KR102035844B1 - Recombinant coryneform microorganism to produce L-tryptophan and method for producing L-tryptophan using the same - Google Patents

Abstract

The present application relates to a genus of Corynebacterium producing recombinant L-tryptophan using genetic engineering techniques, and a method for producing L-tryptophan using the microorganism.

Description

Recombinant coryneform microorganism to produce L-tryptophan and method for producing L-tryptophan using the same}

The present application relates to a genus of Corynebacterium producing recombinant L-tryptophan using genetic engineering techniques, and a method for producing L-tryptophan using the microorganism.

L-Tryptophan is one of the essential amino acids, and has been widely used as a raw material for pharmaceuticals such as feed additives or sap and health food materials. Currently, microbial direct fermentation is mainly used for L-tryptophan production.

The microorganisms used to produce L-tryptophan have been used mainly for the selection of analog-resistant strains through chemical or physical mutations.However, due to the rapid development of genetic recombination technology and molecular-level regulatory mechanisms in the 1990s, genetic engineering techniques were used. Recombinant strains are mainly used.

On the other hand, L-tryptophan production using Escherichia coli has been conducted in various ways, including sugar influx, pentose phosphate pathway, serine biosynthesis, aromatic / tryptophan biosynthesis, and is now leading to industrial production. Microorganisms of the genus Corynebacterium, a commonly recognized as safe (GRAS) strain, have the same advantages as Endotoxin-free and are applied to the production of glutamic acid, lysine, and valine. Inadequately applied to industrial production.

Against this background, in the present application, from the metabolic point of view, tryptophan biosynthesis is enhanced by genetic engineering of the microorganism of the genus Corynebacterium, and at the same time, the erythrose 4-phosphate, which is the first substrate of tryptophan biosynthesis, is increased, thereby significantly reducing tryptophan production capacity. Confirmed to be improved to complete the present application.

JP 1985-176593 A

One object of the present application is to provide a Corynebacterium microorganism that produces L-tryptophan, genetically recombined to overproduce L-tryptophan.

Another object of the present application, the step of culturing the microorganism in the medium; And it provides a method for producing L- tryptophan, comprising the step of recovering L-tryptophan from the cultured microorganism or medium.

This will be described in detail as follows. In addition, each description and embodiment disclosed in this application may apply to each other description and embodiment. That is, all combinations of the various elements disclosed in this application are within the scope of the present application. In addition, the scope of the present application is not to be limited by the specific description described below.

One aspect of the present application for achieving the above object is a gene encoding an ansranilic acid synthase whose N-terminus is composed of the amino acid sequence of SEQ ID 32 and the feedback inhibition is released, and tryptophan operon and transke comprising the same It is directed to a Corynebacterium microorganism that produces L-tryptophan with enhanced expression of the gene encoding tolase.

In another embodiment, the microorganism is modified to express a gene encoding a feedback released anthranilic acid synthetase consisting of an amino acid sequence of SEQ ID NO: 33 and an E. coli-derived tryptophan operon additionally, Provided are Corynebacterium microorganisms that produce L-tryptophan.

As used herein, the term "L-tryptophan" is one of α-amino acids and refers to an aromatic amino acid that is an essential amino acid that is not synthesized in the body and has a chemical formula of C ₁₁ H ₁₂ N ₂ O ₂ .

As used herein, the term "tryptophan operon (Trp operon)" refers to a group of genes encoding enzymes involved in synthesizing tryptophan from chorismic acid (chorismate). Specifically, the tryptophan operon may be tryptophan operon derived from Corynebacterium spp., Or tryptophan operon derived from Escherichia spp. In the microorganisms of the genus Corynebacterium, the genes trpE, trpG, trpD, trpC, trpB, trpA, and in the Escherichia microorganisms, the trpE, trpD, trpC, trpB, and trpA genes are arranged in this order under the promoter and effector. Configure Specifically, the Corynebacterium microorganism may be Corynebacterium glutamicum, Escherichia microorganism may be Escherichia coli. The tryptophan operon may have a known nucleotide sequence, and those skilled in the art can easily obtain the nucleotide sequence of tryptophan operon through a database such as NCBI or Kegg. Ordinary tryptophan operon is actively transcribed to produce sufficient amount of tryptophan as required by the cell, but in the presence of sufficient intracellular tryptophan, a repressor binds to tryptophan, which inactivates the tryptophan operon, thereby inhibiting transcription. . In order to produce an excessive amount of tryptophan in the microorganism, try to suppress the inhibitor of tryptophan operon, and genetic manipulation is required so as not to receive feedback suppression by tryptophan.

Proteins encoded by the tryptophan operon include anthranilate synthase, anthranilate phosphoribosyltransferase, phosphoribosylanthranilate isomerase, and indole-3-glycerol phosphate Synthetic enzymes (indole-3-glycerol phosphate synthase) and tryptophan synthase are known. Specifically, first, anthranilic acid synthase acts on the chorismic acid to synthesize anthranilic acid. Ansranilic acid phosphoribosyl transferase then acts to synthesize phosphoribosyl anthranilic acid (N-(5'-phosphoribosyl) -anthranilate). Subsequently, phosphoribosyl anthranilic acid isomerase and indole-3-glycerol phosphatase act to synthesize indole-3-glycerol phosphate, and tryptophan synthase acts on L-tryptophan Complete the synthesis (Bonggaerts et al ., Metab Eng , 3, 289-300, 2001, Iris Brune et al ., J. Bacteriol., 189 (7): 2720-33, 2007).

In the present application, the "anthranilate synthase" refers to an enzyme that synthesizes anthranilic acid from corrismic acid. The anthranilic acid synthase of the present application may be an anthranilic acid synthase derived from Corynebacterium spp., Or an ansranilic acid synthase derived from Escherichia spp. In the case of coryneglutacum, the proteins produced by the trpE and trpG genes in tryptophan operon are polymerized, and the proteins produced by the trpE and trpD genes in E. coli form polymers to form anthranilic acid synthase.

Ansranilic acid synthase of the present application, unlike wild-type or natural-type protein having its activity, has the feature that the inhibition of feedback by the end product tryptophan, derivatives or analogs thereof is released or desensitized. As used herein, the term "feedback inhibition" means that an end product of an enzyme system inhibits or inhibits a reaction at an early stage of the enzyme system. Therefore, in the case of releasing or desensitizing feedback inhibition of anthranilic acid synthase, it is possible to increase the productivity of tryptophan in comparison with the other cases.

It is apparent that the anthranilic acid synthase in which the feedback inhibition is released is included in the present application as long as the feedback inhibition by tryptophan is released or desensitized. Specifically, the anthranilic acid synthase from which the feedback inhibition is released may be N-terminal of the amino acid sequence of SEQ ID NO: 32, or N-terminal of the amino acid sequence of SEQ ID NO: 33. The amino acid sequence of SEQ ID NO: 32 is a sequence in which serine at position 38 is substituted with arginine, and the amino acid sequence of SEQ ID NO: 33 is a sequence in which proline at position 21 is substituted with serine. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 32 or the amino acid sequence of SEQ ID NO: 33 is a polypeptide having the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33, a polypeptide consisting of the amino acid of SEQ ID NO: 32 or 33, or SEQ ID NO: 32 or 33 It can be used interchangeably with a polypeptide comprising an amino acid sequence of.

The anthranilic acid synthase may have a known amino acid sequence derived from Corynebacterium microorganisms or an amino acid sequence derived from Escherichia genus microorganisms, and those skilled in the art will appreciate Amino acid sequences can be easily obtained. However, in the case of microorganisms of the genus Corynebacterium to eliminate feedback inhibition, as long as the N-terminus includes the amino acid of SEQ ID NO: 32 in the entire amino acid sequence, thereafter, it is a known amino acid sequence or other feedback inhibition. Amino acid sequence with release mutations. For Escherichia microorganisms, the N-terminus from the entire amino acid sequence only includes the amino acid of SEQ ID NO: 33 from the entire amino acid sequence for feedback suppression, after which it may be a known amino acid sequence or an amino acid sequence containing other feedback suppression mutations. Can be.

Although the anthranilic acid synthase in the present application is defined as a polypeptide comprising the amino acid of SEQ ID NO: 32 or SEQ ID NO: 33, the amino acid of SEQ ID NO: 32 or 21, except that the position 38 is arginine It does not exclude mutations that can occur naturally in the amino acid sequence of SEQ ID NO: 33, except for serine, or its latent mutations, and are identical to polypeptides comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. Or it is apparent to those skilled in the art that if the corresponding activity corresponds to the anthranilic acid synthetase of the present application. For example, an ansranilic acid synthase derived from the genus Corynebacterium microorganism of the present application includes that the position 38 is arginine, and the amino acid sequence of SEQ ID NO: 32 or 70%, 80%, 90%, 95 It may be a polypeptide consisting of an amino acid sequence having at least%, or 97% homology. In addition, the anthranilic acid synthetase derived from the genus Escherichia microorganism of the present application includes that the position 21 is serine, and the amino acid sequence of SEQ ID NO: 33 or 70%, 80%, 90%, 95%, or 97% thereof. It may be a polypeptide consisting of an amino acid sequence having the above homology. As long as it is an amino acid sequence having such homology and exhibiting an efficacy corresponding to the polypeptide, it is obvious that a polypeptide having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application.

The term “homology” refers to the percent identity between two polynucleotide or polypeptide moieties. It means the degree of coincidence with a given amino acid sequence or base sequence and can be expressed as a percentage. In this specification, homologous sequences thereof having the same or similar activity as a given amino acid sequence or base sequence are designated as "% homology". Homology between sequences from one moiety to another can be determined by known art. For example, using standard software that calculates parameters such as score, identity and similarity, in particular BLAST 2.0, or by hybridization experiments used under defined stringent conditions Appropriate hybridization conditions, which are defined within the scope of the art, are well known to those skilled in the art, and are well known to those skilled in the art (e.g., J. Sambrook et al. Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York.

The polynucleotide encoding the anthranilic acid synthase may be included without limitation so long as it is a sequence capable of encoding the anthranilic acid synthase.

In addition, the polynucleotide encoding the anthranilic acid synthase is within a range that does not change the amino acid sequence of the polypeptide due to the degeneracy of the codon or in consideration of the codon preferred in the organism to express the polypeptide. In the coding region, various modifications may be made. Meanwhile, the polynucleotide encoding the polypeptide may be a polynucleotide including a polynucleotide sequence having at least 70%, 80%, 90%, 95%, or 97% homology thereto. In addition, if a polynucleotide sequence encoding a polypeptide having such homology and exhibiting substantially the same or equivalent potency as the polypeptide, a polynucleotide sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application. Self-explanatory Probes that can be prepared from known gene sequences, such as the amino acid of SEQ ID NO: 32 or SEQ ID NO: 33, are hydrided under stringent conditions with complementary sequences to all or part of the polynucleotide sequence, for example. Any sequence encoding a polypeptide having the same or similar activity as the polypeptide consisting of the sequence can be included without limitation. The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are described specifically in the literature (eg, J. Sambrook et al., Homology). For example, genes with high homology, 70% or more, 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more Genes having homologous hybridization and hybridization with less homologous genes or 60 ° C, 1 × SSC, 0.1% SDS, specifically 60 ° C, 0.1 The conditions of washing | cleaning once, specifically 2 to 3 times can be enumerated at the salt concentration and temperature corresponded to x SSC, 0.1% SDS, More specifically, 68 degreeC, 0.1 x SSC, 0.1% SDS. Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationship between bases of nucleotides that are hybridizable with each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present application may also include isolated nucleotide fragments that are complementary to the entire sequence as well as substantially similar nucleotide sequences. Specifically, polynucleotides having homology can be detected using hybridization conditions including hybridization steps at Tm values 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. Appropriate stringency to hybridize polynucleotides depends on the length and degree of complementarity of the polynucleotides and variables are well known in the art (see Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).

As used herein, the term “transketolase” refers to xylulose-5-phosphate and erythrose 4-phosphate of fructose-6-phosphate and glyceraldehyde-3-phosphate (E4P, erythorse-4-). phosphate) (Kochetov, GA 1982, Transketolase from yeast, rat liver, and pig liver, Methods Enzymol., 90: 209-23). Tryptophan production originates from the aromatic amino acid metabolic cycle, which begins with the condensation reaction of phosphoenolpyruvate with erythrose 4-phosphate. Therefore, smooth supply of two precursors is essential for improving tryptophan productivity, and it is intended to enhance the expression of the tkt gene for smooth supply of erythrose 4-phosphate as a precursor of tryptophan, which is known to be relatively insufficient in the present application.

If the transketorase of the present application has the above activity, those skilled in the art can obviously use the amino acid sequence having the activity regardless of the origin of the microorganism. Specifically, it may have a known amino acid sequence derived from Corynebacterium microorganisms or an amino acid sequence derived from Escherichia microorganisms, those skilled in the art can easily facilitate the amino acid sequence of transketorase through a database such as NCBI or Kegg Can be obtained. More specifically, the transketorase may be derived from Corynebacterium microorganisms, and even more specifically from Corynebacterium glutamicum.

As used herein, the term "enhanced / enhanced" a gene means a state in which the synthesis of the gene of interest into a functioning protein is increased compared to intrinsic or prior to modification. In the above, “intrinsic” refers to a state in which the parent strain originally had before transformation, when the transformation of microorganisms is caused by genetic variation caused by natural or artificial factors.

Specifically, the expression enhancement of the present application is to increase the intracellular copy number of the gene encoding the protein of interest, to introduce a mutation in the expression control sequence of the chromosomal gene encoding the protein, gene expression on the chromosome encoding the protein A method of replacing a regulatory sequence with a highly active sequence, a method of replacing a gene encoding the protein on a chromosome with a mutated gene to increase the protein activity, and a gene on the chromosome that encodes the protein to enhance the activity of the protein It may be made by any one or more methods selected from the group consisting of a method for introducing a mutation in. More specifically, the enhanced expression of the present application may be a substitution of a strong promoter for the promoter of the gene of interest.

The copy number increase of the gene in the above is not particularly limited, but may be performed in a form operably linked to a vector, or by inserting into a chromosome in a host cell. Specifically, a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the protein of the present application is operably linked, may be introduced into the host cell. Alternatively, a vector capable of inserting the polynucleotide into a chromosome in a host cell to which the polynucleotide is operably linked may be introduced into the chromosome of the host cell. Insertion of the polynucleotide into the chromosome can be by any method known in the art, for example by homologous recombination.

Next, modifying the expression control sequence to increase the expression of the polynucleotide is not particularly limited, but deletion, insertion, non-conservative or conservative substitution or their nucleic acid sequence to further enhance the activity of the expression control sequence. It can be carried out by inducing a variation in sequence in combination or by replacing with a nucleic acid sequence having stronger activity. The expression control sequence may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosomal binding site, a sequence for controlling termination of transcription and translation, and the like.

A strong promoter may be linked to the top of the polynucleotide expression unit instead of the original promoter, but is not limited thereto. Examples of known strong promoters include cj1 to cj7 promoters (Korean Patent No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, P _L promoter, tet promoter, gapA promoter, SPL7 Promoter, SPL13 (sm3) promoter (Korean Patent No. 10-1783170), O2 promoter (Korean Patent No. 10-1632642), tkt promoter and yccA promoter and the like, but is not limited thereto.

In addition, modification of the polynucleotide sequence on the chromosome is not particularly limited, but the mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the polynucleotide sequence. Or by replacing with a polynucleotide sequence that has been modified to have stronger activity.

As used herein, the term "introduction" of a gene refers to the activity of a specific protein as it is expressed in the microorganism, which is not originally possessed by the microorganism, or to enhanced activity compared to the intrinsic or pre-modification activity of the protein. It means to appear. For example, a polynucleotide encoding a specific protein may be introduced into a chromosome in a microorganism, or a vector containing a polynucleotide encoding a specific protein may be introduced into a microorganism to exhibit its activity.

The introduction and enrichment of such protein activity is generally at least 1%, 10%, 25%, 50%, based on the activity or concentration of the protein in the wild type or unmodified microbial strain. 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000% may be increased, but is not limited thereto.

As used herein, the term “microorganism that produces L-tryptophan” refers to a microorganism capable of producing an excess of L-tryptophan from a carbon source in the medium compared to wild-type or unmodified microorganisms. In addition, the microorganism producing L-tryptophan may be a recombinant microorganism. Specifically, if the L-tryptophan can be produced, the type is not particularly limited, but the genus Enterbacter, Escherichia, Erwinia, Serratia, Providencia ( Providencia), Corynebacterium genus and Brevibacterium genus microorganisms. More specifically, it may be a microorganism belonging to the genus Escherichia or Corynebacterium.

More specifically, the microorganism of the genus Corynebacterium may be corynebacterium glutamicum, but the expression of genes encoding tryptophan operon and transketorase is enhanced, resulting in L-tryptophan production. Microorganisms belonging to this growing Corynebacterium genus can be included without limitation.

Another aspect of the present application for achieving the above object is a gene encoding an ansranilic acid synthase with an N-terminus consisting of the amino acid sequence of SEQ ID NO: 32 and the feedback inhibition is released, and a tryptophan operon comprising the same; Culturing Corynebacterium genus microorganisms producing L-tryptophan, in which the expression of the gene encoding the transketorase is enhanced, in a medium; And recovering L-tryptophan from the cultured microorganism or medium.

Ansranilate synthase, tryptophan operon, transketolase, L-tryptophan, and microorganisms having the N-terminus consisting of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33 and whose feedback inhibition is released are as described above.

As used herein, the term "culture" means growing the microorganisms under appropriately controlled environmental conditions. Cultivation process of the present application may be made according to suitable media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the strain selected. Specifically, the culture may be batch, continuous and fed-batch, but is not limited thereto.

In the present application, the term "medium" means a substance mixed with nutrients necessary for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water, which is indispensable for survival and development. Specifically, the medium and other culture conditions used for the culture of the microorganism of the present application can be used without any particular limitation as long as the medium is used for the culture of ordinary microorganisms, the microorganism of the present application is suitable carbon source, nitrogen source, personnel, inorganic It can be cultured in a conventional medium containing a compound, an amino acid and / or vitamins, etc. under aerobic conditions, adjusting the temperature, pH and the like.

In the present application, the carbon source may include carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol, and the like; Alcohols such as sugar alcohols, glycerol, pyruvic acid, lactic acid, citric acid and the like; Amino acids such as organic acids, glutamic acid, methionine, lysine and the like. In addition, natural organic nutrients such as starch hydrolyzate, molasses, blackstrap molasses, rice winters, cassava, sugarcane residue and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (ie, reducing sugars). Carbohydrates, such as molasses), and other appropriate amounts of carbon sources can be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more thereof, but are not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or their degradation products, skim soy cakes or their degradation products Can be used. These nitrogen sources may be used alone or in combination of two or more thereof, but is not limited thereto.

The personnel may include a first potassium phosphate, a second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate and the like may be used, and other amino acids, vitamins and / or suitable precursors may be included. These components or precursors may be added batchwise or continuously to the medium. However, it is not limited to this.

In the present application, during the culturing of microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, and the like can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, in order to maintain the aerobic state of the medium, it is possible to inject oxygen or oxygen-containing gas into the medium or to inject nitrogen, hydrogen or carbon dioxide gas without the injection of gas to maintain the anaerobic and unaerobic state, It is not.

The temperature of the medium may be 20 ° C to 50 ° C, specifically 30 ° C to 37 ° C, but is not limited thereto. The incubation period may continue until the desired amount of useful material is obtained, specifically, may be 10 hours to 100 hours, but is not limited thereto.

The step of recovering tryptophan is to recover the desired L- tryptophan from the medium using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, batch, continuous or fed-batch culture method. Can be. For example, centrifugation, filtration, treatment with crystallized protein precipitation agent (salting method), extraction, ultrasonic crushing, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography Various chromatography such as HPLC, HPLC and the like can be used in combination, but is not limited to these examples.

The recovery step may comprise an additional purification process. The purification process may purify the recovered L-tryptophan using any suitable method known in the art.

The L-tryptophan producing microorganism provided in the present application can enhance the expression of the genes encoding the feedback-released tryptophan operon and transketorase to improve L-tryptophan production in Corynebacterium spp. have. In addition, the combination of the expression of E. coli-derived tryptophan operon in the Corynebacterium genus microorganisms can significantly improve the production of L-tryptophan. Thus, it can be useful industrially, it is possible to provide a Corynebacterium genus microorganisms to enhance the fermentation stability to effectively produce L- tryptophan.

Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

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

Example 1 Trp Operon Expression Enhancement and Feedback-resistant trpE Mutation Application

Since wild-type Corynebacterium glutamicum does not produce L-tryptophan or produces only a very small amount, the biosynthetic pathway necessary for production was tried to be strengthened in wild-type Corynebacterium glutamicum.

In Corynebacterium glutamicum ATCC13869, the operon of the L-tryptophan biosynthesis gene was enhanced through promoter enrichment. In addition, serine, amino acid No. 38 from the N-terminus of TrpE was replaced with Arginine in order to alleviate feedback inhibition of TrpE polypeptide due to improved L-tryptophan production (SEQ ID NO: 32) ( JOURNAL OF BACTERIOLOGY, Nov. 1987, p. 5330-5332).

For this genetic manipulation, the trpE promoter Upstream and trpE 38 Downsteam regions where chromosomal homologous recombination occurs were obtained. Specifically, the trpE promoter upstream region using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, and trpE using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 Gene fragments in the Downsteam region of mutation 38 were obtained by PCR.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 sec denaturation, 60 ° C. 30 sec annealing, and 72 ° C. 60 sec polymerization 30 times. The polymerization was carried out at 72 ° C. for 5 minutes.

PCR was performed using primers of SEQ ID NO: 6 and SEQ ID NO: 7 as a template using a synthetically produced promoter SPL7 (SEQ ID NO: 5, Korean Patent No. 10-1783170).

SEQ ID NO: 5 (SPL7 promoter seq.)

GGCGCTTCATGTCAACAATCTTTAACGTTTTCAAGTTCACAAGTCGTGTTCAAATGGTGACAAGATTGGACACTGTGCTGAATTGGCACCAAGCCCTCATAAATGATAGATCTAAATCGAATATCAATATATGGTCTGTTTATTGGAACGCGTCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGTGCAGAAAGAACAAATAATCGTGAATTTTGGCAGCAACAGCAATTCCTGCTACAATTGAAAACGTGCAAAAGCATAGATTATTGGAGGAGATCAAAACA

As polymerase, Solg ^TM Pfu-X DNA polymerase (SolGent co.) Was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 seconds denaturation, 62 ° C. 30 seconds annealing, and 72 ° C. 30 seconds polymerization. After repeating 30 times, the polymerization was performed at 72 ° C. for 5 minutes.

PCR was performed using primers of SEQ ID NO: 8 and SEQ ID NO: 9 using Corynebacterium glutamicum genomic DNA as a template to obtain fragments before trpE 38 from Corynebacterium glutamicum. .

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 sec. Denaturation, 62 ° C. 30 sec. Annealing, and 72 ° C. 30 sec. Polymerization 30 times. The polymerization was carried out at 72 ° C. for 5 minutes.

The amplified trpE promoter upstream and trpE 38 mutation downstream region, the SPL7 promoter and trpE 38 frontal fragment, and the chromosome transformation vector pDZ (patented 10-0924065) cleaved with SmaI restriction enzyme were obtained from the Gibson assembly ( Recombinant plasmids were obtained by cloning using DG Gibson et al., NATURE METHODS, VOL. 6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method and named pDZ-PSPL7-trpE (S38R). Cloning was performed by mixing Gibson assembly reagent and each of the gene fragments in the calculated moles and then preserving at 50 ° C. for 1 hour.

The pDZ-PSPL7-trpE (S38R) vector was transformed into wild-type Corynebacterium glutamicum ATCC13869 strain by electroporation, followed by a second crossover process, and a promoter of the trp operon on the chromosome. And the amino acid No. 38 of TrpE was replaced with serine to arginine (SEQ ID NO: 32). This genetic manipulation was confirmed by PCR and genome sequencing using SEQ ID NO: 10 and SEQ ID NO: 11, which can amplify the external regions of the homologous recombination upstream and downstream regions where the gene was inserted, and named CA04-8325. It was.

Additionally, to introduce only the trpE S38R variant, the trpE (S38R) upstream region was identified using primers of SEQ ID NO: 1 and SEQ ID NO: 3 with the Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template. Using the primer of SEQ ID NO: 4, a gene fragment of the Downsteam region of the trpE 38 mutation was obtained through PCR.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 sec denaturation, 60 ° C. 30 sec annealing, and 72 ° C. 60 sec polymerization 30 times. The polymerization was carried out at 72 ° C. for 5 minutes.

Recombinant plasmids were obtained by cloning the trpE (S38R) upstream and trpE (S38R) downstream fragments amplified by the above method, and the vector pDZ for chromosomal transformation cleaved with SmaI restriction enzyme using the Gibson assembly method, and the pDZ- Named trpE (S38R). Cloning was performed by mixing Gibson assembly reagent and each of the gene fragments in the calculated moles and then preserving at 50 ° C. for 1 hour.

PDZ-trpE (S38R) vector was transformed into wild-type Corynebacterium glutamicum ATCC13869 strain by electroporation, followed by a crossover process, and amino acid 38 of TrpE was identified on serine on the chromosome. Strains were replaced with those obtained. The genetic manipulation was confirmed by PCR and genome sequencing using SEQ ID NO: 10 and SEQ ID NO: 11, which can amplify the external regions of the homologous upstream and downstream regions where the gene was inserted, and named CA04-8312. It was.

 In order to replace the trp operon promoter with the stronger promoter, the SPL7 promoter, the trpE promoter upstream region was identified using primers of SEQ ID NO: 1 and SEQ ID NO: 2 using the Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template. Using the primers of SEQ ID NO: 8 and SEQ ID NO: 4, gene fragments of the trpE promoter downstream (Downsteam) region were obtained through PCR.

PCR was performed using primers of SEQ ID NO: 6 and SEQ ID NO: 7 using the synthetic promoter SPL7 as a template.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 sec denaturation, 60 ° C. 30 sec annealing, and 72 ° C. 60 sec polymerization 30 times. The polymerization was carried out at 72 ° C. for 5 minutes.

Recombinant plasmids were obtained by cloning the trpE promoter amplified by the above method, the trpE promoter downstream and the SPL7 promoter fragment, and the chromosome transformation vector pDZ digested with SmaI restriction enzyme using the Gibson assembly method. PDZ-SPL7_trpE Named it. Cloning was performed by mixing Gibson assembly reagent and each of the gene fragments in the calculated moles and then preserving at 50 ° C. for 1 hour.

The pDZ-SPL7_trpE vector prepared by the above process was transformed into wild-type Corynebacterium glutamicum ATCC13869 strain by electroporation, and then replaced by the SPL7 promoter, which is a trp operon strong promoter on the chromosome, through a second crossover process. Got. This genetic manipulation was confirmed by PCR and genome sequencing using SEQ ID NO: 10 and SEQ ID NO: 11, which can amplify the external regions of the homologous upstream and downstream regions where the gene was inserted, and named CA04-8315. It was.

The sequence of the primer used in this Example is as follows.

SEQ ID NO: 1 (Pspl7-trpE (S38R) _L-1) TCGAGCTCGGTACCCAAACAACTGCGACGTGTGTC SEQ ID NO: 2 (Pspl7-trpE (S38R) _L-2) CATGAAGCGCCGGTACCTTAATCATTTTTGGGTTC SEQ ID NO: 3 (Pspl7-trpE (S38R) _R-1) GCCCTGTTGGAACGCGCTGATATCACCACCAAGAA SEQ ID NO: 4 (Pspl7-trpE (S38R) _R-2) CTCTAGAGGATCCCCAGATGTCACCGTTGTAAATG SEQ ID NO: 6 (Pspl7-1) CCCAAAAATGATTAAGGTACCGGCGCTTCATGTCA SEQ ID NO: 7 (Pspl7-2) GGGATTCGTGCTCATGATATCTGTTTTGATCTCCTCC SEQ ID NO: 8 (trpE (S38R)-1) ATCAAAACAGATATCATGAGCACGAATCCCCATGT SEQ ID NO: 9
(trpE (S38R)-2) GTGGTGATATCAGCGCGTTCCAACAGGGCTGCATC SEQ ID NO: 10
(Confirm_Pspl7-trpE (S38R)-1) GAAGAAGAGGCTGCAGATG SEQ ID NO: 11 (Confirm_Pspl7-trpE (S38R)-2) GATCAGCGCCATCATGTT

Example 2 tkt Gene Expression Enhancement (Transketolase Expression Enhancement)

In order to smoothly supply erythrose 4-phosphate as a precursor of tryptophan, overexpression of the tkt gene was attempted.

Corynebacterium glutamicum ATCC13869 chromosomal DNA was used as a template for the genetic manipulation, and the upstream region to additionally insert the tkt gene was identified using SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 Gene fragments in the Downsteam region where additional tkt genes were inserted were obtained by performing PCR.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 2 minutes, 95 ° C. 20 seconds denaturation, 62 ° C. 40 seconds annealing, 72 ° C. 30 seconds polymerization, and repeated 27 times. The polymerization was carried out at 72 ° C. for 5 minutes.

In addition, in order to secure the tkt gene and the promoter, PCR was performed on the tkt gene fragment including the tkt promoter using SEQ ID NO: 16 and SEQ ID NO: 17 using chromosomal DNA of Corynebacterium glutamicum ATCC of wild species as a template. Obtained through.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were repeated for 2 minutes at 95 ° C., followed by 95 ° C. 20 seconds denaturation, 62 ° C. 40 seconds annealing, and 72 ° C. 1 minute 20 seconds polymerization. After that, the polymerization was carried out at 72 ° C. for 5 minutes.

The upstream to which the amplified tkt gene is to be inserted and the downstream region to which the tkt gene is to be inserted, the tkt gene including the tkt promoter, and the chromosome transformation vector pDZ cleaved with SmaI restriction enzyme were cloned using the Gibson assembly method. Recombinant plasmids were obtained and named pDZ-Pn-tkt. Cloning was performed by mixing Gibson assembly reagent and each of the gene fragments in the calculated moles and then preserving at 50 ° C. for 1 hour.

The produced pDZ-Pn-tkt vector was transformed into ATCC13869 and CJ04-8325 strains by electroporation, and a second crossover process was performed to obtain a strain in which a tkt gene including a tkt promoter was inserted on a chromosome. The genetic manipulations were confirmed by PCR and genome sequencing using the SEQ ID NO: 18 and SEQ ID NO: 19 primers to amplify the external regions of the upstream and downstream regions of the homologous recombination where the gene was inserted. 8321 and CA04-8352.

The sequence of the primer used in this Example is as follows.

SEQ ID NO: 12
(Pn-tkt_L-1) TCGAGCTCGGTACCCAAACTTTGAGTGGGTGCGTG SEQ ID NO: 13
(Pn-tkt_L-2) TCGAGCTACGAGGGCGGTTCCCAGCCCTTCATTAG SEQ ID NO: 14 (Pn-tkt_R-1) ATTAACGGTTAATTGATTCTGGACGTCATGACTAC SEQ ID NO: 15 (Pn-tkt_R-2) CTCTAGAGGATCCCCGCCTCGATGATGCAGTCGTC SEQ ID NO: 16 (Pn-tkt-1) GAAGGGCTGGGAACCGCCCTCGTAGCTCGAGAGTT SEQ ID NO: 17 (Pn-tkt-2) CATGACGTCCAGAATCAATTAACCGTTAATGGAGTCC SEQ ID NO: 18 (Confirm_Pn-tkt-1) ACCCAGAACCCCAAATTTTC
SEQ ID NO: 19
(Confirm_Pn-tkt-2) TTGAGTTCGACAACTTTGG

Example 3: Tryptophan Production of Corynebacterium Microorganisms

In order to check the tryptophan production of the strains produced in Examples 1 and 2 were cultured in the following manner. Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and shaken at 200 rpm for 20 hours at 30 ° C. Thereafter, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 25 ml of production medium and shake-cultured at 200 rpm for 24 hours. After the incubation, the production amount of L-tryptophan was measured by HPLC.

Species Medium (pH 7.0)

20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1.5 g of urea, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 2000 μg per liter of distilled water

Production Medium (pH 7.0)

Glucose 30 g, (NH ₄ ) ₂ SO ₄ 15 g, MgSO ₄ 7H ₂ O 1.2 g, KH ₂ PO ₄ 1 g, yeast extract 5 g, biotin 900 μg, thiamine hydrochloride 4500 μg, calcium-pantothenic acid 4500 μg, CaCO ₃ 30 g (based on 1 liter of distilled water).

Production of L-Tryptophan Producing Strains from Corynebacterium glutamicum characteristics Glucose Used (g / L) Tryptophan
Production rate (g / L) Tryptophan yield
(* 100 g / g,%) ATCC13869 30 0.00 0.00 CA04-8312 trpE :: trpE (S38R) 30 0.00 0.00 CA04-8315 Pn :: SPL7_trpE 30 0.00 0.00 CA04-8321 :: Pn_tkt 30 0.00 0.00 CA04-8325 Pn :: SPL7_trpE (S38R) 30 0.05 0.16 CA04-8352 SPL7_trpE (S38R)
, :: Pn_tkt 30 0.27 0.90

The results for L-tryptophan production in culture for wild type Corynebacterium, CA04-8312, CA04-8315, CA04-8321, CA04-8325, CA04-8352 are shown in Table 3 above.

Tryptophan production was not confirmed in CA04-8312, a strain-releasing feedback mutation in trpE of wild-type Corynebacterium, CA04-8315 enhanced in tryptophan operon, and CA04-8321 intact-enhanced tkt. However, the CA04-8352 strain, which combines these individual traits, produced about five times improved tryptophan compared to CA04-8325. This means that tryptophan operon enrichment with release of feedback to tryptophan and tkt expression enhancement should be combined to improve tryptophan production. The wild type Corynebacterium microorganisms produce only a very small amount of tryptophan, even if they do not produce tryptophan.

Example 4: Enhancing E. coli-derived tryptophan operon expression

In order to further improve the production of tryptophan, an attempt was made to introduce and enhance expression of additional E. coli-derived tryptophan operon. In order to enhance E. coli-derived tryptophan operon, trpE (SEQ ID NO: 33) and E. coli substituted serine with proline, amino acid 21 of trpE, which eliminated the feedback restriction of the SPL7 promoter, known as a strong promoter, and the E. coli TrpE protein. Enhancement proceeded in the form of trpDCBA (J. Biochem. Mol. Biol. 32, 20-24 (1999)).

For this genetic manipulation, the promoter upstream and trpDCBA Downsteam regions where chromosomal homologous recombination takes place were first obtained. Specifically, using the primers of SEQ ID NO: 20 and SEQ ID NO: 21 using Corynebacterium glutamicum chromosomal DNA as a template, the promoter upstream region was downstream, and the primers of SEQ ID NO: 22 and SEQ ID NO: 23 were downstream ( Gene fragments from the Downsteam region were obtained by PCR.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 sec. Denaturation, 58 ° C. 30 sec. Annealing, and 72 ° C. 60 sec. Polymerization 30 times. The polymerization was carried out at 72 ° C. for 5 minutes.

PCR was performed using primers of SEQ ID NO: 24 and SEQ ID NO: 25 using the synthetically produced promoter SPL7 as a template.

Polymerase was used Solg ^TM Pfu-X DNA polymerase (SolGent co.), PCR amplification conditions were denatured at 95 ℃ for 5 minutes, 95 ℃ 30 seconds denaturation, 58 ℃ 30 seconds annealing, 72 ℃ 60 seconds polymerization After repeating 30 times, the polymerization was performed at 72 ° C. for 5 minutes.

In order to secure nucleotides encoding amino acids 1 to variant 21 of the E. coli trpE gene, PCR was performed using SEQ ID NO: 26 and SEQ ID NO: 27 using E. coli W3110 genomic DNA as a template.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 sec. Denaturation, 58 ° C. 30 sec. Annealing, and 72 ° C. 30 sec. The polymerization was carried out at 72 ° C. for 5 minutes.

In order to secure nucleotides encoding mutant amino acids 21 of the E. coli trpE gene to trpE and trpDCBA including the terminal, PCR was performed using E. coli W3110 genomic DNA as a template.

As polymerase, Solg ^TM Pfu-X DNA polymerase was used, and PCR amplification conditions were denatured at 95 ° C. for 5 minutes, 95 ° C. 30 sec. Denaturation, 58 ° C. 30 sec. Annealing, and 72 ° C. 7 min polymerization 30 times. The polymerization was carried out at 72 ° C. for 10 minutes.

To generate chromosomal homologous recombination, the amplified upstream and downstream regions, the SPL7 promoter, the E. coli trpE P21S front fragment, the terminal and trpDCBA genes containing E. coli trpE P21S, and the chromosomal transformation vector pDZ cleaved with SmaI restriction enzyme Recombinant plasmids were obtained by cloning using the Gibson assembly method and named pDZ-PSPL7-trpE (P21S) DCBA.eco. Cloning was performed by mixing Gibson assembly reagent and each of the gene fragments in the calculated moles and then preserving at 50 ° C. for 1 hour.

The produced pDZ-PSPL7-trpE (P21S) DCBA.eco vector was transformed into wild-type Corynebacterium glutamicum ATCC13869, CJ04-8321 and CJ04-8352 strains by electroporation, followed by a second crossover process, followed by chromosome A strain in which the E. coli trpE (P21S) DCBA operon was inserted in the form of SPL7, a strong promoter, was obtained. The genetic manipulations were confirmed by PCR and genome sequencing using the SEQ ID NO: 30 and SEQ ID NO: 31 primers, which can amplify the external regions of the upstream and downstream regions of the homologous recombination where the gene was inserted. 8316, CA04-8330 and CA04-8357.

The sequence of the primer used in this Example is as follows.

SEQ ID NO: 20 (TY384) ttcgagctcggtacccGATCGAGGCTAACAAGGCA SEQ ID NO: 21 (HR-sp R) GGTACCACTAAACCGGAAGGGCCCTCCTGCTGTACTTTCGACA SEQ ID NO: 22 (trpA-HR F) AGTTAAACTAAACAGGAAGAGCCAGTTAAGGGAC SEQ ID NO: 23 (HR-vector R) gactctagaggatccccATCATGGGAATCCGGCCAT SEQ ID NO: 24 (HR-sp F) GGCCCTTCCGGTTTAGTGGTACCGGCGCTTCATGTCAACAATC SEQ ID NO: 25 (spl7-trpE R) TTTGCATGATATCTGTTTTGATCTCCTCCAATA SEQ ID NO: 26 (spl7-trpE F) CAAAACAGATATCATGCAAACACAAAAACCGAC SEQ ID NO: 27 (trpE P21S R) GAAAAAGCGCGGTGGAATTGTCGCGATAAGCGC SEQ ID NO: 28 (trpE P21S F) CAAAACAGATATCATGCAAACACAAAAACCGAC SEQ ID NO: 29 (trpA-HR R) TCTTCCTGTTTAGTTTAACTGCGCGTCGCCGCTT SEQ ID NO: 30 (Nested 1F) CGTTGACCCAAACATGCTG SEQ ID NO: 31 (Nested 1R) CTTCTTCATTTCGGCTATCG

Example 5 Evaluation of E. coli Tryptophan Operon Enhanced Strains

In order to determine the tryptophan production of the CA04-8316, CA04-8330 and CA04-8357 strains prepared in Example 4 and the CA04-8352 strain prepared in Example 2 was cultured in the following manner. Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and shaken at 200 rpm for 20 hours at 30 ° C. Thereafter, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 25 ml of production medium and shake-cultured at 200 rpm for 24 hours. After the incubation, the production amount of L-tryptophan was measured by HPLC.

Species Medium (pH 7.0)

20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1.5 g of urea, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 2000 μg per liter of distilled water

Production Medium (pH 7.0)

Glucose 30 g, (NH ₄ ) ₂ SO ₄ 15 g, MgSO ₄ 7H ₂ O 1.2 g, KH ₂ PO ₄ 1 g, yeast extract 5 g, biotin 900 μg, thiamine hydrochloride 4500 μg, calcium-pantothenic acid 4500 μg, CaCO ₃ 30 g (based on 1 liter of distilled water).

Tryptophan Production in E. coli Tryptophan Operon-Enhanced Strains characteristics Glucose Used (g / L) Tryptophan
Production rate (g / L) Tryptophan yield
(* 100 g / g,%) ATCC13869 30 0.00 0.00 CA04-8316 :: SPL7_trpE (P21S) DCBA.eco 30 0.03 0.10 CA04-8330 :: Pn_tkt,
:: SPL7_trpE (P21S) DCBA.eco 30 0.32 1.06 CA04-8352 SPL7_trpE (S38R)
, :: Pn_tkt 30 0.27 0.90 CA04-8357 SPL7_trpE (S38R)
, :: Pn_tkt,
:: SPL7_trpE (P21S) DCBA.eco 30 2.72 9.06

The results for L-tryptophan production in cultures for wild type Corynebacterium, CA04-8316, CA04-8330, CA04-8352, CA04-8357 are shown in Table 5 above.

Tryptophan operon expression-enhanced CA04-8316 strain containing trpE released from Escherichia coli in wild-type Corynebacterium produced L-tryptophan at 0.03 g / L, and CA04-8321 enhanced only tkt. The yield of tryptophan of the CA04-8330 strain enhanced with the tryptophan operon from which the E. coli-derived feedback was released was improved to 0.32 g / L. In the case of CA04-8357, which combines all the genetic modifications, tryptophan production was expected to be about 0.3-0.6 g / L, but it produced 2.72 g / L, which is about 10 times better than CA04-8352.

From the above results, it was confirmed that when E. coli-derived tryptophan operon combined with the parent strain Corynebacterium type TKT (transsketolase) and feedback releasing tryptophan operon fortification were combined, the yield was significantly increased. That is, it can be seen that synergistic effects on tryptophan production are large when the feedback-released tryptophan operon derived from E. coli and the feedback-released tryptophan operon derived from corynebacterium are combined with the precursor enhancement.

The strain, CA04-8352, was deposited with KCCM12218P on February 2, 2018 in an international deposit with the Korea Microorganism Conservation Center (KCCM), an international depository institution under the Budapest Treaty.

From the above description, those skilled in the art will appreciate that the present application can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present application should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present application.

Korea Microbial Conservation Center (overseas) KCCM12218P 20180202

<110> CJ CheilJedang Corporation <120> Tryptophan operon-enhanced microorganism and method for producing          L-tryptophan using the same <130> KPA180110-KR <160> 48 <170> KoPatentIn 3.0 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pspl7-trpE (S38R) _L-1 <400> 1 tcgagctcgg tacccaaaca actgcgacgt gtgtc 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pspl7-trpE (S38R) _L-2 <400> 2 catgaagcgc cggtacctta atcatttttg ggttc 35 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> Pspl7-trpE (S38R) _R-1 <400> 3 gccctgttgg aacgcgctga tatcaccacc aagaa 35 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pspl7-trpE (S38R) _R-2 <400> 4 ctctagagga tccccagatg tcaccgttgt aaatg 35 <210> 5 <211> 294 <212> DNA <213> Artificial Sequence <220> 223 spl7 promoter seq. <400> 5 ggcgcttcat gtcaacaatc tttaacgttt tcaagttcac aagtcgtgtt caaatggtga 60 caagattgga cactgtgctg aattggcacc aagccctcat aaatgataga tctaaatcga 120 atatcaatat atggtctgtt tattggaacg cgtcccagtg gctgagacgc atccgctaaa 180 gccccaggaa ccctgtgcag aaagaacaaa taatcgtgaa ttttggcagc aacagcaatt 240 cctgctacaa ttgaaaacgt gcaaaagcat agattattgg aggagatcaa aaca 294 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pspl7-1 <400> 6 cccaaaaatg attaaggtac cggcgcttca tgtca 35 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Pspl7-2 <400> 7 gggattcgtg ctcatgatat ctgttttgat ctcctcc 37 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> trpE (S38R)-1 <400> 8 atcaaaacag atatcatgag cacgaatccc catgt 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> trpE (S38R)-2 <400> 9 gtggtgatat cagcgcgttc caacagggct gcatc 35 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Confirm_Pspl7-trpE (S38R)-1 <400> 10 gaagaagagg ctgcagatg 19 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Confirm_Pspl7-trpE (S38R)-2 <400> 11 gatcagcgcc atcatgtt 18 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pn-tkt_L-1 <400> 12 tcgagctcgg tacccaaact ttgagtgggt gcgtg 35 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pn-tkt_L-2 <400> 13 tcgagctacg agggcggttc ccagcccttc attag 35 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pn-tkt_R-1 <400> 14 attaacggtt aattgattct ggacgtcatg actac 35 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pn-tkt_R-2 <400> 15 ctctagagga tccccgcctc gatgatgcag tcgtc 35 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pn-tkt-1 <400> 16 gaagggctgg gaaccgccct cgtagctcga gagtt 35 <210> 17 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Pn-tkt-2 <400> 17 catgacgtcc agaatcaatt aaccgttaat ggagtcc 37 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Confirm_Pn-tkt-1 <400> 18 acccagaacc ccaaattttc 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Confirm_Pn-tkt-2 <400> 19 ttgagttcga caactttgg 19 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TY384 <400> 20 ttcgagctcg gtacccgatc gaggctaaca aggca 35 <210> 21 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> HR-sp R <400> 21 ggtaccacta aaccggaagg gccctcctgc tgtactttcg aca 43 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> trpA-HR F <400> 22 caaaacagat atcatgcaaa cacaaaaacc gac 33 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HR-vector R <400> 23 gactctagag gatccccatc atgggaatcc ggccat 36 <210> 24 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> HR-sp F <400> 24 ggcccttccg gtttagtggt accggcgctt catgtcaaca atc 43 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> spl7-trpE R <400> 25 tttgcatgat atctgttttg atctcctcca ata 33 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> spl7-trpE F <400> 26 caaaacagat atcatgcaaa cacaaaaacc gac 33 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> trpE P21S R <400> 27 gaaaaagcgc ggtggaattg tcgcgataag cgc 33 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> trpE P21S F <400> 28 caaaacagat atcatgcaaa cacaaaaacc gac 33 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> trpA-HR R <400> 29 tcttcctgtt tagtttaact gcgcgtcgcc gctt 34 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nested 1F <400> 30 cgttgaccca aacatgctg 19 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nested 1R <400> 31 cttcttcatt tcggctatcg 20 <210> 32 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> cgl trpE S38R <400> 32 Met Ser Thr Asn Pro His Val Phe Ser Leu Asp Val Arg Tyr His Glu   1 5 10 15 Asp Ala Ser Ala Leu Phe Ala His Leu Gly Gly Thr Thr Ala Asp Asp              20 25 30 Ala Ala Leu Leu Glu Arg Ala Asp          35 40 <210> 33 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> eco trpE P21S <400> 33 Met Gln Thr Gln Lys Pro Thr Leu Glu Leu Leu Thr Cys Glu Gly Ala   1 5 10 15 Tyr Arg Asp Asn Ser Thr Ala Leu Phe His Gln Leu Cys Gly Asp Arg              20 25 30 Pro Ala Thr Leu Leu Leu Glu Ser          35 40 <210> 34 <211> 6770 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of tryptophan operon-Corynebacterium glutamicum <400> 34 atgagcacga atccccatgt tttctcccta gatgtccgct atcacgagga tgcttctgcg 60 ttgtttgccc acttgggtgg cacaaccgca gatgatgcag ccctgttgga acgcgctgat 120 atcaccacca agaatggtat ttcttccctc gcggtgttga agagttcggt gcgcattacg 180 tgcacgggca acacggtggt aacgcagccg ctgacggact cgggtagggc agtggttgcg 240 cgcctaacac agcagcttgg ccagtacaac accgcagaga acacctttag cttccccgcc 300 tccgatgcgg ttgatgagcg cgagcgcctc accgcaccaa gcaccatcga agtgctgcgc 360 aagttgcagt tcgagtccgg ctacagcgac gcgtccctgc cactgctcat gggcggtttc 420 gcctttgatt tcttagaaac ctttgaaacg ctccccgcag tcgaggaaag cgtcaacact 480 taccccgatt accagttcgt cctcgcggaa atcgtcctgg acatcaatca ccaggaccag 540 accgccaaac tcaccggcgt ctccaacgcc ccaggcgagc tcgaggccga gctcaacaag 600 ctttcattgc ttatcgacgc cgccctcccc gcaaccgaac acgcctacca aaccacccct 660 cacgacggcg acactcttcg cgttgtggct gatattcccg atgctcagtt ccgcactcag 720 atcaatgagc tgaaagaaaa catttacaac ggtgacatct accaagttgt cccggcgcgc 780 actttcaccg caccatgtcc tgatgcattc gctgcttatc tgcagctgcg tgccaccaac 840 ccgtcgccgt acatgttcta tatccgtgga ctcaacgaag gtcgctccta tgaacttttt 900 ggcgcatccc ctgagtccaa cctcaagttc accgctgcta accgtgagct gcagctgtac 960 ccaatcgcag gtacccgccc ccgtggactc aacccagatg gctccatcaa cgatgagcta 1020 gatatccgca atgagttgga tatgcgcact gatgccaaag agatcgcgga gcacaccatg 1080 cttgtcgatc tcgcccgcaa cgacctcgcc cgcgtctcgg tcccagcgtc gcgccgggtt 1140 gcggatcttt tgcaggtgga tcgctattcc cgcgtgatgc acttggtgtc ccgtgtgacg 1200 gcgacgttgg acccagagct tgatgctttg gacgcctatc gggcgtgcat gaatatgggc 1260 acgttgaccg gcgctccgaa gttgcgcgct atggagctgt tgcgcggcgt cgaaaagcgc 1320 aggcgtggtt cttatggtgg ggcagtgggg tacctgcgcg gcaatggcga tatggataat 1380 tgcattgtta ttcgttcggc gtttgtccag gatggtgtgg ctgctgtgca ggctggtgct 1440 ggtgtggtcc gcgattctaa tcctcaatct gaagccgatg agacgttgca caaggcgtat 1500 gccgtgttga atgccattgc gcttgctgct ggttccactt tggaggtcat ccgatgacac 1560 acgttgttct cattgataat cacgattctt ttgtctacaa cctggtggat gcgttcgccg 1620 tggccggtta taagtgcacg gtgttccgca atacggtgcc agttgaaacc attttggcag 1680 ccaacccgga cctgatctgc ctttcacctg gacctggtta ccctgccgat gcgggcaaca 1740 tgatggcgct gatcgagcgc acactcggcc agattccttt actgggtatt tgcctcggct 1800 accaggcact catcgaatac cacggcggca aggttgagcc ttgtggccct gtgcacggca 1860 ccaccgacaa catgatcctt actgatgcag gtgtgcagag ccctgttttt gcaggtcttg 1920 ccactgatgt tgagcctgat catccagaag tcccaggccg caaggttcca attggccgtt 1980 atcactcact gggctgcgtg gttgccccag acggtattga atcattgggc acctgttcct 2040 ctgagattgg tgatgtcatc atggcggcac gcaccaccga tggaaaggcc attggcctgc 2100 agtttcaccc tgagtcagtg ctgagcccaa cgggtcctat cattttgtcc cgctgtgtcg 2160 aacaacttct cgcgaactaa taaaaaggat ttgattcatg acttctccag caacactgaa 2220 agttctcaac gcctacttgg ataaccccac tccaaccctg gaggaggcaa ttgaggtgtt 2280 caccccgctg accgtgggtg aatacgatga cgtgcacatc gcagcgctgc ttgcgaccat 2340 ccgtactcgc ggtgagcagt tcgctgatat tgccggcgct gccaaggcat tcctcgcggc 2400 ggctcgtccg ttcccgatta ctggcgcagg tttgctagat tccgctggca ctggtggcga 2460 cggtgccaac accatcaaca tcaccaccgg cgcatccctg atcgcagcat ccggtggagt 2520 gaagctggtt aagcacggca accgttcggt gagctccaag tccggctccg ccgatgtgct 2580 ggaagcgctg aatattcctt tgggccttga tgtggatcgt gctgtgaagt ggttcgaagc 2640 gtccaacttc accttcctgt tcgcacctgc gtacaaccct gaaattgcgc atgtgcagcc 2700 ggttcgccag gcgctgaaat tccccaccat cttcaacacg cttggaccat tgctgtcccc 2760 ggcgcgcccg gagcgtcaga tcatgggcgt ggccaatgcc aatcatggac agctcatcgc 2820 cgaggtcttc cgcgagttgg gccgtacacg cgcgcttgtt gtgcatggcg caggcaccga 2880 tgagatcgca gtccacggca ccaccttggt gtgggagctt aaagaagacg gcaccatcga 2940 gcattacacc atcgagcctg aggaccttgg ccttggccgc tacacccttg aggatctcgt 3000 gggtggcctc ggcactgaga acgccgaagc tatgcgcgct actttcgcgg gcaccggccc 3060 tgatgcacac cgtgatgcgt tggctgcgtc cgcaggtgcg atgttctatc tcaacggcga 3120 tgtcgactcc ttgaaggatg gtgcacaaaa ggcgctttcc ttgcttgccg acggcaccac 3180 ccaggcatgg ttggccaagc acgaagagat cgattactca gaaaaggagt cttccaatga 3240 ctagtaataa tctgcccacg gtgttggaaa gcatcgtgga gggtcgtcgc ggacacctgg 3300 aggaaattcg cgctcgcatc gctcacgtgg atgtggatgc gcttccaaaa tccacccgct 3360 ctctgttcga ttccctcaac cagggtaggg gaggggcgcg tttcatcatg gagtgcaagt 3420 ccgcatcgcc ttctttggga atgattcgtg agcactacca gccgggtgaa atcgctcgcg 3480 tgtactctcg ctacgccagc ggcatttccg tgctgtgcga gccggatcgt tttggtggcg 3540 attacgatca cctcgctacc gttgccgcta cctctcatct tccggtgctg tgcaaagact 3600 tcatcattga tcctgtccag gtacacgcgg cgcgttactt tggtgctgat gccatcctgc 3660 tcatgctctc tgtgcttgat gatgaagagt acgcagcact cgctgccgag gctgcgcgtt 3720 ttgatctgga tatcctcacc gaggttattg atgaggagga agtcgcccgc gccatcaagc 3780 tgggtgcgaa gatctttggc gtcaaccacc gcaacctgca tgatctgtcc attgatttgg 3840 atcgttcacg tcgcctgtcc aagctcattc cagcagatgc cgtgctcgtg tctgagtctg 3900 gcgtgcgcga taccgaaacc gtccgccagc taggtgggca ctccaatgca ttcctcgttg 3960 gctcccagct gaccagccag gaaaacgtcg atctggcagc ccgcgaattg gtctacggcc 4020 ccaacaaagt ctgcggactc acctcaccaa gtgcagcaca aaccgctcgc gcagcgggtg 4080 cggtctacgg cgggctcatc ttcgaagagg catcgccacg taatgtttca cgtgaaacat 4140 cgcaaaaaat catcgccgca gagcccaacc tgcgctacgt cgcggtcagc cgtcgcacct 4200 ccgggtacaa ggatttgctt gtcgacggca tcttcgccgt acaaatccac gccccactgc 4260 agggcagcgt cgaagcagaa aaggcattga tcgccgccgt tcgtgaagag gttggaccgc 4320 aggtccaggt ctggcgcgcg atctcgatgt ccagcccctt gggggctgaa gtggcagctg 4380 cggtggaggg tgacgtcgat aagctaattc ttgatgccca tgaaggtggc agcggggaag 4440 tattcgactg ggctacggtg ccggccgctg tgaaggcaaa gtctttgctc gcgggaggca 4500 tctctccgga caacgctgcg caggcactcg ctgtgggctg cgcaggttta gacatcaact 4560 ctggcgtgga ataccccgcc ggtgcaggca cgtgggctgg ggcgaaagat gccggcgcgc 4620 tgctgaaaat tttcgcgacc atctccacat tccattacta aaggtttaaa taggatcatg 4680 actgaaaaag aaaacttggg cggctccacg ctgctacctg catacttcgg tgaattcggc 4740 ggccagttcg tcgcggaatc cctcctgcct gctctcgacc agctggagaa ggccttcgtt 4800 gacgcgacca acagcccaga gttccgcgaa gaactcggcg gctacctccg cgattatctc 4860 ggccgcccaa ccccgctgac cgaatgctcc aacctgccac tcgcaggcga aggcaaaggc 4920 tttgcgcgga tcttcctcaa gcgcgaagac ctcgtccacg gcggtgcaca caaaactaac 4980 caggtgatcg gccaggtgct gcttgccaag cgcatgggca aaacccgcat catcgcagag 5040 accggcgcag gccagcacgg caccgccacc gctctcgcat gtgcgctcat gggcctcgag 5100 tgcgttgtct acatgggcgc caaggacgtt gcccgccagc agcccaacgt ctaccgcatg 5160 cagctgcacg gcgcgaaggt catccccgtg gaatctggtt ccggcaccct gaaggacgcc 5220 gtgaatgaag cgctgcgcga ttggaccgca accttccacg agtcccacta ccttctcggc 5280 accgccgccg gcccgcaccc attcccaacc atcgtgcgtg aattccacaa ggtgatctct 5340 gaggaagcca aggcacagat gctagagcgc accggcaagc ttcccgacgt tgtggtcgcc 5400 tgtgtcggtg gtggctccaa cgccatcggc atgttcgcag acttcattga cgatgaaggc 5460 gtagagctcg tcggcgctga gccagccggt gaaggcctcg actccggcaa gcacggcgca 5520 accatcacca acggtcagat cggcatcctg cacggcaccc gttcctacct gatgcgcaac 5580 tccgacggcc aagtggaaga gtcctactcc atctccgccg gacttgatta cccaggcgtc 5640 ggcccacagc acgcacacct gcacgccacc ggccgcgcca cctacgttgg tatcaccgac 5700 gccgaagccc tccaagcatt ccagtacctc gcccgctacg aaggcatcat ccccgcactg 5760 gaatcctcac acgcgttcgc ctacgcactc aagcgcgcca agaccgccga agaggaaggc 5820 cagaacttaa ccatcctcgt ctccctatcc ggccgtggcg acaaggacgt tgaccacgtg 5880 cgccgcaccc tcgaagaaaa tccagaactg atcctgaagg acaaccgatg agccgttacg 5940 acgatctttt tgcacgcctc gacacggcag gggagggcgc ctttgttccc ttcatcatgc 6000 tgagcgaccc ttcaccagag gaggctttcc agatcatctc cacagcaatc gaagctggcg 6060 cagatgcact ggaacttggc gtacctttct ccgacccagt tgccgatggc cccaccgtcg 6120 cggaatccca cctccgcgca ctcgacggcg gcgccaccgt agacagcgca ctcgagcaga 6180 tcaagcgcgt gcgcgcagcc tacccagagg ttcccatcgg aatgctcatc tacggcaacg 6240 ttcctttcac ccgtggcttg gatcgcttct accaagagtt cgctgaagct ggcgcagact 6300 ccatcctcct gccagacgtc ccagtccgcg aaggcgcacc gttttctgca gcagctgcag 6360 cagccggaat tgatcccatt tacatcgctc cggccaacgc cagcgagaaa accctcgagg 6420 gtgtctccgc cgcatcaaag ggctacatct acgccatctc ccgcgacggc gtcaccggca 6480 ccgaacgtga atcatccacc gacggcctgt ccgcagtggt ggacaacatc aagaaatttg 6540 atggcgcacc catcctcttg ggcttcggca tctcatcccc tcagcacgtg gcagacgcga 6600 ttgcagcggg tgcttccggt gcgatcacgg gttccgcgat caccaagatc attgcttccc 6660 actgcgaagg tgagcacccg aacccgtcca ccattcgaga tatggacggt ttgaagaagg 6720 atctcactga gttcatctct gcgatgaagg cagcgaccaa gaaggtttag 6770 <210> 35 <211> 518 <212> PRT <213> Artificial Sequence <220> <223> TrpE-cgl <400> 35 Met Ser Thr Asn Pro His Val Phe Ser Leu Asp Val Arg Tyr His Glu   1 5 10 15 Asp Ala Ser Ala Leu Phe Ala His Leu Gly Gly Thr Thr Ala Asp Asp              20 25 30 Ala Ala Leu Leu Glu Arg Ala Asp Ile Thr Thr Lys Asn Gly Ile Ser          35 40 45 Ser Leu Ala Val Leu Lys Ser Ser Val Arg Ile Thr Cys Thr Gly Asn      50 55 60 Thr Val Val Thr Gln Pro Leu Thr Asp Ser Gly Arg Ala Val Val Ala  65 70 75 80 Arg Leu Thr Gln Gln Leu Gly Gln Tyr Asn Thr Ala Glu Asn Thr Phe                  85 90 95 Ser Phe Pro Ala Ser Asp Ala Val Asp Glu Arg Glu Arg Leu Thr Ala             100 105 110 Pro Ser Thr Ile Glu Val Leu Arg Lys Leu Gln Phe Glu Ser Gly Tyr         115 120 125 Ser Asp Ala Ser Leu Pro Leu Leu Met Gly Gly Phe Ala Phe Asp Phe     130 135 140 Leu Glu Thr Phe Glu Thr Leu Pro Ala Val Glu Glu Ser Val Asn Thr 145 150 155 160 Tyr Pro Asp Tyr Gln Phe Val Leu Ala Glu Ile Val Leu Asp Ile Asn                 165 170 175 His Gln Asp Gln Thr Ala Lys Leu Thr Gly Val Ser Asn Ala Pro Gly             180 185 190 Glu Leu Glu Ala Glu Leu Asn Lys Leu Ser Leu Leu Ile Asp Ala Ala         195 200 205 Leu Pro Ala Thr Glu His Ala Tyr Gln Thr Thr Pro His Asp Gly Asp     210 215 220 Thr Leu Arg Val Val Ala Asp Ile Pro Asp Ala Gln Phe Arg Thr Gln 225 230 235 240 Ile Asn Glu Leu Lys Glu Asn Ile Tyr Asn Gly Asp Ile Tyr Gln Val                 245 250 255 Val Pro Ala Arg Thr Phe Thr Ala Pro Cys Pro Asp Ala Phe Ala Ala             260 265 270 Tyr Leu Gln Leu Arg Ala Thr Asn Pro Ser Pro Tyr Met Phe Tyr Ile         275 280 285 Arg Gly Leu Asn Glu Gly Arg Ser Tyr Glu Leu Phe Gly Ala Ser Pro     290 295 300 Glu Ser Asn Leu Lys Phe Thr Ala Ala Asn Arg Glu Leu Gln Leu Tyr 305 310 315 320 Pro Ile Ala Gly Thr Arg Pro Arg Gly Leu Asn Pro Asp Gly Ser Ile                 325 330 335 Asn Asp Glu Leu Asp Ile Arg Asn Glu Leu Asp Met Arg Thr Asp Ala             340 345 350 Lys Glu Ile Ala Glu His Thr Met Leu Val Asp Leu Ala Arg Asn Asp         355 360 365 Leu Ala Arg Val Ser Val Pro Ala Ser Arg Arg Val Ala Asp Leu Leu     370 375 380 Gln Val Asp Arg Tyr Ser Arg Val Met His Leu Val Ser Arg Val Thr 385 390 395 400 Ala Thr Leu Asp Pro Glu Leu Asp Ala Leu Asp Ala Tyr Arg Ala Cys                 405 410 415 Met Asn Met Gly Thr Leu Thr Gly Ala Pro Lys Leu Arg Ala Met Glu             420 425 430 Leu Leu Arg Gly Val Glu Lys Arg Arg Arg Gly Ser Tyr Gly Gly Ala         435 440 445 Val Gly Tyr Leu Arg Gly Asn Gly Asp Met Asp Asn Cys Ile Val Ile     450 455 460 Arg Ser Ala Phe Val Gln Asp Gly Val Ala Ala Val Gln Ala Gly Ala 465 470 475 480 Gly Val Val Arg Asp Ser Asn Pro Gln Ser Glu Ala Asp Glu Thr Leu                 485 490 495 His Lys Ala Tyr Ala Val Leu Asn Ala Ile Ala Leu Ala Ala Gly Ser             500 505 510 Thr Leu Glu Val Ile Arg         515 <210> 36 <211> 208 <212> PRT <213> Artificial Sequence <220> <223> TrpG-cgl <400> 36 Met Thr His Val Val Leu Ile Asp Asn His Asp Ser Phe Val Tyr Asn   1 5 10 15 Leu Val Asp Ala Phe Ala Val Ala Gly Tyr Lys Cys Thr Val Phe Arg              20 25 30 Asn Thr Val Pro Val Glu Thr Ile Leu Ala Ala Asn Pro Asp Leu Ile          35 40 45 Cys Leu Ser Pro Gly Pro Gly Tyr Pro Ala Asp Ala Gly Asn Met Met      50 55 60 Ala Leu Ile Glu Arg Thr Leu Gly Gln Ile Pro Leu Leu Gly Ile Cys  65 70 75 80 Leu Gly Tyr Gln Ala Leu Ile Glu Tyr His Gly Gly Lys Val Glu Pro                  85 90 95 Cys Gly Pro Val His Gly Thr Thr Asp Asn Met Ile Leu Thr Asp Ala             100 105 110 Gly Val Gln Ser Pro Val Phe Ala Gly Leu Ala Thr Asp Val Glu Pro         115 120 125 Asp His Pro Glu Val Pro Gly Arg Lys Val Pro Ile Gly Arg Tyr His     130 135 140 Ser Leu Gly Cys Val Val Ala Pro Asp Gly Ile Glu Ser Leu Gly Thr 145 150 155 160 Cys Ser Ser Glu Ile Gly Asp Val Ile Met Ala Ala Arg Thr Thr Asp                 165 170 175 Gly Lys Ala Ile Gly Leu Gln Phe His Pro Glu Ser Val Leu Ser Pro             180 185 190 Thr Gly Pro Ile Ile Leu Ser Arg Cys Val Glu Gln Leu Leu Ala Asn         195 200 205 <210> 37 <211> 348 <212> PRT <213> Artificial Sequence <220> <223> TrpD-cgl <400> 37 Met Thr Ser Pro Ala Thr Leu Lys Val Leu Asn Ala Tyr Leu Asp Asn   1 5 10 15 Pro Thr Pro Thr Leu Glu Glu Ala Ile Glu Val Phe Thr Pro Leu Thr              20 25 30 Val Gly Glu Tyr Asp Asp Val His Ile Ala Ala Leu Leu Ala Thr Ile          35 40 45 Arg Thr Arg Gly Glu Gln Phe Ala Asp Ile Ala Gly Ala Ala Lys Ala      50 55 60 Phe Leu Ala Ala Ala Arg Pro Phe Pro Ile Thr Gly Ala Gly Leu Leu  65 70 75 80 Asp Ser Ala Gly Thr Gly Gly Asp Gly Ala Asn Thr Ile Asn Ile Thr                  85 90 95 Thr Gly Ala Ser Leu Ile Ala Ala Ser Gly Gly Val Lys Leu Val Lys             100 105 110 His Gly Asn Arg Ser Val Ser Ser Lys Ser Gly Ser Ala Asp Val Leu         115 120 125 Glu Ala Leu Asn Ile Pro Leu Gly Leu Asp Val Asp Arg Ala Val Lys     130 135 140 Trp Phe Glu Ala Ser Asn Phe Thr Phe Leu Phe Ala Pro Ala Tyr Asn 145 150 155 160 Pro Glu Ile Ala His Val Gln Pro Val Arg Gln Ala Leu Lys Phe Pro                 165 170 175 Thr Ile Phe Asn Thr Leu Gly Pro Leu Leu Ser Pro Ala Arg Pro Glu             180 185 190 Arg Gln Ile Met Gly Val Ala Asn Ala Asn His Gly Gln Leu Ile Ala         195 200 205 Glu Val Phe Arg Glu Leu Gly Arg Thr Arg Ala Leu Val Val His Gly     210 215 220 Ala Gly Thr Asp Glu Ile Ala Val His Gly Thr Thr Leu Val Trp Glu 225 230 235 240 Leu Lys Glu Asp Gly Thr Ile Glu His Tyr Thr Ile Glu Pro Glu Asp                 245 250 255 Leu Gly Leu Gly Arg Tyr Thr Leu Glu Asp Leu Val Gly Gly Leu Gly             260 265 270 Thr Glu Asn Ala Glu Ala Met Arg Ala Thr Phe Ala Gly Thr Gly Pro         275 280 285 Asp Ala His Arg Asp Ala Leu Ala Ala Ser Ala Gly Ala Met Phe Tyr     290 295 300 Leu Asn Gly Asp Val Asp Ser Leu Lys Asp Gly Ala Gln Lys Ala Leu 305 310 315 320 Ser Leu Leu Ala Asp Gly Thr Thr Gln Ala Trp Leu Ala Lys His Glu                 325 330 335 Glu Ile Asp Tyr Ser Glu Lys Glu Ser Ser Asn Asp             340 345 <210> 38 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> trpC-cgl <400> 38 Met Thr Ser Asn Asn Leu Pro Thr Val Leu Glu Ser Ile Val Glu Gly   1 5 10 15 Arg Arg Gly His Leu Glu Glu Ile Arg Ala Arg Ile Ala His Val Asp              20 25 30 Val Asp Ala Leu Pro Lys Ser Thr Arg Ser Leu Phe Asp Ser Leu Asn          35 40 45 Gln Gly Arg Gly Gly Ala Arg Phe Ile Met Glu Cys Lys Ser Ala Ser      50 55 60 Pro Ser Leu Gly Met Ile Arg Glu His Tyr Gln Pro Gly Glu Ile Ala  65 70 75 80 Arg Val Tyr Ser Arg Tyr Ala Ser Gly Ile Ser Val Leu Cys Glu Pro                  85 90 95 Asp Arg Phe Gly Gly Asp Tyr Asp His Leu Ala Thr Val Ala Ala Thr             100 105 110 Ser His Leu Pro Val Leu Cys Lys Asp Phe Ile Ile Asp Pro Val Gln         115 120 125 Val His Ala Ala Arg Tyr Phe Gly Ala Asp Ala Ile Leu Leu Met Leu     130 135 140 Ser Val Leu Asp Asp Glu Glu Tyr Ala Ala Leu Ala Ala Glu Ala Ala 145 150 155 160 Arg Phe Asp Leu Asp Ile Leu Thr Glu Val Ile Asp Glu Glu Glu Val                 165 170 175 Ala Arg Ala Ile Lys Leu Gly Ala Lys Ile Phe Gly Val Asn His Arg             180 185 190 Asn Leu His Asp Leu Ser Ile Asp Leu Asp Arg Ser Arg Arg Leu Ser         195 200 205 Lys Leu Ile Pro Ala Asp Ala Val Leu Val Ser Glu Ser Gly Val Arg     210 215 220 Asp Thr Glu Thr Val Arg Gln Leu Gly Gly His Ser Asn Ala Phe Leu 225 230 235 240 Val Gly Ser Gln Leu Thr Ser Gln Glu Asn Val Asp Leu Ala Ala Arg                 245 250 255 Glu Leu Val Tyr Gly Pro Asn Lys Val Cys Gly Leu Thr Ser Pro Ser             260 265 270 Ala Ala Gln Thr Ala Arg Ala Ala Gly Ala Val Tyr Gly Gly Leu Ile         275 280 285 Phe Glu Glu Ala Ser Pro Arg Asn Val Ser Arg Glu Thr Ser Gln Lys     290 295 300 Ile Ile Ala Ala Glu Pro Asn Leu Arg Tyr Val Ala Val Ser Arg Arg 305 310 315 320 Thr Ser Gly Tyr Lys Asp Leu Leu Val Asp Gly Ile Phe Ala Val Gln                 325 330 335 Ile His Ala Pro Leu Gln Gly Ser Val Glu Ala Glu Lys Ala Leu Ile             340 345 350 Ala Ala Val Arg Glu Glu Val Gly Pro Gln Val Gln Val Trp Arg Ala         355 360 365 Ile Ser Met Ser Ser Pro Leu Gly Ala Glu Val Ala Ala Ala Val Glu     370 375 380 Gly Asp Val Asp Lys Leu Ile Leu Asp Ala His Glu Gly Gly Ser Gly 385 390 395 400 Glu Val Phe Asp Trp Ala Thr Val Pro Ala Ala Val Lys Ala Lys Ser                 405 410 415 Leu Leu Ala Gly Gly Ile Ser Pro Asp Asn Ala Ala Gln Ala Leu Ala             420 425 430 Val Gly Cys Ala Gly Leu Asp Ile Asn Ser Gly Val Glu Tyr Pro Ala         435 440 445 Gly Ala Gly Thr Trp Ala Gly Ala Lys Asp Ala Gly Ala Leu Leu Lys     450 455 460 Ile Phe Ala Thr Ile Ser Thr Phe His Tyr 465 470 <210> 39 <211> 417 <212> PRT <213> Artificial Sequence <220> <223> TrpB-cgl <400> 39 Met Thr Glu Lys Glu Asn Leu Gly Gly Ser Thr Leu Leu Pro Ala Tyr   1 5 10 15 Phe Gly Glu Phe Gly Gly Gln Phe Val Ala Glu Ser Leu Leu Pro Ala              20 25 30 Leu Asp Gln Leu Glu Lys Ala Phe Val Asp Ala Thr Asn Ser Pro Glu          35 40 45 Phe Arg Glu Glu Leu Gly Gly Tyr Leu Arg Asp Tyr Leu Gly Arg Pro      50 55 60 Thr Pro Leu Thr Glu Cys Ser Asn Leu Pro Leu Ala Gly Glu Gly Lys  65 70 75 80 Gly Phe Ala Arg Ile Phe Leu Lys Arg Glu Asp Leu Val His Gly Gly                  85 90 95 Ala His Lys Thr Asn Gln Val Ile Gly Gln Val Leu Leu Ala Lys Arg             100 105 110 Met Gly Lys Thr Arg Ile Ila Ala Glu Thr Gly Ala Gly Gln His Gly         115 120 125 Thr Ala Thr Ala Leu Ala Cys Ala Leu Met Gly Leu Glu Cys Val Val     130 135 140 Tyr Met Gly Ala Lys Asp Val Ala Arg Gln Gln Pro Asn Val Tyr Arg 145 150 155 160 Met Gln Leu His Gly Ala Lys Val Ile Pro Val Glu Ser Gly Ser Gly                 165 170 175 Thr Leu Lys Asp Ala Val Asn Glu Ala Leu Arg Asp Trp Thr Ala Thr             180 185 190 Phe His Glu Ser His Tyr Leu Leu Gly Thr Ala Ala Gly Pro His Pro         195 200 205 Phe Pro Thr Ile Val Arg Glu Phe His Lys Val Ile Ser Glu Glu Ala     210 215 220 Lys Ala Gln Met Leu Glu Arg Thr Gly Lys Leu Pro Asp Val Val Val 225 230 235 240 Ala Cys Val Gly Gly Gly Ser Asn Ala Ile Gly Met Phe Ala Asp Phe                 245 250 255 Ile Asp Asp Glu Gly Val Glu Leu Val Gly Ala Glu Pro Ala Gly Glu             260 265 270 Gly Leu Asp Ser Gly Lys His Gly Ala Thr Ile Thr Asn Gly Gln Ile         275 280 285 Gly Ile Leu His Gly Thr Arg Ser Tyr Leu Met Arg Asn Ser Asp Gly     290 295 300 Gln Val Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu Asp Tyr Pro Gly 305 310 315 320 Val Gly Pro Gln His Ala His Leu His Ala Thr Gly Arg Ala Thr Tyr                 325 330 335 Val Gly Ile Thr Asp Ala Glu Ala Leu Gln Ala Phe Gln Tyr Leu Ala             340 345 350 Arg Tyr Glu Gly Ile Ile Pro Ala Leu Glu Ser Ser His Ala Phe Ala         355 360 365 Tyr Ala Leu Lys Arg Ala Lys Thr Ala Glu Glu Glu Gly Gln Asn Leu     370 375 380 Thr Ile Leu Val Ser Leu Ser Gly Arg Gly Asp Lys Asp Val Asp His 385 390 395 400 Val Arg Arg Thr Leu Glu Glu Asn Pro Glu Leu Ile Leu Lys Asp Asn                 405 410 415 Arg     <210> 40 <211> 280 <212> PRT <213> Artificial Sequence <220> <223> TrpA-cgl <400> 40 Met Ser Arg Tyr Asp Asp Leu Phe Ala Arg Leu Asp Thr Ala Gly Glu   1 5 10 15 Gly Ala Phe Val Pro Phe Ile Met Leu Ser Asp Pro Ser Pro Glu Glu              20 25 30 Ala Phe Gln Ile Ile Ser Thr Ala Ile Glu Ala Gly Ala Asp Ala Leu          35 40 45 Glu Leu Gly Val Pro Phe Ser Asp Pro Val Ala Asp Gly Pro Thr Val      50 55 60 Ala Glu Ser His Leu Arg Ala Leu Asp Gly Gly Ala Thr Val Asp Ser  65 70 75 80 Ala Leu Glu Gln Ile Lys Arg Val Arg Ala Ala Tyr Pro Glu Val Pro                  85 90 95 Ile Gly Met Leu Ile Tyr Gly Asn Val Pro Phe Thr Arg Gly Leu Asp             100 105 110 Arg Phe Tyr Gln Glu Phe Ala Glu Ala Gly Ala Asp Ser Ile Leu Leu         115 120 125 Pro Asp Val Pro Val Arg Glu Gly Ala Pro Phe Ser Ala Ala Ala Ala     130 135 140 Ala Ala Gly Ile Asp Pro Ile Tyr Ile Ala Pro Ala Asn Ala Ser Glu 145 150 155 160 Lys Thr Leu Glu Gly Val Ser Ala Ala Ser Lys Gly Tyr Ile Tyr Ala                 165 170 175 Ile Ser Arg Asp Gly Val Thr Gly Thr Glu Arg Glu Ser Ser Thr Asp             180 185 190 Gly Leu Ser Ala Val Val Asp Asn Ile Lys Lys Phe Asp Gly Ala Pro         195 200 205 Ile Leu Leu Gly Phe Gly Ile Ser Ser Pro Gln His Val Ala Asp Ala     210 215 220 Ile Ala Ala Gly Ala Ser Gly Ala Ile Thr Gly Ser Ala Ile Thr Lys 225 230 235 240 Ile Ile Ala Ser His Cys Glu Gly Glu His Pro Asn Pro Ser Thr Ile                 245 250 255 Arg Asp Met Asp Gly Leu Lys Lys Asp Leu Thr Glu Phe Ile Ser Ala             260 265 270 Met Lys Ala Ala Thr Lys Lys Val         275 280 <210> 41 <211> 2103 <212> DNA <213> Artificial Sequence <220> <223> Corynebacterium glutamicum tkt (polynucleotide) <400> 41 ttgaccacct tgacgctgtc acctgaactt caggcgctca ctgtacgcaa ttacccctct 60 gattggtccg atgtggacac caaggctgta gacactgttc gtgtcctcgc tgcagacgct 120 gtagaaaact gtggctccgg ccacccaggc accgcaatga gcctggctcc ccttgcatac 180 accttgtacc agcgggttat gaacgtagat ccacaggaca ccgactgggc aggccgtgac 240 cgcttcgttc tttcttgtgg ccactcctct ttgacccagt acatccagct ttacttgggt 300 ggattcggcc ttgagatgga tgacctgaag gctctgcgca cctgggattc cttgacccca 360 ggacaccctg agtaccgcca caccaagggc gtagagatca ccactggccc tcttggccag 420 ggtcttgcat ctgcagttgg tatggccatg gctgctcgtc gtgagcgtgg cctattcgac 480 ccaaccgctg ctgagggcga atccccattc gaccaccaca tctacgtcat tgcttctgat 540 ggtgacctgc aggaaggtgt cacctctgag gcatcctcca tcgctggcac ccagcagctg 600 ggcaacctca tcgtgttctg ggatgacaac cgcatctcca tcgaagacaa cactgagatc 660 gctttcaacg aggacgttgt tgctcgttac aaggcttacg gctggcagac cattgaggtt 720 gaggctggcg aggacgttgc agcaatcgaa gctgcagtgg ctgaggctaa gaaggacacc 780 aagcgaccta ccttcatccg cgttcgcacc atcatcggct tcccagctcc aaccatgatg 840 aacaccggtg ctgtgcacgg tgctgctctt ggcgcagctg aggttgcagc aaccaagact 900 gagcttggat tcgatcctga ggctcacttc gcgatcgacg atgaggttat cgctcacacc 960 cgctccctcg cagagcgcgc tgcacagaag aaggctgcat ggcaggtcaa gttcgatgag 1020 tgggcagctg ccaaccctga gaacaaggct ctgttcgatc gcctgaactc ccgtgagctt 1080 ccagcgggct acgctgacga gctcccaaca tgggatgcag atgagaaggg cgtcgcaact 1140 cgtaaggctt ccgaggctgc acttcaggca ctgggcaaga cccttcctga gctgtggggc 1200 ggttccgctg acctcgcagg ttccaacaac accgtgatca agggctcccc ttccttcggc 1260 cctgagtcca tctccaccga gacctggtct gctgagcctt acggccgtaa cctgcacttc 1320 ggtatccgtg agcacgctat gggatccatc ctcaacggca tttccctcca cggtggcacc 1380 cgcccatacg gcggaacctt cctcatcttc tccgactaca tgcgtcctgc agttcgtctt 1440 gcagctctca tggagaccga cgcttactac gtctggaccc acgactccat cggtctgggc 1500 gaagatggcc caacccacca gcctgttgaa accttggctg cactgcgcgc catcccaggt 1560 ctgtccgtcc tgcgtcctgc agatgcgaac gagaccgccc aggcttgggc tgcagcactt 1620 gagtacaagg aaggccctaa gggtcttgca ctaacccgcc agaacattcc tgttctggaa 1680 ggcaccaagg agaaggctgc tgaaggcgtt cgccgcggtg gctacgtcct ggttgagggt 1740 tccaaggaaa ccccagatgt gatcctcatg ggctccggct ccgaggttca gcttgcagtt 1800 aacgctgcga aggctctgga agctgagggc gttgcagctc gcgttgtttc cgttccttgc 1860 atggattggt tccaggagca ggacgcagag tacatcgagt ccgttctgcc tgcagctgtg 1920 accgctcgtg tgtctgttga agctggcatc gcaatgcctt ggtaccgctt cttgggcacc 1980 cagggccgtg ctgtctccct tgagcacttc ggtgcttctg cggattacca gaccctgttt 2040 gagaagttcg gcatcaccac cgatgcagtc gtggcagcgg ccaaggactc cattaacggt 2100 taa 2103 <210> 42 <211> 700 <212> PRT <213> Artificial Sequence <220> <223> Corynebacterium glutamicum tkt (polypeptide) <400> 42 Leu Thr Thr Leu Thr Leu Ser Pro Glu Leu Gln Ala Leu Thr Val Arg   1 5 10 15 Asn Tyr Pro Ser Asp Trp Ser Asp Val Asp Thr Lys Ala Val Asp Thr              20 25 30 Val Arg Val Leu Ala Ala Asp Ala Val Glu Asn Cys Gly Ser Gly His          35 40 45 Pro Gly Thr Ala Met Ser Leu Ala Pro Leu Ala Tyr Thr Leu Tyr Gln      50 55 60 Arg Val Met Asn Val Asp Pro Gln Asp Thr Asp Trp Ala Gly Arg Asp  65 70 75 80 Arg Phe Val Leu Ser Cys Gly His Ser Ser Leu Thr Gln Tyr Ile Gln                  85 90 95 Leu Tyr Leu Gly Gly Phe Gly Leu Glu Met Asp Asp Leu Lys Ala Leu             100 105 110 Arg Thr Trp Asp Ser Leu Thr Pro Gly His Pro Glu Tyr Arg His Thr         115 120 125 Lys Gly Val Glu Ile Thr Thr Gly Pro Leu Gly Gln Gly Leu Ala Ser     130 135 140 Ala Val Gly Met Ala Met Ala Ala Arg Arg Glu Arg Gly Leu Phe Asp 145 150 155 160 Pro Thr Ala Ala Glu Gly Glu Ser Pro Phe Asp His His Ile Tyr Val                 165 170 175 Ile Ala Ser Asp Gly Asp Leu Gln Glu Gly Val Thr Ser Glu Ala Ser             180 185 190 Ser Ile Ala Gly Thr Gln Gln Leu Gly Asn Leu Ile Val Phe Trp Asp         195 200 205 Asp Asn Arg Ile Ser Ile Glu Asp Asn Thr Glu Ile Ala Phe Asn Glu     210 215 220 Asp Val Val Ala Arg Tyr Lys Ala Tyr Gly Trp Gln Thr Ile Glu Val 225 230 235 240 Glu Ala Gly Glu Asp Val Ala Ala Ile Glu Ala Ala Val Ala Glu Ala                 245 250 255 Lys Lys Asp Thr Lys Arg Pro Thr Phe Ile Arg Val Arg Thr Ile Ile             260 265 270 Gly Phe Pro Ala Pro Thr Met Met Asn Thr Gly Ala Val His Gly Ala         275 280 285 Ala Leu Gly Ala Ala Glu Val Ala Ala Thr Lys Thr Glu Leu Gly Phe     290 295 300 Asp Pro Glu Ala His Phe Ala Ile Asp Asp Glu Val Ile Ala His Thr 305 310 315 320 Arg Ser Leu Ala Glu Arg Ala Ala Gln Lys Lys Ala Ala Trp Gln Val                 325 330 335 Lys Phe Asp Glu Trp Ala Ala Ala Asn Pro Glu Asn Lys Ala Leu Phe             340 345 350 Asp Arg Leu Asn Ser Arg Glu Leu Pro Ala Gly Tyr Ala Asp Glu Leu         355 360 365 Pro Thr Trp Asp Ala Asp Glu Lys Gly Val Ala Thr Arg Lys Ala Ser     370 375 380 Glu Ala Ala Leu Gln Ala Leu Gly Lys Thr Leu Pro Glu Leu Trp Gly 385 390 395 400 Gly Ser Ala Asp Leu Ala Gly Ser Asn Asn Thr Val Ile Lys Gly Ser                 405 410 415 Pro Ser Phe Gly Pro Glu Ser Ile Ser Thr Glu Thr Trp Ser Ala Glu             420 425 430 Pro Tyr Gly Arg Asn Leu His Phe Gly Ile Arg Glu His Ala Met Gly         435 440 445 Ser Ile Leu Asn Gly Ile Ser Leu His Gly Gly Thr Arg Pro Tyr Gly     450 455 460 Gly Thr Phe Leu Ile Phe Ser Asp Tyr Met Arg Pro Ala Val Arg Leu 465 470 475 480 Ala Ala Leu Met Glu Thr Asp Ala Tyr Tyr Val Trp Thr His Asp Ser                 485 490 495 Ile Gly Leu Gly Glu Asp Gly Pro Thr His Gln Pro Val Glu Thr Leu             500 505 510 Ala Ala Leu Arg Ala Ile Pro Gly Leu Ser Val Leu Arg Pro Ala Asp         515 520 525 Ala Asn Glu Thr Ala Gln Ala Trp Ala Ala Ala Leu Glu Tyr Lys Glu     530 535 540 Gly Pro Lys Gly Leu Ala Leu Thr Arg Gln Asn Ile Pro Val Leu Glu 545 550 555 560 Gly Thr Lys Glu Lys Ala Ala Glu Gly Val Arg Arg Gly Gly Tyr Val                 565 570 575 Leu Val Glu Gly Ser Lys Glu Thr Pro Asp Val Ile Leu Met Gly Ser             580 585 590 Gly Ser Glu Val Gln Leu Ala Val Asn Ala Ala Lys Ala Leu Glu Ala         595 600 605 Glu Gly Val Ala Ala Arg Val Val Ser Val Pro Cys Met Asp Trp Phe     610 615 620 Gln Glu Gln Asp Ala Glu Tyr Ile Glu Ser Val Leu Pro Ala Ala Val 625 630 635 640 Thr Ala Arg Val Ser Val Glu Ala Gly Ile Ala Met Pro Trp Tyr Arg                 645 650 655 Phe Leu Gly Thr Gln Gly Arg Ala Val Ser Leu Glu His Phe Gly Ala             660 665 670 Ser Ala Asp Tyr Gln Thr Leu Phe Glu Lys Phe Gly Ile Thr Thr Asp         675 680 685 Ala Val Val Ala Ala Ala Lys Asp Ser Ile Asn Gly     690 695 700 <210> 43 <211> 6531 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of tryptophan operon-Escherichia coli <400> 43 atgcaaacac aaaaaccgac tctcgaactg ctaacctgcg aaggcgctta tcgcgacaat 60 tccaccgcgc tttttcacca gttgtgtggg gatcgtccgg caacgctgct gctggaatcc 120 gcagatatcg acagcaaaga tgatttaaaa agcctgctgc tggtagacag tgcgctgcgc 180 attacagctt taggtgacac tgtcacaatc caggcacttt ccggcaacgg cgaagccctc 240 ctggcactac tggataacgc cctgcctgcg ggtgtggaaa gtgaacaatc accaaactgc 300 cgtgtgctgc gcttcccccc tgtcagtcca ctgctggatg aagacgcccg cttatgctcc 360 ctttcggttt ttgacgcttt ccgtttattg cagaatctgt tgaatgtacc gaaggaagaa 420 cgagaagcca tgttcttcgg cggcctgttc tcttatgacc ttgtggcggg atttgaagat 480 ttaccgcaac tgtcagcgga aaataactgc cctgatttct gtttttatct cgctgaaacg 540 ctgatggtga ttgaccatca gaaaaaaagc acccgtattc aggccagcct gtttgctccg 600 aatgaagaag aaaaacaacg tctcactgct cgcctgaacg aactacgtca gcaactgacc 660 gaagccgcgc cgccgctgcc agtggtttcc gtgccgcata tgcgttgtga atgtaatcag 720 agcgatgaag agttcggtgg cgtagtgcgt ttgttgcaaa aagcgattcg cgctggagaa 780 attttccagg tggtgccatc tcgccgtttc tctctgccct gcccgtcacc gctggcggcc 840 tattacgtgc tgaaaaagag taatcccagc ccgtacatgt tttttatgca ggataatgat 900 ttcaccctat ttggcgcgtc gccggaaagc tcgctcaagt atgatgccac cagccgccag 960 attgagatct acccgattgc cggaacacgc ccacgcggtc gtcgcgccga tggttcactg 1020 gacagagatc tcgacagccg tattgaactg gaaatgcgta ccgatcataa agagctgtct 1080 gaacatctga tgctggttga tctcgcccgt aatgatctgg cacgcatttg cacccccggc 1140 agccgctacg tcgccgatct caccaaagtt gaccgttatt cctatgtgat gcacctcgtc 1200 tctcgcgtag tcggcgaact gcgtcacgat cttgacgccc tgcacgctta tcgcgcctgt 1260 atgaatatgg ggacgttaag cggtgcgccg aaagtacgcg ctatgcagtt aattgccgag 1320 gcggaaggtc gtcgccgcgg cagctacggc ggcgcggtag gttatttcac cgcgcatggc 1380 gatctcgaca cctgcattgt gatccgctcg gcgctggtgg aaaacggtat cgccaccgtg 1440 caagcgggtg ctggtgtagt ccttgattct gttccgcagt cggaagccga cgaaacccgt 1500 aacaaagccc gcgctgtact gcgcgctatt gccaccgcgc atcatgcaca ggagactttc 1560 tgatggctga cattctgctg ctcgataata tcgactcttt tacgtacaac ctggcagatc 1620 agttgcgcag caatgggcat aacgtggtga tttaccgcaa ccatattccg gcgcaaacct 1680 taattgaacg cctggcgacc atgagcaatc cggtgctgat gctttctcct ggccccggtg 1740 tgccgagcga agccggttgt atgccggaac tcctcacccg cttgcgtggc aagctgccca 1800 ttattggcat ttgcctcgga catcaggcga ttgtcgaagc ttacgggggc tatgtcggtc 1860 aggcgggcga aattctccac ggtaaagcct ccagcattga acatgacggt caggcgatgt 1920 ttgccggatt aacaaacccg ctgccggtgg cgcgttatca ctcgctggtt ggcagtaaca 1980 ttccggccgg tttaaccatc aacgcccatt ttaatggcat ggtgatggca gtacgtcacg 2040 atgcggatcg cgtttgtgga ttccagttcc atccggaatc cattctcacc acccagggcg 2100 ctcgcctgct ggaacaaacg ctggcctggg cgcagcagaa actagagcca gccaacacgc 2160 tgcaaccgat tctggaaaaa ctgtatcagg cgcagacgct tagccaacaa gaaagccacc 2220 agctgttttc agcggtggtg cgtggcgagc tgaagccgga acaactggcg gcggcgctgg 2280 tgagcatgaa aattcgcggt gagcacccga acgagatcgc cggggcagca accgcgctac 2340 tggaaaacgc agcgccgttc ccgcgcccgg attatctgtt tgctgatatc gtcggtactg 2400 gcggtgacgg cagcaacagt atcaatattt ctaccgccag tgcgtttgtc gccgcggcct 2460 gtgggctgaa agtggcgaaa cacggcaacc gtagcgtctc cagtaaatct ggttcgtccg 2520 atctgctggc ggcgttcggt attaatcttg atatgaacgc cgataaatcg cgccaggcgc 2580 tggatgagtt aggtgtatgt ttcctctttg cgccgaagta tcacaccgga ttccgccacg 2640 cgatgccggt tcgccagcaa ctgaaaaccc gcaccctgtt caatgtgctg gggccattga 2700 ttaacccggc gcatccgccg ctggcgttaa ttggtgttta tagtccggaa ctggtgctgc 2760 cgattgccga aaccttgcgc gtgctggggt atcaacgcgc ggcggtggtg cacagcggcg 2820 ggatggatga agtttcatta cacgcgccga caatcgttgc cgaactgcat gacggcgaaa 2880 ttaaaagcta tcagctcacc gcagaagact ttggcctgac accctaccac caggagcaac 2940 tggcaggcgg aacaccggaa gaaaaccgtg acattttaac acgtttgtta caaggtaaag 3000 gcgacgccgc ccatgaagca gccgtcgctg cgaacgtcgc catgttaatg cgcctgcatg 3060 gccatgaaga tctgcaagcc aatgcgcaaa ccgttcttga ggtactgcgc agtggttccg 3120 cttacgacag agtcaccgca ctggcggcac gagggtaaat gatgcaaacc gttttagcga 3180 aaatcgtcgc agacaaggcg atttgggtag aagcccgcaa acagcagcaa ccgctggcca 3240 gttttcagaa tgaggttcag ccgagcacgc gacattttta tgatgcgcta cagggtgcgc 3300 gcacggcgtt tattctggag tgcaagaaag cgtcgccgtc aaaaggcgtg atccgtgatg 3360 atttcgatcc agcacgcatt gccgccattt ataaacatta cgcttcggca atttcggtgc 3420 tgactgatga gaaatatttt caggggagct ttaatttcct ccccatcgtc agccaaatcg 3480 ccccgcagcc gattttatgt aaagacttca ttatcgaccc ttaccagatc tatctggcgc 3540 gctattacca ggccgatgcc tgcttattaa tgctttcagt actggacgac gaccaatatc 3600 gccagcttgc cgccgtcgct cacagtctgg agatgggggt gctgaccgaa gtcagtaatg 3660 aagaggaaca ggagcgcgcc attgcattgg gagcaaaggt cgttggcatc aacaaccgcg 3720 atctgcgtga tttgtcgatt gatctcaacc gtacccgcga gcttgcgccg aaactggggc 3780 acaacgtgac ggtaatcagc gaatccggca tcaatactta cgctcaggtg cgcgagttaa 3840 gccacttcgc taacggtttt ctgattggtt cggcgttgat ggcccatgac gatttgcacg 3900 ccgccgtgcg ccgggtgttg ctgggtgaga ataaagtatg tggcctgacg cgtgggcaag 3960 atgctaaagc agcttatgac gcgggcgcga tttacggtgg gttgattttt gttgcgacat 4020 caccgcgttg cgtcaacgtt gaacaggcgc aggaagtgat ggctgcggca ccgttgcagt 4080 atgttggcgt gttccgcaat cacgatattg ccgatgtggt ggacaaagct aaggtgttat 4140 cgctggcggc agtgcaactg catggtaatg aagaacagct gtatatcgat acgctgcgtg 4200 aagctctgcc agcacatgtt gccatctgga aagcattaag cgtcggtgaa accctgcccg 4260 cccgcgagtt tcagcacgtt gataaatatg ttttagacaa cggccagggt ggaagcgggc 4320 aacgttttga ctggtcacta ttaaatggtc aatcgcttgg caacgttctg ctggcggggg 4380 gcttaggcgc agataactgc gtggaagcgg cacaaaccgg ctgcgccgga cttgatttta 4440 attctgctgt agagtcgcaa ccgggcatca aagacgcacg tcttttggcc tcggttttcc 4500 agacgctgcg cgcatattaa ggaaaggaac aatgacaaca ttacttaacc cctattttgg 4560 tgagtttggc ggcatgtacg tgccacaaat cctgatgcct gctctgcgcc agctggaaga 4620 agcttttgtc agtgcgcaaa aagatcctga atttcaggct cagttcaacg acctgctgaa 4680 aaactatgcc gggcgtccaa ccgcgctgac caaatgccag aacattacag ccgggacgaa 4740 caccacgctg tatctcaagc gtgaagattt gctgcacggc ggcgcgcata aaactaacca 4800 ggtgctgggg caggcgttgc tggcgaagcg gatgggtaaa accgaaatca tcgccgaaac 4860 cggtgccggt cagcatggcg tggcgtcggc ccttgccagc gccctgctcg gcctgaaatg 4920 ccgtatttat atgggtgcca aagacgttga acgccagtcg cctaacgttt ttcgtatgcg 4980 cttaatgggt gcggaagtga tcccggtgca tagcggttcc gcgacgctga aagatgcctg 5040 taacgaggcg ctgcgcgact ggtccggtag ttacgaaacc gcgcactata tgctgggcac 5100 cgcagctggc ccgcatcctt atccgaccat tgtgcgtgag tttcagcgga tgattggcga 5160 agaaaccaaa gcgcagattc tggaaagaga aggtcgcctg ccggatgccg ttatcgcctg 5220 tgttggcggc ggttcgaatg ccatcggcat gtttgctgat ttcatcaatg aaaccaacgt 5280 cggcctgatt ggtgtggagc caggtggtca cggtatcgaa actggcgagc acggcgcacc 5340 gctaaaacat ggtcgcgtgg gtatctattt cggtatgaaa gcgccgatga tgcaaaccga 5400 agacgggcag attgaagaat cttactccat ctccgccgga ctggatttcc cgtctgtcgg 5460 cccacaacac gcgtatctta acagcactgg acgcgctgat tacgtgtcta ttaccgatga 5520 tgaagccctt gaagccttca aaacgctgtg cctgcacgaa gggatcatcc cggcgctgga 5580 atcctcccac gccctggccc atgcgttgaa aatgatgcgc gaaaacccgg ataaagagca 5640 gctactggtg gttaaccttt ccggtcgcgg cgataaagac atcttcaccg ttcacgatat 5700 tttgaaagca cgaggggaaa tctgatggaa cgctacgaat ctctgtttgc ccagttgaag 5760 gagcgcaaag aaggcgcatt cgttcctttc gtcacgctcg gtgatccggg cattgagcag 5820 tcattgaaaa ttatcgatac gctaattgaa gccggtgctg acgcgctgga gttaggtatc 5880 cccttctccg acccactggc ggatggcccg acgattcaaa acgccactct gcgcgccttt 5940 gcggcaggtg tgactccggc acaatgtttt gaaatgctgg cactgattcg ccagaaacac 6000 ccgaccattc ccattggcct gttgatgtat gccaatctgg tgtttaacaa aggcattgat 6060 gagttttatg cccagtgcga aaaagtcggc gtcgattcgg tgctggttgc cgatgtgcca 6120 gttgaagagt ccgcgccctt ccgccaggcc gcgttgcgtc ataatgtcgc acctatcttc 6180 atctgcccgc caaatgccga tgacgacctg ctgcgccaga tagcctctta cggtcgtggt 6240 tacacctatt tgctgtcacg agcaggcgtg accggcgcag aaaaccgcgc cgcgttaccc 6300 ctcaatcatc tggttgcgaa gctgaaagag tacaacgctg cacctccatt gcagggattt 6360 ggtatttccg ccccggatca ggtaaaagca gcgattgatg caggagctgc gggcgcgatt 6420 tctggttcgg ccattgttaa aatcatcgag caacatatta atgagccaga gaaaatgctg 6480 gcggcactga aagtttttgt acaaccgatg aaagcggcga cgcgcagtta a 6531 <210> 44 <211> 520 <212> PRT <213> Artificial Sequence <220> <223> TrpE-eco <400> 44 Met Gln Thr Gln Lys Pro Thr Leu Glu Leu Leu Thr Cys Glu Gly Ala   1 5 10 15 Tyr Arg Asp Asn Ser Thr Ala Leu Phe His Gln Leu Cys Gly Asp Arg              20 25 30 Pro Ala Thr Leu Leu Leu Glu Ser Ala Asp Ile Asp Ser Lys Asp Asp          35 40 45 Leu Lys Ser Leu Leu Leu Val Asp Ser Ala Leu Arg Ile Thr Ala Leu      50 55 60 Gly Asp Thr Val Thr Ile Gln Ala Leu Ser Gly Asn Gly Glu Ala Leu  65 70 75 80 Leu Ala Leu Leu Asp Asn Ala Leu Pro Ala Gly Val Glu Ser Glu Gln                  85 90 95 Ser Pro Asn Cys Arg Val Leu Arg Phe Pro Pro Val Ser Pro Leu Leu             100 105 110 Asp Glu Asp Ala Arg Leu Cys Ser Leu Ser Val Phe Asp Ala Phe Arg         115 120 125 Leu Leu Gln Asn Leu Leu Asn Val Pro Lys Glu Glu Arg Glu Ala Met     130 135 140 Phe Phe Gly Gly Leu Phe Ser Tyr Asp Leu Val Ala Gly Phe Glu Asp 145 150 155 160 Leu Pro Gln Leu Ser Ala Glu Asn Asn Cys Pro Asp Phe Cys Phe Tyr                 165 170 175 Leu Ala Glu Thr Leu Met Val Ile Asp His Gln Lys Lys Ser Thr Arg             180 185 190 Ile Gln Ala Ser Leu Phe Ala Pro Asn Glu Glu Glu Lys Gln Arg Leu         195 200 205 Thr Ala Arg Leu Asn Glu Leu Arg Gln Gln Leu Thr Glu Ala Ala Pro     210 215 220 Pro Leu Pro Val Val Ser Val Pro His Met Arg Cys Glu Cys Asn Gln 225 230 235 240 Ser Asp Glu Glu Phe Gly Gly Val Val Arg Leu Leu Gln Lys Ala Ile                 245 250 255 Arg Ala Gly Glu Ile Phe Gln Val Val Pro Ser Arg Arg Phe Ser Leu             260 265 270 Pro Cys Pro Ser Pro Leu Ala Ala Tyr Tyr Val Leu Lys Lys Ser Asn         275 280 285 Pro Ser Pro Tyr Met Phe Phe Met Gln Asp Asn Asp Phe Thr Leu Phe     290 295 300 Gly Ala Ser Pro Glu Ser Ser Leu Lys Tyr Asp Ala Thr Ser Arg Gln 305 310 315 320 Ile Glu Ile Tyr Pro Ile Ala Gly Thr Arg Pro Arg Gly Arg Arg Ala                 325 330 335 Asp Gly Ser Leu Asp Arg Asp Leu Asp Ser Arg Ile Glu Leu Glu Met             340 345 350 Arg Thr Asp His Lys Glu Leu Ser Glu His Leu Met Leu Val Asp Leu         355 360 365 Ala Arg Asn Asp Leu Ala Arg Ile Cys Thr Pro Gly Ser Arg Tyr Val     370 375 380 Ala Asp Leu Thr Lys Val Asp Arg Tyr Ser Tyr Val Met His Leu Val 385 390 395 400 Ser Arg Val Val Gly Glu Leu Arg His Asp Leu Asp Ala Leu His Ala                 405 410 415 Tyr Arg Ala Cys Met Asn Met Gly Thr Leu Ser Gly Ala Pro Lys Val             420 425 430 Arg Ala Met Gln Leu Ile Ala Glu Ala Glu Gly Arg Arg Arg Gly Ser         435 440 445 Tyr Gly Gly Ala Val Gly Tyr Phe Thr Ala His Gly Asp Leu Asp Thr     450 455 460 Cys Ile Val Ile Arg Ser Ala Leu Val Glu Asn Gly Ile Ala Thr Val 465 470 475 480 Gln Ala Gly Ala Gly Val Val Leu Asp Ser Val Pro Gln Ser Glu Ala                 485 490 495 Asp Glu Thr Arg Asn Lys Ala Arg Ala Val Leu Arg Ala Ile Ala Thr             500 505 510 Ala His His Ala Gln Glu Thr Phe         515 520 <210> 45 <211> 531 <212> PRT <213> Artificial Sequence <220> <223> TrpD-eco <400> 45 Met Ala Asp Ile Leu Leu Leu Asp Asn Ile Asp Ser Phe Thr Tyr Asn   1 5 10 15 Leu Ala Asp Gln Leu Arg Ser Asn Gly His Asn Val Val Ile Tyr Arg              20 25 30 Asn His Ile Pro Ala Gln Thr Leu Ile Glu Arg Leu Ala Thr Met Ser          35 40 45 Asn Pro Val Leu Met Leu Ser Pro Gly Pro Gly Val Pro Ser Glu Ala      50 55 60 Gly Cys Met Pro Glu Leu Leu Thr Arg Leu Arg Gly Lys Leu Pro Ile  65 70 75 80 Ile Gly Ile Cys Leu Gly His Gln Ala Ile Val Glu Ala Tyr Gly Gly                  85 90 95 Tyr Val Gly Gln Ala Gly Glu Ile Leu His Gly Lys Ala Ser Ser Ile             100 105 110 Glu His Asp Gly Gln Ala Met Phe Ala Gly Leu Thr Asn Pro Leu Pro         115 120 125 Val Ala Arg Tyr His Ser Leu Val Gly Ser Asn Ile Pro Ala Gly Leu     130 135 140 Thr Ile Asn Ala His Phe Asn Gly Met Val Met Ala Val Arg His Asp 145 150 155 160 Ala Asp Arg Val Cys Gly Phe Gln Phe His Pro Glu Ser Ile Leu Thr                 165 170 175 Thr Gln Gly Ala Arg Leu Leu Glu Gln Thr Leu Ala Trp Ala Gln Gln             180 185 190 Lys Leu Glu Pro Ala Asn Thr Leu Gln Pro Ile Leu Glu Lys Leu Tyr         195 200 205 Gln Ala Gln Thr Leu Ser Gln Gln Glu Ser His Gln Leu Phe Ser Ala     210 215 220 Val Val Arg Gly Glu Leu Lys Pro Glu Gln Leu Ala Ala Ala Leu Val 225 230 235 240 Ser Met Lys Ile Arg Gly Glu His Pro Asn Glu Ile Ala Gly Ala Ala                 245 250 255 Thr Ala Leu Leu Glu Asn Ala Ala Pro Phe Pro Arg Pro Asp Tyr Leu             260 265 270 Phe Ala Asp Ile Val Gly Thr Gly Gly Asp Gly Ser Asn Ser Ile Asn         275 280 285 Ile Ser Thr Ala Ser Ala Phe Val Ala Ala Ala Cys Gly Leu Lys Val     290 295 300 Ala Lys His Gly Asn Arg Ser Val Ser Ser Lys Ser Gly Ser Ser Asp 305 310 315 320 Leu Leu Ala Ala Phe Gly Ile Asn Leu Asp Met Asn Ala Asp Lys Ser                 325 330 335 Arg Gln Ala Leu Asp Glu Leu Gly Val Cys Phe Leu Phe Ala Pro Lys             340 345 350 Tyr His Thr Gly Phe Arg His Ala Met Pro Val Arg Gln Gln Leu Lys         355 360 365 Thr Arg Thr Leu Phe Asn Val Leu Gly Pro Leu Ile Asn Pro Ala His     370 375 380 Pro Pro Leu Ala Leu Ile Gly Val Tyr Ser Pro Glu Leu Val Leu Pro 385 390 395 400 Ile Ala Glu Thr Leu Arg Val Leu Gly Tyr Gln Arg Ala Ala Val Val                 405 410 415 His Ser Gly Gly Met Asp Glu Val Ser Leu His Ala Pro Thr Ile Val             420 425 430 Ala Glu Leu His Asp Gly Glu Ile Lys Ser Tyr Gln Leu Thr Ala Glu         435 440 445 Asp Phe Gly Leu Thr Pro Tyr His Gln Glu Gln Leu Ala Gly Gly Thr     450 455 460 Pro Glu Glu Asn Arg Asp Ile Leu Thr Arg Leu Leu Gln Gly Lys Gly 465 470 475 480 Asp Ala Ala His Glu Ala Ala Val Ala Ala Asn Val Ala Met Leu Met                 485 490 495 Arg Leu His Gly His Glu Asp Leu Gln Ala Asn Ala Gln Thr Val Leu             500 505 510 Glu Val Leu Arg Ser Gly Ser Ala Tyr Asp Arg Val Thr Ala Leu Ala         515 520 525 Ala Arg Gly     530 <210> 46 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> TrpC-eco <400> 46 Met Gln Thr Val Leu Ala Lys Ile Val Ala Asp Lys Ala Ile Trp Val   1 5 10 15 Glu Ala Arg Lys Gln Gln Gln Pro Leu Ala Ser Phe Gln Asn Glu Val              20 25 30 Gln Pro Ser Thr Arg His Phe Tyr Asp Ala Leu Gln Gly Ala Arg Thr          35 40 45 Ala Phe Ile Leu Glu Cys Lys Lys Ala Ser Pro Ser Lys Gly Val Ile      50 55 60 Arg Asp Asp Phe Asp Pro Ala Arg Ile Ala Ala Ile Tyr Lys His Tyr  65 70 75 80 Ala Ser Ala Ile Ser Val Leu Thr Asp Glu Lys Tyr Phe Gln Gly Ser                  85 90 95 Phe Asn Phe Leu Pro Ile Val Ser Gln Ile Ala Pro Gln Pro Ile Leu             100 105 110 Cys Lys Asp Phe Ile Ile Asp Pro Tyr Gln Ile Tyr Leu Ala Arg Tyr         115 120 125 Tyr Gln Ala Asp Ala Cys Leu Leu Met Leu Ser Val Leu Asp Asp Asp     130 135 140 Gln Tyr Arg Gln Leu Ala Ala Val Ala His Ser Leu Glu Met Gly Val 145 150 155 160 Leu Thr Glu Val Ser Asn Glu Glu Glu Gln Glu Arg Ala Ile Ala Leu                 165 170 175 Gly Ala Lys Val Val Gly Ile Asn Asn Arg Asp Leu Arg Asp Leu Ser             180 185 190 Ile Asp Leu Asn Arg Thr Arg Glu Leu Ala Pro Lys Leu Gly His Asn         195 200 205 Val Thr Val Ile Ser Glu Ser Gly Ile Asn Thr Tyr Ala Gln Val Arg     210 215 220 Glu Leu Ser His Phe Ala Asn Gly Phe Leu Ile Gly Ser Ala Leu Met 225 230 235 240 Ala His Asp Asp Leu His Ala Ala Val Arg Arg Val Leu Leu Gly Glu                 245 250 255 Asn Lys Val Cys Gly Leu Thr Arg Gly Gln Asp Ala Lys Ala Ala Tyr             260 265 270 Asp Ala Gly Ala Ile Tyr Gly Gly Leu Ile Phe Val Ala Thr Ser Pro         275 280 285 Arg Cys Val Asn Val Glu Gln Ala Gln Glu Val Met Ala Ala Ala Pro     290 295 300 Leu Gln Tyr Val Gly Val Phe Arg Asn His Asp Ile Ala Asp Val Val 305 310 315 320 Asp Lys Ala Lys Val Leu Ser Leu Ala Ala Val Gln Leu His Gly Asn                 325 330 335 Glu Glu Gln Leu Tyr Ile Asp Thr Leu Arg Glu Ala Leu Pro Ala His             340 345 350 Val Ala Ile Trp Lys Ala Leu Ser Val Gly Glu Thr Leu Pro Ala Arg         355 360 365 Glu Phe Gln His Val Asp Lys Tyr Val Leu Asp Asn Gly Gln Gly Gly     370 375 380 Ser Gly Gln Arg Phe Asp Trp Ser Leu Leu Asn Gly Gln Ser Leu Gly 385 390 395 400 Asn Val Leu Leu Ala Gly Gly Leu Gly Ala Asp Asn Cys Val Glu Ala                 405 410 415 Ala Gln Thr Gly Cys Ala Gly Leu Asp Phe Asn Ser Ala Val Glu Ser             420 425 430 Gln Pro Gly Ile Lys Asp Ala Arg Leu Leu Ala Ser Val Phe Gln Thr         435 440 445 Leu Arg Ala Tyr     450 <210> 47 <211> 397 <212> PRT <213> Artificial Sequence <220> <223> trpB-eco <400> 47 Met Thr Thr Leu Leu Asn Pro Tyr Phe Gly Glu Phe Gly Gly Met Tyr   1 5 10 15 Val Pro Gln Ile Leu Met Pro Ala Leu Arg Gln Leu Glu Glu Ala Phe              20 25 30 Val Ser Ala Gln Lys Asp Pro Glu Phe Gln Ala Gln Phe Asn Asp Leu          35 40 45 Leu Lys Asn Tyr Ala Gly Arg Pro Thr Ala Leu Thr Lys Cys Gln Asn      50 55 60 Ile Thr Ala Gly Thr Asn Thr Thr Leu Tyr Leu Lys Arg Glu Asp Leu  65 70 75 80 Leu His Gly Gly Ala His Lys Thr Asn Gln Val Leu Gly Gln Ala Leu                  85 90 95 Leu Ala Lys Arg Met Gly Lys Thr Glu Ile Ile Ala Glu Thr Gly Ala             100 105 110 Gly Gln His Gly Val Ala Ser Ala Leu Ala Ser Ala Leu Leu Gly Leu         115 120 125 Lys Cys Arg Ile Tyr Met Gly Ala Lys Asp Val Glu Arg Gln Ser Pro     130 135 140 Asn Val Phe Arg Met Arg Leu Met Gly Ala Glu Val Ile Pro Val His 145 150 155 160 Ser Gly Ser Ala Thr Leu Lys Asp Ala Cys Asn Glu Ala Leu Arg Asp                 165 170 175 Trp Ser Gly Ser Tyr Glu Thr Ala His Tyr Met Leu Gly Thr Ala Ala             180 185 190 Gly Pro His Pro Tyr Pro Thr Ile Val Arg Glu Phe Gln Arg Met Ile         195 200 205 Gly Glu Glu Thr Lys Ala Gln Ile Leu Glu Arg Glu Gly Arg Leu Pro     210 215 220 Asp Ala Val Ile Ala Cys Val Gly Gly Gly Ser Asn Ala Ile Gly Met 225 230 235 240 Phe Ala Asp Phe Ile Asn Glu Thr Asn Val Gly Leu Ile Gly Val Glu                 245 250 255 Pro Gly Gly His Gly Ile Glu Thr Gly Glu His Gly Ala Pro Leu Lys             260 265 270 His Gly Arg Val Gly Ile Tyr Phe Gly Met Lys Ala Pro Met Met Gln         275 280 285 Thr Glu Asp Gly Gln Ile Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu     290 295 300 Asp Phe Pro Ser Val Gly Pro Gln His Ala Tyr Leu Asn Ser Thr Gly 305 310 315 320 Arg Ala Asp Tyr Val Ser Ile Thr Asp Asp Glu Ala Leu Glu Ala Phe                 325 330 335 Lys Thr Leu Cys Leu His Glu Gly Ile Ile Pro Ala Leu Glu Ser Ser             340 345 350 His Ala Leu Ala His Ala Leu Lys Met Met Arg Glu Asn Pro Asp Lys         355 360 365 Glu Gln Leu Leu Val Val Asn Leu Ser Gly Arg Gly Asp Lys Asp Ile     370 375 380 Phe Thr Val His Asp Ile Leu Lys Ala Arg Gly Glu Ile 385 390 395 <210> 48 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> TrpA-eco <400> 48 Met Glu Arg Tyr Glu Ser Leu Phe Ala Gln Leu Lys Glu Arg Lys Glu   1 5 10 15 Gly Ala Phe Val Pro Phe Val Thr Leu Gly Asp Pro Gly Ile Glu Gln              20 25 30 Ser Leu Lys Ile Ile Asp Thr Leu Ile Glu Ala Gly Ala Asp Ala Leu          35 40 45 Glu Leu Gly Ile Pro Phe Ser Asp Pro Leu Ala Asp Gly Pro Thr Ile      50 55 60 Gln Asn Ala Thr Leu Arg Ala Phe Ala Ala Gly Val Thr Pro Ala Gln  65 70 75 80 Cys Phe Glu Met Leu Ala Leu Ile Arg Gln Lys His Pro Thr Ile Pro                  85 90 95 Ile Gly Leu Leu Met Tyr Ala Asn Leu Val Phe Asn Lys Gly Ile Asp             100 105 110 Glu Phe Tyr Ala Gln Cys Glu Lys Val Gly Val Asp Ser Val Leu Val         115 120 125 Ala Asp Val Pro Val Glu Glu Ser Ala Pro Phe Arg Gln Ala Ala Leu     130 135 140 Arg His Asn Val Ala Pro Ile Phe Ile Cys Pro Pro Asn Ala Asp Asp 145 150 155 160 Asp Leu Leu Arg Gln Ile Ala Ser Tyr Gly Arg Gly Tyr Thr Tyr Leu                 165 170 175 Leu Ser Arg Ala Gly Val Thr Gly Ala Glu Asn Arg Ala Ala Leu Pro             180 185 190 Leu Asn His Leu Val Ala Lys Leu Lys Glu Tyr Asn Ala Ala Pro Pro         195 200 205 Leu Gln Gly Phe Gly Ile Ser Ala Pro Asp Gln Val Lys Ala Ala Ile     210 215 220 Asp Ala Gly Ala Ala Gly Ala Ile Ser Gly Ser Ala Ile Val Lys Ile 225 230 235 240 Ile Glu Gln His Ile Asn Glu Pro Glu Lys Met Leu Ala Ala Leu Lys                 245 250 255 Val Phe Val Gln Pro Met Lys Ala Ala Thr Arg Ser             260 265

Claims (8)

L-enhanced expression of a gene encoding ansranilic acid synthase with an N-terminus consisting of the amino acid sequence of SEQ ID 32, with release of feedback inhibition, and a gene encoding tryptophan operon and transketorase comprising the same A microorganism of the genus Corynebacterium that produces tryptophan.
The method of claim 1, wherein the enhanced tryptophan operon expression corynebacterium microorganisms producing L- tryptophan, wherein the promoter of tryptophan operon is substituted with a promoter with increased expression activity compared to the intrinsic promoter.
The microorganism of claim 1, wherein the microorganism further contains a gene encoding a feedback released anthranilic acid synthetase consisting of an amino acid sequence of SEQ ID NO: 33 and E. coli-derived tryptophan operon including the same, and enhanced expression. A microorganism of the genus Corynebacterium that produces L-tryptophan.
The microorganism of claim 3, wherein the E. coli-derived tryptophan operon produces L-tryptophan, wherein the promoter of tryptophan operon is replaced with a promoter having increased expression activity compared to the intrinsic promoter.
According to claim 1, wherein the microorganism is Corynebacterium glutamicum Corynebacterium microorganisms for producing L-tryptophan.
L-tryptophan with enhanced expression of a gene encoding ansranilic acid synthase with an N-terminus consisting of the amino acid sequence of SEQ ID 32, with release of feedback inhibition, and a tryptophan operon and transketolase comprising the same Culturing the Corynebacterium microorganism producing the medium in the medium; And recovering L-tryptophan from the cultured microorganism or medium.
The method according to claim 6, wherein the microorganism is further modified to express a gene encoding the feedback released anthranilic acid synthase consisting of the amino acid sequence of SEQ ID NO: 33 and E. coli-derived tryptophan operon containing the same Tryptophan production method.
The method of claim 6, wherein the microorganism is Corynebacterium glutamicum.