Art
[1]
The present application is produced with the ability of Corynebacterium (L- isoleucine Corynebacterium relates to a method of producing L- isoleucine used) in the microorganism and this.
BACKGROUND
[2]
Branched-chain amino acids (branched-chain amino acid) is L- Valine, to refer to the three kinds of L- leucine and L- isoleucine, it is industrially used in applications such as food additives, pharmaceutical agents. In particular, L- isoleucine is metabolized in muscles to generate energy, and involved in the production of hemoglobin and the fatigue relief and growth-promoting functions. As a result, has been widely used, such as suaekje agent and a nutrient are being increasingly used in sport nutrition.
[3]
Biosynthesis of L- isoleucine using the microorganism using pyruvate (pyruvate) and 2-keto-butyric acid (2-ketobutyrate) to the precursor is synthesized via the three intermediate metabolite (Jinhwan Park et al ., Appl. Microbial. Biotechnol . 85: 491-506, 2010).
[4]
However, L- isoleucine biosynthesis step of acetonitrile hydroxy L- used Leo threonine di hydrazide to produce a 2-keto acid from other non-kinase (threonine dehydratase, ilvA gene) the next step of the hydroxy acid synthase (acetohydroxy acid synthase, genes ilvBN) are both subject to feedback inhibition by L- isoleucine.
[5]
Therefore, to control the enzymes involved in the biosynthesis via the feedback off by L- isoleucine and it can be seen that an important factor in making an L- isoleucine-producing strain of the yield (Jinhwan Park et al ., Biotechnology Journal, 560 -577, 2010). In addition it should be made smooth supply of the branched-chain amino acid is pyruvic acid because they are generated by the same biosynthetic pathway to (pyruvate), in order to mass-produce L- isoleucine in a high yield of 2-keto acid of the previous step L- threonine ( Republic of Korea Patent No. 10-0823044 call).
[6]
Detailed Description of the Invention
SUMMARY
[7]
The present inventors have While reviewing a novel biosynthetic pathway which does not use the L- threonine as a precursor, introduction of a gene having a sheet ramal rate synthase (citramalate synthase) activity in L- isoleucine biosynthesis pathway L- isoleucine by ensuring that production is improved, thereby completing the present application.
Problem solving means
[8]
The purpose of the present application is to provide a recombinant microorganism having an L- isoleucine biosynthesis pathway is introduced the new production capacity.
[9]
Another object of the present application provides a method of producing L- isoleucine recovering the L- isoleucine from culturing a recombinant microorganism of the new biosynthesis pathway is introduced in the medium, and the microorganism or the medium to.
Effects of the Invention
[10]
Corynebacterium spp with L- isoleucine producing ability of the present application sheet ramal rate synthase activity is introduced into the high-L- isoleucine with new biosynthetic pathway which does not use the L- threonine to yield a precursor It can be produced by.
[11]
Best Mode for Carrying Out the Invention
[12]
In order to achieve the above object, one aspect of the present application is Corynebacterium (with L- isoleucine producing ability, which comprises a protein of the sheet ramal rate synthase activity Corynebacterium provides) spp.
[13]
The term "L- isoleucine" in this application is one of the essential amino acid structurally L- valine, L- leucine with formula HO for the branched-chain amino acids 2 CCH (NH 2 ) CH (CH 3 ) CH 2 CH 3 in It means a L- amino acids.
[14]
"Having L- isoleucine producing ability" term in the present application refers to it refers to the ability to accumulate L- isoleucine in the medium or the microorganism when the microorganism is cultivated in a culture medium. These L- isoleucine producing ability may be a property of, or enhancing or improving the properties imparted by the strains held by the wild type strain.
[15]
For the recombinant mutant, such as for example, the micro-organisms strengthen the auxotrophic mutant strain, analogue-resistant mutant strain, or metabolism control mutant strain of acquisition, or L- isoleucine biosynthesis-related enzyme activity in order to have an L- isoleucine producing ability the method used for transformation of a microorganism, such as produced can be applied alone or in combination.
[16]
In addition, L- isoleucine case to enhance the biosynthesis-related enzyme activity, gene, gene specifically ilvG, ilvM, ilvE, ilvD and ilvA involved in L- isoleucine biosynthesis is enhanced will be enhanced in combination with either alone or in combination of two or more can. The ilvG, ilvM, ilvE, ilvD and ilvA genes are each acetonitrile hydroxy acid synthase of the isozyme II large subunit, small subunit, transaminase, dihydroxy-acid having hydrazide hydratase and threonine deaminase of It encrypts the first.
[17]
The term "sheet ramal rate synthase (EC2.3.1.182)" in this application is an enzyme catalyzing the process of converting the acetyl -CoA and pyruvate from a sheet ramal rate and coenzyme A (coenzyme A). The enzyme meth furnace knife Lactococcus Jana when ( Methanocaldococcus jannaschii ) (Howell et al , J Bacteriol 181:.. 331-333, 1999), Leptospira interrogating elegans ( Leptospira interogans ) (Xu . Et al ., J Bacteriol 186: 5400-5409, 2004), Geo bakteo seolpu redu sense ( Geobacter sulfurreducens are found in fungi and the like) of the isoleucine-threonine biosynthesis-involved in the non-path-dependent (Risso's et al , J Bacteriol 190: 2266-.. 2274, 2008). The enzyme has a high specificity for pyruvate as a substrate, are known to be typically inhibited by isoleucine.
[18]
The term "protein having a sheet ramal rate synthase activity" in the present application, and "the gene encoding the" include any protein, and any gene encoding it has a sheet ramal rate synthase activity as described above may be included, without limitation, .
[19]
Specifically, the protein of the sheet ramal rate synthase activity can be a meta-no knife Rhodococcus origin, and may be more specifically, meta-no knife Lactococcus Jana when derived. The sheet ramal rate synthase is SEQ ID NO: having the amino acid sequence represented by 1, cimA gene encoding the SEQ ID NO: having the nucleotide sequence represented by 2. Since depending on the species or strain of microorganism there is a difference in the amino acid sequence of a protein showing the activity, or a functionally equivalent plurality of nucleic acids because of the degeneracy (degeneracy) of the genetic code, can encode any desired protein, It is not limited to the disclosed SEQ ID NOS.
[20]
Further, substantially in SEQ ID NO: 1 in the amino acid sequence with 70% or more, for example 80% or more, more specifically, a more specifically more than 90%, is over 95%, even more specifically over 98% of Bi If the protein having a substantially sheet ramal rate synthase activity as the amino acid sequence shown can be included in the scope of this application without limitation.
[21]
If the amino acid sequence having the biological activity the same as or correspond to the protein of Figure 1, even if some sequence having a deletion, modified, substituted or added in the amino acid sequence also: Further, substantially the sequence number as the sequence having the sequence homology with included in the scope of the present application is obvious.
[22]
In addition, the gene encoding the synthase sheet ramal rate is SEQ ID NO: may have the nucleotide sequence encoding the amino acid sequence represented by 1. The polynucleotide in consideration of the codon-preferred organisms intended to express the result or the protein to the degeneracy (degeneracy) of the codon, it is possible within a range that does not alter the amino acid sequence of the protein made various changes to the coding region . The polynucleotide sequence, for example SEQ ID NO: can have a polynucleotide sequence of Figure 2, this is 80% homology, specifically 90% or more, and more specifically may have a nucleotide sequence at least 99%. But it is not limited thereto.
[23]
In addition, SEQ ID NO: In order to obtain the said sheet ramal rate synthase activity is enhanced variants: the present application as a variant of the protein of 1 provides the CimA (M) mutant library obtained by using error-prone PCR.
[24]
Specifically, CimA (M) mutant library according to the present application
[25]
CimA has the amino acid sequence of 3 (M) m1: SEQ ID NO: (specifically, SEQ ID NO: can be coded from a polynucleotide having the base sequence of 4);
[26]
CimA has the amino acid sequence of the 5 (M) m2: SEQ ID NO: (specifically, SEQ ID NO: can be coded from a polynucleotide having the base sequence of the 6);
[27]
SEQ ID NO: 7 amino acid sequence has a CimA (M) of m3 (more specifically, SEQ ID NO: can be coded from a polynucleotide having a base sequence of 8);
[28]
CimA has the amino acid sequence of the 9 (M) m4: SEQ ID NO: (specifically, SEQ ID NO: can be coded from a polynucleotide having a base sequence of 10);
[29]
CimA has the amino acid sequence of a 11 (M) m5: SEQ ID NO: (specifically, SEQ ID NO: can be coded from a polynucleotide having a base sequence of 12);
[30]
CimA (M) has the amino acid sequence of 13 m6: SEQ ID NO: (specifically, SEQ ID NO: can be coded from a polynucleotide having a base sequence of 14); And
[31]
CimA (M) has the amino acid sequence of 15 m7: SEQ ID NO: may comprise (more specifically, SEQ ID NO: can be coded from a polynucleotide having a base sequence of 16).
[32]
In addition, a substantially substantially as the amino acid sequence shown by the SEQ ID NO of amino acid sequence and specifically 80% or more, more specifically, more specifically more than 90%, is over 95%, even more particularly at least 99% homologous If the protein having the activity of the seat ramal rate synthase variants it may be included within the scope of this application without limitation.
[33]
The term "homology" in the present application refers to the degree of matching the given amino acid sequence or nucleotide sequence and can be expressed as a percentage. As used herein, a homologous sequence thereof having the same or similar activity with a given amino acid sequence or nucleotide sequence is represented by the "% homology". For example, the sequence by Southern hybridization experiment under the score (score), identities (identity) and similarity (similarity) standard software, stringent conditions specifically used, define the BLAST 2.0 calculating a parameter (parameter), such as (such as, Sambrook be confirmed by comparing, and the appropriate hybridization conditions to be defined is the technical range and et al can be determined by: a Laboratory Manual 1989 See, Molecular Cloning.), well known to a person skilled in the art.
[34]
In a specific exemplary embodiment, the microorganism of the present application meta furnace knife Lactococcus Jana when derived from SEQ ID NO: consisting of 3, 5, 7, 9, 11, 13 and 15: SEQ ID NO: as a sheet ramal rate synthase and variants thereof of 1 It can include a gene encoding a protein of amino acid sequence selected from the group. More specifically, it is possible to express a gene of interest by transforming a recombinant vector containing the gene into the microorganism, respectively.
[35]
The term "recombinant vector" used in this application refers to a DNA preparation containing the nucleotide sequence of the polynucleotide encoding the desired protein operably linked to suitable control sequences so as to express the desired protein in a suitable host. The control sequences may include any operator sequence, sequences that control the termination of the sequence, and a transcription and translation encoding a suitable mRNA ribosome-binding site for regulating the promoter, such that transcription can initiate transcription. The recombinant vector may be suitable after the host cell transformed into, irrespective of the replication of the host genome, or function, it can be integrated into the genome itself.
[36]
The recombinant vector used in the present application as long as it can replicate in a host cell is not particularly limited, and may be manufactured by using any vector known in the art. Examples of the normal vector to be used may be a naturally occurring or recombinant plasmid of the state, cosmid, virus and bacteriophage. For example, the phage vector or course as mid vector may be used. PWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, etc. Charon4A, and Charon21A, pBR series, pUC system, pBluescriptII system as plasmid vector It may be used based pGEM, pTZ-based, such as pCL and pET-based system. Available vectors in this application is not particularly limited and may be a known expression vector. Specifically, it may be used pECCG117, pDZ, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC vector or the like.
[37]
It can be introduced into the polynucleotide encoding the target protein in the chromosome via a vector for chromosomal insertion in the host cell, for example. Introduced into the chromosome of the polynucleotide is, for any method, for example, known in the art, it may be made by homologous recombination, but is not limited thereto.
[38]
The term "transgenic" in this application is meant to allow the protein to the polynucleotide encoding the expression in a host cell by introducing the recombinant vector comprising the polynucleotide encoding the target protein in the host cell. If, as long as the transformed polynucleotide can be expressed in a host cell, is inserted in the chromosome of the host cell it can be positioned with or without all of the location, or in addition to any chromosome.
[39]
In addition, it means that the term "operably linked to" the promoter sequence and the gene rea sequence to initiate and mediate the transcription of the polynucleotide encoding the target protein of the present application in the above is operatively connected to.
[40]
[41]
On the other hand, Corynebacterium in specific embodiments of the present application Solarium spp is additionally activated the enhanced-isopropyl malate dehydrogenase (3-isopropylmalate dehydrogenase) and isopropyl maleate di Hydra other dehydratase (3-isopropylmalate dehydratase ) may include.
[42]
The term in the present application, "enhanced activity of the protein" means that the activity of the protein which increases the activity compared to the wild-type or mutant former state. Specifically, the art increase in the inherent gene activity encoding a protein, within or amplification of the intrinsic gene from an external factor, deletion of the inhibitory regulator of the genes, gene copy increase in the number, and gene introduction from the outside, deformation of the expression control sequence , it may include that the activity increased by a particular promoter and gene replacement or modification of protein activity by the increase in mutation.
[43]
Specifically, in this application isopropyl malate dehydrogenase or isopropyl maleate di-hydrazide the other is to strengthen the activity of the protein kinase
[44]
1) increasing the copy number of the polynucleotide encoding the protein,
[45]
2) transformation of the expression control sequences to increase the expression of the polynucleotide,
[46]
3) modification of the polynucleotide sequence on the chromosome so that the activity is enhanced in the enzyme, deletion of the inhibitory regulatory elements of the gene expression, or
[47]
4) but can be performed in the method is selected from a combination thereof, is not particularly limited.
[48]
Increasing the copy number of the polynucleotide in the above is not specifically limited to, or carried out in operably linked to a vector form, can be carried out by being inserted into the chromosome in the host cell. Specifically, either the vector capable of a polynucleotide encoding a protein of the present application operatively connected to, and independent of replication and host functions may be performed by being introduced into the host cell, the polynucleotide is operably linked to, a host a vector capable of inserting the polynucleotide into the chromosome is introduced into a host cell in the cell by being can be carried out in a way to increase the copy number of the polynucleotide within the chromosome of the host cell.
[49]
Next, it is to transform the expression control sequences, the expression of the polynucleotide to increase, particularly for but not limited to, a nucleic acid sequence so as to further enhance the activity of the expression control sequence deletion, insertion, Vivo wholly or conservative substitution or a carried out in combination to induce a mutation on the sequence, or may be performed by replacing as a nucleic acid sequence having a stronger activity. The expression control sequences, include especially useful for but not limited to, promoters, operator sequences, such as sequences that control the termination of the sequence, the transcription and translation coding for a ribosome binding site.
[50]
There is a strong heterologous promoters rather than the native promoter upper portion of the polynucleotide expression units can be connected, and examples of strong promoters are the promoters cj7 (Republic of Korea Patent No. 10-0620092 and No. WO 2006/065095), EF-Tu promoter, groEL promoter, and the like aceA or aceB promoter, it is possible to more preferably improve the expression of the polynucleotide is operably linked to a Corynebacterium promoter and operation of cj7 derived promoter coding for the enzyme.
[51]
In addition, deformation of the polynucleotide sequence on the chromosome is not particularly limited to, variations on the polynucleotide deletions to the nucleic acid sequence so as to further enhance the sequence activity, insertion, Vivo wholly or conservative substitution or expression control by a combination of SEQ ID NO: performed by inducing, or may be performed by replacing as in the polynucleotide sequence improved so as to have a stronger activity.
[52]
[53]
The term "an isopropyl malate dehydrogenase" in the present application, and "di-isopropyl maleate hydratase other kinase" is an enzyme involved in valine biosynthetic pathway L- leucine, L- isoleucine and L-, these active protein and the gene encoding it has can be included without any protein, and any gene encoding the enzyme having the activity as described above limits.
[54]
Specifically, the dehydrogenase (EC1.1.1.85) isopropyl maleate in this application may be a genus Corynebacterium microorganism-derived, more specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ) derived from one can. On the other hand, isopropyl malate dehydrogenase of the present application is SEQ ID NO: may be one having an amino acid sequence represented by 17. Further, substantially in SEQ ID NO: the amino acid sequence with 70% or more of 17, specifically, 80% or more, more specifically, a more specifically more than 90%, is over 95%, even more specifically more than 99% of Bi If substantially protein of isopropyl malate dehydrogenase activity as the amino acid sequence shown can be included in the scope of this application without limitation.
[55]
Further, the gene coding for the isopropyl malate dehydrogenase is SEQ ID NO: may have the nucleotide sequence encoding the amino acid sequence represented by 17. Since depending on the species or strain of microorganism there is a difference in the amino acid sequence of a protein showing the activity, or a functionally equivalent plurality of nucleic acids because of the degeneracy (degeneracy) of the genetic code, can encode any desired protein, the genes include, but are not limited to the disclosed sequence number, for example, SEQ ID nO: may have a base sequence of 18, this is 80% homology, specifically 90% or more, a base sequence at least more specifically, 99% It may have.
[56]
In addition, the end-isopropyl in the present application rate de Hydra other kinase (EC4.2.1.33) can be a microorganism of the genus Corynebacterium origin, more specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ) derived from one can. On the other hand, isopropyl maleate di-hydrazide another kinase of the present application is composed of two subunits (small / large subunits), each of which is SEQ ID NO: may be one having an amino acid sequence represented by 21: 19 and SEQ ID NO: . Further, substantially in SEQ ID NO: 19 and the amino acid sequence with 70% or more of 21, specifically, 80% or more, more specifically 90% or more, still more specifically at least 95%, even more specifically more than 99% of the If substantially protein of di-isopropyl maleate hydratase other dehydratase activity as the amino acid sequence shown by Bi it may be included within the scope of this application without limitation.
[57]
In addition, the gene encoding the di-isopropyl maleate hydratase other kinase is SEQ ID NO: may have the nucleotide sequence encoding the amino acid sequence shown in 21: 19 and SEQ ID NO. Since depending on the species or strain of microorganism there is a difference in the amino acid sequence of a protein showing the activity, or a functionally equivalent plurality of nucleic acids because of the degeneracy (degeneracy) of the genetic code, can encode any desired protein, the gene is not limited to the disclosed SEQ ID NOS. For example, SEQ ID NO: 20 and SEQ ID NO: may have a base sequence of 22, this is 80% homology, specifically 90% or more, and more specifically may have a nucleotide sequence at least 99%.
[58]
[59]
On the other hand, Corynebacterium in specific embodiments of the present application Solarium spp may include enzymes involved in the enhanced L- isoleucine biosynthesis pathway further activity.
[60]
The term "enzyme involved in L- isoleucine biosynthesis pathway" in the present application, the aspartate-kinase (aspartate kinase, the gene lysC), aspartate semi-aldehyde dehydrogenase -β- (aspartate-β-semialdehyde dehydrogenase, asd gene), homoserine dehydrogenase, threonine di Hydra other kinase (homoserine dehydrogenase, hom gene), homoserine kinase (homoserine kinase, thrB gene), threonine synthase (threonine synthase, thrC gene) ( threonine dehydratase, ilvA gene), amino-transferase (aminotransferase, ilvE gene) but not the like can be included. However, this limitation.
[61]
[62]
On the other hand, the genus Corynebacterium microorganism in specific embodiments of the present application may include additionally pyruvate dehydrogenase (pyruvate dehydrogenase) inactivated.
[63]
The term "pyruvate dehydrogenase (EC1.2.4.1)" in the present application is a pyruvate acetyl -CoA and CO 2 is an enzyme that converts a. In the present application it may be the aceE gene coding for the enzyme in order to increase the supply of acetyl -CoA used as a precursor for the production of the gene cimA deficient strain.
[64]
Specifically, the pyruvate dehydrogenase (EC1.2.4.1) in the present application may be a genus Corynebacterium microorganism-derived, more specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ) can be derived have. On the other hand, pie base of the present application bait dehydrogenase is SEQ ID NO: may be one having an amino acid sequence represented by 25. Further, substantially in SEQ ID NO: amino acid sequence with 70% or more of 25, specifically, 80% or more, more specifically, more specifically more than 90%, is over 95%, even more particularly at least 99% homologous If substantially protein having a pyruvate dehydrogenase activity as the amino acid sequence shown can be included in the scope of this application without limitation.
[65]
In addition, a gene coding for the pyruvate dehydrogenase kinase D is SEQ ID NO: may have the nucleotide sequence encoding the amino acid sequence represented by 25. Since depending on the species or strain of microorganism there is a difference in the amino acid sequence of a protein showing the activity, or a functionally equivalent plurality of nucleic acids because of the degeneracy (degeneracy) of the genetic code, can encode any desired protein, the gene is not limited to the disclosed SEQ ID NOS. For example, SEQ ID NO: can have a base sequence of 26, and this is 80% homology, specifically 90% or more, and more specifically may have a nucleotide sequence at least 99%.
[66]
The term "inactivated" in this application is, it means that the gene encoding the polypeptide, or not expressed, represented a decrease of in gene expression caused by defective expression activity, or even the expression does not produce the polypeptide in the functional .
[67]
In addition, it means that not only will you have a gene that encodes a polypeptide that is active fire completely contained nothing that expression level is not significantly reduced substantially expressed. Thus, the gene inactivation is complete (knock-out) or in part (e. G., Mutant genes gene represents a low-order-type gene (hypomorph) or this partial reduction in affecting activity that represents less than the normal level of expression of It may be the product of a).
[68]
Furthermore, the present application that the polypeptide of the "inactivation" has the activity of the polypeptide relative to the unmodified strain, as well as reduction in its activity also includes the case is removed. Specifically, threonine di inactivation of Hydra other kinase in the present application,
[69]
1) Some or all of the deletion of the polynucleotide encoding the protein,
[70]
2) transformation of the expression control sequence to reduce the expression of the polynucleotide,
[71]
3) modification of the polynucleotide sequence on the chromosome so that the activity of the protein of weakening, or
[72]
4) but can be performed in the method is selected from a combination thereof, is not particularly limited.
[73]
Method for deleting a part or all of a polynucleotide encoding a protein from above by replacing the polynucleotide encoding my intrinsic target protein chromosome through a bacterial vector for in chromosome inserted into a portion the nucleic acid sequence is deleted polynucleotides or marker gene It can be carried out. The "part" is different according to the kind of a polynucleotide but, more specifically, from 1 to 300, and more specifically, from 1 to 100, even more specifically, 1 to 50 days more.
[74]
Next, a method of modifying the expression control sequence to reduce the expression of the polynucleotide is, but not particularly limited to, a nucleic acid sequence to further weaken the activity of the expression control sequence deletion, insertion, Vivo wholly or conservative substitution or a combination of the two performed by inducing a mutation on the expression control sequence, or may be carried out by replacing a nucleic acid sequence having a weaker activity. The expression control sequence may comprise a sequence that has a sequence control, and termination of transcription and decryption encoding a promoter, operator sequence, a ribosome binding site, and the like.
[75]
In addition, the method of modifying the polynucleotide sequence on the chromosome is carried out by inducing a sequence variation on the polynucleotide sequence so as to further weaken the activity of the protein by deletion, insertion, Vivo wholly or conservative substitution or a combination thereof, or, more weak It can be performed by replacing into the polynucleotide sequence so as to have improved activity and, and the like.
[76]
Microorganism having an isoleucine-producing ability In a further specific embodiment of the present application can be included in the scope of the present application, without limitation, if the micro-organisms that can be expressed is introduced into a protein of the sheet ramal rate synthase activity. Examples of Escherichia genus ( Escherichia sp.), Shigella genus ( Shigella sp.), In bakteo a sheet ( Citrobacter sp.), Salmonella in ( Salmonella sp.), Enterococcus bakteo in ( Enterobacter sp.) Yeosi California in ( Yersinia SP.), keurep when Ella in ( Klebsiella SP.), air Winiah in ( Erwinia SP.), the genus Corynebacterium ( of Corynebacterium SP.), Brevibacterium genus ( Brevibacterium SP.), Lactobacillus genus ( Lactobacillus sp.), celecoxib grandma eggplant in ( Selenomanas sp.), Vibrio genus ( Vibrio sp.), Pseudomonas species ( Pseudomonas sp.), Streptomyces sheath in ( Streptomyces sp.), in oh Kano bacteria ( Arcanobacterium sp. ), an alkali jeneseu in ( Alcaligenes may be a microorganism belonging to sp, etc.). More specifically, a microorganism is a microorganism belonging to the genus Corynebacterium of the present application, but no more specifically, Corynebacterium glutamicum, or limited.
[77]
[78]
Another aspect of the present application
[79]
Culturing a microorganism of the genus Corynebacterium, which comprises a protein of the sheet ramal rate synthase (citramalate synthase) activity in the medium, and
[80]
Provides, L- isoleucine producing recovering the L- isoleucine from the medium or the microorganism.
[81]
In the method, the sheet ramal rate synthase Corynebacterium spp Gene encoding a protein having the activity are as previously described.
[82]
Briefly, the genus Corynebacterium microorganism meth furnace knife Rhodococcus ( Methanocaldococcus ) may be a gene is introduced for encoding the sheet ramal rate synthase derived, specifically, SEQ ID NO: 1, 3, 5, 7 , 9, 11, may be a gene coding for the introduction of sheet ramal rate synthase having an amino acid sequence selected from the group consisting of 13 and 15.
[83]
In addition, the genus Corynebacterium microorganism of the present application is an the addition of isopropyl malate dehydrogenase (3-isopropylmalate dehydrogenase) and the activity of the isopropyl maleate di Hydra other dehydratase (3-isopropylmalate dehydratase) enhance be can. Further, the genus Corynebacterium microorganism may be one of the deactivation of the activated additionally pyruvate dehydrogenase (pyruvate dehydrogenase).
[84]
Specifically, the microorganism of the genus Corynebacterium according to one example of the present application can be a Corynebacterium kumil Gene encoding the sheet ramal rate synthase.
[85]
In the method, the method comprising: culturing the microorganism of Corynebacterium genus according to the present application, and may be particularly, but not limited thereto, carried out by the known batch culture method, the continuous culture method, a fed-batch culture method.
[86]
At this time, the culture conditions, particularly for but not limited to, a basic compound to an appropriate pH using: (phosphoric acid or sulfuric acid for example) (for example, pH 5 to 9, in particular (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound It can be adjusted to pH 6 to 8). It is also possible to use anti-foaming agents such as fatty acid polyglycol ester can suppress foam generation.
[87]
Oxygen or an oxygen-containing gas mixture is introduced to the culture to maintain aerobic conditions, and the culture temperature is 20 to 45 ℃, specifically, it is possible to maintain the 25 to 40 ℃. Culture is continued until the amount of the desired L- isoleucine obtained with a maximum, it can be incubated for typically 10 to 160 hours to achieve this object. L- isoleucine-cost by the production culture may be secreted into the culture medium or remains in the cell.
[88]
In addition, the medium for the culture to be used per a carbon source and a carbohydrate (such as glucose, sucrose trehalose, lactose, fructose, maltose, know three, starch and cellulose), maintenance, and fat (such as soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (for example, glycerol and ethanol) and organic acids (e.g. acetic acid) can be used by using individually or mixed, such as , however, and the like.
[89]
The nitrogen source may include nitrogen-containing organic compounds (e.g., peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean bakbun and urea), or inorganic compounds (e.g., ammonium ammonium ammonium sulfate, chloride, phosphate, ammonium carbonate and ammonium nitrate) may be used either individually or using a mixture or the like. However, the embodiment is not limited thereto.
[90]
As a source of phosphate, but it is not available for use, or a mixture of monobasic potassium phosphate, potassium susoyi, such as the corresponding sodium-containing salt thereto separately, like. In addition, other metal salts (such as magnesium sulfate or iron sulfate), essential amino acids, and growth, such as Vitamins, but may include a promoting material, and the like.
[91]
A method of producing an isoleucine in the concrete embodiments of the present application may be the acetate is added to the feed in the four corridor step of culturing the microorganism of the genus tumefaciens.
[92]
In the method, the method for recovery of L- isoleucine production in the culture step is a culture method, for example, batch, continuous or fed-batch to the art according to the culture method using an appropriate method known in the field of interest from the culture broth the amino acid can be collected. For example, but are centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, etc. can be used, it not limited to these.
[93]
[94]
Another aspect of the present application
[95]
It provides a sheet ramal rate synthase (synthase citramalate) isoleucine production in Corynebacterium spp, including a protein of the active application.
[96]
In the above applications, the sheet ramal rate synthase Corynebacterium spp Gene encoding a protein having the activity are as previously described.
[97]
As described above, the application of L- isoleucine genus Corynebacterium microorganism having a production capability is sheet ramal rate synthase activity is introduced through the new L- biosynthetic pathway which does not use the L- threonine to the precursor and the isoleucine can be produced in high yield.
[98]
Mode for the Invention
[99]
Example 1: Meta-no knife Lactococcus derived cimA making a recombinant vector containing the gene
[100]
[101]
<1-1> Meta-no knife caucus derived cimA preparation of gene fragments
[102]
[103]
To obtain a 1476 bp fragment containing the open reading frame (ORF) of the gene cimA (NC_000909.1) encoding the sheet ramal rate synthase, Zeno mixer - using a tip system (Genomic-tip system, Qiagen) No meta Lactococcus Jana knife when ( Methanocaldococcus jannaschii , genomic DNA was extracted from) DSM 2661.
[104]
Meta furnace knife Lactococcus Jana when DSM 2661 derived sheet ramal rate synthase is SEQ ID NO: 491 has an amino acid sequence represented by 1, cimA gene encoding the SEQ ID NO: having the nucleotide sequence represented by 2.
[105]
And to the gDNA as a template derived from the SEQ ID NO: The PCR was performed using primer pairs 35 and 36. The PCR reactions were denatured at 95 ℃ for 30 seconds, elongation at 56 ℃ process in 30 seconds annealing and 72 ℃ 60 seconds was carried out 30 times repeatedly.
[106]
After the PCR product amplified therefrom by electrophoresis in 1.0% agarose gel was obtained by eluting the band of the desired size, it was named as "cimA (M) fragment".
[107]
[108]
Primer cimA-5-NdeI (SEQ ID NO: 35):
[109]
5'-GCATCATATGATGGTAAGGATATTC-3'
[110]
Primer cimA-3-XbaI (SEQ ID NO: 36):
[111]
CGATCTAGATTAATTCAACAACATGTT-5'-3 '
[112]
[113]
<1-2> A recombinant vector p117- cj7 - the cimA (M) produced
[114]
[115]
A known genus Corynebacterium microorganism-derived p117-gfp cj7-containing promoter cj7 of PCR was carried out by a template (Republic of Korea Patent No. 10-0620092 call). Wherein 'p117' E. coli (Corynebacterium shuttle vector E. coli - Corynebacterium shuttle vector) is pECCG117: represents a (Biotechnology Letters 13 (10) 721-726 , 1991). The PCR reaction to SEQ ID NO: 27 and 28 were performed in modified 95 ℃ for 30 seconds by using the primer pair, at 56 ℃ in 20 seconds annealing and 72 seconds elongation process ℃ 30 to 30 iterations.
[116]
The PCR product amplified therefrom after electrophoresis on 1.0% agarose gel was obtained by eluting the band of 323 bp in size, it was named as "fragment cj7".
[117]
[118]
Primer Pcj7-5-KpnI (SEQ ID NO: 27):
[119]
5'-GATGGTACCACCCCAGAAACATCCCAGC-3'
[120]
Pcj7-3 NdeI primer (SEQ ID NO: 28):
[121]
5'-CGATCATATGGAGTGTTTCCTTTCGTTGGG-3'
[122]
[123]
A cimA (M) fragment prepared in the above embodiments cj7 fragment prepared in the above Example <1-1> as a template, and SEQ ID NO: Using the primer pair 27 and 36 were subjected to PCR fusion (sewing). PCR reactions were denatured at 95 ℃ for 30 seconds, elongation at 56 ℃ process in 30 seconds annealing and 72 ℃ 60 seconds was carried out 30 times repeatedly.
[124]
After the PCR product amplified therefrom by electrophoresis in 1.0% agarose gel was obtained by eluting the band of 1799 bp in size, it was named as "cj7-cimA (M) fragment".
[125]
[126]
It was cj7-cimA (M) after the treatment the fragments with restriction enzymes KpnI and XbaI, the linear p117 fragments and ligation (ligation) treated with the same restriction enzyme obtained above.
[127]
Was converted transformed with the recombinant vector produced therefrom by heat shock into E. coli DH5α cells (the same or less heat shock transformation,), and blotted them on solid media containing LB kanamycin 25 μg / ml and incubated overnight at 37 ℃. Wherein the one colony was cultured platinum containing 25 μg / ml kanamycin for was inoculated to 3 ml LB liquid medium overnight and then, using a plasmid mini peuraep Kit (Qiagen, catalog # 27 104, the same hereinafter) to recover a plasmid DNA .
[128]
Whether the production of recombinant vector was confirmed by treatment with restriction enzymes KpnI and XbaI, to SEQ ID NO: 29 and 30 primers with modified from 95 ℃ 30 seconds, at 56 ℃ in 30 seconds annealing and 72 ℃ a 90-second elongation process of clones were confirmed by performing PCR under the conditions of repeated 30 times. A recombinant vector obtained therefrom was named "p117-cj7-cimA (M)".
[129]
[130]
Primer 117-F (SEQ ID NO: 29):
[131]
5'-CCACAGCCGACAGGATGGTGA-3'
[132]
Primer 117-R (SEQ ID NO: 30):
[133]
5 'CTCAGGGTGTAGCGGTTCGGT-3'
[134]
[135]
<1-3> Preparation of 2-keto-butyric acid leuBCD gene fragments which is involved in the biosynthetic pathway
[136]
[137]
To 2-keto-butyric acid biosynthesis gene enhanced the production of recombinant vector, Corey four of isopropyl tumefaciens in microbial malate dehydrogenase (3-isopropylmalate dehydrogenase) and di-isopropyl maleate hydratase other kinase (3- the isopropylmalate dehydratase) was prepared as follows leuBCD coding gene fragment.
[138]
First, the isopropyl malate dehydrogenase (EC1.1.1.85), Genoa mixer to obtain a 1359 bp fragment containing the ORF of the leuB gene encoding - using a tip system (Qiagen) Corynebacterium Tommy glutamicum ( Corynebacterium glutamicum ) , genomic DNA was extracted from ATCC13032.
[139]
Corynebacterium glutamicum isopropyl malate dehydrogenase of the ATCC13032-derived SEQ ID NO: has a 340 amino acid sequence represented by 17, leuB gene encoding the SEQ ID NO: having the nucleotide sequence represented by 18 .
[140]
And to the gDNA as a template derived from the SEQ ID NO: The PCR was performed using primer pairs 31 and 32. The PCR reactions were denatured at 95 ℃ for 30 seconds, elongation at 56 ℃ process in 30 seconds annealing and 72 ℃ 60 seconds was carried out 30 times repeatedly.
[141]
After the PCR product amplified therefrom by electrophoresis in 1.0% agarose gel was obtained by eluting the band of the desired size, it was named as "fragment leuB".
[142]
[143]
Primer leuB-5-cimA (SEQ ID NO: 31):
[144]
5'-TTGTTGAATTAATCTAGAGGTGACACCCCAGTGG-3'
[145]
Primer leuB-3-leuC (SEQ ID NO: 32):
[146]
5'-TCGCAGCTGCCACCGATATTTAGCTTTGCAGCGC-3'
[147]
[148]
Isopropyl maleate di Hydra other kinase (EC4.2.1.33) PCR using the gDNA of Corynebacterium glutamicum ATCC13032 as a template to obtain a 2078 bp fragment containing the ORF of the gene encoding the leuCD were .
[149]
Sequence has a nucleotide sequence described in 20 therefrom: leuC gene isopropyl maleate di to code the large subunits (large subunit) of Hydra other kinase, Corynebacterium glutamicum leuC gene of ATCC13032-derived SEQ ID NO: No: it encodes a polypeptide having the amino acid sequence represented by 19. Also, leuD gene isopropyl maleate D in coding small subunit (small subunit) of Hydra other kinase, Corynebacterium glutamicum leuD gene of ATCC13032-derived SEQ ID NO: having the nucleotide sequence represented by 22 which from SEQ ID NO: it encodes a polypeptide having the amino acid sequence represented by 21.
[150]
The PCR reaction to SEQ ID NO: 33 and 34 were performed in modified 95 ℃ for 30 seconds by using the primer pair, 56 ℃ in 30 seconds annealing and 72 seconds elongation ℃ 30 to 80 times in the repetition process.
[151]
After the PCR product amplified therefrom by electrophoresis in 1.0% agarose gel was obtained by eluting the band of the desired size, it was named as "fragment leuCD".
[152]
[153]
Primer leuC-5-leuB (SEQ ID NO: 33):
[154]
GCGCTGCAAAGCTAAATATCGGTGGCAGCTGCGA-5'-3 '
[155]
Primer leuD-3-XbaI (SEQ ID NO: 34):
[156]
5'-TGGCGGCCGCTCTAGAGCTTTCGCTATCAGACTG-3'
[157]
[158]
The leuB fragment leuCD fragment obtained above as a template and SEQ ID NO: was subjected to fusion PCR using the primer pair 31 and 34. PCR reactions were denatured at 95 ℃ for 30 seconds, elongation at 56 ℃ process in 30 seconds annealing and 72 ℃ 120 seconds was carried out 30 times repeatedly.
[159]
After the PCR product amplified therefrom by electrophoresis in 1.0% agarose gel was obtained by eluting the band of 3437 bp in size, it was named as "fragment leuBCD".
[160]
[161]
<1-4> Preparation of recombinant vectors p117-cj7-cimA (M) -leuBCD
[162]
[163]
To 2-keto-butyric acid biosynthesis gene enhanced the production of recombinant vector, Example <1-3> process the recombinant vector p117-cj7-cimA (M) prepared in Example <1-2> with XbaI, and treated with a leuBCD fragment obtained from the restriction enzyme XbaI was then ligated to the two fragments.
[164]
It was converted transformed with the recombinant vector produced therefrom by heat shock into E. coli DH5α cells, and plated it on LB solid medium containing kanamycin 25 μg / ml and incubated overnight at 37 ℃. The colonies which were incubated platinum containing 25 μg / ml kanamycin was inoculated on the 3 ml LB liquid medium and plasmid DNA was recovered using the After culturing overnight, plasmid mini kit peuraep.
[165]
Whether the production of recombinant vector with restriction enzymes was confirmed by treatment with XbaI, the SEQ ID NO: 29 and a primer pair to 30 modified on the 95 ℃ 30 seconds, at 56 ℃ in 30 seconds annealing and 72 ℃ 30 to 180 seconds of elongation process times clones were confirmed by performing PCR under the condition of repeating. A recombinant vector obtained therefrom was named "p117-cj7-cimA (M) -leuBCD".

Claims
[Claim 1]
A method of producing L- isoleucine, comprising: sheet ramal rate synthase (synthase citramalate) culturing a microorganism of the genus Corynebacterium, which comprises a protein which is active in the medium, and from the microorganisms or culture medium L - recovering the isoleucine.
[Claim 2]
The method of claim 1, wherein the sheet ramal rate synthase meta furnace knife Rhodococcus genus (genus Methanocaldococcus how a) derived from a microorganism, producing L- isoleucine.
[Claim 3]
The method of claim 1, wherein the sheet ramal rate synthase SEQ ID NO: method has the amino acid sequence is selected from 1, 3, 5, 7, 9, 11, 13 and 15 the group consisting of, producing L- isoleucine .
[Claim 4]
The method of claim 1, wherein the genus Corynebacterium microorganism is additionally activated the enhanced-isopropyl malate dehydrogenase (3-isopropylmalate dehydrogenase) and isopropyl maleate di Hydra other dehydratase (3-isopropylmalate dehydratase) the a method for producing L- isoleucine included.
[Claim 5]
The method of claim 1 wherein producing, L- isoleucine including the genus Corynebacterium microorganism further inactivation of pyruvate dehydrogenase (pyruvate dehydrogenase).
[Claim 6]
The method of claim 1 wherein producing, L- isoleucine the genus Corynebacterium which acetate is fed further in the step of culturing the microorganism.
[Claim 7]
The method of claim 1, wherein the microorganism of the genus Corynebacterium, Corynebacterium glutamicum ( Corynebacterium glutamicum method for producing a, L- isoleucine).
[Claim 8]
Comprising a protein of the sheet ramal rate synthase activity, L- isoleucine genus Corynebacterium microorganism having a production capability.
[Claim 9]
9. The method of claim 8 wherein the sheet ramal rate synthase SEQ ID NO: 1, 3, 5, 7, 9, 11, has the amino acid sequence selected from the group consisting of 13 and 15, having the L- isoleucine producing ability Corynebacterium spp.
[Claim 10]
9. The method of claim 8 wherein the microorganism is further activity is enhanced isopropyl malate dehydrogenase and di-isopropyl maleate hydratase other protein kinase, L- isophthalic in Corynebacterium having a leucine-producing ability, including microbe.
[Claim 11]
9. The method of claim 8 wherein the microorganism is further inactivation of pyruvate dehydrogenase, L- isoleucine genus Corynebacterium microorganism having a production capability including the.
[Claim 12]
9. The method of claim 8 wherein the microorganism is Corynebacterium glutamicum of, L- isoleucine genus Corynebacterium microorganism having a production capability.