A 5-hydroxytryptophan production strain and its construction method and application

Abstract

The present invention provides a 5-hydroxytryptophan production strain and a construction method and application thereof. The strain has a clear genetic background and is effective in producing 5-hydroxytryptophan. E.coli HTP11 Subtilis subtilis spores pureF bsu K316Q Mutated gene, upregulated strain E.coli HTP11 of guaB , guaA , gmk , ndk The transcription level of the gene was increased by heterologous introduction of the gapX Gene, missing aromatic amino acid transporter serB , tnaB , aroP Gene, up-regulated inner membrane efflux transporter yhjV The transcription level of the gene was increased and electroporated into the pTm-serB plasmid, which could stably provide 5-hydroxytryptophan, the direct precursor of 5-hydroxytryptophan, and had a high efficiency of 5-hydroxytryptophan output.

Description

Technical Field

[0001] The invention relates to the field of compound biotechnology production, in particular to a 5-hydroxytryptophan production strain and a construction method and application thereof. Background Art

[0002] 5-Hydroxytryptophan (5-HTP), chemically known as 5-hydroxy-3-indolyl-α-alanine, is a precursor for the biosynthesis of the neurotransmitter serotonin and can effectively treat a variety of diseases, such as depression, fibromyalgia, obesity, insomnia and chronic headaches. In recent years, with the exploration of the function of 5-HTP, 5-HTP has become one of the most commercially valuable small-variety amino acids.

[0003] my country is the main producer of 5-HTP, and the natural product synthesis method is still the main production method of 5-HTP. This method uses the seeds of the African plant Ghana as raw materials, and after a series of complex processes, it finally obtains pure 5-HTP. Although it can obtain a high-purity 5-HTP product, its raw material cost is high, and the environmental pressure of plant extraction is relatively large; the enzyme catalysis method requires the addition of the precursor tryptophan, and the enzyme catalysis process requires the participation of coenzymes, so the cost is relatively high.

[0004] At present, direct microbial fermentation has become the first choice to replace current production methods because of its advantages such as low cost, simple process and easy industrialization, and needs to be further developed and utilized. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a 5-hydroxytryptophan producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned 5-hydroxytryptophan producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned 5-hydroxytryptophan producing strain.

[0008] In order to solve the above technical problems, the technical solution of the present invention is:

[0009] A 5-hydroxytryptophan production strain, which is a strain E. coli HTP20 , which is an artificially modified Escherichia coli with a specific genotype ( E.coli W3110 ): In strain E.coli HTP11 Subtilis subtilis spore-derived genes purF bsu K316Q Mutated gene, upregulated strain E.coli HTP11 of guaB , guaA , gmk ,ndk The transcription level of the gene was increased by heterologous introduction of the gapX Gene, missing aromatic amino acid transporter serB , tnaB , aroP Gene, up-regulated inner membrane efflux transporter yhjV The transcription level of the gene was measured and electroporated into the pTm-serB plasmid.

[0010] Preferably, the above-mentioned 5-hydroxytryptophan producing strain, the starting strain E. coli HTP11 The strain in patent publication number CN115948311A E.coli HTP11 .

[0011] Preferably, the 5-hydroxytryptophan producing strain comprises the subtilis spore-derived purF bsu K316Q The mutant gene was integrated into the genome and the expression was enhanced by using the trc promoter; gapX The gene is integrated into the genome and the expression is enhanced by using the trc promoter; guaB , guaA , yhjV Genes are overexpressed on the genome and the expression is enhanced by using the trc promoter; gmk , ndk The gene was overexpressed in the genome and the expression was enhanced by using the lac promoter; serB , tnaB , aroP Gene.

[0012] Preferably, the above-mentioned 5-hydroxytryptophan producing strain, purF bsu K316Q The mutant gene underwent site-directed mutagenesis, where the base at position 946 was changed from A to C, resulting in the amino acid residue at position 316 being changed from lysine (K) to glutamine (Q).

[0013] Preferably, in the above-mentioned 5-hydroxytryptophan producing strain, the nucleotide sequence of the lac promoter is shown in the sequence listing SEQ ID NO.1, the nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.2, and the purF bsu K316Q The nucleotide sequence of the mutant gene is shown in the sequence table SEQ ID NO.3. gapX The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.4. guaB The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.5. guaAThe nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.6. gmk The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.7. ndk The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.8. yhjV The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.9, serB The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.10. tnaB The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.11. aroP The nucleotide sequence of the gene is shown in the sequence listing as SEQ ID NO.12, and the nucleotide sequence of the pTm-serB plasmid is shown in the sequence listing as SEQ ID NO.13.

[0014] The construction method of the above 5-hydroxytryptophan production strain is to E.coli HTP11 As the starting strain (chassis strain), gene editing was performed using metabolic engineering methods. The specific steps are as follows:

[0015] (1) A gene encoding an amidophosphoribosyltransferase from Bacillus subtilis was integrated into the genome purF bsu K316Q The mutant gene was driven by the trc promoter;

[0016] (2) The E. coli-derived guaB , guaA gene, and driven by the trc promoter;

[0017] (3) The E. coli-derived gmk , ndk gene, and driven by the lac promoter;

[0018] (4) A gene encoding 3-phosphoglyceraldehyde dehydrogenase from Corynebacterium glutamicum was integrated into the genome. gapX gene, and driven by the trc promoter;

[0019] (5) The gene encoding phosphoserine phosphatase was knocked out in the genome serB Gene;

[0020] (6) The gene encoding the tryptophan transporter was knocked out in the genome tnaB, aroP Gene;

[0021] (7) The gene encoding the efflux protein is integrated into the genome yhjV Gene;

[0022] (8) Electroporate the pTm-serB plasmid into the transformed strain in step (7).

[0023] Application of the above 5-hydroxytryptophan producing strain in fermentation production of 5-hydroxytryptophan.

[0024] Preferably, the above-mentioned 5-hydroxytryptophan production strain is used to produce 5-hydroxytryptophan by fermentation in a fermenter, and the specific method is as follows:

[0025] (1) Slant culture: Take the 5-hydroxytryptophan producing strain and inoculate it on the slant medium as the first generation slant culture, and culture it at 36-37℃ for 12-14h; take the colony of the first generation slant culture and inoculate it on a new slant medium as the second generation slant culture, and culture it at 36-37℃ for 10-12h;

[0026] (2) Seed culture: Wash the second-generation slant with sterile water and transfer it to the seed culture medium. The culture temperature is 34-36°C. The bioreactor is controlled to automatically add ammonia solution to maintain the pH of the seed solution at 7.0±0.2, the dissolved oxygen value at 40-50%, and the OD of the seed solution at 1. 600nm When the temperature reaches 15-20, the next step of fermentation culture is carried out;

[0027] (3) Fermentation culture: The inoculation amount is 20%, the culture temperature is 34-36°C, and the bioreactor is controlled to automatically add ammonia solution to maintain the seed liquid pH at 6.2-7.2 and the dissolved oxygen value at 30%-50%.

[0028] Preferably, in the application of the above-mentioned 5-hydroxytryptophan producing strain, the slant culture medium used in step (1) is a universal LB solid culture medium.

[0029] Preferably, in the application of the above-mentioned 5-hydroxytryptophan producing strain, the seed culture medium used in step (2) is: 20-40 g / L glucose, 4-6 g / L yeast extract powder, 2-5 g / L ammonium sulfate, 1-3 g / L potassium dihydrogen phosphate, 0.5-2 g / L anhydrous magnesium sulfate, 20-40 mg / L ferrous sulfate heptahydrate, 10-20 mg / L manganese sulfate monohydrate, V H 0.1-0.5 mg / L, V B1 0.5-1mg / L, the rest is water.

[0030] Preferably, in the application of the above-mentioned 5-hydroxytryptophan production strain, the fermentation medium used in step (3) is: 20-40 g / L glucose, 3-6 g / L yeast extract powder, 2-5 g / L peptone, 4-8 g / L ammonium sulfate, 3-6 g / L potassium dihydrogen phosphate, 2-4 g / L anhydrous magnesium sulfate, 40-60 mg / L ferrous sulfate heptahydrate, 20-40 mg / L manganese sulfate monohydrate, V H0.1-0.5mg / L, V B1 0.5-1 mg / L, the rest is water.

[0031] The above culture media can be prepared using standard methods.

[0032] Beneficial effects:

[0033] The above 5-hydroxytryptophan producing strain has a clear genetic background. E.coli HTP11 On the basis of can stably provide 5-hydroxytryptophan direct precursor 5-hydroxytryptophan, and E.coli HTP11 In the tryptophan hydroxylation module, the coenzyme supply system is optimized, including the supply of synthetic raw materials and reducing power, the stability of the plasmid system is optimized, and it has efficient 5-hydroxytryptophan output capacity. Specifically:

[0034] First, 5-hydroxytryptophan producing bacteria E.coli HTP11 On this basis, the coenzyme tetrahydropterin supply system was optimized, which increased the carbon flux of the GTP synthesis pathway, the raw material for tetrahydropterin synthesis, made the tryptophan hydroxylase coenzyme expression system efficient, and enhanced the reducing power supply of the tetrahydropterin cycle pathway;

[0035] Then, the tryptophan supply system and plasmid system of the engineered bacteria were coupled and optimized. By coupling the tryptophan supply system and the plasmid system, the supply of precursor tryptophan and the stability of hydroxylated tryptophan of the strain were improved, which was specifically manifested in the knockout of the strain genome. serB Gene, resulting in auxotrophy of serine, a precursor for tryptophan synthesis, serB Gene complementation on plasmid, i.e. serB The gene acts as a non-antibiotic selection tag, and the high copy number of the plasmid enhances the carbon flux of the serine synthesis pathway, ensuring an adequate supply of serine, a precursor of the tryptophan synthesis pathway. During the fermentation process, due to the screening pressure of the non-antibiotic selection tag, there is no need to add antibiotics to ensure the stability of plasmid replication;

[0036] Finally, the export system of 5-hydroxytryptophan was modified. By knocking out the aromatic amino acid internal transport protein and overexpressing the efflux protein, 5-hydroxytryptophan could be quickly exported to the extracellular space, thereby improving the ability of the engineered bacteria to transport 5-hydroxytryptophan to the extracellular space. The 5-hydroxytryptophan engineered strain finally constructed uses glucose as the carbon source, and the fermentation process does not require the addition of 5-hydroxytryptophan precursors or antibiotics, and has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the map of plasmid pTm-serB.

[0038] Figure 2 5-HTP production strainE. coli HTP20 Fermentation process curve. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0040] The percentage sign "%" involved in the embodiments, unless otherwise specified, refers to mass percentage. The percentage of a solution refers to the number of grams of a solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0041] The corresponding promoters and genes in the examples are shown in the sequence table. The primers used in the construction of the strains involved are shown in Table 1.

[0042] Table 1 Primers involved in strain construction

[0043] Primer Sequence number Sequence ycdN-US SEQ ID NO.14 GATTTTGACGCCACCAACACC ycdN-UA SEQ ID NO.15 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCATC ycdN-DS SEQ ID NO.16 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGG ycdN-DA SEQ ID NO.17 CATATCGTATTCGCCAGGCTG purF-3 SEQ ID NO.18 GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCTTGCTGAAATCAAAGGCTT purF1-1C-A SEQ ID NO.19 GCCTGTTGCCTCTGCATAGCCGATCGCCGCTGAAATACTGGAAACCGG purF1-2C-S SEQ ID NO.20 CGGCTATGCAGAGGCAACAGGCATTCCGTATGAGCTTGGCTTAATCCAAAACCGTTATGTTGG purF1-3-A SEQ ID NO.21 GTGTCCAAGTGTCAATGCCG purF1-3-S SEQ ID NO.22 CGGCATTGACACTTGGACACAT purF-4 SEQ ID NO.23 GATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATTTGGTTAATACTGCTTCTTTTACGTGAGG pGRB-ycdN-s SEQ ID NO.24 AGTCCTAGGTATAATACTAGTCATTACTGCGTGAAGGCGCGGTTTTAGAGCTAGAA pGRB-ycdN-a SEQ ID NO.25 TTCTAGCTCTAAAACCGCGCCTTCACGCAGTAATGACTAGTATTATACCTAGGACT yjiT-US SEQ ID NO.26 AATAGTTGTTGCCGCCTGAGT yjiT-UA SEQ ID NO.27 TGTGTGAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAACAGGCAGCAAAGTCCC yjiT-DS SEQ ID NO.28 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAGCACTACCTGTGAAGGGATGT yjiT-DA SEQ ID NO.29 CAGGGCTTCCACAGTCACAAT guaA-trc-s SEQ ID NO.30 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGACGGAAAACATTCATAAG guaA-trc-a SEQ ID NO.31 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCATTCCCACTCAATGGTA pGRB-yjiT-s SEQ ID NO.32 AGTCCTAGGTATACTAGTAGGGATTATGAACGGCAATGGTTTTAGAGCTAGAA pGRB-yjiT-a SEQ ID NO.33 TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAGTATTATACCTAGGACT yjiP-US SEQ ID NO.34 GCCATACCGCCAGCAAGAT yjiP-UA SEQ ID NO.35 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCAGATATTCCCCTTTCCACC yjiP-DS SEQ ID NO.36 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACGGATGACAAACGCAAAGC yjiP-DA SEQ ID NO.37 AAAGGCGGATTTTTACTGTGGA guaB-trc-s SEQ ID NO.38 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCTACGTATCGCTAAAGAAG guaB-trc-a SEQ ID NO.39 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCAGGAGCCCAGACGGTAG pGRB-yjiP-s SEQ ID NO.40 AGTCCTAGGTATAATACTAGTCTTTGTCGATGAAAAATTGCGTTTTAGAGCTAGAA pGRB-yjiP-a SEQ ID NO.41 TTCTAGCTCTAAAACGCAATTTTTCATCGACAAAGACTAGTATTATACCTAGGACT ylbE-US SEQ ID NO.42 ACCCAACCTTACGCAACCAG ylbE-UA SEQ ID NO.43 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT ylbE-DS SEQ ID NO.44 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA ylbE-DA SEQ ID NO.45 GGCGTAACTCAGCAGGCAG gmk-lac-s SEQ ID NO.46 TTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACTCACACAGGAAACAGACCATGGCTCAAGGCACGCTTTAT gmk-lac-a SEQ ID NO.47 TTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAGTCATAGTAATCCAGCAACTCTTGTG pGRB-ylbE-s SEQ ID NO.48 AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA pGRB-ylbE-a SEQ ID NO.49 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT ΔserB-US SEQ ID NO.50 ATGCCTAACATTACCTGGTGCGA ΔserB-UA SEQ ID NO.51 GTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCCGGCGTGCGCAGGTGCGGG ΔserB-DS SEQ ID NO.52 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGATTGCCTCCGGCGGCT ΔserB-DA SEQ ID NO.53 TTACTTCTGATTCAGGCTGCCTGA ndk-lac-s SEQ ID NO.54 TTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACTCACACAGGAAACAGACCATGGCTATTGAACGTACT ndk-lac-a SEQ ID NO.55 TGTGCCCGCGCACCCGTTAACAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG pGRB-serB-s SEQ ID NO.56 AGTCCTAGGTATAATACTAGTGTAACCGAACGGGCGATGCGGTTTTAGAGCTAGAA pGRB-serB-a SEQ ID NO.57 TTCTAGCTCTAAAACCGCATCGCCCGTTCGGTTACACTAGTATTATACCTAGGACT ΔtnaB-US SEQ ID NO.58 CTGCTGCGTTTAACCATTCC ΔtnaB-UA SEQ ID NO.59 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCGGCAAGATCAACAGGTAAA ΔtnaB-DS SEQ ID NO.60 ACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAGACCGTTTCTTCGCCATTTAA ΔtnaB-DA SEQ ID NO.61 AATCAGTGGCACTTTGGCATG gapX-trc-s SEQ ID NO.62 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACGCACAACCACAAGGACT gapX-trc-a SEQ ID NO.63 CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTATAGTACGGCTGGCTGCCT pGRB-tnaB-s SEQ ID NO.64 AGTCCTAGGTATAATACTAGTCGACGCCACCAGCTCCACTGGTTTTAGAGCTAGAA pGRB-tnaB-a SEQ ID NO.65 TTCTAGCTCTAAAACCAGTGGAGCTGGTGGCGTCGACTAGTATTATACCTAGGACT ΔaroP-US SEQ ID NO.66 CAGCACGGCGAGCAGCTAAAGCGC ΔaroP-UA SEQ ID NO.67 GTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCGGCAGAAACCCAGGTGGGG ΔaroP-DS SEQ ID NO.68 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTGTCGATAAACGTGGTGT ΔaroP-DA SEQ ID NO.69 CTGTTTAAAGAGAAAACCGCCAAAG yhjV-trc-s SEQ ID NO.70 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCAGCACAACACACTATCGA yhjV-trc-a SEQ ID NO.71 ACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACTCAATCAGCTTCAACAG pGRB-aroP-s SEQ ID NO.72 AGTCCTAGGTATACTAGTCGTTAGCAACCTGTGGGATCGTTTTAGAGCTAGAA pGRB-aroP-a SEQ ID NO.73 TTCTAGCTCTAAAACGATCCCACAGGTTGCTAACGACTAGTATTATACCTAGGACT TM2-S SEQ ID NO.74 ACAGCAAATGGGTCGCGGATCCATGAAACTGGAAGATGTACCGTGG TM2-A SEQ ID NO.75 GTGCTCGAGTGCGGCCGCATTATGTATCTTTCAAAATTTCGATACTCTG TM2-line-S SEQ ID NO.76 CAGAGTATCGAAATTTTGAAAGATACATAATGCGGCCGCACTCGAGCAC TM2-line-A SEQ ID NO.77 CCACGGTACATCTTCCAGTTTCATGGATCCGCGACCCATTTGCTGT serB-line-S SEQ ID NO.78 TGAAATTGTTATCCGCTCACAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAACGGGATCTCGACGCTCTCC serB-line-A SEQ ID NO.79 TCTCAGGCAGCCTGAATCAGAAGTAAGCGCAACGCCAATTAATGTAAGT serB-S SEQ ID NO.80 ACTTACATTAATTGCGTTGCGCTTACTTCTGATTCAGGCTGCCTGAGA serB-A SEQ ID NO.81 TCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGCCTAACATTACCTGGTGCGA

[0044] The gene editing method used in the gene manipulation in the embodiment refers to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.), and the professional terms involved in the present invention can be explained in the article unless otherwise noted. "Knockout" as referred to in the present invention refers to the inactivation of the target gene, and "introduction" refers to the insertion of the exogenous gene into the genome of the engineered bacteria after connecting it to the promoter and terminator.

[0045] Example 1

[0046] This example is intended to illustrate the strain E. coli HTP20 In particular, if there is a method for operating a gene of the same type in the embodiments, it will only be provided once and annotated without further elaboration.

[0047] ①In ycdN Pseudogene loci controlled by the trc promoter purF bsu fbr Gene overexpression:

[0048] by E.coliW3110The genome was used as a template, and ycdN-US / ycdN-UA and ycdN-DS / ycdN-DA were used to amplify the upstream and downstream homology arms, respectively. Then, the mutant nucleotide sequence was designed on purF1-1C-A, purF1-2C-S, purF1-3-A, and purF1-3-S, and purF1-1C-A / purF1-2C-S, purF1-3-A / purF1-3-S had homologous sequences. The genome of Bacillus subtilis was used as a template, and primers purF-3 / purF1-1C-A, purF1-2C-S / purF1-3-A, and purF1-3-S / purF-4 were used to perform PCR amplification to obtain fragment 1, fragment 2, and fragment 3, respectively. Then, fragment 1, fragment 2, and fragment 3 were used as templates, and primers purF-3 / purF-4 were used to perform overlapping PCR to obtain the target fragment P trc - purF bsu fbr ; Using the upstream and downstream homology arms and target gene fragments as templates, ycdN-US and ycdN-DA as primers, overlapping fragments were obtained by overlapping PCR amplification; using pGRB-ycdN-s and pGRB-ycdN-a as primers, annealing to obtain gRNA fragments, and connecting them with the pGRB vector to obtain pGRB-ycdN; preparing E.coli HTP11 Electrotransform competent cells, electrotransform the target fragment and pGRB-ycdN into competent cells, and screen positive transformants to obtain strains E.coli HTP13 .

[0049] ②In yjiT Pseudogene loci controlled by the trc promoter guaA Gene overexpression:

[0050] by E.coliW3110 The genome was used as a template, and yjiT-US / yjiT-UA and yjiT-DS / yjiT-DA, guaA-trc-s / guaA-trc-a were used to amplify the upstream and downstream homology arms and the target gene fragments by PCR, and then, the upstream and downstream homology arms and the target gene fragments were used as templates, and yjiT-US and yjiT-DA were used as primers to amplify the overlapping fragments by overlapping PCR; pGRB-yjiT-s and pGRB-yjiT-a were used as primers to anneal the gRNA fragments, and they were connected to the pGRB vector to obtain pGRB-yjiT; preparation E. coli HTP13 Electrotransform competent cells, electrotransform the target fragment and pGRB-yjiT into competent cells, and screen positive transformants to obtain strains E. coli HTP14 .

[0051] ③In yjiP Pseudogene loci controlled by the trc promoter guaB Gene Expression:

[0052] The same operation method as in ② is used, except that the primers used are yjiP-US / yjiP-UA, yjiP-DS / yjiP-DA, guaB-trc-s / guaB-trc-a, pGRB-yjiP-s, pGRB-yjiP-a. The competent cells are E. coli HTP14 , obtain strains E.coli HTP15 .

[0053] ④In ylbe Pseudogene loci are controlled by the lac promoter gmk Gene Expression:

[0054] The same operation method as in ② is used, except that the primers used are ylbE-US / ylbE-UA, ylbE-DS / ylbE-DA, gmk-lac-s / gmk-lac-a, pGRB-ylbE-s, pGRB-ylbE-a. The competent cells are E. coli HTP15 , obtain strains E. coli HTP16 .

[0055] ⑤In serB Gene locus controlled by lac promoter ndk Gene Expression:

[0056] The same operation method as in ② is used, except that the primers used are ΔserB-US / ΔserB-UA, ΔserB-DS / ΔserB-DA, ndk-lac-s / ndk-lac-a, pGRB-serB-s, pGRB-serB-a. The competent cells are E. coli HTP16 , obtain strains E.coli HTP17 .

[0057] ⑥In tnaB Gene locus controlled by trc promoter gapX Gene Expression:

[0058] The operation method is the same as in ②, except that the primers used are ΔtnaB-US / ΔtnaB-UA, ΔtnaB-DS / ΔtnaB-DA, gapX-trc-s / gapX-trc-a, pGRB-tnaB-s, pGRB-tnaB-a. The competent cells are E. coli HTP17 , obtain strains E.coli HTP18 .

[0059] ⑦InaroP Gene locus controlled by trc promoter yhjV Gene Expression:

[0060] The same operation method as in ② is used, except that the primers used are ΔaroP-US / ΔaroP-UA, ΔaroP-DS / ΔaroP-DA, yhjV-trc-s / yhjV-trc-a, pGRB-aroP-s, pGRB-aroP-a. The competent cells are E. coli HTP18 , obtain strains E.coli HTP19 .

[0061] ⑧ Preparation E. coli HTP19 Electrotransform competent cells, electrotransform plasmid pTm-serB into competent cells, and select positive transformants to obtain E.coli HTP20 .

[0062] Example 2

[0063] This example is intended to illustrate the construction method of plasmid pTm-serB in Example 1. The plasmid pTm-serB map is shown in Figure 1 , refer to CN115948311A for genes and promoters, and the specific steps are as follows:

[0064] ⑨ Using the artificial synthetic gene TM2 as a template and TM2-S and TM2-A as primers, the TM2 fragment with homology to the pet-28a linear vector was amplified; using the pet-28a empty plasmid (purchased from Yixiang Biotechnology (Tianjin) Co., Ltd.) as a template and TM2-line-S and TM2-line-A as primers, the pet-28a linear vector with homology to the TM2 fragment was amplified, and the TM2 gene fragment and the linear vector were connected by recombinase to obtain a plasmid carrying the TM2 gene.

[0065] ⑩ has the same operation method as in step ⑨, except that the primers used are serB-S / serB-A, serB-line-S / serB-line-A, and the plasmid template used is the plasmid constructed in step ⑨.

[0066] Any recombinase can be used to connect the linearized vector and the gene fragment. In this embodiment, the ClonExpress® rapid cloning technology of Nanjing Novogene Biotechnology Co., Ltd. is used to construct the overexpression plasmid. The nucleotide sequence of the plasmid pTm-serB finally obtained is shown in the sequence table SEQ ID NO.13.

[0067] Example 3

[0068] Using the strain described in Example 1 E.coli HTP20The specific steps of fermentation production of 5-hydroxytryptophan in a 5L mechanically stirred fermenter are as follows:

[0069] (1) Slant culture: Take the 5-hydroxytryptophan producing strain and inoculate it on the slant medium as the first generation slant culture, and culture it at 36-37℃ for 12 hours; the slant medium should be the universal LB solid medium; take the colony of the first generation slant culture and inoculate it on the new slant medium as the second generation slant culture, and culture it at 36-37℃ for 10 hours;

[0070] (2) Seed culture: The bacterial moss in the second generation slant was washed with sterile water and transferred to the seed culture medium. The culture temperature was 36°C. A 5L bioreactor was used to control the automatic flow addition of 25% ammonia solution to maintain the pH of the seed solution at 7.0±0.2, the dissolved oxygen value at 40-50%, and the OD of the seed solution at 1.5-2.0. 600nm When the pH value reaches 20, the next step of fermentation culture is carried out. The seed culture medium used is: glucose 30 g / L, yeast extract powder 4 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 3 g / L, anhydrous magnesium sulfate 1 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate monohydrate 10 mg / L, V H 0.5 mg / L, V B1 0.5 mg / L, the rest is water;

[0071] (3) Fermentation culture: the inoculation amount was 20%, the culture temperature was 36°C, and the pH of the seed solution was maintained at 7.0±0.2 by automatically adding 25% ammonia solution through the bioreactor control, and the dissolved oxygen value was 30%-50%. The fermentation medium used was: glucose 20 g / L, yeast extract 4 g / L, peptone 2 g / L, ammonium sulfate 4 g / L, potassium dihydrogen phosphate 3 g / L, anhydrous magnesium sulfate 2 g / L, ferrous sulfate heptahydrate 60 mg / L, manganese sulfate monohydrate 30 mg / L, V H 0.5 mg / L, V B1 1 mg / L, and the rest is water; when the glucose in the culture medium is consumed, 80% (m / v) glucose solution is added to maintain the glucose concentration in the fermentation medium at 0.1-1g / L; the fermentation period is 32 h.

[0072] like Figure 2 As shown in the figure, the production of 5-hydroxytryptophan in a 5 L fermenter reached 16.8 g / L after 32 h of fermentation. E. coli HTP20 It is a production strain that synthesizes 5-hydroxytryptophan from scratch using glucose as raw material. It does not require the addition of precursor 5-hydroxytryptophan or antibiotics and has obvious technical advantages.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A 5-hydroxytryptophan producing strain, characterized in that: In strain E.coli HTP11 Subtilis subtilis spore-derived genes F bsu K316Q Mutated genes, upregulated strains E.coli HTP11 of guaB , guA , gmk , ndk The transcription level of the gene was increased by heterologous introduction of the gapX Gene, missing serB Gene, tnaB Gene, aroP Gene, up-regulated inner membrane efflux transporter Yj Y The transcription level of the gene was measured by electroporation into the pTm-serB plasmid. E. coli HTP11 The strain in patent publication number CN115948311A E.coli HTP11 , F bsu K316Q The nucleotide sequence of the mutant gene is shown in the sequence table SEQ ID NO.

3. gapX The nucleotide sequence of the gene is shown in the sequence listing as SEQ ID NO.4, and the nucleotide sequence of the pTm-serB plasmid is shown in the sequence listing as SEQ ID NO.

13.

2. The 5-hydroxytryptophan producing strain according to claim 1, characterized in that: Subtilis subtilis spores F bsu K316Q The mutant gene was integrated into the genome and the expression was enhanced by using the trc promoter; gapX The gene is integrated into the genome and the expression is enhanced by using the trc promoter; guaB Gene, guA Gene, Yj Y Genes are overexpressed on the genome and the expression is enhanced by using the trc promoter; gmk Gene, ndk Genes are overexpressed on the genome and the expression is enhanced using the lac promoter; Knockout strain genome serB Gene, tnB Gene, aroP Gene.

3. The 5-hydroxytryptophan producing strain according to claim 2, characterized in that: The nucleotide sequence of the lac promoter is shown in the sequence listing SEQ ID NO.1, the nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.2, guaB The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.

5. guA The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.

6. gmk The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.

7. ndk The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.

8. Yj Y The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.

9. serB The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.

10. tnB The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.

11. aroP The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

12.

4. The method for constructing the 5-hydroxytryptophan producing strain according to any one of claims 1 to 3, characterized in that: by E. coli HTP11 As a starting strain, the specific steps are as follows: (1) The genome is integrated with a gene encoding a gene derived from Bacillus subtilis F bsu K316Q The mutant gene was driven by the trc promoter; (2) The E. coli-derived guaB Gene, guA gene, and driven by the trc promoter; (3) The E. coli-derived gmk Gene, ndk gene, and driven by the lac promoter; (4) A gene encoding 3-phosphoglyceraldehyde dehydrogenase from Corynebacterium glutamicum was integrated into the genome. gapX gene, and driven by the trc promoter; (5) The gene encoding phosphoserine phosphatase was knocked out in the genome serB Gene; (6) The gene encoding the tryptophan transporter was knocked out in the genome tnB Gene 、aroP Gene; (7) The gene encoding the efflux protein is integrated into the genome Yj Y Gene; (8) Electroporate the pTm-serB plasmid into the transformed strain in step (7).

5. Use of the 5-hydroxytryptophan producing strain according to any one of claims 1 to 3 in the fermentation production of 5-hydroxytryptophan.

6. The use according to claim 5, characterized in that: The method for producing 5-hydroxytryptophan by fermentation in a fermenter is as follows: (1) Slant culture: Take the 5-hydroxytryptophan producing strain and inoculate it on the slant medium as the first generation slant culture, and culture it at 36-37℃ for 12-14h; take the colony of the first generation slant culture and inoculate it on a new slant medium as the second generation slant culture, and culture it at 36-37℃ for 10-12h; (2) Seed culture: Wash the second-generation slant with sterile water and transfer it to the seed culture medium. The culture temperature is 34-36°C. The bioreactor is controlled to automatically add ammonia solution to maintain the pH of the seed solution at 7.0±0.2, the dissolved oxygen value at 40-50%, and the OD of the seed solution at 1. 600nm When the temperature reaches 15-20, the next step of fermentation culture is carried out; (3) Fermentation culture: The inoculation amount is 20%, the culture temperature is 34-36°C, and the bioreactor is controlled to automatically add ammonia solution to maintain the seed liquid pH at 6.2-7.2 and the dissolved oxygen value at 30%-50%.

7. The use according to claim 6, characterized in that: The slant culture medium used in step (1) is a universal LB solid culture medium.

8. The use according to claim 6, characterized in that: The seed culture medium used in step (2) is: 20-40 g / L glucose, 4-6 g / L yeast extract powder, 2-5 g / L ammonium sulfate, 1-3 g / L potassium dihydrogen phosphate, 0.5-2 g / L anhydrous magnesium sulfate, 20-40 mg / L ferrous sulfate heptahydrate, 10-20 mg / L manganese sulfate monohydrate, V H 0.1-0.5 mg / L, V B1 0.5-1 mg / L, the rest is water.

9. The use according to claim 6, characterized in that: The fermentation medium used in step (3) is: 20-40 g / L glucose, 3-6 g / L yeast extract powder, 2-5 g / L peptone, 4-8 g / L ammonium sulfate, 3-6 g / L potassium dihydrogen phosphate, 2-4 g / L anhydrous magnesium sulfate, 40-60 mg / L ferrous sulfate heptahydrate, 20-40 mg / L manganese sulfate monohydrate, V H 0.1-0.5 mg / L, V B1 0.5-1 mg / L, the rest is water.

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