JP3975470B2 - Method for producing L-isoleucine by fermentation - Google Patents

Description

【０１５４】

【０１５５】

【０１５６】

【０１５７】

【０１５８】

【０１５９】

【図面の簡単な説明】
【図１】プラスミドｐＨＳＧＳＫの構築手順を示す。
図１で用いられている略記号のうち、ＨはＤＮＡ上に存在するＨｉｎｄIII 切断部位を示す。同様にＰはＰｓｔＩ切断部位を、ＫはＫｐｎＩ切断部位を、ＢはＢａｍＨＩ切断部位を、ＸはＸｂａＩ切断部位を、ＳｍはＳｍａＩ切断部位を示す。＆は制限酵素による再切断が不能であることを示す。
Ｐ→はプロモーターを示す。ＳＤはシャイン・ダルガノ配列を示す。ＡＴＧは翻訳開始コドンを示す。ＴＧＡは翻訳終始コドンを示す。
Ａｐは形質転換株にアンピシリン耐性を付与するマーカー遺伝子である。Ｃｍは形質転換株にクロラムフェニコール耐性を付与するマーカー遺伝子である。
【図２】ｉｌｖＧＭＥＤＡオペロンのアテニュエーションに必要な領域を除去したｉｌｖＧＭ遺伝子を造成し、プラスミドｐｄＧＭ１を構築する手順を示す。
図２で用いられている略記号は、図１で用いられている略記号と同じ意味のものである。
【図３】プラスミドｐＭＷＧＭＡ２の構築手順を示す。
図３で用いられている略記号は、図１で用いられている略記号と同じ意味のものである。
【図４】プラスミドｐＭＷＤ５の構築手順を示す。
図４で用いられている略記号は、図１で用いられている略記号と同じ意味のものであるが、加えて、ＳはＳａｌＩ切断部位を示す。また、ｐａｒはプラスミドの安定化領域を示す。[0001]
[Industrial application fields]
The present invention relates to a method for producing L-isoleucine by a fermentation method and an Escherichia bacterium used in the production method. L-isoleucine is an essential amino acid for humans and animals, and is mainly useful for pharmaceutical use including nutrients.
[0002]
[Prior art]
Since isoleucine has two asymmetric carbons, it is difficult to produce only the L form industrially at low cost by the method using chemical synthesis. A method for producing L-isoleucine by bioconversion using a precursor of L-isoleucine biosynthesis such as α-aminobutyric acid and α-ketobutyric acid as a raw material is also known. However, these methods are expensive, and are not advantageous methods for producing L-isoleucine at an industrially low cost.
[0003]
The following is known as a method for producing L-isoleucine by direct fermentation using an inexpensive carbon source and nitrogen source. 1) A method using a mutant strain of Corynebacterium, Serratia or Escherichia resistant to an antagonist of L-isoleucine as an L-isoleucine producing strain (Japanese Patent Publication Nos. 51-21077 and 52-4629) 2) Escherichia or Coryne in which threonine deaminase or acetohydroxy acid synthase, which is a key enzyme for biosynthesis of L-isoleucine, is enhanced by recombinant DNA technology, is disclosed in Japanese Patent Publication Nos. 56-29998 and 56-3035. A method in which a microorganism belonging to the genus Bacteria is used as an L-isoleucine-producing strain (JP-A-2-458, JP-A-2-42988, etc.).
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for producing L-isoleucine at a higher yield and at a lower cost by using a fermentation method.
[0005]
[Means for Solving the Problems]
The present inventors have provided a thrABC operon containing a thrA gene encoding aspartokinase I-homoserine dehydrogenase I substantially desensitized by L-threonine, and threonine substantially desensitized by L-isoleucine. L. isoleucine is produced using an Escherichia bacterium having L-isoleucine productivity, characterized by retaining an ilvGMEDA operon containing the ilvA gene encoding deaminase and excluding the region necessary for attenuation. We succeeded in manufacturing at a higher yield and lower cost.
[0006]
The present invention relates to a thrABC operon containing a thrA gene encoding aspartokinase I-homoserine dehydrogenase I substantially desensitized by L-threonine, and threonine deaminase substantially desensitized by L-isoleucine. The present invention provides an Escherichia bacterium having L-isoleucine productivity, characterized in that it retains the ilvGMEDA operon containing the coding ilvA gene and from which the region necessary for attenuation is removed.
The Escherichia bacterium of the present invention can retain the thrABC operon and the ilvGMEDA operon on a plasmid.
The bacterium belonging to the genus Escherichia of the present invention can hold two types of plasmids: a plasmid (A) carrying the thrABC operon and a plasmid (B) carrying the ilvGMEDA operon.
Specific examples of the ilvGMEDA operon include those having a DNA sequence from which DNA sequences Nos. 953 to 1160 are deleted from SEQ ID NO: 1 in Sequence Listing.
[0007]
A specific example of the plasmid (A) is pVIC40, and a specific example of the plasmid (B) is pMWD5.
A preferred Escherichia bacterium is Escherichia coli.
A more preferred Escherichia bacterium of the present invention is a thrABC that lacks the thrC gene, can grow in the presence of 5 mg / ml of L-threonine, lacks threonine dehydrogenase activity, and the ilvA gene has a leaky mutation. This is Escherichia coli in which an operon and the ilvGMEDA operon are introduced. Specific examples thereof include Escherichia coli AJ12919 strain.
[0008]
The present invention also provides a thrABC operon comprising a thrA gene encoding aspartokinase I-homoserine dehydrogenase I substantially desensitized by L-threonine, and an aspart that is substantially desensitized by L-lysine. Retaining the lysC gene encoding kinase III and the ilvGMEDA operon containing the ilvA gene encoding threonine deaminase substantially desensitized by L-isoleucine and having a region necessary for attenuation removed. The present invention provides a bacterium belonging to the genus Escherichia having L-isoleucine productivity.
The bacterium belonging to the genus Escherichia of the present invention can retain the thrABC operon, the lysC gene, and the ilvGMEDA operon on a plasmid.
[0009]
The bacterium belonging to the genus Escherichia of the present invention can hold two types of plasmids: a plasmid (C) carrying the thrABC operon and the lysC gene and a plasmid (B) carrying the ilvGMEDA operon.
Specific examples of the ilvGMEDA operon include those having a DNA sequence from which DNA sequences Nos. 953 to 1160 are deleted from SEQ ID NO: 1 in Sequence Listing.
A specific example of the plasmid (C) is pVICLC * 80A, and a specific example of the plasmid (B) is pMWD5.
A preferred Escherichia bacterium is Escherichia coli.
A more preferred Escherichia bacterium of the present invention is a thrABC that lacks the thrC gene, can grow in the presence of 5 mg / ml of L-threonine, lacks threonine dehydrogenase activity, and the ilvA gene has a leaky mutation. Escherichia coli into which an operon, the lysC gene, and the ilvGMEDA operon have been introduced. A specific example thereof is Escherichia coli AJ13100 strain.
[0010]
The present invention further comprises culturing the aforementioned Escherichia bacterium in a medium, producing and accumulating L-isoleucine in the medium, and collecting L-isoleucine from the medium, and a method for producing L-isoleucine by a fermentation method Is to provide.
Hereinafter, in the present specification, the thrA gene encoding aspartokinase I-homoserine dehydrogenase I in which inhibition by L-threonine has been substantially eliminated may be referred to as a “released thrA gene”. In addition, the thrABC operon containing the thrA gene encoding aspartokinase I-homoserine dehydrogenase I, which is substantially desensitized by L-threonine, may be referred to as a “released thrABC operon”.
[0011]
In the present specification, an ilvA gene encoding a threonine deaminase from which inhibition by L-isoleucine has been substantially released is sometimes referred to as a “released ilvA gene”. In addition, the ilvGMEDA operon containing the ilvA gene (released ilvA gene) encoding threonine deaminase whose inhibition by L-isoleucine is substantially released may be referred to as a “released ilvGMEDA operon”. The ilvGMEDA operon containing the release-type ilvA gene and from which a region necessary for attenuation is removed may be referred to as a “fully release-type ilvGMEDA operon”.
In the present specification, aspartokinase I-homoserine dehydrogenase I may be abbreviated as “AKI-HDI”. Further, threonine deaminase may be abbreviated as “TD”.
[0012]
In the present specification, aspartokinase I-homoserine dehydrogenase I in which inhibition by L-threonine is substantially released is sometimes referred to as “released AKI-HDI”. In addition, threonine deaminase from which inhibition by L-isoleucine has been substantially eliminated may be referred to as “released TD”.
Hereinafter, the present invention will be described in detail.
[0013]
<1. ThrABC operon containing thrA gene whose inhibition by L-threonine is substantially released>
[0014]
The thrABC operon containing the thrA gene encoding aspartokinase I-homoserine dehydrogenase I, which is substantially inhibited from being inhibited by L-threonine, that is, the released thrABC operon is wild-type thrABC in that the thrA gene contained therein has a mutation. Different from operons. The mutation means a mutation that cancels feedback inhibition by L-threonine for AKI-HDI encoded by the thrA gene.
[0015]
A method for obtaining such a release-type thrABC operon is as follows. First, DNA containing the wild-type thrABC operon is subjected to in vitro mutation treatment, and the DNA after mutation treatment and a vector DNA suitable for the host are ligated to obtain recombinant DNA. If a recombinant DNA is introduced into a host microorganism to obtain a transformant, and a transformant that has been expressed so as to express a release type AKI-HDI is selected, the transformant becomes a release type thrABC operon. Holding. In addition, DNA containing the wild-type thrABC operon is ligated with a vector DNA compatible with the host to obtain recombinant DNA, and then the recombinant DNA is subjected to in vitro mutation treatment, and the recombinant DNA after mutation treatment is used as a host microorganism. Even if a transformant is obtained by introduction and a transformant that has been expressed so as to express the release type AKI-HDI is selected, the transformant retains the release type thrABC operon.
[0016]
After a microorganism that produces wild-type AKI-HDI is subjected to mutation treatment to create a mutant strain that produces release-type AKI-HDI, a release-type thrABC operon may be obtained from the mutant strain. Alternatively, after transforming a transformant introduced with a recombinant DNA linked with a wild-type thrABC operon to create a mutant that produces mutant AKI-HDI, a recombinant DNA is produced from the mutant. If recovered, a release-type thrABC operon may be created on the same DNA.
[0017]
Examples of the drug for in vitro mutation treatment of DNA include hydroxylamine. Hydroxylamine is a chemical mutation treatment agent that causes a mutation from cytosine to thymine by changing cytosine to N4-hydroxycytosine. When the microorganism itself is subjected to mutation treatment, treatment with a mutation agent usually used for artificial mutation such as ultraviolet irradiation or N-methyl-N′-nitro-N-nitrosoguanidine (NTG) is performed.
Examples of the thrABC operon include those derived from bacteria belonging to the genus Escherichia, particularly the thrABC operon derived from Escherichia coli (hereinafter sometimes referred to as “E. coli”).
[0018]
When the thrABC operon derived from bacteria belonging to the genus Escherichia is used, any microorganisms belonging to the genus Escherichia may be used as the donor bacteria for the DNA containing the wild-type thrABC operon. Specifically, those listed in the book by Neidhardt et al. (Neidhardt, F. C. et. Al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, table 1) can be used. For example, E.coli JM109 strain and MC1061 strain can be mentioned. When a wild strain is used as a donor bacterium for DNA containing the thrABC operon, DNA containing the wild-type thrABC operon can be obtained.
[0019]
(1) Acquisition of wild-type thrABC operon
Hereinafter, preparation examples of DNA containing the thrABC operon will be described. First, E. coli having wild type AKI-HDI, for example, MC1061 strain is cultured to obtain a culture. In order to culture the microorganism, it may be cultured by a normal solid culture method, but it is preferable to employ a liquid culture method in consideration of the efficiency in collecting bacteria. As the medium, a medium obtained by adding sodium chloride (NaCl) to yeast extract, peptone, tryptone, or meat extract is used. Specifically, L-broth (Bacto tryptone 1%, Bacto yeast extract 0.5%, NaCl 0.5%, glucose 0.1%, pH = 7.2). The initial pH of the medium is suitably adjusted to 6-8. Further, the culture is performed at 30 to 42 ° C., preferably around 37 ° C. for 4 to 24 hours, by aeration and agitation deep culture, shaking culture or stationary culture.
[0020]
The culture thus obtained is centrifuged at, for example, 3,000 r.p.m. for 5 minutes to obtain cells of E. coli MC1061 strain. From these cells, for example, by the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619, (1963)), the method of KS Kirby (Biochem. J., 64, 405, (1956)), etc. Chromosomal DNA can be obtained.
[0021]
In order to isolate the thrABC operon from the chromosomal DNA thus obtained, a chromosomal DNA library is prepared. First, chromosomal DNA is partially decomposed with an appropriate restriction enzyme to obtain a mixture of various fragments. For example, Sau3AI is digested by acting on chromosomal DNA for various times (1 minute to 2 hours) at a temperature of 30 ° C. or higher, preferably 37 ° C. and an enzyme concentration of 1 to 10 units / ml.
[0022]
Subsequently, the cleaved chromosomal DNA fragment is ligated to a vector DNA capable of autonomous replication in Escherichia bacterial cells to produce a recombinant DNA. Specifically, a restriction enzyme that produces the same base sequence as the restriction enzyme Sau3AI used for cleaving chromosomal DNA, such as BamHI, is at least 30 hours at a temperature of 30 ° C. or more and an enzyme concentration of 1 to 100 units / ml. Preferably, it is allowed to act on vector DNA for 1 to 3 hours to completely digest it and cleave it. Next, the chromosomal DNA fragment mixture obtained as described above and the cleaved vector DNA are mixed, and this is mixed with a DNA ligase, preferably T4 DNA ligase, at a temperature of 4-16 ° C. and an enzyme concentration of 1-100 units / ml. Recombinant DNA is obtained by allowing to act for 1 hour or more, preferably 6 to 24 hours under the conditions.
[0023]
The obtained recombinant DNA is used to transform Escherichia microorganisms such as Escherichia coli K-12 strain or E. coli Gif106M1 strain (thrA1101, metLM1000, lysC1001, ilvA, argH). Convert to make a chromosomal DNA library. This transformation can be accomplished by DM Morrison's method (Methods in Enzymology 68, 326, 1979) or by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159). (1970)) etc. The E. coli Gif106M1 strain is available from the E. coli Genetic Stock Center (Connecticut, USA).
[0024]
From the obtained chromosomal DNA library, a strain having increased aspartokinase activity or a strain having complemented auxotrophy due to an aspartokinase gene deficiency is selected, and a strain having a recombinant DNA containing the thrA gene Get.
[0025]
In order to confirm whether the candidate strain having the recombinant DNA containing the thrA gene retains the recombinant DNA cloned in the thrA gene, a cell extract is prepared from the candidate strain, and a crude enzyme solution is added from the cell extract. This can be achieved by preparing and confirming that aspartokinase activity is increased. The enzyme activity of aspartokinase can be measured by the method of Stattman et al. (Stadtman, ER, Cohen, GN, LeBras, G., and Robichon-Szulmajster, H., J. Biol. Chem., 236 , 2033 (1961)).
[0026]
Moreover, since the aspartokinase-deficient mutant is auxotrophic for L-lysine, L-threonine, L-methinion and diaminopimelate, when the aspartokinase-deficient mutant is used as a host, L-lysine, L-threonine A thrA gene by isolating a strain capable of growing on a minimal medium containing no L-methinion and diaminopimelic acid, or on a medium not containing homoserine and diaminopimelic acid, and recovering the recombinant DNA from the strain. DNA fragments containing can be obtained.
[0027]
However, E. coli has three types of aspartokinase, and there are also three types of genes encoding aspartokinase. Therefore, in order to confirm whether or not the target thrA gene has been isolated, it is preferable to analyze and confirm its base sequence or restriction enzyme cleavage pattern. The base sequence of the thrA gene of E. coli has already been reported (Katinka, M et al., Proc. Natl. Acad. Sci. U.S.A., 77, 5730 (1980)).
[0028]
A recombinant DNA in which a DNA containing the thrA gene is inserted into a vector DNA from the above strain is obtained by, for example, the method of P. Guerry et al. (J. Bacteriol., 116, 1064, (1973)) or the method of DB Clewell. (J. Bacteriol., 110, 667, (1972)).
[0029]
In order to isolate the full length of the thrABC operon, it is necessary to select again a transformant carrying a recombinant DNA containing the full length of the thrABC operon from the chromosomal DNA library. That is, selection by colony hybridization is performed using the obtained thrA gene as a probe. The colony hybridization method follows a conventional method (Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989)).
[0030]
In order to confirm whether or not the full length of the thrABC operon was isolated, the isolated candidate DNA was introduced into a strain lacking the thrB gene to confirm that the thrB gene deficiency was recovered and further isolated. A candidate DNA may be introduced into a strain lacking the thrC gene to confirm that the thrC gene deficiency is recovered. As a strain lacking the thrB gene, there is an E. coli YA73 strain (thrB1000, thi-1, relA1, λ-, spoT1). As a strain lacking the thrC gene, there is E. coli Gif41 strain (thrC1001, thi-1, relA1, λ-, spoT1). The E. coli YA73 strain and E. coli Gif41 strain are available from the E. coli Genetic Stock Center (Connecticut, USA).
[0031]
In addition, in order to confirm whether or not the full length of the thrABC operon has been isolated, the base sequence or restriction enzyme cleavage pattern of the isolated candidate DNA is analyzed and confirmed. The nucleotide sequence of the thrB gene of E. coli has already been reported (Cossart, P et al., Nucleic Acids Res., 9, 339 (1981)), and the nucleotide sequence of the thrC gene of E. coli has already been reported. (Parsot, C et al., Nucleic Acids Res., 11, 7331 (1983)).
[0032]
The wild-type thrABC operon was obtained by preparing a chromosomal DNA from a strain having the wild-type thrABC operon on the chromosome by the method of Saito, Miura, etc., and using the polymerase chain reaction method (PCR: Polymerase chain reaction; White, TJ et al; Trends Genet. 5, 185 (1989)), and can also be performed by amplifying the thrABC operon. A DNA primer used for the amplification reaction is complementary to both 3 'ends of a DNA duplex containing the entire region or a partial region of the thrABC operon. When only a partial region of the thrABC operon is amplified, it is necessary to screen a DNA fragment containing the entire region from the chromosomal DNA library using the DNA fragment as a probe. When the entire region of the thrABC operon is amplified, the PCR reaction solution containing the amplified thrABC operon-containing DNA fragment is subjected to agarose gel electrophoresis, and then the desired DNA fragment is extracted to contain the thrABC operon. DNA fragments can be recovered.
[0033]
As a DNA primer, for example, an appropriate primer may be prepared based on a sequence known in E. coli. The base sequence of the thrA gene of E. coli is reported in Katinka, M et al., Proc. Natl. Acad. Sci. USA, 77, 5730 (1980), and the base sequence of the thrB gene of E. coli is Cossart. , P et al., Nucleic Acids Res., 9, 339 (1981), and the nucleotide sequence of the thrC gene of E. coli is Parsot, C et al., Nucleic Acids Res., 11, 7331 (1983). ).
[0034]
The primer DNA is synthesized by a conventional method such as the phosphoramidide method (see Tetrahedron Letters, 22, 1859 (1981)) using a commercially available DNA synthesizer (for example, DNA synthesizer model 380B manufactured by Applied Biosystems). be able to. The PCR reaction was carried out using a commercially available PCR reaction apparatus (Perkin Elmer DNA thermal cycler PJ2000 type or the like), Taq DNA polymerase (supplied by Takara Shuzo Co., Ltd.), and a method designated by the supplier. Can be done according to.
[0035]
The thrABC operon amplified by the PCR method is connected to a vector DNA capable of autonomous replication in Escherichia bacterial cells and introduced into Escherichia bacterial cells, thereby facilitating operations such as introducing mutations into the thrA gene. . The vector DNA used, the transformation method, and the method for confirming the presence of the thrABC operon are the same as described above.
[0036]
(2) Introduction of mutation into thrABC operon
The thrABC operon obtained as described above may be subjected to mutations such as substitution, insertion and deletion of amino acid residues by recombinant PCR (Higuchi, R., 61, PCR Technology (Erlich, HA Eds , Stockton press (1989))), site-directed mutagenesis (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et.al., Meth. In Enzymol ., 154, 367 (1987)). When these methods are used, the target mutation can be caused at the target site.
[0037]
Moreover, according to the method of chemically synthesizing the target gene, mutations to the target site or random mutations can be introduced.
Furthermore, there is a method (Hashimoto, T. and Sekiguchi, M., J. Bacteriol., 159, 1039 (1984)) in which the thrABC operon on the chromosome or plasmid is directly treated with hydroxylamine. Alternatively, a method of irradiating an bacterium belonging to the genus Escherichia having a thrABC operon with ultraviolet rays or a method of treating with a chemical agent such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid may be used. According to these methods, mutations can be introduced at random.
[0038]
As a method for selecting the released thrABC operon, first, a recombinant DNA comprising a DNA fragment containing the thrABC operon and a vector DNA is directly mutated with hydroxylamine or the like, and for example, E. coli Gif106M1 is transformed. . Next, the transformed strain is cultured in a minimal medium such as M9 containing an antagonist of L-threonine, such as α-amino-β-hydroxyvaleric acid (AHV). A strain carrying a recombinant DNA containing the wild-type thrABC operon can synthesize L-homoserine and diaminopimelic acid (DAP) because AKI-HDI expressed by the recombinant DNA is inhibited by AHV. Eliminates growth. In contrast, a strain carrying a recombinant DNA containing a thrABC operon containing a thrA gene encoding AKI-HDI that has been desensitized by L-threonine is released by the thrA gene encoded in the recombinant DNA. Since type AKI-HDI is not inhibited by AHV, it should be able to grow on minimal medium supplemented with AHV. By utilizing this phenomenon, a strain whose growth is resistant to AHV, that is, a strain carrying a recombinant DNA containing a thrABC operon containing a released thrA gene whose inhibition is released can be selected.
[0039]
The released thrABC operon thus obtained is introduced into a suitable host microorganism as a recombinant DNA and expressed to carry AKI-HDI in which feedback inhibition is released, and the L-threonine biosynthesis encoded by the thrABC operon. Microorganisms with enhanced expression of synthetic enzymes can be obtained. As the host, microorganisms belonging to the genus Escherichia are preferable, and examples thereof include Escherichia coli (E. coli).
[0040]
Alternatively, a release-type thrABC operon extracted from the recombinant DNA and inserted into another vector DNA may be used. The vector DNA that can be used in the present invention is preferably a plasmid vector DNA, and examples thereof include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, and pMW218. In addition, phage DNA vectors can be used.
[0041]
Furthermore, in order to efficiently perform the expression of the release-type thrABC operon, the transcription control region (operator or attenuator) located upstream of the release-type thrABC operon may be removed. Alternatively, other promoters that work in microorganisms such as lac, trp, and PL may be linked, and a promoter unique to the thrABC operon may be amplified and used.
[0042]
In addition, as described above, the thrABC operon inserted into a vector DNA capable of autonomous replication may be introduced into the host and retained in the host as extrachromosomal DNA such as a plasmid. Daction, transposon (Berg, DE and Berg, CM, Bio / Technol., 1, 417 (1983)), Mu phage (JP-A-2-109985) or homologous recombination (Experiments in Molecular Genetics, Cold Spring Habor Lab. (1972)) may be incorporated into the chromosome of the host microorganism.
[0043]
<2. IlvGMEDA operon containing the ilvA gene whose inhibition by L-isoleucine is substantially released and the region necessary for attenuation is removed>
The ilvGMEDA operon containing the ilvA gene encoding threonine deaminase substantially desensitized by L-isoleucine, that is, the released ilvGMEDA operon differs from the wild type ilvGMEDA operon in that the contained ilvA gene has a mutation. This mutation means a mutation that cancels feedback inhibition by L-isoleucine on TD encoded by the ilvA gene.
[0044]
The ilvGMEDA operon containing the ilvA gene encoding threonine deaminase substantially desensitized by L-isoleucine and having the region necessary for attenuation removed, ie, the completely released ilvGMEDA operon is added to the released ilvGMEDA operon. Thus, it is different from the wild type ilvGMEDA operon or the release type ilvGMEDA operon in that a mutation is introduced so that no attenuation occurs. This mutation includes not only a part of the attenuator existing in the 5 'upstream region of the ilvGMEDA operon, but a part or a part of the attenuator, and a part that inserts a new DNA fragment inside the attenuator.
[0045]
The method for obtaining the release type ilvGMEDA operon is as follows. First, DNA containing the wild type ilvGMEDA operon is subjected to in vitro mutation treatment, and the DNA after mutation treatment and a vector DNA suitable for the host are ligated to obtain recombinant DNA. If a recombinant DNA is introduced into a host microorganism to obtain a transformant, and a transformant that has been expressed so as to express a release TD is selected, the transformant retains the release ilvGMEDA operon. is doing. In addition, DNA containing the wild type ilvGMEDA operon is ligated with vector DNA suitable for the host to obtain recombinant DNA, and then the recombinant DNA is subjected to in vitro mutation treatment, and the recombinant DNA after mutation treatment is used as a host microorganism. Even when a transformant is obtained by introduction and a transformant that has been expressed so as to express the release TD is selected, the transformant retains the release ilvGMEDA operon.
[0046]
After mutating a microorganism that produces wild-type TD and creating a mutant strain that produces release-type TD, the release-type ilvGMEDA operon may be obtained from the mutant strain. Alternatively, after transforming a transformant introduced with a recombinant DNA to which the wild-type ilvGMEDA operon has been ligated to create a mutant strain producing mutant TD, the recombinant DNA can be recovered from the mutant strain. For example, a release-type ilvGMEDA operon may be created on the same DNA.
[0047]
Examples of the drug for in vitro mutation treatment of DNA include hydroxylamine. Hydroxylamine is a chemical mutation treatment agent that causes a mutation from cytosine to thymine by changing cytosine to N4-hydroxycytosine. When the microorganism itself is subjected to mutation treatment, treatment with a mutation agent usually used for artificial mutation such as ultraviolet irradiation or N-methyl-N′-nitro-N-nitrosoguanidine (NTG) is performed.
Examples of the ilvGMEDA operon include those derived from bacteria belonging to the genus Escherichia, in particular, the ilvGMEDA operon derived from E. coli.
[0048]
When the ilvGMEDA operon derived from the genus Escherichia is used, any microorganisms belonging to the genus Escherichia may be used as the donor bacteria for the DNA containing the wild type ilvGMEDA operon. Specifically, those listed in the book by Neidhardt et al. (Neidhardt, F.C. et.al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, table 1) can be used. When a wild strain is used as a donor strain for DNA containing the ilvGMEDA operon, DNA containing the wild type ilvGMEDA operon can be obtained.
[0049]
However, when E. coli is used as a DNA donor for the wild type ilvGMEDA operon, the K12 wild strain does not express active acetohydroxyacid synthase isozyme II (AHASII) because the ilvG gene has a frameshift mutation. (Proc. Natl. Acad. Sci. USA 78, 922, 1981). Therefore, when the K12 strain is used as a DNA donor bacterium, a mutant strain whose frame is returned so that the activity of acetohydroxy acid synthase II encoded by the ilvG gene is restored is used, and then the mutant strain is used as a DNA donor bacterium. There is a need. Alternatively, only the ilvG gene may be isolated using E. coli other than the strain derived from the K12 strain as a DNA donor and introduced into the ilvGMEDA operon derived from the K12 strain. That is, the ilvMEDA part is isolated using the K12 strain as a DNA donor, and only the ilvG gene is isolated using E. coli other than the strain derived from the K12 strain as a DNA donor, and both are ligated to form the full-length ilvGMEDA operon. The isozyme II (AHASII) of acetohydroxy acid synthase is composed of two large and small subunits, and the large subunit is encoded by the ilvG gene. The small subunit is encoded by the ilvM gene.
[0050]
The method for obtaining the fully released ilvGMEDA operon is as follows.
The position and base sequence of the attenuator existing in the 5 'upstream region of the ilvGMEDA operon has been reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). By using the released ilvGMEDA operon as a starting material, the fully released ilvGMEDA operon is obtained by preparing the ilvGMEDA operon from which the attenuator is completely removed.
[0051]
The base sequence described as SEQ ID NO: 1 in the sequence listing discloses a region necessary for attenuation. The DNA sequence from 999th to 1007 codes for the continuous part of valine residues present in the leader peptide, and the DNA sequence from 1008th to 1016th codes for the continuous part of isoleucine residues present in the leader peptide. To do. The DNA sequence from 1081 to 1104 encodes the portion that forms a row-independent terminator-like stem / loop in the attenuator.
[0052]
When a sufficient amount of L-isoleucine is present in the cell, transcription by RNA polymerase is terminated because a low-independent terminator-like stem / loop is formed by RNA transcribed from the 1081st to 1104th DNA sequences. The ilvGMEDA operon is not expressed.
[0053]
When L-isoleucine is deficient in the cell, the tRNA to which isoleucine is bound is deficient in the cell, and translation by ribosomes stagnates in a continuous portion of isoleucine residues present in the region encoding the leader peptide. As a result, mRNA forms a new three-dimensional structure, resulting in the formation of a low-independent terminator-like stem / loop formed by RNA transcribed from the 1081st to 1104th DNA sequences. It will not be done. For this reason, transcription by RNA polymerase continues and the ilvGMEDA operon is expressed.
Therefore, in order to remove the region necessary for attenuation, the DNA sequence from the 1008th to the 1016th sequence or the DNA sequence from the 1081st to the 1104th sequence of the sequence disclosed in SEQ ID NO: 1 in the sequence listing is removed. Just do it.
[0054]
By the way, removing the region necessary for attenuation means that a mutation has been introduced so that attenuation does not occur. Therefore, the mutation is not limited to the one that removes all attenuators existing in the 5 'upstream region of the ilvGMEDA operon. In short, it may be a mutation that prevents the attenuator from forming a row-independent terminator-like stem / loop. It may be a mutation that prevents a continuous part of isoleucine residues from being included in the leader peptide. This is because the attenuation does not work in either case.
[0055]
That is, this mutation includes not only a part of the attenuator existing in the 5 'upstream region of the ilvGMEDA operon, but a part of or a part near the attenuator, and a part that inserts a new DNA fragment inside the attenuator.
[0056]
The ilvGMEDA operon containing the ilvA gene encoding threonine deaminase whose inhibition by L-isoleucine has been substantially released and from which the region necessary for attenuation is removed, ie, the completely released ilvGMEDA operon is a wild-type ilvGMEDA operon. After the mutation was introduced to prevent attenuation, the mutation was introduced into the ilvA gene contained in the ilvGMEDA operon, and the inhibition of L-isoleucine on the threonine deaminase encoded by the ilvA gene was substantially reduced. It can also be prepared to be released.
[0057]
(1) Acquisition of wild type ilvGMEDA operon
In order to obtain DNA containing the ilvGMEDA operon, a method of obtaining the ilvGM gene, the ilvE gene, the ilvD gene and the ilvA gene and linking them can be considered.
[0058]
First, E. coli with wild-type TD For example, E. coli K12 strain, E. coli W3110 strain, E. coli MC1061 strain (the above three strains have ilvG caused frameshift), E. coli MI162 Strain (thr-10, car-94, λ-, relA1, ilvG603, thi-1) and E. coli B strain (the above two strains have normal ilvG) to obtain a culture . In order to culture the microorganism, it may be cultured by a normal solid culture method, but it is preferable to employ a liquid culture method in consideration of the efficiency in collecting bacteria. As the medium, a medium obtained by adding sodium chloride (NaCl) to yeast extract, peptone, tryptone, or meat extract is used. Specifically, L-broth (Bacto tryptone 1%, Bacto yeast extract 0.5%, NaCl 0.5%, glucose 0.1%, pH = 7.2). The initial pH of the medium is suitably adjusted to 6-8. Further, the culture is performed at 30 to 42 ° C., preferably around 37 ° C. for 4 to 24 hours, by aeration and agitation deep culture, shaking culture or stationary culture. The E. coli MI162 strain is available from the E. coli Genetic Stock Center (Connecticut, USA). The index number of this stock is CGSC5919. Detailed properties of this strain are described in Mol. Gen. Genet. 143, 243 (1976), J. Bacteriol., 149, 294 (1982).
[0059]
The culture thus obtained is centrifuged at, for example, 3,000 r.p.m. for 5 minutes to obtain E. coli cells. From this cell, chromosomal DNA can be obtained by the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)), the method of KS Kirby (Biochem. J., 64, 405 (1956)), etc. Can be obtained.
[0060]
In order to isolate the ilvGMEDA operon from the chromosomal DNA thus obtained, a chromosomal DNA library is prepared. First, chromosomal DNA is partially decomposed with an appropriate restriction enzyme to obtain a mixture of various fragments. For example, Sau3AI is digested by acting on chromosomal DNA for various times (1 minute to 2 hours) at a temperature of 30 ° C. or higher, preferably 37 ° C. and an enzyme concentration of 1 to 10 units / ml.
[0061]
Subsequently, the cleaved chromosomal DNA fragment is ligated to a vector DNA capable of autonomous replication in Escherichia bacterial cells to produce a recombinant DNA. Specifically, a restriction enzyme that produces the same base sequence as the restriction enzyme Sau3AI used for cleaving chromosomal DNA, such as BamHI, is at least 30 hours at a temperature of 30 ° C. or more and an enzyme concentration of 1 to 100 units / ml. Preferably, it is allowed to act on vector DNA for 1 to 3 hours to completely digest it and cleave it. Next, the chromosomal DNA fragment mixture obtained as described above and the cleaved vector DNA are mixed, and this is mixed with a DNA ligase, preferably T4 DNA ligase, at a temperature of 4-16 ° C. and an enzyme concentration of 1-100 units / ml. Recombinant DNA is obtained by allowing to act for 1 hour or more, preferably 6 to 24 hours under the conditions.
[0062]
Using the obtained recombinant DNA, microorganisms belonging to the genus Escherichia such as E. coli MI262 (leuB6, ilvI614, ilvH612, λ-, relA1, spoT1, ilvB619, ilvG603, ilvG605 (am), thi-1) Acetohydroxy acid synthase deficient mutant, E. coli AB2070 (proA2, trp-3, hisG4, ilvE12, metE46, thi-1, ara-9, lacY1 or lacZ4, galK2, malA1, mtl-1, rpsL8 or rpsL9 , ton-1, tsx-3, λR, λ-, supE44) or E. coli AB1280 strain (hisG1, ilvD16, metB1, argH1, thi-1, ara-13, lacY1 or dihydroxy acid dehydratase-deficient mutants such as lacZ4, gal-6, xyl-7, mtl-2, malA1, rpsL8,9 or 17, tonA2, λR, λ-, supE44), E. coli AB1255 strain (thi-1 , ilvA201, argH1, metB1, hisG1, lacY1 or lacZ4, malA1, mtl-2, xyl-7, ara-13, gal-6, rpsL8,9 or 17, tonA2, tsx-5, λR, λ-, supE44) A chromosomal DNA library by transforming a threonine deaminase-deficient mutant such as . This transformation can be accomplished by the DM Morrison method (Methods in Enzymology 68, 326, 1979) or by treating recipient cells with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and the like. The E. coli MI262 strain is available from the E. coli Genetic Stock Center (Connecticut, USA). The index number of this stock is CGSC5769. The detailed properties of this strain are described in Mol. Gen. Genet., 156, 1 (1977). The E. coli AB2070 strain is available from the E. coli Genetic Stock Center (Connecticut, USA). The index number of this stock is CGSC2070. The detailed properties of this strain are described in J. Bacteriol., 109, 730 (1972). The E. coli AB1280 strain is available from the E. coli Genetic Stock Center (Connecticut, USA). The index number of this stock is CGSC1280. The E. coli AB1255 strain is available from the E. coli Genetic Stock Center (Connecticut, USA). The index number of this stock is CGSC1255. The detailed properties of this strain are described in J. Bacteriol., 109, 730 (1972).
[0063]
By the way, since the whole nucleotide sequence of the ilvGMEDA operon has been clarified (Nucleic Acids Res. 15, 2137 (1987)), the target gene exists by selecting a specific restriction enzyme and digesting the chromosomal DNA. The DNA fragment can be of a specific length. It is more efficient to create a chromosomal DNA library using this recombinant DNA by ligating only a DNA fragment having this specific length to a vector DNA, and the target gene exists. DNA fragments to be obtained can be obtained.
[0064]
From the obtained chromosomal DNA library, a strain with increased acetohydroxy acid synthase activity or a strain with complemented auxotrophy due to acetohydroxy acid synthase gene deficiency is selected, and a recombinant DNA containing the ilvGM gene is selected. A strain is obtained.
From the obtained chromosomal DNA library, a strain having increased transaminase B activity or a strain having complemented auxotrophy due to transaminase B gene deficiency is selected to obtain a strain having a recombinant DNA containing the ilvE gene. .
[0065]
From the obtained chromosomal DNA library, a strain having increased dihydroxy acid dehydratase activity or a strain having complemented auxotrophy due to dihydroxy acid dehydratase gene deficiency is selected, and a strain having a recombinant DNA containing the ilvD gene Get.
[0066]
From the obtained chromosomal DNA library, a strain with increased threonine deaminase activity or a strain with complemented auxotrophy due to threonine deaminase gene deficiency is selected to obtain a strain having a recombinant DNA containing the ilvA gene. .
[0067]
In order to confirm whether a candidate strain having a recombinant DNA containing the ilvGM gene retains the recombinant DNA into which the ilvGM gene has been cloned, a cell extract is prepared from the candidate strain, and a crude enzyme solution is used from the cell extract. This can be achieved by preparing and confirming that acetohydroxy acid synthase activity is increased. A method for measuring the enzyme activity of acetohydroxy acid synthase can be performed by the method of M. D. Felice et al. (Methods in Enzymology 166, 241).
[0068]
In addition, since acetohydroxy acid synthase-deficient mutant strains are isoleucine, leucine and valine-requiring mutants, when acetohydroxy acid synthase-deficient mutant strains are used as hosts, they can grow on a minimal medium that does not contain valine. And a DNA fragment containing the ilvGM gene can be obtained by recovering the recombinant DNA from the strain.
[0069]
Alternatively, the base sequence of DNA containing the ilvGM gene has already been reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can also be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence in the report.
[0070]
As described above, a mutation has occurred in the open reading frame of the ilvG gene of E. coli K12. As a result, a frame shift occurs, and the translation ends because a stop codon appears in the middle. That is, the stop codon appears at 982 to 984th from the start codon ATG (1st to 3rd) of the ilvG gene. Therefore, when the ilvGM gene obtained from the same strain is used, it is necessary to restore the mutated portion to normal by the site-directed mutagenesis method. For example, in the ilvG gene (ilvG603) of Escherichia coli MI162 strain, two base pairs of TG are inserted before the termination codon TGA at the 982 to 984th positions, and the frame returns to normal. Others are described in detail in Fig. 2 of J. Bacteriol. 149, 294 (1982).
[0071]
A method for confirming whether a candidate strain having a recombinant DNA containing the ilvE gene retains the recombinant DNA into which the ilvE gene has been cloned is as follows. Since a transaminase B-deficient mutant is isoleucine-requiring, when a transaminase B-deficient mutant is used as a host, a strain that can grow on a minimal medium that does not contain isoleucine is isolated, and a recombinant DNA is isolated from the strain. Can be obtained to obtain a DNA fragment containing the ilvE gene.
[0072]
Alternatively, the base sequence of DNA containing the ilvE gene has already been reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can also be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence in the report.
[0073]
A method for confirming whether a candidate strain having a recombinant DNA containing the ilvD gene retains the recombinant DNA into which the ilvD gene has been cloned is as follows. Since the dihydroxy acid dehydratase-deficient mutant strain is isoleucine, leucine, and valine-requiring mutants, when the dihydroxy acid dehydratase-deficient mutant strain is used as a host, isolate the strain that can grow on a medium not containing valine, A DNA fragment containing the ilvD gene can be obtained by recovering the recombinant DNA from the strain.
[0074]
Alternatively, the base sequence of DNA containing the ilvD gene has already been reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can also be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence in the report.
[0075]
In order to confirm whether a candidate strain having a recombinant DNA containing the ilvA gene retains the recombinant DNA into which the ilvA gene has been cloned, a cell extract is prepared from the candidate strain, and a crude enzyme solution is added thereto. It can be achieved by preparing and confirming that threonine deaminase activity is increased. A method for measuring the enzyme activity of threonine deaminase is described in Methods in Enzymology 17B, 555 (1971).
[0076]
In addition, since a threonine deaminase-deficient mutant is isoleucine-requiring, when a threonine deaminase-deficient mutant is used as a host, a strain that can grow on a minimal medium not containing isoleucine is isolated and assembled from the strain. By recovering the replacement DNA, a DNA fragment containing the ilvA gene can be obtained.
[0077]
Alternatively, the base sequence of DNA containing the ilvA gene has already been reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)). Therefore, it can also be confirmed by isolating the recombinant DNA from the candidate strain, decoding its base sequence, and comparing it with the base sequence in the report.
From each of the above strains, recombinant DNA can be obtained by the method of P. Guerry et al. (J. Bacteriol., 116, 1064 (1973)), the method of DB Clewell (J. Bacteriol., 110, 667 (1972)), etc. It can be isolated.
[0078]
In order to isolate the full length of the ilvGMEDA operon, a DNA fragment having the ilvGM gene, a DNA fragment having the ilvE gene, a DNA fragment having the ilvD gene, and a DNA fragment having the ilvA gene are ligated. When ligating, the entire nucleotide sequence of the ilvGMEDA operon already reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)) can be referred to.
[0079]
The wild type ilvGMEDA operon was obtained by preparing a chromosomal DNA from a strain having the wild type ilvGMEDA operon on the chromosome by the method of Saito, Miura, etc., and using the polymerase chain reaction method (PCR: Polymerase chain reaction; White, TJ et al; Trends Genet. 5, 185 (1989)), and can also be performed by amplifying the ilvGMEDA operon. A DNA primer used for the amplification reaction is complementary to both 3 'ends of a DNA duplex containing the entire region or a partial region of the ilvGMEDA operon. When only a partial region of the ilvGMEDA operon is amplified, it is necessary to screen a DNA fragment containing the entire region from the chromosomal DNA library using the DNA fragment as a probe. When the entire region of the ilvGMEDA operon is amplified, the PCR reaction solution containing the amplified ilvGMEDA operon-containing DNA fragment is subjected to agarose gel electrophoresis, and then the desired DNA fragment is extracted to contain the ilvGMEDA operon. DNA fragments can be recovered.
To prepare a DNA primer, the entire nucleotide sequence of E. coli ilvGMEDA operon, which has already been reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)), can be referred to.
[0080]
The primer DNA is synthesized by a conventional method such as the phosphoramidide method (see Tetrahedron Letters, 22, 1859 (1981)) using a commercially available DNA synthesizer (for example, DNA synthesizer model 380B manufactured by Applied Biosystems). be able to. The PCR reaction was carried out using a commercially available PCR reaction apparatus (Perkin Elmer DNA thermal cycler PJ2000 type or the like), Taq DNA polymerase (supplied by Takara Shuzo Co., Ltd.), and a method designated by the supplier. Can be done according to.
[0081]
The ilvGMEDA operon amplified by PCR is connected to a vector DNA capable of autonomous replication in Escherichia bacterial cells and introduced into Escherichia bacterial cells, and is necessary for the operation of introducing mutations into the ilvA gene and attenuation. This makes it easy to perform operations such as removing a region. The vector DNA used, the transformation method, and the method for confirming the presence of the ilvGMEDA operon are the same as described above.
[0082]
(2) Introduction of mutations into the ilvGMEDA operon
The ilvGMEDA operon obtained as described above may be subjected to mutation such as substitution, insertion and deletion of amino acid residues by recombinant PCR (Higuchi, R., 61, PCR Technology (Erlich, HA Eds , Stockton press (1989))), site-directed mutagenesis (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et.al., Meth. In Enzymol ., 154, 367 (1987)). When these methods are used, the target mutation can be caused at the target site.
Moreover, according to the method of chemically synthesizing the target gene, mutations to the target site or random mutations can be introduced.
[0083]
Furthermore, there is a method (Hashimoto, T. and Sekiguchi, M., J. Bacteriol., 159, 1039 (1984)) in which the ilvGMEDA operon on the chromosome or plasmid is directly treated with hydroxylamine. Alternatively, a method of irradiating a bacterium belonging to the genus Escherichia having the ilvGMEDA operon with ultraviolet rays or a method of treating with a chemical agent such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid may be used. According to these methods, mutations can be introduced at random.
[0084]
A method for selecting a DNA fragment having a released ilvA gene is as follows. First, a recombinant DNA composed of a DNA fragment having the ilvA gene and a vector DNA is directly mutated with hydroxylamine or the like to transform, for example, E. coli strain W3110. Next, when the transformed strain is cultured in a minimal medium containing an appropriate amount of glycyl-L-leucine, such as M9, for example, an L-isoleucine antagonist, the strain carrying the recombinant DNA having the wild type ilvA gene is In some cases, isoleucine cannot be synthesized and growth can be suppressed (the use of a small copy of the vector is preferable because the influence of the gene-doseage effect can be reduced). In contrast, a strain carrying a recombinant DNA having an ilvA gene encoding TD that has been desensitized by L-isoleucine synthesizes an excessive amount of L-isoleucine even when glycyl-L-leucine is present. Should be able to grow on minimal medium supplemented with glycyl-L-leucine. By utilizing this phenomenon, a strain whose growth is resistant to glycyl-L-leucine, that is, a strain carrying a recombinant DNA having a released ilvA gene whose inhibition has been released can be selected.
[0085]
The thus obtained release-type ilvGMEDA operon containing the release-type ilvA gene is introduced into a suitable host microorganism as a recombinant DNA and expressed to carry the TD whose feedback inhibition has been released, and the L- encoded by the ilvGMEDA operon. Microorganisms with enhanced expression of isoleucine biosynthetic enzymes can be obtained. As the host, microorganisms belonging to the genus Escherichia are preferable, and examples thereof include Escherichia coli (E. coli).
[0086]
Alternatively, a release-type ilvGMEDA operon may be taken out from the recombinant DNA and inserted into another vector DNA. The vector DNA that can be used in the present invention is preferably a plasmid vector DNA, and examples thereof include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, and pMW218. In addition, phage DNA vectors can be used.
[0087]
Furthermore, in order to efficiently perform the expression of the release type ilvGMEDA operon, the transcription control region (operator or attenuator) located upstream of the release type ilvGMEDA operon may be removed. Alternatively, other promoters working in microorganisms such as lac, trp, and PL may be linked, and a promoter specific to the ilvGMEDA operon may be amplified and used.
[0088]
Further, as described above, the release-type ilvGMEDA operon inserted into a vector DNA capable of autonomous replication may be introduced into the host and retained in the host as extrachromosomal DNA such as a plasmid. Daction, transposon (Berg, DE and Berg, CM, Bio / Technol., 1, 417 (1983)), Mu phage (JP-A-2-109985) or homologous recombination (Experiments in Molecular Genetics, Cold Spring Habor Lab. (1972)) may be incorporated into the chromosome of the host microorganism.
[0089]
(3) Removal of the area required for attenuation of the ilvGMEDA operon
In order to remove the region required for attenuation from the ilvGMEDA operon, that is, to introduce a mutation that does not cause attenuation in the ilvGMEDA operon, all the attenuators existing in the 5 'upstream region of the ilvGMEDA operon Alternatively, in addition to those that remove the vicinity, those that insert a new DNA fragment inside the attenuator are also included. Here, the “attenuator” is a base sequence that forms a row-independent terminator-like stem / loop. For example, it is a portion from the 1081st position to the 1104th position of the base sequence described in SEQ ID NO: 1 in the sequence listing.
[0090]
To remove the attenuator, prepare a DNA fragment upstream of the attenuator part of the ilvGMEDA operon, and prepare a DNA fragment of the ilvGMEDA operon downstream of the attenuator part. What is necessary is just to connect. For example, a DNA fragment upstream of the attenuator part of the ilvGMEDA operon can be prepared by cleaving a DNA fragment containing the full length ilvGMEDA operon with an appropriate restriction enzyme. Alternatively, a DNA fragment upstream of the attenuator part of the ilvGMEDA operon may be amplified by the PCR method. The primer DNA used in the PCR method has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)), and has already been reported by G. Coppola et al. (Gene 97, 21 (1991)). ) Chemical synthesis may be performed based on the base sequence. Furthermore, a DNA fragment upstream of the attenuator part of the ilvGMEDA operon may be chemically synthesized.
A method for preparing a DNA fragment in the downstream region of the attenuator part of the ilvGMEDA operon is also the same.
[0091]
The fully-released ilvGMEDA operon may be obtained by preparing the ilvGMEDA operon from which the attenuator is partially or in the vicinity by using the release-type ilvGMEDA operon as a starting material. Since the position and base sequence of the attenuator has already been reported by R. P. Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)), the DNA to be removed is determined based on this sequence.
[0092]
The DNA to be removed contains a portion of DNA necessary for the attenuator to form a row-independent terminator-like stem / loop, or a region encoding a continuous isoleucine residue located upstream of the stem / loop. A suitable DNA portion or a DNA portion containing both is preferred. In order to remove a part of or the vicinity of the attenuator, a DNA fragment upstream of the DNA part to be removed of the ilvGMEDA operon is prepared, and a part of the DNA downstream of the ilvGMEDA operon to be removed is prepared. What is necessary is just to prepare a DNA fragment and to connect both. For example, a DNA fragment upstream of the DNA portion to be removed in the ilvGMEDA operon can be prepared by cleaving a DNA fragment containing the full length ilvGMEDA operon with an appropriate restriction enzyme. Alternatively, a DNA fragment upstream of the DNA portion to be removed in the ilvGMEDA operon may be amplified by PCR. The primer DNA used in the PCR method has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)), and has already been reported by G. Coppola et al. (Gene 97, 21 (1991)). ) Chemical synthesis may be performed based on the base sequence. Furthermore, you may chemically synthesize | combine the DNA fragment of an upstream region from the DNA part removed among ilvGMEDA operons.
The method for preparing a DNA fragment in the downstream region of the DNA portion to be removed in the ilvGMEDA operon is also the same.
[0093]
A completely released ilvGMEDA operon may be obtained by preparing an ilvGMEDA operon in which a new DNA fragment is inserted into the attenuator using the released ilvGMEDA operon as a starting material. Since the position and base sequence of the attenuator has already been reported by RP Lawther et al. Or G. Coppola et al., The insertion position of the new DNA fragment to be inserted and the DNA fragment to be inserted are inserted based on this sequence. The base sequence is determined.
[0094]
The new DNA fragment to be inserted contains the DNA part necessary for the attenuator to form a row-independent terminator-like stem / loop, or a continuous isoleucine residue located upstream of the stem / loop. It is preferably inserted into the DNA portion to be encoded. This is because, as a result of the insertion, the attenuator may not be able to form a row-independent terminator-like stem / loop, which is expected to cause the attenuator to fail.
[0095]
The nucleotide sequence of the new DNA fragment to be inserted is designed so that when inserted, the attenuator does not form a row-independent terminator-like stem / loop, and when inserted, upstream of the stem / loop. It is preferable that the isoleucine residue is not located next to each other.
[0096]
In order to insert a new DNA fragment into the attenuator, a DNA fragment upstream of the DNA portion of the ilvGMEDA operon into which the new DNA fragment is inserted is prepared, and a new DNA fragment of the ilvGMEDA operon is prepared. What is necessary is just to prepare the DNA fragment of the downstream region rather than the DNA part into which a fragment is inserted, to prepare a new DNA fragment to be further inserted, and to connect the three parties. For example, a DNA fragment upstream of the DNA portion into which a new DNA fragment is inserted in the ilvGMEDA operon can be prepared by cleaving a DNA fragment containing the full length ilvGMEDA operon with an appropriate restriction enzyme. Alternatively, a DNA fragment upstream of the DNA portion into which a new DNA fragment is inserted in the ilvGMEDA operon may be amplified by PCR. The primer DNA used for the PCR method has already been reported by RP Lawther et al. (Nucleic Acids Res. 15, 2137 (1987)), and already reported by G. Coppola et al. (Gene 97, 21 (1991)). ) Chemical synthesis may be performed based on the base sequence. Furthermore, a DNA fragment upstream of the DNA portion into which a new DNA fragment is inserted in the ilvGMEDA operon may be chemically synthesized.
The method for preparing a DNA fragment downstream from the DNA portion into which a new DNA fragment is inserted in the ilvGMEDA operon is also the same.
[0097]
New DNA fragments to be inserted can be prepared by chemical synthesis.
[0098]
In addition, a DNA fragment in the upstream region from the DNA portion into which the new DNA fragment is inserted in the ilvGMEDA operon or a DNA fragment in the downstream region from the DNA portion into which the new DNA fragment is inserted in the ilvGMEDA operon is obtained by PCR. When amplifying, a new DNA fragment inserted into the primer DNA can be linked. For example, in the ilvGMEDA operon, one strand of the new DNA fragment to be inserted is linked to the 3 ′ DNA primer used for amplifying the DNA fragment upstream of the DNA portion into which the new DNA fragment is inserted. . Similarly, in the ilvGMEDA operon, the complementary strand of the new DNA fragment to be inserted is linked to the 5 ′ DNA primer used for amplifying the DNA fragment downstream from the DNA portion into which the new DNA fragment is inserted. . Two kinds of DNA fragments amplified using these primers are ligated.
[0099]
The completely released ilvGMEDA operon thus obtained is introduced into a suitable host microorganism as a recombinant DNA, and has a TD whose feedback inhibition has been released by expressing it, and since no attenuation occurs, the ilvGMEDA operon Microorganisms in which the expression of the encoded L-isoleucine biosynthetic enzyme group is further enhanced can be obtained. As the host, microorganisms belonging to the genus Escherichia are preferable, and examples thereof include Escherichia coli (E. coli).
[0100]
Alternatively, a completely released ilvGMEDA operon may be taken out from the recombinant DNA and inserted into another vector DNA. The vector DNA that can be used in the present invention is preferably a plasmid vector DNA, and examples thereof include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, and pMW218. In addition, phage DNA vectors can be used.
[0101]
Furthermore, in order to efficiently express the fully released ilvGMEDA operon, other promoters working in microorganisms such as lac, trp, and PL may be linked upstream of the fully released ilvGMEDA operon. These promoters may be amplified and used.
[0102]
In addition, as described above, a completely released ilvGMEDA operon inserted into a vector DNA capable of autonomous replication may be introduced into the host and retained in the host as extrachromosomal DNA such as a plasmid. Transduction, transposon (Berg, DE and Berg, CM, Bio / Technol., 1, 417 (1983)), Mu phage (JP-A-2-109985) or homologous recombination (Experiments in Molecular Genetics, Cold Spring Habor Lab (1972)) may be incorporated into the chromosome of the host microorganism.
[0103]
Any microorganism may be used as the donor microorganism for the DNA containing the gene encoding AKIII (lysC) as long as it belongs to the genus Escherichia. Specifically, those listed in the book by Neidhardt et al. (Neidhardt, F.C. et, al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) can be used. Examples include E. coli JM109 strain and MG1061 strain.
[0104]
When a wild strain is used as a donor bacterium for DNA containing the gene encoding AKIII (lysC), DNA containing the wild-type AKIII gene can be obtained. AKIII gene (lysC) in which a mutation was introduced into this gene and feedback inhibition by L-lysine was released^*) Can be obtained by employing an in vitro mutation treatment method in which DNA is directly treated with hydroxylamine. Hydroxylamine converts cytosine to N^Four-Chemical mutation treatment agent that causes C → T mutation by changing to hydroxycytosine.
In addition, in order to obtain a DNA containing the AKIII gene in which feedback inhibition by L-lysine is released, it is possible to obtain by using a mutant strain in which feedback inhibition by L-lysine for AKIII activity is released as a DNA donor. it can. The mutant strain is obtained from, for example, a cell group of mutant strains subjected to a usual mutation treatment method, ultraviolet irradiation, or treatment with a mutation agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG). be able to.
[0105]
Next, preparation of DNA containing a gene encoding AKIII (lysC) will be described.
First, E. coli having wild type lysC, for example, MC1061 strain is cultured to obtain a culture. In order to culture the microorganism, it may be cultured by a normal solid culture method, but it is preferable to adopt a liquid culture method from the viewpoint of collection efficiency. In addition, as the medium, for example, yeast extract, peptone, meat extract, corn sweeper, or one or more nitrogen sources such as soybean or wheat leachate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, chloride One or more inorganic salts such as sodium, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate are added, and further, saccharide raw materials, vitamins and the like are added as necessary. The initial pH of the medium is suitably adjusted to 7-8. The culture is performed at 30 to 42 ° C., preferably around 37 ° C. for 4 to 24 hours, usually by stirring deep culture, shaking culture, stationary culture, or the like. The culture thus obtained is centrifuged at, for example, 3,000 r.p.m. for 5 minutes to obtain cells of E. coli MC1061 strain.
From this bacterial cell, for example, the method of Saito et al. (Biochem. Biophys. Acta.,72, 619, (1963)), the method of K. S. Kirby (Biochem. J., 64, 405, (1956)) and the like, and thereby chromosomal DNA can be obtained.
[0106]
In the method of isolating the lysC gene, the chromosomal DNA obtained by the above method is cleaved with an appropriate restriction enzyme. Subsequently, a vector DNA that can grow in Escherichia bacteria cells is ligated, and the resulting recombinant DNA is used to transform an Escherichia microorganism aspartokinase I, II, III all-deficient mutant, such as GT3. (Gene library), a strain that can grow on a minimal medium containing no lysine is isolated, and from this, recombinant DNA of the lysC gene can be separated.
[0107]
Specifically, chromosomal DNA is digested with a restriction enzyme, such as Sau3AI, at a temperature of 30 ° C. or higher, preferably 37 ° C., at an enzyme temperature of 1 to 10 units / ml for various times (1 minute to 2 hours) Partially decompose to obtain a mixture of various chromosomal DNA fragments. A restriction enzyme that produces the same base sequence as the restriction enzyme Sau3AI used for cleaving the chromosomal DNA, such as BamHI, is added to vector DNA that can grow in Escherichia bacteria cells at a temperature of 30 ° C. or higher and an enzyme concentration of 1 to 100 units / unit. It is allowed to act on ml for 1 hour or longer, preferably 1 to 3 hours to obtain a digested and cleaved DNA by complete digestion. Next, a mixture containing a DNA fragment containing the lysC gene derived from E. coli MC1061 obtained as described above and a cleaved vector DNA are mixed, and this is mixed with a DNA ligase, preferably T4 DNA ligase. The recombinant DNA is obtained by reacting at a temperature of 4 to 16 ° C. and an enzyme concentration of 1 to 100 units for 1 hour or longer, preferably 6 to 24 hours.
[0108]
The vector DNA that can be used in the present invention is preferably a psramid vector DNA, and examples thereof include pUC19, pUC18, pBR322, pHSG299, pHSG399, and RSF1010. In addition, phage DNA vectors can also be used. In order to efficiently express the target useful gene, a promoter that works in microorganisms such as lac, trp, and PL may be used. In this case, the recombinant DNA includes a transposon (Berg, DE and Berg, CM, Bio / Technol., 1,417 (1983)), Mu phage (Japanese Patent Laid-Open No. Hei 2-109985) or homology. Those incorporated into the chromosome by a method using recombination (Experiments in Molecular Genetics, Cold Spring Habor Lab. (1972)) are also included.
[0109]
Using this recombinant DNA, for example, Escherichia coli K-12 strain, preferably GT3 strain, etc. is transformed to have lysC gene recombinant DNA from strains with increased AK activity or strains supplemented with auxotrophy. A strain is obtained. This transformation can be achieved by DMMorrison's method (Methods in Enzymology 68, 326, 1979) or by treating recipient cells with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol.,53, 159 (1970)). A recombinant DNA in which a DNA containing the lysC gene is inserted into a vector DNA from the above strain is obtained by, for example, the method of P. Guerry et al.116, 1064, (1973)), D.B.Clewell's method (J. Bacteriol.,110, 667, (1972)).
[0110]
To confirm whether the candidate strain actually retains the cloned recombinant DNA, prepare a cell extract and prepare a crude enzyme solution from it to confirm aspartokinase activity. Can be achieved. The enzyme activity of aspartokinase can be measured by the method of Stattman et al. (Stadtman, E.R., Cohen, G.N., LeBras, G., and Robichon-Szulmajster, H., J. Biol. Chem.,236, 2033 (1961)).
[0111]
Alternatively, the lysC gene can be obtained by amplifying the lysC gene from the chromosomal DNA obtained by the method of Saito et al., Etc. by PCR (polymerase chain reaction; White, TJ et al; Trends Genet., 5, 185 (1989)). You can also do it. A DNA primer used for amplification is complementary to both 3 'ends of a DNA duplex containing the entire or partial region of the lysC gene. When only a partial region of the lysC gene is amplified, it is necessary to screen a DNA fragment containing the entire region from the gene library using the DNA fragment as a primer. When the entire region is amplified, the DNA fragment containing the lysC gene can be recovered by subjecting the DNA fragment to agarose gel electrophoresis and then cutting out the target band.
[0112]
Examples of the DNA primer include a sequence known in E. coti (Cassan, M., Parsot, C., Cohen, G.N., and Patte, J.C., J. Biol. Chem.,261, 1052 (1986)), but the region of 1347base encoding the lysC gene can be amplified. 5′-CTTCCCTTGTGCCAAGGCTG-3 ′ (SEQ ID NO: 6 in the sequence listing below) and 5′-GAATTCCTTTGCGAGCAG Two primers of −3 ′ (SEQ ID NO: 7 in the sequence listing below) are suitable. DNA synthesis can be performed according to a conventional method using “DNA synthesizer model 3808” manufactured by Applied Biosystems, using the phosphoamidide method (see Tetrahedron Letters, 22, 1859 (1981)). The PCR reaction can be performed using “DNA thermal cycler PJ2000 type” manufactured by Takara Shuzo Co., Ltd. using Taq DNA polymerase according to the method specified by the supplier.
[0113]
The lysC gene amplified by the PCR method is connected to a vector DNA that can grow in Escherichia bacterium cells, and is introduced into Escherichia bacterium cells. The vector DNA used, the transformation method, and the method for confirming lysC are the same as described above.
As a method for carrying out mutation such as amino acid substitution, insertion and deletion in the obtained lysC, a recombinant PCR method (Higuchi, R., 61, PCR Technology (Erlich, HA Eds., Stockton Press (1989))), site Specific mutation method (Kramer, W. and Frits, HJ, Meth. In Enzymol.,154,350 (1987)) and Kunkel, T.A. et.al., Meth. In Enzymol.,154,367 (1987)). When these methods are used, the target mutation can be caused at the target site. Furthermore, a method of directly treating a target gene DNA on a chromosome or plasmid with hydroxylamine (Hashimoto, T. and Sekiguchi, M., J. Bacteriol.,159, 1039 (1984)) or a method for chemically synthesizing a target gene by irradiating a cell containing the DNA with an ultraviolet ray irradiation method or treatment with a chemical agent such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid. Etc., mutations can be introduced at random.
[0114]
Uninhibited mutant gene lysC^*As a method for obtaining the above, first, the mutated recombinant DNA is transformed into an AK-deficient strain such as E. coli GT3 strain. Next, the transformed strain is cultured in a minimal medium containing a significant amount of lysine, for example, M9. In the strain carrying the wild-type lysC-containing plasmid, since only AK is inhibited by lysine, threonine, isoleucine, methionine, and diaminopimelic acid (DAP) cannot be synthesized and growth is suppressed. In contrast, lysC, which was released from inhibition by lysine^*  A plasmid-bearing strain with a should be able to grow on minimal medium supplemented with significant amounts of lysine. By utilizing this phenomenon, a strain whose growth is resistant to lysine or lysine analog S-2-aminoethylcysteine (AEC), that is, lysC whose inhibition has been released^*A plasmid-bearing strain having can be selected.
[0115]
By introducing the mutated gene thus obtained into a suitable microorganism (host) as a recombinant DNA and expressing it, a microorganism having AK in which feedback inhibition is released can be obtained.
In order to produce threonine, the obtained lysC gene or mutant lysC gene (lysC^* ) Is introduced and amplified, the wild strain of the genus Escherichia described above can be mentioned, but in addition to this, the replication origin of the recombinant vector DNA constructed here and the lysC gene or mutant lysC gene (lysC gene)^* ) Function, the recombinant vector DNA is replicable and the lysC gene or the mutant lysC gene (lysC^* ) Can be used as a host. The most preferred host is E. coli B-3996 strain.
[0116]
<3. Production of L-isoleucine according to the present invention>
An Escherichia bacterium transformed by introducing the released thrABC operon obtained as described above and further retaining the released ilvGMEDA operon is cultured in a suitable medium, and L-isoleucine is added to the culture. L-isoleucine can be produced efficiently by accumulating production and collecting L-isoleucine from the culture. That is, L-isoleucine can be efficiently produced by retaining both the release-type thrABC operon and the release-type ilvGMEDA operon in bacteria belonging to the genus Escherichia.
[0117]
Furthermore, an Escherichia bacterium transformed by introducing the release-type thrABC operon obtained as described above and further retaining the complete release-type ilvGMEDA operon is cultured in a suitable medium, By producing and accumulating L-isoleucine and collecting L-isoleucine from the culture, L-isoleucine can be produced more efficiently. That is, L-isoleucine can be produced more efficiently by allowing both the release-type thrABC operon and the complete release-type ilvGMEDA operon to be retained in Escherichia bacteria.
[0118]
Examples of the Escherichia bacterium that retains the release thrABC operon include Escherichia bacterium that has been transformed by incorporating a DNA containing the release thrABC operon into chromosomal DNA, or a vector capable of autonomous replication in the DNA and Escherichia bacterium cells. Examples include bacteria belonging to the genus Escherichia transformed by introducing recombinant DNA obtained by ligating DNA into cells.
[0119]
On the other hand, in order to transform by introducing a DNA containing the release type ilvGMEDA operon into a bacterium belonging to the genus Escherichia, the DNA containing the release type ilvGMEDA operon may be incorporated into the chromosomal DNA for transformation, including the release type ilvGMEDA operon. You may transform by introduce | transducing the recombinant DNA formed by ligating DNA and the vector DNA which can replicate autonomously in an Escherichia bacterium cell into a cell.
[0120]
Furthermore, in order to transform by introducing a DNA containing a completely released ilvGMEDA operon into a bacterium belonging to the genus Escherichia, the DNA containing the completely released ilvGMEDA operon may be incorporated into a chromosomal DNA for transformation. You may transform by introduce | transducing the recombinant DNA formed by ligating DNA containing an operon and the vector DNA which can replicate autonomously in the bacterium of the genus Escherichia into the cell.
[0121]
When both the released thrABC operon and the released ilvGMEDA operon, or both the released thrABC operon and the fully released ilvGMEDA operon are introduced into a bacterium belonging to the genus Escherichia, both operons are incorporated into the chromosomal DNA of the genus Escherichia. Or may be retained as extrachromosomal DNA on a single or separate plasmid in the cell. Furthermore, one of each operon may be incorporated and retained on the chromosomal DNA, and the other operon may be retained as extrachromosomal DNA on the plasmid in the cell. When the release-type thrABC operon and the release-type ilvGMEDA operon or the fully-release type ilvGMEDA operon are carried in the Escherichia bacterium in the form of separate plasmids, different types are used so that the two plasmids do not exhibit incompatibility. It preferably has a replication origin.
[0122]
When DNA containing the released thrABC operon and DNA containing the released ilvGMEDA operon are introduced into bacteria belonging to the genus Escherichia, the order of introduction of both DNAs is not limited.
When introducing a DNA containing the released thrABC operon and a DNA containing the fully released ilvGMEDA operon into a bacterium belonging to the genus Escherichia, the order of introduction of both DNAs is not limited.
[0123]
As the Escherichia bacterium used as a host, a promoter of a released thrABC operon, a released ilvGMEDA operon, a fully released ilvGMEDA operon or another promoter for expressing these genes functions in the cell, and When either of them is introduced as extrachromosomal DNA onto a plasmid, any DNA replication origin can be used as long as it can function and replicate in the cell. When the release-type thrABC operon and the release-type ilvGMEDA operon or the complete-release type ilvGMEDA operon are provided in the Escherichia bacterium in the form of separate plasmids, the two origins of replication of the plasmids must function together.
[0124]
Production of L-isoleucine by the microorganism according to the present invention can be carried out by a usual microorganism culture method.
The medium used may be a synthetic medium or a natural medium as long as it contains a carbon source, a nitrogen source, and an inorganic substance, and if necessary, contains an appropriate amount of nutrient source required for growth by the strain used.
[0125]
Examples of the carbon source include various carbohydrates including glucose and various organic acids including pyruvic acid. Moreover, alcohols such as ethanol and glycerol can be used depending on the assimilation property of the microorganism to be used.
As the nitrogen source, ammonia, ammonium salts of various acids such as ammonium sulfate, amines and other nitrogen compounds, and natural nitrogen sources such as peptone, soybean hydrolyzate, and fermentation cell decomposition product can be used.
Examples of inorganic substances include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, and calcium carbonate.
[0126]
The culture is performed under aerobic conditions such as shaking culture and aeration stirring culture, and the culture temperature is 20 to 40 ° C, preferably 30 to 38 ° C. The pH of the medium is in the range of 5-9, preferably in the range of 6.5-7.2. The pH of the medium is adjusted with ammonia, calcium carbonate, various acids, various bases, buffer solutions, and the like.
Usually, L-isoleucine accumulates in the culture medium by culturing for 1 to 3 days.
After completion of the culture, solids such as microbial cells are removed from the culture solution by centrifugation or membrane separation, and L-isoleucine can be recovered by ion exchange, concentration, crystallization, or the like.
[0127]
【Example】
Examples of the present invention are shown below.
Example 1 Construction of pMWD5
Chromosomal DNA was extracted from Escherichia coli MI162 strain. The chromosomal DNA was cleaved with the restriction enzyme HindIII. The HindIII-HindIII DNA fragment containing the ilvGM gene has been found to be 4.8 kb long. Therefore, a HindIII-HindIII DNA fragment having a length of about 4.8 kb was ligated with a DNA fragment obtained by cleaving plasmid vector pBR322 purchased from Takara Shuzo with HindIII. The obtained DNA mixture was introduced into Escherichia coli MI262 strain which is an acetohydroxy acid synthase deficient strain. A strain that was transformed and complemented with a trait lacking acetohydroxy acid synthase was selected, and a plasmid possessed by the strain was isolated. As a result of analyzing the structure of the plasmid, a 4.8 kb DNA fragment containing the ilvGM gene was inserted into the HindIII site of pBR322. This plasmid was named pBRGM7.
[0128]
Gene 97, 21, (1991), Proc. Natl. Acad. Sci. USA 78, 922, (1981) and J. Bacteriol. 149, 294, (1982). Synthetic oligonucleotide DNAs described in Sequence Listing SEQ ID NO: 2 and SEQ ID NO: 3 were synthesized. DNA amplification was performed by PCR using both DNAs as primers and chromosomal DNA of MI162 strain as a template. The DNA fragment to be amplified is a DNA fragment having the 25th to 952nd sequences in the base sequence set forth in SEQ ID NO: 1 in the sequence listing. This fragment is referred to as fragment (A).
[0129]
Similarly, the base sequences reported in Gene 97, 21, (1991), Proc. Natl. Acad. Sci. USA 78, 922, (1981) and J. Bacteriol. 149, 294, (1982) are referenced. Thus, synthetic oligonucleotide DNAs described in SEQ ID NO: 4 and SEQ ID NO: 5 were synthesized. DNA amplification was performed by PCR using both DNAs as primers and chromosomal DNA of MI162 strain as a template. The DNA fragment to be amplified is a DNA fragment having the 1161st to 2421st sequences among the base sequences described in SEQ ID NO: 1 in the Sequence Listing. This fragment is referred to as fragment (B).
[0130]
A large fragment obtained by digesting fragment (A) with SmaI and a DNA fragment obtained by digesting pUC18 (Takara Shuzo) with SmaI were ligated to prepare plasmid pUCA. A large fragment obtained by digesting fragment (B) with KpnI and a large fragment obtained by digesting pHSG399 (Takara Shuzo) with HincII and KpnI were ligated to produce plasmid pHSGB.
[0131]
Plasmid pUCA was digested with KpnI, the cut end was blunted using a large fragment of DNA polymerase I (Klenow fragment), and further digested with PstI, and finally a DNA fragment containing fragment (A) was isolated. . Plasmid pHSGB was digested with HindIII, the blunt end was blunted with a large fragment of DNA polymerase I (Klenow fragment), and further digested with PstI, and finally a DNA fragment containing fragment (B) was isolated. . Both DNA fragments were ligated to produce plasmid pHSGSK.
[0132]
The SmaI-KpnI fragment derived from fragment (A) and fragment (B) mounted on pHSGSK was designated as fragment (C). Fragment (C) corresponds to a fragment obtained by cleaving a 4.8 kb HindIII-HindIII fragment containing the ilvGM gene with SmaI and KpnI, and includes a promoter, an SD sequence and an upstream region of the ilvG gene. It lacks about 0.2 kb of arrangement leading to the nuator region. The procedure for constructing pHSGSK is summarized in FIG.
[0133]
A fragment (C) was obtained by digesting the plasmid pHSGSK with SmaI and KpnI, and a large DNA fragment was obtained by digesting the plasmid pBRGM7 with SmaI and KpnI. The resulting plasmid was named pdGM1. The 4.6 kb HindIII-HindIII fragment containing the ilvGM gene loaded on pdGM1 lacks the region necessary for attenuation. The ilvGM gene lacking the region necessary for attenuation is expressed as “ΔattGM” in this example and the drawings. The procedure for constructing pdGM1 is summarized in FIG.
[0134]
The plasmid pDRIA4 described in JP-A-2-458 is a shuttle vector pDR1120 that can autonomously replicate in Escherichia bacteria and autonomously replicates in Brevibacterium bacteria, and is substantially inhibited by L-isoleucine. It is prepared by ligating a BamHI-BamHI fragment containing the ilvA gene encoding the released threonine deaminase. The BamHI-BamHI fragment is described as 2.3 kb in Japanese Patent Laid-Open No. 2-458, but it is now known to be 2.75 kb. The plasmid pDRIA4 is present outside the chromosomal DNA of Brevibacterium flavum AJ12358 (FERM BP-5087) or Brevibacterium flavum AJ12359 (FERM BP-5088). Plasmid pDRIA4 can be prepared from these strains by conventional methods.
[0135]
A HindIII-BamHI fragment containing the ilvA gene encoding threonine deaminase from which inhibition by L-isoleucine was substantially eliminated from the BamHI-BamHI 2.3 kb DNA fragment on the plasmid pDRIA4 was prepared, and the vector pMW119 (manufactured by Nippon Gene Co., Ltd.) was prepared. ) Was ligated with a DNA fragment obtained by digestion with HindIII and BamHI. The plasmid thus prepared was named pMWA1. The ilvA gene encoding threonine deaminase substantially desensitized by L-isoleucine may be expressed as “A *” in this example and drawings.
[0136]
A DNA fragment obtained by cleaving plasmid pMWA1 with HindIII and a DNA fragment containing the ilvGM gene obtained by cleaving plasmid pdGM1 with HindIII were ligated. By analyzing the position of the restriction enzyme recognition site present on the plasmid, the one in which the transcription direction of the ilvGM gene and the transcription direction of the ilvA gene were the same direction was selected, and this was designated as plasmid pMWGMA2. The procedure for constructing pMWGMA2 is summarized in FIG.
[0137]
Chromosomal DNA of Escherichia coli MI162 strain was prepared, and this was cleaved with SalI and PstI to prepare a DNA fragment mixture. On the other hand, vector pUC19 (Takara Shuzo) was cleaved with SalI and PstI to prepare a DNA fragment. The DNA fragment mixture was ligated with the DNA fragment obtained by cleaving pUC19 to obtain a DNA mixture. This DNA mixture was introduced into the AB2070 strain, which is a transaminase B-deficient strain, and a strain that was transformed to restore the requirement for branched-chain amino acids was selected. When a plasmid was prepared from this strain, a DNA fragment obtained by cutting plasmid pUC19 with SalI and PstI was linked to a SalI-PstI DNA fragment containing the ilvE gene. This plasmid was named pUCE1.
[0138]
pMWGMA2 was partially digested with HindIII to prepare a DNA fragment mixture. On the other hand, pUCE1 was cleaved with HindIII to prepare a 1.7 kb HindIII-HindIII DNA fragment containing part of the ilvE gene and part of the ilvD gene. A dihydroxy acid dehydratase (ilvD gene product) -deficient strain AB1280 was transformed using a DNA mixture obtained by linking the two, and the transformed strain that lost the requirement for branched-chain amino acids was selected. When a plasmid was prepared from the transformant, a DNA fragment obtained by cutting pMWGMA2 only at the HindIII site existing between ΔattGM and A *, a part of the ilvE gene derived from pUCE1, and the ilvD gene A 1.7 kb HindIII-HindIII DNA fragment containing a portion of the ilvGMEDA operon was regenerated. The plasmid thus obtained was named pMWD5. The procedure for constructing pMWD5 is summarized in FIG.
[0139]
The plasmid pMWD5 obtained as described above uses pMW119 as a vector, contains the ilvA gene encoding TD that has been substantially desensitized by L-isoleucine, and a region necessary for attenuation is removed. This is a plasmid carrying the ilvGMEDA operon.
[0140]
(Example 2 Construction of L-isoleucine producing strain)
The plasmid pMWD5 obtained in Example 1 was converted to the L-threonine producing bacterium Escherichia coli B-3996 described in JP-T-3-501682 and USP 5,175,107 by the conventional rubidium chloride method. Introduced by Among the strains transformed with pMWD5, transformants that were simultaneously resistant to streptomycin 100 μg / ml and ampicillin 100 μg / ml were selected. The obtained transformant was designated as AJ12919 strain.
[0141]
The L-threonine-producing bacterium Escherichia coli B-3996 has been deposited with the USSR Antibiotics Research Institute under the registration number 1867. The B-3996 host strain is the Escherichia coli TDH-6 strain, which lacks the thrC gene, has saccharose utilization, can grow in the presence of 5 mg / ml L-threonine, and lacks threonine dehydrogenase. However, the ilvA gene has a leaky mutation. The B-3996 strain retains the plasmid pVIC40 obtained by mounting the thrABC operon containing the thrA gene encoding AKI-HDI, which is substantially desensitized by L-threonine, mounted on a vector derived from the broad host range vector RSF1010. is doing. Therefore, the Escherichia coli AJ12919 strain carrying the plasmid pVIC40 and the plasmid pNWD5 has a thrABC operon containing the thrA gene encoding AKI-HDI that has been substantially desensitized by L-threonine, and is inhibited by L-isoleucine. This is a strain that retains the ilvGMEDA operon containing the ilvA gene encoding TD that has been substantially released and from which the region necessary for attenuation is removed, by a plasmid vector.
[0142]
(Example 3 L-isoleucine production test by AJ12919 strain (1))
The L-isoleucine fermentation production test of the AJ12919 strain obtained in Example 2 was performed. AJ12919 strain was applied to a medium composed of 1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar, streptomycin 100 μg / ml, and ampicillin 100 μg / ml. After culturing for 24 hours, a part of the culture medium was put into a 20 ml fermentation medium (glucose 4%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, first sulfate sulfate. Iron heptahydrate 0.001%, manganese sulfate pentahydrate 0.001%, yeast extract 0.2%, calcium carbonate 3%, pH 7.0), and cultured with shaking at 37 ° C. for 24 hours. The L-isoleucine and L-threonine concentrations in the culture supernatant from which the cells were removed were measured by high performance liquid chromatography. The results are shown in Table 1.
[0143]
[Table 1]
Strain   L-isoleucine concentration ( g / L)  L-threonine concentration ( g / L )
AJ12919   10    0
[0144]
(Example 4 L-isoleucine production test by AJ12919 strain (2))
After culturing AJ12919 strain on an agar medium similar to Example 3 at 37 ° C. for 24 hours, the strain AJ12919 was inoculated into a 1-liter fermentor containing 300 ml of fermentation medium having the same composition as Example 3, and stirred at 400 rpm and aeration volume of 300 ml. The seed culture solution was obtained by culturing at 37 ° C. for 16 hours at / min. 30 ml of the obtained seed culture solution was glucose 6%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 0.001%, Inoculate a 1 liter fermentor containing 250 ml of fermentation medium having a composition of manganese sulfate pentahydrate 0.001%, yeast extract 0.2%, pH 7.0, medium at 37 ° C. and aeration rate 300 ml / min. Cultivation was performed for 23 hours while supplying glucose as appropriate and controlling the pH at around 7.0 with ammonia gas while controlling the number of agitation so that the concentration of dissolved oxygen was 5% or more. The L-isoleucine concentration in the culture supernatant from which the cells were removed was measured by high performance liquid chromatography. As a result, the amount of L-isoleucine accumulated in the culture supernatant was 39 g / L.
[0145]
(Example 5 Construction of L-isoleucine producing strain (2))
International Publication No. WO94 / 11517 discloses an L-threonine producing strain B-3996 strain deposited under the registration number 1867 at the former USSR Antibiotics Research Institute. And the inhibition by L-lysine deposited under the registration number FERM P-13136 (international deposit number FERM BP-4462) at the Institute of Biotechnology, Japan Institute of Industrial Science and Technology. A lysC gene-carrying plasmid (pLLC * 80) that encodes AKIII that has been released, and a thrABC operon containing a thrA gene that encodes AKI-HDI that has been substantially desensitized by L-threonine, and L-lysine Which encodes AKIII substantially uninhibited by A method for constructing a plasmid pVICLC * 80A in which a sC gene is loaded on a vector derived from the broad host range vector RSF1010, and introducing the plasmid pVICLC * 80A into the host B-399 strain (TDH-6 strain) of the B-3996 strain And L-threonine producing bacteria.
[0146]
The plasmid pMWD5 obtained in Example 1 was introduced into the L-threonine producing bacterium in which the plasmid pVICLC * 80A was introduced into the host B-399 strain (TDH-6 strain) of the B-3996 strain by the conventional rubidium chloride method. did. Among the strains transformed with pMWD5, transformants that were simultaneously resistant to streptomycin 100 μg / ml and ampicillin 100 μg / ml were selected. The obtained transformant was designated as AJ13100 strain.
[0147]
The host strain of AJ13100 strain is Escherichia coli TDH-6 strain (B-399 strain), which lacks the thrC gene, has sucrose utilization, and can grow in the presence of 5 mg / ml L-threonine. The dehydrogenase is deleted and the ilvA gene has a leaky mutation. The AJ13100 strain has a thrABC operon containing a thrA gene encoding AKI-HDI that is substantially desensitized by L-threonine, and a lysC gene encoding AKIII that is substantially desensitized by L-lysine. The plasmid pVICLC * 80A mounted on the vector derived from the broad host range vector RSF1010 and the plasmid pMWD5 obtained in Example 1 are retained. Accordingly, the Escherichia coli AJ13100 strain encodes the thrABC operon containing the thrA gene encoding AKI-HDI that is substantially desensitized by L-threonine, and AKIII that is substantially desensitized by L-lysine. A lysC gene and an ilvGMEDA operon containing the ilvA gene encoding TD that has been substantially desensitized by L-isoleucine and from which a region necessary for attenuation is removed is retained by a plasmid vector.
[0148]
(Example 6 L-isoleucine production test by AJ13100 strain)
The L-isoleucine fermentation production test of AJ13100 strain obtained in Example 5 was performed. AJ13100 strain was applied to a medium composed of 1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar, 100 μg / ml streptomycin, 100 μg / ml ampicillin, After culturing for 24 hours, a part of the culture medium was put into a 20 ml fermentation medium (glucose 4%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, first sulfate sulfate. Iron heptahydrate 0.001%, manganese sulfate pentahydrate 0.001%, yeast extract 0.2%, calcium carbonate 3%, pH 7.0), and cultured with shaking at 37 ° C. for 24 hours. The AJ12919 strain obtained in Example 2 was also cultured in the same manner. The L-isoleucine and L-threonine concentrations in the culture supernatant from which the cells were removed were measured by high performance liquid chromatography. The results are shown in Table 2.
[0149]
[Table 2]
Strain   L-isoleucine concentration ( g / L)  L-threonine concentration ( g / L )
AJ12919 10 0
AJ13100   12    0
[0150]
【The invention's effect】
The bacterium belonging to the genus Escherichia having L-isoleucine productivity according to the present invention is a plasmid having a plurality of copies of the thrABC operon containing the thrA gene encoding aspartokinase I-homoserine dehydrogenase I substantially desensitized by inhibition by L-threonine. Since it is mounted and amplified, a sufficient amount of L-threonine, which is a precursor of L-isoleucine, is biosynthesized. Furthermore, the ilvGMEDA operon containing the ilvA gene encoding threonine deaminase whose inhibition by L-isoleucine was substantially released and from which the region necessary for attenuation was removed was mounted on a multiple copy plasmid and amplified. Therefore, L-threonine is efficiently converted to L-isoleucine.
[0151]
When the ilvGMEDA operon containing the ilvA gene encoding threonine deaminase substantially desensitized by L-isoleucine and having the region necessary for attenuation removed is used, the ilvGM gene, ilvE gene, ilvD gene and ilvA gene L-isoleucine biosynthetic enzyme group derived from is produced in a well-balanced manner. Transcription is performed efficiently because it is performed by the original promoter.
[0152]
In general, by using a multicopy plasmid to increase the gene dosage of the ilvGMEDA operon containing the ilvA gene encoding threonine deaminase substantially desensitized by L-isoleucine, the transcription level of the ilvGMEDA operon is negative. The control is released and the productivity of L-isoleucine appears to be sufficiently improved. However, the present invention succeeded in improving the productivity of L-isoleucine more unexpectedly by removing the region necessary for attenuation.
[0153]
[Sequence Listing]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[Brief description of the drawings]
FIG. 1 shows the construction procedure of plasmid pHSGSK.
Of the abbreviations used in FIG. 1, H indicates a HindIII cleavage site present on DNA. Similarly, P represents a PstI cleavage site, K represents a KpnI cleavage site, B represents a BamHI cleavage site, X represents an XbaI cleavage site, and Sm represents an SmaI cleavage site. & Indicates that re-cleavage with a restriction enzyme is impossible.
P → represents a promoter. SD indicates the Shine-Dalgarno sequence. ATG represents a translation initiation codon. TGA indicates a translation termination codon.
Ap is a marker gene that confers ampicillin resistance to the transformed strain. Cm is a marker gene that confers chloramphenicol resistance to the transformed strain.
FIG. 2 shows a procedure for constructing a plasmid pdGM1 by constructing an ilvGM gene from which a region necessary for attenuation of the ilvGMEDA operon is removed.
The abbreviations used in FIG. 2 have the same meaning as the abbreviations used in FIG.
FIG. 3 shows the construction procedure of plasmid pMWGMA2.
The abbreviations used in FIG. 3 have the same meaning as the abbreviations used in FIG.
FIG. 4 shows the procedure for constructing plasmid pMWD5.
The abbreviations used in FIG. 4 have the same meaning as the abbreviations used in FIG. 1, but in addition, S indicates a SalI cleavage site. Moreover, par shows the stabilization area | region of a plasmid.

Claims (16)

  ThrABC operon containing thrA gene encoding aspartokinase I-homoserine dehydrogenase I de-inhibited by L-threonine and ilvA gene encoding threonine deaminase de-inhibited by L-isoleucine and attenuation A bacterium belonging to the genus Escherichia having L-isoleucine productivity, characterized in that it retains the ilvGMEDA operon from which a necessary region has been removed.   The bacterium belonging to the genus Escherichia according to claim 1, wherein the thrABC operon and the ilvGMEDA operon are retained on a plasmid.   The bacterium belonging to the genus Escherichia according to claim 2, wherein two types of plasmids are retained: a plasmid (A) carrying the thrABC operon and a plasmid (B) carrying the ilvGMEDA operon.   The bacterium belonging to the genus Escherichia according to any one of claims 1 to 3, wherein the ilvGMEDA operon has a DNA sequence in which DNA sequences 953 to 1160 are deleted from SEQ ID NO: 1 in the sequence listing. .   The Escherichia bacterium according to claim 4, wherein the plasmid (A) is pVIC40 and the plasmid (B) is pMWD5 among the two kinds of plasmids.   The bacterium belonging to the genus Escherichia according to any one of claims 1 to 5, wherein the bacterium belonging to the genus Escherichia is Escherichia coli.   The thrABC operon and the ilvGMEDA operon were introduced into a host strain that lacks the thrC gene, can grow in the presence of 5 mg / ml L-threonine, lacks threonine dehydrogenase activity, and the ilvA gene has a leaky mutation The bacterium belonging to the genus Escherichia according to claim 6, wherein the bacterium belongs to the genus Escherichia. A fermentation characterized by culturing the Escherichia bacterium according to any one of claims 1 to 7 in a medium, producing and accumulating L-isoleucine in the medium, and collecting L-isoleucine from the medium. A method for producing L-isoleucine by the method.   A thrABC operon containing a thrA gene encoding aspartokinase I-homoserine dehydrogenase I de-inhibited by L-threonine; a lysC gene encoding aspartokinase III de-inhibited by L-lysine; A bacterium belonging to the genus Escherichia having L-isoleucine productivity, comprising an ilvGMEDA operon containing an ilvA gene encoding threonine deaminase that has been desensitized by isoleucine and having a region necessary for attenuation removed. . The bacterium belonging to the genus Escherichia according to claim 9 , wherein the thrABC operon, the lysC gene, and the ilvGMEDA operon are retained on a plasmid. The bacterium belonging to the genus Escherichia according to claim 10 , wherein two types of plasmids are retained: a plasmid (C) carrying the thrABC operon and the lysC gene, and a plasmid (B) carrying the ilvGMEDA operon. . The bacterium belonging to the genus Escherichia according to any one of claims 9 to 11 , wherein the ilvGMEDA operon has a DNA sequence in which DNA sequences 953 to 1160 of the DNA sequence shown in SEQ ID NO: 1 are deleted. . The Escherichia bacterium according to claim 12 , wherein, of the two kinds of plasmids, the plasmid (C) is pVICLC * 80A and the plasmid (B) is pMWD5. The bacterium belonging to the genus Escherichia according to any one of claims 9 to 13 , wherein the bacterium belonging to the genus Escherichia is Escherichia coli. The thrABC operon, the lysC gene, and the ilvGMEDA operon are deficient in the thrC gene, can grow in the presence of 5 mg / ml L-threonine, lack threonine dehydrogenase activity, and the ilvA gene has a leaky mutation The bacterium belonging to the genus Escherichia according to claim 14 , which has been introduced into a strain. A fermentation comprising culturing the Escherichia bacterium according to any one of claims 9 to 15 in a medium, causing L-isoleucine to be produced and accumulated in the medium, and collecting L-isoleucine from the medium. A method for producing L-isoleucine by the method.

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