An engineered strain for producing 5-hydroxytryptophan, a construction method and application thereof

Abstract

The application discloses an engineering strain for producing 5-hydroxytryptophan and a construction method and application thereof, and the construction steps of the engineering strain are as follows: a recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH is constructed; the plasmid is introduced into escherichia coli or vibrio natriegens to obtain an engineering strain for producing 5-hydroxytryptophan; the engineering strain for producing 5-hydroxytryptophan is fermented to obtain 5-hydroxytryptophan, and the production efficiency is high, and the production cost is reduced; and the conversion efficiency of L-tryptophan as a substrate is above 88.6%.

Description

Technical Field

[0001] This invention relates to an engineered strain for producing 5-hydroxytryptophan, its construction method, and its application, belonging to the field of biotechnology. Background Technology

[0002] 5-Hydroxytryptophan (5-HTP) is an amino acid. In mammals and humans, it serves as a precursor to the neurotransmitter serotonin (5-HT), and subsequently, melatonin. Currently, 5-HTP has been shown to regulate mood, sleep, and behavior, and is effective in treating various conditions such as depression, fibromyalgia, and obesity. Therefore, 5-HTP has broad application prospects in the pharmaceutical and health supplement industries.

[0003] The production methods of 5-HTP include chemical methods, natural extraction, and biological methods. Chemical synthesis of 5-HTP suffers from harsh reaction conditions and poor substrate regioselectivity. Currently, commercial production of 5-HTP mainly involves extraction from Griffonia simplicifolia seeds (seeds of a woody climbing shrub primarily growing in Africa). However, Griffonia simplicifolia seed production is limited by season and region, resulting in scarce sources and high extraction costs, thus keeping the prices of 5-HTP-based pharmaceuticals and health products high. Besides the two methods mentioned above, microbial production of 5-HTP is considered a very attractive approach because it enables green catalytic production under mild conditions. Therefore, constructing an engineered strain capable of effectively catalyzing the production of 5-HTP is of great significance for the microbial fermentation synthesis of 5-HTP and for reducing its cost. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an engineered strain for producing 5-hydroxytryptophan.

[0005] A second objective of this invention is to provide a method for constructing an engineered strain that produces 5-hydroxytryptophan.

[0006] A third objective of this invention is to provide an application of the engineered strain described above for catalyzing the production of 5-hydroxytryptophan.

[0007] The technical solution of this invention is summarized as follows: A method for constructing an engineered strain that produces 5-hydroxytryptophan includes the following steps: (a) The FolE gene and pET-28a plasmid were digested with restriction endonuclease NcoI / BamHI. The resulting fragments were ligated with T4 DNA ligase to obtain the recombinant plasmid pET-28a-FolE. The FolE gene is an abbreviation for GTP cyclic hydrolase I gene, as shown in SEQ ID No. 1; (b) Using PTPS-RBS-BamHI-F and PTPS-R as upstream and downstream primers, and PTPS gene as template, PCR amplification was performed to obtain PTPS gene fragment; Using SPR-PTPS-F and SPR-XhoI-R as upstream and downstream primers, PCR amplification was performed with the SPR gene as a template to obtain the SPR gene fragment; the PTPS gene fragment and the SPR gene fragment were then assembled by PCR using the overlap method to obtain the PTPS-SPR gene fragment. The PTPS-SPR gene fragment and the recombinant plasmid pET-28a-FolE were digested with restriction endonucleases BamHI / XhoI. The resulting fragments were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-FolE-PTPS-SPR. The PTPS gene is an abbreviation for codon-optimized 6-pyruvyltetrahydrobiopterin synthase gene, shown in SEQ ID No. 2; the nucleotide sequence of PTPS-RBS-BamHI-F is shown in SEQ ID No. 10; and the nucleotide sequence of PTPS-R is shown in SEQ ID No. 11. The SPR gene is an abbreviation for codon-optimized metoprolol reductase gene, shown in SEQ ID No. 3; the nucleotide sequence of SPR-PTPS-F is shown in SEQ ID No. 12; the nucleotide sequence of SPR-XhoI-R is shown in SEQ ID No. 13. (c) The recombinant plasmid pET-28a-FolE-PTPS-SPR and PACYC-Duet1 plasmid were digested with restriction endonucleases NcoI / XhoI to obtain the FolE-PTPS-SPR fragment and the digested PACYC-Duet1 plasmid, respectively; the FolE-PTPS-SPR fragment and the digested PACYC-Duet1 plasmid were ligated with T4 DNA ligase to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR. (d) Using PCD-NdeI-F and PCD-R as upstream and downstream primers, PCR amplification was performed using the PCD gene as a template to obtain the PCD gene fragment; using DHPR-RBS-F and DHPR-KpnI-R as upstream and downstream primers, PCR amplification was performed using the DHPR gene as a template to obtain the DHPR gene fragment; the PCD gene fragment and the DHPR gene fragment were then PCR-assembled using the overlap method to obtain the PCD-DHPR gene fragment. The PCD-DHPR gene fragment and the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR were digested with restriction endonucleases NdeI / KpnI. The resulting fragments were ligated using T4 DNA ligase to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR. The PCD gene is an abbreviation for codon-optimized pterinolamine dehydratase gene, as shown in SEQ ID No. 4; the nucleotide sequence of PCD-NdeI-F is shown in SEQ ID No. 14; and the nucleotide sequence of PCD-R is shown in SEQ ID No. 15. The DHPR gene is an abbreviation for codon-optimized dihydropterin reductase gene, as shown in SEQ ID No. 5; the nucleotide sequence of DHPR-RBS-F is shown in SEQ ID No. 16; and the nucleotide sequence of DHPR-KpnI-R is shown in SEQ ID No. 17. (e) The TPH1 gene and pET-28a plasmid were digested with restriction endonuclease BamHI / NotI to obtain fragments; the fragments were ligated with T4 DNA ligase to obtain the recombinant plasmid pET-28a-TPH1. The TPH1 gene is an abbreviation for codon-optimized tryptophan hydroxylase gene, as shown in SEQ ID No. 6; (f) The GDH gene and recombinant plasmid pET-28a-TPH1 were digested with restriction endonuclease NotI / XhoI. The resulting fragments were ligated with T4 DNA ligase to obtain the recombinant plasmid pET-28a-TPH1-GDH. The GDH gene is short for codon-optimized glucose dehydrogenase gene; as shown in SEQ ID No. 7; (g) Using the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR obtained in step (d) as a template, and Dieling-F and Dieling-R as upstream and downstream primers, PCR amplification was performed to obtain the PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR gene fragment; the nucleotide sequence of Dieling-F is shown in SEQ ID No. 23; the nucleotide sequence of Dieling-R is shown in SEQ ID No. 22; Using the recombinant plasmid pET-28a-TPH1-GDH obtained in step (f) as a template, and using TPH1-GDH-F2 and TPH1-GDH-R2 as upstream and downstream primers, PCR amplification was performed to obtain the TPH1-GDH gene fragment; the nucleotide sequence of TPH1-GDH-F2 is shown in SEQ ID No. 24; the nucleotide sequence of TPH1-GDH-R2 is shown in SEQ ID No. 25. The TPH1-GDH gene fragment and the PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR gene fragment were seamlessly recombined using a seamless cloning kit to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH. (h) Proceed in one of the following two ways: Method 1: The recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH is introduced into Escherichia coli or sodium-dependent Vibrio to obtain an engineered strain that produces 5-hydroxytryptophan. Method 2: Using the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH as a template, PCR amplification was performed using MPSPDTG-F and MPSPDTG-R as upstream and downstream primers to obtain the FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH gene fragment; then, using the pBE2RS plasmid as a template, PCR amplification was performed using pBE2RS-F and pBE2RS-R as upstream and downstream primers to obtain the pBE2RS gene fragment. The nucleotide sequence of MPSPDTG-F is shown in SEQ ID No. 26; the nucleotide sequence of MPSPDTG-R is shown in SEQ ID No. 27. The nucleotide sequence of pBE2RS-F is shown in SEQ ID No. 29; the nucleotide sequence of pBE2RS-R is shown in SEQ ID No. 28. The FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH gene fragment and the pBE2RS gene fragment were seamlessly recombined using a seamless cloning kit to obtain the recombinant plasmid pBE2RS-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH; the recombinant plasmid pBE2RS-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH was introduced into Bacillus subtilis to obtain another engineered strain that produces 5-hydroxytryptophan.

[0008] The engineered strain that produces 5-hydroxytryptophan was constructed using the above method.

[0009] Applications of the engineered strains mentioned above that catalyze the production of 5-hydroxytryptophan.

[0010] Advantages of this invention: Experiments have shown that the 5-hydroxytryptophan produced by the engineered strain of this invention has high production efficiency and low production cost; the conversion efficiency using L-tryptophan as a substrate is over 88.6%. Detailed Implementation

[0011] The Escherichia coli BL21(DE3) used in this invention is Escherichia coli (CGMCC 1.12883) purchased from Beijing TransGen Biotech Co., Ltd. Other Escherichia coli with different numbers can also be used in this invention.

[0012] The sodium-dependent Vibrio used in this invention is, for example, CGMCC 1.8755, which was purchased from Beijing TransGen Biotech Co., Ltd. Other sodium-dependent Vibrio species can also be used in this invention.

[0013] The Bacillus subtilis used in this invention is Bacillus subtilis (CGMCC 1.821) as an example, which was purchased from Beijing TransGen Biotech Co., Ltd. Other Bacillus subtilis with different serial numbers can also be used in this invention.

[0014] The seamless cloning kit, purchased from Beijing TransGen Biotech Co., Ltd., was used as an example. Seamless recombination was performed according to the instructions.

[0015] Example 1 The recombinant plasmid involves seven genes, and their sequence information is as follows: According to the source Escherichia coli The gene sequence of the GTP cyclic hydrolase I gene is referred to as the FolE gene (SEQ ID No. 1). According to the source Homo sapiens The gene sequence of the 6-pyruvyltetrahydrobiopterin synthase gene was obtained by codon optimization for Escherichia coli, resulting in the codon-optimized 6-pyruvyltetrahydrobiopterin synthase gene, abbreviated as PTPS gene (SEQ ID No. 2). According to the source Homo sapiens The gene sequence of the metoprolol reductase gene was obtained by codon optimization for Escherichia coli, resulting in the codon-optimized metoprolol reductase gene, abbreviated as SPR gene (SEQ ID No. 3). According to the source Homo sapiensThe gene sequence of the pterinolamine dehydratase gene was obtained by codon optimization for Escherichia coli, resulting in the codon-optimized pterinolamine dehydratase gene, abbreviated as PCD gene (SEQ ID No. 4). According to the source Homo sapiens The gene sequence of the dihydropterin reductase gene was obtained by codon optimization for Escherichia coli, resulting in the codon-optimized dihydropterin reductase gene, abbreviated as DHPR gene (SEQ ID No. 5). According to the source Homo sapiens The gene sequence of the tryptophan hydroxylase gene was obtained by codon optimization for Escherichia coli, resulting in the codon-optimized tryptophan hydroxylase gene, abbreviated as TPH1 (SEQ ID No. 6). According to the source Bacillus subtilis The gene sequence of glucose dehydrogenase was obtained by codon optimization for Escherichia coli, resulting in the codon-optimized glucose dehydrogenase gene, abbreviated as GDH (SEQ ID No. 7).

[0016] The codon optimization described above is for Escherichia coli. Codon optimization can also be performed for Vibrio natriureticis or Bacillus subtilis to complete this invention.

[0017] The aforementioned codon optimization and gene synthesis were commissioned to Suzhou Genewise Biotechnology Co., Ltd.

[0018] Table 1. PCR primers required for constructing recombinant plasmids

[0019] Using the sequences in Table 1 as upstream and downstream primers, FolE-NcoI-F (SEQ ID No. 8) and FolE-BamHI-R (SEQ ID No. 9), PCR amplification was performed using the FolE gene as a template to obtain the gene fragment with sequence SEQ ID No. 1. Using the sequences in Table 1 with PTPS-RBS-BamHI-F (SEQ ID No. 10) and PTPS-R (SEQ ID No. 11) as upstream and downstream primers, PCR amplification was performed using the PTPS gene as a template to obtain the gene fragment with sequence SEQ ID No. 2; Using the sequences in Table 1 as upstream and downstream primers, SPR-PTPS-F (SEQ ID No. 12) and SPR-XhoI-R (SEQ ID No. 13), PCR amplification was performed using the SPR gene as a template to obtain the gene fragment with sequence SEQ ID No. 3. Using the sequences PCD-NdeI-F (SEQ ID No. 14) and PCD-R (SEQ ID No. 15) in Table 1 as upstream and downstream primers, PCR amplification was performed with the PCD gene as a template to obtain the gene fragment with sequence SEQ ID No. 4; Using the sequences DHPR-RBS-F (SEQ ID No. 16) and DHPR-KpnI-R (SEQ ID No. 17) in Table 1 as upstream and downstream primers, PCR amplification was performed using the DHPR gene as a template to obtain the gene fragment with sequence SEQ ID No. 5; Using the sequences TPH1-pET28a-BamHI-F (SEQ ID No. 18) and TPH1-Not1-R (SEQ ID No. 19) in Table 1 as upstream and downstream primers, PCR amplification was performed with the TPH1 gene as a template to obtain the gene fragment with sequence SEQ ID No. 6; Using the sequences GDH-RBS-NotI-F (SEQ ID No. 20) and Kucao-GDH-XhoI-R (SEQ ID No. 21) in Table 1 as upstream and downstream primers, PCR amplification was performed with the GDH gene as a template to obtain the gene fragment with sequence SEQ ID No. 7.

[0020] The PCR reaction conditions for the above seven genes are shown in Table 2: Table 2. PCR conditions for 7 genes in recombinant plasmids

[0021] Example 2 The construction of an engineered strain for producing 5-hydroxytryptophan includes the following steps: (a) The FolE gene and the commercially available pET-28a plasmid were digested with the restriction endonuclease NcoI / BamHI and incubated in a 37°C water bath for 6 h. The digested gene products were subjected to agarose gel electrophoresis, and the correct gene fragments were recovered using a gel recovery kit from Beijing TransGen Biotech Co., Ltd. The digested FolE gene and the digested pET-28a plasmid were obtained. The fragments were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-FolE. The FolE gene is an abbreviation for GTP cyclic hydrolase I gene, as shown in SEQ ID No. 1; (b) Using PTPS-RBS-BamHI-F and PTPS-R as upstream and downstream primers, and PTPS gene as template, PCR amplification was performed to obtain PTPS gene fragment; Using SPR-PTPS-F and SPR-XhoI-R as upstream and downstream primers, PCR amplification was performed with the SPR gene as a template to obtain the SPR gene fragment; the PTPS gene fragment and the SPR gene fragment were then assembled by PCR using the overlap method to obtain the PTPS-SPR gene fragment. The PTPS-SPR gene fragment and the recombinant plasmid pET-28a-FolE were digested with restriction endonucleases BamHI / XhoI. The resulting fragments were then ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-FolE-PTPS-SPR. The PTPS gene is an abbreviation for codon-optimized 6-pyruvyltetrahydrobiopterin synthase gene, shown in SEQ ID No. 2; the nucleotide sequence of PTPS-RBS-BamHI-F is shown in SEQ ID No. 10; and the nucleotide sequence of PTPS-R is shown in SEQ ID No. 11. The SPR gene is an abbreviation for codon-optimized metoprolol reductase gene, shown in SEQ ID No. 3; the nucleotide sequence of SPR-PTPS-F is shown in SEQ ID No. 12; the nucleotide sequence of SPR-XhoI-R is shown in SEQ ID No. 13. (c) The recombinant plasmid pET-28a-FolE-PTPS-SPR and PACYC-Duet1 plasmid (commercial) were digested with restriction endonucleases NcoI / XhoI to obtain the FolE-PTPS-SPR fragment and the digested PACYC-Duet1 plasmid, respectively; the FolE-PTPS-SPR fragment and the digested PACYC-Duet1 plasmid were ligated with T4 DNA ligase to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR; (d) Using PCD-NdeI-F and PCD-R as upstream and downstream primers, PCR amplification was performed using the PCD gene as a template to obtain the PCD gene fragment; using DHPR-RBS-F and DHPR-KpnI-R as upstream and downstream primers, PCR amplification was performed using the DHPR gene as a template to obtain the DHPR gene fragment; the PCD gene fragment and the DHPR gene fragment were then PCR-assembled using the overlap method to obtain the PCD-DHPR gene fragment. The PCD-DHPR gene fragment and the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR were digested with restriction endonucleases NdeI / KpnI. The resulting fragments were ligated using T4 DNA ligase to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR. The PCD gene is an abbreviation for codon-optimized pterinolamine dehydratase gene, as shown in SEQ ID No. 4; the nucleotide sequence of PCD-NdeI-F is shown in SEQ ID No. 14; and the nucleotide sequence of PCD-R is shown in SEQ ID No. 15. The DHPR gene is an abbreviation for codon-optimized dihydropterin reductase gene, as shown in SEQ ID No. 5; the nucleotide sequence of DHPR-RBS-F is shown in SEQ ID No. 16; and the nucleotide sequence of DHPR-KpnI-R is shown in SEQ ID No. 17. (e) The TPH1 gene and pET-28a plasmid were digested using the restriction endonuclease BamHI / NotI, and the reaction was incubated in a 37°C water bath for 6 h. Afterwards, the digested gene products were subjected to agarose gel electrophoresis, and the correct gene fragments were recovered using a gel recovery kit from Beijing TransGen Biotech Co., Ltd., yielding the digested TPH1 gene and the digested pET-28a plasmid, respectively. The digested TPH1 gene and the digested pET-28a plasmid were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-TPH1. The TPH1 gene is short for codon-optimized tryptophan hydroxylase gene; as shown in SEQ ID No. 6; (f) The GDH gene and recombinant plasmid pET-28a-TPH1 were cut using restriction endonuclease NotI / XhoI to obtain the cut GDH gene and the cut recombinant plasmid pET-28a-TPH1, respectively. The cut GDH gene and the cut recombinant plasmid pET-28a-TPH1 were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-TPH1-GDH. The GDH gene is short for codon-optimized glucose dehydrogenase gene; as shown in SEQ ID No. 7; (g) Using the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR obtained in step (d) as a template, and Dieling-F and Dieling-R as upstream and downstream primers, PCR amplification was performed to obtain the PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR gene fragment; the nucleotide sequence of Dieling-F is shown in SEQ ID No. 23; the nucleotide sequence of Dieling-R is shown in SEQ ID No. 22; Using the recombinant plasmid pET-28a-TPH1-GDH obtained in step (f) as a template, and with the nucleotide sequences shown in TPH1-GDH-F2 and TPH1-GDH-R2 as upstream and downstream primers, PCR amplification was performed to obtain the TPH1-GDH gene fragment; the nucleotide sequence of TPH1-GDH-F2 is shown in SEQ ID No. 24; the nucleotide sequence of TPH1-GDH-R2 is shown in SEQ ID No. 25. The TPH1-GDH gene fragment and the PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR gene fragment were seamlessly recombined using a seamless cloning kit to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH. (h) The recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH was transformed into Escherichia coli (CGMCC 1.12883) BL21(DE3) competent cells. The competent cells with the introduced plasmid were spread on the surface of chloramphenicol-resistant LB solid plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L chloramphenicol, 20 g / L agar) and incubated at 37°C for 12 h. After that, single colonies were picked and placed in chloramphenicol-resistant LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L chloramphenicol) and incubated at 37°C and 220 rpm for 12 h. The resulting bacterial cells were an engineered strain-1 that produces 5-hydroxytryptophan.

[0022] Example 3 The construction of an engineered strain for producing 5-hydroxytryptophan includes the following steps: Steps (a), (b), (c), (d), (e), (f), and (g) are the same as steps (a), (b), (c), (d), (e), (f), and (g) in Example 2. (h) The recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH was introduced into competent cells of *C. 1.8755* using electroporation. The cells infused with the plasmid were then spread on LB3 solid plates containing chloramphenicol resistance (10 g / L peptone, 5 g / L yeast extract, 30 g / L sodium chloride, 50 mg / L chloramphenicol, 20 g / L agar) and incubated at 37°C for 12 h. Single colonies were then picked and placed in LB3 liquid medium containing chloramphenicol resistance (10 g / L peptone, 5 g / L yeast extract, 30 g / L sodium chloride, 50 mg / L chloramphenicol) and incubated at 37°C and 220 rpm for 8 h. The resulting bacterial cells were an engineered strain-2 that produces 5-hydroxytryptophan.

[0023] Example 4 The construction of an engineered strain for producing 5-hydroxytryptophan includes the following steps: Steps (a), (b), (c), (d), (e), (f), and (g) are the same as steps (a), (b), (c), (d), (e), (f), and (g) in Example 2. (h) Using the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH as a template, PCR amplification was performed using MPSPDTG-F and MPSPDTG- as upstream and downstream primers to obtain the FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH gene fragment; then using the pBE2RS plasmid as a template, PCR amplification was performed using pBE2RS-F and pBE2RS-R as upstream and downstream primers to obtain the pBE2RS gene fragment; The nucleotide sequence of MPSPDTG-F is shown in SEQ ID No. 26; the nucleotide sequence of MPSPDTG-R is shown in SEQ ID No. 27. The pBE2RS-F nucleotide sequence is shown in SEQ ID No. 29; the pBE2RS-R nucleotide sequence is shown in SEQ ID No. 28; The FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH gene fragment obtained by the above PCR amplification was seamlessly recombined with the pBE2RS gene fragment using a seamless cloning kit to obtain the recombinant plasmid pBE2RS-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH. The recombinant plasmid pBE2RS-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH was introduced into competent Bacillus subtilis (CGMCC 1.821) cells via electroporation. The cells infused with the plasmid were then plated on LB agar plates containing ampicillin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L ampicillin, 20 g / L agar) and incubated at 37°C for 12 h. Single colonies were then picked and placed in LB liquid medium containing ampicillin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L ampicillin) and incubated at 37°C and 220 rpm for 8 h. The resulting bacterial cells were an engineered strain-3 that produces 5-hydroxytryptophan.

[0024] Example 5 An engineered strain-1 for producing 5-hydroxytryptophan catalyzes the production of 5-hydroxytryptophan: An engineered strain-1 producing 5-hydroxytryptophan was cultured in chloramphenicol-resistant LB liquid medium at 37°C and 220 rpm until OD200 was reached. 600 When the concentration was 0.8, whey powder with a final concentration of 50 g / L and L-tryptophan with a final concentration of 5 g / L were added. The culture temperature was adjusted to 30℃, 150 rpm, and cultured for 48 h. The concentration of 5-hydroxytryptophan (5-HTP) was 4.21 g / L, and the L-tryptophan conversion rate was 93.6%.

[0025] Example 6 An engineered strain-2 for producing 5-hydroxytryptophan catalyzes the production of 5-hydroxytryptophan: An engineered strain-2 producing 5-hydroxytryptophan was cultured in chloramphenicol-resistant LB3 liquid medium at 37°C and 220 rpm until OD200 was reached. 600 When the concentration was 0.8, whey powder with a final concentration of 70 g / L and L-tryptophan with a final concentration of 5 g / L were added. The culture temperature was adjusted to 30℃, 150 rpm, and cultured for 48 h. The 5-HTP concentration was 4.33 g / L, and the L-tryptophan conversion rate was 96.5%.

[0026] Example 7 An engineered strain-3 catalyzes the production of 5-hydroxytryptophan: An engineered strain-3 producing 5-hydroxytryptophan was cultured in ampicillin-resistant LB liquid medium at 37°C and 220 rpm until OD200 was reached. 600When the concentration was 0.8, whey powder with a final concentration of 70 g / L and L-tryptophan with a final concentration of 5 g / L were added. The culture temperature was adjusted to 30℃, 150 rpm, and cultured for 48 h. The 5-HTP concentration was 4.15 g / L, and the L-tryptophan conversion rate was 88.6%.

Claims

1. A method for constructing an engineered strain that produces 5-hydroxytryptophan, characterized in that: Includes the following steps: (a) The FolE gene and pET-28a plasmid were digested with restriction endonuclease NcoI / BamHI. The resulting fragments were ligated with T4 DNA ligase to obtain the recombinant plasmid pET-28a-FolE. The FolE gene is an abbreviation for GTP cyclic hydrolase I gene, as shown in SEQ ID No. 1; (b) Using PTPS-RBS-BamHI-F and PTPS-R as upstream and downstream primers, and PTPS gene as template, PCR amplification was performed to obtain PTPS gene fragments; Using SPR-PTPS-F and SPR-XhoI-R as upstream and downstream primers, PCR amplification was performed with the SPR gene as a template to obtain the SPR gene fragment; the PTPS gene fragment and the SPR gene fragment were then assembled by PCR using the overlap method to obtain the PTPS-SPR gene fragment. The PTPS-SPR gene fragment and the recombinant plasmid pET-28a-FolE were digested with restriction endonucleases BamHI / XhoI. The resulting fragments were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-FolE-PTPS-SPR. The PTPS gene is an abbreviation for codon-optimized 6-pyruvyltetrahydrobiopterin synthase gene, shown in SEQ ID No. 2; the nucleotide sequence of PTPS-RBS-BamHI-F is shown in SEQ ID No. 10; and the nucleotide sequence of PTPS-R is shown in SEQ ID No.

11. The SPR gene is an abbreviation for codon-optimized metoprolol reductase gene, shown in SEQ ID No. 3; the nucleotide sequence of SPR-PTPS-F is shown in SEQ ID No. 12; the nucleotide sequence of SPR-XhoI-R is shown in SEQ ID No.

13. (c) The recombinant plasmid pET-28a-FolE-PTPS-SPR and PACYC-Duet1 plasmid were digested with restriction endonucleases NcoI / XhoI to obtain the FolE-PTPS-SPR fragment and the digested PACYC-Duet1 plasmid, respectively; the FolE-PTPS-SPR fragment and the digested PACYC-Duet1 plasmid were ligated with T4 DNA ligase to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR. (d) Using PCD-NdeI-F and PCD-R as upstream and downstream primers, PCR amplification was performed using the PCD gene as a template to obtain the PCD gene fragment; using DHPR-RBS-F and DHPR-KpnI-R as upstream and downstream primers, PCR amplification was performed using the DHPR gene as a template to obtain the DHPR gene fragment; the PCD gene fragment and the DHPR gene fragment were then PCR-assembled using the overlap method to obtain the PCD-DHPR gene fragment. The PCD-DHPR gene fragment and the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR were digested with restriction endonucleases NdeI / KpnI. The resulting fragments were ligated using T4 DNA ligase to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR. The PCD gene is an abbreviation for codon-optimized pterinolamine dehydratase gene, as shown in SEQ ID No. 4; the nucleotide sequence of PCD-NdeI-F is shown in SEQ ID No. 14; and the nucleotide sequence of PCD-R is shown in SEQ ID No.

15. The DHPR gene is an abbreviation for codon-optimized dihydropterin reductase gene, as shown in SEQ ID No. 5; the nucleotide sequence of DHPR-RBS-F is shown in SEQ ID No. 16; and the nucleotide sequence of DHPR-KpnI-R is shown in SEQ ID No.

17. (e) The TPH1 gene and pET-28a plasmid were digested with restriction endonuclease BamHI / NotI to obtain fragments; the fragments were ligated with T4 DNA ligase to obtain the recombinant plasmid pET-28a-TPH1. The TPH1 gene is an abbreviation for codon-optimized tryptophan hydroxylase gene, as shown in SEQ ID No. 6; (f) The GDH gene and recombinant plasmid pET-28a-TPH1 were digested with restriction endonuclease NotI / XhoI. The resulting fragments were ligated with T4 DNA ligase to obtain the recombinant plasmid pET-28a-TPH1-GDH. The GDH gene is short for codon-optimized glucose dehydrogenase gene; as shown in SEQ ID No. 7; (g) Using the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR obtained in step (d) as a template, and Dieling-F and Dieling-R as upstream and downstream primers, PCR amplification was performed to obtain the PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR gene fragment; the nucleotide sequence of Dieling-F is shown in SEQ ID No. 23; the nucleotide sequence of Dieling-R is shown in SEQ ID No. 22; Using the recombinant plasmid pET-28a-TPH1-GDH obtained in step (f) as a template, and using TPH1-GDH-F2 and TPH1-GDH-R2 as upstream and downstream primers, PCR amplification was performed to obtain the TPH1-GDH gene fragment; the nucleotide sequence of TPH1-GDH-F2 is shown in SEQ ID No. 24; the nucleotide sequence of TPH1-GDH-R2 is shown in SEQ ID No.

25. The TPH1-GDH gene fragment and the PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR gene fragment were seamlessly recombined using a seamless cloning kit to obtain the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH. (h) Proceed in one of the following two ways: Method 1: The recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH is introduced into Escherichia coli or sodium-dependent Vibrio to obtain an engineered strain that produces 5-hydroxytryptophan. Method 2: Using the recombinant plasmid PACYC-Duet1-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH as a template, PCR amplification was performed using MPSPDTG-F and MPSPDTG-R as upstream and downstream primers to obtain the FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH gene fragment; then, using the pBE2RS plasmid as a template, PCR amplification was performed using pBE2RS-F and pBE2RS-R as upstream and downstream primers to obtain the pBE2RS gene fragment. The nucleotide sequence of MPSPDTG-F is shown in SEQ ID No. 26; the nucleotide sequence of MPSPDTG-R is shown in SEQ ID No.

27. The nucleotide sequence of pBE2RS-F is shown in SEQ ID No. 29; the nucleotide sequence of pBE2RS-R is shown in SEQ ID No.

28. The FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH gene fragment and the pBE2RS gene fragment were seamlessly recombined using a seamless cloning kit to obtain the recombinant plasmid pBE2RS-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH; the recombinant plasmid pBE2RS-FolE-PTPS-SPR-PCD-DHPR-TPH1-GDH was introduced into Bacillus subtilis to obtain an engineered strain that produces 5-hydroxytryptophan.

2. An engineered strain for producing 5-hydroxytryptophan constructed by the method of claim 1.

3. The application of the engineered strain for producing 5-hydroxytryptophan according to claim 2 in catalytic production of 5-hydroxytryptophan.

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