[1]Herein is a microorganism comprising this polypeptide or with an acyl transferase activity; N- acetyl-producing composition of the -L- methionine comprising the polypeptide or microorganism; A method of producing N- acetyl -L- methionine using the polypeptide or microorganism. In addition, the present application relates to an expression vector comprising the polynucleotide and the polynucleotide coding for the polypeptide.
BACKGROUND
[2]
N- acetyl-methionine is similar to the effect of methionine and the methionine derivative, it is possible to reduce the methionine specific effort it is possible to add a large amount of methionine compared to when added to food. In the case of animals with a rumen when used as a feed additive is methionine line used by the rumen microorganisms, while does not absorb the animals, protected amino acids are absorbed and then reaches the sheet passes through the rumen for N- acetyl methionine (rumen- a protected amino acid). Decomposition resistance in the rumen of the N- acetyl -DL- methionine is a known bar (Amos et al., Journal of Animal Science, 1974, 39 (3), pp. 612-617). Therefore, the production of N- acetyl-methionine has an industrial value, particularly because the feed additive is desirable to provide a L-amino acid has a high absorption rate is fast in vivo utilization when used as, research and development for the N- acetyl -L- methionine this is required. In the case of an enzyme reported to be the conversion of methionine to existing as N- acetyl-methionine is the only YncA of Escherichia coli (US 8,143,031 B2). In the case of the above document, hayeoteul determine the use of indirectly-acetyl coenzyme A (acetyl-coA) through the DTNB assay to measure the activity of YncA only, failed to confirm the production of the actual N- acetyl-methionine. In addition, it is not the identified map bar produced the actual N- acetyl-methionine by a transformant transformed by the YncA.
Detailed Description of the Invention
SUMMARY
[3]
The inventors have an ongoing effort to produce the N- acetyl -L- methionine via microbial fermentation or enzymatic reaction, to explore the N- acetyl -L- methionine producing microorganism in capability was identify acyl transferase of the 6 new species, present invention by using a novel acyl transferase or a microorganism expressing them of the present invention confirmed that this can be compared to known acyl transferase producing N- acetyl -L- methionine of high density and economic was completed.
Problem solving means
[4]
One object of the present application is to provide a microorganism having the sequence number 1 to any of amino acid sequence or the activity of the acyl transferase comprising a polypeptide represented by the amino acid sequence with at least 90% homology of the SEQ ID NO: 6 will be.
[5]
It is another object of the present application is to provide a polypeptide shown by amino acid sequence having any one of amino acid sequence, or the 90% or more of homology with the active transition acetyl, SEQ ID NO: 1 to SEQ ID NO: 6 to L- methionine.
[6]
It is another object of the present application is to provide a polynucleotide encoding the polypeptide.
[7]
It is another object of the present application is to provide an expression vector including the polynucleotide.
[8]
It is another object of the present application is (i) the microorganism or a culture thereof; (Ii) said polypeptide; Or to provide a composition for preparing the N- acetyl -L- methionine from, L- methionine, including combinations thereof, as an active ingredient.
[9]
It is another object of the present application is (i) the microorganism or a culture thereof; (Ii) said polypeptide; Or to provide a method of using a combination thereof comprising the step of acetylating the L- methionine, N- acetyl -L- methionine.
Effects of the Invention
[10]
Microorganism containing a novel acyl transferase of the present application is the microorganism of the present application bar, which is enhanced in the acyl transferase activity it can be efficiently used to by the acetyl L- methionine producing N- acetyl -L- methionine.
Best Mode for Carrying Out the Invention
[11]
, One aspect of the present application for achieving the above object has any of the SEQ ID NO: 1 to SEQ ID NO: 6 one amino acid sequence or the acyl transferase activity comprising a polypeptide represented by the amino acid sequence with at least 90% homologous It provides a microorganism having.
[12]
[13]
As used herein, the term "acyl transferase (acyltransferase)" refers to an enzyme having the activity to transfer from a donor to an acyl group (acyl group) to the receptor. The donor herein include, but not a limitation, which provides for an acyl receptor, and may be specifically acetyl coenzyme A (acetyl-coA). In addition, although herein, the receptor is not limited as long as it can be an acyl group from a donor, it can be specifically L- methionine.
[14]
Specifically, the acyl transferase may be in the genus Pseudomonas, genus Bacillus, Enterobacter genus, Pseudomonas Vibrio genus, Yarrow subtotal, or the genus Corynebacterium origin, more specifically, Pseudomonas footage is, Bacillus subtilis, Enterobacter sp. 638, pseudo Vibrio sp. FO-BEG1, Yaroslavl WIA Mathematica license can be repositories or Corynebacterium glutamicum origin.
[15]
Specifically, the acyl transferase may be a polypeptide having any of the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6. In addition, SEQ ID NO: 1 to the SEQ ID NO: 6 of any of the amino acid sequence with 70% or more, particularly 80% or more, more particularly having at least 90% homologous, substantially any of SEQ ID NO: 1 to SEQ ID NO: 6 may be a polypeptide having an amino acid sequence having one of the identical or corresponding to the acyl transferase activity and the amino acid sequence. Or substantially in SEQ ID NO: 1 to at SEQ ID NO: 6 one amino acid sequence having one of the identical or corresponding to the acyl transferase activity and the amino acid sequence of some sequences are deleted, modified, substituted or added in the amino acid as a sequence having such homology it is a sequence which, in the case thereof, it is obvious also included in the scope of the present application.
[16]
In the amino acid sequence or nucleotide sequence of the gene encoding the term "homology" refers to a polypeptide, as used herein, of a base or amino acid residue between After alignment (align) so that the best possible match between the positive sequence in a specific comparison region sequence It refers to the same degree. If homology is sufficiently high expression product of the gene it may have the same or similar activity. % Of the sequence identity may be determined using the sequence comparison program known, there may be mentioned BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc., for example.
[17]
[18]
As used herein, the term "microorganism having acyl transferase activity" refers to a microorganism producing the acyl transferase out within the organism, and / or organism. Specifically, the number of microorganisms of the present disclosure includes a polypeptide having any of the amino acid sequence of said SEQ ID NO: 1 to SEQ ID NO: 6, and pass it to an acyl-bar, having a receptor activity of the acyl transferase. Specifically, the microorganism of the present application can produce an N- acetyl -L- methionine import acetyl transition to the active L- methionine. N- acetyl -L- methionine and N- acetyl-methionine herein are used interchangeably.
[19]
Further, the microorganism of the present application include microorganisms as well as microorganisms that are implicitly included in a polypeptide having any of the amino acid sequence of said SEQ ID NO: 1 to SEQ ID NO: 6, the activity of the polypeptide compared to their intrinsic activity . That is, the production capacity of an acyl transferase can be enhanced or imparted by the natural or artificial mutants or species improved. "Strengthening" of the term active polypeptides as used herein, means to hold the micro-organisms and improve the active polypeptide in. Enhance the activity of the polypeptide is not as long as the activity of each polypeptide, such as strengthening of the activity of the target polypeptide can be strengthened bibyeon natural in its natural state, or the polypeptide in this state, limited. For example, i) increasing the copy number of the polynucleotide coding for the respective polypeptide, ii) transformation of the expression control sequences to increase the expression of the polynucleotide, iii) the polynucleotide sequence on the chromosome so that enhance the activity of the respective polypeptide modifications and iv) may be performed by a method selected from the group consisting of a combination thereof. Specifically, the improvement in the upstream of the nucleotide sequence encoding the respective poly how to insert a polynucleotide containing a base sequence encoding the peptide in a chromosome, a method of introducing microorganisms by introducing the polynucleotide in a vector system, each polypeptide introducing a promoter that is the active, or made of a method of introducing a variant of the nucleotide sequence to a method of introducing each of the polypeptide gave a mutation to a promoter, the method of modifying the base sequence of the 5'-UTR and coding region for each polypeptide It may be performed by a method selected from the group, but
[20]
Further, the microorganism having an acyl transferase activity in the present application may be included regardless the one derived from a microorganism having an acyl transferase activity. An example in S. Cherry teeth ( Escherichia sp.), The genus Corynebacterium ( Corynebacterium sp.), Mai in process as Saccharomyces (Saccharomyces sp.) Or Yarrow subtotal in ( Yarrowia may be sp.), And more specifically to E. coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), a My process serenity busy as Saccharomyces ( Saccharomyces cerevisiae ), or Yarrow subtotal lipoic Lai urticae ( Yarrowia lipolytica may be a), but is not limited thereto.
[21]
Further, the microorganism having an acyl transfer activity to herein may be a microorganism with improved cell membrane permeability of the donor and / or acceptor. Method of increasing the permeability of the cell membrane may be using existing known methods, but can be specifically to repeat the step of freezing and thawing the microorganism is not limited to this.
[22]
Further, the microorganism having an acyl transferase activity in the present application can produce an acyl group transfer product by addition of a substrate involved in the biosynthetic pathway of the donor in a microorganism, such as glucose, instead of the acyl donor to the medium. Specifically, to produce the N- acetyl -L- methionine by culturing in glucose instead of acetyl coenzyme A was added culture medium a microorganism of the present application can.
[23]
Further, the microorganism having an acyl transferase activity in the present application but can be a biosynthetic receptor acyl group is transferred from the donor, but not limited thereto. Specifically, the number of microorganisms of the present application is ah brought L- methionine-producing ability of the receptor of the group even if the culture medium in the L- methionine is not added to produce an N- acetyl -L- methionine can.
[24]
Further, the microorganism having an acyl transferase activity in the present application is a conventional known for the biosynthesis-related routes or related mechanisms, such as a substrate source strength related mechanisms to promote N- acetyl -L- methionine-producing ability, independent of the acyl transferase Additionally, the mutation may be a mutation introducing microorganisms.
[25]
[26]
One aspect of the present application provides one of which is active in the acyl transferase, SEQ ID NO: 1 to SEQ ID NO: 6 one amino acid sequence or the polypeptide represented by the amino acid sequence with more than 90% homology. Specifically, the activity of the acyl transferase may be an acetyl transfer activity for the L- methionine.
[27]
The polypeptide as defined above.
[28]
[29]
One aspect of the present application provides polynucleotides encoding any of which is active in the acyl transferase, SEQ ID NO: 1 to SEQ ID NO: 6 one amino acid sequence or the polypeptide represented by the amino acid sequence with at least 90% homologous . The polypeptide as defined above.
[30]
For example, the polynucleotide may be a any one of the nucleotide sequence of SEQ ID NO: 7 to SEQ ID NO: 12, but is not limited thereto. In addition, the polynucleotides due to the degeneracy (genetic code degeneracy) of the genetic code are included in the base sequence and also variants thereof encoding the same amino acid sequence herein. For example, it may be, which is modified to have the optimum codon depending on the microorganism used.
[31]
Specifically, an amino acid sequence having the nucleotide sequence with 70% or more, specifically 80% or greater, more specifically has a greater than 90% homologous, substantially the activity of the transferase acyl be the same as or corresponds to the nucleotide sequence It may be a nucleotide sequence encoding. Or which can be prepared from the gene sequence of the known probe, for example, by Hydride Chemistry under the complementary sequences and stringent conditions to all or a portion of the nucleotide sequence may be a sequence encoding poly peptayideueul having the activity of an acyl transferase . And forming the "stringent conditions" refers to a so-called specific hybrid, refers to a condition non-specific hybrid is not formed. For example the expressed, homology between high gene, for example, 80% or more, specifically, more specifically at least 90%, at least 95%, more specifically 97% or more, particularly Specifically, more than 99% of the hybrid gene having the same sex between shoes, rather homologous conditions do not hybridize low gene together, or the 60 ℃ washing condition of an ordinary Southern hybridization, and 1 × SSC, 0.1% SDS, specifically 60 ℃, 0.1 × SSC, with 0.1% SDS, more specifically, in the salt concentration and temperature corresponding to 68 ℃, 0.1 × SSC, 0.1% SDS, 1 time, and specifically may be exemplified by conditions under which washing twice or three times ( Sambrook et al, Molecular Cloning:.. A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)).
[32]
The probe used for the hybridization may be a part of the complementary sequence of the base sequence. These probes, with the oligonucleotides constructed based on the known sequences as primers, can be constructed by PCR, a gene fragment containing these sequences as the template. For example, as the probe, it may be a gene fragment of 300bp long. More specifically, as a probe, in the case of using a gene fragment of about 300bp length, as the washing conditions of hybridization, 50 ℃, are exemplified a 2 × SSC and 0.1% SDS.
[33]
Genes used herein, including the polypeptide sequence and a promoter sequence to which they are coded can be obtained from a known database. Examples, but can be obtained in the NCBI GenBank etc., not limited to this.
[34]
[35]
One aspect of the present application provides an expression vector comprising the polynucleotide.
[36]
Expression vector comprising a polynucleotide of the present application refers to a polynucleotide construct that contains essential regulatory elements as an expression vector capable of expressing the desired protein, the polynucleotide insert operably linked to the expression in a suitable host cell. By the transfection of the recombinant vector into a host cell producing conversion (transformation) or transfection (transfection), it is possible to obtain the desired protein, which.
[37]
Expression vector comprising a polynucleotide provided herein is not particularly limited to, Escherichia coli-derived plasmid (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis ( Bacillus subtilis ) - derived plasmid (pUB110 and pTP5), yeast- the resulting plasmid containing the (YEp13, YEp24 and YCp50) and Agrobacterium mediated transformation Ti- plasmid that can be used for conversion. Specific examples of phage DNA is a phage λ- (Charon4A, Charon21A, EMBL3, EMBL4 , λgt10, λgt11 and λZAP). In addition, retroviruses (retrovirus), adenovirus (adenovirus) or vaccinia virus (vaccinia virus) animal viruses, insect viruses such as baculovirus and the virus (baculovirus), double stranded plant viruses such as (eg, CaMV), single-stranded viruses or it is a viral vector derived from a mini-virus can also be used.
[38]
As well as a vector of the present invention, nucleic acid expression activating protein (e.g., B42 same) is connected to the fusion plasmid (containing the fusion plasmid, for example, pJG4-5) may be used. Also, to facilitate purification of the desired protein is recovered in the present invention, the plasmid vector may further comprise a different sequence, as needed. This plasmid contains the fusion, and the like GST, GFP, His-tag, Myc-tag with a tag (tag), but it is not a fusion plasmid of the present invention by the above examples only.
[39]
Further, in the production of the fusion protein, and may include a chromatography step, the fusion protein can be purified by a particular affinity chromatography. For example, glutathione -S- transferase I when a fusion is the case can be used a substrate of glutathione of the enzyme, hexa-histidine is used by using an Ni-NTA resin binding His- column (Novagen, USA) to a desired protein, a can be easily recovered.
[40]
In order to insert the polynucleotide into a vector of the present application, a method can be used to cut and then inserted into restriction sites or cloning site of an appropriate vector DNA a purified DNA with an appropriate restriction enzyme.
[41]
A polynucleotide encoding a polypeptide of the present disclosure may be operably linked to the vector. In addition to vectors of the present application is a nucleic acid of a promoter and present enhancer (enhancer) and the sheath element (cis element), splicing signals (splicing signal), a poly A additional signal (poly A addition signal), selection markers (selection marker), such as, Lai bojom binding sequence (ribosome binding sequence, SD sequence) may further comprise the like. Examples of the selection marker, chloramphenicol resistance nucleic acid, the ampicillin resistance nucleotide, dihydro folate reductase (dihydrofolate reductase), neomycin, but may have to be used such as resistance nucleic acid, being a further component which is connected to be operated by the above examples limit not. in the present invention the term "transfection" is the introduction of the foreign DNA to be enabling replication by DNA or chromosomal integration as a factor of chromosome completed by introducing the DNA into a host into a cell artificially causing genetic changes It refers to the phenomenon.
[42]
May be introduced into a portion of an expression vector or the expression vector comprising a polynucleotide encoding a polypeptide of the present application within a host cell transfected the switch, in which a portion of the expression vector is, the activity of the acyl transferase above in a host cell to grant means a part of an expression vector comprising a polynucleotide portion encoding a polypeptide of the present application. For example, but it is the T-DNA of the Ti plasmid that is transferred into the host cell in Agrobacterium bacteria mediated transformation method, without being limited thereto.
[43]
Transfection method of the present application may be used and any transformation method can be easily carried out according to conventional methods in the art. In general, transformation methods, the CaCl 2 precipitation, CaCl 2 Hanahan method, electroporation method (electroporation), the calcium phosphate precipitation method with improved efficiency by using a reducing substance of DMSO (dimethyl sulfoxide) in the method, the protoplast fusion method, a silicon carbide there is a stirring method using a fiber, Agrobacterium-mediated bacterial transformation method, transfection method using PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation method and the like. A method for transforming a vector containing a polynucleotide encoding a polypeptide of the present application is not limited to the above examples, it may be used without transformation or transfection methods commonly used in the art limitations.
[44]
The type of host to be used for manufacturing transformants transformed cells herein, but is not particularly limited as long as that to express a polynucleotide of the present application. Specific examples of hosts that can be used in the present invention is Escherichia coli ( E. coli Escherichia (such as) Escherichia ) bacteria belonging to the genus; Bacillus subtilis ( Bacillus subtilis , Bacillus (such as) Bacillus ) bacteria belonging to the genus; Pseudomonas footage is ( Pseudomonas putida Pseudomonas (such) Pseudomonas ) bacteria belonging to the genus; Celebi jiae as Saccharomyces Mai Seth ( Saccharomyces cerevisiae ), Ski investigation Seth Caro My pombe ( Schizosaccharomyces pombe yeast like); There are animal cells, plant cells and insect cells. Specific examples of the specific examples of E. coli strains that can be used in the present invention CL41 (DE3), BL21 or HB101, Bacillus subtilis strain has WB700 or LKS87.
[45]
The expression vector is introduced into a transformant containing the polynucleotide of the present disclosure may be transformed in the form of transformed cells or organisms.
[46]
Herein is a promoter, it may also be used by any promoter to express a polynucleotide of the present application in a host. For example, trp promoter, lac promoter, PL promoter or PR promoter of E. coli or phage, such as-derived promoter; T7 promoter, such as E. coli infected with phage-a (Korea Laid-Open Publication No. 10-2004-0107215 No.) derived promoter, CaMV35S, MAS or histone promoter, cj7 promoter may be used. In addition, the promoter can also be used in artificially modified such as the tac promoter.
[47]
[48]
One aspect of the present application: (i) the microorganism or a culture of the present application; (Ii) a polypeptide of the present application; Or it provides a method of manufacturing an N- acetyl -L- methionine comprising the step of acetylating the L- methionine using a combination of the two.
[49]
Specifically, the production method (i) or a microorganism culture of the present application; (Ii) a polypeptide of the present application; Or a step of acetylation of L- methionine, by using a combination thereof; And it may include the step of recovering the acetylated L- methionine in, N- acetyl -L- methionine.
[50]
[51]
Herein, the term "culture" is meant that the growth in the environmental conditions of suitably adjusting the microorganism. Culturing process of the present application may be made in accordance with the appropriate culture medium and culture conditions known in the art. This culturing process may be used by those skilled in the art easily adjusted according to the selected strain. In the method, the method comprising culturing the microorganism, 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. Medium, and other cultures used for the culture of microorganisms conditions of the present application, if the medium used for the culture of ordinary microorganisms, but can whichever used without particular limitations, specifically, appropriate to the microorganism of the present carbon source, a nitrogen source, personnel, inorganic compounds, in the usual medium containing such amino acids and / or vitamins it can be cultured while controlling the temperature, pH, etc. under aerobic conditions.
[52]
Carbohydrates such herein as the carbon source are glucose, fructose, sucrose, maltose, mannitol, sorbitol; Alcohols such as sugar alcohols, glycerol, pyruvic acid, lactic acid, citric acid; It may include amino acids such as organic acids, glutamic acid, methionine, lysine, but not limited to this. In addition, starch hydrolyzate, molasses, black strap molasses, rice winter, Casa member, sugar cane waste, and corn steep liquor can be used an organic nutrient source of such natural, specifically, the switch to glucose and sterilized pretreated molasses (that is, a reducing sugar the molasses), and the carbohydrate can be used as such, can be variously used for any other suitable amount of carbon sources, without limitation. These carbon sources can be used alone or in combination of two or more.
[53]
The inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, carbonate facial titanium, ammonium nitrate wish; Organic nitrogen sources such as amino acids, such as glutamic acid, methionine, glutamine, peptone, NZ- amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, defatted soybean cake or its degradation products It can be used. These nitrogen sources can be used alone or two or more of them, but this can be used in combination, but is not limited thereto.
[54]
The personnel in the phosphoric acid first, potassium phosphate, potassium 2, or its corresponding sodium-containing salts can be included such as. Inorganic compounds include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, may be used such as calcium carbonate and more than one amino acid, may be included are vitamins and / or appropriate precursors, but is not limited thereto. These media or precursors may be added batchwise or continuously to the culture, but is not limited thereto.
[55]
As used herein, by a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid was added in an appropriate manner in the culture during the culturing of the microorganism, it is possible to adjust the pH of water culture. In addition, during the culture it is possible to suppress the air bubbles generated by using antifoaming agents such as fatty acid polyglycol ester. Furthermore, it can be injected without injection of the gas or of nitrogen, hydrogen or carbon dioxide gas in order to maintain the culture, oxygen or oxygen-containing gas injected into or anaerobic conditions and Miho group into the water in order to maintain aerobic condition of water culture.
[56]
Incubation temperature of water, but can be different in accordance with the present microorganism, and specifically, 20 to 50 ℃ ℃, more specifically, 25 ℃ to 40 ℃, but is not limited thereto. The incubation period may be continued until it is obtained the production of the desired useful substance, specifically, it may be a 10 hours to 100 hours but not limited thereto.

Claims

[Claim 1]SEQ ID NO: 1 to SEQ ID NO: 6 or the amino acid sequence of any one of microorganisms having an activity of acyl transferase comprising a polypeptide represented by the amino acid sequence with 90% or more of homology.
[Claim 2]
According to claim 1, wherein the microorganism is S. Cherry teeth in ( Escherichia sp.), The genus Corynebacterium ( Corynebacterium sp.), Mai in process as Saccharomyces ( Saccharomyces sp.) And Yarrow subtotal in ( Yarrowia a sp.) microorganism selected from the group consisting of.
[Claim 3]
The method of claim 1, wherein the microorganism is a microorganism producing N- acetyl -L- methionine import acetyl transition to the active L- methionine.
[Claim 4]
Having an activity to transfer an acetyl L- methionine, SEQ ID NO: 1 to SEQ ID NO: 6 in which one of the amino acid sequence or the polypeptide represented by the amino acid sequence with more than 90% homology.
[Claim 5]
Claim 4 of the polynucleotide encoding the polypeptide.
[Claim 6]
The expression vector containing the polynucleotide of Claim 5.
[Claim 7]
(I) any one of claim 1 to claim 3, wherein the microorganism of or a culture; (Ii) the polypeptide of claim 4; Or a combination thereof as an active ingredient, N- acetyl -L- methionine from the composition for preparing L- methionine.
[Claim 8]
(I) any one of claim 1 to claim 3, wherein the microorganism of or a culture; (Ii) the polypeptide of claim 4; Alternatively, the method for producing N- acetyl -L- methionine comprising the step of acetylating the L- methionine using a combination of the two.
[Claim 9]
10. The method of claim 8, wherein the method further comprising the step of recovering the acetylated L- methionine in, N- acetyl -L- methionine.