CN114875087A - Method for synthesizing 5-hydroxy beta-indolyl alanine by using beta-indolyl alanine as substrate and application thereof - Google Patents

Abstract

The invention provides a method for synthesizing 5-hydroxy β-indolyl alanine using β-indolyl alanine as a substrate and its application. The 5-hydroxy β-indolyl alanine high-yielding strain provided by the invention is used for the biological method of synthesizing 5-hydroxy β-indolyl alanine by using β-indolyl alanine as a substrate, 5 -Hydroxyβ-indolylalanine was synthesized in high yield. Through the construction of the BH4 regeneration system and the mining of new BH4 regeneration enzymes, the invention strengthens the regeneration ability of the engineering strain BH4, especially the simultaneous action of the double BH4 regeneration system, and significantly improves the biosynthesis of 5-hydroxy β-indolyl Alanine production.

Description

A method for synthesizing 5-hydroxy β-indolyl alanine using β-indolyl alanine as a substrate and its application

technical field

The invention relates to the field of biotechnology, in particular to a method for synthesizing 5-hydroxy β-indolyl alanine using β-indolyl alanine as a substrate and application thereof.

Background technique

5-Hydroxyβ-indolylalanine is widely present in the seeds of leguminous plants, among which the seeds of the African plant Ghana have the highest content. The basic process route of extracting and purifying 5-hydroxy β-indolylalanine from Ghana seeds is: Ghana seeds crushing - extraction - filtration and centrifugation - physical decolorization - vacuum concentration - crystallization, recrystallization - vacuum drying. However, as the number of African Ghana trees continues to decrease, it is becoming more and more difficult to obtain 5-hydroxyβ-indolylalanine by traditional extraction methods. In recent years, the market demand for 5-hydroxy β-indolylalanine is increasing, and it is difficult to meet the market demand by relying on natural product extraction. More and more companies have begun to study 5-hydroxy from chemical synthesis and biological fermentation. Production of β-indolylalanine.

Most of the reported synthetic methods for the biosynthesis of 5-hydroxyβ-indolylalanine firstly synthesize β-indolylalanine and then hydroxylate it to generate 5-hydroxyβ-indolylalanine acid. The biosynthesis of β-indolylalanine is relatively mature. However, the hydroxylation of β-indolylalanine to 5-hydroxyβ-indolylalanine requires the participation of BH4 as a cofactor, and BH4 is difficult to store and expensive, which makes industrial production by adding additional BH4. 5-Hydroxyβ-indolylalanine becomes impossible.

However, there is no BH4 synthesis pathway in microorganisms, and in order to synthesize in microorganisms, the addition of exogenous genes must be used. The invention improves the output of 5-hydroxy β-indolylalanine by strengthening the BH4 synthesis and regeneration ability of the engineering bacteria and through the application of the double BH4 regeneration system.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide high-producing strains of 5-hydroxyβ-indolylalanine, construct and biosynthesize 5-hydroxyβ-indolylalanine with β-indolylalanine as a substrate sour method.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is as follows:

Provided is a method for synthesizing 5-hydroxy β-indolyl alanine by biological method using β-indolyl alanine as a substrate, comprising the following steps:

1) utilizing the described synthetic 5-hydroxy β-indolyl alanine protein coding gene or the recombinant genetic engineering bacteria capable of expressing the synthetic β-indolyl alanine protein coding gene, with β-indolyl alanine Acid as a substrate, biofermentatively synthesizes 5-hydroxyβ-indolylalanine, and the protein encoding gene capable of expressing and synthesizing 5-hydroxyβ-indolylalanine includes β-indolylalanine production. 5-Hydroxyβ-indolylalanine pathway key enzyme gene TPH2; BH4 synthesis gene FOLE, PTPS, SPR; BH4 regeneration enzyme genes include PCD gene, qBH2-producing BH4-regenerating enzyme gene QDPR and BH2-producing BH4-regenerating enzyme gene DHFR;

2) 5-hydroxyβ-indolylalanine is isolated from the system of 1).

According to the above scheme, the nucleotide sequence of the PCD gene is shown in SEQ ID NO: 5, and the nucleotide sequence of the qBH2-producing BH4 regenase gene QDPR is shown in any sequence in SEQ ID NO: 6-10; BH2 produces BH4 The nucleotide sequence of the regenase gene DHFR is shown in any one of SEQ ID NOs: 11-15.

Preferably, the nucleotide sequence of the BH2-producing BH4 regenase gene QDPR is shown in any sequence in SEQ ID NO: 6 and SEQ ID NO: 8; the nucleotide sequence of the BH2-producing BH4 regenase gene DHFR is shown in SEQ ID NO: 8 ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

More preferably, the nucleotide sequence of the BH2-producing BH4 regenase gene QDPR is shown in SEQ ID NO: 8; the nucleotide sequence of the BH2-producing BH4 regenase gene DHFR is shown in SEQ ID NO: 14.

According to the above scheme, the nucleotide sequence of the key enzyme gene TPH2 in the pathway of β-indolylalanine producing 5-hydroxyβ-indolylalanine is shown in SEQ ID NO: 1;

The nucleotide sequences of the key enzyme genes FOLE, PTPS and SPR of the BH4 synthesis pathway are shown in SEQ ID NOs: 2-4.

According to the above scheme, the step 1) is to transform the constructed recombinant bacteria into 5-hydroxy β using β-indolyl alanine as a substrate after the activation and cultivation of the constructed recombinant bacteria. -Indolylalanine.

Specifically, the above method is as follows: after the constructed recombinant genetically engineered bacteria are activated and cultured in LB medium, respectively, a seed liquid is obtained, the seed liquid is transferred to a fermentation medium, after fermentation, IPTG is added for induction, and β-indole is added. 5-hydroxyβ-indolylalanine is produced by fermentation.

Further, the seed solution OD600=5;

The seed medium (mass percentage) is 1% tryptone, 1% sodium chloride and 0.5% yeast extract;

The culture conditions of the seed solution are 28-40°C, 160-230rpm, preferably 37°C, 225rpm;

The ratio of the seed liquor to the fermentation medium is 1%-10%, and the preferred ratio is 2%;

The composition (mass percentage) of the fermentation medium is: 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH ₂ PO ₄ and 1.64% K ₂ HPO ₄ ·3H ₂ O;

The fermentation time before the induction is 1-5h, preferably 2h;

The final concentration of the IPTG is 0.1-10 mM, preferably 1 mM;

The condition for inducing expression is 16-37°C, preferably 30°C;

The β-indolylalanine concentration of the reaction system is 1-50g/L, preferably 10g/L;

The fermentation time is 18-72h, preferably 24-66h, more preferably 36-48h.

The present invention also provides a BH4 regenase gene, the BH4 regenase gene includes the BH2-producing BH4 regenase gene DHFR, and the nucleotide sequence is shown in any of SEQ ID NOs: 11-15.

According to the above scheme, the BH4 regenase gene further includes PCD gene, qBH2 produces BH4 regenase gene QDPR, the nucleotide sequence of PCD gene is as shown in SEQ ID NO: 5, and qBH2 produces the nucleus of BH4 regenase-based QDPR. The nucleotide sequence is shown in any of SEQ ID NOs: 6-10.

The present invention also provides an engineering bacterium for synthesizing 5-hydroxy β-indolyl alanine by biological method using β-indolyl alanine as a substrate, comprising synthesizing 5-hydroxy β-indolyl alanine protein The coding gene, the synthetic 5-hydroxy β-indolyl alanine protein coding gene includes the key enzyme gene TPH2 of the β-indolyl alanine producing 5-hydroxy β-indolyl alanine pathway; BH4 synthesis Gene FOLE, PTPS, SPR; the above-mentioned BH4 regeneration enzyme gene.

The present invention also provides a method for constructing the above-mentioned genetically engineered bacteria: the key enzyme gene TPH2 of the β-indolylalanine-producing 5-hydroxyβ-indolylalanine pathway, and the BH4 synthetic genes FOLE, PTPS and SPR are put into The same plasmid is tandemly expressed; the BH4 regenase genes PCD, QDPR and DHFR are put into the same plasmid for tandem expression; the above-mentioned plasmids are co-transformed into host cells to obtain recombinant genetically engineered bacteria.

Further, the host cell is an Escherichia coli host cell, preferably BL21(DE3).

Beneficial effects of the present invention:

The present invention provides 5-hydroxy β-indolyl alanine high-yielding strain by constructing double BH4 regeneration system engineering bacteria, which is used for biological synthesis of 5-hydroxy β-indolyl alanine by using β-indolyl alanine as a substrate. Indolylalanine, 5-hydroxyβ-indolylalanine was synthesized in high yield.

Further screening of qBH2-producing BH4 regenase gene QDPR (dihydropterin reductase) and BH2-producing BH4 reductase gene DHFR (dihydrofolate reductase) isoenzyme combination from different species can further significantly improve the biological Yield of synthesis of 5-hydroxyβ-indolylalanine.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

Figure 1: Plasmid map of pET28a-H2FPS

Figure 2: pACYCDuet-PQ plasmid map

Figure 3: pACYCDuet-PQF plasmid map

Figure 4: Schematic diagram of the synthesis of 5-hydroxyβ-indolylalanine in the double BH4 regeneration system using β-indolylalanine as a substrate;

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail, which detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention.

It should be understood that the terms described in the present invention are only used to describe particular embodiments, and are not used to limit the present invention. Additionally, for numerical ranges in the present disclosure, it should be understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 relates. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also 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 invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the present invention. The description and examples of the present application are only exemplary.

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

As a host for exogenous gene expression, Escherichia coli has a clear genetic background, simple technical operation, simple culture conditions, and large-scale fermentation economy, which has attracted much attention. At present, Escherichia coli is the most widely used and successful expression system, and is often the first choice for high-efficiency expression. The following starts from Escherichia coli, and realizes efficient and large-scale industrial production of 5-hydroxyβ-indolylalanine by means of a fermentation method by transforming Escherichia coli.

Example 1 Construction and screening of single BH4 regeneration system engineering bacteria

Tandem expression of key enzyme gene TPH2 in β-indolylalanine hydroxylation pathway; key enzyme FOLE, PTPS and SPR in BH4 synthesis pathway.

The TPH2, FOLE, PTPS and SPR genes were synthesized and inserted between the NdeI and XhoI sites of the vector plasmid pET28a(+) to obtain pET28a-TPH2, pET28a-FOLE, pET28a-PTPS and pET28a-SPR plasmids.

Using F1 and R1 as primers, the pET28a-TPH2 plasmid was used as the template to clone the pET28a plasmid vector containing the TPH2 gene; using F2 and R2 as the primers, the plasmid pET28a-FOLE was used as the template to clone to obtain the FOLE gene; using F3 and R3 as the primers, the plasmid pET28a was cloned - PTPS was used as template to clone to obtain PTPS gene; F4 and R4 were used as primers, plasmid pET28a-SPR was used as template to clone to obtain SPR gene.

The above fragments were linked together by a seamless cloning kit to form plasmid pET28a-H2FPS (as shown in Figure 1)

Table 1 Primer sequence 1

Tandem expression of key enzyme genes PCD and QDPR in BH4 regeneration pathway.

BH3OH generates qBH2 gene PCD and 5 kinds of qBH2 from different sources to produce BH4 regenerating enzyme-based QDPR genes, which are synthesized through codon optimization to obtain Q1, Q2, Q3, Q4, Q5 (sequences are shown in SEQ ID NO: 6-10 in turn. ), put PCD and Q1-Q5 between the NcoI and AflII sites of the pACYCDuet plasmid to obtain pACYCDuet-PCD, pACYCDuet-Q1, pACYCDuet-Q2, pACYCDuet-Q3, pACYCDuet-Q4, pACYCDuet-Q5 plasmids.

Using pACYCDuet-P-F and pACYCDuet-P-R primers, pACYCDuet-PCD plasmid as template, cloned to obtain pACYCDuet plasmid vector containing PCD gene (sequence shown as SEQ ID NO: 5); with Q1-F, Q1-R, Q2-F , Q2-R, Q3-F, Q3-R, Q4-F, Q4-R, Q5-F, Q5-R primers, respectively using plasmids pACYCDuet-Q1, pACYCDuet-Q2, pACYCDuet-Q3, pACYCDuet-Q4, pACYCDuet -Q5 is the template clone to obtain the Q1-Q5 gene fragment.

The above Q1-Q5 gene fragments were respectively connected with the pACYCDuet plasmid vector fragment containing the PCD gene through a seamless cloning kit to form plasmids pACYCDuet-PQ1, pACYCDuet-PQ2, pACYCDuet-PQ3, pACYCDuet-PQ4, pACYCDuet-PQ5. (Picture 2)

Table 1 Primer sequence 1

Construction and Screening of 5-Hydroxyβ-Indolylalanine-Producing Strain with Single BH4 Regenerating Enzyme

The plasmid pET28a-H2FPS and pACYCDuet-PQ1, pACYCDuet-PQ2, pACYCDuet-PQ3, pACYCDuet-PQ4, pACYCDuet-PQ5 were electrotransformed into Escherichia coli BL21 (DE3) competent cells to obtain engineering bacteria KC011, KC012, KC013, KC014, KC015. The engineering bacteria KC011, KC012, KC013, KC014 and KC015 were respectively cultured in the seed medium containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol antibiotics for 10 h to obtain seed liquid, the OD ₆₀₀ = 5. The seed medium (mass percentage) is 1% tryptone, 1% sodium chloride and 0.5% yeast extract, and the culture condition of the seed liquid is 37° C., 225 rpm; 2% of the above seed liquid is inoculated To the fermentation medium (mass percentage) containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH ₂ PO ₄ and 1.64% K ₂ HPO ₄ ·3H ₂ O; fermentation culture for 2h Afterwards, the final concentration of 1 mM IPTG was added to induce expression, the temperature for inducing expression was 30°C, and the final concentration of 10 g/L β-indolylalanine was added. Fermentation conversion, using high performance liquid chromatography to detect the production of 5-hydroxyβ-indolylalanine.

The results are shown in the table, among which: the initial rate of 5-hydroxyβ-indolylalanine production by engineering bacteria KC011 and KC013 was higher than that of other strains, and the engineering bacteria KC013 continued to produce 5-hydroxyβ-indolylalanine It was better than KC011, indicating that the QDPR numbered Q3 had the best ability to regenerate BH4.

Table 2: Comparison of yields of different QDPR strains

Embodiment 2 Construction and screening of double BH4 regeneration system engineering bacteria

The above-mentioned BH4 regeneration key genes QDPR and DHFR were further combined to form a double BH4 regeneration system, and the effect of screening the engineering bacteria of these different gene combinations to synthesize 5-hydroxyβ-indolylalanine was evaluated. Specifically, the double BH4 regeneration system was constructed by taking the PCD gene shown in SEQ ID NO: 5 and the QDPR gene shown in SEQ ID NO: 8 as examples.

Based on the double BH4 regeneration system, the synthesis route of 5-hydroxyβ-indolylalanine using β-indolylalanine as a substrate is shown in Figure 4.

Tandem expression of key genes PCD, QDPR and different DHFRs in the dual BH4 regeneration system:

DHFR genes from different sources were selected and synthesized by codon optimization to obtain F1-F5 (the sequences are shown in SEQ ID NOs: 11-15 in sequence), and the F1-F5 genes were inserted into the pACYCDuet plasmid between the NcoI and AflII sites to obtain pACYCDuet -F1, pACYCDuet-F2, pACYCDuet-F3, pACYCDuet-F4, pACYCDuet-F5, plasmid.

Using pACYCDuet-P-F and pACYCDuet-PQ3-R as primers and pACYCDuet-PQ3 plasmid as template, clone the pACYCDuet plasmid vector containing PCD and Q3 genes; use F1-F, F1-R, F2-F, F2-R, F3- F, F3-R, F4-F, F4-R, F5-F, F5-R are primers, respectively cloned using plasmids pACYCDuet-F1, pACYCDuet-F2, pACYCDuet-F3, pACYCDuet-F4, pACYCDuet-F5 as templates Obtain F1-F5 gene fragments.

The F1-F5 gene fragment and the pACYCDuet plasmid vector containing the PCD and Q3 genes were linked together by a seamless cloning kit to form plasmids pACYCDuet-PQ3F1, pACYCDuet-PQ3F2, pACYCDuet-PQ3F3, pACYCDuet-PQ3F4, pACYCDuet-PQ3F5. (As shown in Figure 3) Table 3 Primer Sequence 2

Construction and Screening of 5-Hydroxyβ-Indolylalanine-Producing Strain with Double BH4 Regenerating Enzyme

The plasmid pET28a-H2FPS and pACYCDuet-PQ3F1, pACYCDuet-PQ3F2, pACYCDuet-PQ3F3, pACYCDuet-PQ3F4, pACYCDuet-PQ3F5 were electrotransformed into E. coli BL21 (DE3) competent cells to obtain engineering bacteria KC021, KC022, KC023, KC024, KC025. The engineering bacteria KC021, KC022, KC023, KC024, and KC025 were cultured in the seed medium containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol antibiotics for 10 hours, respectively, to obtain a seed liquid with an OD ₆₀₀ = 5. The seed medium (mass percentage) is 1% tryptone, 1% sodium chloride and 0.5% yeast extract, and the culture condition of the seed liquid is 37° C., 225 rpm; 2% of the above seed liquid is inoculated To the fermentation medium (mass percentage) containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH ₂ PO ₄ and 1.64% K ₂ HPO ₄ ·3H ₂ O; fermentation culture for 2h Afterwards, the final concentration of 1 mM IPTG was added to induce expression, the temperature for inducing expression was 30°C, and the final concentration of 10 g/L β-indolylalanine was added. Fermentation conversion, using high performance liquid chromatography to detect the production of 5-hydroxyβ-indolylalanine.

The results are shown in the table, among which: the initial rate of 5-hydroxyβ-indolylalanine production by engineering bacteria KC021, KC022, KC023 and KC024 was higher than that of other strains, while the engineering bacteria KC023 and KC024 continued to produce 5-hydroxyβ- Indolylalanine is better than KC021 and KC022, and it is determined that the combination of DHFR and Q3 numbered F4 has the best ability to regenerate BH4, which can ensure the continuous and efficient production of the product.

Table 4: Production comparison of different DHFR strains

In the present invention, the β-indolyl alanine is obtained through the coordination and construction of the β-indolyl alanine hydroxylation gene TPH2; the BH4 synthesis genes FOLE, PTPS, SPR; the BH4 regeneration gene PCD, QDPR and DHFR. The engineering bacterium for synthesizing 5-hydroxyβ-indolylalanine by substrate biological method is used for synthesizing 5-hydroxyβ-indolylalanine by biological method using β-indolylalanine as substrate.

Further through the screening of the key enzyme gene QDPR of the BH4 regeneration pathway and the combined screening of the new key enzyme gene DHFR of the BH4 regeneration pathway, the constructed dual BH4 regeneration system significantly improved the biosynthesis of 5-hydroxyβ-indolylalanine. . It provides conditions for efficient large-scale industrial production of 5-hydroxyβ-indolylalanine.

sequence listing

<110> Hebei Weida Kang Biotechnology Co., Ltd.

<120> Method for synthesizing 5-hydroxyβ-indolylalanine with β-indolylalanine as substrate and its application

<130> 1

<160> 47

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1038

<212> DNA

<213> artificial sequence()

<400> 1

atgaaactgg aggatgttcc gtggttcccg cgtaagatta gcgagctgga taagtgcagc 60

caccgtgttc tgatgtatgg tagcgaactg gacgcggatc acccgggttt taaggacaac 120

gtgtaccgtc agcgtcgtaa atatttcgtg gatgttgcga tgggttacaa gtatggccaa 180

ccgatcccgc gtgttgaata caccgaggaa gaaaccaaga cctggggtgt ggtttttcgt 240

gaactgagca aactgtaccc gacccacgcg tgccgtgagt atctgaagat tttctgcctg 300

ctgaccaaat actgcggcta tcgtgaagac aacgtgccgc agctggagga tgttagcatg 360

tttctgaagg agcgtagcgg tttcaccgtg cgtccggttg cgggttacct gagcccgcgt 420

gactttctgg cgggtctggc gtaccgtgtt ttccactgca cccaatatat ccgtcacggc 480

agcgacccgc tgtatacccc ggaaccggat acctgccacg agctgctggg tcacgttccg 540

ctgctggcgg atccgaaatt cgcgcagttt agccaagaaa ttggtctggc gagcctgggt 600

gcgagcgatg aggatgtgca gaagctggcg acctgctact tctttaccat cgaattcggt 660

ctgtgcaaac aggaaggtca actgcgtgcg tatggtgcgg gcctgctgag cagcattggc 720

gaactgaagc acgcgctgag cgacaaggcg tgcgtgaaag cgtttgatcc gaaaaccacc 780

tgcctgcagg aatgcctgat caccaccttc caagaagcgt actttgttag cgagagcttc 840

gaagaggcga aggagaaaat gcgtgacttc gcgaagagca tcacccgtcc gttcagcgtg 900

tactttaacc cgtataccca gagcatcgaa attctgaaag acacccgtag cattgagaac 960

gtggttcaag atctgcgtag cgacctgaac accgtttgcg atgcgctgaa taaaatgaac 1020

caatatctgg gtatctaa 1038

<210> 2

<211> 669

<212> DNA

<213> artificial sequence()

<400> 2

atgccatcac tcagtaaaga agcggccctg gttcatgaag cgttagttgc gcgaggactg 60

gaaacaccgc tgcgcccgcc cgtgcatgaa atggataacg aaacgcgcaa aagccttatt 120

gctggtcata tgaccgaaat catgcagctg ctgaatctcg acctggctga tgacagtttg 180

atggaaacgc cgcatcgcat cgctaaaatg tatgtcgatg aaattttctc cggtctggat 240

tacgccaatt tcccgaaaat caccctcatt gaaaacaaaa tgaaggtcga tgaaatggtc 300

accgtgcgcg atatcactct gaccagcacc tgtgaacacc attttgttac catcgatggc 360

aaagcgacgg tggcctatat cccgaaagat tcggtgatcg gtctgtcaaa aattaaccgc 420

attgtgcagt tctttgccca gcgtccgcag gtgcaggaac gtctgacgca gcaaattctt 480

attgcgctac aaacgctgct gggcaccaat aacgtggctg tctcgatcga cgcggtgcat 540

tactgcgtga aggcgcgtgg catccgcgat gcaaccagtg ccacgacaac gacctctctt 600

ggtggattgt tcaaatccag tcagaatacg cgccacaaat ttctgcgcgc tgtgcgtcat 660

cacaactga 669

<210> 3

<211> 435

<212> DNA

<213> artificial sequence()

<400> 3

atgaatgcgg cggtgggcct gcgtcgtcgt gcgcgtctga gccgtctggt tagctttagc 60

gcgagccatc gtctgcacag cccgagcctg agcgcggagg aaaacctgaa ggttttcggt 120

aaatgcaaca acccgaacgg tcacggccac aactacaagg tggttgtgac catccacggc 180

gagattgacc cggttaccgg catggtgatg aacctgaccg atctgaaaga atatatggag 240

gaagcgatca tgaagccgct ggaccacaaa aacctggacc tggatgttcc gtactttgcg 300

gatgttgtga gcaccaccga gaacgttgcg gtgtatattt gggaaaacct gcagcgtctg 360

ctgccggtgg gcgcgctgta taaggtgaaa gtgtatgaaa ccgacaacaa catcgtggtg 420

tataagggtg aataa 435

<210> 4

<211> 810

<212> DNA

<213> artificial sequence()

<400> 4

atgaccatga ttaccccgag cctgggccgt ggccgtctgg gttgcgcggt gtgcgttctg 60

accggtgcga gccgtggttt tggccgtgcg ctggcgccgc agctggcggg tctgctgagc 120

ccgggcagcg tgctgctgct gagcgcgcgt agcgactcca tgctgcgtca gctgaaagag 180

gagctgtgca cccagcaacc gggtctgcaa gtggttctgg cggcggcgga tctgggtacc 240

gagagcggcg tgcagcaact gctgagcgcg gttcgtgagc tgccgcgtcc ggaacgtctg 300

caacgtctgc tgctgatcaa caacgcgggt accctgggtg atgtgagcaa gggcttcctg 360

aacattaacg atctggcgga agttaacaac tactgggcgc tgaacctgac cagcatgttg 420

tgcctgacca ccggtaccct gaacgcgttc agcaacagcc cgggcctgag caaaaccgtg 480

gttaacatca gcagcctgtg cgcgctgcag ccgtttaagg gttggggcct gtactgcgcg 540

ggtaaagcgg cgcgtgacat gctgtatcaa gtgctggcgg ttgaggaacc gagcgtgcgt 600

gttctgagct atgcgccggg tccgctggac accaacatgc agcaactggc gcgtgaaacc 660

agcatggacc cggaactgcg tagccgtctg cagaagctga acagcgaggg tgaactggtt 720

gattgcggca ccagcgcgca aaaactgctg agcctgctgc aacgcgatac ctttcagagc 780

ggtgcgcacg tggactttta cgacatctaa 810

<210> 5

<211> 315

<212> DNA

<213> artificial sequence()

<400> 5

atggcaggca aagcgcaccg tctgtctgcg gaagaacgcg atcagctgct gccgaacctg 60

cgtgcagtgg gctggaacga actggaaggt cgcgatgcta tcttcaaaca attccacttt 120

aaggatttca accgtgcgtt cggcttcatg actcgtgttg cactgcaagc agaaaaactg 180

gatcatcacc cggaatggtt caacgtttac aacaaagtac atattaccct gagcacccac 240

gaatgcgcag gtctgtctga acgcgatatc aacctggcgt ccttcatcga acaggttgcg 300

gtttccatga cctga 315

<210> 6

<211> 735

<212> DNA

<213> artificial sequence()

<400> 6

atggcagcag cagcagcagc aggtgaagct cgtcgtgttc tggtttacgg tggtcgtggt 60

gcactgggca gccgctgcgt tcaggcattc cgtgcgcgta actggtgggt ggcttccgtg 120

gacgttgtcg agaacgaaga ggcgtccgca tccattatcg ttaaaatgac cgattccttc 180

accgaacagg cagatcaggt tactgccgaa gttggcaaac tgctgggtga agaaaaagta 240

gatgcgatcc tgtgtgttgc tggtggctgg gctggtggta acgccaaaag caaatccctg 300

ttcaaaaact gtgatctgat gtggaaacag tctatctgga cctctactat ttctagccat 360

ctggcgacta agcacctgaa agaaggtggt ctgctgaccc tggccggtgc caaagcagct 420

ctggatggta cgccgggtat gattggttac ggcatggcta aaggcgctgt acatcagctg 480

tgccagtccc tggcgggtaa aaactctggc atgccaccgg gtgctgcggc gatcgcggtg 540

ctgccggtga ccctggatac cccgatgaat cgtaaaagca tgccggaagc ggatttctct 600

tcttggacgc cgctggaatt tctggtagaa acgttccacg attggattac cggtaaaaac 660

cgtccgtctt ccggtagcct gatccaggta gtgacgaccg aaggtcgcac ggaactgacg 720

ccagcctatt tttga 735

<210> 7

<211> 726

<212> DNA

<213> artificial sequence()

<400> 7

atggcggcga gcggcgaagc gcgccgcgtg ctggtgtatg gcggccgcgg cgcgctgggc 60

agccgctgcg tgcaggcgtt tcgcgcgcgc aactggtggg tggcgagcat tgatgtggtg 120

gaaaacgaag aagcgagcgc gagcgtgatt gtgaaaatga ccgatagctt taccgaacag 180

gcggatcagg tgaccgcgga agtgggcaaa ctgctgggcg atcagaaagt ggatgcgatt 240

ctgtgcgtgg cgggcggctg ggcgggcggc aacgcgaaaa gcaaaagcct gtttaaaaac 300

tgcgatctga tgtggaaaca gagcatttgg accagcacca ttagcagcca tctggcgacc 360

aaacatctga aagaaggcgg cctgctgacc ctggcgggcg cgaaagcggc gctggatggc 420

accccgggca tgattggcta tggcatggcg aaaggcgcgg tgcatcagct gtgccagagc 480

ctggcgggca aaaacagcgg catgccgagc ggcgcggcgg cgattgcggt gctgccggtg 540

accctggata ccccgatgaa ccgcaaaagc atgccggaag cggattttag cagctggacc 600

ccgctggaat ttctggtgga aacctttcat gattggatta ccggcaacaa acgcccgaac 660

agcggcagcc tgattcaggt ggtgaccacc gatggcaaaa ccgaactgac cccggcgtat 720

ttttaa 726

<210> 8

<211> 654

<212> DNA

<213> artificial sequence()

<400> 8

atggatatta tcagcgttgc gctgaaacgt catagcacca aagcctttga tgcaagcaaa 60

aagctgaccc cggaacaggc agaacagatt aaaacgctgc tgcagtatag cccgagcagc 120

accaacagcc agccgtggca ttttattgtc gcaagcaccg aagaaggtaa agcacgtgtt 180

gcaaaaagcg cagcaggtaa ttatgttttt aatgaacgta aaatgctgga tgcaagccat 240

gtggttgtat tttgtgcaaa aaccgcaatg gatgatgtgt ggctgaaact ggttgttgat 300

caggaagatg cagatggccg ttttgccacc ccggaagcca aagcagcaaa tgataaaggt 360

cgtaaatttt ttgcagatat gcatcgtaaa gatttacatg atgatgcaga atggatggca 420

aaacaggtat atctgaatgt tggtaacttt ctgctgggtg ttgcagcact gggtctggat 480

gccgttccga ttgaaggttt tgatgcagca attctggatg cagaatttgg tctgaaagaa 540

aaaggttata cctccctggt tgttgttcct gttggtcatc attcagttga agattttaat 600

gcaaccctgc cgaaatctcg tctgccgcag aatattacac tgacggaagt ttaa 654

<210> 9

<211> 627

<212> DNA

<213> artificial sequence()

<400> 9

atggcgaccg cggcgggcga agcgcgccgc gtgctggtgt atggcggccg cggcgcgctg 60

ggcagccagt gcgtgcaggc gtttcgcgcg cgcaactggt gggtggcgag cattgatctg 120

gtggaaaacg aagaagcgag cgcgaacgtg ctggtgaaaa tgaccgatag ctttaccgaa 180

caggcggatc aggtgaccgc ggatattggc cagctgctgg gcgcggaaaa agtggatgcg 240

attctgtgcg tggcgggcgg ctgggcgggc ggcagcgcga aaagcaaaag cctgtttaaa 300

aactgcgatc tgatgtggaa acagagcatg tggaccagca ccattagcag ccatctggcg 360

accaaacatc tgaaagaagg cggcctgctg accctgaccg gcgcgaaagc ggcgctggat 420

ggcaccccgg gcatgattgg ctatggcatg gcgaaaggcg cggtgcatca gctgtgccgc 480

agcctggcgg gcaaagatag cggcatgccg gcgggcagcg cggcgattgc ggtgctgccg 540

gtgaccctgg ataccccgat gaaccgcaaa agcatgccga aagcggattt tagcagctgg 600

accccgctgg aatttctggt ggaataa 627

<210> 10

<211> 708

<212> DNA

<213> artificial sequence()

<400> 10

atgagcgcgg gccgcgtggt gatttatggc ggcaaaggcg cgctgggcag cgcgtgcgtg 60

gatcatttta aagcgaacaa ctattgggtg ggcagcattg atctgaccga aaacgaaaaa 120

gcggatgtga gcattgtggt gccgcgcgat gcgagctggg tggaacagga agaaaccgtg 180

gtgagcaaag tgggcgaaag cctggcgggc gaaaaactgg atgcggtgat ttgcgtggcg 240

ggcggctggg cgggcggcaa cgcgaaaaaa gatctggcga aaaacgcgga tctgatgtgg 300

aaacagagcg tgctgaccag cgcgattagc gcggcggtgg cggcgcagca tctgaaagcg 360

ggcggcctgc tggcgctgac cggcgcgaaa ccggcgctgg aaggcacccc gggcatgatt 420

ggctatggca tggcgaaagc ggcggtgcat cagctgaccc gcagcctggg cgcggaaaaa 480

agcggcctgc cggcgggcag cctggcggtg agcattctgc cggtgaccct ggataccccg 540

atgaaccgca aatggatgcc ggatgcggat tttggcacct ggaccccgct gaccgaagtg 600

gcgggcctgt ttctgaaatg gacccaggat caggaacgcc cgaaaaccgg cagcctgctg 660

cagctgatta ccaccaacgg cattacccag ctgattgcgg cggaataa 708

<210> 11

<211> 564

<212> DNA

<213> artificial sequence()

<400> 11

atggtgggca gcctgaactg cattgtggcg gtgagccaga acatgggcat tggcaaaaac 60

ggcgatctgc cgtggccgcc gctgcgcaac gaatttcgct attttcagcg catgaccacc 120

accagcagcg tggaaggcaa acagaacctg gtgattatgg gcaaaaaaac ctggtttagc 180

attccggaaa aaaaccgccc gctgaaaggc cgcattaacc tggtgctgag ccgcgaactg 240

aaagaaccgc cgcagggcgc gcattttctg agccgcagcc tggatgatgc gctgaaactg 300

accgaacagc cggaactggc gaacaaagtg gatatggtgt ggattgtggg cggcagcagc 360

gtgtataaag aagcgatgaa ccatccgggc catctgaaac tgtttgtgac ccgcattatg 420

caggattttg aaagcgatac ctttttttccg gaaattgatc tggaaaaata taaactgctg 480

ccggaatatc cgggcgtgct gagcgatgtg caggaagaaa aaggcattaa atataaattt 540

gaagtgtatg aaaaaaacga ttaa 564

<210> 12

<211> 564

<212> DNA

<213> artificial sequence()

<400> 12

atggtgcgcc cgctgaactg cattgtggcg gtgagccaga acatgggcat tggcaaaaac 60

ggcgatctgc cgtggccgct gctgcgcaac gaatttaaat attttcagcg catgaccacc 120

accagcagcg tggaaggcaa acagaacctg gtgattatgg gccgcaaaac ctggtttagc 180

attccggaaa aaaaccgccc gctgaaagat cgcattaaca ttgtgctgag ccgcgaactg 240

aaagaaccgc cgcagggcgc gcattttctg gcgaaaagcc tggatgatgc gctgaaactg 300

attgaacagc cggaactggc gagcaaagtg gatatggtgt gggtggtggg cggcagcagc 360

gtgtatcagg aagcgatgaa ccagccgggc catctgcgcc tgtttgtgac ccgcattatg 420

caggaatttg aaagcgatac ctttttttccg gaaattgatc tggaaaaata taaactgctg 480

ccggaatatc cgggcgtgct gagcgaaatt caggaagaaa aaggcattaa atataaattt 540

gaagtgtatg aaaaaaaaga ttaa 564

<210> 13

<211> 480

<212> DNA

<213> artificial sequence()

<400> 13

atgatcagtc tgattgcggc gttagcggta gatcgcgtta tcggcatgga aaacgccatg 60

ccgtggaacc tgcctgccga tctcgcctgg tttaaacgca acaccttaaa taaacccgtg 120

attatgggcc gccatacctg ggaatcaatc ggtcgtccgt tgccaggacg caaaaatatt 180

atcctcagca gtcaaccggg tacggacgat cgcgtaacgt gggtgaagtc ggtggatgaa 240

gccatcgcgg cgtgtggtga cgtaccagaa atcatggtga ttggcggcgg tcgcgtttat 300

gaacagttct tgccaaaagc gcaaaaactg tatctgacgc atatcgacgc agaagtggaa 360

ggcgacaccc atttcccgga ttacgagccg gatgactggg aatcggtatt cagcgaattc 420

cacgatgctg atgcgcagaa ctctcacagc tattgctttg agattctgga gcggcggtaa 480

<210> 14

<211> 573

<212> DNA

<213> artificial sequence()

<400> 14

atggtgcgca gcctgaacag cattgtggcg gtgtgccaga acatgggcat tggcaaagat 60

ggcaacctgc cgtggccgcc gctgcgcaac gaatataaat attttcagcg catgaccagc 120

accagccatg tggaaggcaa acagaacgcg gtgattatgg gcaaaaaaac ctggtttagc 180

attccggaaa aaaaccgccc gctgaaagat cgcattaaca ttgtgctgag ccgcgaactg 240

aaagaagcgc cgaaaggcgc gcattatctg agcaaaagcc tggatgatgc gctggcgctg 300

ctggatagcc cggaactgaa aagcaaagtg gatatggtgt ggattgtggg cggcaccgcg 360

gtgtataaag cggcgatgga aaaaccgatt aaccatcgcc tgtttgtgac ccgcattctg 420

catgaatttg aaagcgatac cttttttccg gaaattgatt ataaagattt taaactgctg 480

accgaatatc cgggcgtgcc ggcggatatt caggaagaaa acggcattca gtataaattt 540

gaagtgtatc agaaaagcgt gctggcgcag taa 573

<210> 15

<211> 489

<212> DNA

<213> artificial sequence()

<400> 15

atgaccgtga gcattattgt ggcgattgat gaaaacaaag cgattggcaa aaacaaccag 60

ctgctgtggc atctgccgaa cgatctgaaa ttttttaaaa aaaccaccag cggccatacc 120

attattatgg gccgcaaaac ctttgatagc attggcaaag cgctgccgaa ccgccgcaac 180

attgtgatta gccgcaacaa aaacctgaaa attgaaggcg cggaagtgta tagcagcatt 240

gatcaggcgc tgaacacctg caaaaacgaa caggaagtgt ttattattgg cggcgcggaa 300

atttataaac aggcggaacc gattaccgat aaattttata ttaccaaagt gcatcatcag 360

tttgatgcgg atacctttttt taacaacctg aacctgaacg aactgaacga aatttggcgc 420

gaagaaaacc atgcggatga acgccatctg tatgattata cctttctgat tctggaaaaa 480

cgcaaataa 489

<210> 16

<211> 44

<212> DNA

<213> artificial sequence()

<400> 16

caaagcccga aaggaagctg agttggctgc tgccaccgct gagc 44

<210> 17

<211> 45

<212> DNA

<213> artificial sequence()

<400> 17

ctcgagaaga aggagatata catatgccat cactcagtaa agaag 45

<210> 18

<211> 58

<212> DNA

<213> artificial sequence()

<400> 18

caggaaacag ctatgaccat gattaccaat agcatgaata tgaatgcggc ggtgggcc 58

<210> 19

<211> 45

<212> DNA

<213> artificial sequence()

<400> 19

ggatcccagg aaacagctat gaccatgatt accccgagcc tgggc 45

<210> 20

<211> 45

<212> DNA

<213> artificial sequence()

<400> 20

atgtatatct ccttcttctc gagttagata cccagatatt ggttc 45

<210> 21

<211> 59

<212> DNA

<213> artificial sequence()

<400> 21

attcatgcta ttggtaatca tggtcatagc tgtttcctgt cagttgtgat gacgcacag 59

<210> 22

<211> 46

<212> DNA

<213> artificial sequence()

<400> 22

agctgtttcc tgggatcctt attcaccctt atacaccacg atgttg 46

<210> 23

<211> 45

<212> DNA

<213> artificial sequence()

<400> 23

tcctttcggg ctttgttaga tgtcgtaaaa gtccacgtgc gcacc 45

<210> 24

<211> 45

<212> DNA

<213> artificial sequence()

<400> 24

ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccc 45

<210> 25

<211> 45

<212> DNA

<213> artificial sequence()

<400> 25

atgtatatct ccttcttaga tccgcggccg ctcaggtcat ggaaa 45

<210> 26

<211> 45

<212> DNA

<213> artificial sequence()

<400> 26

gcggccgcgg atctaagaag gagatataca tatggcagca gcagc 45

<210> 27

<211> 45

<212> DNA

<213> artificial sequence()

<400> 27

agcagcctag gttaatcaaa aataggctgg cgtcagttcc gtgcg 45

<210> 28

<211> 45

<212> DNA

<213> 28

<400> 28

gaagggagata tacatatggc ggcgagcggc gaagcgcgcc gcgtg 45

<210> 29

<211> 46

<212> DNA

<213> artificial sequence()

<400> 29

agcagcctag gttaattaaa aatacgccgg ggtcagttcg gttttg 46

<210> 30

<211> 44

<212> DNA

<213> artificial sequence()

<400> 30

gaagggagata tacatatgga tattatcagc gttgcgctga aacg 44

<210> 31

<211> 45

<212> DNA

<213> artificial sequence()

<400> 31

agcagcctag gttaattaaa cttccgtcag tgtaatattc tgcgg 45

<210> 32

<211> 42

<212> DNA

<213> artificial sequence()

<400> 32

gaagggagata tacatatggc gaccgcggcg ggcgaagcgc gc 42

<210> 33

<211> 44

<212> DNA

<213> artificial sequence()

<400> 33

agcagcctag gttaattatt ccaccagaaa ttccagcggg gtcc 44

<210> 34

<211> 45

<212> DNA

<213> artificial sequence()

<400> 34

gaagggagata tacatatgag cgcgggccgc gtggtgattt atggc 45

<210> 35

<211> 45

<212> DNA

<213> artificial sequence()

<400> 35

agcagcctag gttaattatt ccgccgcaat cagctgggta atgcc 45

<210> 36

<211> 45

<212> DNA

<213> artificial sequence()

<400> 36

ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccc 45

<210> 37

<211> 47

<212> DNA

<213> artificial sequence()

<400> 37

atgtatatct ccttcttact agttcaaaaa taggctggcg tcagttc 47

<210> 38

<211> 45

<212> DNA

<213> artificial sequence()

<400> 38

gaagggagata tacatatggt gggcagcctg aactgcattg tggcg 45

<210> 39

<211> 39

<212> DNA

<213> artificial sequence()

<400> 39

agcagcctag gttaattaat cgtttttttc atacacttc 39

<210> 40

<211> 45

<212> DNA

<213> artificial sequence()

<400> 40

gaagggagata tacatatggt gcgcccgctg aactgcattg tggcg 45

<210> 41

<211> 39

<212> DNA

<213> artificial sequence()

<400> 41

agcagcctag gttaattaat ctttttttttc atacacttc 39

<210> 42

<211> 46

<212> DNA

<213> artificial sequence()

<400> 42

gaagggagata tacatatgat cagtctgatt gcggcgttag cggtag 46

<210> 43

<211> 46

<212> DNA

<213> artificial sequence()

<400> 43

agcagcctag gttaattacc gccgctccag aatctcaaag caatag 46

<210> 44

<211> 45

<212> DNA

<213> artificial sequence()

<400> 44

gaaggagata tacatatggt gcgcagcctg aacagcattg tggcg 45

<210> 45

<211> 45

<212> DNA

<213> artificial sequence()

<400> 45

agcagcctag gttaattact gcgccagcac gcttttctga tacac 45

<210> 46

<211> 43

<212> DNA

<213> artificial sequence()

<400> 46

gaagggagata tacatatgac cgtgagcatt attgtggcga ttg 43

<210> 47

<211> 44

<212> DNA

<213> artificial sequence()

<400> 47

agcagcctag gttaattatt tgcgtttttc cagaatcaga aagg 44

Claims (12)

1. A method for synthesizing 5-hydroxy beta-indolyl alanine by using beta-indolyl alanine as substrate biological method is characterized in that: the method comprises the following steps:

1) the 5-hydroxy beta-indolyl alanine protein coding gene or the recombinant gene engineering bacterium capable of expressing and synthesizing the beta-indolyl alanine protein coding gene is utilized to biologically ferment and synthesize the 5-hydroxy beta-indolyl alanine by taking the beta-indolyl alanine as a substrate, and the gene capable of expressing and synthesizing the 5-hydroxy beta-indolyl alanine protein coding gene comprises a key enzyme gene TPH2 in a way that the beta-indolyl alanine generates the 5-hydroxy beta-indolyl alanine; BH4 synthetic genes FOLE, PTPS, SPR; the BH4 regenerant genes comprise PCD genes, BH4 regenerant genes QDPR produced by qBH2 and BH4 regenerant genes DHFR produced by BH 2;

2) separating 5-hydroxy beta-indolyl alanine from the system in 1).

2. The method of claim 1, wherein: the nucleotide sequence of the PCD gene is shown as SEQ ID NO. 5, and the nucleotide sequence of the BH 4-producing regenerant gene QDPR produced by qBH2 is shown as any one sequence of SEQ ID NO. 6-10; the nucleotide sequence of BH 4-producing regenerant gene DHFR of BH2 is shown as any sequence in SEQ ID NO. 11-15.

3. The method of claim 1, wherein: the nucleotide sequence of BH 2-produced BH 4-produced regenerase gene QDPR is shown as any sequence of SEQ ID NO. 6 and SEQ ID NO. 8; the nucleotide sequence of BH 2-produced BH 4-produced regenerase gene DHFR is shown as any sequence of SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.

4. The method of claim 1, wherein: the nucleotide sequence of BH 2-produced BH 4-produced regenerase gene QDPR is shown as SEQ ID NO. 8; the nucleotide sequence of BH 4-producing regenerant gene DHFR of BH2 is shown as SEQ ID NO. 14.

5. The method of claim 1, wherein: the nucleotide sequence of a key enzyme gene TPH2 of a pathway for producing 5-hydroxy beta-indolylalanine from beta-indolylalanine is shown as SEQ ID NO. 1;

the nucleotide sequences of key enzyme genes FOLE, PTPS and SPR in the synthetic pathway of BH4 are shown in SEQ ID NO. 2-4.

6. The method of claim 1, wherein: the step 1) is to transfer the constructed recombinant bacteria to a fermentation culture medium for induction expression after activation culture, and convert the recombinant bacteria to generate 5-hydroxy beta-indolyl alanine by taking beta-indolyl alanine as a substrate.

7. The method of claim 1, wherein: the method comprises the following steps: respectively carrying out activated culture on the constructed recombinant genetic engineering bacteria by an LB culture medium to obtain a seed solution, transferring the seed solution to a fermentation culture medium, adding IPTG (isopropyl-beta-indolyl-alanine) for induction after fermentation, adding beta-indolyl-alanine, and carrying out fermentation conversion to generate 5-hydroxy beta-indolyl-alanine.

8. The method of claim 1, wherein: the seed liquid OD600 is 5;

the seed culture medium comprises (by mass) 1% of tryptone, 1% of sodium chloride and 0.5% of yeast extract;

the culture condition of the seed liquid is 28-40 ℃, 160-230 rpm;

the proportion of the seed liquid to the fermentation medium is 1-10%;

the fermentation medium comprises the following components in percentage by mass: 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH ₂ PO ₄ And 1.64% K ₂ HPO ₄ ·3H ₂ O；

The fermentation time before induction is 1-5 h;

the final concentration of IPTG is 0.1-10 mM;

the condition for inducing expression is 16-37 ℃;

the concentration of the beta-indolyl alanine in the reaction system is 1-50 g/L;

the fermentation time is 18-72h, preferably 24-66 h.

A BH4 regenerase gene characterized by: the BH4 regenerant gene comprises BH 2-produced BH 4-containing regenerant gene DHFR, and the nucleotide sequence is shown as any sequence in SEQ ID NO. 11-15.

10. The BH4 regenerase gene according to claim 9, wherein: the BH4 regenerant gene further comprises a PCD gene and a BH 4-producing regenerant gene QDPR produced by qBH2, wherein the nucleotide sequence of the PCD gene is shown as SEQ ID NO. 5, and the nucleotide sequence of the BH 4-producing regenerant gene QDPR produced by qBH2 is shown as any one of SEQ ID NO. 6-10.

11. An engineering bacterium for synthesizing 5-hydroxy beta-indolyl alanine by using beta-indolyl alanine as a substrate biological method comprises a coding gene for synthesizing 5-hydroxy beta-indolyl alanine protein, wherein the coding gene for synthesizing 5-hydroxy beta-indolyl alanine protein comprises a key enzyme gene TPH2 for producing 5-hydroxy beta-indolyl alanine by beta-indolyl alanine; BH4 synthetic genes FOLE, PTPS, SPR; the BH 4-regenerating enzyme gene of claim 9 or claim 10.

12. A method for constructing a genetically engineered bacterium according to claim 11, wherein the method comprises the following steps: key enzyme genes TPH2 and BH4 synthetic genes FOLE, PTPS and SPR in the process of producing 5-hydroxy beta-indolylalanine by beta-indolylalanine are put into the same plasmid for serial expression; BH4 regenerase genes PCD, QDPR and DHFR are put into the same plasmid to be expressed in series; and (3) co-transforming the plasmids into host cells to obtain the recombinant gene engineering bacteria.

Images