-described information on the entire nucleotide sequence of the chromosomal DNA and the homologous recombination technique, or a chromosomal DNA region of about 20 kbp which can be deleted is covered. Mutant strain libraries and the like have been prepared (Non Patent Literatures 3 and 4).

The E. coli yhdA gene is known to be located at 73.27 minutes on the chromosomal DNA and encodes a protein consisting of 646 amino acids, but the function of the protein is not known (Non-Patent Document 5). ).
http://www.tigr.org/tdb/mdb/mdbcomplete.html J. Bacteriol. (Journal of Bacteriology), 180, 2063 (1998) Protein Nucleic Acid Enzyme, 46, 2386, 2001 Nature Biotechnol., 20, 1018 (2002) Science, 277, 1453 (1997)

  An object of the present invention is to provide a microorganism strain with improved growth, and a method for producing a useful substance using the microorganism.

The present invention relates to the following (1) to (7).
(1) A microorganism having a chromosomal DNA in which part or all of the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 has been deleted, and having improved growth compared to a parent strain having a chromosomal DNA having no deletion.
(2) From a parent strain having a chromosomal DNA in which a part or all of DNA having homology of 80% or more with the DNA consisting of the base sequence represented by SEQ ID NO: 1 is deficient, and having a chromosomal DNA having no deficiency. Microorganisms with improved growth.

(3) a DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 on a chromosomal DNA; A microorganism having improved growth over a parent strain having, on a chromosomal DNA, a DNA encoding a protein having the amino acid sequence to be obtained.
(4) The microorganism according to (1) to (3), wherein the microorganism is a microorganism belonging to the genus Escherichia .

(5) a microorganism belonging to the genus Escherichia is a microorganism of the above (4) which is Escherichia coli.
(6) The microorganism according to any one of (1) to (5) is cultured in a medium, a useful substance is produced and accumulated in the culture, and the useful substance is collected from the culture. Method for producing useful substances.

(7) The method according to (6) above, wherein the useful substance is a useful substance selected from the group consisting of proteins, amino acids, nucleic acids, vitamins, sugars, organic acids, and lipids.
Hereinafter, the present invention will be described in detail.

  According to the present invention, it is possible to provide a microorganism having improved growth and a method for producing a useful substance using the microorganism.

The microorganism of the present invention has a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, or a homology of 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with the DNA. Any microorganism may be used as long as it has a chromosomal DNA in which part or all of the DNA has been deleted, and has improved growth from a parent strain having the chromosomal DNA without the deletion. , for example, it can be mentioned, such as a microorganism belonging to the genus Escherichia, as a microorganism belonging to the genus Escherichia, and the like Escherichia coli.

Examples of the Escherichia coli, can be mentioned Escherichia coli having a chromosomal DNA which some or all of the DNA deficient represented by SEQ ID NO: 1.
The DNA having a homology of 80% or more with the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 refers to a homologous gene of the gene having the nucleotide sequence represented by SEQ ID NO: 1, It can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like using all or a part of the DNA having the base sequence represented by No. 1 as a probe.

  Specifically, after performing hybridization at 65 ° C. in the presence of 1.0 mol / l sodium chloride using a filter on which DNA derived from colonies or plaques has been immobilized, a 0.1 to 2-fold concentration of SSC solution (1 A double-concentration SSC solution has a composition of 150 mmol / l sodium chloride and 15 mmol / l sodium citrate), and the filter can be identified by washing the filter at 65 ° C. Hybridization is performed according to the method described in Molecular Cloning, Third Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995). It can be performed according to it.

Further, by using the base sequence represented by SEQ ID NO: 1 as a query and searching a chromosomal DNA of various microorganisms or a database of a gene library, the DNA consisting of the base sequence represented by SEQ ID NO: 1 DNA having homology can be specified.
The homology between the amino acid sequence and the base sequence can be determined by, for example, the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (1993)] by Karlin and Altschul and FASTA [Methods Enzymol., 183 , 63 (1990)]. Can be determined using Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215 , 403 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, parameters are, for example, Score = 100 and wordlength = 12. When an amino acid sequence is analyzed by BLASTX based on BLAST, parameters are set to, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

  DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or DNA having a part or all of the DNA having 80% or more homology with the DNA is DNA having the nucleotide sequence represented by SEQ ID NO: 1 Or a DNA having 80% or more homology with the DNA, a mutant DNA having a base sequence in which one or more bases have been deleted, substituted or added, and a microorganism having a chromosomal DNA having the mutant DNA. A DNA which is a microorganism having improved growth over a parent strain having a chromosomal DNA not having the mutant DNA.

  Specifically, in the DNA having the nucleotide sequence represented by SEQ ID NO: 1 or the DNA having the homology of 80% or more with the DNA having the nucleotide sequence represented by SEQ ID NO: 1, any one or more bases, Preferably, DNAs deficient in any 10 to 1500 bases, more preferably 100 to 1000 bases, and still more preferably 200 to 500 bases can be mentioned. preferable.

Further, by introducing a site-specific mutation into a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a DNA having 80% or more homology with the DNA, one or more bases are deleted in the DNA. A DNA having a substituted or added base sequence, wherein the amino acid sequence encoded by the mutant DNA is different from the amino acid sequence encoded by the DNA before site-directed mutagenesis. I can give it. Introduction of site-specific mutations is described in Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular Cloning Third Edition), Current Protocols in Molecular Biology, John Wiley & Sons ( 1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research, 10 , 6487 (1982), Proc. Natl. Acad. Sci. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Acad. Sci. USA, 82 , 488 (1985), and the like.

The number of bases to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a well-known method such as the above-described site-directed mutagenesis, and is one to several tens. The number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
The microorganism having improved growth from the parent strain having the chromosomal DNA not having the mutant DNA is a microorganism in which a defect has been introduced and a microorganism which is a parent strain in which the defect has not been introduced, cultured in the same medium for the same time. When performed, it refers to a microorganism having more cells than the parent strain. The culturing time may be any time as long as the time can accurately compare the number of cells of the microorganism having the chromosomal DNA deficient in the DNA and the parent strain of the microorganism, but is preferably in the latter half of the logarithmic growth phase, more preferably The time to reach the stationary phase can be increased. The above-mentioned parent strain refers to the original strain subjected to the above-mentioned deletion, and even if the parent strain is a wild-type microorganism, an industrially useful mutant, cell fusion strain, transduced strain or gene It may be a recombinant strain created using recombinant technology.

Further, the microorganism of the present invention has, on a chromosomal DNA, a DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and Any microorganism may be used as long as it is a microorganism having improved growth over a parent strain having a DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 on chromosomal DNA, and the microorganism may be, for example, a genus Escherichia . microorganisms, such as it is possible to increase the belonging to, as a microorganism belonging to the genus Escherichia, and the like Escherichia coli.

As the Escherichia coli, it can be mentioned Escherichia coli having a chromosomal DNA which some or all of the DNA encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 is missing.
A microorganism having on the chromosomal DNA a DNA encoding the amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 encodes the amino acid sequence represented by SEQ ID NO: 2 The DNA can be obtained by introducing a site-specific mutation into the DNA. The introduction of site-specific mutations is described in Molecular Cloning, Third Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10 , 6487 (1982), Proc. Natl. Acad. Sci. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Acad. Sci. USA, 82 , 488 (1985), etc. be able to.

The number of amino acids to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a well-known method such as the above-described site-directed mutagenesis, and is 1 to several tens, The number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
In addition, the deletion, substitution, or addition of one or more amino acids means that there is a deletion, substitution, or addition of one or more amino acid residues at an arbitrary position in the same sequence. Substitution or addition may occur at the same time, and a microorganism having a DNA encoding a protein having a deleted, substituted or added amino acid sequence on chromosomal DNA may be a protein having the amino acid sequence before deletion, substitution or addition. Any amino acid may be deleted, substituted or added as long as the growth of the microorganism is higher than that of the parent strain of the microorganism having the encoding DNA on the chromosomal DNA. Regardless of natural type. As natural amino acid residues, L-alanine, L-asparagine, L-aspartic 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.

  A microorganism having improved growth from a parent strain having a DNA encoding a protein consisting of an amino acid sequence having no deletion, substitution or addition of the above amino acids on chromosomal DNA is defined as having a deletion, substitution or addition of one or more amino acids. When the obtained microorganism and a parent strain of the microorganism are cultured in the same medium for the same period of time, this refers to a microorganism having more cells than the parent strain. The culturing time may be any time as long as it can accurately compare the log of the microorganism into which the deletion, substitution or addition of one or more amino acids has been introduced and the parent strain of the microorganism, but is preferably a logarithmic growth phase. The time to reach the latter half, more preferably the stationary phase, can be increased. The parent strain refers to the original strain subjected to the deletion, substitution, or addition of the amino acid. Even if the parent strain is a wild-type microorganism, an industrially useful mutant strain, cell fusion The strain may be a strain, a transduced strain, or a recombinant strain created using a gene recombination technique.

As a method for producing the microorganism of the present invention, 1) a gene-deficient strain library in which each gene on the chromosomal DNA of the microorganism is exhaustively destroyed, or a deletion-capable chromosomal DNA region of about 20 kbp is exhaustively deleted. Escherichia coli in which all or a part of the yhd A gene has been deleted by using, for example, a method for selecting a strain having improved growth from the parent strain from the parent strain using the defective mutant library. 3) Using a homologous recombination method or the like, a DNA encoding an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the protein encoded by the yhdA gene method for producing Escherichia coli having the, 4) phase by using a homologous recombination method, on the chromosomal DNA, the Escherichia coli YhdA A method for producing a microorganism in which all or part of a gene having 80% or more homology with a gene is deleted can be used.

The parent strain referred to in the method 1) refers to the original strain which has been subjected to the above-described gene disruption or chromosomal DNA region deletion treatment. Even if the parent strain is a wild-type microorganism, an industrially useful modified strain The strain may be a strain, a cell fusion strain, a transduced strain, or a recombinant strain created using a gene recombination technique.
As a method for preparing the gene or region-deficient strain library of the above 1), DNA containing the terminal sequence of the gene or region to be deleted is subcloned, and then a DNA fragment sandwiching a drug resistance gene is constructed with the DNA. And a method for constructing a gene or region-deficient strain library by introducing the DNA fragment into chromosomal DNA by homologous recombination twice [protein nucleic acid enzyme, 46 , 2386 (2001)].

By using the above-described gene or region-deficient strain library as a screening source, it is possible to observe the effects on growth of only a clearly defined disruption of one gene or group of genes, as in the case of using a mutagen. Phenomena in which unknown mutations adversely affect growth can be excluded.
As a specific method for selecting a strain with improved growth, a method of inoculating each strain of the gene or region-deficient strain library into a complete medium containing glucose and comparing the growth with the parent strain can be mentioned. For example, when the Escherichia coli wild-type strain W3110 is used as a parent strain, the growth can be compared using anti-biotic medium 3 (manufactured by Difco) as a complete medium containing glucose.

As a method of comparing the growth, the parent strain and the culture of the library when each strain is cultured in a liquid medium, the absorbance of 580 to 660 nm, preferably 660 nm is measured, and the parent strain and the culture of each strain of the library are measured. A method for comparing the absorbances can be given.
By selecting a strain that shows higher turbidity than the parent strain when cultured for the same time, a strain with improved growth over the parent strain can be obtained.

Next, the gene deficiency of each of the strains selected by the above method is transformed into the same strain different from the parent strain by the transduction method. Examples of transduction methods include phage transduction [Ann. Rev. Genetics, 2 , 245 (1968)] and a method using natural competence [Molecular Biological Method for Bacillus, 33 , John Wiley & Sons Ltd. (1990)]. I can give it. When Escherichia coli is used as the microorganism, a transduction method using P1 phage is preferred. The strain into which each defect is introduced is preferably a wild-type strain, but a strain having a mutation may be used as long as it can introduce each of the defects. For example, when Escherichia coli W3110 strain is used as a parent strain, there can be mentioned a method in which a gene deficiency of a selected strain with improved growth is introduced into a wild-type strain Escherichia coli MG1655 strain by a transduction method using P1 phage. .

The strain library prepared by transducing each defect into a wild-type strain as described above is again subjected to screening using turbidity as an index, and the absorbance of the culture solution when cultured for the same time is higher than that of the wild-type strain. By selecting high strains, final growth-improved strains can be obtained.
The microorganisms obtained by the above method are represented by Escherichia coli having a chromosomal DNA lacking between b3252 and b3254 and between b2236 and b2259, and b3252 by the b number notation described in Science 277 , 1453 (1997). Escherichia coli in which only the gene (hereinafter referred to as the yhdA gene) is deleted and disrupted.

Examples of the method 2) include a method using Escherichia coli capable of performing homologous recombination on a chromosome with linear DNA.
Examples of the linear DNA include a linear DNA in which DNAs having homology with the terminal sequence of the yhdA gene are arranged at both ends of a chloramphenicol resistance gene.
The linear DNA is introduced into Escherichia coli capable of homologous recombination on the chromosome by the linear DNA by a method such as electroporation using a competent cell, and a chloramphenicol-resistant strain is selected. Thus, Escherichia coli deficient in the yhdA gene can be obtained.

  In the method 3), one or more amino acids are deleted or substituted in the amino acid sequence encoded by introducing a site-specific mutation into a DNA having the nucleotide sequence represented by SEQ ID NO: 1. Alternatively, by obtaining a mutant DNA encoding the added amino acid sequence and using a homologous recombination method, a DNA having the base sequence represented by SEQ ID NO: 1 has a chromosomal DNA substituted with the mutant DNA. Examples of the method include a method for producing a microorganism.

  According to the above method 4), using a DNA having the base sequence represented by SEQ ID NO: 1 as a query, the homology with the DNA is determined to be 80% or more, preferably 85%, from the base sequence database of the chromosomal DNA of each microorganism. % Or more, more preferably 90% or more, and still more preferably 95% or more. A method of identifying a gene having homology and producing a microorganism deficient in the gene using the homologous recombination method established in each microorganism. Can be given.

The production method of the present invention relates to a method for producing a useful substance, comprising culturing the microorganism of the present invention in a medium, producing and accumulating the useful substance in a culture, and collecting the useful substance from the culture. .
The useful substance is not particularly limited as long as it is a useful substance produced using a microorganism, and examples thereof include proteins, amino acids, nucleic acids, vitamins, sugars, organic acids, and lipids.

Cultivation of the microorganism used in the production method of the present invention can be performed according to the usual method described below.
The culture medium for culturing the microorganism used in the production method of the present invention includes a carbon source that can be assimilated by the microorganism, a nitrogen source, inorganic salts, and the like. Any of the synthetic media may be used.

The carbon source may be any as long as the microorganism can assimilate, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, organic acids such as propionic acid, ethanol, Alcohols such as propanol can be used.
Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal, and soybean meal. A hydrolyzate of soybean meal, various fermentation cells, and digests thereof can be used.

As the inorganic salt, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 40 ° C, and the culturing time is usually 16 hours to 7 days. The pH during the culturing is preferably maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

When useful substances are generated and accumulated outside the cells, after the culture is completed, precipitates such as cells are removed from the culture, and an ion exchange method, a concentration method, a salting-out method, and the like are used in combination. A useful substance of interest can be isolated and purified from the culture.
When the useful substance is produced and accumulated in the cells, the cells are recovered from the culture after the culture, and then crushed by an appropriate method such as a mechanical or chemical method. By using an ion exchange treatment, a concentration method, a salting-out method, and the like, the intended useful substance can be isolated and purified from the cell lysate.

  Hereinafter, the present invention will be described in detail with reference to Examples.

Primary Screening of Chromosome-Partially Deleted Strain A wild-type strain MG1655 was used as a parent strain, and a mutant library lacking a portion on the chromosome was prepared according to the method described in Protein Nucleic Acid Enzyme, 46 , 2386 (2001).
The glycerol stock of each strain of the mutant library and wild-type strains W3110 strain and MG1655 strain was prepared by adding an antibiotic medium 3 agar plate medium (0.15% meat extract, 0.15%) containing 50 μg / ml diaminopimelic acid. 15% yeast extract, 0.5% peptone, 0.1% glucose, 0.35% sodium chloride, 0.132% dipotassium hydrogen phosphate, 1.5% agar, pH 7.0), and apply to 30 ° C. Overnight. Approximately 1 μl of the grown bacteria was scraped off with an ase, and 8 ml of an antibiotic medium 3 liquid medium containing 50 μg / ml diaminopimelic acid (0.15% meat extract, 0.15% yeast extract, 0.5% peptone, 0.1% glucose, 0.35% sodium chloride, 0.132% dipotassium hydrogen phosphate, pH 7.0), and cultured with shaking at 30 ° C overnight to prepare a pre-culture solution.

  80 μl of the preculture was inoculated into 8 ml of Antibiotic Medium 3 liquid medium containing 50 μg / ml diaminopimelic acid, and the absorbance at 660 nm was measured every hour after inoculation using a colorimeter, The absorbance at the time of culturing for 20 to 24 hours was defined as final turbidity. After the seventh hour, the absorbance when the culture solution was diluted 10-fold was also measured, and used for calculating turbidity as needed. Table 1 shows the results. In addition, the turbidity with the turbidity of the wild-type strain W3110 strain after culturing overnight as 100% was shown as the final attained turbidity. As a result, as shown in Table 1, strains having the following five deletion regions were obtained as strains having a turbidity higher than that of the W3110 strain by 10% or more. The above culture evaluation was performed by culturing each strain in five independent test tubes in two independent experiments, and the results shown in Table 1 were obtained in all cultures.

Introduction of the deletion region into another strain strain by P1 phage and culture evaluation Each chromosomal defect of the growth-improving candidate strain obtained in Example 1 was determined according to the method described in Ann. Rev. Genetics, 2 , 245 (1968). Wild type strain W3110 was transduced using P1 phage.
Since the drug resistance marker gene of each deficient strain in the chromosome-deficient library prepared in Example 1 is a kanamycin resistance gene, the wild-type strain W3110 can be transduced by the P1 phage transduction method using kanamycin resistance as an index. .

  First, P1 phage was prepared from each defective strain by the following method. Each defective strain was treated with an LB liquid medium containing 20 mg / l kanamycin [10 g / l bactotryptone (Difco), 5 g / l bactoeast extract (Difco), 5 g / l sodium chloride, 1 ml / l 1 mol / l sodium hydroxide] at 30 ° C. overnight, then 0.5 ml of each defective strain culture medium, 1.4 ml of fresh LB liquid medium and 100 μl of 0.1 mol / l calcium chloride Mix with the solution.

  After shaking the mixed solution at 30 ° C. for 5 to 6 hours, 400 μl of the mixed solution was transferred to a sterilized tube. After adding 2 μl of the P1 phage stock solution and keeping the mixture at 37 ° C. for 10 minutes, the soft agar solution (10 g / l bactotripton, 5 g / l bactoeast extract, 5 mmol / l calcium chloride) kept at 50 ° C. 3 ml of 1 ml / l 1 mol / l sodium hydroxide, 3 g / l agar) was added, and the mixture was stirred well, and the mixture was stirred well on a Ca-LB agar plate (10 g / l bactotryptone, 5 g / l bactoeast extract, 5 mmol). / L calcium chloride, 1 ml / l 1 mol / l sodium hydroxide, 15 g / l agar, plate diameter 15 cm). After culturing the plate at 37 ° C. for 7 hours, the soft agar portion was finely crushed and collected with a spreader and centrifuged at 1500 g at 4 ° C. for 10 minutes. 100 μl of chloroform was added to 1.5 ml of the supernatant, and stored at 4 ° C. overnight. The next day, the solution was centrifuged at 15000 g for 2 minutes, and the supernatant was stored at 4 ° C. as a phage stock.

  Next, the W3110 strain was transformed using the obtained phage. 200 μl of a culture of the W3110 strain cultured in a Ca-LB liquid medium at 30 ° C. for 4 hours was collected, and 1 μl of a phage stock was added. After keeping the temperature at 37 ° C. for 10 minutes, 5 ml of LB liquid medium and 200 μl of 1 mol / l sodium citrate solution were added and stirred. After centrifugation at 1500 g at 25 ° C. for 10 minutes, the supernatant was discarded, and 1 ml of LB liquid medium and 10 μl of 1 mol / l sodium citrate solution were added to the precipitated cells, followed by incubation at 30 ° C. for 2 hours. 100 μl of the solution was spread on an antibiotic medium 3 agar plate containing 20 mg / l kanamycin, and cultured at 30 ° C. overnight. By applying 24 colonies appearing for each of the defective introduced strains to an antibiotic medium 3 agar plate medium containing 20 mg / l kanamycin, confirming the drug sensitivity thereof, and selecting a kanamycin resistant strain, The W3110 strain in which each defective region was transduced was obtained. These strains were cultured in the same manner as in Example 1, and the culture was evaluated. As a result, as shown in Table 2, strains having the following two deletion regions were obtained as strains exhibiting a turbidity increase of 10% or more than those of the wild-type strain W3110. The above culture evaluation was performed by culturing each strain in five independent test tubes in two independent experiments, and the results shown in Table 2 were obtained in all cultures.

Preparation of Each Gene-Disrupted Strain in OCL-25-Deficient Region and Evaluation of Culture There were three putative gene regions (CDS) in the defective region OCL-25 selected in Example 2. Therefore, a strain in which each CDS was disrupted by a drug resistance gene was prepared, and the growth in liquid culture was measured using turbidity as an index.
Deletion strains of each gene contained in the OCL-25 region were prepared as follows. According to the method described in Example 2, Escherichia coli KM22 strain capable of homologous recombination on chromosome with linear DNA [Δ (recCptr recB recD) :: Plac-red kan), Gene, 246 , 321 (2000)] Thus, P1 phage was prepared. According to the method described in Example 2, a chromosomal region [Δ (recCptr recB recD) :: Plac-red kan] enabling homologous recombination on a chromosome with linear DNA was transferred to a wild-type strain W3110 by the P1 transduction method. After transduction, a W3110red strain was obtained.

Next, a DNA fragment for disrupting the yhdA gene (b3252) was obtained by PCR using pHSG398 plasmid DNA (manufactured by Takara Shuzo) as a template (FIG. 1). For PCR, LA-Taq manufactured by Takara Shuzo Co., Ltd. was used, and a reaction solution having a composition described in the instruction manual was prepared in accordance with the instruction manual. The PCR was performed by incubating 50 μl of the reaction solution at 94 ° C. for 1 minute, then performing a cycle of 98 ° C. for 20 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 3 minutes 30 times, and then incubating at 72 ° C. for 10 minutes. Performed under reaction conditions. As a template DNA, 40 ng of purified pHSG398 plasmid DNA was used. As the primer DNA for PCR, a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 was used as a sense strand primer, and a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 4 was used as an antisense strand primer. The 20 mer at the 3 'end of each of the primer DNAs was designed to hybridize to both ends of the chloramphenicol resistance gene in pHSG398. Also, the 5'-end 40mer of each primer was designed to have homology with the sequence of the end of the yhdA gene to be destroyed. 10 pmol of each of the primers thus designed was added to the above reaction solution. By the above PCR, a DNA fragment containing the chloramphenicol resistance gene (cat gene) and having a region homologous to the terminal 40 bases of the yhdA gene at both ends was obtained. This DNA fragment was purified using QIA quick PCR Purification Kit (manufactured by QIAGEN) to obtain 50 μl of a DNA solution.

The DNA in this solution was recovered as a precipitate by the ethanol precipitation method, and the whole amount was added to 40 μl of a competent cell solution of the W3110red strain to perform transformation. The method for preparing the competent cell of the W3110red strain was in accordance with the method described in Gene, 246 , 321 (2000). In the transformation, the purified DNA was dissolved in a small amount of pure water (about several μl), added to a competent cell solution (40 μl), and placed in a 0.1 cm cuvette (BioRad) at 1.8 KV and 25 μF. It was performed by electroporation under the conditions. 1 ml of SOC liquid medium (20 g / l bactotryptone, 5 g / l bacto yeast extract, 0.5 g / l sodium chloride, 0.2 ml / l 5 mol / l sodium hydroxide, 20 ml / l 1 mol / l glucose After culturing at 37 ° C. for 2 hours using 10 ml / l of 1 mol / l magnesium chloride and 10 ml / l of 1 mol / l magnesium sulfate, the total amount of cells collected by centrifugation was reduced to 15 μg / ml of chloramphenicol. LB agar plate containing 1.5% agar in an LB liquid medium (hereinafter abbreviated as LBcm agar medium plate), and cultured at 37 ° C. overnight to obtain a chloramphenicol-resistant strain. .

Was replaced to remove chloramphenicol resistance gene yhdA gene as described above, it was obtained W3110ΔRyhdA strain (Fig. 1). In addition, strains from which each gene of b3253 (yhdH) and b3254 were deleted (referred to as W3110ΔyhdH strain and W3110Δb3254 strain, respectively) were produced by the same method (FIG. 2). The W3110ΔRyhdA strain strain (W3110derutaeru25 strain) were deleted at the same time the three genes of strains remove yhdA gene (W3110derutayhdA Ltd.) and from yhdA to b3254 inserted a chloramphenicol resistance gene in the direction opposite to the was also prepared ( (Fig. 2).

  Table 3 shows the primers used in preparing each of the defective strains.

  Each strain produced as described above was cultured in the same manner as in Example 1, and the culture was evaluated. Table 4 shows the results. The above culture evaluation was performed by culturing each strain in five independent test tubes in two independent experiments, and the results shown in Table 4 were obtained in all cultures.

Culture evaluation of yhdA- deficient strain using synthetic medium Culture evaluation of W3110ΔRyhdA strain and W3110ΔL25 strain prepared in Example 3 was performed using a synthetic medium. The wild type strain W3110 strain was used as a control.
The glycerol stock of each strain was spread on an antibiotic medium 3 agar plate containing 50 μg / ml diaminopimelic acid, and cultured at 30 ° C. overnight. Approximately 1 μl of the grown bacteria is scraped off with an ase, inoculated into 8 ml of Antibiotic Medium 3 liquid medium containing 50 μg / ml diaminopimelic acid, and shake-cultured at 30 ° C. overnight to prepare a preculture solution. did. Add 300 μl of the preculture to 30 ml of minimal liquid medium (1.2% glucose, 0.6% disodium phosphate, 0.3% potassium dihydrogen phosphate, 0.05% sodium chloride, 0.1% Ammonium chloride, 2 mmol / l magnesium sulfate heptahydrate, 12.5 μmol / l iron sulfate, 100 μmol / l calcium chloride). Immediately after inoculation and at 12, 16, 20, 24, 36, 40, 44 and 48 hours from the start of the culture, the culture was sampled, and the absorbance at 660 nm (FIG. 3) and the amount of protein per culture (FIG. 4) were determined. It was measured. The measurement of the protein amount was performed by the following method.

  The cells recovered from 100 μl of the culture solution by centrifugation (10,000 g × 10 minutes) are washed with 1 ml of pure water and then centrifuged again (10,000 g × 10 minutes) to obtain the obtained cells. Was suspended in 160 μl of pure water, 40 μl of a 5% SDS solution was added, the mixture was heated at 100 ° C. for 3 minutes, returned to room temperature, and 800 μl of a 1% SDS solution was added. Using this solution, the total protein derived from the cells was quantified using a DC protein assay kit manufactured by Bio-Rad, and the amount of protein per medium was calculated. Bovine IgG was used as a standard protein for preparing a calibration curve. As a result, as shown in FIG. 3, each of the defective strains had a turbidity of about 20% at 36 hours of cultivation, and as shown in FIG. it was high.

Production of Proline Using OCL-25 Region-Deficient Strain According to the method described in Example 2, P1 phage was prepared from Escherichia coli KM22 strain capable of homologous recombination on chromosome with linear DNA. In addition, according to the method described in Example 2, a chromosomal region [Δ (recCptr recB recD) :: Plac-red kan] that enables homologous recombination on a chromosome with linear DNA was subjected to P1 transduction to obtain a wild-type strain. W3110 was transduced to obtain a W3110red strain.

Next, a W3110red_tet strain, which is a strain obtained by replacing the kanamycin resistance gene and the tetracycline resistance gene of the W3110red strain by the following method, was prepared.
Using the chromosomal DNA (10 ng) of the W3110red strain as a template and a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 13 or 14 as a primer set (50 pmol each), instructions attached to LA-Taq (Takara Shuzo) A PCR reaction solution was prepared according to the procedure described above. The PCR was carried out under the conditions that the temperature was kept at 94 ° C. for 2 minutes, a cycle of 94 ° C. for 15 seconds, 55 ° C. for 20 seconds, 68 ° C. for 3 minutes was repeated 30 times, and then the temperature was kept at 72 ° C. for 10 minutes.

After the amplified DNA fragment was separated by agarose gel electrophoresis, it was cut out from the gel and purified by a conventional method (hereinafter, referred to as DNA fragment 1).
In addition, using the chromosomal DNA (10 ng) of the CGSC7465 strain (obtained from the Coli Genetic Stock Center of Yale University, USA) having the Tn10 transposon on the chromosome as a template, a DNA consisting of the nucleotide sequences represented by SEQ ID NOS: 15 and 16 was used as a primer set. (50 pmol each), and a PCR reaction solution was prepared according to the instructions attached to LA-Taq. The PCR was performed under the conditions that the temperature was kept at 94 ° C. for 2 minutes, the cycle of 94 ° C. for 15 seconds, 55 ° C. for 20 seconds, 68 ° C. for 90 seconds was repeated 30 times, and then the temperature was kept at 72 ° C. for 10 minutes.

After the amplified DNA fragment was separated by agarose gel electrophoresis, it was cut out from the gel and purified by a conventional method (hereinafter, referred to as DNA fragment 2).
Using the chromosomal DNA (10 ng) of the W3110red strain as a template and a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 17 or 18 as a primer set (50 pmol each), PCR reaction was performed according to the instructions attached to LA-Taq. A liquid was prepared. The PCR was carried out under the conditions that the temperature was kept at 94 ° C. for 2 minutes, a cycle of 94 ° C. for 15 seconds, 55 ° C. for 20 seconds, 68 ° C. for 3 minutes was repeated 30 times, and then the temperature was kept at 72 ° C. for 10 minutes.

After the amplified DNA fragment was separated by agarose gel electrophoresis, it was cut out from the gel and purified by a conventional method (hereinafter, referred to as DNA fragment 3).
Next, using the DNA fragments 1 to 3 (10 ng each) obtained above as a template, a DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 19 and 20 was used as a primer set (50 pmol each), and LA-Taq was used. A PCR reaction solution was prepared according to the attached instructions. The PCR was carried out under the conditions that the temperature was kept at 94 ° C. for 2 minutes, a cycle of 94 ° C. for 15 seconds, 55 ° C. for 20 seconds, 68 ° C. for 3 minutes was repeated 30 times, and then the temperature was kept at 72 ° C. for 10 minutes.

After the amplified DNA fragment was separated by agarose gel electrophoresis, it was cut out from the gel and purified by a conventional method (hereinafter, referred to as DNA fragment 4). The DNA fragment has a sequence homologous to the kanamycin resistance gene at both ends and has a tetracycline resistance gene at the center.
1 μg of the DNA fragment 4 was added to 30 μl of a competent cell solution of the W3110red strain to perform transformation. Competent cells of the W3110red strain were prepared according to the method described in Gene, 246 , 321 (2000). Transformation was performed by electroporation using a 0.1 cm cuvette (manufactured by BioRad) at 1.8 KV and 25 μF. The cells subjected to the transformation were 1 ml of an SOB liquid medium containing 2 mmol / l IPTG [20 g / l bactotryptone (manufactured by Difco), 5 g / l bactoeast extract (manufactured by Difco), 0.5 g / l 1 ml / l sodium hydroxide, 0.2 ml / l 5 mol / l sodium hydroxide, 10 ml / l 1 mol / l magnesium chloride, 10 ml / l 1 mol / l magnesium sulfate] at 30 ° C. for 3 hours, and then 100 μl Was spread on an LB agar plate (1.5% agar in LB liquid medium) containing 20 μg / ml tetracycline, and cultured at 37 ° C. overnight. As the tetracycline-resistant strain, a target transformant W3110red_tet strain in which the kanamycin-resistant gene on the chromosome was replaced with the tetracycline-resistant gene was obtained.

Next, a proline-producing strain was prepared using the W3110red strain as a parent strain by the following method.
First, using the W3110red strain as a parent strain, a strain W3110red ΔputA :: cat strain in which the proline-degrading enzyme structural gene putA was disrupted was prepared. A DNA fragment containing the chloramphenicol resistance gene was amplified by PCR using the pHSG398 plasmid DNA as a template and the DNA consisting of the nucleotide sequences represented by SEQ ID NOS: 21 and 22 as a primer set. PCR was performed using EX-Taq manufactured by Takara Shuzo Co., Ltd. to prepare 100 μl of a reaction solution containing 20 ng of purified plasmid DNA and 50 pmol of each primer according to the instructions attached to EX-Taq. After incubating at 95 ° C. for 1 minute, 94 ° C. After repeating a cycle of 30 seconds at 64 ° C. for 30 seconds and 72 ° C. for 1 minute 30 times, the temperature was kept at 72 ° C. for 3 minutes. The entire amount of the reaction solution was purified using the QIA quick PCR Purification Kit to obtain 30 μl of a DNA solution. To decompose the plasmid pHSG398 traces next contaminating, prepared according to the instructions of the restriction enzyme accompanying the reaction solution 100μl which each restriction enzyme pst I and bgl I on DNA whole solution was added in 10 U, 37 ° C. , And the whole amount was purified using QIA quick PCR Purification Kit to obtain 30 µl of a DNA solution.

Next, by using 0.1 μl of the DNA solution as a template and a PCR using a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 23 or 24 as a primer set, the chloramphenicol resistance gene was held at the center, A DNA fragment having the 5 ′ end of the putA gene (DNA consisting of the base sequence represented by SEQ ID NO: 25) and the 3 ′ end (DNA consisting of the base sequence represented by SEQ ID NO: 26) was amplified. In the PCR, 100 μl of a reaction solution containing 0.1 μl of the purified template DNA (containing about 10 ng of DNA) and 50 pmol of each primer was prepared using EX-Taq according to the instructions attached to the EX-Taq, and the mixture was prepared at 95 ° C. After the temperature was maintained for 30 minutes, the cycle of 94 ° C. for 30 seconds, 51 ° C. for 30 seconds, and 72 ° C. for 1 minute was repeated 30 times, and then the temperature was maintained at 72 ° C. for 3 minutes. The whole reaction solution was purified using the QIA quick PCR Purification Kit.

Next, 1 μg of the DNA was added to 30 μl of a competent cell solution of the W3110red strain to perform transformation. Transformation of the W3110red strain was performed by electroporation under the same conditions as described above.
100 μl of the transformed cell solution was spread on an LB agar plate containing chloramphenicol at 50 μg / ml (LB liquid medium containing 1.5% agar; hereinafter, abbreviated as LBcm agar plate). Cultivated overnight at ° C. As the chloramphenicol-resistant strain, a target transformant W3110red ΔputA :: cat strain in which the putA gene on the chromosome was replaced with the chloramphenicol-resistant gene was obtained.

Next, using the W3110red ΔputA :: cat strain as a parent strain, a PK strain in which the proBA operon was disrupted was prepared. A DNA fragment containing a gentamicin resistance gene was amplified by PCR using plasmid pFastBACdual (manufactured by Invitrogen) as a template and a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 27 or 28 as a primer set. The PCR was performed by preparing 25 μl of a reaction solution containing 10 ng of pFastBACdual and 20 pmol of each primer according to the instructions attached to LA-Taq, keeping the mixture at 94 ° C. for 3 minutes, then at 94 ° C. for 15 seconds, at 55 ° C. for 20 seconds, at 68 ° C. After repeating the 1 minute cycle 30 times, the test was performed under the condition that the temperature was kept at 72 ° C. for 10 minutes. After the completion of the reaction, the entire amount of the reaction solution was purified using the QIA quick PCR Purification Kit to obtain 50 μl of a DNA solution.

Next, by using 1 μl of the DNA solution as a template and a PCR using a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 29 or 30 as a primer set, the gentamicin resistance gene was placed at the center, and the 5 ′ of the proBA operon was placed at both ends. A DNA fragment having a terminal (DNA consisting of the base sequence represented by SEQ ID NO: 31) and a 3 ′ end (DNA consisting of the base sequence represented by SEQ ID NO: 32) was amplified. In the PCR, 25 μl of a reaction solution containing 1 μl of the purified template DNA (containing about 10 ng of DNA) and 20 pmol of each primer was prepared according to the instructions attached to LA-Taq, and kept at 94 ° C. for 3 minutes. After repeating a cycle of 55 ° C. for 20 seconds and 68 ° C. for 1 minute 30 times, the temperature was kept at 72 ° C. for 10 minutes. The entire amount of the reaction solution was purified using the QIA quick PCR Purification Kit (hereinafter, referred to as DNA fragment 5).

In addition, a 1 kbp region at each of the 5 ′ upstream and 3 ′ downstream of the proBA operon was prepared by PCR using chromosomal DNA as a template. For the preparation of the 5 'upstream region (hereinafter, referred to as DNA fragment 6), a primer set was used for DNA consisting of the nucleotide sequences represented by SEQ ID NO: 33 and SEQ ID NO: 34. For the preparation of the 3 ′ downstream region (hereinafter referred to as DNA fragment 7), a DNA consisting of the nucleotide sequences represented by SEQ ID NO: 35 and SEQ ID NO: 36 was used as a primer set. Each PCR was performed under the following conditions except for the primer set. That is, using the chromosomal DNA of the W3110red strain (10 ng) as a template, 25 μl of a reaction solution containing 20 pmol of each primer was prepared according to the instructions attached to LA-Taq, kept at 94 ° C. for 3 minutes, and then incubated at 94 ° C. for 15 seconds. After repeating a cycle of 55 ° C. for 20 seconds and 68 ° C. for 1 minute 30 times, the temperature was kept at 72 ° C. for 10 minutes. The whole reaction solution was purified using the QIA quick PCR Purification Kit.

  Next, PCR was performed using the DNA fragments 5 to 7 obtained above as a template to prepare a product obtained by ligating the three fragments. That is, using DNA fragments 5 to 7 (10 ng each) as a template, 25 μl of a PCR reaction solution containing 20 pmol of each of the primers shown in SEQ ID NO: 33 and SEQ ID NO: 36 was prepared according to the instructions attached to LA-Taq, and was prepared at 94 ° C. After maintaining the temperature for 3 minutes, a cycle of 94 ° C. for 15 seconds, 55 ° C. for 20 seconds, and 68 ° C. for 3 minutes was repeated 30 times, and then the conditions were maintained at 72 ° C. for 10 minutes.

After the amplified DNA fragment was separated by agarose gel electrophoresis, it was cut out from the gel and purified (hereinafter, referred to as DNA fragment 8). The DNA fragment 8 has a sequence homologous to the 1 kbp region 5 ′ upstream of the proBA operon at one end, a gentamicin resistance gene at the center, and 1 kbp 3 ′ downstream of the proBA operon at the other end. This is a DNA fragment having a sequence homologous to the region.

Transformation was performed by adding 1 μg of the purified DNA fragment 8 to 50 μl of a competent cell solution of the W3110red ΔputA :: cat strain. Competent cells of the W3110red ΔputA :: cat strain were prepared in the same manner as in the preparation of the W3110red competent cells described above. Transformation was performed by the same method as the above-mentioned method for transforming the W3110red strain. However, an LB agar plate containing 5 μg / ml gentamicin was used as a selective medium for the transformant.

As a gentamicin-resistant strain, a target transformant PK strain in which the proBA operon on the chromosome was replaced with a gentamicin-resistant gene was obtained.
M9 minimal agar medium (1.5% agar, 1.2% glucose, 0.6% disodium phosphate, 0.3% potassium dihydrogen phosphate, 0.05% sodium chloride, 0.1% ammonium chloride, Apply PK strain to each of M9 minimal agar medium containing 2 mmol / l magnesium sulfate heptahydrate, 12.5 μmol / l iron sulfate, 100 μmol / l calcium chloride) and proline (8 mg / l). The cells were cultured overnight and examined for growth. The cells grew in the minimal medium containing proline, but did not grow in the minimal medium containing no proline, confirming that the PK strain showed a clear proline requirement.

Next, a proline-producing mutant PKB74 strain was prepared by the following method by introducing a mutant proBA operon into the PK strain.
A 4 kbp DNA fragment containing the proBA operon was amplified by PCR using the chromosomal DNA of the W3110red strain as a template and DNA having the nucleotide sequence represented by SEQ ID NO: 33 or 36 as a primer set. The PCR was performed by preparing 25 μl of a reaction solution containing 10 ng of chromosomal DNA and 20 pmol of each primer according to the instructions attached to LA-Taq, keeping the mixture at 94 ° C. for 3 minutes, then at 94 ° C. for 15 seconds, 55 ° C. for 20 seconds, and 68 ° C. After repeating a cycle of 4 minutes for 30 times, the temperature was kept at 72 ° C. for 10 minutes.

The amplified DNA fragment was separated by agarose gel electrophoresis, cut out from the gel by a conventional method, purified, and then converted to plasmid pPCR-Script Amp using a commercially available kit (Qiagen PCR-Script Cloning Kit). It was cloned to generate the plasmid pPCR proBA.
Using the pPCR proBA as a template, the previously reported point mutation proB74 [L. Csonka, and et al: Gene, 64, 199-205 (1988)] was introduced into proBA in vitro using the Qiagen QuickChange Kit. . In vitro mutagenesis using the QuickChange Kit was performed according to the attached instructions to prepare a plasmid pPCR proB74 in which the above mutation was introduced into the proB gene in the proBA operon. The nucleotide sequence of the primer DNA having the mutant sequence used for introducing the mutation point is shown in SEQ ID NO: 37.

Next, after pPCR proB74 was cleaved with the restriction enzyme BsrGI , a DNA fragment containing the mutant proBA operon was separated by agarose electrophoresis and recovered and purified from the gel.
2 μg of the purified DNA fragment containing the mutant proBA operon was added to 50 μl of competent cells of a PK strain prepared according to the method described in Gene, 246 , 321 (2000) to perform transformation. Transformation was performed by the electroporation method described above. However, the selection medium for the transformant was performed on an M9 minimal agar medium, and a target proline-producing mutant strain PKB74 was obtained by selecting a strain that did not require proline.

The PKB74 strain has a kanamycin resistance gene (kan) on its chromosome [Δ (recCptr recB recD) :: Plac-red kan]. The kanamycin resistance gene was replaced with a tetracycline resistance gene by the following method.
Using the W3110red_tet strain prepared above, a P1 transducing phage was prepared. The phage was used to transform PKB74 strain. Transformants were selected on an LB agar plate medium containing 20 mg / l tetracycline. By selecting a tetracycline-resistant strain, a PKB74_tet strain in which the kanamycin-resistant gene was replaced with the tetracycline-resistant gene was obtained.

  Next, various chromosomal deletions were introduced into the PKB74_tet strain. A P1 transducing phage was obtained from the partially chromosome-deleted strain OCL-25 by the above method, and the PKB74_tet strain was transformed using the phage. Then, a kanamycin-resistant strain was transformed on an LB agar plate medium containing 20 mg / l kanamycin. By selection, a strain ΔOCL-25 in which the OCL-25 region of the PKB74_tet strain was deleted was obtained.

  The inoculum of the PKB74_tet strain and the ΔOCL-25 strain obtained above was transformed into an LB agar medium (15 g / l agar, 10 g / l bactotryptone, 5 g / l bactoeast extract, 5 g / l sodium chloride, 1 ml / l of 1 mol / l). 1 sodium hydroxide) and cultured at 30 ° C. overnight. Sufficiently grown colonies were scraped, inoculated into test tubes each containing 5 ml of LB liquid medium, and cultured with shaking at 30 ° C. for 20 hours. Add 500 μl of the culture solution to 50 ml of Med7 liquid medium (3% glucose, 0.8% peptone, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.002% The cells were inoculated into a 300 ml Erlenmeyer flask containing calcium chloride, 1% calcium carbonate, 1% ammonium sulfate, 0.2% sodium chloride, and 0.03% iron sulfate) and cultured at 30 ° C with shaking.

  Cultures were sampled over time, the cultures were centrifuged, and the supernatant was diluted 1000-fold. The proline concentration in the culture solution was quantified by analyzing the diluent with an amino acid direct analysis system DXa-500 manufactured by Dionex. As a result, it was found that the ΔOCL-25 strain had an increased amount of proline production as compared with the parent strain PKB74_tet. Table 5 shows the results.

SEQ ID NO: 3—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 4—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 5—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 6—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 7—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 8-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 9-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 10—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 11—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 12—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 13-Description of artificial sequence: synthetic DNA
SEQ ID NO: 14-Description of artificial sequence: synthetic DNA
SEQ ID NO: 15-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 16-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 17—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 18—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 19-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 20—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 21—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 22—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 23—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 24—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 25-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 26-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 27—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 28-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 29—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 30-Description of artificial sequence: synthetic DNA
SEQ ID NO: 31—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 32—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 33—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 34—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 35-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 36-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 37—Description of Artificial Sequence: Synthetic DNA

FIG. 1 is a diagram showing a method for preparing a W3110ΔRyhdA strain in which the yhdA gene on the chromosomal DNA has been replaced and deleted with a chloramphenicol resistance gene. In the figure, open lines indicate DNAs present at both ends of the yhdA gene on the chromosomal DNA or DNAs homologous to the DNA. 2, Escherichia coli W3110 strain is wild-type strain, substitutionally is deficient yhdA gene chloramphenicol resistance gene in Escherichia coli W3110ΔRyhdA strain substitutionally deficient, the yhdH gene chloramphenicol resistance gene and Escherichia coli W3110ΔyhdH strain, E. coli b3254 gene chloramphenicol resistance gene substitutionally was deficient E. coli W3110Δb3254 strain, E. and coli W3110derutaRyhdA strain inserting a chloramphenicol resistance gene in the reverse direction FIG. 2 shows the structure of chromosomal DNA of the E. coli W3110ΔyhdA strain in which the yhdA gene has been deleted and the E. coli W3110ΔL25 strain in which the three genes from yhdA to b3254 have been simultaneously deleted with the chloramphenicol resistance gene. . In the figure, open arrows and open lines indicate chloramphenicol resistance genes. FIG. 3 is a diagram showing the growth of Escherichia coli W3110ΔRyhdA strain (△), E. coli W3110ΔL25 strain (◆), and E. coli W3110 strain (●) when cultured in a synthetic medium. The vertical axis represents the absorbance at 660 nm, and the horizontal axis represents the culture time. FIG. 4 is a diagram showing the protein concentration per culture solution when the Escherichia coli W3110ΔRyhdA strain (△), the E. coli W3110ΔL25 strain (◆), and the E. coli W3110 strain (●) are cultured in a synthetic medium. The vertical axis represents the protein concentration, and the horizontal axis represents the culture time.

Explanation of reference numerals

cat: Chloramphenicol resistance gene
lacZ: β- galactosidase gene W3110: Escherichia coli W3110 strain ΔyhdA: Escherichia coli W3110ΔyhdA stock ΔRyhdA: Escherichia coli W3110ΔRyhdA stock ΔyhdH: Escherichia coli W3110ΔyhdH stock Δb3254: Escherichia coli W3110Δb3254 share ΔL25: Escherichia coli W3110ΔL25 shares

Claims (7)

A microorganism having a chromosomal DNA in which part or all of the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 has been deleted, and having improved growth compared to a parent strain having a chromosomal DNA having no deletion. A DNA having a homology of 80% or more with the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 has a chromosomal DNA partially or completely deficient, and has improved growth compared to a parent strain having a chromosomal DNA having no deficiency. Microorganisms. In the amino acid sequence represented by SEQ ID NO: 2, a DNA encoding a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added is present on chromosomal DNA, and the amino acid represented by SEQ ID NO: 2 A microorganism having improved growth compared to a parent strain having a DNA encoding a protein having a sequence on chromosomal DNA. The microorganism according to any one of claims 1 to 3, wherein the microorganism is a microorganism belonging to the genus Escherichia . The microorganism according to claim 4, wherein the microorganism belonging to the genus Escherichia is Escherichia coli . A method for producing a useful substance, comprising culturing the microorganism according to claim 1 in a medium, producing and accumulating the useful substance in a culture, and collecting the useful substance from the culture. Law. 7. The method according to claim 6, wherein the useful substance is a useful substance selected from the group consisting of proteins, amino acids, nucleic acids, vitamins, sugars, organic acids, and lipids.

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