Specification
Title of the invention: Novel branched-chain amino acid aminotransferase variant and leucine production method using the same
technical field
[One]
The present application relates to a novel branched-chain amino acid aminotransferase variant and a leucine production method using the same.
[2]
background
[3]
Branched-chain amino acids refer to three types of valine, leucine, and isoleucine, and are known to be mainly metabolized in muscles and used as an energy source during activity. As it is known that branched-chain amino acids play an important role in maintaining and increasing muscle during activity, its usage is increasing. In particular, leucine is a kind of essential amino acid and is widely used in medicines, foods, feed additives, and industrial drugs.
[4]
On the other hand, the production of leucine using microorganisms is mainly made through microorganisms of the genus Escherichia or microorganisms of the genus Corynebacterium (US 2020-0032305 A1), and goes through several steps from pyruvic acid to ketoisocaproate (2-ketoisocaproate) ) is known to be biosynthesized as a precursor. However, since enzymes used for leucine synthesis are equally used in the biosynthesis of branched-chain amino acids, it is difficult to industrially mass-produce one branched-chain amino acid through fermentation.
[5]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[6]
As a result of diligent efforts to develop a leucine production method with a higher yield than the prior art, the present inventors have discovered a branched-chain amino acid aminotransferase (bcaT) variant that increases leucine production ability, and submitted the present application. completed.
[7]
means of solving the problem
[8]
The object of the present application is a branched amino acid aminotransferase (branched amino acid aminotransferase) variant in which the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; a polynucleotide encoding it; a vector containing it; It is to provide a microorganism containing it.
[9]
Another object of the present application is to provide a leucine production method comprising the step of culturing the microorganism in a medium.
[10]
Effects of the Invention
[11]
Since the branched-chain amino acid aminotransferase mutant of the present application increases the leucine-producing ability compared to the wild-type, it can be widely used for more efficient mass production of leucine.
[12]
Best mode for carrying out the invention
[13]
A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the specific descriptions described below.
[14]
In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Also, such equivalents are intended to be covered by this application.
[15]
[16]
One aspect of the present application provides a branched amino acid aminotransferase (branched amino acid aminotransferase) variant in which the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid can do.
[17]
Specifically, the present application may provide a branched chain amino acid aminotransferase variant comprising one or more substitutions in the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution is the 156th amino acid from the N-terminus to an amino acid other than valine. It may include substituted ones. The 'other amino acid' is not limited as long as it is an amino acid other than the 156th amino acid of SEQ ID NO: 1, valine. Specifically, the variant may be a protein in which the 156th amino acid is substituted with a nonpolar amino acid in the amino acid sequence of SEQ ID NO: 1, and more specifically, the variant has the 156th valine in the amino acid sequence of SEQ ID NO: 1 alanine It may be a branched chain amino acid aminotransferase variant substituted with, but is not limited thereto.
[18]
As used herein, the term "branched-chain amino acid aminotransferase (bcaT)" is an enzyme involved in the biosynthesis of branched-chain amino acids, and in the present application, the branched-chain amino acid aminotransferase is "bcaT", "trans It may be used interchangeably with "transaminase B", or "ilvE". In addition, the branched chain amino acid aminotransferase may be one encoded by the ilvE gene, but is not limited thereto.
[19]
In microorganisms, leucine is derived from pyruvic acid to acetolactic acid, dihydroxy isovaleric acid, ketoisovaleric acid, 2-isopropylmalic acid, 3-iso It is known to be biosynthesized via isopropylmalic acid and ketoisocaproic acid. In addition, these biosynthetic processes are acetohydroxy acid synthase, acetohydroxyacid isomeroreductase, dihydroxy acid dehydratase, isopropyl malic acid synthase ( Catalyzed by enzymes such as isopropylmalic acid synthase, isopropylmalic acid dehydratase, isopropylmalic acid dehydrogenase, branched amino acid aminotransferase to be biosynthesized
[20]
However, since branched-chain amino acid aminotransferase is also involved in the biosynthesis of valine and isoleucine as well as leucine, there has been a problem in industrially manufacturing one branched-chain amino acid through fermentation by manipulating the enzyme.
[21]
In the present application, the branched-chain amino acid aminotransferase may have its sequence obtained from GenBank of NCBI, a known database, but is not limited thereto, and branched-chain amino acid aminotransferase is secured based on various methods well-known in the art. can do. Examples of the method include gene synthesis technology including codon optimization to secure enzymes with high efficiency in microorganisms of the genus Corynebacterium, which are commonly used for enzyme expression, or bioinformatics methods based on large-scale genome information of microorganisms. There is a screening method for useful enzyme resources by, but is not limited thereto.
[22]
The branched-chain amino acid aminotransferase to be mutated in the present application may be a branched-chain amino acid aminotransferase derived from Corynebacterium sp., specifically, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 It may be a /protein, but a polypeptide/protein having the same activity may be included without limitation. As an example, it may include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more homology or identity therewith, but is not limited thereto. Specifically, the branched chain amino acid aminotransferase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to SEQ ID NO: 1. can do. In addition, if a polypeptide/protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added is also a polypeptide/protein that has such homology or identity and exhibits branched-chain amino acid aminotransferase activity, the polypeptide to be mutated in the present application It is obvious that it is included within the scope of /protein.
[23]
That is, even if it is described in the present application as 'a protein or polypeptide having an amino acid sequence described in a specific SEQ ID NO:' or a 'protein or polypeptide consisting of an amino acid sequence described in a specific SEQ ID NO:', a protein consisting of the amino acid sequence of the corresponding SEQ ID NO: It is apparent that a protein or polypeptide having an amino acid sequence in which some sequences are deleted, modified, substituted or added may also be used in the present application, provided that it has the same or corresponding activity as the polypeptide. For example, even if some sequence of 'a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1' is deleted, modified or substituted, or the polypeptide contains an added amino acid sequence, it is the same as or It is obvious that if it has the corresponding activity, it may belong to the 'polypeptide consisting of the amino acid sequence of SEQ ID NO: 1'.
[24]
As used herein, the term "variant" means that one or more amino acids differ from the recited sequence in conservative substitution and/or modification, but the function of the protein ( functions) or properties (properties) are maintained. A variant differs from an identified sequence by several amino acid substitutions, deletions or additions. Such variants can generally be identified by modifying one of the polypeptide sequences and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. In addition, some variants may include variants in which one or more portions such as an N-terminal leader sequence or a transmembrane domain have been removed. Other variants contain portions from the N- and/or C-terminus of the mature protein.removed variants may be included. As used herein, the term "conservative substitution" may refer to substituting one amino acid with another amino acid having similar structural and/or chemical properties. Such variants may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the polypeptide.
[25]
In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a variant, or variant polypeptide, is conjugated with a signal (or leader) sequence at the N-terminus of the protein involved in the transfer of the protein co-translationally or post-translationally. can do. The variants may also be conjugated with other sequences or linkers to identify, purify, or synthesize the polypeptide.
[26]
[27]
The term "variant" includes terms such as mutant, modified, mutated protein, mutant polypeptide, mutant, and mutant protein (in English, modified, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.) It may be used, and if it is a term used in a mutated sense, it is not limited thereto. For the purposes of the present application, the variant may have a branched-chain amino acid aminotransferase activity that is changed to have enhanced leucine-producing ability.
[28]
That is, in the present application, "branched-chain amino acid aminotransferase variant" refers to one or more amino acid substitutions in the amino acid sequence of a polypeptide having branched-chain amino acid aminotransferase activity, and the term "branched-chain amino acid aminotransferase variant" means It can be used interchangeably with "polypeptide having branched chain amino acid aminotransferase activity". In addition, the term "branched chain amino acid aminotransferase variant" is a variant branched chain amino acid aminotransferase protein, variant bcaT, variant bcaT protein, bcaT variant, variant transaminase B (transaminase B) protein, variant Transaminase B, transaminase B mutant, mutant ilvE protein, mutant ilvE, ilvE mutant, etc. may be used interchangeably, but as long as the term is used in the mutated meaning as described above, it is not limited thereto.
[29]
The 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, branched amino acid aminotransferase variants have branched chain amino acid aminotransferase activity It is apparent that in the amino acid sequence of a polypeptide having For example, the variant may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% homology or identity to the sequence of SEQ ID NO: 1 in a sequence of SEQ ID NO: 1 It may include a sequence in which the amino acid at the position corresponding to the 156th position from the N-terminus is substituted with another amino acid.
[30]
[31]
The 'other amino acid' may be an amino acid other than the amino acid corresponding to the 156th amino acid of SEQ ID NO: 1. Specifically, the variant may be one in which the amino acid corresponding to the 156th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with a non-polar amino acid having structural and/or chemical properties similar to those of alanine. More specifically, the variant may be one in which the amino acid corresponding to amino acid 156 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, but is not limited thereto.
[32]
[33]
As used herein, the term “corresponding position” refers to an amino acid residue at a position listed in a protein or polypeptide, or an amino acid residue similar to, identical to, or homologous to a residue listed in a protein or polypeptide. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.
[34]
[35]
In the present application, specific numbering may be used for amino acid residue positions in proteins used in this application. For example, by aligning the polypeptide sequence of the protein of the present application with the target protein to be compared, it is possible to renumber the position corresponding to the amino acid residue position of the protein of the present application.
[36]
[37]
The branched-chain amino acid aminotransferase mutant of the present application may have an activity to increase the leucine-producing ability in a microorganism as compared to a wild-type or unmutated branched-chain amino acid aminotransferase protein.
[38]
The branched chain amino acid aminotransferase variant has the amino acid sequence of SEQ ID NO: 1 in which the 156th valine (V: valine) amino acid residue from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, more specifically, alanine and 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% or more homology or identity, and more specifically, 90% or more homology or identity with SEQ ID NO: 1 may be to have
[39]
In one embodiment, the variant may consist of SEQ ID NO: 3, but is not limited thereto. In addition, in the branched chain amino acid aminotransferase variant, the amino acid corresponding to the 156th position from the N-terminus in SEQ ID NO: 3 is fixed (that is, the amino acid at the position corresponding to the 156th amino acid of SEQ ID NO: 3 is, SEQ ID NO: 3 is alanine identically to the 156th amino acid of SEQ ID NO: 3 and 70% or more, specifically 80%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% or more homology or identity to an amino acid sequence with or more, but is not limited thereto. In addition, if an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the protein, a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added in addition to the amino acid sequence at position 156 is also within the scope of the present application. The inclusion is obvious.
[40]
[41]
As used herein, the term 'homology' or 'identity' refers to the degree to which two given amino acid sequences or nucleotide sequences are related to each other and may be expressed as a percentage.
[42]
The terms homology and identity can often be used interchangeably.
[43]
Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences under moderate or high stringent conditions generally contain at least about 50%, 60%, 70%, 80% of the total or full-length of the sequence. or more than 90% hybrid. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.
[44]
Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA" program. or, as performed in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the EMBOSS package, The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine. (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073) For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW, can be used to determine homology, similarity or identity.
[45]
Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. 48: 443 by comparing sequence information using a GAP computer program. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). The default parameters for GAP programs are (1) binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the term "homology" or "identity" may refer to a relevance between sequences.
[46]
[47]
Another aspect of the present application may provide a polynucleotide encoding the branched chain amino acid aminotransferase variant.
[48]
In addition, another aspect of the present application may provide a vector including the polynucleotide.
[49]
As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, the branched chain amino acid amino It may be a polynucleotide fragment encoding a transferase variant.
[50]
The polynucleotide encoding the branched-chain amino acid aminotransferase variant of the present application may be included without limitation as long as it is a polynucleotide sequence encoding the branched-chain amino acid aminotransferase variant of the present application. The polynucleotide encoding the branched-chain amino acid aminotransferase variant may be included without limitation as long as it is a sequence encoding a variant in which the 156th amino acid is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 1. Specifically, it may be a polynucleotide sequence encoding a variant in which the 156th amino acid is substituted with alanine in the amino acid sequence of SEQ ID NO: 1. For example, the polynucleotide encoding the branched-chain amino acid aminotransferase variant of the present application may be a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. More specifically, it may be composed of the polynucleotide sequence of SEQ ID NO: 4, but is not limited thereto.
[51]
In the polynucleotide, various modifications can be made in the coding region within a range that does not change the amino acid sequence of the protein due to the degeneracy of the codon or in consideration of the codon preferred in the organism to express the protein. . Therefore, it is obvious that a polynucleotide that can be translated into a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or a polypeptide having homology or identity thereto may also be included due to codon degeneracy.
[52]
In addition, the 156th amino acid in the amino acid sequence of SEQ ID NO: 1 is changed to another amino acid by hydridation under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the nucleotide sequence. Any sequence encoding a substituted branched-chain amino acid aminotransferase variant may be included without limitation.
[53]
The "stringent condition" may be a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989). For example, genes having high homology or identity, 80% or more, 85% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, In particular, the conditions in which genes having 99% or more homology or identity hybridize with each other and genes with lower homology or identity do not hybridize, or at 60° C., which is a washing condition for normal Southern hybridization. 1 X SSC, 0.1% SDS, specifically at a salt concentration and temperature equivalent to 60°C, 0.1 X SSC, 0.1% SDS, more specifically 68°C, 0.1 X SSC, 0.1% SDS, once, specifically The conditions for washing 2 to 3 times can be exemplified.
[54]
Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include isolated nucleic acid fragments that are complementary to substantially similar nucleic acid sequences as well as the entire sequence.
[55]
Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be 60 °C , 63 °C or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
[56]
The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[57]
[58]
As used herein, the term "vector" refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the variant protein of interest operably linked to a suitable regulatory sequence so that the variant protein of interest can be expressed in a suitable host. can be The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
[59]
The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used, but is not limited thereto.
[60]
For example, a polynucleotide encoding a target mutant protein in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. The polynucleotide may further include a selection marker for confirming whether or not the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and a selectable phenotype such as drug resistance, auxotrophic tolerance, resistance to cytotoxic agents, or expression of a surface variant protein. Markers that give ? may be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
[61]
[62]
Another aspect of the present application is the branched chain amino acid aminotransferase variant; a polynucleotide encoding the variant; And it can provide a microorganism comprising any one or more of the vector containing the polynucleotide.
[63]
Specifically, the microorganism may be a microorganism prepared by transformation with a vector containing a polynucleotide encoding a mutant, but is not limited thereto.
[64]
In the present application, the term "transformation" may refer to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. As long as the polynucleotide can be introduced and expressed into a host cell, it may be introduced in any form. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette is usually a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site and a translation terminator.

Claims
[Claim 1]
Branched-chain amino acid aminotransferase (branc) in which the 156th valine (V: valine) amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acidhed amino acid aminotransferase) variants.
[Claim 2]
The branched chain amino acid aminotransferase variant according to claim 1, wherein the valine at position 156 is substituted with alanine.
[Claim 3]
The branched chain amino acid aminotransferase variant according to claim 1, wherein the variant has at least 90% homology or identity with the amino acid sequence of SEQ ID NO: 1.
[Claim 4]
The branched chain amino acid aminotransferase variant according to claim 1, wherein the variant consists of the amino acid sequence of SEQ ID NO: 3.
[Claim 5]
A polynucleotide encoding the variant of any one of claims 1 to 4.
[Claim 6]
A vector comprising the polynucleotide of claim 5 .
[Claim 7]
A variant of any one of claims 1 to 4; a polynucleotide encoding the variant; And, comprising any one or more of the vector comprising the polynucleotide, Corynebacterium ( Corynebacterium sp.) genus microorganism.
[Claim 8]
The microorganism of the genus Corynebacterium sp. according to claim 7, wherein the microorganism of the genus Corynebacterium produces leucine.
[Claim 9]
The microorganism of the genus Corynebacterium according to claim 7, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
[Claim 10]
A method for producing leucine, comprising the step of culturing the microorganism of claim 7 in a medium.
[Claim 11]
The method of claim 10, further comprising the step of isolating or recovering leucine from the cultured microorganism or medium.