Method for biosynthesizing 5-hydroxy-β-indolylalanine using glucose as substrate

Abstract

The present invention provides a method for biosynthesizing 5-hydroxy-β-indolylalanine using glucose as a substrate. The present invention utilizes the gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine or a recombinant genetic engineering bacterium capable of expressing the gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine to synthesize 5-hydroxy-β-indolylalanine; and the yield of 5-hydroxy-β-indolylalanine is increased by organically integrating multiple pathways of different pathways. The process is easy to implement, the conditions are easy to control, and it is suitable for promoting industrial production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a novel method for synthesizing 5-hydroxy beta-indolyl alanine by biological method using glucose as substrate. Background Art

[0002] 5-Hydroxy-β-indolylalanine (5-hydroxytryptophan, 5-HTP) was discovered in 1958. It is an amino acid derivative formed by the oxidation of β-indolylalanine (tryptophan, one of the essential amino acids for the human body) by β-indolylalanine hydroxylase (tryptophan hydroxylase, TPH, also known as β-indolylalanine monooxygenase, tryptophan 5-monooxygenase).

[0003] 5-Hydroxy-β-indolylalanine is widely found in the seeds of leguminous plants, among which the highest content is found in the seeds of the African plant Griffonia. The leaves and seeds of the Griffonia tree have been used as medicines by people in Africa to treat wounds, kidney diseases and enema since ancient times. The ointment made from its bark can also treat skin diseases. Its main active ingredient is 5-Hydroxy-β-indolylalanine.

[0004] my country uses the alcohol method to extract 5-hydroxy-β-indolylalanine. The basic process route for extracting and refining 5-hydroxy-β-indolylalanine from Ghana seeds is: Ghana seed crushing - extraction - filtration and centrifugation - physical decolorization - vacuum concentration - crystallization, recrystallization - vacuum drying. In the whole process, only alcohol and water are generally used for extraction, avoiding the pollution of organic solvents. In addition, high-purity 5-hydroxy-β-indolylalanine can also be obtained from Ghana seeds through ion exchange resin adsorption separation and ultrafiltration membrane separation.

[0005] However, as the number of African Ghanaian trees continues to decrease, it is becoming increasingly difficult to obtain 5-hydroxy-β-indolylalanine by traditional extraction methods. 5-hydroxy-β-indolylalanine, as a natural medicine for resisting depression and relieving affective disorders in the human neurometabolism pathway, has seen an increasing market demand in recent years. It is difficult to meet market demand by simply extracting natural products. More and more companies are beginning to study the production of 5-hydroxy-β-indolylalanine from aspects such as chemical synthesis and biological fermentation.

[0006] Most of the methods reported so far for the biosynthesis of 5-hydroxy-β-indolylalanine are to synthesize β-indolylalanine first and then hydroxylate it to generate 5-hydroxy-β-indolylalanine. The biosynthesis of β-indolylalanine is relatively mature, but due to the heterologous expression of β-indolylalanine hydroxylases from humans and other sources in prokaryotic hosts such as Escherichia coli, the hydroxylation efficiency is low and the hydroxylation conditions are harsh, resulting in a low yield of 5-hydroxy-β-indolylalanine, which cannot meet the needs of industrial production. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a 5-hydroxy β-indolyl alanine high-yielding strain, a construction method and a method for biosynthesizing 5-hydroxy β-indolyl alanine using glucose as a substrate, so as to realize efficient large-scale industrial production of 5-hydroxy β-indolyl alanine by fermentation.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for biosynthesizing 5-hydroxy-β-indolylalanine using glucose as a substrate is provided, comprising the following steps:

[0010] 1) Using the gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine or the recombinant genetically engineered bacteria capable of expressing the gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine, taking glucose as a substrate, biofermenting and synthesizing 5-hydroxy-β-indolylalanine, the gene encoding the protein capable of expressing the protein for synthesizing 5-hydroxy-β-indolylalanine includes TPH2, a key enzyme in the hydroxylation pathway of β-indolylalanine; nagGH, a key enzyme in the hydroxylation pathway of anthranilate; trpEDCBA, a key enzyme in the β-indolylalanine pathway; folE, PTPS, and SPR genes, key enzymes in the BH4 synthesis pathway;

[0011] 2) 5-hydroxyβ-indolylalanine is separated from the system of 1).

[0012] The above synthetic 5-hydroxy-β-indolylalanine protein encoding gene expresses the following protein, and the protein has any of the following amino acid sequences:

[0013] a: The amino acid sequence is as shown in SEQ ID NO: 07-17, specifically: the amino acid sequence of TPH2, a key enzyme in the β-indolylalanine hydroxylation pathway, is shown in SEQ ID NO: 07; the amino acid sequence of nagGH, a key enzyme in the anthranilate hydroxylation pathway, is shown in SEQ ID NO: 08-09; the amino acid sequence of trpEDCBA, a key enzyme in the β-indolylalanine pathway, is shown in SEQ ID NO: 10-14 (the amino acid sequences of the five enzymes trpE, trpD, trpC, trpB, and trpA are shown in SEQ ID NO: 10-14, respectively); the amino acid sequences of folE, PTPS, and SPR genes, key enzymes in the BH4 synthesis pathway, are shown in SEQ ID NO: 15-17, respectively.

[0014] or b: having a sequence identity of at least 95% with the amino acid sequences described in a.

[0015] Further, it has a sequence identity of more than 98% with the amino acid sequence described in a; more preferably, it has a sequence identity of more than 99% with the amino acid sequence described in a;

[0016] or c: formed by substituting, adding or deleting one or more amino acid residues at the C-terminus and / or N-terminus of each amino acid sequence described in a;

[0017] Furthermore, a derivative protein having the function of the protein is formed by adding or deleting 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues at the C-terminus and / or N-terminus of the amino acid sequence described in a.

[0018] According to the above scheme, the nucleotide sequence of the TPH2 gene, a key enzyme in the β-indolylalanine hydroxylation pathway, is shown in SEQ ID NO: 01; the nagGH, a key enzyme in the anthranilate hydroxylation pathway, is shown in SEQ ID NO: 02; the trpEDCBA, a key enzyme in the β-indolylalanine pathway, is shown in SEQ ID NO: 03; the nucleotide sequences of the folE, PTPS, and SPR genes, key enzymes in the BH4 synthesis pathway, are shown in SEQ ID NOs: 04-06, respectively.

[0019] According to the above scheme, the specific steps of step 2) are: the recombinant bacteria constructed in step 1) are activated and cultured in LB medium, and then transferred to a fermenter to induce enzyme expression, and then 5-hydroxy β-indolyl alanine is synthesized from scratch using glucose as a substrate. Specifically, the constructed recombinant genetically engineered bacteria are activated and cultured in LB medium to obtain seed liquid, and the seed liquid is transferred to a fermenter, and fermented until OD600 grows to 1-50, IPTG is added to induce conversion and batch fed fermentation is started to synthesize 5-hydroxy β-indolyl alanine from scratch using glucose as a substrate.

[0020] Furthermore, the seed solution OD 600 =5-10;

[0021] The culture conditions of the seed solution are 28-40°C, 160-230rpm, preferably 37°C, 225rpm;

[0022] The seed solution is cultured for 10-15 hours, preferably 12 hours;

[0023] The ratio of the seed liquid to the fermentation medium is 1%-10%, preferably 5%;

[0024] The seed culture medium (mass percentage) is 1% tryptone, 1% sodium chloride and 0.5% yeast extract;

[0025] The fermentation medium is composed of: 24 g / L yeast powder, 12 g / L peptone, 16.43 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, and 10 g / L glucose.

[0026] The culture conditions of the fermentation medium are 28-40°C, 160-500rpm, preferably 37°C, 300rpm;

[0027] The pH during the fermentation is controlled at 5-9, preferably 7.0;

[0028] The induced OD 600 1-50, preferably 10;

[0029] The final concentration of IPTG is 0.1-10 mM, preferably 1 mM;

[0030] The induction expression condition is 16-37°C, preferably 30°C;

[0031] The glucose concentration of the reaction system is 1-100 g / L, preferably 10 g / L;

[0032] The fermentation time is 18-72 hours, preferably 24-66 hours, and more preferably 48-60 hours.

[0033] The present invention also provides a recombinant vector comprising the genes encoding all enzymes for synthesizing 5-hydroxy β-indolyl alanine. The sequences of the genes encoding all enzymes for synthesizing 5-hydroxy β-indolyl alanine are shown in SEQ ID NOs: 1-6.

[0034] The present invention also provides a recombinant genetically engineered bacterium for biosynthesizing 5-hydroxy-β-indolylalanine using glucose as a substrate. The recombinant genetically engineered bacterium is a recombinant genetically engineered bacterium comprising a recombinant vector encoding a gene for synthesizing 5-hydroxy-β-indolylalanine enzyme as shown in SEQ ID NO: 1-6, or a recombinant genetically engineered bacterium obtained by integrating part of the enzyme genes into the genome of a host cell for expression, and expressing the remaining enzyme genes in a plasmid and then transferring them into the aforementioned host cell.

[0035] According to the above scheme, the key enzymes folE, PTPS, and SPR genes of the BH4 synthesis pathway are expressed on the genome of the host cell, and other target enzyme genes are expressed in plasmids and then transferred into the host cell whose genome expresses the key enzymes folE, PTPS, and SPR genes of the BH4 synthesis pathway.

[0036] Provided is a method for constructing a recombinant genetically engineered bacterium: expressing the key enzyme TPH2 gene of the β-indolylalanine hydroxylation pathway, the key enzyme nagGH of the anthranilate hydroxylation pathway, and the key enzyme trpEDCBA enzyme gene on the β-indolylalanine pathway in a plasmid to obtain a recombinant vector; and transferring the recombinant vector into a host cell expressing the key enzyme folE, PTPS, and SPR genes of the BH4 synthesis pathway on the genome to obtain a recombinant genetically engineered bacterium.

[0037] The method for constructing the recombinant genetically engineered bacteria of the present invention specifically comprises the following steps:

[0038] The TPH2 gene, a key enzyme in the β-indolylalanine hydroxylation pathway, and the nagGH gene, a key enzyme in the anthranilic acid hydroxylation pathway, are placed in the same plasmid for tandem expression; the trpEDCBA gene, a key enzyme in the β-indolylalanine pathway, is placed in the same plasmid for tandem expression; the above plasmids are transformed into host cells together to obtain recombinant genetically engineered bacteria.

[0039] Furthermore, the host cell is an Escherichia coli host cell, preferably a BL21 (DE3), ΔtrpR (folE, PTPS, SPR), ΔtnaA Escherichia coli host cell; that is, BL21 (DE3) is used as the starting strain, the trpR gene on the genome is replaced with folE, PTPS, SPR genes, and the tnaA gene is knocked out.

[0040] A specific implementation method provided by the present invention is:

[0041] Transformation of host bacteria: Starting from strain BL21 (DE3), using FPS / trpR-F, FPS / trpR-R as primers and pET28a-FPS plasmid as template, we obtained folE, PTPS, and SPR gene fragments with upstream and downstream homologous arms of trpR, and electroporated this fragment into BL21 competent cells to replace the trpR gene on the genome, and PCR screened positive clones. Named 5HT-09.

[0042] Then, tnaAup-F, tnaAup-R, tnaAdown-F, tnaAdown-R were used as primers, and BL21 (DE3) genome was used as template to obtain the upstream fragment of tnaA gene and the downstream fragment of tnaA gene. The two fragments were connected by fusion PCR using tnaAup-F and tnaAdown-R as primers to obtain the ΔtnaA fragment. This fragment was electrotransferred into 5HT-09 competent cells to replace the genomic tnaA gene, and positive clones were screened by PCR. It was named 5HT-10.

[0043] The present invention also provides the use of the above-mentioned genes encoding all enzymes for synthesizing 5-hydroxy β-indolyl alanine, the recombinant vector or the genetically engineered bacteria in producing enzymes in the pathway for synthesizing 5-hydroxy β-indolyl alanine.

[0044] The present invention also provides the use of all enzyme encoding genes for synthesizing 5-hydroxy-β-indolylalanine, the recombinant vector or the recombinant genetic engineering bacteria in producing 5-hydroxy-β-indolylalanine.

[0045] The invention also provides a series of enzymes for synthesizing 5-hydroxy beta-indolyl alanine, expression plasmids of several enzymes, several expression hosts and recombinant genetic engineering strains for synthesizing 5-hydroxy beta-indolyl alanine.

[0046] Beneficial effects of the present invention:

[0047] (1) The present invention discloses a recombinant vector and a recombinant genetic engineering bacterium containing a series of enzyme encoding genes for synthesizing 5-hydroxy β-indolyl alanine. The series of enzyme encoding genes encoding the synthesized 5-hydroxy β-indolyl alanine in the recombinant vector are expressed by fermentation culture to obtain a series of enzymes for synthesizing 5-hydroxy β-indolyl alanine. The process is easy to implement, the conditions are easy to control, and it is suitable for promoting industrial production.

[0048] (2) The present invention organically integrates multiple pathways of different synthetic pathways and enhances the expression of key enzymes in the pathway of producing 5-hydroxy-β-indolylalanine from glucose, thereby increasing the yield of 5-hydroxy-β-indolylalanine. This provides data support for the industrialization of producing 5-hydroxy-β-indolylalanine from glucose, improving its production efficiency and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 :Plasmid pET28a-H2GH map

[0051] Figure 2 :Plasmid pACYCDuet-TRP Map DETAILED DESCRIPTION

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0055] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0056] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0057] Example 1 Construction of plasmid

[0058] Tandem expression of key enzymes in different hydroxylation pathways:

[0059] After codon optimization, TPH2, a key enzyme in the β-indolylalanine hydroxylation pathway, and nagGH, a key enzyme in the anthranilate hydroxylation pathway, were inserted into the NdeI and XhoI sites of the pET28a(+) plasmid to obtain pET28a-TPH2 and pET28a-nagGH plasmids, respectively.

[0060] Using H2-F and H2-R as primers and pET28a-TPH2 plasmid as template, the pET28a plasmid vector containing the TPH2 gene was cloned; using nagGH-F and nagGH-R as primers and plasmid pET28a-nagGH as template, the nagGH gene was cloned; the above two fragments were connected together using a seamless cloning kit to form plasmid pET28a-H2GH.

[0061] Construction of expression plasmids for key enzymes in the β-indolylalanine pathway:

[0062] Using trp-F and trp-R as primers and BL21 (DE3) genome as a template, the trpEDCBA gene fragment was cloned; using pACYCDuet-F and pACYCDuet-R as primers and pACYCDuet empty plasmid as a template, the trpEDCBA gene was inserted between the NcoI and EcoRI sites of the pACYCDuet plasmid to obtain the pACYCDuet-TRP plasmid.

[0063] Table 1 Primer sequence 1

[0064]

[0065] Example 2 Transformation of host bacteria

[0066] Acquisition of BH4 synthesis gene: folE, PTPS, SPR, key enzymes in BH4 synthesis pathway, codon optimization, artificial synthesis of optimized folE, PTPS, SPR gene sequences, as shown in SEQ ID No: 1-3, folE, PTPS, SPR genes were inserted between NdeI and XhoI sites of pET28a(+) plasmid to obtain pET28a-FPS expression vector;

[0067] Starting from strain BL21 (DE3), using FPS / trpR-F, FPS / trpR-R as primers and pET28a-FPS plasmid as template, folE, PTPS, and SPR gene fragments with upstream and downstream homologous arms of trpR were obtained. This fragment was electrotransferred into BL21 competent cells to replace the trpR gene on the genome, and positive clones were screened out by PCR. Named 5HT-09. Then, using tnaAup-F, tnaAup-R, tnaAdown-F, and tnaAdown-R as primers and BL21 (DE3) genome as template, the upstream fragment of tnaA gene and the downstream fragment of tnaA gene were obtained. The two fragments were connected by fusion PCR using tnaAup-F and tnaAdown-R as primers to obtain the ΔtnaA fragment. This fragment was electrotransferred into 5HT-09 competent cells to replace the genomic tnaA gene, and positive clones were screened out by PCR. Named 5HT-10.

[0068] Table 2 Primer sequence 2

[0069]

[0070]

[0071] Example 3 Construction and fermentation of a strain producing 5-hydroxy-β-indolylalanine from glucose

[0072] The plasmids pET28a-HG and pACYCDuet-TRP in Example 1 were electroporated into the host cells 5HT-10 (BL21 (DE3), ΔtrpR (folE, PTPS, SPR), ΔtnaA) in Example 2 to obtain the recombinant engineered bacteria 5HT108. The engineering bacteria 5HT108, a single colony activated by solid LB medium plate was inoculated into a 500mL seed bottle containing 100mL liquid LB medium (1% peptone, 0.5% yeast powder, 1% sodium chloride, the rest is water, pH 7.0), 50mg / L kanamycin and 34mg / L chloramphenicol were added, and the culture was shaken at 37°C and 220rpm for 12h; the inoculation amount was inoculated into 3L fermentation medium according to 5%, wherein the components of the fermentation medium were: 24g / L yeast powder, 12g / L peptone, 16.43g / L dipotassium hydrogen phosphate, 2.31g / L potassium dihydrogen phosphate, and 10g / L glucose. 50mg / L kanamycin and 34mg / L chloramphenicol were added, the fermentation temperature was 37°C, the rotation speed was 300rpm, and when OD600 grew to 10, 0.1mM IPTG was added to induce conversion and start batch fed fermentation. The batch fermentation temperature was 30°C, ammonia was used to control the pH to 7.0, and glucose was fed in batches to control its concentration to 10 g / L. The production of products was detected during the fermentation process. The fermentation time was generally selected to be 48-60 hours, and the yield reached the maximum at 56 hours of fermentation. Finally, the 5HT108 strain could ferment up to 25 g / L of 5-hydroxy-β-indolylalanine, and the remaining β-indolylalanine was less than 1 g / L.

[0073] The traditional method of synthesizing 5-hydroxy β-indolyl alanine by a single biological pathway has low yield due to factors such as low efficiency of the hydroxylation step and harsh conditions, and a large amount of β-indolyl alanine is left over, making it difficult to separate and purify the product. For this reason, the prior art generally adopts a weakened β-indolyl alanine synthesis, which can alleviate the surplus to a certain extent, but also reduces the yield of the final product 5-hydroxy β-indolyl alanine.

[0074] The present invention uses glucose as a substrate to synthesize an intermediate product of anthranilic acid on the path of 5-hydroxy-β-indolylalanine through 5-hydroxylation reaction, cyclization reaction, polymerization reaction, and simultaneously synthesizes the target product through cyclization reaction, polymerization reaction, and 5-hydroxylation reaction, designs a multi-path synthesis route, and synthesizes 5-hydroxy-β-indolylalanine through multi-path organic integration of different synthesis paths, thereby achieving the purpose of significantly improving the yield of 5-hydroxy-β-indolylalanine, overcoming the fusion problem of multi-path synthesis, and being able to control β-indolylalanine within a concentration that is easy to separate. It has an important impact on the industrialization of 5-hydroxy-β-indolylalanine. It provides a new idea for the industrialization of 5-hydroxy-β-indolylalanine produced from glucose, improving its production efficiency, and reducing production costs.

[0075] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention. SEQUENCE LISTING <110> Hebei Vidakang Biotechnology Co., Ltd. <120> Method for biosynthesizing 5-hydroxy-β-indolylalanine using glucose as substrate and its application <160> 17 <170> PatentIn version 3.3 <210> 1 <211> 1473 <212> DNA <213> Synthesis <400> 1 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> 2 <211> 1760 <212> DNA <213> Synthetic <400> 2 atgagtgaac cccaacgatt aaaacccgtg tttccccaag atccgaaatg gccgggcgaa 60 ggtagcagcc gcgttccctt ctgggcctac acccgcgaag acctgtacaa gcgcgaattg 120 gagcgcctgt tctatgcaaa ccactggtgc tatgtaggcc tggaagccga gattccgaat 180 ccaggcgact tcaagcgaac ggtgatcggt gagcgctcgg tcatcatggt gcgtgatccg 240 gatggcggca tcaacgtggt ggagaacgtc tgcgcccacc gtggcatgcg cttttgccgc 300 gagcgccacg gcaacgccaa ggacttcttc tgcccctacc accagtggaa ctacagcctc 360 aagggtgacc tgcagggcgt gcccttccgc cgtggcgtca aggaggacgg caaggtcaac 420 ggcggcatgc ccaaggactt caaactcgaa gaacacggcc tgaccaagct caaggtggcc 480 gcccgaggcg gtgcagtgtt tgcctcttt gaccacgatg tcgagccctt cgagggttc 540 ctgggcccaa ccatcctgca ttacttcgac cgcgtcttca acggccgcaa gctcaagatc 600 ctgggctacc gccgccagcg catcccgggc aactggaagc tgatgcagga gaatcaag 660 gacccctacc acccgggcct gctgcacacc tggttctcga ccttcgggct ctggcgcgcc 720 gacaacaagt cggaactgaa gatggacgcc aagttccgcc acgccgcaat gatctccacg 780 cgcggtcagg gcggcaagaa cgaggaggtc gtgtccggcg tggacagctt caaggaacag 840 atgaaggtga acgacccgcg cctgctcgac atcgtgcccg agccctggtg gggcggtccg 900 actgcggtga tgaccacgat cttccccagc gtgatcatcc agcagcaggt caacagcgta 960 tcgacccgcc acatccagcc caacggtcac ggctccttcg atttcgtctg gacccacttc 1020 ggcttcgagg acgacaacga ggagtggacc cagcgccgcc tgatccaggc caacctgttc 1080 gggccggcgg gcttcgtgtc ggccgatgac ggcgaggtga tcgagtggtc gcaggaaggc 1140 tttgagcaaa aaccgacgca ccgcaccgtg atcgagatgg gcggtcacga aatcggcgac 1200 acggaccaca tggtcaccga gacgctgatc cgcggcatgt acgactactg gcgcaaggtg 1260 atgggggaat aaacatggtc gacttcaaaa cctatttcga actgctgaac ctgtacagcg 1320 actacgccat ggtgtgcgac tccgccaatt gggagaagtg gcctgatttc ttcatcgaga 1380 ccggcaccta ccgcctgcag ccgcgcgaga actttgagca gggcctgccg ctgtgcctgc 1440 tggcgctgga gagcaaggca atgatccgcg accgggtgta cggcgtcaag gaaaccatgt 1500 accacgaccc ctactaccag cgccacatcg tgggcacgcc gcgcgtgctg tcagtggagc 1560 gtgacgcgga tggcgagcgc atcaccgccg aagccagcta tgccgtgatt cgcaccaagt 1620 acgacggcga ttccacgatt ttcaacgccg gctattaccg agacgtgatc gtgcgcacgc 1680 ccgagggcct caagctgaag tcgcgcctgt gcgtttacga cagcgaaatg atccccaact 1740 ctgtgatcta tccaatctga 1760 <210> 3 <211> 6531 <212> DNA <213> Escherichia coli <400> 3 atgcaaacac aaaaccgac tctcgaacta ctaacctgcg aaggcgctta tcgcgacaac 60 ccgaccgcgc tttttcacca gttgtgtggg gatcgtccgg caacgctgct gctggaatcc 120 gcagatatcg acagcaaaga tgatttaaaa agcctgctac tggtagacag tgcgctgcgc 180 attacagcat taggtgacac tgtcacaatt aaggcgttat ccggcaatgg tgaagccctg 240 ctggcactac tggataacgc cctgcctgcg ggtgtggaaa atgaacaatc accaaactgc 300 cgtgtgctgc gcttcccccc tgtcagtcca ctgctggatg aagacgctcg cttatgctcc 360 cttcggtttt ttgacgcttt ccgtttattg cagaatctgt tgaatgtacc gaaggaagaa 420 cgagaagcca tgttcttcgg cggcctgttc tcttatgacc ttgtggcggg atttgaagat 480 ttaccgcaac tgtcagcgga aaataactgc cctgatttct gtttttatct cgctgaaacg 540 ctgatggtga ttgaccatca gaaaaaaagc acccgtattc aggccagcct gtttgctccg 600 aatgaagaag aaaaacaacg tctcactgct cgcctgaacg aactacgtca gcaactgacc 660 gaagccgcgc cgccgctgcc agtggtttcc gtgccgcata tgcgttgtga atgtaatcag 720 agcgatgaag agttcggtgg cgtagtgcgt ttgttgcaaa aagcgattcg cgctggagaa 780 attttccagg tggtgccatc tcgccgtttc tctctgccct gcccgtcacc gctggcggcc 840 tattacgtgc tgaaaaagag taatcccagc ccgtacatgt tttttatgca ggataatgat 900 ttcaccctat ttggcgcgtc gccggaaagc tcgctcaagt atgatgccac cagccgccag 960 attgagatct acccgattgc cggaacacgc ccacgcggtc gtcgcgccga tggttcactg 1020 gacagagatc tcgacagccg tattgaactg gaaatgcgta ccgatcataa agagctgtct 1080 gaacatctga tgctggttga tctcgcccgt aatgatctgg cacgcatttg cacccccggc 1140 agccgctacg tcgccgatct caccaaagtt gaccgttatt cctatgtgat gcacctcgtc 1200 tctcgcgtag tcggcgaact gcgtcacgat cttgacgccc tgcacgctta tcgcgcctgt 1260 atgaatatgg ggacgttaag cggtgcgccg aaagtacgcg ctatgcagtt aattgccgag 1320 gcggaaggtc gtcgccgcgg cagctacggc ggcgcggtag gttatttcac cgcgcatggc 1380 gatctcgaca cctacattgt gatccgctcg gcgctggtgg aaaacggtat cgccaccgtg 1440 caagcgggtg ctggtgtagt ccttgattct gttccgcagt cggaagccga cgaaacccgt 1500 aacaaagccc gcgctgtact gcgcgctatt gccaccgcgc atcatgcaca ggagactttc 1560 tgatggctga cattctgctg ctcgataata tcgactcttt tacgtacaac ctggcagatc 1620 agttgcgcag caatgggcat aacgtggtga tttaccgcaa ccatattccg gcgcaaacct 1680 taattgaacg cctggcgacc atgagcaatc cggtgctgat gctttctcct ggccccggtg 1740 tgccgagcga agccggttgt atgccggaac tcctcacccg cttgcgtggc aagctgccca 1800 ttattggcat ttgcctcgga catcaggcga ttgtcgaagc ttacgggggc tatgtcggtc 1860 aggcgggcga aattctccac ggtaaagcct ccagcattga acatgacggt caggcgatgt 1920 ttgccggatt aacaaacccg ctgccggtgg cgcgttatca ctcgctggtt ggcagtaaca 1980 ttccggccgg tttaaccatc aacgcccatt ttaatggcat ggtgatggca gtacgtcacg 2040 atgcggatcg cgtttgtgga ttccagttcc atccggaatc cattctcacc acccagggcg 2100 ctcgcctgct ggaaaacg ctggcctggg cgcagcagaa actagagcca gccaacacgc 2160 tgcaaccgat tctggaaaaa ctgtatcagg cgcagacgct tagccaacaa gaaagccacc 2220 agctgttttc agcggtggtg cgtggcgagc tgaagccgga aacactggcg gcggcgctgg 2280 tgagcatgaa aattcgcggt gagcacccga acgagatcgc cggggcagca accgcgctac 2340 tggaaaacgc agcgccgttc ccgcgcccgg attatctgtt tgctgatatc gtcggtactg 2400 gcggtgacgg cagcaacagt atcaatattt ctaccgccag tgccgtttgtc gccgcggcct 2460 gtgggctgaa agtggcgaaa cacggcaacc gtagcgtctc cagtaaatct ggttcgtccg 2520 atctgctggc ggcgttcggt attaatcttg atatgaacgc cgataaatcg cgccaggcgc 2580 tggatgagtt aggtgtatgt ttcctctttg cgccgaagta tcacaccgga ttccgccacg 2640 cgatgccggt tcgccagcaa ctgaaaaccc gcaccctgtt caatgtgctg gggccattga 2700 ttaacccggc gcatccgccg ctggcgttaa ttggtgttta tagtccggaa ctggtgctgc 2760 cgattgccga aaccttgcgc gtgctggggt atcaacgcgc ggcggtggtg cacagcggcg 2820 ggatggatga agtttcatta cacgcgccga caatcgttgc cgaactgcat gacggcgaaa 2880 ttaaaagcta tcagctcacc gcagaagact ttggcctgac accctaccac caggagcaac 2940 tggcaggcgg aacaccggaa gaaaaccgtg acattttaac acgtttgtta caaggtaaag 3000 gcgacgccgc ccatgaagca gccgtcgctg cgaacgtcgc catgttaatg cgcctgcatg 3060 gccatgaaga tctgcaagcc aatgcgcaaa ccgttcttga ggtactgcgc agtggttccg 3120 cttacgacag agtcaccgca ctggcggcac gagggtaaat gatgcaaacc gttttagcga 3180 aaatcgtcgc agacaaggcg atttgggtag aagcccgcaa acagcagcaa ccgctggcca 3240 gttttcagaa tgaggttcag ccgagcacgc gacattttta tgatgcgcta cagggtgcgc 3300 gcacggcgtt tattctggag tgcaagaaag cgtcgccgtc aaaaggcgtg atccgtgatg 3360 atttcgatcc agcacgcatt gccgccattt ataaacatta cgcttcggca atttcggtgc 3420 tgactgatga gaaatatttt caggggagct ttaatttcct ccccatcgtc agccaaatcg 3480 ccccgcagcc gattttatgt aaagacttca ttatcgaccc ttaccagatc tatctggcgc 3540 gctattacca ggccgatgcc tgctttata tgctttcagt actggatgac gaccaatatc 3600 gccagcttgc cgccgtcgct cacagtctgg agatggggt gctgaccgaa gtcagtaatg 3660 aaggagaaca ggagcgcgcc attgcattgg gagcaaaggt cgttggcatc aacaaccgcg 3720 atctgcgtga tttgtcgatt gatctcaacc gtacccgcga gcttgcgccg aaactggggc 3780 3840 gccacttgc taacggttt ctgattggtt cggcgttgat ggcccatgac gatttgcacg 3900 ccgccgtgcg ccgggtgttg ctgggtgaga ataaagtg tggcctgacg cgtgggcaag 3960 atgctaaagc agcttatgac gcgggcgcga tttacggtgg gttgattttt gttgcgacat 4020 caccgcgttg cgtcaacgtt gaacaggcgc aggaagtgat ggctgcggca ccgttgcagt 4080 atgttggcgt gttccgcaat cacgatattg ccgatgtggt ggacaaagct aaggtgttat 4140 cgctggcggc agtgcaactg catggtaatg aagaacagct gtatatcgat acgctgcgtg 4200 aagctctgcc agcacatgtt gccatctgga aagcattaag cgtcggtgaa accctgcccg cccgcgagtt tcagcacgtt gataaatatg ttttagacaa cggccagggt ggaagcgggc aacgttttga ctggtcacta ttaaatggtc aatcgcttgg caacgttctg ctggcggggg gcttaggcgc agataactgc gtggaagcgg cacaaaccgg ctgcgccgga cttgatttta attctgctgt agagtcgcaa ccgggcatca aagacgcacg tctttttggcc tcggttttcc 4500. agacgctgcg cgcatattaa ggaaggac aatgacaaca ttacttaacc cctattttgg tgagtttggc ggcatgtacg tgccacaaat cctgatgcct gctctgcgcc agctggaaga 4620. agcttttgtc agtgcgcaaa aagatcctga atttcaggct cagttcaacg acctgctgaa aaactatgcc gggcgtccaa ccgcgctgac caaatgccag aacattacag ccgggacgaa caccacgctg tatctcaagc gtgaagattt gctgcacggc ggcgcgcata aaactaacca ggtgctgggg caggcgttgc tggcgaagcg gatgggtaa accgaaatca tcgccgaaac cggtgccggt cagcatggcg tggcgtcggc ccttgccagc gccctgctcg gcctgaaatg 4920 ccgtatttat atgggtgcca aagacgttga acgccagtcg cctaacgttt ttcgtatgcg 4980 cttaatgggt gcggaagtga tcccggtgca tagcggttcc gcgacgctga aagatgcctg 5040 taacgaggcg ctgcgcgact ggtccggtag ttacgaaacc gcgcactata tgctgggcac 5100 cgcagctggc ccgcatcctt atccgaccat tgtgcgtgag tttcagcgga tgattggcga 5160 agaaaccaaa gcgcagattc tggaaagaga aggtcgcctg ccggatgccg ttatcgcctg 5220 tgttggcggc ggttcgaatg ccatcggcat gtttgctgat ttcatcaatg aaaccaacgt 5280 cggcctgatt ggtgtggagc caggtggtca cggtatcgaa actggcgagc acggcgcacc 5340 gctaaaacat ggtcgcgtgg gtatctattt cggtatgaaa gcgccgatga tgcaaaccga 5400 agacgggcag attgaagaat cttactccat ctccgccgga ctggatttcc cgtctgtcgg 5460 cccacaacac gcgtatctta acagcactgg acgcgctgat tacgtgtcta ttaccgatga 5520 tgaagccctt gaagccttca aaacgctgtg cctgcacgaa gggatcatcc cggcgctgga 5580 atcctcccac gccctggccc atgcgttgaa aatgatgcgc gaaaacccgg ataaagagca 5640 gctactggtg gttaaccttt ccggtcgcgg cgataaagac atcttcaccg ttcacgatat 5700 tttgaaagca cgagggggaaa tctgatggaa cgctacgaat ctctgtttgc ccagttgaag 5760 gagcgcaaag aaggcgcatt cgttcctttc gtcacgctcg gtgatccggg cattgagcag 5820 tcattgaaaa ttatcgatac gctaattgaa gccggtgctg acgcgctgga gttaggtatc 5880 cccttctccg acccactggc ggatggcccg acgattcaaa acgccactct gcgcgccttt 5940 gcggcaggtg tgactccggc acaatgtttt gaaatgctgg cactgattcg ccagaaacac 6000 ccgaccattc ccattggcct gttgatgtat gccaatctgg tgtttaacaa aggcattgat 6060 gagttttatg cccagtgcga aaaagtcggc gtcgattcgg tgctggttgc cgatgtgcca 6120 gttgaagagt ccgcgccctt ccgccaggcc gcgttgcgtc ataatgtcgc acctatcttc 6180 atctgcccgc caaatgccga tgacgacctg ctgcgccaga tagcctctta cggtcgtggt 6240 tacacctatt tgctgtcacg agcaggcgtg accggcgcag aaaaccgcgc cgcgttaccc 6300 ctcaatcatc tggttgcgaa gctgaaagag tacaacgctg cacctccatt gcagggattt 6360 ggtatttccg ccccggatca ggtaaaagca gcgattgatg caggagctgc gggcgcgatt 6420 tctggttcgg ccattgttaa aatcatcgag caacatatta atgagccaga gaaaatgctg 6480 gcggcactga aagtttttgt acaaccgatg aaagcggcga cgcgcagtta a 6531 <210> 4 <211> 573 <212> DNA <213> Synthetic <400> 4 atgaaagaag tgaacaaaga acagatcgaa caggcagtgc gtcagattct ggaagcaatc 60 ggtgaagatc cgaatcgcga aggtctgctg gataccccga aacgcgttgc caaaatgtat 120 gcagaagttt ttagcggtct gaacgaagat ccgaaagaac attttcagac aatttttggt 180 gaaaaccatg aagaactggt gctggttaaa gatattgcat ttcatagtat gtgcgaacat 240 catctggttc cgttttatgg taaagcacat gtggcatata ttccacgtgg tggtaaagta 300 acaggtctga gcaaactggc ccgtgcagtt gaagcagttg caaaacgtcc gcagctgcag 360 gaacgtatta ccagcacaat cgcagaaagc attgtcgaaa ccctggaccc tcatggtgtg 420 atggttgttg ttgaagcaga acacatgtgt atgacaatgc gcggtgtccg taaaccaggt 480 gcaaaaaccg ttaccagcgc agtgcgcggt gtttttaaag atgatgccgc agcacgtgca 540 gaagttctgg aacatattaa acgccaggat taa 573 <210> 5 <211> 438 <212> DNA <213> Synthetic <400> 5 atgagcaccg aaggtggtgg tcgccgctgt caggcgcagg ttagccgccg tattagcttt 60 agcgcgagcc atcgtctgta ttccaaattt ctgagcgatg aagaaaacct gaaactgttt 120 ggtaaatgta ataacccgaa tggtcatggt cataattata aagttgtggt gaccgttcat 180 ggtgaaattg atcctgccac cggtatggtc atgaatctgg cagatttaaa aaaatatatg 240 gaagaagcaa ttatgcagcc gctggatcat aaaaatctgg atatggatgt tccgtatttt 300 gcagatgttg ttagcaccac cgaaaatgtt gcagtttata tttgggataa tctgcagaaa 360 gttctgccgg ttggtgttct gtataaagtt aaagtttatg aaaccgataa taatattgtt 420 gtttataaag gtgaataa 438 <210> 6 <211> 786 <212> DNA <213> Synthetic <400> 6 atggaaggtg gtctgggtcg tgccgtttgt ctgctgacgg gtgcaagccg tggttttggt 60 cgtacactgg caccgctgct ggcgagcctg ctgagccctg gtagcgttct ggttctgagc 120 gcacgtaatg atgaagcact gcgtcagctg gaagcagaac tgggtgcaga acgtagtggt 180 ctgcgcgttg ttcgtgttcc ggcagattta ggtgcagaag caggtctgca gcagctgctg 240 ggtgcactgc gtgaactgcc tcgtcctaaa ggtctgcagc gtctgctgct gattaataat 300 gcaggtagtc tgggtgatgt tagcaaaggt tttgtagatt taagcgattc tactcaggtt 360 aataattatt gggccctgaa tctgacgagt atgctgtgtc tgacttctag cgtactgaaa 420 gcatttcctg atagtccggg tctgaatcgt accgtggtta atatttccag cctgtgtgca 480 ctgcagccgt ttaaaggctg ggcactgtat tgtgccggta aagcagcacg tgatatgctg 540 tttcaggttc tggcactgga agaaccaaat gttcgtgttc tgaattatgc tccgggtccg 600 ctggatacgg atatgcagca gctggcgcgt gaaacatcag ttgatcctga tatgcgtaaa 660 ggtctgcagg aactgaaagc aaaaggtaaa ctggtggatt gtaaagttag cgcacagaaa 720 ctgctgagcc tgctggaaaa agatgaattt aaaagtggtg cacatgtgga tttttatgat 780 aaataa 786 <210> 7 <211> 490 <212> PRT <213> Synthetic <400> 7 MQPAMMMFSS KYWARRGFSL DSAVPEEHQL LGSSTLNKPN SGKNDDKGNK GSSKREAATE 60 SGKTAVVFSL KNEVGGLVKA LRLFQEKRVN MVHIESRKSR RRSSEVEIFV DCECGKTEFN 120 ELIQLLKFQT TIVTLNPPEN IWTEEEELED VPWFPRKISE LDKCSHRVLM YGSELDADHP 180 GFKDNVYRQR RKYFVDVAMG YKYGQPIPRV EYTEEETKTW GVVFRELSKL YPTHACREYL 240 KNFPLLTKYC GYREDNVPQL EDVSMFLKER SGFTVRPVAG YLSPRDFLAG LAYRVFHCTQ 300 YIRHGSDPLY TPEPDTCHEL LGHVPLLADP KFAQFSQEIG LASLGASDED VQKLATCYFF 360 TIEFGLCKQE GQLRAYGAGL LSSIGELKHA LSDKACVKAF DPKTTCLQEC LITTFQEAYF 420 VSESFEEAKE KMRDFAKSIT RPFSVYFNPY TQSIEILKDT RSIENVVQDL RSDLNTVCDA 480 LNKMNQYLGI 490 <210> 8 <211> 423 <212> PRT <213> Synthetic <400> 8 MSEPQRLKPV FPQDPKWPGE GSSRVPFWAY TREDLYKREL ERLFYANHWC YVGLEAEIPN 60 PGDFKRTVIG ERSVIMVRDP DGGINVVENV CAHRGMRFCR ERHGNAKDFF CPYHQWNYSL 120 KGDLQGVPFR RGVKQDGKVN GGMPKDFKLE EHGLTKLKVA ARGGAVFASF DHDVEPFEEF 180 LGPTILHYFD RVFNGRKLKI LGYRRQRIPG NWKLMQENIK DPYHPGLLHT WFSTFGLWRA 240 DNKSELKMDA KFRHAAMIST RGQGGKNEEV VSGVDSFKEQ MKVNDPRLLD IVPEPWWGGP 300 TAVMTTIFPS VIIQQQVNSV STRHIQPNGH GSFDFVWTHF GFEDDNEEWT QRRLIQANLF 360 GPAGFVSADD GEVIEWSQEG FEQKPTHRTV IEMGGHEIGD TDHMVTETLI RGMYDYWRKV 420 MGE 423 <210> 9 <211> 161 <212> PRT <213> Synthetic <400> 9 MVDFKTYFEL LNLYSDYAMV CDSANWEKWP DFFIETGTYR LQPRENFEQG LPLCLLALES 60 KAMIRDRVYG VKETMYHDPY YQRHIVGTPR VLSVERDADG ERITAEASYA VIRTKYDGDS 120 TIFNAGYYRD VIVRTPEGLK LKSRLCVYDS EMIPNSVIYP I 161 <210> 10 <211> 520 <212> PRT <h2 style=";text-align:left;direction:ltr"><213> Escherichia coli<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> MQTQKPTLEL LTCEGAYRDN PTALFHQLCG DRPATLLLES ADIDSKDDLK SLLLVDSALR 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ITALGDTVTI KALSGNGEAL LALLDNALPA GVENEQSPNC RVLRFPPVSP LLDEDARLCS 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LSVFDAFRLL QNLLNVPKEE REAMFFGGLF SYDLVAGFED LPQLSAENNC PDFCFYLAET 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LMVIDHQKKS TRIQASLFAP NEEEKQRLTA RLNELRQQLT EAAPPLPVVS VPHMRCECNQ 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SDEEFGGVVR LLQKAIRAGE IFQVVPSRRF SLPCPSPLAA YYVLKKSNPS PYMFFMQDND 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> FTLFGASPES SLKYDATSRQ IEIYPIAGTR PRGRRADGSL DRDLDSRIEL EMRTDHKELS 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> EHLMLVDLAR NDLARICTPG SRYVADLTKV DRYSYVMHLV SRVVGELRHD LDALHAYRAC 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> MNMGTLSGAP KVRAMQLIAE AEGRRRGSYG GAVGYFTAHG DLDTYIVIRS ALVENGIATV 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> QAGAGVVLDS VPQSEADETR NKARAVLRAI ATAHHAQETF 520<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 531<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Escherichia coli<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> MADILLLDNI DSFTYNLADQ LRSNGHNVVI YRNHIPAQTL IERLATMSNP VLMLSPGPGV 60<h2 style=";text-align:left;direction:ltr"> PSEAGCMPEL LTRLRGKLPI IGICLGHQAI VEAYGGYVGQ AGEILHGKAS SIEHDGQAMF 120 AGLTNPLPVA RYHSLVGSNI PAGLTINAHF NGMVMAVRHD ADRVCGFQFH PESILTTQGA 180 RLLEQTLAWA QQKLEPANTL QPILEKLYQA QTLSQQESHQ LFSAVVRGEL KPEQLAAALV 240 SMKIRGEHPN EIAGAATALL ENAAPFPRPD YLFADIVGTG GDGSNSINIS TASAFVAAAC 300 GLKVAKHGNR SVSSKSGSSD LLAAFGINLD MNADKSRQAL DELGVCFLFA PKYHTGFRHA 360 MPVRQQLKTR TLFNVLGPLI NPAHPPLALI GVYSPELVLP IAETLRVLGY QRAAVVHSGG 420 MDEVSLHAPT IVAELHDGEI KSYQLTAEDF GLTPYHQEQL AGGTPEENRD ILTRLLQGKG 480 DAAHEAAVAA NVAMLMRLHG HEDLQANAQT VLEVLRSGSA YDRVTALAAR G 531 <210> 12 <211> 453 <212> PRT <213> Escherichia coli <400> 12 MMQTVLAKIV ADKAIWVEAR KQQQPLASFQ NEVQPSTRHF YDALQGARTA FILECKKASP 60 SKGVIRDDFD PARIAAIYKH YASAISVLTD EKYFQGSFNF LPIVSQIAPQ PILCKDFIID 120 PYQIYLARYY QADACLLMLS VLDDDQYRQL AAVAHSLEMG VLTEVSNEEE QERAIALGAK 180 VVGINNRDLR DLSIDLNRTR ELAPKLGHNV TVISESGINT YAQVRELSHF ANGFLIGSAL 240 MAHDDLHAAV RRVLLGENKV CGLTRGQDAK AAYDAGAIYG GLIFVATSPR CVNVEQAQEV 300 MAAAPLQYVG VFRNHDIADV VDKAKVLSLA AVQLHGNEEQ LYIDTLREAL PAHVAIWKAL 360 SVGETLPARE FQHVDKYVLD NGQGGSGQRF DWSLLNGQSL GNVLLAGGLG ADNCVEAAQT 420 GCAGLDFNSA VESQPGIKDA RLLASVFQTL RAY 453 <210> 13 <211> 397 <212> PRT <213> Escherichia coli <400> 13 MTTLLNPYFG EFGGMYVPQI LMPALRQLEE AFVSAQKDPE FQAQFNDLLK NYAGRPTALT 60 KCQNITAGTN TTLYLKREDL LHGGAHKTNQ VLGQALLAKR MGKTEIIAET GAGQHGVASA 120 LASALLGLKC RIYMGAKDVE RQSPNVFRMR LMGAEVIPVH SGSATLKDAC NEALRDWSGS 180 YETAHYMLGT AAGPHPYPTI VREFQRMIGE ETKAQILERE GRLPDAVIAC VGGGSNAIGM 240 FADFINETNV GLIGVEPGGH GIETGEHGAP LKHGRVGIYF GMKAPMMQTE DGQIEESYSI 300 SAGLDFPSVG PQHAYLNSTG RADYVSITDD EALEAFKTLC LHEGIIPALE SSHALAHALK 360 MMRENPDKEQ LLVVNLSGRG DKDIFTVHDI LKARGEI 397 <210> 14 <211> 268 <212> PRT <213> Escherichia coli <400> 14 MERYESLFAQ LKERKEGAFV PFVTLGDPGI EQSLKIIDTL IEAGADALEL GIPFSDPLAD 60 GPTIQNATLR AFAAGVTPAQ CFEMLALIRQ KHPTIPIGLL MYANLVFNKG IDEFYAQCEK 120 VGVDSVLVAD VPVEESAPFR QAALRHNVAP IFICPPNADD DLLRQIASYG RGYTYLLSRA 180 GVTGAENRAA LPLNHLVAKL KEYNAAPPLQ GFGISAPDQV KAAIDAGAAG AISGSAIVKI 240 IEQHINEPEK MLAALKVFVQ PMKAATRS 268 <210> 15 <211> 190 <212> PRT <213> Synthetic <400> 15 MKEVNKEQIE QAVRQILEAI GEDPNREGLL DTPKRVAKMY AEVFSGLNED PKEHFQTIFG 60 ENHEELVLVK DIAFHSMCEH HLVPFYGKAH VAYIPRGGKV TGLSKLARAV EAVAKRPQLQ 120 ERITSTIAES IVETLDPHGV MVVVEAEHMC MTMRGVRKPG AKTVTSAVRG VFKDDAAARA 180 EVLEHIKRQD 190 <210> 16 <211> 145 <212> PRT <213> Synthetic <400> 16 MSTEGGGRRC QAQVSRRISF SASHRLYSKF LSDEENLKLF GKCNNPNGHG HNYKVVVTVH 60 GEIDPATGMV MNLADLKKYM EEAIMQPLDH KNLDMDVPYF ADVVSTTENV AVYIWDNLQK 120 VLPVGVLYKV KVYETDNNIV VYKGE 145 <210> 17 <211> 261 <212> PRT <213> Synthetic <400> 17 MEGGLGRAVC LLTGASRGFG RTLAPLLASL LSPGSVLVLS ARNDEALRQL EAELGAERSG 60 LRVVRVPADL GAEAGLQQLL GALRELPRPK GLQRLLLINN AGSLGDVSKG FVDLSDSTQV 120 NNYWALNLTS MLCLTSSVLK AFPDSPGLNR TVVNISSLCA LQPFKGWALY CAGKAARDML 180 FQVLALEEPN VRVLNYAPGP LDTDMQQLAR ETSVDPDMRK GLQELKAKGK LVDCKVSAQK 240 LLSLLEKDEF KSGAHVDFYD K 261

Claims

1. A method for biosynthesizing 5-hydroxy-β-indolylalanine using glucose as a substrate, comprising the following steps: 1) Using the gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine or the recombinant genetically engineered bacteria capable of expressing the gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine, 5-hydroxy-β-indolylalanine is synthesized by biological fermentation with glucose as a substrate. The gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine includes TPH2, a key enzyme in the hydroxylation pathway of β-indolylalanine; nagGH, a key enzyme in the hydroxylation pathway of anthranilate; trpEDCBA, a key enzyme in the β-indolylalanine pathway; and folE, PTPS, and SPR, key enzymes in the BH4 synthesis pathway; 2) 5-hydroxy-β-indolylalanine is separated from the system of 1), and the gene encoding the synthetic 5-hydroxy-β-indolylalanine protein expresses the following proteins, and the amino acid sequences of the proteins are the sequences shown in SEQ ID NOs: 07-17, specifically: the amino acid sequence of TPH2, a key enzyme in the β-indolylalanine hydroxylation pathway, is shown in SEQ ID NOs: 07; the amino acid sequence of nagGH, a key enzyme in the anthranilate hydroxylation pathway, is shown in SEQ ID NOs: 08-09; the amino acid sequences of trpE, trpD, trpC, trpB, and trpA, five key enzymes in the β-indolylalanine pathway, are shown in SEQ ID NOs: 10-14, respectively; the amino acid sequences of folE, PTPS, and SPR genes, key enzymes in the BH4 synthesis pathway, are shown in SEQ ID NOs: 15-17, respectively.

2. The synthesis method according to claim 1, characterized in that: The nucleotide sequence of the TPH2 gene, a key enzyme in the β-indolylalanine hydroxylation pathway, is shown in SEQ ID NO: 01; the nagGH, a key enzyme in the anthranilate hydroxylation pathway, is shown in SEQ ID NO: 02; the trpEDCBA, a key enzyme in the β-indolylalanine pathway, is shown in SEQ ID NO: 03; the nucleotide sequences of the folE, PTPS, and SPR genes, key enzymes in the BH4 synthesis pathway, are shown in SEQ ID NOs: 04-06, respectively.

3. The synthesis method according to claim 1, characterized in that: Step 2) is: the recombinant bacteria constructed in step 1) are activated and cultured in LB medium, transferred to a fermenter for induction of expression, and then 5-hydroxy-β-indolylalanine is synthesized from scratch using glucose as a substrate.

4. The synthesis method according to claim 3, characterized in that: The constructed recombinant genetic engineering bacteria were activated and cultured in LB medium to obtain seed liquid, which was then transferred to a fermentation tank. Fermentation to OD 600 When the cells grew to 1-50, IPTG was added to induce conversion and fed-batch fermentation was started to synthesize 5-hydroxy-β-indolylalanine from scratch using glucose as a substrate.

5. The synthesis method according to claim 4, characterized in that: The seed solution OD 600 =5-10; The ratio of the seed liquid to the fermentation medium is 1%-10%; The fermentation culture is carried out under the conditions of 28-40°C and 160-500rpm; The pH value during the fermentation is controlled at 5-9; The final concentration of IPTG is 0.1-10 mM; The conditions for inducing expression were 16-37°C; The glucose concentration in the reaction system is 1-100 g / L; The fermentation time is 18-72h.

6. A recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium is a recombinant genetically engineered bacterium comprising a recombinant vector encoding a synthetic 5-hydroxy-β-indolylalanase as shown in SEQ ID NO: 1-6, or a recombinant genetically engineered bacterium obtained by integrating part of the enzyme gene into the genome of a host cell for expression, and then expressing the remaining enzyme gene in a plasmid and then transferring it into the aforementioned host cell.

7. The recombinant genetically engineered bacterium according to claim 6, characterized in that: The genes of folE, PTPS, and SPR, key enzymes in the BH4 synthesis pathway, are expressed on the genome of the host cell, and other target enzyme genes are expressed in plasmids and then transferred into the host cell whose genome expresses the genes of folE, PTPS, and SPR, key enzymes in the BH4 synthesis pathway.

8. The method for constructing a recombinant genetically engineered bacterium according to claim 6, characterized in that: The key enzyme TPH2 gene of the β-indolylalanine hydroxylation pathway, the key enzyme nagGH gene of the anthranilic acid hydroxylation pathway and the key enzyme trpEDCBA gene on the β-indolylalanine pathway are expressed in a plasmid to obtain a recombinant vector; the recombinant vector is transferred into a host cell expressing the key enzyme folE, PTPS, and SPR genes of the BH4 synthesis pathway on the genome to obtain a recombinant genetically engineered bacterium.

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