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  • C12Y203/01001—Amino-acid N-acetyltransferase (2.3.1.1)

Definitions

• N-Acetylmethionine which is a derivative of methionine, has similar efficacy to methionine, but it can reduce methionine-specific flavors and can be added in a large amount compared to methionine when added to foods.
• methionine when methionine is used as a feed additive, it is first used by rumen microorganisms and thus is not absorbed by the animals, whereas N-acetylmethionine is a rumen-protected amino acid that is absorbed after passing through the rumen and reaching the intestine.
• Another object of the present disclosure is to provide a polypeptide having an acetyltransferase activity, the polypeptide being represented by an amino acid sequence of any one of SEQ ID NOS. 1 to 6 or an amino acid sequence having 90% or more homology to the amino acid sequence.
• Still another object of the present disclosure is to provide an expression vector including the polynucleotide.
• acyltransferase used in the present disclosure refers to an enzyme having an activity of transferring an acyl group from a donor to a receptor.
• the donor is not limited as long as it can provide an acyl group to a receptor, but may specifically be acetyl coenzyme A (acetyl-CoA).
• the receptor is not limited as long as it can receive an acyl group from a donor, but may specifically be L-methionine.
• the acyltransferase may be derived from genus Pseudomonas , genus Bacillus , genus Enterobacter , genus Pseudovibrio , genus Yarrowia , or genus Corynebacterium . More specifically, the acyltransferase may be derived from Pseudomonas putida, Bacillus subtilis, Enterobacter sp. 638 , Pseudovibrio sp. FO-BEG1, Yarrowia lipolytica , or Corynebacterium glutamicum.
• the enhancement of polypeptide activity may be performed by a method selected from a method of inserting a polynucleotide including a nucleotide sequence encoding each polypeptide into a chromosome, a method of introducing the polynucleotide into a microorganism through a vector system, a method of introducing a promoter exhibiting an improved activity upstream of a base sequence encoding each polypeptide or introducing each of the mutated polypeptides into a promoter, a method of modifying the nucleotide sequence in the 5′-UTR region, and a method of introducing a mutant of the base sequence encoding each polypeptide, but the present disclosure is not limited thereto.
• the microorganism having an acyltransferase activity may biosynthesize a receptor to which an acyl group is transferred from a donor, but the present disclosure is not limited thereto.
• the microorganism of the present disclosure may produce N-acetyl-L-methionine even when it is cultured in a medium to which L-methionine is not added because it has an ability of producing L-methionine that is an acceptor of an acyl group.
• the polynucleotide may be a base sequence encoding an amino acid sequence having 70% or more, 80% or more, or 90% or more homology to the above base sequence and having an acyltransferase activity substantially the same as or corresponding to that of the above base sequence.
• the polynucleotide may be a probe that can be prepared from a known gene sequence, for example, it may be a sequence encoding a polypeptide having an acyltransferase activity by hybridization with a complementary sequence to all or part of the base sequence under stringent conditions.
• stringent conditions refer to conditions where a specific hybrid is formed and a non-specific hybrid is not formed.
• the expression vector including the polynucleotide according to the present disclosure is an expression vector capable of expressing a target protein in a suitable host cell, and refers to a polynucleotide product including an essential control element operably linked so as to express a polynucleotide insert.
• Target proteins may be obtained by transforming or transfecting the prepared recombination vector in the host cell.
• the expression vector including the polynucleotide according to the present disclosure is not particularly limited, but includes Escherichia coli -derived plasmids (pYG601BR322, pBR325, pUC118, and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24, and YCp50), and Ti-plasmids that can be used for agrobacterium -mediated transformation.
• phage DNA include ⁇ -phages (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11, and lambda ZAP).
• the fusion protein may be purified by affinity chromatography.
• glutathione-S-transferase which is a substrate of this enzyme, may be used.
• glutathione which is a substrate of this enzyme.
• hexahistidine a desired target protein can be easily recovered using a Ni-NTA His-conjugated resin column (Novagen, USA).
• An expression vector including a polynucleotide encoding the polypeptide of the present disclosure or a part of the expression vector may be introduced into a host cell.
• a part of the expression vector refers to a portion of the expression vector, the portion including a portion of the polynucleotide encoding the polypeptide of the present disclosure so as to impart the acyltransferase activity into the host cell.
• T-DNA of Ti-plasmid transferred into the host cell in agrobacterium -mediated transformation may be exemplified, but the present disclosure is not limited thereto.
• the pH of a culture may be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid to a culture in an appropriate manner during the culturing of microorganisms. Further, during the culturing of microorganisms, the formation of bubbles may be suppressed by using a defoaming agent such as aliphatic polyglycol ester. Further, oxygen or oxygen-containing gas may be injected into a culture in order to maintain the aerobic state of the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected into the culture in order to maintain the anaerobic and non-aerobic states of the culture without injecting the gas.
• a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid
• targeted N-acetyl-L-methionine may be recovered from the culture solution using a suitable method known in the related art according to the method of culturing the microorganism of the present disclosure, for example, a batch culture method, a continuous culture method, or a fed-batch culture method.
• a suitable method known in the related art for example, a batch culture method, a continuous culture method, or a fed-batch culture method.
• centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, and the like may be used, and a combination of suitable methods known in the art may be used.
• Pseudomonas putida, Bacillus subtilis, Enterobacter sp. 638, or Yarrowia lipolytica PO1f was inoculated in a 14 mL disposable culture tube containing 3 mL of a liquid YPD medium (1% yeast extract, 2% bacto-peptone, 2% glucose), and was shake-cultured for 24 hours under conditions of 30° C. and 200 rpm to obtain a seed culture solution.
• a liquid YPD medium 1% yeast extract, 2% bacto-peptone, 2% glucose
• the culture solution was left in an oven at 50° C. overnight to be concentrated, and 1 ⁇ L of the concentrated culture solution was analyzed using thin layer chromatography. 10 mM of a N-acetyl-L-methionine reagent (Sigma Aldrich 01310) was used as a control group. The same spot as the control group was confirmed from the culture liquid concentrate of the above six kinds of microorganisms.
• polypeptides are polypeptides having novel acyltransferase activity lower than the homology of a conventional acyltransferase, and the microorganisms including these polypeptides are also microorganisms having novel acyltransferase activity which has not been previously known.
• Pseudomonas -derived, Bacillus -derived, Enterobacter -derived, and Pseudovibrio -derived novel acyltransferase genes (SEQ ID NOS. 7, 8, 9, and 10) were synthesized as codons optimized for Escherichia coli based on amino acid sequences (SEQ ID NOS. 1, 2, 3 and 4) of the respective enzymes.
• the culture solution was centrifuged to obtain cell pellets, the cell pellets were frozen in a refrigerator at ⁇ 20° C. The processes of remelting the fully frozen cell pellets at room temperature and refreezing these cell pellets were repeated three times to impart high permeability to the cell membrane.
• the culture solution was resuspended to a total volume of 5 mL by adding a 50 mM phosphate buffer (pH 7.0) to concentrate the culture solution.
• Example 4-2-1 Preparation of Acyltransferase Overexpression Vector for Introducing Microorganism of Genus Corynebacterium
• the gene based on base sequence 11 was polymerized through PCR using the vector pUCtk-ylmat of Example 1 as a template and using primers 13 and 14.
• the gene based on base sequence 12 was polymerized through PCR using the vector pUCtk-cgmat of Example 1 as a template and using primers 15 and 16.
• the colonies transformed with the vector into which the gene was inserted were selected by PCR, and then plasmids were obtained using a generally known plasmid extraction method.
• the obtained plasmids were named pECCG117-Pcj7-ppmat, pECCG117-Pcj7-bsmat, pECCG117-Pcj7-entmat, pECCG117-Pcj7-pvmat, pECCG117-Pcj7-ylmat, and pECCG117-Pcj7-cgmat.
• the six kinds of amplified acyltransferase polynucleotides were treated with restriction enzymes SpeI and XhoI to obtain DNA fragments. After six DNA fragments were obtained, they are linked to the SpeI and XhoI sites of the p414ADH vector, which is a shuttle vector of Escherichia and Saccharomyces , to be transformed into Escherichia coli DH5a, and were smeared in an LB solid medium containing ampicillin (100 mg/L).
• the colonies transformed with the vector into which the gene was inserted were selected by PCR, and then plasmids were obtained using a plasmid miniprep kit (Bioneer, Korea).
• the obtained plasmids were named p414ADH-ppmat, p414ADH-bsmat, p414ADH-entmat, p414ADH-pvmat, p414ADH-ylmat, and p414ADH-cgmat.
• Example 4-3-2 Preparation of Strain for Introducing Novel Acyltransferase Overexpression Vector and Confirmation of N-Acetyl-L-Methionine Production Capacity
• the vectors were introduced into the Saccharomyces cerevisiae CEN.PK2-1D, and then smeared in a YPD plate medium (1% yeast extract, 2% bacto-peptone, 2% glucose), and cultured at 30° C. for 24 hours to obtain strains.
• the obtained strains were named ScCEN/p414ADH-ppmat, ScCEN/p414ADH-bsmat, ScCEN/p414ADH-entmat, ScCEN/p414ADH-pvmat, ScCEN/p414ADH-ylmat, ScCEN/p414ADH-cgmat, and ScCEN/p414ADH.
• the gene based on base sequence 11 was polymerized through PCR using the vector pUCtk-ylmat of Example 1 as a template and using primers 37 and 38.
• the gene based on base sequence 12 was polymerized through PCR using the vector pUCtk-cgmat of Example 1 as a template and using primers 39 and 40.
• the PCR was performed by conducting denaturation at 94° C. for 5 minutes, repeating denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds and polymerization at 72° C. for 1 minute 30 times, and then conducting a polymerization reaction at 72° C. for 7 minutes.

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