Methods for producing l-tryptophan by enhancing prephenate dehydratase activity

Abstract

To provide a method for producing L-tryptophane by enhancing prephenate dehydratase activity, and a microorganism that produces L-tryptophan with enhanced prephenate dehydratase activity.SOLUTION: Provided herein is a method for producing L-tryptophane comprising culturing a microorganism that produces L-tryptophan with enhanced prephenate dehydratase activity. Also provided is a composition for tryptophan production comprising a microorganism that produces L-tryptophan with enhanced prephenate dehydratase activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present application relates to a method for producing L-tryptophan by enhancing prephenate dehydratase (PheA) activity. [Background technology]

[0002] L-tryptophan is an essential amino acid and has been widely used as a feed additive, a raw material for medicines such as infusions, and an ingredient in health foods. Currently, direct fermentation using microorganisms is the most widely used method for producing L-tryptophan.

[0003] In the early days, microorganisms used for L-tryptophan production were mainly strains that showed resistance to L-tryptophan analogues through selection of chemical or physical mutations. However, with the rapid development of recombinant gene technology in the 1990s and the discovery of various regulatory mechanisms at the molecular level, recombinant strains using genetic engineering techniques are now mainly used.

[0004] On the other hand, in recombinant L-tryptophan producing strains, the phenylalanine (Phe) or tyrosine (Tyr) biosynthetic pathways, which are in the competing pathway with chorismate, have generally been deleted or weakened to maximize the fermentation yield of tryptophan [J Ind Microbiol Biotechnol. 2011 Dec;38(12):1921-9], [Appl Environ Microbiol. 1999 Jun;65(6):2497-502].

[0005] However, L-tryptophan producing strains that require phenylalanine or tyrosine do not require both amino acids (phenylalanine or tyrosine) during the growth phase and production phase. The difficulty of adjusting the amount of cyclic amines (amino acids, tyrosine, etc.) used, and the difficulty of producing large quantities There was additional cost for production and difficulties in preparing the main and feed media due to the low solubility of both the above amino acids (phenylalanine and tyrosine).

[0006] To solve this problem, the present inventors have created a Corynebacterium strain that produces high yields of L-tryptophan without deficiency or weakening of the phenylalanine or tyrosine pathway from a wild-type Corynebacterium strain (US Publication US 2020-0063219 A1). When using the Corynebacterium strain that produces high yields of L-tryptophan, it was observed that there was no accumulation of phenylalanine in the culture medium during culture in a high-concentration fermenter, and tyrosine was produced at a level of 0.2 g / L at the end of the culture. However, the above strain had a problem in that L-tryptophan production was not maximized due to the production of anthranilate in the latter half of the culture. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Publication US 2020-0063219 A1 [Patent Document 2] US Register Publication US 8426577 [Patent Document 3] United States Register Publication US 7662943 B2 [Patent Document 4] WO2019-164346 A1 [Patent Document 5] Korean Patent No. 10-1968317 [Non-patent literature]

[0008] [Non-Patent Document 1] J Ind Microbiol Biotechnol. 2011 Dec;38(12):1921-9 [Non-Patent Document 2] Appl Environ Microbiol. 1999 Jun;65(6):2497-502 [Non-Patent Document 3] Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873 (1993) [Non-Patent Document 4] Methods Enzymol., 183, 63, 1990 [Non-Patent Document 5] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-Patent Document 6] FM Ausubel et al., Current Protocols in Molecular Biology [Non-Patent Document 7] Introduction to Biotechnology and Genetic Engineering, AJ Nair., 2008 [Non-Patent Document 8] Gene, 145 (1994) 69-73 [Non-Patent Document 9] DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix [Non-Patent Document 10] Appl. Microbiol.Biotechnol. (1999) 52:541-545 Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors further corrected the biosynthetic partitioning between phenylalanine and tyrosine from prephenate by enhancing the activity of prephenate dehydratase (PheA) in the Corynebacterium strain producing high yields of L-tryptophan. This correction of aromatic amino acid production in a competitive pathway not only controls the final amount of phenylalanine or tyrosine produced in the culture medium, but also reduces the production of anthranilate in the latter half of the culture, resulting in a dramatic increase in L-tryptophan production, and thus completed the present application. [Means for solving the problem]

[0010] The present application relates to a method for the preparation of a compound having enhanced prephenate dehydratase activity. The present invention provides a microorganism capable of producing L-tryptophan.

[0011] The present application provides a method for producing L-tryptophan, comprising the step of culturing an L-tryptophan-producing microorganism having enhanced prephenate dehydratase activity in a medium.

[0012] The present application provides a composition for producing L-tryptophan, comprising an L-tryptophan-producing microorganism having enhanced prephenate dehydratase activity. Effect of the Invention

[0013] The L-tryptophan producing microorganism of the present application having enhanced prephenate dehydratase activity can minimize the accumulation of anthranilate and produce L-tryptophan with high efficiency. In an embodiment of the present invention, for example, the following items are provided: (Item 1) L-Tryptophan with enhanced prephenate dehydratase activity A tryptophan-producing microorganism. (Item 2) 2. The microorganism according to item 1, wherein the prephenate dehydratase comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity thereto. (Item 3) 2. The microorganism described in item 1, wherein the enhanced activity is achieved by increasing the copy number of the gene encoding the prephenate dehydratase or by replacing the gene promoter with a strong promoter. (Item 4) 2. The microorganism according to item 1, wherein the microorganism is a Corynebacterium sp. (Item 5) The microorganism is Corynebacterium glutamicum. 5. The microorganism according to item 4, (Item 6) A method for producing L-tryptophan, comprising the step of culturing an L-tryptophan-producing microorganism having enhanced prephenate dehydratase activity in a medium. (Item 7) 7. The method for producing L-tryptophan according to Item 6, further comprising recovering L-tryptophan from the culture medium or the microorganism. (Item 8) A composition for producing L-tryptophan, comprising an L-tryptophan producing microorganism having enhanced prephenate dehydratase activity. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram of the PDCM2 plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each different description and embodiment. That is, all combinations of the various elements disclosed in this application belong to the scope of this application. Also, the following specific description is not considered to limit the scope of this application.

[0016] One aspect of the present application is a method for producing a compound having prephenate dehydratase activity. To provide an enhanced L-tryptophan producing microorganism.

[0017] The term "L-tryptophan" in this application refers to one of 20 α-amino acids, and is an essential amino acid that is not biosynthesized in many organisms, including humans. Tryptophan is known to act primarily as a biochemical precursor, from which various substances are synthesized, such as neurotransmitters such as serotonin, neurohormones such as melatonin, niacin, and oxine.

[0018] L-tryptophan is synthesized from chorismate (chorismic acid), and the group of genes encoding the enzymes involved in this process is known as the tryptophan operon (Trp operon). The tryptophan operon is known to contain a structural gene and an expression regulatory region. Normally, the tryptophan operon is actively transcribed so that the cell can produce sufficient tryptophan to meet its demand. However, when there is sufficient intracellular tryptophan, a repressor binds to the tryptophan and inactivates the tryptophan operon, resulting in transcription being suppressed. The tryptophan operon can be derived from various microorganisms, such as Corynebacterium microorganisms and Escherichia microorganisms. The "expression regulatory region" of the tryptophan operon means a site that is present upstream of the structural genes constituting the tryptophan operon and can regulate the expression of the structural genes. The structural genes constituting the tryptophan operon in Corynebacterium microorganisms may be composed of trpE, trpG, trpD, trpC, trpB, and trpA genes, and the structural genes constituting the tryptophan operon in Escherichia microorganisms may be composed of trpE, trpD, trpC, trpB, and trpA genes. The expression regulatory region of the tryptophan operon may be present upstream of trpE at the 5' position of the tryptophan operon structural gene. Specifically, it includes a tryptophan regulator (trp regulator; trpR), a promoter (trp promoter), an operator (trp operator), a tryptophan leader peptide (trp leader peptide; trp L), and a tryptophan attenuator (trp attenuator), excluding a structural gene that can constitute a tryptophan operon. More specifically, a promoter (trp promoter), an operator (trp operator), a tryptophan leader peptide (trp leader peptide; trp L), and a tryptophan It may also include an attenuator (trp attenuator).

[0019] In the present application, the term "prephenate dehydratase (hereinafter, "PheA")" refers to an enzyme that dehydrates L-phenylalanine in chorismate or prephenate. It is an enzyme in the pathway that produces tyrosine and is known to be an enzyme in a competitive step with the tyrosine biosynthetic pathway. The gene encoding the protein may be, for example, the pheA gene, but is not limited thereto, and the pheA gene may be regulated by the tryptophan operon. In the present application, the term "pheA gene" may be used interchangeably with "gene encoding prephenate dehydratase" and "pheA gene."

[0020] The PheA may have, consist of, or contain the amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto. The sequence of SEQ ID NO: 1 can be confirmed in the publicly known database, NCBI Genbank.

[0021] Specifically, the PheA may be SEQ ID NO: 1 and / or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the above SEQ ID NO: 1. It is clear that PheA having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, or added is also included within the scope of the present application, so long as the amino acid sequence has such homology or identity and exhibits a function corresponding to the above PheA.

[0022] As used herein, the terms "homology and identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0023] Conserved polynucleotide or polypeptide sequence homology or identity is determined by standard sequence algorithms, and may be used with default gap penalties established by the program used. Substantially homologous or identical sequences can hybridize generally to at least about 50%, 60%, 70%, 80%, or 90% or more of the entire sequence or full length under medium or high stringent conditions. Hybridization also takes into account polynucleotides that contain degenerate codons in place of codons in the polynucleotide.

[0024] Homology or identity to the above polypeptide or polynucleotide sequences can be determined, for example, by the literature algorithm BLAST [see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)], or FASTA by Pearson (see Methods Enzymol., 183, 63, 1990). Based on such an algorithm, BLAST For example, programs called BLASTN and BLASTX have been developed (see: http: / / www.ncbi.nlm.nih.gov). In addition, whether any amino acid or polynucleotide sequence has homology, similarity or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and appropriate hybridization conditions defined are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology).

[0025] The term "microorganism capable of producing L-tryptophan" in the present application means a microorganism naturally having an ability to produce L-tryptophan or a microorganism in which the ability to produce L-tryptophan has been imparted to a parent strain that does not have the ability to produce L-tryptophan. Specifically, the microorganism may be, but is not limited to, a microorganism capable of producing L-tryptophan with enhanced PheA activity.

[0026] Specifically, the "microorganism producing L-tryptophan" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. More specifically, it is a microorganism in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and may be a microorganism in which a genetic mutation has occurred for the production of the desired L-tryptophan or in which the L-tryptophan production activity has been enhanced. For the purposes of this application, the L-tryptophan-producing microorganism is characterized by enhanced PheA activity and increased ability to produce the desired L-tryptophan, and may be, but is not limited to, a genetically modified microorganism or a recombinant microorganism.

[0027] In the present application, the term "enhanced activity" of a protein means that the activity of the protein is increased compared to the intrinsic activity. The above-mentioned "intrinsic activity" means the activity of a specific protein that a parent strain or a non-transformed microorganism originally had before the trait change, when the trait is changed by a genetic mutation due to natural or artificial factors. This may be used interchangeably with "activity before transformation." The term "increased" activity of a protein compared to the intrinsic activity means that the activity of a specific protein is improved compared to the activity of a specific protein that a parent strain or a non-transformed microorganism originally had before the trait change.

[0028] The "increase in activity" can be achieved by introducing an exogenous protein or enhancing the activity of an endogenous protein, specifically, by enhancing the activity of an endogenous protein. Whether or not the activity of the protein is enhanced can be confirmed by an increase in the activity level, expression level, or amount of a product produced from the protein.

[0029] In the present application, the protein to be the subject of the above-mentioned activity enhancement, i.e., the target protein, may be PheA, but is not limited thereto.

[0030] In the present application, the product produced from the protein may be, but is not limited to, L-tryptophan.

[0031] The activity of the protein can be enhanced by various methods well known in the art, and is not limited as long as the activity of the target protein can be enhanced compared to that of the microorganism before transformation. The method may be, but is not limited to, genetic engineering or protein engineering.

[0032] The method for enhancing protein activity using genetic engineering can be, for example, 1) an increase in the intracellular copy number of the gene encoding the protein; 2) A method of replacing the gene expression regulatory sequence on the chromosome encoding the protein with a sequence having a strong activity; 3) A method for modifying the base sequence of the initiation codon or 5'-UTR region of the protein so as to increase the activity of the protein. 4) A method for modifying a polynucleotide sequence on a chromosome so as to increase the activity of the protein; 5) introduction of an exogenous polynucleotide exhibiting the activity of the above protein or a codon-optimized mutant polynucleotide of the above polynucleotide; or 6) The above methods may be combined, but are not limited to these.

[0033] The method of enhancing the activity of a protein using the protein engineering can be carried out, for example, by analyzing the tertiary structure of a protein and selectively modifying or chemically modifying exposed sites, but is not limited thereto.

[0034] The above 1) increase in the copy number of a gene encoding a protein in a cell can be achieved by any method known in the art, for example, by introducing into a host cell a vector to which a gene encoding the protein is operably linked, the vector being capable of replicating and functioning independently of a host, or by introducing into a host cell a vector to which the gene is operably linked, the vector being capable of inserting the gene into a chromosome in the host cell, but is not limited thereto.

[0035] In this application, the term "vector" refers to a DNA construct that contains a polynucleotide sequence encoding a protein of interest in a compatible host in a form operably linked to a regulatory sequence suitable for expressing the protein of interest. The expression regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate and function independently of the host genome, or can be integrated into the genome itself.

[0036] The vector used in the present application is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, the vector usable in the present application may be pDCM2 (FIG. 1, SEQ ID NO: 3) constructed for the insertion and exchange of genes in the Corynebacterium chromosome, but is not particularly limited thereto, and any known expression vector can be used.

[0037] In this application, the term "transformation" refers to introducing a recombinant vector containing 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. As long as the transformed polynucleotide can be expressed in the host cell, it includes all of these, regardless of whether it is located within the host cell's chromosome by insertion or extrachromosomally. The above transformation method includes any method for introducing a nucleic acid into a cell, and can be performed by selecting a suitable standard technique according to the host cell, as known in the art. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, etc. Microinjection, Polyethylene glycol (PEG) method, DEAE-dextran method, These include, but are not limited to, the cationic liposome method and the lithium acetate-DMSO method.

[0038] In addition, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence or an expression control region that initiates and mediates the transcription of the polynucleotide encoding the target protein of the present application. The operable linkage can be produced using a gene recombination technique known in the art, and the site-specific DNA cleavage and ligation can be produced using cleavage and ligation enzymes known in the art, but is not limited thereto.

[0039] The method of replacing the expression regulatory sequence of a gene on a chromosome that encodes a protein with a sequence having a stronger activity can be carried out by any method known in the art, for example, by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory sequence, or by replacing the expression regulatory sequence with a nucleic acid sequence having a stronger activity. The expression regulatory sequence can include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc. Specifically, the method can be, but is not limited to, linking a strong heterologous promoter instead of the original promoter.

[0040] Examples of known strong promoters include mutant lysC promoter (US Patent Publication US 8426577), CJ7 promoter (US Patent Publication US 7662943 B2), CJ1 promoter (US Patent Publication US 7662943 B2), lac promoter, Trp promoter, trc promoter, etc. The strong promoter may include, but is not limited to, the promoter, tac promoter, lambda phage PR promoter, PL promoter, and tet promoter. Specifically, the strong promoter usable in the present application may be PlysCm1 (SEQ ID NO: 4) produced by modifying a part of the sequence of the mutant lysc promoter (US Patent Publication US 8426577), but is not particularly limited thereto, and any publicly known promoter may be used.

[0041] The method for modifying the start codon or 5'-UTR region of a protein as described above in 3) may be any method known in the art, for example, a method for replacing the endogenous start codon of the protein with another start codon that has a higher protein expression rate than the endogenous start codon, but is not limited thereto.

[0042] The method of modifying the polynucleotide sequence on the chromosome so as to increase the activity of the protein (4) can be carried out by any method known in the art, for example, by inducing mutations in the nucleic acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the polynucleotide sequence, or by replacing the polynucleotide sequence with an improved polynucleotide sequence so as to have stronger activity. Specifically, the replacement may be, but is not limited to, inserting the gene into the chromosome by homologous recombination.

[0043] The vector used in this case is a selection marker to check whether the vector is inserted into the chromosome or not. The selection marker can further comprise a selection marker that is capable of detecting the presence of a selection marker in a cell transformed with the vector. The purpose of the present invention is to select, i.e., to confirm whether or not the gene to be introduced is inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein can be used, but is not limited to these. In an environment treated with a selective agent, only cells that express the selection marker survive or show other phenotypes, allowing the selection of transformed cells.

[0044] The introduction of a foreign polynucleotide exhibiting the activity of the above protein 5) can be carried out by any method known in the art, for example, by introducing a foreign polynucleotide encoding a protein exhibiting the same / similar activity as the above protein, or a codon-optimized mutant polynucleotide thereof, into a host cell. The above foreign polynucleotide can be used without any restriction on its origin or sequence, as long as it exhibits the same / similar activity as the above protein. In addition, the introduced foreign polynucleotide can be introduced into a host cell with its codons optimized so that optimized transcription and translation are performed in the host cell. The above introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell to produce a protein and increase its activity.

[0045] Finally, 6) the combination of the above methods can be carried out by simultaneously applying any one or more of the above methods 1) to 5).

[0046] Such enhanced activity of a protein may be, but is not limited to, an increase in the activity or concentration of the corresponding protein based on the activity or concentration of the protein expressed in a wild-type or unmodified microbial strain, or an increase in the amount of a product produced from the protein. In this application, the term "unmodified strain" or "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a naturally occurring strain itself or a strain before its characteristics are changed due to genetic mutation caused by artificial factors. In this application, the change in characteristics may be an enhanced activity of PheA. The above "unmodified strain" or "unmodified microorganism" may be used interchangeably with "non-mutated strain", "non-mutated strain", "non-mutated microorganism", "non-mutated microorganism" or "reference microorganism".

[0047] In the present application, the reference microorganism is not particularly limited as long as it is a microorganism that produces L-tryptophan, and includes, without limitation, mutant strains having enhanced L-tryptophan production ability compared to the wild type. Examples thereof include, but are not limited to, wild-type Corynebacterium glutamicum ATCC13869 strain, CJ04-8321 strain (PCT Publication WO WO2019-164346 A1), or strains obtained by adding one or more genetic modifications to the above strains in order to enhance the L-tryptophan biosynthetic pathway.

[0048] The one or more genetic modifications may be, for example, any one or more genetic modifications selected from the following: overexpressing the activity of the L-tryptophan operon; improving the supply and efficiency of L-tryptophan precursors; improving the excretion of L-tryptophan; and weakening or inactivating the activity of competitive pathway genes, directional pathway regulators of the L-tryptophan operon, L-tryptophan influx genes, and L-tryptophan influx and degradation genes, but are not limited thereto.

[0049] The genetic modification for overexpressing the activity of the L-tryptophan operon may be, for example, i) strengthening the promoter of the L-tryptophan biosynthesis gene operon, ii) eliminating the feedback inhibition of the TrpE protein associated with the improvement of production in the L-tryptophan operon, or ii) strengthening the promoter of the L-tryptophan biosynthesis gene operon. Specifically, i) may be strengthening the promoter of the L-tryptophan biosynthesis gene operon by replacing it with a strong promoter SPL7, ii) may be introducing trpE(P21S)DCBA or trpE(S38R)DCBA, which is an L-tryptophan operon having a feedback-limited trpE trait, and iii) may be strengthening the promoter of the L-tryptophan biosynthesis gene operon by replacing it with a strong promoter SPL7, but is not limited thereto.

[0050] The genetic modification that improves the supply and efficiency of L-tryptophan precursors provides a continuous supply of L-tryptophan precursors, such as E4P (erythorse-4-phosphate), and energy. This may involve enhancing the expression of related genes for efficient utilization of energy, specifically, introducing a gene encoding tkt (transketolase) or enhancing its expression, but is not limited thereto.

[0051] The genetic modification for improving the excretion of L-tryptophan may be, for example, the introduction of an exogenous membrane protein for improving the excretion of L-tryptophan, specifically, a membrane protein derived from Herbaspirillum rhizosphaerae. Alternatively, the gene encoding the is not limited to.

[0052] The strain to which one or more genetic modifications have been added may be, for example, CA04-8325 (US Published Application US 2020-0063219 A1) in which trpE(S38R)DCBA, an L-tryptophan operon containing a strong promoter SPL7 and having a feedback-limited trpE trait, has been introduced into the ATCC13869 strain, CA04-8352 (PCT Publication WO WO2019-164346 A1) in which the tkt gene has been inserted into the CA04-8325 strain, or CA04-8405 strain (US Published Application US 2020-0063219 A1) produced by introducing a gene encoding a membrane protein derived from Herbaspirillum rhizosphaerae into the CJ04-8352 strain, but is not limited thereto.

[0053] For the purpose of this application, the above-mentioned L-tryptophan-producing microorganism may be any microorganism that has enhanced PheA activity by the above-mentioned method and is capable of producing L-tryptophan. In this application, the above-mentioned "L-tryptophan-producing microorganism" may be used interchangeably with "L-tryptophan-producing microorganism" and "microorganism capable of producing L-tryptophan."

[0054] Examples of the microorganism include those of the genera Corynebacterium, Escherichia, Enterobacter, Erwinia, Serratia, Providencia, and Brevibacterium. and specifically, the microorganism may be a microorganism belonging to the genus Corynebacterium. It may be a microorganism.

[0055] More specifically, the genus Corynebacterium is Corynebacterium Corynebacterium glutamicum, Corynebacterium ammoniac Corynebacterium ammoniagenes, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium porphyra Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, etc., and Corynebacterium glutamicum. Microorganisms belonging to the genus Corynebacterium may be included without limitation.

[0056] Another aspect of the present application provides a method for producing L-tryptophan, comprising the step of culturing an L-tryptophan producing microorganism having enhanced prephenate dehydratase activity in a medium.

[0057] The above-mentioned prephenate dehydratase, the activity enhancement, and the microorganism producing L-tryptophan are as described above.

[0058] In the above method, the step of culturing the microorganism may be performed by, but is not limited to, a known batch culture method, continuous culture method, fed-batch culture method, etc. In this case, the culture conditions are not particularly limited to, but may be adjusted to an appropriate pH (e.g., pH 5-9, specifically pH 6-8, most specifically pH 7.0) using a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid), and aerobic conditions may be maintained by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20-45°C, specifically 25-40°C, and the culture may be performed for about 10-160 hours, but is not limited thereto. The amino acid produced by the above culture may be secreted into the medium or remain in the cells.

[0059] In addition, the culture medium used may contain, as carbon sources, sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower seed 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), which may be used individually or in combination, but are not limited thereto. As nitrogen sources, nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn steeping liquid, soybean meal flour, and urea), or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), which may be used individually or in combination, but are not limited thereto. As phosphorus sources, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts, which may be used individually or in combination, but are not limited thereto. The medium may also contain other essential growth promoters such as metal salts (eg, magnesium sulfate or ferrous sulfate), amino acids, and vitamins.

[0060] The method according to the present application may further comprise a step of recovering L-tryptophan from the culture medium or the microorganism. The method of recovering the amino acid produced in the above culture step of the present application may be to collect the amino acid of interest from the culture medium using a suitable method known in the art depending on the culture method. For example, centrifugation, Separation, filtration, anion exchange chromatography, crystallization, HPLC, and the like may be used, and the desired amino acid can be recovered from the medium or the microorganism using suitable methods known in the art.

[0061] The recovery step may also include a purification step and may be carried out using any suitable method known in the art. Thus, the recovered amino acids may be in a purified form or may be a microbial fermentation broth containing the amino acids (Introduction to Biotechnology and Genetic Engineering, AJ Nair., 2008). Before or after the above recovery step, suitable methods known in the art can be added to efficiently recover the target amino acid.

[0062] Another aspect of the present application provides a composition for producing L-tryptophan, comprising an L-tryptophan producing microorganism having enhanced prephenate dehydratase activity. The above-mentioned prephenate dehydratase, the activity enhancement, and the microorganism producing L-tryptophan are as described above.

[0063] The composition for producing L-tryptophan includes the pheA gene encoding the PheA, and may include a structure for enhancing the PheA or pheA gene without limitation. Specifically, the structure may be in a form contained in a vector so as to express an operably linked gene in a host cell into which it is introduced, as described above. Specifically, the expression of the pheA gene, which is a gene encoding the prephenate dehydratase, may be enhanced through an increase in the copy number of the gene or an exchange with a strong promoter.

[0064] Another aspect of the present application provides a use of the composition for producing L-tryptophan.

[0065] The present application will be described in more detail with reference to examples below. However, these examples are for illustrative purposes only and the scope of the present application is not limited to these examples.

[0066] Example 1: Plasmid construction A plasmid (pDCM2, FIG. 1, SEQ ID NO: 3) for inserting and exchanging genes in the Corynebacterium chromosome was designed and synthesized using the gene synthesis service of Bionix Co., Ltd. The plasmid was designed to contain a restriction enzyme that is easy to use for cloning, referring to a well-known paper related to the sacB system [Gene, 145 (1994) 69-73]. The pDCM2 thus synthesized The plasmid has the following characteristics:

[0067] 1) It has a replication origin that functions only in E. coli. It is capable of self-replication, but Corynebacterium has the property of being unable to self-replicate. 2) It has a kanamycin resistance gene as a selection marker. 3) It has the levan sucrose gene (sacB) as a secondary positive selection marker. 4) The final strain does not contain any genetic information derived from the pDCM2 plasmid.

[0068] Example 2: Construction of plasmid for enhancing prephenate dehydratase Enhancement of prephenate dehydratase (pheA) activity To achieve this, a mutant lysC promoter (registered US patent application Ser. No. 201102363) was used, which is known to be a strong promoter. Based on US 8426577 B2), a part of the sequence was modified to design the promoter as SEQ ID NO: 4, which was then synthesized using the gene synthesis service of Bionix Co., Ltd., and named the promoter PlysCm1. Using the PlysCm1 promoter, a plasmid was constructed that enhances prephenate dehydratase activity by additionally inserting the pheA gene or by replacing the wild-type promoter of the pheA gene with PlysCm1.

[0069] Example 2-1: Construction of plasmid for gene insertion In order to insert the pheA gene using the PlysCm1 promoter, the chromosomal DNA of wild-type Corynebacterium glutamicum ATCC13869 was used as a template, and the upstream region where homologous recombination occurs on the chromosome was cloned using the primer pair of SEQ ID NO: 5 and SEQ ID NO: 6, as shown in SEQ ID NO: 7 and SEQ ID NO: 8. After amplifying the downstream region using primer pairs, The primer sequences used are shown in Table 1 below.

[0070] [Table 1]

[0071] PCR was performed to obtain the above fragment. The polymerase was Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 4 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0072] In addition, a PlysCm1 promoter fragment was obtained using SEQ ID NO: 9 and SEQ ID NO: 10 with the previously synthesized SEQ ID NO: 4 as a template. Additionally, a pheA gene fragment (SEQ ID NO: 2) was obtained using SEQ ID NO: 11 and SEQ ID NO: 12 with the wild-type Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template. The primer sequences used here are shown in Table 2 below.

[0073] [Table 2]

[0074] Polymerase is Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 4 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0075] The upstream and downstream fragments of the region where homologous recombination occurs on the chromosome obtained in the above process, the PlysCm1 promoter fragment, the pheA gene fragment, and the chromosomal transformation vector pDCM2 cut with SmaI restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). A plasmid was obtained and named pDCM2-Tn::PlysCm1_pheA.

[0076] Example 2-2: Plasmid construction for promoter replacement We attempted to construct a plasmid that enhances prephenate dehydratase activity by replacing the wild-type promoter of the pheA gene with PlysCm1. Specifically, the wild-type Corynebacterium glutamicum (ATCC13869) chromosomal DNA was used as a template to construct the plasmid with the sequence of SEQ ID NO: 1. The primer pair of SEQ ID NO:13 and SEQ ID NO:14 was used to generate the wild-type promoter upstream of the pheA gene, where homologous recombination occurs on the chromosome. A gene fragment containing both the PlysCm1 promoter and its downstream was obtained using primers SEQ ID NO: 15 and SEQ ID NO: 16 with the previously constructed pDCM2-Tn::PlysCm1_pheA plasmid as a template. The primer sequences used are shown in Table 3 below.

[0077] [Table 3]

[0078] To obtain the above fragment, the polymerase Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 4 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0079] The upstream fragment of the pheA promoter, the promoter and downstream fragment containing PlysCm1, and the chromosomal transformation vector pDCM2 cut with SmaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDCM2-Pn::PlysCm1_pheA.

[0080] Example 3: Construction of strain with enhanced prephenate dehydratase expression and confirmation of tryptophan production Membrane protein from Herbaspirillum rhizosphaerae The gene encoding the protein (accession number NZ_LFLU01000012.1) was cloned into the tryptophan-producing strain The CA04-8405 strain (KCCM12099P, US Patent Publication US 2020-0063219 A1), which was prepared by introducing pDCM2-Tn::PlysCm1_pheA prepared in Example 2-1 above into the CA04-8352 strain (Korean Patent Registration No. 10-1968317), was transformed using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and then a strain in which the PlysCm1_pheA gene was additionally inserted was obtained through a secondary crossover process. The insertion of the gene was confirmed through PCR amplification and genome sequencing using a primer pair of SEQ ID NO: 17 and SEQ ID NO: 18, which can amplify the external sites of the upstream and downstream regions of the homologous recombination, respectively. The strain in which the gene was inserted was named CM05-9157. The primer sequences used here are as shown in Table 4 below.

[0081] [Table 4]

[0082] In the same manner as above, the CA04-8405 strain was transformed with pDCM2-Pn::PlysCm1_pheA prepared in Example 2-2 above by electroporation, and then a secondary crossover process was performed to obtain a strain in which the wild-type pheA promoter was replaced with the PlysCm1 promoter. The promoter replacement was confirmed through PCR amplification and genome sequencing using a primer pair of SEQ ID NO: 19 and SEQ ID NO: 20, which can amplify the external sites of the upstream and downstream regions of the homologous recombination, respectively. The strain in which the promoter was replaced was named CM05-9158. The primer sequences used are shown in Table 5 below.

[0083] [Table 5]

[0084] In order to confirm the tryptophan production of the CM05-9157 and CM05-9158 strains prepared through the above process, the CA04-8405 strain was used as a control and the cultivation and tryptophan production were compared as follows. Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. After cultivation, three new 250 ml corner baffle flasks containing 25 ml of production medium were prepared for each strain, and 1 ml of the above seed culture was inoculated into each flask and cultured at 30°C for 24 hours with shaking at 200 rpm. After the completion of the shaking cultivation, the amount of L-tryptophan produced was measured using HPLC.

[0085] Seed medium (pH 7.0) Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4 7H2O 0.5g, biotin 100μg, thiamine HCl 1000μg, calcium pantothenate 2000μg, nicotinamide 2000μg (based on 1 liter of distilled water)

[0086] <Production medium (pH 7.0)> Glucose 30g, (NH4)2SO4 15g, MgSO47H2O 1.2g, KH2PO4 1g, yeast extract 5g, biotin 900μg, thiamine hydrochloride 4500μg, calcium pantothenate 4500μg, CaCO3 30g (based on 1 liter of distilled water).

[0087] After culturing CA04-8405, CM05-9157 and CM05-9158 strains with enhanced pheA expression, the results of L-tryptophan production in the medium are shown in Table 6 below.

[0088] [Table 6]

[0089] As a result of culturing the CM05-9157 and CM05-9158 strains with enhanced pheA expression, 1.93 and 1.94 g / L of L-tryptophan were produced, respectively. This is an increase of about 0.37 g / L compared to the control CA04-8405 strain, and the fermentation yield was improved by about 23-24%. In addition, it was confirmed that the enhanced pheA expression reduced the production of anthranilate, which resulted in an increase in tryptophan production.

[0090] The above CM05-9157 strain was internationally deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on February 20, 2020, and was assigned the deposit number KCCM12670P.

[0091] From the above description, a person skilled in the art to which the present invention pertains will understand that the present application may be implemented in other specific forms without changing the technical idea or essential features of the present application. In this regard, it should be understood that the above described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

[0092] [ka]

Claims

[Claim 1] The invention described in this specification.

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