CN117924441A - Novel variant of DNA-binding transcription regulatory factor MalT and method for producing L-aromatic amino acid using the same - Google Patents

Novel variant of DNA-binding transcription regulatory factor MalT and method for producing L-aromatic amino acid using the same Download PDF

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CN117924441A
CN117924441A CN202311382643.2A CN202311382643A CN117924441A CN 117924441 A CN117924441 A CN 117924441A CN 202311382643 A CN202311382643 A CN 202311382643A CN 117924441 A CN117924441 A CN 117924441A
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amino acid
malt
dna
corynebacterium
aromatic amino
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金贤英
乔伊·车
曹永一
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Daesang Corp
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Daesang Corp
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Abstract

The present invention relates to a novel variant of a DNA-binding transcription regulatory factor MalT, which changes the activity of a protein by substituting one or more amino acids constituting the amino acid sequence of the DNA-binding transcription regulatory factor MalT, thereby enabling efficient production of L-tryptophan, L-phenylalanine or L-tyrosine by a recombinant microorganism comprising the variant, and a method for producing an L-aromatic amino acid using the same.

Description

Novel variant of DNA-binding transcription regulatory factor MalT and method for producing L-aromatic amino acid using the same
Technical Field
The present invention relates to a novel variant of DNA-binding transcription regulatory factor MalT and a method for producing L-aromatic amino acid using the same.
Background
Amino acids are classified into hydrophobic, hydrophilic, basic and acidic amino acids according to the nature of side chains, and amino acids having benzene rings therein are called aromatic amino acids. Among the aromatic amino acids, phenylalanine, tyrosine and tryptophan are essential amino acids that cannot be synthesized in living bodies, and belong to the high value-added industry that forms a market of $3000 million each year worldwide.
The production of aromatic amino acids can be carried out using wild-type strains obtained in a natural state or variant strains modified in such a manner as to enhance the amino acid productivity. In recent years, in order to improve the production efficiency of aromatic amino acids, genetic recombination techniques have been applied to microorganisms such as E.coli and coryneform bacteria which are used in many cases for producing useful substances such as L-amino acids, and various recombinant strains or variants having excellent L-aromatic amino acid productivity have been developed, and a method for producing L-aromatic amino acids using the same. In particular, the following attempts were made: the production of the corresponding amino acid is increased by targeting genes such as enzymes, transcription factors, transport proteins, etc. involved in the biosynthesis pathway of the L-aromatic amino acid, or inducing mutation in promoters regulating their expression. However, since the types of proteins such as enzymes, transcription factors, transport proteins, etc., which are directly or indirectly involved in the production of L-aromatic amino acids, are several tens of kinds, a great deal of research is actually required as to whether the L-aromatic amino acid production capacity is increased or not according to the activity change of such proteins.
Prior art literature
Patent literature
Korean patent No. 10-1830002
Disclosure of Invention
The object of the present invention is to provide variants of the DNA-binding transcriptional regulator MalT.
In addition, it is an object of the present invention to provide polynucleotides encoding the above variants.
In addition, it is an object of the present invention to provide a transformant comprising the above variant or polynucleotide.
The present invention also provides a method for producing an L-aromatic amino acid using the transformant.
In one embodiment of the present invention, there is provided a DNA-binding transcription regulatory factor MalT variant comprising the amino acid sequence of SEQ ID NO. 4, wherein glycine 315 (Gly) in the amino acid sequence of SEQ ID NO. 2 is replaced with serine (Ser).
The "DNA-binding transcription regulatory factor MalT (DNA-binding transcriptional regulator MalT)" used in the present invention positively regulates transcription of maltose regulon (regulon) of a gene product responsible for absorption and catabolism of maltooligosaccharide (malto-oligosacharide). The DNA-binding transcription regulatory factor MalT in the present invention may be a polypeptide encoded by the MalT gene and having the activity of the DNA-binding transcription regulatory factor MalT, but is not limited thereto.
The sequence information of the nucleic acid and protein of the above DNA-binding transcription regulatory factor MalT can be obtained by a known sequence database (for example, genBank, uniProt).
According to one embodiment of the present invention, the DNA-binding transcription regulatory factor MalT may be encoded by the base sequence of SEQ ID NO. 1 or may be constituted by the amino acid sequence of SEQ ID NO. 2.
The amino acid sequence of the DNA-binding transcription regulatory factor MalT according to the invention or the base sequence encoding it may comprise a base sequence or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the respective sequences. The term "homology" or "identity" as used herein refers to the percentage of identity (%) between two sequences when the base sequence or amino acid sequence to be used as a reference is aligned with any other base sequence or amino acid sequence so as to correspond to the maximum extent for analysis.
According to one embodiment of the present invention, the DNA-binding transcription regulatory factor MalT or a gene encoding the same may be derived from wild-type Escherichia coli (ESCHERICHIA COLI).
As used herein, "variant" refers to a polypeptide in which one or more amino acids at the N-terminal, C-terminal and/or within the amino acid sequence of a particular gene are conservatively substituted (conservative substitution), deleted (modified), altered (modified) or added to differ from the amino acid sequence prior to mutation, but maintain functions or properties. As used herein, "conservative substitutions" refer to the substitution of one amino acid for another that is structurally and/or chemically similar, with little or no effect on the activity of the protein or polypeptide. The amino acid is selected from alanine (Ala), isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met), asparagine (Asn), cysteine (Cys), glutamine (Gln), serine (Ser), threonine (Thr), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), histidine (His), lysine (Lys), glycine (Gly) and proline (Pro).
In addition, variants include variants with more than one N-terminal leader sequence or a portion of the transmembrane domain (transmembrane domain) removed, or variants with a portion of the N-and/or C-terminal of the mature protein (protein) removed.
Such variants may have increased (enhanced) or unchanged or decreased (attenuated) ability compared to the protein prior to mutation. Herein, "adding or enhancing" includes the following cases: the activity of the protein itself is increased as compared with the protein before mutation; in the case where the overall intracellular enzyme activity level is high compared with that of the wild-type strain or the strain expressing the protein before mutation due to increased expression or increased translation of the gene encoding the protein, etc.; and combinations thereof. Furthermore, "reducing or weakening" includes the following: the activity of the protein itself is reduced as compared with the protein before mutation; the expression of a gene encoding a protein is inhibited, translation is inhibited, or the like, so that the overall intracellular enzymatic activity is lower than that of the wild-type strain or the strain expressing the protein before mutation; and combinations thereof. In the present invention, variants may be used in combination with variants, alterations, variant polypeptides, variant proteins, variants, and the like.
The DNA-binding transcription regulatory factor MalT variant according to the invention may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence of SEQ ID NO. 4, provided that the function or property of the variant is maintained.
Another aspect of the present invention provides a polynucleotide encoding a MalT variant of the above DNA-binding transcriptional regulator.
The "polynucleotide (polynucleotide)" used in the present invention is a polymer (polymer) of nucleotides in which nucleotide monomers (monomers) are linked in a chain form by covalent bonds, and is a DNA or RNA strand of a predetermined length or more, more specifically, a polynucleotide fragment encoding the protein variant.
The above polynucleotide contains a base sequence encoding the amino acid sequence of SEQ ID NO. 4, for example, may contain a base sequence of SEQ ID NO. 3.
Another aspect of the present invention provides a vector comprising a polynucleotide encoding a MalT variant of the DNA-binding transcriptional regulator described above.
In addition, another embodiment of the present invention provides a transformant comprising the above-mentioned DNA-binding transcription regulatory factor MalT variant or polynucleotide.
The term "vector" as used herein refers to any type of nucleic acid sequence transport structure used as a means for delivering and expressing a target gene into a host cell. Unless otherwise indicated, such vectors may refer to the insertion of a supported nucleic acid sequence into a host cell gene for expression and/or expression separately. Such vectors include the necessary regulatory elements operably linked for expression of the gene insert, "operably linked (operably linked)" means that the gene of interest and its regulatory sequences are functionally bound to each other and linked in a manner that enables gene expression, "regulatory elements" include promoters for carrying out transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate termination of transcription and translation.
The vector used in the present invention is not particularly limited as long as it can replicate in a host cell, and any vector known in the art can be used. Examples of the vector include plasmids, cosmids, viruses, and phages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, we15, M13, λmbl3, λmbl4, λ IXII, λ ASHII, λapii, λt10, λt11, charon4A, charon a, etc., and as plasmid vectors, pBR system, pUC system, pbluescript ii system, pGEM system, pTZ system, pCL system, pET system, etc., are available, but not limited thereto.
The above vectors may be representatively constructed as vectors for cloning or vectors for expression. The vector for expression may use a conventional vector used in the art for expressing a foreign gene or protein in a plant, animal or microorganism, and may be constructed by various methods well known in the art.
The "recombinant vector" used in the present invention may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and prokaryotic cells are used as a host, a strong promoter (e.g., plλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter) that allows transcription to proceed generally contains a ribosome binding site for translation initiation and transcription/translation termination sequences. When eukaryotic cells are used as hosts, the replication origins to be initiated in eukaryotic cells contained in the vector include, but are not limited to, f1 replication origins, SV40 replication origins, pMB1 replication origins, adenovirus replication origins, AAV replication origins, BBV replication origins, and the like. In addition, promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV) may be utilized, and typically have polyadenylation sequences as transcription termination sequences.
The recombinant vector may include a selection marker for selecting transformants (host cells) transformed with the vector, and only cells expressing the selection marker may survive in the medium treated with the selection marker, thereby enabling selection of the transformed cells. The selection markers include, but are not limited to, ampicillin, kanamycin, streptomycin, and chloramphenicol, as typical examples.
By inserting the recombinant vector into a host cell, a transformant can be produced, which can be obtained by introducing the recombinant vector into an appropriate host cell. The host cell is a cell in which the above-described expression vector can be stably and continuously cloned or expressed, and any host cell known in the art can be used.
When a prokaryotic cell is transformed to produce a recombinant microorganism, a strain of Escherichia coli such as E.coli JM109、E.coli BL21、E.coli RR1、E.coli LE392、E.coli B、E.coli X 1776、E.coli W3110、E.coli XL1-Blue can be used as a host cell; a corynebacterium strain; bacillus strains such as bacillus subtilis and bacillus thuringiensis; various intestinal bacteria and strains such as Salmonella typhimurium, serratia marcescens and Pseudomonas species, etc., but are not limited thereto.
In the case of transforming eukaryotic cells for the production of recombinant microorganisms, yeasts (e.g., saccharomyces cerevisiae), insect cells, plant cells, and animal cells, for example, sp2/0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, hepG2, huh7, 3T3, RIN, MDCK cell lines, etc., can be used as host cells, but are not limited thereto.
The term "transformation" as used herein refers to a phenomenon in which a foreign DNA is introduced into a host cell to artificially cause a gene change, and the term "transformant (transformant)" refers to a host cell into which a foreign DNA is introduced and in which the expression of a target gene is stably maintained.
In the above transformation, an appropriate vector introduction technique is selected according to the host cell, so that the target gene or a recombinant vector comprising the same can be expressed in the host cell. For example, the vector introduction may be performed by electroporation (electroporation), thermal shock (heat-shock), calcium phosphate (CaPO 4) precipitation, calcium chloride (CaCl 2) precipitation, microinjection (microinjection), polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited thereto. The transformed gene may be included as long as it can be expressed in the host cell, and is not limited to insertion into the chromosome of the host cell or to being located extrachromosomally.
The above transformant includes a cell transfected, transformed or infected with the recombinant vector according to the present invention in an organism or in a test tube, and may be used as the same term as a recombinant host cell, a recombinant cell or a recombinant microorganism.
According to one embodiment of the present invention, the transformant may be an Escherichia strain or a Corynebacterium strain.
The strain of the genus Escherichia may be, but not limited to, escherichia coli (ESCHERICHIA COLI), escherichia ibutescens (ESCHERICHIA ALBERTII), escherichia blattae (ESCHERICHIA BLATTAE), escherichia fries (ESCHERICHIA FERGUSONII), escherichia hertz (ESCHERICHIA HERMANNII), or Escherichia wound (ESCHERICHIA VULNERIS).
The corynebacterium strain may be Corynebacterium glutamicum (Corynebacterium glutamicum), lactobacillus plantarum (Corynebacterium crudilactis), corynebacterium desert (Corynebacterium deserti), corynebacterium broom (Corynebacterium callunae), corynebacterium threonae (Corynebacterium suranareeae), corynebacterium oleae (Corynebacterium lubricantis), corynebacterium canal Sang Bangzhuang (Corynebacterium doosanense), corynebacterium hepaticum (Corynebacterium efficiens), corynebacterium Ubbelopsis (Corynebacterium uterequi), corynebacterium parvum (Corynebacterium stationis), corynebacterium parvum (Corynebacterium pacaense), corynebacterium mirabilis (Corynebacterium singulare), corynebacterium humicola (Corynebacterium humireducens), corynebacterium maritimum (Corynebacterium marinum), corynebacterium salt tolerance (Corynebacterium halotolerans), corynebacterium pterans (Corynebacterium spheniscorum), corynebacterium freudenreichii (Corynebacterium freiburgense), corynebacterium striatum (Corynebacterium striatum), corynebacterium canis (Corynebacterium canis), corynebacterium ammoniagenes (Corynebacterium ammoniagenes), corynebacterium renifolium (Corynebacterium renale), corynebacterium pollutes (Corynebacterium pollutisoli), corynebacterium hanensis (Corynebacterium imitans), corynebacterium reesei (Corynebacterium caspium), corynebacterium testiclae (Corynebacterium testudinoris), corynebacterium pseudolarium (Corynebacaterium pseudopelargi) or Corynebacterium flavum (Corynebacterium flavescens), but is not limited thereto.
The transformant of the present invention may be a strain comprising the above-mentioned DNA-binding transcription regulatory factor MalT variant or a polynucleotide encoding the same, or a vector comprising the same; a strain expressing the above DNA-binding transcription regulatory factor MalT variant or polynucleotide; or a strain having an activity to the above-mentioned DNA-binding transcription regulatory factor MalT variant, but is not limited thereto.
The transformant of the present invention may include other protein variants or genetic variations in addition to the above-mentioned DNA-binding transcription regulatory factor MalT variants.
According to one embodiment of the present invention, the transformant may have L-aromatic amino acid productivity.
The transformant may naturally have L-aromatic amino acid productivity or may be artificially provided with L-aromatic amino acid productivity.
According to one embodiment of the present invention, the transformant has an improved L-aromatic amino acid productivity because the activity of the DNA-binding transcription regulatory factor MalT is altered.
As used herein, "increased productivity" means that the productivity of L-aromatic amino acids is increased as compared to the parent strain. The parent strain refers to a wild-type strain or a mutant strain to be mutated, and includes a subject to be mutated directly or a subject to be transformed by a recombinant vector or the like. In the present invention, the parent strain may be a wild-type escherichia strain or a corynebacterium strain or an escherichia strain or a corynebacterium strain mutated from a wild-type.
The transformant according to the present invention shows an increased L-aromatic amino acid productivity as compared with a strain comprising a protein before mutation (parent strain) by introducing a variant of the DNA-binding transcription regulatory factor MalT, whereby the activity of the DNA-binding transcription regulatory factor MalT is altered. More specifically, the above transformant may increase the L-aromatic amino acid production by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% as compared with the parent strain, or may increase it by 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold or 10-fold, but is not limited thereto. As an example, a transformant comprising the above-mentioned DNA-binding transcription regulatory factor MalT variant may have an L-aromatic productivity increased by 5% or more, specifically, 5 to 50% (preferably 7 to 40%) as compared with the parent strain.
The composition comprising the transformant according to the present invention can be used as a composition for producing an L-aromatic amino acid.
Another aspect of the present invention provides a method for producing an L-aromatic amino acid, comprising the steps of: a step of culturing the transformant in a medium; and recovering the L-aromatic amino acid from the transformant or a medium in which the transformant is cultured.
The above-mentioned culture may be carried out according to a suitable medium and culture conditions known in the art, and the medium and culture conditions may be easily adjusted and used by those skilled in the art. Specifically, the medium may be a liquid medium, but is not limited thereto. The cultivation method may include, for example, batch cultivation (batch cultivation), continuous cultivation (continuous culture), fed-batch cultivation (fed-batch cultivation), or a combination thereof, but is not limited thereto.
According to one embodiment of the invention, the above-mentioned culture medium must meet the requirements of the particular strain in a suitable manner, and can be suitably deformed by a person skilled in the art. For the culture medium of the strain of Escherichia or the strain of Corynebacterium, reference may be made to well-known document (Manual of Methods for General Bacteriology.American Society for Bacteriology.Washington D.C.,USA,1981),, but is not limited thereto.
According to one embodiment of the present invention, the medium may contain various carbon sources, nitrogen sources and trace element components. As the carbon source which can be used, there are included sugars such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose and the like and carbohydrates; oil and fat such as soybean oil, sunflower seed oil, castor seed oil, coconut oil and the like; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerin and ethanol; organic acids such as acetic acid. These substances may be used alone or in the form of a mixture, but are not limited thereto. As nitrogen sources that can be used, peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be included. The nitrogen source may also be used alone or in the form of a mixture, but is not limited thereto. As the supply source of phosphorus that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts may be included, but are not limited thereto. The medium may contain a metal salt such as magnesium sulfate or iron sulfate required for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be contained. In addition, precursors suitable for the culture medium may be used. The above-mentioned medium or individual components may be added to the culture broth in batches or continuously by an appropriate means during the culture, but are not limited thereto.
According to an embodiment of the present invention, during the cultivation, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the microorganism culture solution in an appropriate manner to adjust the pH of the culture solution. In addition, during the culture, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble generation. Further, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture. The temperature of the culture solution may be generally 20℃to 45℃and, for example, may be 25℃to 40 ℃. The incubation time may be continued until the desired throughput of the useful substance is obtained, for example, may be 10 to 160 hours.
According to one embodiment of the present invention, in the above-mentioned step of recovering an L-aromatic amino acid from a transformant to be cultured or a medium in which the transformant is cultured, the produced L-aromatic amino acid may be collected or recovered from the medium according to a culture method and by using a suitable method known in the art. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), and the like may be used, but are not limited thereto.
According to one embodiment of the present invention, in the step of recovering an L-aromatic amino acid, the medium may be centrifuged at a low speed to remove biomass, and the obtained supernatant may be separated by ion exchange chromatography.
According to one embodiment of the present invention, the step of recovering the L-aromatic amino acid may include a step of purifying the L-aromatic amino acid.
According to one embodiment of the present invention, the L-aromatic amino acid may be 1 or more selected from the group consisting of L-tryptophan, L-phenylalanine and L-tyrosine.
The variant of the DNA-binding transcription regulatory factor MalT according to the present invention changes the protein activity by substitution of one or more amino acids in the amino acid sequence constituting the DNA-binding transcription regulatory factor MalT, so that L-tryptophan, L-phenylalanine or L-tyrosine can be efficiently produced by a recombinant microorganism comprising the variant.
Drawings
FIG. 1 shows the structure of a plasmid pDSG according to an embodiment of the present invention.
FIG. 2 shows the structure of plasmid pDS9 according to an embodiment of the present invention.
Detailed Description
The present invention will be described in more detail below. However, the description is merely illustrative for the purpose of facilitating understanding of the present invention, and the scope of the present invention is not limited to such illustrative description.
Example 1 preparation of strains expressing DNA-binding transcription regulator MalT variants
In order to confirm the effect of a variant (SEQ ID NO: 4) in which glycine 315 (Gly) in the amino acid sequence (SEQ ID NO: 2) of the DNA-binding transcription regulatory factor MalT was replaced with serine (Ser) on the production of L-aromatic amino acids, a vector expressing the above DNA-binding transcription regulatory factor MalT variant and a strain into which the above vector was introduced were produced. For gene insertion of the DNA-binding transcription regulator MalT variant in the strain, plasmids pDSG and pDS9 were used and made as follows.
The above plasmid pDSG (SEQ ID NO: 23) has an origin of replication which functions only in E.coli, and has an ampicillin (ampicillin) resistance gene and a guide RNA (gRNA) expression mechanism. The above plasmid pDS9 (SEQ ID NO: 24) has an origin of replication which functions only in E.coli, has a kanamycin (kanamycin) resistance gene, a lambda Red gene (exo, bet and gam) expression system and a CAS9 expression mechanism derived from Streptococcus pyogenes (Streptococcus pyogenes).
1-1 Preparation of vector pDSG-malT (Gly 315 Ser) for transformation
PCR was performed using E.coli (ESCHERICHIA COLI) MG1655 (KCTC 14419 BP) gDNA as a template and using the primer set of primer 7 and primer 9 and the primer set of primer 8 and primer 10, thereby obtaining an upstream (upstream) fragment of malT of amino acid variation No. 315 in the amino acid sequence of DNA-binding transcription regulatory factor MalT. Then, PCR was performed using the primer pair of primer 11 and primer 13 and the primer pair of primer 12 and primer 14 with escherichia coli MG1655 (KCTC 14419 BP) gDNA as a template, and a downstream (downstream) fragment of MalT having No. 315 amino acid mutation in the amino acid sequence of DNA-binding transcription regulatory factor MalT was obtained. The upstream (upstream) and downstream (downstream) fragments contain a codon sequence for translation of Gly 315 to Ser in the amino acid sequence of the DNA-binding transcription regulator MalT. Here, TAKARA PRIMESTAR Max DNApolymerase, PCR was used as the polymerase, and the denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds and polymerization at 72℃for 10 seconds were repeated 30 times.
PCR was performed using the plasmid pDSG as a template and the primer pair of primer 3 and primer 5, the primer pair of primer 4 and primer 6, the primer pair of primer 15 and primer 1, and the primer pair of primer 16 and primer 2, thereby obtaining 4 pDSG gene fragments. Each gene fragment contained a gRNA sequence targeted to Gly of malT. The gRNA selects the NGG pre 20mer of the sequence to be mutated. Here, TAKARA PRIMESTAR Max DNApolymerase, PCR was used as the polymerase, and the denaturation at 95℃for 10 seconds, annealing at 57℃for 15 seconds and polymerization at 72℃for 15 seconds were repeated 30 times.
Then, the obtained upstream (upstream) and downstream (downstream) fragments of malT and 4 pDSG fragments were cloned by a self-assembled cloning method (BioTechniques 51:55-56 (July 2011)), thereby obtaining a recombinant plasmid, which was named pDSG-malT (Gly 315 Ser).
1-2 Production of L-tryptophan or L-phenylalanine producing Strain into which MalT variant MalT (Gly 315 Ser) of DNA-binding transcription regulatory factor was introduced
Coli (ESCHERICHIA COLI) KCCM13013P and KCCM10016 were used as parent strains for the production of L-tryptophan-producing strains and L-phenylalanine-producing strains, respectively.
PDS9 plasmid was transformed 1 time into each parent strain, and after culturing in LB-Km (LB liquid medium (Luria Bertani broth) containing 25g/L and kanamycin (kanamycin) containing 50 mg/L) solid medium, colonies resistant to kanamycin (kanamycin) were selected. The pDSG-malT (Gly 315 Ser) plasmid was transformed 2 times into the selected transformant, cultured in LB-Amp & Km (LB liquid medium (Luria Bertani broth) containing 25g/L, 100mg/L ampicillin (ampicillin) and 50mg/L kanamycin (kanamycin)) solid medium, colonies resistant to ampicillin (ampicillin) and kanamycin (kanamycin) were selected, and fragments were obtained by colony PCR using the primer pair of primer 17 and primer 18. Here, TAKARA PRIMESTAR Max DNApolymerase, PCR was used as the polymerase, and the denaturation at 95℃for 10 seconds, annealing at 57℃for 10 seconds, and polymerization at 72℃for 15 seconds were repeated 30 times. The obtained fragment was sequenced by using the primer pair of the primer 17 and the primer 18 to confirm the sequence.
The 2 transformants were subjected to 7 subcultures in LB liquid medium, and colonies were selected in LB solid medium. Each colony was picked (picking) and cultured in LB, LB-Amp and LB-Km solid medium. Colonies that grew in LB solid medium and did not grow in LB-Amp and LB-Km solid medium were selected. The strains selected as described above were named KCCM13013P_malT (Gly 315 Ser) and KCCM10016_malT (Gly 315 Ser), respectively.
The primer sequences used in example 1 are shown in Table 1 below.
[ Table 1]
Primer name Sequence number Primer sequence (5 '-3')
Primer 1 SEQ ID NO:5 caattttattatagtaattgactattatac
Primer 2 SEQ ID NO:6 TCATCCATCCcaattttattatagtaattgactattatac
Primer 3 SEQ ID NO:7 GGATGGATGATACCAGCGAGgttttagagctagaaatagc
Primer 4 SEQ ID NO:8 TACCAGCGAGgttttagagctagaaatagc
Primer 5 SEQ ID NO:9 gagcctgtcggcctacctgct
Primer 6 SEQ ID NO:10 cggccggcatgagcctgtcg
Primer 7 SEQ ID NO:11 atgccggccgACCCATCAGGAAGCGAAGCA
Primer 8 SEQ ID NO:12 ACCCATCAGGAAGCGAAGCAGTT
Primer 9 SEQ ID NO:13 ATCCGCTGTAAAAACAGCCCCTGAC
Primer 10 SEQ ID NO:14 GGTATCATCCATCCGCTGTAAAAAC
Primer 11 SEQ ID NO:15 GGATGATACCAGCGAGTGGTTCTGCTATCA
Primer 12 SEQ ID NO:16 AGCGAGTGGTTCTGCTATCACCCGC
Primer 13 SEQ ID NO:17 GTTAACTTCGCCGTAGCGATGTTGG
Primer 14 SEQ ID NO:18 CTAGCAGGGTGTTAACTTCGCCGTA
Primer 15 SEQ ID NO:19 ACCCTGCTAGgagctcctgaaaatctcgataac
Primer 16 SEQ ID NO:20 gagctcctgaaaatctcgataac
Primer 17 SEQ ID NO:21 CATTGCCAATCTGCGTGTTC
Primer 18 SEQ ID NO:22 ACCATCGTTAATCGCCACCT
Experimental example 1 evaluation of L-aromatic amino acid production ability of Strain introduced with DNA-binding transcription regulatory factor MalT variant
The L-tryptophan or L-phenylalanine production capacity of the parent strain (KCCM 13013P and KCCM 10016) and the strain (KCCM 13013P_malT (Gly 3 15 Ser) and KCCM10016_malT (Gly 3 15 Ser)) into which the DNA-binding transcription regulatory factor MalT variant was introduced was compared.
Each strain (parent strain or variant strain) was inoculated 1% by volume to a flask containing 10m1 of the tryptophan-producing medium or phenylalanine-producing medium of Table 2, and cultured with shaking at 37℃and 200rpm for 72 hours. After the completion of the culture, the concentration of L-tryptophan or L-phenylalanine in the medium was measured by HPLC (Agilent), and the results are shown in tables 3 and 4 below, respectively.
[ Table 2]
[ Table 3]
[ Table 4]
As shown in tables 3 and 4 above, it was confirmed that the variant strains KCCM13013P_malT (Gly 315 Ser) and KCCM10016_malT (Gly 315 Ser) into which the DNA-binding transcription regulatory factor MalT variant was introduced had been substituted with serine due to glycine 315 in the amino acid sequence of the DNA-binding transcription regulatory factor MalT, thereby improving L-tryptophan and L-phenylalanine production by 14.8% and 10.6%, respectively, as compared with the parent strain.
The present invention has been studied so far, focusing on its preferred embodiments. Those skilled in the art to which the invention pertains will appreciate that the invention may be practiced in modified forms without deviating from the essential characteristics of the invention. Accordingly, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is indicated in the claims rather than in the foregoing description and all differences within the scope equivalent thereto are intended to be included in the present invention.
[ Preservation information ]
1. Preservation agency name: korean culture collection for classical cultures (KCTC)
Preservation agency address: korean full Roth Malay Kogyi Ipsin-gil 181, 56212 Korea institute of vital engineering (Korea Research Institute of Bioscience and Biotechnology, KRIBB)
Deposit number: KCTC14419BP
The preservation date: 20201228
Classification naming of biological materials: coli (ESCHERICHIA COLI)
2. Preservation agency name: korean microorganism collection center (KCCM)
Preservation agency address: hongji inner 2A road No. 45 academic circle building, post code 03641 in the Siemens gate district of Korea head city
Deposit number: KCCM13013P
The preservation date: 20210622
Classification naming of biological materials: coli (ESCHERICHIA COLI)
3. Preservation agency name: korean microorganism collection center (KCCM)
Preservation agency address: hongji-1-hole, 361-221, academic circle building, zip code 120-091 in the Siemens of Korea head city
Deposit number: KCCM10016
The preservation date: 19921024
Classification naming of biological materials: coli (ESCHERICHIA COLI).

Claims (7)

1. A DNA-binding transcription regulatory factor MalT variant consisting of the amino acid sequence of SEQ ID No. 4 is obtained by substituting glycine 315 in the amino acid sequence of SEQ ID No. 2, gly, with serine, ser.
2. A polynucleotide encoding the variant of claim 1.
3. A transformant comprising the variant of claim 1 or the polynucleotide of claim 2.
4. The transformant according to claim 3, wherein the transformant is an Escherichia (ES CHERICHIA) genus strain or a Corynebacterium (Corynebacterium) genus strain.
5. The transformant according to claim 3, wherein the transformant has L-aromatic amino acid productivity.
6. A method for producing an L-aromatic amino acid, comprising the steps of:
a step of culturing the transformant according to claim 3 in a medium; and
Recovering the L-aromatic amino acid from the transformant or a medium in which the transformant is cultured.
7. The method according to claim 6, wherein the L-aromatic amino acid is 1 or more selected from the group consisting of L-tryptophan, L-phenylalanine and L-tyrosine.
CN202311382643.2A 2022-10-24 2023-10-24 Novel variant of DNA-binding transcription regulatory factor MalT and method for producing L-aromatic amino acid using the same Pending CN117924441A (en)

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