阅读说明：本技术 乙酰羟酸合酶变体、包含其的微生物和用其生产l-支链氨基酸的方法 (Acetohydroxyacid synthase variants, microorganisms comprising the same, and methods for producing L-branched amino acids using the same ) 是由 全爱智 宋秉哲 李智惠 金宗贤 金蕙园 于 2018-07-10 设计创作，主要内容包括：本公开涉及新的乙酰羟酸合酶、包含其的微生物或利用其生产L-支链氨基酸的方法。(The present disclosure relates to a novel acetohydroxyacid synthase, a microorganism comprising the same, or a method for producing an L-branched amino acid using the same.)

1. Acetohydroxy acid synthase variants in which the amino acid 503 (tryptophan) from the N-terminus is replaced by glutamine, asparagine or leucine in the acetohydroxy acid synthase large subunit (acetolactate synthase large subunit; IlvB protein) of the amino acid sequence SEQ ID NO: 1.

2. The acetohydroxyacid synthase variant according to claim 1, wherein said acetohydroxyacid synthase variant consists of the amino acid sequence of any one of SEQ id nos 31 to 33.

3. A polynucleotide encoding an acetohydroxyacid synthase variant according to claim 1 or 2.

4. A vector comprising a polynucleotide encoding an acetohydroxyacid synthase variant according to claim 1 or 2.

5. A transformant in which the vector of claim 4 is introduced.

6. A microorganism of the genus Corynebacterium (Corynebacterium) that produces L-branched-chain amino acids, comprising at least one of the following: the acetohydroxyacid synthase variant according to claim 1 or 2; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

7. The L-branched-chain amino acid-producing microorganism according to claim 6, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum (Corynebacterium glutamicum).

8. The L-branched-chain amino acid-producing microorganism according to claim 6, wherein the L-branched-chain amino acid is L-valine or L-leucine.

9. A method for producing an L-branched amino acid, comprising:

(a) culturing the L-branched amino acid-producing microorganism according to claim 6 in a medium; and

(b) recovering the L-branched amino acid from the microorganism or culture medium in step (a).

10. The method for producing an L-branched amino acid according to claim 9, wherein the L-branched amino acid is L-valine or L-leucine.

Technical Field

The present disclosure relates to novel acetohydroxyacid synthase variants and uses thereof, and in particular, acetohydroxyacid synthase variants, microorganisms containing the same, or methods of producing L-branched amino acids.

Background

Branched-chain amino acids (e.g., L-valine, L-leucine, and L-isoleucine) are known to increase protein levels in individuals and to have an important role as an energy source during exercise, and thus are widely used in medicines, foods, and the like. Regarding the biosynthesis of branched-chain amino acids, the same enzymes are used in parallel biosynthetic pathways, and thus it is difficult to produce a single kind of branched-chain amino acid on an industrial scale by fermentation. In the preparation of branched-chain amino acids, the role of acetohydroxyacid synthase (i.e., the first enzyme in the biosynthesis of branched-chain amino acids) is of primary importance; however, previous studies on acetohydroxyacid synthase have focused mainly on the release of feedback inhibition due to modification of acetohydroxyacid synthase small subunit (IlvN Protein) (Protein ExprPurif.2015 5 months; 109:106-12, US2014-0335574, US2009-496475, US2006-303888, US2008-245610), thus revealing a serious shortage of relevant studies.

Acetohydroxy acid synthase is an enzyme having the function of producing acetolactate from two molecules of pyruvate and 2-acetyl-2-hydroxy-butyrate from ketobutyrate and pyruvate. Acetohydroxyacid synthase catalyzes the decarboxylation of pyruvate and the condensation reaction with another molecule of pyruvate to produce acetolactate, which is a precursor of valine and leucine; or catalyze pyruvate decarboxylation and condensation reactions with 2-ketobutyric acid to produce acetohydroxybutanoate, which is the precursor of isoleucine. Acetohydroxyacid synthase is therefore a very important enzyme involved in the initial biosynthesis of L-branched amino acids.

Disclosure of Invention

Technical problem

The present inventors have made efforts to efficiently produce L-branched amino acids, and thus they developed large subunit variants. Then, the present inventors confirmed that L-branched amino acids are produced in high yield from microorganisms containing the variant, thereby completing the present disclosure.

Technical scheme

It is an object of the present disclosure to provide acetohydroxyacid synthase variants.

It is another object of the present disclosure to provide a polynucleotide encoding an acetohydroxyacid synthase variant, a vector containing the polynucleotide, and a transformant into which the vector is introduced.

It is still another object of the present disclosure to provide a microorganism producing an L-branched-chain amino acid, wherein the microorganism contains the acetohydroxyacid synthase variant or has the vector introduced therein.

It is yet another object of the present disclosure to provide a method for producing an L-branched amino acid, comprising: culturing a microorganism producing an L-branched chain amino acid in a medium; and recovering the L-branched-chain amino acid from the microorganism or the medium thereof.

Advantageous effects

When the activity of acetohydroxyacid synthase variants according to the present disclosure is introduced into a microorganism, the microorganism can significantly improve the ability to produce L-branched amino acids. Therefore, the microorganism can be widely used for mass production of L-branched amino acids.

Best mode for carrying out the invention

To achieve the above objects, the present disclosure provides, in one aspect, acetohydroxyacid synthase variants in which the 96 th amino acid (i.e., threonine) is substituted with an amino acid other than threonine, the 503 th amino acid (i.e., tryptophan) is substituted with an amino acid other than tryptophan, or both the 96 th amino acid (i.e., threonine) and the 503 th amino acid (i.e., tryptophan) are substituted with another amino acid in the acetohydroxyacid synthase large subunit (i.e., acetolactate synthase large subunit; IlvB protein).

Specifically, the large subunit of acetohydroxyacid synthase can have the amino acid sequence of SEQ ID NO. 1. More specifically, the acetohydroxyacid synthase variant can be an acetohydroxyacid synthase variant that: wherein, in the amino acid sequence of SEQ ID NO. 1, the 96 th amino acid (i.e., threonine) or the 503 th amino acid (i.e., tryptophan) from the N-terminus thereof is substituted with another amino acid; or the amino acid at position 96 (i.e., threonine) and the amino acid at position 503 (i.e., tryptophan) are each substituted with another amino acid.

As used herein, the term "acetohydroxyacid synthase" refers to an enzyme that is involved in the biosynthesis of L-branched amino acids, and which can be involved in the first step of the biosynthesis of L-branched amino acids. Specifically, acetohydroxyacid synthase can catalyze pyruvate decarboxylation and condensation reactions with another molecule of pyruvate to produce acetolactate (i.e., a precursor of valine), or pyruvate decarboxylation and condensation reactions with 2-ketobutyrate to produce acetohydroxybutanoate (i.e., a precursor of isoleucine). Specifically, starting from acetolactate, L-valine is biosynthesized by sequential reactions catalyzed by acetohydroxy acid isomeroreductase (isomeroreductase), dihydroxy acid dehydratase and transaminase B. In addition, starting from acetolactate, L-leucine is biosynthesized as a final product by sequential reactions catalyzed by acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, 2-isopropylmalate synthase, isopropylmalate isomerase, 3-isopropylmalate dehydrogenase and transaminase B. Meanwhile, starting from acetohydroxy butyrate, L-isoleucine is biosynthesized as a final product through a sequential reaction catalyzed by acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase and transaminase B. Acetohydroxyacid synthases are therefore important enzymes in the biosynthetic pathway of L-branched amino acids.

Acetohydroxyacid synthase is encoded by two genes, i.e., ilvB and ilvN. The ilvB gene encodes the large subunit of acetohydroxyacid synthase (IlvB), while the ilvN gene encodes the small subunit of acetohydroxyacid synthase (IlvN).

In the present disclosure, the acetohydroxyacid synthase may be an enzyme derived from a microorganism of the genus Corynebacterium (Corynebacterium), and specifically from Corynebacterium glutamicum (Corynebacterium glutamicum). More specifically, as the bulky subunit of acetohydroxyacid synthase, any protein having an activity of the IlvB protein and having 70% or more, specifically 80% or more, more specifically 85% or more, even more specifically 90% or more, and even more specifically 95% homology or identity with the amino acid sequence of SEQ ID NO. 1, and the amino acid sequence of SEQ ID NO. 1 can be included without limitation. In addition, due to codon degeneracy, a polynucleotide encoding a protein having an IlvB protein activity may be variously modified in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region in consideration of codons preferred in an organism expressing the protein. The nucleotide sequence may be included without limitation as long as it encodes the amino acid sequence of SEQ ID NO. 1, and specifically, it may be a nucleotide sequence encoded by the nucleotide sequence of SEQ ID NO. 2.

As used herein, the term "acetohydroxyacid synthase variant" refers to a protein in which one or more amino acids in the amino acid sequence of the acetohydroxyacid synthase protein are modified (e.g., added, deleted, or substituted). Specifically, acetohydroxyacid synthase variants are proteins in which a modification of the disclosure results in a significant increase in their activity as compared to their wild type or prior to modification.

As used herein, the term "modification" refers to conventional methods for improving enzymes, and any method known in the art may be used without limitation, including strategies such as rational design (rational design) and directed evolution (directive resolution). For example, strategies for rational design include methods of specifying amino acids at specific positions (site-directed mutagenesis or site-specific mutagenesis) and the like, and strategies for directed evolution include methods of inducing random mutagenesis and the like. In addition, the modification may be one induced by natural mutation without external manipulation. Specifically, the acetohydroxyacid synthase variant can be an isolated variant, a recombinant protein, or a non-naturally occurring variant, but the acetohydroxyacid synthase variant is not limited thereto.

The acetohydroxyacid synthase variants of the present disclosure can be, in particular, an IlvB protein having the amino acid sequence of SEQ ID NO:1, wherein amino acid 96 (threonine) or amino acid 503 (tryptophan) is mutated from its N-terminus; or the 96 th amino acid (threonine) and the 503 th amino acid (tryptophan) are simultaneously substituted with another amino acid, but the acetohydroxy acid synthase variant is not limited thereto. For example, an acetohydroxyacid synthase variant of the present disclosure can be an IlvB protein in which amino acid 96 (threonine) is substituted with serine, cysteine, or alanine, or amino acid 503 (tryptophan) is substituted with glutamine, asparagine, or leucine. In addition, it is apparent that any acetohydroxyacid synthase variant having an amino acid sequence in which the amino acid at position 96 or the amino acid at position 503 is substituted with another amino acid and a partial amino acid sequence is deleted, modified, substituted or added can exhibit the same or corresponding activity as the acetohydroxyacid synthase variant of the present disclosure.

Further, acetohydroxyacid synthase variant large subunits themselves, acetohydroxyacid synthases including acetohydroxyacid synthase variant large subunits, and acetohydroxyacid synthases including acetohydroxyacid synthase variant large subunits and small subunits, having the above-described modifications, may all be included within the scope of the acetohydroxyacid synthase variants of the present disclosure, but the acetohydroxyacid synthase variants are not limited thereto.

In the present disclosure, it was confirmed that the production amount of L-branched amino acids can be increased by substituting amino acid at position 96 and amino acid at position 503 of acetohydroxyacid synthase protein with various other amino acids, and thus it was confirmed that the amino acid positions at position 96 and position 503 are important in the modification of acetohydroxyacid synthase protein involved in the production of L-branched amino acids. However, since the substituted amino acids in the embodiments of the present disclosure are only representative embodiments showing the effects of the present disclosure, the scope of the present disclosure should not be limited to these embodiments, and it is apparent that acetohydroxyacid synthase variants can have effects corresponding to those described in the embodiments when the 96 th amino acid (threonine) is substituted with an amino acid other than threonine, the 503 th amino acid (tryptophan) is substituted with an amino acid other than tryptophan, or both the 96 th amino acid and the 503 th amino acid are substituted with different amino acids.

In addition, the acetohydroxy acid synthase variants of the present disclosure can have an amino acid sequence shown by any one of SEQ ID NOS:28 to 33, but the amino acid sequence of the acetohydroxy acid synthase variants is not limited thereto. In addition, any polypeptides having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology or identity to the above-described amino acid sequences can be included without limitation, so long as the polypeptides have substantially the same or corresponding activity as the activity of the acetohydroxyacid synthase variants by including the modifications of the present disclosure.

Homology and identity refer to the degree of relatedness between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage.

The terms "homology" and "identity" are generally used interchangeably with each other.

Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, and can be combined with default gap penalties (default gap penalties) established by the program to be used. Substantially, a homologous or identical sequence can hybridize under moderately or highly stringent conditions along its entire sequence or along at least about 50%, about 60%, about 70%, about 80%, or about 90% of its entire length. With respect to the polynucleotides to be hybridized, polynucleotides comprising degenerate codons instead of codons are also contemplated.

Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined, for example, by known computer algorithms (e.g., the "FASTA" program) using default parameters (e.g., in Pearson et al (1988) (Proc. Natl. Acad. Sci. USA 85: 2444)). Alternatively, it may be determined by using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.biol.48: 443) performed in the Needleman program of EMBOSS package (EMBOSS: the European Molecular Biology Open Software Suite, Rice et al, 2000, trends Genet.16:276-277) (5.0.0 or higher) (including the GCG program package (Deterux, J.et al, nucleic acids Research 12:387(1984)), BLASTP, BLASTN, FASTA (Atschul, [ S. ] [ F. et al, J.Molec Biol 215]: 403: 1990), ide Compounds, Hunting J.Biosic, [ ED. ], SantereJ. ] [ C. ] ED., J.Biotechnology [ C. ] or Natalem [ C.J.J. ] homology, for example, Mat.8: Mat.J.Biotechnology (see No., USA, C.D.D.J.D..

The homology, similarity, or identity of a polynucleotide or polypeptide can be determined by comparing sequence information using the GAP computer program (e.g., Needleman et al (1970), JMol Biol 48:443) disclosed in Smith and Waterman, adv.Appl.Math (1981)2: 482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar divided by the total number of symbols for the shorter of the two sequences. Default parameters for the GAP program may include: (1) gribskov et al (1986) Nucl. acids Res.14:6745 unary comparison matrix (containing identity value Of 1 And non-identity value Of 0) And weighted comparison matrix (or EDNAFULL (EMBOSS version Of NCBI NUC 4.4) substitution matrix) such as disclosed by Schwartz And Dayhoff, eds., Atlas Of Protein Sequence And Structure, National biological Research Foundation, pp.353-358 (1979); (2) a penalty of 3.0 per gap and an additional 0.10 penalty per symbol per gap (or gap open penalty of 10, gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the terms "homology" or "identity" represent the relatedness between sequences.

Another aspect of the disclosure provides polynucleotides encoding the acetohydroxyacid synthase variants of the disclosure.

As used herein, the term "polynucleotide" has a meaning including DNA or RNA molecules, and nucleotides as its basic building block (building block) include not only natural nucleotides but also analogues in which sugar or base regions are modified. In the present disclosure, the polynucleotide may be a polynucleotide isolated from a cell or a polynucleotide artificially synthesized, but the polynucleotide is not limited thereto.

Polynucleotides encoding the acetohydroxyacid synthase variants of the present disclosure can include, without limitation, any nucleotide sequence encoding a protein having the activity of the acetohydroxyacid synthase variants of the present disclosure. In particular, various modifications may be made in the coding region of the protein within the range that does not change the amino acid sequence of the protein due to codon degeneracy or codons preferred in view of the microorganism expressing the protein. The polynucleotide may include, without limitation, any nucleotide sequence encoding the amino acid sequence of SEQ ID NOS:28 to 33, and specifically, a nucleotide sequence having the nucleotide sequence of SEQ ID NOS:34 to 39. In addition, any polypeptides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% homology or identity to the above-described amino acid sequences can also be included without limitation, as long as these polypeptides have substantially the same or corresponding activity as acetohydroxyacid synthase variants due to codon degeneracy, through modifications including the present disclosure.

Alternatively, any sequence encoding a protein having the activity of a protein consisting of the amino acid sequence of SEQ ID NOS:28 to 33 may be included without limitation by hybridizing to a probe, which can be prepared from a known gene sequence (for example, a sequence complementary to all or part of a nucleotide sequence), under stringent conditions.

"stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are described in detail in the literature (e.g., j.sambrook et al, supra). Stringent conditions can include conditions under which genes having a high degree of homology or identity (e.g., genes having at least 80%, specifically at least 85%, more specifically at least 90%, even more specifically at least 95%, even more specifically at least 97%, or most specifically at least 99%) can hybridize to each other; conditions under which genes having lower homology or identity cannot hybridize to each other; or as conditions for washing conditions commonly used for Southern hybridization (for example, salt concentration and temperature corresponding to 60 ℃,1 XSSC, 0.1% SDS; specifically 60 ℃, 0.1 XSSC, 0.1% SDS; more specifically 68 ℃, 0.1 XSSC, 0.1% SDS; once, specifically two or three times).

Hybridization requires that the two nucleotides have complementary sequences, although depending on the stringency of the hybridization, mismatches between bases may be possible. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the disclosure may also include isolated nucleic acid fragments that are complementary to the entire sequence as well as substantially similar nucleic acid sequences.

Specifically, polynucleotides having homology or identity may be utilized to include T_mHybridization conditions for the hybridization step at 55 ℃ or by using the above conditions. In addition, T_mThe value may be 60 ℃, 63 ℃ or65 ℃, but is not limited thereto and may be appropriately controlled by those skilled in the art according to the purpose. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and variables are well known in the art (see, Sambrook et al, supra, 9.50 to 9.51, 11.7 to 11.8).

Yet another aspect of the disclosure provides a vector comprising a polynucleotide encoding a modified acetohydroxyacid synthase variant of the disclosure.

As used herein, the term "vector" refers to any vector used for cloning and/or transferring nucleotides into a host cell. The vector may be a replicon that effects replication of the fragment(s) in combination with other DNA fragment(s). A "replicon" refers to any genetic unit that serves as a self-replicating unit (i.e., that can replicate through self-regulation) for DNA replication in vivo. In particular, the vector may be a plasmid, phage, cosmid, chromosome or virus, in a natural or recombinant state. For example, as a phage vector or cosmid vector, pWE15, M13, λ MBL3, λ MBL4, λ ixi, λ ASHII, λ APII, λ t10, λ t11, Charon4A, Charon21A, etc. can be used, and as a plasmid vector, those based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. can be used. The vector that can be used in the present disclosure is not particularly limited, and any known expression vector can be used. In addition, the vector may include a transposon or an artificial chromosome.

In the present disclosure, the vector is not particularly limited as long as it comprises a polynucleotide encoding the acetohydroxyacid synthase variants of the present disclosure. The vector may be a vector that can replicate and/or express the nucleic acid molecule in eukaryotic or prokaryotic cells, including mammalian cells (e.g., cells of human, monkey, rabbit, rat, hamster, mouse, etc.), plant cells, yeast cells, insect cells, and bacterial cells (e.g., e.coli, etc.), and in particular, may be a vector operably linked to a suitable promoter such that the polynucleotide can be expressed in a host cell and including at least one selectable marker.

In addition, as used herein, the term "operably linked" refers to a functional linkage between a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a protein of interest of the present disclosure and the gene sequences described above.

Yet another aspect of the present disclosure provides a transformant into which the vector of the present disclosure is introduced.

In the present disclosure, the transformant can be any transformable cell into which the above-described vector can be introduced and in which the acetohydroxyacid synthase variants of the present disclosure can be expressed. Specifically, the transformant may be any transformed bacterial cell belonging to the genus Escherichia (Escherichia), Corynebacterium, Streptomyces (Streptomyces), Brevibacterium (Brevibacterium), Serratia (Serratia), Providencia (Providencia), Salmonella typhimurium (Salmonella typhimurium), or the like; a yeast cell; fungal cells such as Pichia pastoris; transformed insect (e.g., Drosophila (Drosophila), Spodoptera (Spodoptera) Sf9, etc.) cells; cells of transformed animals (e.g., Chinese Hamster Ovary (CHO), SP2/0 (mouse myeloma), human lymphoblastoid COS, NSO (mouse myeloma), 293T, Bowmelanoma (bowmelanoma), HT-1080, Baby Hamster Kidney (BHK), Human Embryonic Kidney (HEK), PERC.6 (human retinal cell)); or a transformed plant cell, but the transformant is not limited thereto.

Yet another aspect of the present disclosure provides a microorganism producing L-branched chain amino acids, wherein the microorganism contains an acetohydroxyacid synthase variant or has introduced therein a vector containing a polynucleotide encoding the variant.

As used herein, the term "L-branched amino acid" refers to an amino acid having a branched alkyl group on a side chain, and includes valine, leucine, and isoleucine. Specifically, in the present disclosure, the L-branched amino acid may be L-valine or L-leucine, but is not limited thereto.

As used herein, the term "microorganism" includes all wild-type microorganisms and microorganisms that are genetically modified naturally or artificially, and it is a concept including all microorganisms in which a specific mechanism is attenuated or enhanced by insertion of an exogenous gene or enhancement or attenuation of activity of an endogenous gene. Microorganisms refer to all microorganisms that can express acetohydroxyacid synthase variants of the present disclosure. Specifically, the microorganism may be Corynebacterium glutamicum, Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Brevibacterium lactofermentum (Brevibacterium lactofermentum), Brevibacterium flavum (Brevibacterium flavum), Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Corynebacterium thermoaminogenes (Corynebacterium efficiens), and the like, and more specifically, Corynebacterium glutamicum, but is not limited thereto.

As used herein, the term "L-branched amino acid-producing microorganism" may refer to a natural microorganism or a modified microorganism having the ability to produce L-branched amino acids by modification, and specifically may refer to a non-naturally occurring recombinant microorganism, but the microorganism is not limited thereto. The microorganism producing L-branched amino acids is a microorganism containing the acetohydroxyacid synthase variant of the present disclosure or a vector containing a polynucleotide encoding the variant introduced therein, and the microorganism can have a significantly increased ability to produce L-branched amino acids as compared with a wild-type microorganism, a microorganism containing a native-type acetohydroxyacid synthase protein, a non-modified microorganism containing an acetohydroxyacid synthase protein, and a microorganism not containing an acetohydroxyacid synthase protein.

Yet another aspect of the present disclosure provides a method of producing an L-branched amino acid, comprising: culturing a microorganism producing L-branched chain amino acids; and recovering the L-branched amino acid from the microorganism or the culture medium in the above step.

As used herein, the term "culturing" refers to culturing a microorganism under artificially controlled environmental conditions. In the present disclosure, the method for producing an L-branched amino acid using a microorganism capable of producing an L-branched amino acid can be performed by a method widely known in the art. Specifically, the cultivation may be performed in a batch process (batch process), a fed-batch process (fed-batch process), or a repeated fed-batch process, but the batch process is not limited thereto.

The medium used for the cultivation must meet the requirements of the particular strain used. For example, media suitable for culturing Corynebacterium strains are known in the art (e.g., Manual of Methods for General Bacteriology by American Society for Bacteriology, Washington D.C., USA, 1981).

Sugar sources that can be used in the medium may be sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, starch, and cellulose); oils and lipids (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil); fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid); alcohols (e.g., glycerol and ethanol); and organic acids (e.g., acetic acid). These materials may be used independently or in combination, but the manner of use is not limited thereto.

Examples of the nitrogen source that can be used in the medium may include peptone, yeast extract, meat extract (meal juice), malt extract, corn steep liquor, soybean flour (soybean meal), and urea, or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate). These nitrogen sources may also be used independently or in combination, but the mode of use is not limited thereto.

The phosphorus source that can be used in the culture medium can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium-containing salts. In addition, the medium may contain metal salts required for cell growth. Further, materials necessary for growth (e.g., amino acids and vitamins) may be used in addition to the above materials. In addition, precursors suitable for the culture medium may be used. The above-mentioned raw materials may be sufficiently added to the culture in a batch or continuous manner during the culture, but the addition method is not limited thereto.

The pH of the culture can be adjusted in an appropriate manner using basic compounds (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or acidic compounds (e.g., phosphoric acid or sulfuric acid). In addition, a defoaming agent (e.g., fatty acid polyglycol ester) may be used to prevent foam generation. Oxygen or oxygen-containing gas (e.g., air) may be injected into the culture to maintain aerobic conditions of the culture. The temperature of the culture may generally be in the range of 20 ℃ to 45 ℃, and specifically 25 ℃ to 40 ℃. The culture may be continued until a maximum amount of L-branched amino acids is produced, and specifically 10 to 160 hours. The L-branched amino acid may be released into a medium or contained in a cell, but is not limited thereto.

Methods for recovering L-branched-chain amino acids from microorganisms or cultures may include those well known in the art; for example, centrifugation, filtration, treatment with a protein crystal precipitant (salting-out method), extraction, ultrasonication (ultrasounddisruption), ultrafiltration, dialysis, various chromatographies (e.g., molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, and a combination thereof may be employed, but the method is not limited thereto. In addition, the step of recovering the L-branched amino acid may further comprise a purification process, and the purification process may be performed using an appropriate method known in the art.

Detailed Description

The present disclosure will be described in more detail below with reference to the following examples. However, these embodiments are for illustrative purposes only, and the present disclosure is not intended to be limited by these embodiments.