Patent Application
20110111458
The present invention relates to an E. coli mutant strain having chromosomal DNA shorter than that of a wild-type E. coli strain and a process for producing a useful substance using such mutant strain.
All nucleotide sequences of the chromosomal DNAs of E. coli have been elucidated (Non-Patent Documents 1 and 2). Also, a method wherein a given gene or a given region on chromosomal DNA of E. coli is deleted by homologous recombination has been known (Non-Patent Document 3).
As reduced-genome strains defective in plural regions on the chromosome, MDS43 strain defective in 708 kbp (non-patent document 4), Δ16 strain defective in 1377 kbp (non-patent document 5) and Step28 strain defective in 1027 kbp (patent document 1) are already known.
MDS43 strains are known to grow equivalently to wild-type Escherichia coli strain MG1655, which is a parental strain, in a minimal medium or LB medium. In addition, Step28 strain shows an increased bacterial cell amount on termination of culture as compared to parental strain W3110red strain free of defective region on the chromosome, and is known to have a high ATP regeneration activity. Moreover, threonine production using Step28T strain, wherein threonine biosynthetic gene is reinforced, shows improved threonine productivity as compared to the use of W3110red strain.
However, Δ16 strain is known to show low growth rate as compared to the parental strain, MG1655 strain. Thus, the phenotype of a reduced-genome strain varies depending on the strain, and how the deletion of a chromosomal region exceeding 1000 kbp changes the properties of Escherichia coli is difficult to predict. Therefore, Escherichia coli defective in a region of not less than 1000 kbp in the chromosomal DNA does not necessarily show useful properties.
In addition, an E. coli mutant, which lacks regions of 1040 kbp or more on chromosomal DNA and a process for producing a useful substance using such mutant have not yet been known.
It is an object of the present invention to provide an industrially useful E. coli mutant strain that lacks regions of 1040 kbp or more on chromosomal DNA and a process for producing a useful substance using such mutant.
The present invention relates to the following (1)-(28).
The present invention can provide an E. coli mutant, the amount of E. coli cells of which, after a given period of culture, is larger than that of a wild-type E. coli strain, and a process for producing a useful substance using such mutant.
In the present description, the wild-type E. coli strains may be any E. coli strains that are found in natural populations at the highest frequency. Examples thereof include the E. coli W3110 strain (ATCC27325) and the E. coli MG1655 strain (ATCC47076) that belong to the E. coli K-12 strain, the ATCC11303 strain that belongs to the E. coli B strain, and the ATCC9637 strain that belongs to the E. coli W strain.
The nucleotide sequence as shown in SEQ ID NO: 1 in the present specification is the full-length nucleotide sequence of chromosomal DNA of the E. coli W3110 strain registered as of Aug. 16, 2007 on the internet site URL::http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore &id=89106884. The above-mentioned nucleotide sequence shown in the internet site is a nucleotide sequence of Accession No. AC—000091 in NCBI (National Center for Biothechnology Information) GenBank.
In addition, the name of each gene on the Escherichia coli chromosome, which is used in the above-mentioned internet site, is used in Table 1 and Table 2 of the present description.
In Table 1, the gene shown by the gene name ykgM(JW5034) refers to nucleotides 311598 to 311738 of the nucleotide sequence as shown in SEQ ID NO: 1, the gene shown by the gene name ykgM(JW5035) refers to nucleotides 311738 to 312001 of the nucleotide sequence as shown in SEQ ID NO: 1, the gene shown by the gene name phnE(JW4064) refers to nucleotides 4327121 to 4327489 of the nucleotide sequence, and the gene shown by the gene name phnE(JW4065) refers to nucleotides 4327339 to 4327959 of the nucleotide sequence as shown in SEQ ID NO: 1.
The term chromosomal DNA that contains genes refers to chromosomal DNA that contains a specific structural gene and a region containing a specific regulatory function such as a promoter, operator or the like.
In the present specification, the term chromosomal DNA that does not contain gene refers to chromosomal DNA that does not contain a part of or an entire structural gene of a given gene or chromosomal DNA that does not contain a region having a specific regulatory function, such as a promoter and/or operator of such gene.
The chromosomal DNA that does not contain part of a structural gene is DNA that does not contain part of a structural gene, and a functional protein is not transcribed and expressed from the structural gene. Examples thereof include DNA that has no initiation codon due to a lack of the 5′ end region of the structural gene and DNA that lacks most of the 3′ end region, such as 10% or more, preferably 20% or more, more preferably 30% or more, and further preferably 40% or more of the entire 3′ end region. Chromosomal DNA that does not contain two adjacent genes may be chromosomal DNA that does not contain DNA consisting of the nucleotide sequence located between such genes.
The homologous gene refers to a gene that shows sequence homology of the nucleotide sequence of the gene in the Escherichia coli species, or a gene encoding a protein having an amino acid sequence showing sequence homology. Showing sequence homology means that a nucleotide sequence shows sequence similarity of not less than 85%, preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 98%, particularly preferably not less than 99%, or an amino acid sequence shows sequence similarity of not less than 80%, preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 98%, particularly preferably not less than 99%.
The E. coli mutant strain having a chromosomal DNA containing not more than 10% of a particular set of genes or homologous gene thereof means an E. coli mutant strain having a chromosomal DNA lacking not less than 90%, preferably not less than 95%, more preferably not less than 98%, still more preferably not less than 99%, of a gene selected from a set of genes or homologous gene thereof.
The DNA equivalent to DNA comprising a nucleotide sequence represented by given nucleotide numbers in the nucleotide sequence as shown in SEQ ID NO: 1 may be any DNA, as far as it is substantially the same as DNA having a nucleotide sequence represented by such given nucleotide numbers of an E. coli species in terms of the composition of the gene contained and the position on chromosomal DNA. When compositions of genes contained in DNA are substantially the same as each other, genes contained in such DNA are composed of genes identical or homologous to each other, and arranged in the same order. In that case, nucleotide sequences may differ among genes. When the DNA having a nucleotide sequence represented by given nucleotide numbers contains an insertion sequence, however, such insertion sequence may be absent in DNA equivalent to the aforementioned DNA.
Substantially the same positions on chromosomal DNA refers to the following conditions. When adenine of the initiation codon of the thrL gene on E. coli chromosomal DNA is designated as No. 190 and the nucleotide sequence of chromosomal DNA is numbered along the coding chain of the thrL gene, differences between such nucleotide numbers and the given nucleotide numbers are 50 or smaller, preferably 30 or smaller, more preferably 10 or smaller, and further preferably 5 or smaller. When the DNA having a nucleotide sequence represented by given nucleotide numbers contains an insertion sequence, however, DNA equivalent to the aforementioned DNA does not necessarily contain such insertion sequence. Thus, such insertion sequence and nucleotide numbers may be different. An example of DNA equivalent to DNA having a nucleotide sequence represented by given nucleotide numbers of the nucleotide sequence as shown in SEQ ID NO: 1 is DNA having sequence similarity of 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 98% or more, and particularly preferably 99% or more with the nucleotide sequence represented by given nucleotide numbers.
Sequence similarity of amino acid sequence or nucleotide sequence can be determined with the utilization of the BLAST algorithm of Karlin and Altschul (Pro. Natl. Acad. Sci., U.S.A., 90, 5873, 1993) or the FASTA algorithm (Methods Enzymol., 183, 63, 1990). Based on the BLAST algorithm, programs referred to as BLASTN or BLASTX have been developed (J. Mol. Biol., 215, 403, 1990). When analyzing nucleotide sequences by BLASTN based on BLAST, the score parameter is set to 100 and the word length parameter is set to 12, for example. When analyzing amino acid sequences via BLASTX based on BLAST, the score parameter is set to 50 and the word length parameter is set to 3, for example. When using BLAST and Gapped BLAST programs, the default parameters thereof are used. Specific procedures of such analytical methods are known (http://www.ncbi.nlm.nih.gov.).
1. E. coli Mutant Strain of the Present Invention
The E. coli mutant strain of the present invention may be (1) any E. coli mutant strain comprising chromosomal DNA that comprise any gene excluding the genes [2] that are present on chromosomal DNA of a wild-type E. coli strain or any E. coli mutant strain comprising chromosomal DNA that does not comprise any gene excluding the genes [1], provided that it comprises the genes [1], and does not comprise the genes [2] and having chromosomal DNA that is at least 1040 kbp shorter than that of a wild-type E. coli strain,
A preferable example of the E. coli mutant strain of the present invention is an E. coli mutant strain comprising chromosomal DNA that comprises DNA consisting of the nucleotide sequence of the genes of Table 1 and the above-mentioned [1] in the nucleotide sequences shown in SEQ ID NO: 1 or a DNA equivalent thereto, does not contain DNA consisting of the nucleotide sequence of chpA gene or DNA equivalent thereto, and does not contain DNA consisting of the nucleotide sequences of [3] chpA gene, uspG gene, ybdR gene, rnk gene, rna gene, citT gene, citG gene, citX gene, citF gene, citE gene, citD gene, citC gene, citA gene, citB gene, dcuC gene, crcA gene, cspE gene, crcB gene, ybeH gene, ybeM gene, yqcA gene, yqcB gene, yqcC gene, syd gene, yqcD gene, ygdH gene, sdaC gene, sdaB gene, exo gene, fucO gene, fucA gene, fucP gene, fucI gene, fucK gene, fucU gene, fucR gene, ygdE gene, ygdD gene, gcvA gene, ygdI gene, csdA gene, ygdK gene, araD gene, araA gene, araB gene, araC gene, yabI gene, thiQ gene, thiP gene, tbpA gene, sgrR gene, setA gene, yhfZ gene, yhfY gene, yhfX gene, yhfW gene, php gene, yhfU gene, yhfT gene, yhfS gene, frlR gene, frlD gene, frlC gene, frlB gene, frlA gene, yhfL gene, pncA gene, ydjE gene, ydjF gene, ydjG gene, ydjH gene, ydjI gene, ydjJ gene, ydjK gene, ydjL gene, yeaC gene, yeaA gene, ade gene, yicO gene, yicN gene, yicM gene, yicS gene, nlpA gene, yicL gene, setC gene, ygjI gene, ygjJ gene, ygjK gene, fadH gene, ygjM gene, ygjN gene, ygjO gene, ygjP gene, ygjQ gene, ygjR gene, alx gene, sstT gene, ygjV gene, yrhB gene, yrhA gene, yhhZ gene, yhhY gene, yhhX gene, yhhW gene, yeaK gene, yoaI gene, yeaL gene, yeaM gene, yeaN gene, yeaO gene, yoaF gene, yeaP gene, yeaQ gene, yoaG gene, yeaR gene, yeaS gene, yeaT gene, yeaU gene, yeaV gene, yeaW gene, yeaX gene, hyfA gene, hyfB gene, hyfC gene, hyfD gene, hyfE gene, hyfF gene, hyfG gene, hyfH gene, hyfI gene, hyfJ gene, hyfR gene, focB gene or homologous gene thereof, or DNA equivalent thereto. More preferred is
In addition, (23) an E. coli mutant strain having a chromosomal DNA comprising the genes [1] or homologous gene thereof of (1), and not more than 10% of the genes [2] of (1) and the genes [3] below or homologous gene thereof, [3] chpA gene, uspG gene, ybdR gene, rnk gene, rna gene, citT gene, citG gene, citX gene, citF gene, citE gene, citD gene, citC gene, citA gene, citB gene, dcuC gene, crcA gene, cspE gene, crcB gene, ybeH gene, ybeM gene, yqcA gene, yqcB gene, yqcC gene, syd gene, yqcD gene, ygdH gene, sdaC gene, sdaB gene, exo gene, fucO gene, fucA gene, fucP gene, fucI gene, fucK gene, fucU gene, fucR gene, ygdE gene, ygdD gene, gcvA gene, ygdI gene, csdA gene, ygdK gene, araD gene, araA gene, araB gene, araC gene, yabI gene, thiQ gene, thiP gene, tbpA gene, sgrR gene, setA gene, yhfZ gene, yhfY gene, yhfX gene, yhfW gene, php gene, yhfU gene, yhfT gene, yhfS gene, frlR gene, frlD gene, frlC gene, frlB gene, frlA gene, yhfL gene, pncA gene, ydjE gene, ydjF gene, ydjG gene, ydjH gene, ydjI gene, ydjJ gene, ydjK gene, ydjL gene, yeaC gene, yeaA gene, ade gene, yicO gene, yicN gene, yicM gene, yicS gene, nlpA gene, yicL gene, setC gene, ygjI gene, ygjJ gene, ygjK gene, fadH gene, ygjM gene, ygjN gene, ygjO gene, ygjP gene, ygjQ gene, ygjR gene, alx gene, sstT gene, ygjV gene, yrhB gene, yrhA gene, yhhZ gene, yhhY gene, yhhX gene, yhhW gene, yeaK gene, yoaI gene, yeaL gene, yeaM gene, yeaN gene, yeaO gene, yoaF gene, yeaP gene, yeaQ gene, yoaG gene, yeaR gene, yeaS gene, yeaT gene, yeaU gene, yeaV gene, yeaW gene, yeaX gene, hyfA gene, hyfB gene, hyfC gene, hyfD gene, hyfE gene, hyfF gene, hyfG gene, hyfH gene, hyfI gene, hyfJ gene, hyfR gene, focB gene
is also the E. coli mutant strain of the present invention.
Further, the E. coli mutant strain of the present invention includes;
The E. coli mutant strains described in (1) to (23) above appear to lack parts of or entire genes that encode proteins that are not directly involved in biosynthesis of useful substances but indirectly suppress generation of such useful substances. Such E. coli mutant strains and the mutant strain of (24) above are useful as E. coli mutant strains for producing useful substances.
The E. coli mutant strain of the present invention may be any E. coli mutant strain, provided that it belongs to Escherichia coli. It is preferably a mutant strain of the E. coli K-12 strain, the E. coli B strain, or the E. coli W strain, more preferably a mutant strain of the E. coli K-12 strain, further preferably a mutant strain of the E. coli W3110 strain (ATCC27325) or the E. coli MG1655 strain (ATCC47076), and particularly preferably the E. coli W3110 strain (ATCC27325), for example.
2. Method for Producing the E. coli Mutant Strain of the Present Invention
The E. coli mutant strain of the present invention can be obtained by any method, provided that it allows such mutant strain to be obtained. For example, the following method wherein a given gene or DNA on E. coli chromosomal DNA is deleted may be employed.
A gene or DNA on E. coli chromosomal DNA can be deleted via a method utilizing homologous recombination, for example. An example of a general method utilizing homologous recombination is a method involving the use of plasmids for homologous recombination that can be prepared by ligating DNA fragments located at both outer sides of the gene or DNA that is intended to be deleted on E. coli chromosomal DNA to plasmid DNA having a drug resistance gene that cannot be autonomously replicated in such E. coli.
After the plasmids for homologous recombination are introduced into an E. coli cell by a conventional method, transformants into which the plasmids for homologous recombination have been integrated to chromosomal DNA by homologous recombination are selected using the drug resistance as an indicator. The obtained transformants are cultured in a medium that does not contain the aforementioned drug for several hours to 1 day, thereafter the cultures are spread on an agar medium that contains the drug and to an agar medium that does not contain the drug, and strains that do not grow in the former medium but grow in the latter medium are selected. Thus, strains in which the second homologous recombination has taken place on the chromosomal DNA can be obtained. By determining the nucleotide sequence of a region of the chromosomal DNA in which the gene or DNA to be deleted was present, the introduction of deletion of the target gene or DNA on chromosomal DNA can be confirmed.
An example of a method utilizing homologous recombination, and thereby effectively introducing deletion, substitution or addition of nucleotide(s) into multiple genes, is a method involving the use of a linear DNA.
Specifically, a linear DNA that contains DNA fragments existing at both outside regions of a gene or DNA to be deleted on chromosomal DNA is incorporated into a cell to cause homologous recombination between the chromosomal DNA and the introduced linear DNA. This method can be applied to any microorganisms, provided that such Escherichia coli effectively incorporate a linear DNA. Such microorganisms include preferably Escherichia coli that expresses λ phage-derived recombinant proteins (Red recombination system).
An example of Escherichia coli that expresses the λRed recombination system is Escherichia coli JM101 possessing pKD46, which is plasmid DNA having the gene of the λ Red recombination system (available from the E. coli Genetic Stock Center, Yale University, U.S.A.).
Examples of DNAs that can be used for homologous recombination include:
Any drug resistance genes can be used, provided that such genes impart drug resistance to a drug to which E. coli shows sensitivity. For example, the drug resistance genes include kanamycin resistance genes, chloramphenicol resistance genes, gentamicin resistance genes, spectinomycin resistance genes, tetracycline resistance genes, ampicillin resistance genes, and the like.
The genes that can be used for negative selection are genes that are lethal to E. coli under given culture conditions, when such genes are expressed in E. coli. Examples of such genes include sacB genes derived from the microorganism which belongs to the genus Bacillus [Appl. Environ. Microbiol., 59, 1361-1366, 1993] and rpsL genes derived from the microorganism which belongs to the genus Escherichia [Genomics, 72, 99-104, 2001].
DNA that exists at both ends of the aforementioned linear DNA which is located at both outside regions of a chromosomal DNA into which introduction of substitution or deletion of nucleotide(s) is intended, or DNA homologous thereto, is oriented on the linear DNA in the same direction as the direction thereof on the chromosomal DNA. The length thereof is preferably about 10 bp to 100 bp, more preferably about 20 bp to 50 bp, and further preferably about 30 bp to 40 bp.
The nucleotide sequence that is recognized by yeast-derived Flp recombinase may be a nucleotide sequence that is recognized by yeast-derived Flp recombinase and has a nucleotide sequence which catalyze homologous recombination, provided that the aforementioned protein recognizes such nucleotide sequence and catalyzes homologous recombination of such nucleotide sequence.
A DNA homologous thereto refers to a DNA which has identity to the extent that homologous recombination takes place between the aforementioned linear DNA and a target region on a chromosomal DNA. Specifically, it refers to DNA having 80% or more, preferably 90% or more, more preferably 95% or more, and further preferably 100% homology.
Homology of the nucleotide sequences can be determined using the aforementioned programs such as BLAST or FASTA.
The aforementioned linear DNA fragment can be prepared by PCR. Also, DNA containing the linear DNA can be constructed on a plasmid and the linear DNA of interest can then be obtained by restriction enzyme treatment.
Deletion, substitution, or addition of nucleotide(s) can be introduced into chromosomal DNAs of E. coli by any of the following methods 1 to 4:
A method of introducing a linear DNA into a host microorganism that is employed in the above methods can be any method as long as the DNA can be introduced into the microorganism. Examples thereof include a method involving the use of calcium ions [Proc. Natl. Acad. Sci., U.S.A., 69, 2110, 1972], the protoplast method [Japanese Published Unexamined Patent Application No. 2483942/88 (1988)], and electroporation [Nucleic Acids Res., 16, 6127, 1988].
By using the DNA comprising at a site around the center thereof any gene to be inserted into a chromosomal DNA as the DNA that is used in method 2 or method 3 [2], drug resistance genes or the like can be deleted, and at the same time, any genes can be introduced into the chromosomal DNA.
According to the methods 2 to 4 above, foreign genes, such as drug resistance genes and genes that can be used for negative selection, do not remain in the chromosomal DNA of the transformant which is finally obtained. With the utilization of such methods, accordingly, the procedures of Method 2, Methods 3 [1] to [3] or method 4 [1] [2] can be repeated by using the same drug resistance genes and genes that can be used for negative selection. This enables the easy production of E. coli having deletion of gene or DNA at two or more different regions on a chromosomal DNA.
The amounts of cells of the E. coli mutant strains obtained by such method are greater than those of wild-type E. coli strains after a given period of culture. This can be confirmed by, for example, culturing the wild-type parental E. coli strains and the mutant strains using M9 minimal medium that is composed of 1% glucose, 0.6% disodium phosphate, 0.3% potassium dihydrogenphosphate, 0.05% sodium chloride, 0.1% ammonium chloride, 2 mmol/l of magnesium sulfate heptahydrate, 10 mg/l of iron sulfate and 100 μmol/l of calcium chloride, and assaying absorption of culture products at 660 nm at a given frequency, or assaying an ATP content per unit of medium via a conventional technique.
Also, the E. coli mutant strain of the present invention can be produced by a method wherein a gene or DNA on chromosomal. DNA of E. coli capable of producing a useful substance is deleted or a method wherein the capacity for producing a useful substance is imparted to E. coli from which a gene or DNA on chromosomal DNA has been deleted.
E. coli capable of producing a useful substance may be any E. coli, provided that it is capable of producing one or more types of useful substances. Examples of such E. coli include a strain isolated from nature that is capable of producing a useful substance, and E. coli to which the ability to produce a desirable useful substance has been artificially imparted by known methods.
Examples of such known methods include:
When useful substances are amino acids in the specific methods (a) to (e) above, for example, method (a) is described in Agric. Biol. Chem., 43, 105-111, 1979, J. Bacteriol., 110, 761-763, 1972, and Appl. Microbiol. Biotechnol., 39, 318-323, 1993; method (b) is described in Agric. Biol. Chem., 43, 105-111, 1979 and J. Bacteriol., 110, 761-763, 1972; method (c) is described in Appl. Microbiol. Biotechnol., 39, 318-323, 1993 and Agric. Biol. Chem., 39, 371-377, 1987; method (d) is described in Appl. Environ. Microbiol., 38, 181-190, 1979 and Agric. Biol. Chem., 42, 1773-1778, 1978; and method (e) is described in Agric. Biol. Chem., 36, 1675-1684, 1972, Agric. Biol. Chem., 41, 109-116, 1977, Agric. Biol. Chem., 37, 2013-2023, 1973, and Agric. Biol. Chem., 51, 2089-2094, 1987. With reference to these literatures, microorganisms capable of forming and accumulating various amino acids can be produced.
Also, many examples of a method for producing microorganisms capable of forming and accumulating amino acids by any of or two or more of methods (a) to (e) above or a combination thereof are described in Biotechnology 2nd ed., vol. 6, Products of Primary Metabolism (VCH Verlagsgesellschaft mbH, Weinheim, 1996), section 14a or 14b, Advances in Biochemical Engineering/Biotechnology 79, 1-35, 2003, or Amino san Hakko (Amino acid fermentation), Japan Scientific Societies Press, Hiroshi Aida et al. (1986). In addition, many reports have been made concerning a specific method for producing microorganisms capable of forming and accumulating amino acids. Examples thereof include Japanese Published Unexamined Patent Application No. 2003-164297, Agric. Biol. Chem., 39, 153-160, 1975, Agric. Biol. Chem., 39, 1149-1153, 1975, Japanese Published Unexamined Patent Application No. 13599/83(1983), J. Gen. Appl. Microbiol., 4, 272-283, 1958, Japanese Published Unexamined Patent Application No. 94985/88(1988), Agric. Biol. Chem., 37, 2013-2023, 1973, WO 97/15673, Japanese Published Unexamined Patent Application No. 18596/81(1981), Japanese Published Unexamined Patent Application No. 144092/81(1981), and Japanese Published Unexamined Patent Application No. 2003-511086. With reference to these literatures, E. coli mutants capable of producing one or more kinds of amino acids can be produced. Also, E. coli capable of producing other useful substance can be produced by reinforcement of enzymes for biosynthesis of said useful substance, and modification of biosynthesis regulatory mechanism, excretion mechanism and the like in the same way as in the production of E. coli mutant strain capable of producing an amino acid.
The present invention relates to a method for producing useful substances characterized by culturing the E. coli mutant strain of the present invention in a medium so as to form and accumulate such useful substances in the culture and recovering the useful substances from the culture.
Useful substances are not particularly limited, provided that such substances can be produced with the use of the E. coli mutant strain of the present invention. Examples thereof include proteins, amino acids, nucleic acids, vitamins, saccharides, organic acids, and lipids. Examples of proteins include inosine kinase, glutamate 5-kinase (EC 2.7.2.11), glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.41), pyrroline-5-carboxylate reductase (EC 1.5.1.2), and human granulocyte-colony-stimulating factors. An example of peptide is glutathione. Examples of amino acids include L-alanine, glycine, L-glutamine, L-glutamic acid, L-asparagine, L-aspartic acid, L-lysine, L-methionine, L-threonine, L-leucine, L-valine, L-isoleucine, L-proline, L-histidine, L-arginine, L-tyrosine, L-tryptophane, L-phenylalanine, L-serine, L-cysteine, L-3-hydroxyproline, and L-4-hydroxyproline. Examples of nucleic acids include adenosine triphosphate, inosine, guanosine, inosinic acid, and guanylic acid. Examples of vitamins include riboflavin, thiamine, and ascorbic acid. Examples of saccharides include xylose and xylitol. Examples of lipids include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).
The method of producing a useful substance of the present invention also includes a method of producing a useful substance using an enzyme reaction requiring ATP, which comprises using the E. coli mutant strain of the present invention to produce an enzyme protein catalyzing the enzyme reaction and supplying, from the E. coli mutant strain of the present invention, ATP necessary for maintaining progress of the reaction catalyzed by the enzyme protein.
The E. coli mutant strain that is used for the production method of the present invention can be cultured in accordance with a conventional method as described below.
A medium for culturing E. coli mutant strain which is used for the production method of the present invention may be natural or synthetic medium as long as it contains carbon sources, nitrogen sources, inorganic salts, etc. assimilable by the microorganisms and the microorganisms can be efficiently cultured therein.
Any carbon sources assimilable by the microorganisms can be used. As carbon sources, carbohydrates such as glucose, fructose, sucrose, molasses containing any of such substances, starch, and starch hydrolysate; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol, and the like can be used.
As nitrogen sources, ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean cake and hydrolysate of soybean cake, and various fermentation microorganisms and digests thereof, and the like can be used.
As inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, iron(I) sulfate, manganese sulfate, copper sulfate, and calcium carbonate, and the like can be used.
Culturing is carried out under aerobic conditions such as shaking culture or submerged aeration agitation culture. Culturing temperature is preferably 15 to 40° C., and culture time is generally 16 hours to 7 days. During culturing, the pH is preferably maintained at 3.0 to 9.0. The pH is adjusted with an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like.
When useful substances are formed and accumulated extracellularly, precipitates such as cells are removed from the culture after the completion of culturing, and target useful substances can be isolated and purified from the culture via combined use of ion-exchange treatment, concentration, salting out, and other means.
When useful substances are formed and accumulated intracellularly, cells are recovered from the culture after the completion of culturing and then disrupted via adequate means such as mechanical or chemical means. Target useful substances can be isolated and purified from the solution of disrupted cells via combined use of ion-exchange treatment, concentration, salting out, and other means.
According to the chromosome processing methods described in JP-A-2004-166576 and WO2006/57341, namely, a method using sacB gene encoding levansucrase derived from Bucillus subtilis, reduced-genome strains S1-S10 strains defective in the regions containing the genes shown in the following Table 2 on the chromosome of the Escherichia coli W3110 strain were prepared from the Step28 strain (WO2006/57341) defective in the 1026.7 kbp region on the chromosome of W3110red strain (WO2006/57341), which is a strain wherein the recombination system of Escherichia coli K-12 lineage strain W3110 strain has been replaced by a Red recombination system derived from λ phage. According to the method, primer DNAs are designed based on the nucleotide sequences on both ends of the region desired to be defective on the chromosomal DNA and, using these primer DNAs and chromosomal DNA as a template, PCR reaction is performed to amplify DNA fragment, using which any region on the chromosomal DNA can be eliminated.
Then, the growth was compared among the W3110red strain, reduced-genome strain S28 strain (WO2006/57341) and S1-S10 strains. The gene group wherein step28 strain is defective on the chromosomal DNA is shown in Table 2.
Using the strains indicated in Table 2, a growth test was performed. For the growth test, each strain was spread on an LB agar medium (LB medium [Bacto Tryptone (manufactured by Difco) 10 g/l, yeast extract (manufactured by Difco) 10 g/l, sodium chloride 5 g/l] added with Bacto Agar (manufactured by Difco) (15 g/l) and solidified as a plate) containing 30 mg/l kanamycin, and cultured at 37° C. overnight to give bacterial cells. A small amount of respective bacterial cells grown on the LB agar medium was seeded on 5 ml of an LB medium containing 30 mg/l kanamycin and cultured at 37° C. overnight to give a culture. 300 μl of the culture was seeded on 30 ml of a is growth test medium (11.28 g/l 5×M9 premixed salts, 100 mM MOPS buffer, 6% glucose, 1% yeast extract, 2 mM MgSO4, 0.1 mM CaCl2, 5 mg/l FeSO4, pH 6.8, 30 mg/l kanamycin), and cultured with shaking for 24 hr at 30° C. The turbidity of each reduced-genome strain after culture was determined by measuring the absorbance at 660 nm. The turbidity of each reduced-genome strain based on that of the W3110red strain as 100 and the total deletion length of the chromosomal DNA of each reduced-genome strain are shown in Table 3.
The ATP production activity of W3110red strain, Step28 strain and S10 strain was each measured by the following method. Each strain was spread on an LB agar medium, and cultured at 30° C. overnight. A small amount of the bacterial cells grown on the LB agar medium was scraped and seeded in a 96 well deep well plate containing LB medium (1 ml) containing 3% glucose, and cultured at 30° C. for 24 hr. The turbidity after culture was determined by measuring the absorbance at 590 nm. In addition, the ATP production activity per culture medium was measured by the method described in J. Biomol. Screen., 11, 310 (2006). The ATP production activity of Step28 strain and S10 strain per culture medium based on that of the W3110red strain per culture medium as 100 is shown in Table 4. The S10 strain showed a remarkably improved ATP production activity per culture medium.
A strain, wherein the λ Red recombination system retained by the step28 strain and S10 strain was substituted by a wild-type recBCD recombination system, was prepared as follows.
The ygdB gene located near the chromosome where the λRed gene group is present is substituted by a DNA fragment containing chloramphenicol resistance gene and levansucrase gene by the method described in JP-A-2004-166576. The P1 phage obtained from the wild-type strain was transduced into the prepared strain to give a sucrose resistant strain, whereby a strain wherein the vicinity of ygdB gene was substituted by a wild-type chromosome fragment (including recBCD gene group) is obtained. The recBCD strains prepared from the Step28 strain and S10 strain were designated as S28W strain and S10W strain, respectively.
Then, the lacI gene was reintegrated with the S28W strain and S10W strain by the following method. Using the method described in JP-A-2004-166576, a DNA fragment containing chloramphenicol resistance gene and levansucrase gene was inserted into the W3110red strain chromosome while simultaneously deleting the region from the yafW gene to the yahM gene described in Table 1. P1 phage was prepared from said strain, transduced into the S28W strain and S10W strain to give a chloramphenicol resistant strain, whereby lacI revertant, wherein a lacI gene near the marker gene was transduced, was obtained. A marker portion containing chloramphenicol resistance gene and levansucrase gene was removed from the obtained lacI gene revertant by the method described in JP-A-2004-166576. The finally prepared lacI gene revertants derived from S28W strain and S10W strain were designated as S28W_lacI strain and S10W_lacI strain.
Then, using the method described in Appl. Environ. Microbiol., 73, 7657 (2007), a xylitol-producing gene group was introduced into each of the W3110 strain, S28W_lacI strain and S10W_lacI strain to give strains imparted with conversion capacity from xylose to xylitol. Each of the prepared strains was seeded on a medium (30 ml) containing M9 minimal medium added with 6% glucose, 8% xylose, 1% yeast extract, 0.1 mM IPTG, 100 mM MOPS buffer, and cultured with shaking in a 300 ml baffled flask at 30° C., 250 rpm. The xylitol amount in a culture medium at 46 hr culture was measured. As a result, 14.3, 13.5, 20.6 g/l of xylitol was produced in the culture medium by culturing xylitol-producing strains with W3110 strain, S28W_lacI strain and S10W_lacI strain as parental strains, respectively. The productivity of xylitol was the highest in the strain derived the S10 strain.
Using the E. coli mutant strain of the present invention as a host cell, a useful substance can be produced efficiently.
| Number | Date | Country | Kind |
|---|---|---|---|
| 068793/2008 | Mar 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/055267 | 3/18/2009 | WO | 00 | 9/16/2010 |
