CN112522222B

CN112522222B - Novel tryptophan hydroxylase mutant and application thereof

Info

Publication number: CN112522222B
Application number: CN202011547701.9A
Authority: CN
Inventors: 赵云现; 杨志彬; 李爱涛; 赵晶; 展全乐; 崔金旺
Original assignee: Hebei Weidakang Biotechnology Co ltd
Current assignee: Hebei weidakang Biotechnology Co.,Ltd.
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-12-07
Anticipated expiration: 2040-12-24
Also published as: CN112522222A

Abstract

The invention discloses a novel tryptophan hydroxylase mutant and application thereof, belonging to the technical field of biology. The amino acid sequence of the tryptophan hydroxylase mutant is represented by SEQ ID NO: 1, mutation formation of the sequence shown in the specification; the site of the sequence mutation is selected from one or more of the following amino acid residue sites: 185 bits, 195 bits, 197 bits, 200 bits, 217 bits, 316 bits, 357 bits, 396 bits, 398 bits, 399 bits, 446 bits, and/or 447 bits. According to the invention, the 5-hydroxytryptophan can be obtained by constructing the recombinant engineering bacteria of the tryptophan hydroxylase 2(TPH2) mutant, fermenting and catalyzing by tryptophan, and compared with wild engineering bacteria, the yield of 5-hydroxytryptophan generated by catalyzing tryptophan by the tryptophan hydroxylase mutant is obviously improved, so that a basis is provided for reducing the using amount of the catalyst and finally reducing the production cost, and further the industrial production of the 5-hydroxytryptophan is favorably realized.

Description

Novel tryptophan hydroxylase mutant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a novel tryptophan hydroxylase mutant and application thereof.

Background

5-hydroxytryptophan (5-hydroxytryptophan, 5-HTP) was discovered in 1958 as an amino acid derivative formed by oxidation of tryptophan (tryptophan, one of the essential amino acids in the human body) by tryptophan hydroxylase (TPH, also known as tryptophan monooxygenase, tryptophan 5-monooxygenase).

5-hydroxytryptophan can generate neurotransmitter 5-hydroxytryptamine (serotonin, 5-HT, also called serotonin) through decarboxylation in human metabolism, and has the effects of relieving symptoms of depression, insomnia, inappetence, weight disorder and the like in a human body. 5-hydroxytryptamine is distributed in large amounts in the brain and nerves and is a precursor of melatonin synthesis. In humans, melatonin is associated with sleep. In the neurology medicine, the action mechanism of Prozac is to slow down the consumption of 5-hydroxytryptophan, so that 5-hydroxytryptophan is continuously produced by human body, and the mood and the sleep of the patient are kept normal. On the other hand, direct intake of more 5-hydroxytryptophan is more effective and healthy. In addition, 5-hydroxytryptophan can not cause dry mouth and hyposexuality caused by antidepressant drug. Therefore, 5-hydroxytryptophan can be a natural substitute of the medicine, and the high-efficiency synthesis of the medicine is particularly important in modern medicine and is widely concerned by people.

5-hydroxytryptophan is abundantly present in seeds of the Gardnerella tree. The leaves and seeds of the Gardner tree have been used as medicaments for treating wounds, nephropathy, enema and the like by people in Africa since ancient times, the ointment prepared from the bark of the Gardner tree can also treat skin diseases, and the main active ingredient of the ointment is 5-hydroxytryptophan. 5-hydroxytryptophan used in the pharmaceutical industry at present is mostly extracted from gana seeds, and is extracted by a hydrothermal method, an alcohol method or an ultrasonic method. Liu Dai Lin et al adopt resin adsorption method to separate 5-hydroxytryptophan from Griffonia simplicifolia seed, the extraction rate is about 7 times of unit content in raw material. However, as the number of African gardner trees is decreasing, it becomes more and more difficult to obtain 5-hydroxytryptophan by traditional extraction methods, which also forces people to find an alternative method for synthesizing 5-hydroxytryptophan.

With the continuous decrease of the resources such as Gardner and the like and the continuous emphasis of people on the green and environment-friendly synthesis process, the biological catalysis or fermentation method for synthesizing 5-hydroxytryptophan by catalyzing tryptophan by tryptophan hydroxylase gradually draws attention of people. The synthesis efficiency of tryptophan in engineering bacteria is improved by adopting genetic engineering and metabolic engineering methods, and 5-hydroxytryptophan is obtained by catalysis of tryptophan hydroxylase.

Two different tryptophan hydroxylase genes exist in humans, located on chromosomes 11 and 12, respectively, and encode two proteins (TPH1 and TPH 2). TPH1 is a highly homologous protein to TPH2 with 71% sequence identity. TPH1 is expressed mainly in peripheral tissues (periphery) and pineal, whereas TPH2 is expressed mainly in the brain.

Studies report that overexpression of TPH1 or TPH2 in E.coli can catalyze tryptophan to synthesize 5-hydroxytryptophan. However, due to poor solubility expression of tryptophan hydroxylase in escherichia coli, low hydroxylase activity and the like, the yield of 5-hydroxytryptophan is low, and the requirement of industrial production cannot be met. The tryptophan hydroxylase is used as a key enzyme for catalyzing tryptophan to produce 5-hydroxytryptophan, the activity of the tryptophan hydroxylase is improved, the yield of 5-hydroxytryptophan can be improved, the using amount of a catalyst is reduced, or the reaction time is shortened, so that the production cost is reduced, and the industrial production of 5-hydroxytryptophan is greatly influenced.

Disclosure of Invention

The invention aims to provide a novel tryptophan hydroxylase mutant and application thereof, aiming at solving the problems in the prior art, the tryptophan hydroxylase mutant is obtained by mutating a specific site of a wild-type tryptophan hydroxylase, and the yield of 5-hydroxytryptophan produced by catalyzing tryptophan by using the mutant is obviously improved.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a tryptophan hydroxylase mutant, which comprises an amino acid sequence as shown in any one of the following items:

a: the amino acid sequence of the tryptophan hydroxylase mutant is represented by SEQ ID NO: 1, mutating the sequence shown in the figure; the site of the sequence mutation is selected from one or more of the following amino acid residue sites: 185 bits, 195 bits, 197 bits, 200 bits, 217 bits, 316 bits, 357 bits, 396 bits, 398 bits, 399 bits, 446 bits, and/or 447 bits;

b: the amino acid sequence of the tryptophan hydroxylase mutant has at least 95 percent of sequence identity with the amino acid sequence of a;

further, the amino acid sequence of the tryptophan hydroxylase mutant has more than 98 percent of sequence identity with the amino acid sequence of the a; more preferably, the tryptophan hydroxylase mutant has an amino acid sequence that has 99% or more sequence identity with the amino acid sequence of a;

c: the amino acid sequence of the tryptophan hydroxylase mutant is formed by substituting, adding or deleting one or more amino acid residues at the C terminal and/or the N terminal of the amino acid sequence of a;

further, the amino acid sequence of the tryptophan hydroxylase mutant is formed by adding or deleting 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, and most preferably 1 to 3 amino acid residues to or from the C-terminal and/or N-terminal of the amino acid sequence described in a.

All the amino acid mutants have the amino acid sequences shown as SEQ ID NO: 1 to catalyze the generation of 5-hydroxytryptophan from tryptophan.

Preferably, the sequence mutation in a is selected from the following amino acid residue position mutation and the amino acid residue position mutation is shown as follows:

185 bits: pro, Ala;

195 bits: ala, Val, Leu, Ile;

197 bit: ile, Leu, Val, Met, Ala, Phe;

200 bits: gln, Asn;

217 bit: ile, Leu, Val, Met, Ala, Phe;

316 bit: cys;

357 bit: ile, Leu, Val, Met, Ala, Phe;

396: glu and Asp;

398 bits: arg and Lys;

399 bits: pro;

446 bit: arg and Lys;

447 bits: tyr, Trp, Phe.

185 bits: pro;

195 bits: ala;

197 bit: ile;

200 bits: gln;

217 bit: ile;

316 bit: cys;

357 bit: ile;

396: glu;

398 bits: arg;

399 bits: pro;

446 bit: arg;

447 bits: tyr.

The invention also provides a gene for coding the tryptophan hydroxylase mutant.

The invention also provides a recombinant vector containing the coding gene of the tryptophan hydroxylase mutant.

The invention also provides a recombinant gene engineering bacterium, which is obtained by transferring the recombinant vector into a host cell.

Preferably, said host cell comprises said recombinant vector or its genome into which said tryptophan hydroxylase mutant encoding gene has been integrated.

The invention also provides the application of the tryptophan hydroxylase mutant, the recombinant vector or the genetic engineering bacterium in producing the tryptophan hydroxylase.

The invention also provides the application of the tryptophan hydroxylase mutant, the recombinant vector or the genetic engineering bacterium in producing 5-hydroxytryptophan.

The invention also provides a method for producing 5-hydroxytryptophan, which comprises the following steps:

1) producing 5-hydroxytryptophan by using the tryptophan hydroxylase mutant or the recombinant genetic engineering bacteria;

2) 5-hydroxytryptophan is separated from the system in 1).

The invention discloses the following technical effects:

the invention discloses a novel tryptophan hydroxylase mutant which is prepared by carrying out the following steps of: 1 to obtain a plurality of mutants, and the obtained mutants have the amino acid sequences similar to those shown in SEQ ID NO: the function of the wild-type tryptophan hydroxylase shown in the formula 1 for catalyzing tryptophan to produce 5-hydroxytryptophan.

The invention also discloses a recombinant vector containing the coding gene of the tryptophan hydroxylase mutant and recombinant gene engineering bacteria. And expressing the gene coding the tryptophan hydroxylase mutant in the recombinant vector by a fermentation culture mode to obtain the tryptophan hydroxylase. The process is easy to implement, the conditions are easy to control, and the method is suitable for popularization of industrial production.

The inventor finds that the tryptophan hydroxylase mutant with improved activity can be obtained by mutating a specific site of the wild-type tryptophan hydroxylase, and experiments prove that the yield of 5-hydroxytryptophan produced by the tryptophan hydroxylase mutant can be improved by 1.4 times compared with the wild-type tryptophan hydroxylase, so that the invention can achieve the purposes of reducing the catalyst dosage and finally reducing the production cost, and simultaneously provides a basis for developing excellent tryptophan hydroxylase and promoting the industrial production of 5-hydroxytryptophan.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a map of the pET28a-TPH2 expression plasmid;

FIG. 2 shows the result of PCR-verified transformation of TPH2 wild-type engineering bacteria; lane 1: DNA Marker, lane 2: PCR verification of TPH2 wild type engineering strain;

FIG. 3 shows the result of PCR-verified transformation of the recombinant engineered bacteria of TPH2 mutant; lane 1: DNA Marker; lane 2: mutant 1; lane 3: mutant 2; lane 4: mutant 3; lane 5: mutant 4; lane 6: mutant 5; lane 7: mutant 6; lane 8: mutant 7; lane 9: mutant 8; lane 10: mutant 9;

FIG. 4 is an L-tryptophan standard HPLC chromatogram;

FIG. 5 is an HPLC chromatogram of 5-hydroxytryptophan standard.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

It is known to the person skilled in the art that if an enzyme is mutated in order to obtain a mutant with improved activity, it is crucial to find a site where the activity can be improved after the mutation. In the present invention, the amino acid sequence is as shown in SEQ ID NO: 1, to obtain a tryptophan hydroxylase mutant with improved activity.

The inventor has found that in SEQ ID NO: 1 can improve the yield of 5-hydroxytryptophan by mutating at one or more of the following sites of the amino acid sequence shown in the specification: 185 bits, 195 bits, 197 bits, 200 bits, 217 bits, 316 bits, 357 bits, 396 bits, 398 bits, 399 bits, 446 bits, or/and 447 bits.

In the present invention, the terms "tryptophan hydroxylase" or "tryptophan hydroxylase mutant" or "tryptophan hydroxylase of the invention" are used with the same meaning, and are used interchangeably herein, to mean a polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 1, and the tryptophan hydroxylase mutant obtained by mutating at one or more of the above-mentioned sites is a tryptophan hydroxylase having an activity of catalyzing the production of 5-hydroxytryptophan from tryptophan and having an increased 5-hydroxytryptophan production.

In view of the teachings of the present invention and the prior art, it will also be apparent to those skilled in the art that "tryptophan hydroxylase mutants of the present invention" shall also include variants thereof that have the same or similar function as the "tryptophan hydroxylase mutants of the present invention" but differ in amino acid sequence by a small amount from the tryptophan hydroxylase amino acid sequence of the examples of the present invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 30, preferably 1 to 10, more preferably 1 to 6, still more preferably 1 to 3, most preferably 1) amino acids, and addition of one or more (usually up to 30, preferably up to 10, more preferably up to 6 or 3) amino acids at the C-terminus and/or N-terminus. For example, it is well known to those skilled in the art that substitutions with amino acids of similar or analogous properties, e.g., isoleucine and leucine, do not alter the function of the resulting protein. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus, such as a 6-His tag added for ease of isolation, will not generally alter the function of the resulting protein.

It will also be understood by those skilled in the art that the variant forms of "tryptophan hydroxylase of the invention" described herein do not include reversion to the wild-type tryptophan hydroxylase by mutation; in other words, the mutant forms of the tryptophan hydroxylase mutants of the present invention are obtained by further mutation based on the tryptophan hydroxylase mutants obtained in the examples of the present invention, and correspond to the amino acid sequences shown in SEQ ID NO: 1, the amino acid residues at positions 185, 195, 197, 200, 217, 316, 357, 396, 398, 399, 446 and/or 447 are the same as those in the amino acid sequence of the tryptophan hydroxylase obtained in the examples of the present invention.

The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" means a position in two sequences that corresponds to a specified position in the other sequence after alignment by homology or sequence identity. Thus, if a 6-His tag is added to one end of the amino acid sequence of the tryptophan hydroxylase obtained in the examples of the present invention, the mutant obtained has a sequence corresponding to SEQ ID NO: 1 may be position 191 of the amino acid sequence shown in 1.

In particular embodiments, the homology or sequence identity may be 90% or more, preferably 95% to 98%, most preferably 99% or more.

Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: computer Molecular Biology (computerized Molecular Biology), Lesk, a.m. ed, oxford university press, new york, 1988; biological calculation: informatics and genomic Projects (Biocomputing: information and Genome Projects), Smith, d.w. eds, academic press, new york, 1993; computer Analysis of Sequence Data (Computer Analysis of Sequence Data), first part, Griffin, a.m. and Griffin, h.g. eds, Humana Press, new jersey, 1994; sequence Analysis in MolecuLar Biology (Sequence Analysis in MolecuLar Lar Biology), von Heinje, G., academic Press, 1987 and Sequence Analysis primers (Sequence Analysis Primer), Gribskov, M. and Devereux, J. eds M Stockton Press, New York, 1991 and Carllo, H. and Lipman, D., SIAM J. applied Math.48: 1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: GCG package (Devereux, J. et al, 1984), BLAST, Muscle, MAFFT and Clustal. The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altsch. mu.L, S. et al, NCBI NLM NIH Bethesda, Md.20894; Altsch. mu.L, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Variants of the polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with DNA encoding a "tryptophan hydroxylase mutant of the invention". The invention also includes other polypeptides, such as fusion proteins comprising a "tryptophan hydroxylase mutant of the invention" or a fragment thereof. In addition to almost full-length polypeptides, the invention also encompasses active fragments of the "tryptophan hydroxylase mutants of the invention". Typically, the fragment has at least about 20 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the amino acid sequence of the "tryptophan hydroxylase mutant of the invention".

The invention also provides analogs of "tryptophan hydroxylase". These analogs may differ from the native "tryptophan hydroxylase mutants of the invention" by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide such as acetylation, glycosylation or carboxylation, in vivo or in vitro. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, a conservative variant polypeptide of "tryptophan hydroxylase" refers to a polypeptide in which at most 20, preferably at most 10, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties, as compared with the amino acid sequence of the tryptophan hydroxylase mutant in the present embodiment, but the conservative variant polypeptide still has the same or similar activity as the tryptophan hydroxylase mutant in the present embodiment, i.e., the activity of catalyzing tryptophan to produce 5-hydroxytryptophan, and the yield of 5-hydroxytryptophan is significantly increased.

Thus, in view of the teachings of the present invention and the prior art, one skilled in the art can generate conservatively variant mutants by making amino acid substitutions as shown, for example, in Table 1 below.

TABLE 1

In view of this, in a specific embodiment, the amino acid sequence of the tryptophan hydroxylase of the invention has the following amino acid residues at one or more positions selected from the group consisting of:

185 bits: pro, Ala; 195 bits: ala, Val, Leu, Ile; 197 bit: ile, Leu, Val, Met, Ala, Phe; 200 bits: gln, Asn; 217 bit: ile, Leu, Val, Met, Ala, Phe; 316 bit: cys; 357 bit: ile, Leu, Val, Met, Ala, Phe; 396: glu and Asp; 398 bits: arg and Lys; 399 bits: pro; 446 bit: arg and Lys; 447 bits: tyr, Trp, Phe. In a preferred embodiment, the amino acid sequence of the tryptophan hydroxylase of the invention has the following amino acid residues at one or more positions selected from the group consisting of: 185 bits: pro; 195 bits: ala; 197 bit: ile; 200 bits: gln; 217 bit: ile; 316 bit: cys; 357 bit: ile; 396: glu; 398 bits: arg; 399 bits: pro; 446 bit: arg; 447 bits: tyr.

The protein of the present invention may be a recombinant protein, a natural protein, a synthetic protein, preferably a recombinant protein. The proteins of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells). Depending on the host used in the recombinant production protocol, the protein of the invention may be glycosylated or may be non-glycosylated. The proteins of the invention may or may not also include an initial methionine residue.

It will be understood by those skilled in the art that the "tryptophan hydroxylase mutants" of the present invention also include fragments, derivatives and analogs of the "tryptophan hydroxylase mutants". A fragment, derivative or analogue of a polypeptide of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

In view of the prior art in this field and the teaching of the present invention, the active fragment of the tryptophan hydroxylase of the invention can be easily obtained by those skilled in the art. For example, a biologically active fragment of a "tryptophan hydroxylase mutant" is defined herein as a fragment of a "tryptophan hydroxylase mutant" that retains all or part of the function of the full-length "tryptophan hydroxylase mutant". Typically, the biologically active fragment retains at least 50% of the activity of the full-length "tryptophan hydroxylase mutant". Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99% or 100% of the activity of the full-length "tryptophan hydroxylase mutant".

Based on the teaching of the present invention and the prior art, those skilled in the art can also understand that the tryptophan hydroxylase of the present invention can be prepared into other utilization forms such as immobilized enzymes.

The invention also provides a polynucleotide sequence encoding the tryptophan hydroxylase mutant or a degenerate variant thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the nucleotide sequence encoding the tryptophan hydroxylase mutants of the embodiments of the invention or degenerate variants. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a tryptophan hydroxylase mutant according to the claims of the present invention, but differs from the nucleotide sequence encoding the tryptophan hydroxylase mutant in the examples of the present invention.

In the present invention, the polynucleotide sequence encoding the "tryptophan hydroxylase mutant" may be inserted into a recombinant expression vector or genome. The term "expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

The skilled person can use well-known methods to construct expression vectors comprising DNA sequences encoding "tryptophan hydroxylase mutants" and appropriate transcription/translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cells described herein include host cells comprising the above-described recombinant vector or having integrated on its genome the coding sequence of the "tryptophan hydroxylase mutant" of the invention. The host cell or the strain can efficiently express the novel tryptophan hydroxylase with high catalytic performance, thereby improving the level of producing 5-hydroxytryptophan.

The host cell of the invention may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells. In particular embodiments, the strains include, but are not limited to: coli (e), Corynebacterium glutamicum (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis). In a preferred embodiment, the strain is escherichia coli (e.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation may also be usedThe method of electroporation is performed. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. The recombinant polypeptide in the above method may be constitutively expressed or conditionally expressed, for example: after the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell disruption by osmosis, sonication, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

In view of the teachings of the present invention and the prior art, one of ordinary skill in the art will appreciate that the tryptophan hydroxylase of the present invention, as well as the coding sequence, expression vector, and host cell thereof, can be used to catalyze the decarboxylation of tryptophan to produce 5-hydroxytryptophan.

On the basis, the invention also provides a method for catalyzing the decarboxylation of tryptophan to generate 5-hydroxytryptophan by using the tryptophan hydroxylase, the expression vector or the host cell. For example, in a specific embodiment, 5-hydroxytryptophan can be produced by culturing a host cell comprising an expression vector of the present invention or a coding sequence of a tryptophan hydroxylase mutant of the present invention integrated on its genome or by using a tryptophan hydroxylase of the present invention to catalyze tryptophan; the resulting 5-hydroxytryptophan is then obtained from the catalytic system.

The tryptophan used for catalyzing the production of the 5-hydroxytryptophan can be the tryptophan produced by the host cell and can also be the tryptophan added from an external source.

The inventors have extensively and deeply studied and unexpectedly found that mutation of a specific site of a wild-type tryptophan hydroxylase can result in a tryptophan hydroxylase mutant with improved activity, thereby providing a basis for development of an excellent tryptophan hydroxylase and further production of 5-hydroxytryptophan. The present invention has been completed based on this finding. The acquisition and use of the tryptophan hydroxylase mutants will be described below with reference to specific examples.

EXAMPLE 1 construction of TPH2 wild-type Strain

The human TPH2 gene was codon optimized, chemically synthesized by biologies (shanghai), and TPH2 gene was inserted between NdeI and XhoI sites of pET28a (+) plasmid by biologies (shanghai) to obtain pET28a-TPH2 expression vector (as shown in fig. 1), which was transformed into e.coli DH5 α. Through sequencing verification, plasmids in positive clones are extracted and transformed into E.coli BL21(DE3), and E.coli BL21(DE3)/pET28a-TPH2 wild-type engineering strains are obtained.

And (3) strain identification: after the wild type engineered strain E.coli BL21(DE3)/pET28a-TPH2 was activated by a solid LB medium plate, a single colony was picked up and inoculated into a 500mL seed bottle containing 100mL of a liquid LB medium (peptone 1%, yeast powder 0.5%, sodium chloride 1%, pH 7.0), 50mg/L kanamycin was added, shaking-cultured at 37 ℃ and 220rpm overnight, and identified by seed PCR.

PCR amplification System (10. mu.L): 1 μ L of bacterial liquid, 0.5 μ L of upstream and downstream primers, 5 μ L of 2 XT 5 SuperPCR Mix (Colony) (purchased from Beijing Okagaku), ddH₂O 3μL。

The PCR reaction conditions were 98 ℃ for 3min, 28 cycles (98 ℃ for 10s, 58 ℃ for 10s, 72 ℃ for 15s), and 72 ℃ for 3 min.

Wherein, the primer sequence is as follows:

an upstream primer F: 5'-ATGCAACCGGCCATGATGATG-3', respectively;

a downstream primer R: 5'-TTAGATACCAAGATATTGGTTCATTTTGTTCAGCG-3' are provided.

As shown in FIG. 2, the results showed that the foreign gene TPH2 was successfully transferred into the wild-type engineered strain.

Example 2 obtaining of TPH2 mutant Strain

Through rational analysis and design of TPH2 gene (shown as SEQ ID NO: 14), 9 single-point or double-point mutants are determined, site-directed mutagenesis is carried out on TPH2 by adopting a homologous recombination method (shown as table 2), each mutant is designed with a pair of primers, constructed pET28a-TPH2 wild-type plasmid is used as a template, corresponding primers are respectively adopted for PCR amplification (shown as table 3), and the amplification products are sequenced and identified to respectively obtain 9 mutant sequences such as SEQ ID NO: 15-SEQ ID NO: 23. after gene expression, the 185 th asparagine of TPH2 is mutated into proline, the 195 th valine is mutated into alanine, the 197 th valine is mutated into isoleucine, the 200 th glycine is mutated into asparagine, the 217 th threonine is mutated into isoleucine, the 316 th threonine is mutated into cysteine, the 357 th cysteine is mutated into isoleucine, the 396 th cysteine is mutated into glutamic acid, the 398 th lysine is mutated into arginine, the 399 th alanine is mutated into proline, the 446 th tyrosine is mutated into arginine, and the 447 th phenylalanine is mutated into tyrosine.

PCR amplification System (50. mu.L): mu.L of template, 2.5. mu.L of each of the upstream and downstream primers, 5. mu.L of dNTPs, 5. mu.L of 10 XKOD Buffer, 0.5. mu.L of KOD DNA polymerase, ddH₂O 38.5μL。

The PCR reaction conditions are as follows: 95 ℃ for 5min, 25 cycles (95 ℃ for 30s, 56 ℃ for 30s, 68 ℃ for 30s), 68 ℃ for 10 min.

TABLE 2TPH2 mutants

TABLE 3 primer sequences

Purifying and recovering PCR products by using a glue recovery kit (purchased from Biotech limited of Beijing Ongzhike); and then using the recovered PCR product as a primer to carry out second-step PCR amplification, and digesting the obtained PCR product by using Dpn I to remove the template. Transforming the transformant into E.coli DH5 alpha, sequencing the obtained transformant by Beijing engine family, sequencing a correct plasmid, and transforming the transformant into E.coli BL21(DE3) by a chemical method, which specifically comprises the following steps: (1) take out the stored e.coli BL21(DE3) competent cells, lyse on ice; (2) add 3. mu.L (about 1ng) of the mutant product per tube, mix gently, and stand on ice for 30 min; (3) placing the tube in a constant-temperature water bath at 42 ℃ for heat shock for 45 seconds; taking out the tube and placing on ice for 2 min; 0.5mL of LB medium incubated at 37 ℃ was added to each tube, shaking-cultured at 37 ℃ for 1 hour, and the culture was applied to LB plates with kanamycin resistance and cultured overnight in an inverted state at 37 ℃. Recombinant engineered strains of TPH2 mutant, E.coli BL21(DE3)/pET28-N41P, E.coli BL21(DE3)/pET28-V51A/V53I, E.coli BL21(DE3)/pET28-G56N, E.coli BL21(DE3)/pET28-T73I, E.coli BL21(DE3)/pET 3-T172 3, E.coli BL 3 (DE3)/pET 3-C252 3, E.coli BL 3 (DE3)/pET 3-K3/36255/A36255 and E.coli BL 3 (DE3)/pET 3/3F 303 are obtained respectively.

And (3) strain identification: the recombinant engineered strains of the 9 TPH2 mutants obtained above were strain-identified by the same method as that of PCR identification of the wild-type engineered strain of example 1.

As shown in FIG. 3, the results are shown in FIG. 3, E.coli BL21(DE3)/pET28-N41P, E.coli BL21(DE3)/pET28-V51A/V53I, E.coli BL21(DE3)/pET28-G56N, E.coli BL21(DE3)/pET28-T73I, E.coli BL21(DE3)/pET28-T172 28, E.coli BL 28 (DE 28)/pET 28-C213 28, E.coli BL 28 (DE 28)/pET 28-C252 28, E.coli BL 28 (DE 28)/pET 28-K28/A36255 and E.coli BL 72 (DE 28)/pET 28-K28/F254/A36255/28/A36255, E.coli BL 28 (DE 28)/pET 28-K28/F302/F303, and E.coli BL 28/28, and TPH 6, mutant, and mutant thereof are respectively successfully constructed and the mutants, and the mutants are respectively.

EXAMPLE 3 production of 5-hydroxytryptophan by TPH2 wild-type and mutant recombinant engineered strains

1) Thallus culture of recombinant engineering strain and expression of tryptophan hydroxylase

The recombinant engineering bacteria of each TPH2 mutant obtained in example 2 were taken, and a single colony activated by a solid LB medium plate was inoculated into a 500mL seed bottle containing 100mL of a liquid LB medium (peptone 1%, yeast powder 0.5%, sodium chloride 1%, balance water, pH 7.0), 50mg/L kanamycin was added, and shaking culture was performed at 37 ℃ and 220rpm for 12 hours; 50mg/L kanamycin was added to 500mL of 100mL TB fermentation medium (24 g/L yeast powder, 12g/L peptone, 16.43g/L dipotassium hydrogen phosphate, 2.31g/L potassium dihydrogen phosphate, 5g/L glycerol) at 5% inoculum size, at 37 deg.C, 220rpm, OD₆₀₀When the growth reaches 0.6-0.8, 0.1mM IPTG is supplemented to induce and express tryptophan hydroxylase for 20 h.

2) Production of 5-hydroxytryptophan

The whole cell catalysis is carried out in a 50mL reaction bottle, the concentration of the substrate tryptophan is about 10g/L, the addition amount of the cofactor (5,6,7, 8-tetrahydro-2-amino-6- (1, 2-dihydroxypropyl) -4(1h) -pteridinone) is 1g/L, the addition amount of the recombinant engineering bacteria is 15 percent of the wet weight of the cells, the reaction temperature is 30 ℃, and the reaction time is 2h, so as to obtain the catalytic reaction solution. The catalytic reaction solution was diluted with deionized water by a suitable amount, centrifuged at 12000 Xg for 3min, and the supernatant was filtered through a 0.22 μm organic filter, and then the contents of L-tryptophan and 5-hydroxytryptophan were measured by Shimadzu LAT-20A High Performance Liquid Chromatography (HPLC), and the change in the production of 5-hydroxytryptophan was recorded.

Chromatographic conditions, chromatographic column: shimadzustein C18, 35 ℃; the mobile phase is methanol: 50mM phosphate buffer (pH 3.0) ═ 1: 9; the flow rate is 1.0mL/min, and the sample loading quantity is 10 mu L; the detection wavelength was 275 nm. The peak time of L-tryptophan is 14.0-14.5 (as shown in figure 4), and the peak time of 5-hydroxytryptophan is 5.3-5.7 (as shown in figure 5).

The reaction solution is detected by High Performance Liquid Chromatography (HPLC), and the result shows that the TPH2 mutant recombinant engineering bacteria can obtain 5-hydroxytryptophan (5-HTP) with improved activity after fermentation and tryptophan induction, and the peak-off time is 5.3-5.7. The yield of 5-hydroxytryptophan produced by the wild engineering bacteria and the recombinant engineering bacteria is calculated by adopting an internal standard method, and as shown in table 4, compared with the wild TPH2, the yield of the N41P mutant is 1.1 times that of the wild type; the yield of the V51A/V53I mutant is 1.4 times that of the wild type; the yield of the G56N mutant was 1.1-fold that of the wild type; the yield of the T73I mutant was 1.2 times that of the wild type; the yield of the T172C mutant was 1.2-fold that of the wild type; the yield of the C213I mutant was 1.2-fold that of the wild type; the yield of the C252E mutant was 1.1-fold that of the wild type; the yield of the K254R/A255P mutant is 1.1 times that of the wild type; the yield of the Y302R/F303Y mutant was 1.1-fold higher than that of the wild type.

TABLE 4 production ratios of hydroxytryptophan from different recombinant engineered bacteria

It can be seen that the activity of the tryptophan hydroxylase mutant obtained by mutating the specific site of the wild-type tryptophan hydroxylase is remarkably improved, as shown in table 4, the yield of 5-hydroxytryptophan can be remarkably increased, the yield of 5-HTP produced by tryptophan catalyzed by the constructed recombinant engineering bacterium E.coli BL21(DE3)/pET28-V51A/V53I is remarkably improved and is 1.4 times that of the wild-type tryptophan, and the conversion rate can reach 72.8%. The method provides data support for the industrialization of producing 5-hydroxytryptophan by using tryptophan hydroxylase to catalyze tryptophan, improving the production efficiency and reducing the production cost.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> Baodingiy Biotechnology Ltd

<120> a novel tryptophan hydroxylase mutant and application thereof

<160> 23

<170> SIPOSequenceListing 1.0

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Met Gln Pro Ala Met Met Met Phe Ser Ser Lys Tyr Trp Ala Arg Arg

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Gly Phe Ser Leu Asp Ser Ala Val Pro Glu Glu His Gln Leu Leu Gly

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Ser Ser Thr Leu Asn Lys Pro Asn Ser Gly Lys Asn Asp Asp Lys Gly

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Asn Lys Gly Ser Ser Lys Arg Glu Ala Ala Thr Glu Ser Gly Lys Thr

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Ala Val Val Phe Ser Leu Lys Asn Glu Val Gly Gly Leu Val Lys Ala

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Leu Arg Leu Phe Gln Glu Lys Arg Val Asn Met Val His Ile Glu Ser

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Arg Lys Ser Arg Arg Arg Ser Ser Glu Val Glu Ile Phe Val Asp Cys

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Glu Cys Gly Lys Thr Glu Phe Asn Glu Leu Ile Gln Leu Leu Lys Phe

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Gln Thr Thr Ile Val Thr Leu Asn Pro Pro Glu Asn Ile Trp Thr Glu

130 135 140

Glu Glu Glu Leu Glu Asp Val Pro Trp Phe Pro Arg Lys Ile Ser Glu

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Leu Asp Lys Cys Ser His Arg Val Leu Met Tyr Gly Ser Glu Leu Asp

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Ala Asp His Pro Gly Phe Lys Asp Asn Val Tyr Arg Gln Arg Arg Lys

180 185 190

Tyr Phe Val Asp Val Ala Met Gly Tyr Lys Tyr Gly Gln Pro Ile Pro

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Arg Val Glu Tyr Thr Glu Glu Glu Thr Lys Thr Trp Gly Val Val Phe

210 215 220

Arg Glu Leu Ser Lys Leu Tyr Pro Thr His Ala Cys Arg Glu Tyr Leu

225 230 235 240

Lys Asn Phe Pro Leu Leu Thr Lys Tyr Cys Gly Tyr Arg Glu Asp Asn

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Val Pro Gln Leu Glu Asp Val Ser Met Phe Leu Lys Glu Arg Ser Gly

260 265 270

Phe Thr Val Arg Pro Val Ala Gly Tyr Leu Ser Pro Arg Asp Phe Leu

275 280 285

Ala Gly Leu Ala Tyr Arg Val Phe His Cys Thr Gln Tyr Ile Arg His

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Gly Ser Asp Pro Leu Tyr Thr Pro Glu Pro Asp Thr Cys His Glu Leu

305 310 315 320

Leu Gly His Val Pro Leu Leu Ala Asp Pro Lys Phe Ala Gln Phe Ser

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Gln Glu Ile Gly Leu Ala Ser Leu Gly Ala Ser Asp Glu Asp Val Gln

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Lys Leu Ala Thr Cys Tyr Phe Phe Thr Ile Glu Phe Gly Leu Cys Lys

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Gln Glu Gly Gln Leu Arg Ala Tyr Gly Ala Gly Leu Leu Ser Ser Ile

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Gly Glu Leu Lys His Ala Leu Ser Asp Lys Ala Cys Val Lys Ala Phe

385 390 395 400

Asp Pro Lys Thr Thr Cys Leu Gln Glu Cys Leu Ile Thr Thr Phe Gln

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Glu Ala Tyr Phe Val Ser Glu Ser Phe Glu Glu Ala Lys Glu Lys Met

420 425 430

Arg Asp Phe Ala Lys Ser Ile Thr Arg Pro Phe Ser Val Tyr Phe Asn

435 440 445

Pro Tyr Thr Gln Ser Ile Glu Ile Leu Lys Asp Thr Arg Ser Ile Glu

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Asn Val Val Gln Asp Leu Arg Ser Asp Leu Asn Thr Val Cys Asp Ala

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Leu Asn Lys Met Asn Gln Tyr Leu Gly Ile

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<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ccgggtttta aagatccggt ttatcgtcag cgtcgcaa 38

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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cgtcgcaagt attttgcgga tattgcaatg ggttacaa 38

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<212> DNA

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gttgatgttg caatgaatta caaatacggt cagccgat 38

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tataccgaag aagaaattaa aacctggggt gttgtttt 38

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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actccggaac cggattgctg tcatgaactg ctgggtca 38

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cagaaactgg caaccattta tttctttacc attgaatt 38

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gtccggttgc aggttatctg agt 23

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcggcacatg acccagcagt tcatg 25

<210> 10

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gtgtatacgg attgaaataa acgctaaacg gacggg 36

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<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atcaaatgct ttaacttctg ctttatcgct cagtgcat 38

<210> 12

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggttttcgga tcaaacggac gaacacatgc tttatcgc 38

<210> 13

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctgtgtatac ggattataac gaacgctaaa cggacggg 38

<210> 14

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 15

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag atccggttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 16

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgcggatat tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 17

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgaat 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 18

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaat taaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 19

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggattgctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 20

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaaccat ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 21

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcagaagt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 22

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt tcgtccgttt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgtttattt caatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

<210> 23

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atgcaaccgg ccatgatgat gttcagctcc aaatattggg ctcgtcgtgg tttctctctg 60

gactctgctg ttccggaaga acaccagctg ctgggctcct ctactctgaa caaaccgaac 120

tccggtaaaa acgatgacaa aggcaacaag ggtagctcta aacgcgaagc agccacggag 180

tctggcaaaa ccgcggtagt tttttccctg aagaacgaag tgggcggtct ggttaaagca 240

ctgcgcctgt tccaggagaa acgtgtcaat atggtacaca tcgaaagccg taaatcccgc 300

cgtcgctcct ccgaagtgga aatctttgtt gactgcgaat gtggtaaaac cgaatttaac 360

gagctgattc agctgctgaa attccagacc actattgtca ccctgaaccc accagagaac 420

atctggactg aagaagaaga actggaagat gttccgtggt ttccgcgtaa aattagcgaa 480

ctggataaat gtagccatcg tgttctgatg tatggtagtg aactggatgc agatcatccg 540

ggttttaaag ataatgttta tcgtcagcgt cgcaagtatt ttgttgatgt tgcaatgggt 600

tacaaatacg gtcagccgat tccgcgtgtt gaatataccg aagaagaaac caaaacctgg 660

ggtgttgttt ttcgtgaact gagcaaactg tatccgacac atgcctgtcg tgaatatctg 720

aaaaactttc cgctgctgac caaatattgt ggttatcgtg aagataacgt tccgcagtta 780

gaagatgtta gcatgtttct gaaagaacgc agcggtttta ccgttcgtcc ggttgcaggt 840

tatctgagtc cgcgtgattt tctggcaggt ctggcatatc gtgtttttca ttgtacccag 900

tatattcgcc atggtagcga tccgctgtat actccggaac cggatacctg tcatgaactg 960

ctgggtcatg tgccgctgct ggcagatccg aaatttgcac agtttagcca agaaattggt 1020

ctggcaagcc tgggtgcaag tgatgaagat gtgcagaaac tggcaacctg ttatttcttt 1080

accattgaat ttggcctgtg caaacaagag ggtcagctgc gtgcctatgg tgcaggtctg 1140

ctgagcagca ttggtgaact gaaacatgca ctgagcgata aagcatgtgt taaagcattt 1200

gatccgaaaa ccacctgtct gcaagaatgt ctgattacca cctttcaaga agcctatttc 1260

gttagcgaaa gctttgaaga ggccaaagaa aaaatgcgcg attttgccaa aagcattacc 1320

cgtccgttta gcgttcgtta taatccgtat acacagagca tcgagatcct gaaagatacc 1380

cgtagcattg aaaatgtggt gcaagacctg cgttccgatc tgaacaccgt atgcgacgcg 1440

ctgaacaaaa tgaaccaata tcttggtatc taa 1473

Claims

1. A tryptophan hydroxylase mutant, wherein the amino acid sequence of the tryptophan hydroxylase mutant consists of the amino acid sequence shown as SEQ ID NO: 1, the amino acid residue at the position 316 of the sequence is mutated into Cys.

2. A gene encoding the tryptophan hydroxylase mutant according to claim 1.

3. A recombinant vector comprising a gene encoding the tryptophan hydroxylase mutant according to claim 2.

4. A recombinant genetically engineered bacterium obtained by transferring the recombinant vector according to claim 3 into a host cell.

5. The recombinant genetically engineered bacterium of claim 4, wherein the host cell comprises the recombinant vector of claim 3 or a gene encoding the tryptophan hydroxylase mutant of claim 2 integrated into the genome thereof.

6. Use of the tryptophan hydroxylase mutant according to claim 1, the recombinant vector according to claim 3 or the recombinant genetically engineered bacterium according to claim 4 or 5 for producing tryptophan hydroxylase.

7. Use of the tryptophan hydroxylase mutant according to claim 1, the recombinant vector according to claim 3 or the recombinant genetically engineered bacterium according to claim 4 or 5 for producing 5-hydroxytryptophan.

8. A method for producing 5-hydroxytryptophan, comprising the steps of:

1) producing 5-hydroxytryptophan by using the tryptophan hydroxylase mutant as claimed in claim 1 or the recombinant genetically engineered bacterium as claimed in claim 4 or 5;

2) 5-hydroxytryptophan is separated from the system in 1).